Download 1 General IntroductIon Pulmonary arterIal

1
General Introduction
Pulmonary Arterial
Hypertension and Right
Heart Failure
M.L. Handoko1,2, A. Vonk-Noordegraaf2, W.J.
Paulus1
Departments of 1Physiology and 2Pulmonology,
VU University Medical Center / Institute for
Cardiovascular Research, Amsterdam, The
Netherlands
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Pulmonary hypertension is a hemodynamic and pathophysiological condition, defined as a mean
pulmonary artery pressure of 25 mmHg or higher at rest, as assessed by right heart catheteriza-
Chapter 1
Pulmonary arterial hypertension
tion.1,2 Although pulmonary hypertension, secondary to congestive heart failure or COPD, is
commonly observed in patients, its primary form - pulmonary arterial hypertension (throughout
this thesis abbreviated as PH) - is quite rare, with an estimated prevalence of 15 per million.3 PH
mostly affects women (male/female ratio of 1:2) and may develop at all ages (peak-prevalence
at age of 50 years).3 The aetiology of PH is unknown, but some genetic risk factors have been
identified (BMPR2-mutations), and PH has also been associated with congenital heart disease,
connective tissue diseases, drugs and toxins (anorexigens), HIV infection, portal hypertension,
and hemoglobinopathies.1,2
PH is characterized by excessive remodeling of the pulmonary vasculature, resulting in
increased pulmonary vascular resistance and right ventricular (RV) afterload, which leads to
extensive RV remodeling and ultimately to right heart failure and premature death (Figure 1).
Unfortunately, no definite cure exists. Nevertheless, in the last decade several new drugs were
introduced for the treatment of PH. By different pharmacological actions, prostacyclines, endothelin receptor antagonists and phosphodiesterase-5 inhibitors selectively lower pulmonary
vascular resistance, thereby secondarily improving right heart function, resulting in significant
clinical improvement.4 However, even under maximal treatment, the prognosis of PH-patients
remains grim, with a 5-year survival of about 50%.2
Figure 1
Pulmonary arterial hypertension is characterized by excessive pulmonary vascular remodeling, resulting in narrowing
of the pulmonary vessels. This leads to increased pulmonary vascular resistance and right ventricular (RV) afterload, RV
remodeling and ultimately to right heart failure.
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General introduction
Prognostic importance of right ventricular (dys)function
and remodeling in pulmonary arterial hypertension
The degree of symptoms (i.a. breathlessness, fatigue, peripheral edema) and survival in patients
with PH are strongly related to the severity of RV dysfunction.1,2 The first PH-registry with
prospective follow-up already identified mean pulmonary artery pressure, right atrial pressure
and cardiac index as independent prognostic variables, all indices of RV function.5 Of note,
indices that reflect pulmonary arterial function and vascular integrity (like pulmonary vascular resistance and diffusion capacity) were of limited prognostic value in this study.5 In other
words, it was not the extent of the pulmonary vascular remodeling per se, but maladaptation
of the right ventricle itself that leads to premature death in PH.6 These observations have been
confirmed by others,7 and are currently still valid, regardless of the introduction of several new
PH-medications since.8,9
Right heart failure was first presumed an inevitable consequence of PH, and therefore not
considered a potential target for therapy. However, recently it has been recognized that the
adequacy of RV compensatory response (i.e. preservation of stroke volume) is quite variable
amongst individuals,9 and this has triggered the interest for therapies that could directly improve
right heart function.10,11
Therapy for right heart failure: parallels with left heart
failure treatments?
Currently, no therapy exists that aims to directly improve right heart function. Extensive RV
remodeling (hypertrophy and dilatation) with neurohumoral activation,12,13 and (inter)ventricular dyssynchrony14,15 are often observed in patients with PH and right heart failure (Figure 2).
Therefore, intervening in processes underlying RV remodeling, and diminishing of ventricular
dyssynchrony might offer clinical benefit.
In “left” heart failure (heart failure related to impaired left ventricular (LV) function), comparable therapeutic strategies have been demonstrated very successful in multiple large welldesigned clinical trials. The beneficial effects of β-blockers (often combined with ACE-inhibitors
and aldosteron antagonists) in left heart failure are well-established, as well as the role of exercise
training.16,17 These interventions (partially) neutralize the detrimental effects of chronic overactivation of the β-adrenergic system, which results in reversed LV remodeling, improved LV
function, and a significant reduction of cardiac-related morbidity and mortality. Comparable
results have been achieved with cardiac resynchronization therapy.16,17 Here, advanced biventricular pacemakers technology is used to synchronize LV contraction, resulting in improved
LV function.
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Chapter 1
Figure 2
Mid-ventricular short-axis magnetic resonance images of a healthy control subject (A–C) and a patient with severe
pulmonary arterial hypertension (mean pulmonary arterial pressure: 76 mmHg; D–F). Images were acquired at
end-diastole, end-systole, and ~100 ms after end-systole (early diatole). The right ventricle (RV) of the PH-patient
is hypertrophied and dilated, (inter)ventricular dyssynchrony is present, most easily appreciated by bulging of the
interventricular septum into the left ventricle (arrow in F) at early-diastole.20
It is tempting to extrapolate the therapeutic recommendations for left heart failure to PH-induced
right heart failure, even though there are important structural, functional and developmental
differences between the left and right ventricle, and important differences in etiology between
left and right heart failure (ischemic vs. pressure-overload, respectively).6 Well-established left
heart failure therapies have never been tested in the setting of PH and right heart failure. In this
thesis, we therefore investigated the therapeutic potential of exercise training, β-blocker therapy
and resynchronization therapy (RV-pacing) for PH-induced right heart failure.
The monocrotaline rat model for pulmonary arterial
hypertension
Experimental animal studies are often the only way to comprehensively test novel treatment
strategies. By providing a proof-of-concept, experimental studies facilitate the identification of
promising new therapies, which are subsequently investigated in (PH-)patients.
In this thesis, we used the monocrotaline rat model, a well-established model for PH and
right heart failure.18,19 In this model, PH is induced by a single subcutaneous injection of
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General introduction
monocrotaline. In its native form, monocrotaline reacts little with living tissue, but becomes
activated by liver cytochromes to monocrotaline-pyrrole (MCT-P), a highly reactive and instable
metabolite, with a short half-life of only a few seconds. MCT-P is temporarily stabilized by binding to erythrocytes. During gas-exchange in the lungs, MCT-P detaches from its carrier, where
it “selectively” damages the lung endothelium. In the following weeks, gradual but extensive
pulmonary vascular remodeling occurs, resulting in a marked increase in RV afterload. This triggers RV remodeling – including RV hypertrophy, dilatation with neurohumoral activation, and
ventricular dyssynchrony – which eventually leads to overt right heart failure, like clinical PH.
Aim and outline of this thesis
In this thesis, several novel treatment strategies for right heart failure were tested in an experimental model of PH, inspired by lessons from the left heart.
In Chapter 2, we characterized our model, and demonstrated that hemodynamic alterations
during the progression of PH in rats can be comprehensively monitored over time, by the combined use of echocardiography and a refined RV pressure-telemetry technique.
In Chapter 3, we studied the effects of exercise training in a rat model using two distinct clinical
phenotypes of PH, as the role of exercise training in current clinical management of patients with
PH is unclear. In particular, it is uncertain if exercise training is beneficial for all PH-patients,
including patients with right heart failure.
In Chapter 4, β-blocker therapy was evaluated. Beta-blocker therapy is a well-established
treatment for left heart failure, but strongly discouraged in PH, due to its negative inotropic
and chronotropic effects. We hypothesized that β-blocker therapy in PH is tolerated, if carefully
introduced, and could have favourable effects on mortality and cardiac function.
In Chapter 5, the effects of RV-pacing on right heart function were studied in an isolated heart
preparation. At end-stage PH, (inter)ventricular dyssynchrony is often observed. This results in
inefficient pumping of the heart, which may be partially restored by resynchronization therapy.
Finally in Chapter 6, the results presented in this thesis are placed in a broader perspective.
More specifically, the relevance of well-established therapies for left heart failure are discussed as
novel therapeutic strategies for right heart failure secondary to PH, that warrants further clinical
investigation.
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S, Gibbs JS, Gomez-Sanchez MA, Jondeau G, Klepetko W, Opitz C, Peacock A, Rubin L, Zellweger
M, Simonneau G. Guidelines for the diagnosis and treatment of pulmonary hypertension. Eur Respir
J 2009;34:1219-1263.
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Chapter 1
2. McLaughlin VV, Archer SL, Badesch DB, Barst RJ, Farber HW, Lindner JR, Mathier MA, McGoon
MD, Park MH, Rosenson RS, Rubin LJ, Tapson VF, Varga J. ACCF/AHA 2009 expert consensus
document on pulmonary hypertension a report of the American College of Cardiology Foundation
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Pulmonary Hypertension Association. J Am Coll Cardiol 2009;53:1573-1619.
3. Humbert M, Sitbon O, Chaouat A, Bertocchi M, Habib G, Gressin V, Yaici A, Weitzenblum E,
Cordier JF, Chabot F, Dromer C, Pison C, Reynaud-Gaubert M, Haloun A, Laurent M, Hachulla
E, Simonneau G. Pulmonary arterial hypertension in France: results from a national registry. Am J
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4. Humbert M, Sitbon O, Simonneau G. Treatment of pulmonary arterial hypertension. N Engl J Med
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5. D’Alonzo GE, Barst RJ, Ayres SM, Bergofsky EH, Brundage BH, Detre KM, Fishman AP, Goldring
RM, Groves BM, Kernis JT. Survival in patients with primary pulmonary hypertension: results from
a national prospective registry. Ann Intern Med 1991;115:343-349.
6. Handoko ML, de Man FS, Allaart CP, Paulus WJ, Westerhof N, Vonk-Noordegraaf A. Perspectives on
novel therapeutic strategies for right heart failure in pulmonary arterial hypertension: lessons from
the left heart. Eur Respir Rev 2010;19:72-82.
7. Sandoval J, Bauerle O, Palomar A, Gomez A, Martinez-Guerra ML, Beltran M, Guerrero ML. Survival
in primary pulmonary hypertension: validation of a prognostic equation. Circulation 1994;89:17331744.
8. Sitbon O, Humbert M, Nunes H, Parent F, Garcia G, Herve P, Rainisio M, Simonneau G. Long-term
intravenous epoprostenol infusion in primary pulmonary hypertension: prognostic factors and
survival. J Am Coll Cardiol 2002;40:780-788.
9. van Wolferen SA, Marcus JT, Boonstra A, Marques KM, Bronzwaer JG, Spreeuwenberg MD, Postmus
PE, Vonk-Noordegraaf A. Prognostic value of right ventricular mass, volume, and function in idiopathic pulmonary arterial hypertension. Eur Heart J 2007;28:1250-1257.
10. Voelkel NF, Quaife RA, Leinwand LA, Barst RJ, McGoon MD, Meldrum DR, Dupuis J, Long CS,
Rubin LJ, Smart FW, Suzuki YJ, Gladwin M, Denholm EM, Gail DB. Right ventricular function
and failure: report of a National Heart, Lung, and Blood Institute working group on cellular and
molecular mechanisms of right heart failure. Circulation 2006;114:1883-1891.
11. Ghofrani HA, Barst RJ, Benza RL, Champion HC, Fagan KA, Grimminger F, Humbert M, Simonneau
G, Stewart DJ, Ventura C, Rubin LJ. Future perspectives for the treatment of pulmonary arterial
hypertension. J Am Coll Cardiol 2009;54:S108-S117.
12. Kurzyna M, Torbicki A. Neurohormonal modulation in right ventricular failure. Eur Heart J (Suppl)
2007;9:H35-H40.
13. Schrier RW, Bansal S. Pulmonary hypertension, right ventricular failure, and kidney: different from
left ventricular failure? Clin J Am Soc Nephrol 2008;3:1232-1237.
14. Marcus JT, Gan CT, Zwanenburg JJ, Boonstra A, Allaart CP, Gotte MJ, Vonk-Noordegraaf A. Interventricular mechanical asynchrony in pulmonary arterial hypertension: left-to-right delay in peak
shortening is related to right ventricular overload and left ventricular underfilling. J Am Coll Cardiol
2008;51:750-757.
15. Dohi K, Onishi K, Gorcsan J III, Lopez-Candales A, Takamura T, Ota S, Yamada N, Ito M. Role of
radial strain and displacement imaging to quantify wall motion dyssynchrony in patients with left
ventricular mechanical dyssynchrony and chronic right ventricular pressure overload. Am J Cardiol
2008;101:1206-1212.
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General introduction
16. Hunt SA, Abraham WT, Chin MH, Feldman AM, Francis GS, Ganiats TG, Jessup M, Konstam MA,
Mancini DM, Michl K, Oates JA, Rahko PS, Silver MA, Stevenson LW, Yancy CW. 2009 focused
update incorporated into the ACC/AHA 2005 Guidelines for the Diagnosis and Management of
Heart Failure in Adults: a report of the American College of Cardiology Foundation/American Heart
Association Task Force on Practice Guidelines: developed in collaboration with the International
Society for Heart and Lung Transplantation. Circulation 2009;119:e391-e479.
17. Dickstein K, Cohen-Solal A, Filippatos G, McMurray JJ, Ponikowski P, Poole-Wilson PA, Stromberg
A, van Veldhuisen DJ, Atar D, Hoes AW, Keren A, Mebazaa A, Nieminen M, Priori SG, Swedberg
K, Vahanian A, Camm J, De CR, Dean V, Dickstein K, Filippatos G, Funck-Brentano C, Hellemans
I, Kristensen SD, McGregor K, Sechtem U, Silber S, Tendera M, Widimsky P, Zamorano JL. ESC
Guidelines for the diagnosis and treatment of acute and chronic heart failure 2008: the Task Force
for the Diagnosis and Treatment of Acute and Chronic Heart Failure 2008 of the European Society
of Cardiology. Developed in collaboration with the Heart Failure Association of the ESC (HFA) and
endorsed by the European Society of Intensive Care Medicine (ESICM). Eur Heart J 2008;29:23882442.
18. Stenmark KR, Meyrick B, Galie N, Mooi WJ, McMurtry IF. Animal models of pulmonary arterial
hypertension: the hope for etiological discovery and pharmacological cure. Am J Physiol Lung Cell
Mol Physiol. 2009;297:L1013-L1032.
19. Wilson DW, Segall HJ, Pan LC, Lame MW, Estep JE, Morin D. Mechanisms and pathology of monocrotaline pulmonary toxicity. Crit Rev Toxicol 1992;22:307-325.
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2
A Refined RadioTelemetry Technique to
Monitor Right Ventricle
or Pulmonary Artery
Pressures in Rats:
A Useful Tool in
Pulmonary Hypertension
Research
M.L. Handoko1,2, I. Schalij1,2, K. Kramer3, A.
Sebkhi4, P.E. Postmus2, W.J. van der Laarse1,
W.J. Paulus1, A.Vonk-Noordegraaf2
Departments of 1Physiology and 2Pulmonology,
VU University Medical Center / Institute for
Cardiovascular Research, Amsterdam, the
Netherlands. 3Department of Health, Safety and
Environmental Affairs, VU University, Amsterdam,
the Netherlands. 4Section on Clinical Pharmacology,
Faculty of Medicine, Imperial College, Hammersmith
Hospital, London, United Kingdom
Pfugers Archiv 2008; 455: 951-959
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RV pressure-telemetry
ABSTRACT
Implantable radio-telemetry methodology, allowing for continuous recording of pulmonary hemodynamic, has previously been used to assess effects of therapy on development and treatment
of pulmonary arterial hypertension. In the original procedure, rats were subjected to invasive
thoracic surgery, which imposes significant stress that may disturb critical aspects of the cardiovascular system and delay recovery. In the present study, we describe and compare the original
trans-thoracic approach with a new, simpler trans-diaphragm approach for catheter placement,
which avoids the need for surgical invasion of the thorax.
Satisfactory overall success rates up to 75% were achieved in both approaches, and right ventricular pressures, heart and respiratory rates normalized within two weeks. However, recovery
was significantly faster in trans-diaphragm than in trans-thoracic operated animals (6.4 ±0.5 vs.
9.5 ±1.1 days, respectively; p < 0.05). Stable right ventricular pressures were recorded for more
than four months and pressure changes, induced by monocrotaline or pulmonary embolisms,
were readily detected.
The data demonstrate that right ventricular pressure-telemetry is a practicable procedure and
a useful tool in pulmonary hypertension research in rats, especially when used in combination
with echocardiography. We conclude that the described trans-diaphragm approach should be
considered as the method of choice, for it is less invasive and simpler to perform.
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INTRODUCTION
Over the last decade, new medical treatments became available for the treatment of pulmonary
arterial hypertension (PH) patients, and this development set off a renewed interest in the
pathophysiology and hemodynamics of the pulmonary circulation.1-3 Experimental PH-studies
(RV) pressure in anesthetized, open-chest animals undergoing artificial respiration, which does
not allow determining changes of pressures in time. Measuring pulmonary hemodynamic have
improved considerably since RV pressure-telemetry was firstly introduced by Hess, et al.,4 as it
allows for continuous monitoring of RV or PA pressures, without introducing artifacts caused
Chapter 2
are still largely confined to single measurements of pulmonary arterial (PA) or right ventricular
by stress or anesthetics.5 Moreover, animals fitted with telemetry can serve as their own controls,
thus permitting the use of smaller groups of animals than is possible with the single measurement approach in anesthetized animals.5 Although many investigators have embraced this new
technology with enthusiasm and despite its proven potential, studies actually using this methodology in rats are scarce.4,6-9
One possible explanation for this discrepancy is that the original procedure was only described
briefly before,4 complicating its implementation for potential new users. Secondly, the described
trans-thoracic approach involves RV catheterization through complex and highly invasive thoracotomy.4 In the present study, we hypothesized that RV catheterization performed via an opening
in the diaphragm, thereby omitting a thoracotomy, may be a simpler, less invasive alternative.
In the present report, we provide a detailed description of the alternative trans-diaphragm approach together with a comparison with the original trans-thoracic approach. Furthermore, we
illustrate its usefulness when studying the pulmonary circulation, by presenting novel comprehensive pulmonary and cardiac hemodynamic data during the development of monocrotalineinduced PH.
METHODS
All experiments were approved by the Institutional Animal Care and Use Committee of the VU
University, and were conducted in accordance with the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes, and the Dutch
Animal Experimentation Act.
Animals
Thirty-eight male Wistar rats were used (250-300 g; Harlan, Horst, the Netherlands), of which
33 were operated and 5 served as controls (no surgery). The animals were allowed to adapt to
their new environment for at least one week before surgery. The animals were conventionally
housed in pairs under controlled conditions (temperature 21-22 °C; humidity 60-65 %; 12:12 hr
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RV pressure-telemetry
light-dark cycle) and had free access to filtered water and standard rat chow (Global 2016, Harlan
Teklad, Blackthorn-Bicester, England).
Telemetry system
An implantable radio-telemetry system for blood pressure measurements for small laboratory
animals was used, comprising a radio-transmitter (TA11PA-C40) fitted with a 10 cm long catheter (Data Science International (DSI), St. Paul MN). Transmitters were turned on the day before
implantation and stored in sterilized saline, following the manufacturer’s instructions (http://
www.datasci.com/information/index.asp).
The pressure was monitored continuously during surgery to ensure proper position of the tip
of the pressure-catheter. After successful implantation of the transmitter, RV or PA pressures and
locomotor activity were recorded for 10 seconds every 5 minutes. From the pressure recordings
mean, systolic and diastolic pressure, heart rate, respiratory rate and circadian rhythm were
subsequently derived (Dataquest A.R.T. software 4.0, DSI).
Pre-operative care and anesthesia
All animals received pre-surgical intramuscular buprenorphine analgesia (0.10 mg/kg; ScheringPlough, Maarssen, the Netherlands). For general anesthesia, isoflurane (2.0% in 1:1 O2/air mix;
Pharmachemie, Haarlem, the Netherlands) was used via induction chamber and tracheal intubation (16-G x 51 mm Teflon tube; ventilator settings: breathing frequency 80 /min, pressures 9/0
cmH2O, inspiratory/expiratory ratio 1:1). For extra local analgesia, lidocaine spray (100 mg/ml;
AstraZeneca, Zoetermeer, the Netherlands) was applied at the surgical region. Animals were
maintained under anesthesia for an average time of about 80 minutes.
Animals were placed on a heating pad to maintain body temperature, and positioned in dorsal
recumbency. Hydromellose drops (0.3%; Ratiopharm, Zaandam, the Netherlands) were applied
to prevent drying of the eyes. Following shaving and disinfection with 70% ethanol of the chest
and abdomen, animals were covered with sterile incision foil and the animals were then covered
with sterile incision foil (Opraflex, Lohmann & Rauscher, Almere, the Netherlands). A surgical
microscope was used (magnification 16-64x; Carl Zeiss, Sliedrecht, the Netherlands) for optimal
view.
Surgery: trans-thoracic approach
Implantation of the telemetry transmitter in the abdomen
The abdominal cavity was accessed via a 5-cm midline laparotomy, starting just below the xiphoid
process. The transmitter was placed in the peritoneal cavity, with the catheter pointing caudally
to prevent liver injury and tissue reaction. The abdominal cavity was covered with gauzes soaked
in warm saline and left open until the end of the procedure.
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Routing of the pressure-catheter to the right ventricle
The heart was exposed by means of a left thoracotomy performed at the sixth intercostal space
and midclavicular line. The thorax was opened with blunt scissors, respecting the anatomy of
the overlying muscle layers and cautiously avoiding injury to the lungs. The pressure-catheter
was then tunnelled subcutaneously from the peritoneal cavity to the opening in the thorax and
heart through a wider, 2 x 2 cm window. The pericardium was then opened with two dressing
forceps.
Catheterization of the right ventricle
Chapter 2
temporarily laid aside. Four individual small hooks were used to retract the ribs and expose the
A superficial purse-string (6-0 Prolene, Ethicon, St-Stevens-Woluwe, Belgium) was placed on
the right ventricle free wall, near the apex. Through the purse-string, the right ventricle was
punctured with a 19-G syringe needle in the direction of the RV outflow tract, avoiding any
coronary vessel. After removing the needle, the site was wiped with a sterile cotton stick. The
tip of the catheter was then carefully inserted into the right ventricle, using a vessel canulation
forceps (0.5-1.0 mm outer diameter, Fine Science Tools, Heidelberg, Germany), avoiding loss
of gel from the tip at all times (Figure 1A). Live pressure waveform trace confirmed proper RV
catheterization (RV systolic/diastolic pressures are typically ~25/1 mmHg). Depending on the
protocol, the pressure-catheter was subsequently advanced for about 2 cm more, positioning the
tip of the catheter beyond the pulmonary valves. Again, live pressure waveform trace confirmed
correct positioning of the pressure-catheter in the pulmonary artery (PA systolic/diastolic pressures are typically ~25/10 mmHg; Figure 2A). Finally, the catheter was fixed by the purse-string
and a small drop of tissue adhesive at the site of insertion (10 μl dispensed by a pipette; Vetbond,
3M, St Paul MN).
Closing of the chest and abdomen
After the catheterization, blood clots if any were removed from the pleural cavity with a moist
cotton stick. The ribs were approximated with two individual sutures (5-0 Vicryl, Ethicon). To
promote full expansion of the lungs, a small burst of positive pressure was applied (maximally 10
cmH2O for 1 second). A chest tube (18-G), used as air outlet, was removed after proper thorax
excursions were observed. The chest muscles were laid back in layers (no suturing necessary).
Then, the transmitter was sutured to the abdominal muscle (5-0 Perma-Hand, Ethicon), incorporating the suture rib of the device. The abdominal wall was sutured in a running subcuticular
pattern (5-0 Vicryl). Finally, the skin of the chest and abdomen was closed with a running suture
(5-0 Vicryl).
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RV pressure-telemetry
Figure 1
A) Trans-thoracic approach: a left thoracotomy was performed, and the pressure-catheter was subcutaneously tunnelled
and then inserted into the right ventricle (arrow). Shown is a detailed view of an opened thorax on the catheterised
heart. B) Trans-diaphragm approach: via a laparotomy the diaphragm was opened, and the pressure-catheter was
inserted into the right ventricle (arrow). The catheterized heart can be seen behind the opened diaphragm.
Figure 2
A
B
A) Live pressure recordings while advancing the pressure-catheter into the pulmonary artery. Notice the stepwise
increase in diastolic pressures when maneuvering the catheter beyond the pulmonary valves (arrow), whereas the
systolic pressures remained unchanged. B) Live pressure recordings while intravenously injecting a bolus of microspheres
(arrow; 19 μm, 1.5 million/100g). Upon embolization a significant increase in systolic, diastolic and developed RV
pressures was observed. Furthermore, in both recordings the effect of breathing is noticeable.
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Surgery: trans-diaphragm approach
Opening of abdomen and the diaphragm
The abdominal cavity was accessed via a 5-cm midline laparotomy, with the abdominal wall
retracted by two individual hooks. To expose the diaphragm, a retraction suture was used to lift
with wet gazes, and the liver was gently pushed down by the weight of a blade holder. A small
midline incision in the diaphragm was then made, with small sharp scissors, from the xiphoid
process until the tendinous part of the diaphragmatic membrane. The two sides of the diaphragm
were held aside with two retraction sutures, providing optimal exposure of the heart (Figure 1B).
Chapter 2
the xiphoid process during the whole procedure. The visceral organs were covered temporarily
Catheterization of the right ventricle
RV catheterization was performed similarly as described above. In short, the pericardium
was opened by two dressing forceps, and a purse-string was placed just right of the apex. The
pressure-catheter was then inserted into the right ventricle through a small puncture made with
a syringe needle and fixed in place.
Closing the diaphragm and abdomen
After catheterization, blood clots in the thorax if any were removed. The diaphragm was closed
with a running suture (5-0 Vicryl), starting from the ventral side, with the pressure-catheter
sticking out the diaphragm at the dorsal end of the incision. After that, the retraction suture
through the xiphoid process was removed. To promote full expansion of the lungs, a small burst
of positive pressure was applied, as described above, with the chest tube between the sutures of
the diaphragm, until proper thorax excursions were visually confirmed. To further secure closure
of the diaphragm, a small amount of tissue adhesive (Vetbond, 3M) was applied. Finally, the
transmitter was implanted in the peritoneal cavity, fixed to the abdominal wall with the catheter
facing caudally, and the abdomen was closed, as described above.
Post-operative care
To compensate for loss of fluids, all animals received warm sterile saline at the end of the procedure (5 ml intraperitoneal). Following final skin closure, the animals were allowed to regain
consciousness.
In the first 24 hours of recovery, the animals were housed in individual cages. Each cage was
placed halfway on a heating pad, in such a way that half of the cage was maintained above it
and the other at room temperature. During this period, the animals were monitored three
times, received post-surgical analgesia when clinically indicated (buprenorphine 0.10 mg/kg
subcutaneous), and were provided with drinking gel pads and softened rat chow. After 24 hours,
the animals were conventionally housed in pairs for a full recovery and they were inspected
and weighted daily. Animals were considered fully recovered from surgery when their appear23
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RV pressure-telemetry
ance and behavior were normal, their surgical wounds healed, their pre-surgical body weight
regained, when the pressures normalized and circadian rhythm was restored. The study period
was ended after four months.
Experimental protocol
To induce acute and chronic pulmonary hypertension, 4 animal received an intravenous bolus
injection of microspheres10 (tail; 1.5 million /100g body mass in sterile saline; polystyrene, mean
diameter 19 µm (#7520), Duke Scientific, Fremont CA, USA), and 3 animals monocrotaline subcutaneous11 (80 mg/kg in sterile saline; Sigma-Aldrich, Zwijndrecht, the Netherlands), respectively. Experimental interventions were performed no earlier than two weeks after the surgery.
RV echocardiography
After a two-week recovery, trans-thoracic echocardiographic measurements (ProSound SSD4000 system equipped with a 13-Mhz linear transducer (UST-5542), Aloka, Tokyo, Japan) were
performed on anesthetized but spontaneously breathing rats (isoflurane 2.0% in 1:1 O2/air mix),
according to the standards of the American Society of Echocardiography.12,13 To minimize the
effects of isoflurane on cardiac function, the time-under-anesthesia was set to a maximum of
fifteen minutes.14
The measurements were compared with untreated rats to check for possible adverse effects
related to the procedure. Cardiac output, tricuspid annular plane systolic excursion (TAPSE), RV
wall thickness, and RV end-diastolic diameter were measured, as described before by Hardziyenka, et al.15 TAPSE is a parameter for RV systolic function, which is measured in the apical
four-chamber view of the heart and expresses the displacement of the lateral tricuspid annulus
towards the apex during systole.12
Estimation of pulmonary vascular resistance and RV power output
Additional echocardiographic measurements were performed in monocrotaline-treated rats, and
untreated but operated controls, just before the injection, two weeks after injection and when
the first clinical signs of RV heart failure developed (as indicated by weight loss, dyspnea, and
lethargy, after which the rats were euthanized). Pulmonary vascular resistance (PVR) and RV
power output (RV-power) were estimated, by combining telemetric pressure-data and echocardiographic flow-data.
PVR was estimated by Poiseuille’s law:16,17
PVR ≈ mean PAP / cardiac output ≈ (0.61 * systolic RV pressure + 2 mmHg) / cardiac output.
RV-power was estimated using a simplified pressure-volume analysis, assuming rectangular
pressure-volume loops:16,18
RV-power ≈ systolic RV pressure * stroke volume * heart rate.
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Autopsy
To investigate potential effects of chronic implantation of the telemetry transmitters, the animals
were euthanized at the end of the study protocol by exsanguination under isoflurane (4.0% in
1:1 O2/air mix). Macroscopic examination of RV, lungs, diaphragm and intercostal muscles were
performed, and organ weights (heart, lungs, liver, spleen, kidneys) were measured and compared
analyses.
Statistical analysis
All measurements, unless otherwise stated, are presented as mean ± standard error of the mean
Chapter 2
with controls. Animals treated with monocrotaline or microspheres were excluded from these
(SEM). Normal distribution was verified and Student’s t-test, ANOVA or Fisher Exact test were
used, when appropriate (SPSS 13.0, SPSS, Chicago IL, USA); p < 0.05 was considered statistically
significant.
RESULTS
Success rate
Seventeen animals were operated using the trans-thoracic approach, and 16 animals were operated using the trans-diaphragm approach.
In the trans-thoracic group, 8 animals showed stable pressure signals for the whole study period
of four months. Five animals did not recover from surgery and 4 animals developed instable
pressure signals, caused by clot formation inside the pressure-catheter within two weeks of
recovery. Of the 9 failed procedures, 4 occurred in the first four attempts.
In the trans-diaphragm group, 9 animals showed stable pressure signal for the whole study
period of four months. Six animals did not recover from surgery and 1 animal developed instable pressure signals, caused by clot formation inside the pressure-catheter within two weeks
of recovery. As in the trans-thoracic group, 4 of the failures occurred in the first four attempts.
By discarding the first four animals in each procedure, as we consider this the learning curve,
the overall success rate was 8 of 13 (62%) for the trans-thoracic approach and 9 of 12 (75%) for
the trans-diaphragm approach.
Recovery from surgery
Within a week post surgery, heart and respiratory rates and circadian rhythm normalized in
all rats, and no differences were observed between the two approaches (Table 1). Although all
animals regained their pre-surgical body weight within two weeks, full recovery was significantly
faster in the trans-diaphragm than the trans-thoracic-operated animals (6.4 ±0.5 vs. 9.5 ±1.1
days, respectively; p < 0.05).
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RV pressure-telemetry
Table 1Main results of this study, all values in mean ±SEM
Trans-thoracic approach
Trans-diaphragm
approach
Controls
(n = 17)
(n = 16)
(n = 5)
8 / 13 ( 62%)
7 / 9 (79%)
n.r.
BW to pre-surgical level (days)d
9.5 ±1.1
6.4 ±0.5 *
n.r.
Heart rate (bpm)e
362 ±4
359 ±6
350-400 b 22,23
Respiratory rate (rpm)e
90 ±2
91 ±4
80-100 b 23-25
Circadian rhythm (days)e
3.4 ±0.4
3.7 ±0.6
n.r.
RVSP (mmHg)
25 ±1
24 ±1
21-26 b 8
RVEDP (mmHg)
1.7 ±0.3
2.2 ±0.2
1.0-5.0 b 26
Cardiac output (ml/min)
107 ±5
116 ±7
110 ±5
TAPSE (mm)
3.6 ±0.1
3.7 ±0.2
3.4 ±0.1
RVWT (mm)
1.0 ±0.1
1.0 ±0.1
0.9 ±0.1
RVEDD (mm)
3.6 ±0.1
3.7 ±0.1
3.6 ±0.1
Heart
1.4 ±0.1
1.4 ±0.2
1.3 ±0.1
Lungs
1.5 ±0.1
1.5 ±0.2
1.5 ±0.2
Liver
15.4 ±0.5
15.0 ±0.6
15.1 ±0.5
Spleen
0.65 ±0.03
0.68 ±0.04
0.64 ±0.04
Kidneys
2.4 ±0.2
2.3 ±0.2
2.3 ±0.1
Success ratea
Recovery
Pressurese
RV echocardiographyd
Autopsyf, organ weights (g)
*: p<0.05; n, number of animals; n.r., not relevant.
a
: First four attempts were discarded; b: normal values for Wistar rats, awake and at rest, measured by radio-telemetry; c:
normal values for Wistar rats, measured during acute pressure measurements under anesthesia; d: analyses on surviving
animals only; e: analyses on successfully operated animals only (stable signal); f: analyses on surviving and untreated
animals only (no microspheres or monocrotaline).
Abbreviations: BM, body mass; RVSP, RV systolic pressure; RVEDP, RV end-diastolic pressure; TAPSE, tricuspid annular
plane systolic excursion; RVWT, RV wall thickness; RVEDD, RV end-diastolic diameter.
RV pressures normalized in all animals within two weeks after surgery (RV systolic/diastolic
pressures ~25/1 mmHg), irrespective of the method used (Table 1). PA systolic/diastolic pressures were estimated ~25/10 mmHg (Figure 2A). Activity had no significant effect on the stability
of the pressure signals.
Echocardiography revealed normal cardiac output (~110 ml/min), TAPSE (~3.5 mm), RV wall
thickness (~1.0 mm), and RV end-diastolic diameter (~3.6 mm) in all rats, and no differences
were seen between the differently operated animals and controls (Table 1). Furthermore, other
signs of RV dysfunction, like pericardial effusion and tricuspid regurgitation, were not present
in any animal.
Acute pressure effects of pulmonary embolization by microspheres
RV-telemetry could readily detect acute changes in RV pressure, induced by a bolus injection of
microspheres (Figure 2B). Upon embolization, RV systolic pressures increased from 21 ±1 to 38
±3 mmHg (p < 0.01) and RV diastolic pressures increased from 2 ±1 to 7 ±2 mmHg (p < 0.05).
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A
B
C
D
E
F
Chapter 2
Figure 3
Hemodynamic changes during the development of monocrotaline-induced pulmonary arterial hypertension. Four weeks
after the rats received monocrotaline, clinical signs of right heart failure developed. During this period, systolic pressures
continued to rise (A), while cardiac output could not be maintained (B). The presence of RV remodeling was indicated by
increased RV wall thickness (C) and RV end-diastolic diameter (D). The pulmonary vascular resistance continued to rise
as well (E), while at four weeks, the increase of the RV power output was inadequate to compensate for the dramatically
increased afterload (F).
All values are in mean ±SEM, *: p<0.05; **: p<0.01 vs. to control (post-hoc analyses). Please note that sometimes the
icons conceal the error bars. Abbreviations: MCT (dashed lines): monocrotaline-treated rats; Control (solid lines): control
group (n=3 for both groups). RVSP, RV systolic pressure; RVWT, RV wall thickness; RVEDD, RV end-diastolic diameter; PVR,
pulmonary vascular resistance; RV-power, RV power output.
Hemodynamic changes during the development of monocrotaline-induced
pulmonary arterial hypertension
Changes in hemodynamic parameters during the development of monocrotaline-induced PH
are shown in Figure 3 & Figure 4. Four weeks after monocrotaline-injection, the first clinical
signs of right heart failure developed. During this period, there was a gradual rise in RV systolic
pressure, from 22 ±2 mmHg at baseline, to 38 ±4 mmHg at two weeks, and 72 ±5 mmHg at four
weeks (p < 0.01 vs. control). At four weeks, cardiac output dropped dramatically, from 100 ±7 ml/
min at baseline, to 107 ±8 at two weeks, and 52 ±4 ml/min at four weeks (p < 0.01 vs. control).
27
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RV pressure-telemetry
Figure 4
A
C
B
The development of body mass, RV pressures systolic and cardiac output (triangles) are shown for monocrotaline-treated
rats (injected at day 45; arrow) for a total registration time of 70 days. After 12 days the pre-surgical level of the body
mass was reached, and increased until heart failure developed (A). There is a gradual rise in RV systolic pressures after
injection of monocrotaline, cardiac output remained stable until right heart failure developed (B). The vertical dotted
lines mark the time points: two weeks after recovery of the procedure (day 14), when monocrotaline was injected (day
45), two weeks after injection (day 59), and at sacrifice (day 70) when right heart failure developed. RV pressure recording
remained stable during the whole study period (C). Pressure waveforms of the monocrotaline-treated rat are shown for
day 14, day 45, day 59, and day 70.
In addition, the poor hemodynamic condition of the monocrotaline -treated rats at four weeks
was confirmed by a simultaneous decline in TAPSE from 3.6 ±0.1 mm during the first two weeks
to 1.8 ±0.2 mm at four weeks (p < 0.01 vs. control). During the development of PH, the right
ventricle remodeled initially by increasing RV wall thickness (1.0 ±0.1 mm at baseline, 1.1 ±0.1
mm at two weeks, and 1.3 ±0.1 mm at four weeks; p < 0.01 vs. control), but subsequently by
increasing RV end-diastolic diameters as well (3.5 ±0.1 mm at baseline, 3.6 ±0.1 mm at two
weeks, and 7.0 ±0.5 mm at four weeks; p < 0.01 vs. control).
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During the development of PH, PVR increased continuously, from 1.2 ±0.2 *109 N.m-5.s at
baseline, to 1.9 ±0.2 *109 N.m-5.s at two weeks, and 7.9 ±0.9 *109 N.m-5.s at four weeks (p < 0.01 vs.
control). RV-power was increased at two weeks, but no further increase was seen at four weeks (4.9
±0.7 mW at baseline, 9.1 ±0.9 mW at two weeks, 8.3 ±0.7 mW at four weeks; p < 0.01 vs. controls).
Chronic implantation of the telemetry transmitter in both approaches did not result in any differences in organ weight as compared to control (Table 1), and no apparent pulmonary embolisms
were seen.
Autopsy revealed only local fibrosis of the right ventricle near the insertion point of the catheter
Chapter 2
Autopsy
(Figure 5A). In the trans-thoracic approach, animals showed normal healing of the intercostal
muscles. In the trans-diaphragm approach, no herniation or dehiscence of the diaphragm were
seen in any animal, and often the liver was partially accreted to the healed wound of the diaphragm (Figure 5B).
Figure 5
A
B
A) Autopsy revealed fibrosis only in the near proximity of the insertion point of the pressure-catheter (arrow). B) In the
trans-diaphragm-operated animals, no signs of herniation or dehiscence was seen, and often the liver was partially
accreted to the healed diaphragm. Via a laparotomy the diaphragm was exposed: on the right side, the liver was partially
accreted. One can also observe the pressure-catheter perforating the diaphragm (arrow).
DISCUSSION
The present study describes in detail two surgical techniques to monitor RV and/or PA pressures
over time by radio-telemetry in rats. To our best knowledge, we are the first research group
to describe the trans-diaphragm approach and its comparison with the previously used transthoracic approach. We have demonstrated that:
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RV pressure-telemetry
1) In our hands, both the trans-diaphragm as well as the trans-thoracic approach have satisfactory success rates, especially when considering the complexity of the procedures;
2) The time-to-recovery was significantly shorter with the trans-diaphragm approach than the
trans-thoracic approach;
3) Measured physiological parameters recovered fully in both methods and there were no
permanent detrimental effects on the right ventricle, intercostal muscles or diaphragm;
4) Using RV-telemetry, acute and chronic pressure changes in the pulmonary circulation can
be readily detected, and;
5) RV pressure-telemetry, in combination with echocardiography, allow thorough monitoring
of pulmonary and cardiac hemodynamic changes over time.
Fully recovered animals showed normal RV structure, as expressed by RV wall thickness and
RV end-diastolic diameter, and normal function, as expressed by cardiac output and TAPSE.
Also, in both approaches, RV pressure normalized shortly after surgery, and autopsy revealed
only minor fibrosis in the direct proximity of the catheter. Therefore, permanent detrimental
effects of RV pressure-telemetry on the right ventricle can be excluded for both techniques. In
addition, examination of other organs revealed no abnormalities, as was described by others.4
In animals operated using the trans-diaphragm approach, macroscopic examination revealed
normal wound healing of the diaphragm. Although we have not tested the strength of the healed
diaphragm, others did not find significant differences between a normal (untouched) and a
sutured diaphragm.19 The natural history of small defects in the diaphragm is relatively benign,
as they almost always heal spontaneously,20 especially when the diaphragm is fixated by other
organs.21 Furthermore, the observation of a normal respiratory rate seen in the recovered animals
confirms that the trans-diaphragm approach is safe and a good alternative for the trans-thoracic
approach.
In our experience, insertion of the pressure-catheter into the RV and its advance further into
the pulmonary artery when desired is comparatively easy to perform in the trans-diaphragm
approach. This relative ease of RV catheterization is reflected in the higher success rate compared
to the trans-thoracic approach, and might be well explained by the more caudal and inferior
direction by which the catheter enters the heart. Approaching the heart in this manner entails
minimal manipulation and maneuvering of the catheter during the process of RV catheterization, resulting in a lower incidence of clot formation inside the catheter. This complication occurred more frequently in trans-thoracic-operated animals as a direct consequence of excessive
maneuvering and accidental squeezing of the catheter causing it to lose or displace some of its
anti-thrombolitic gel. The strongest advocate of the trans-diaphragm approach over the transthoracic approach, however, is the significantly faster recovery of the animals. Prompt recovery
from surgery is particularly important in rats with monocrotaline-induced PH because of low
survival rates beyond four weeks after monocrotaline-injection.
In this study, we used echocardiography to validate RV pressure-telemetry in vivo. Additionally, through combination of pressure-data, obtained by RV-telemetry, with echo-Doppler flow
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data, we were able to study the hemodynamics of the pulmonary circulation in greater detail.16
Monocrotaline-induced pulmonary and cardiac remodeling, at the same time as PVR, RV wall
thickness and RV end-diastolic diameter increased. Cardiac adaptation, to overcome the increasing afterload, became inadequate by the fourth week of monocrotaline-injection, in such that
there was no further increase in RV-power to maintain adequate cardiac output while RV systolic
In the present study, we have focused on the refinement of the RV-telemetry technique in rats,
but this does not exclude its applicability in mice and other rodents. Indeed, in their recent study,
Schwenke et al. reported long-term monitoring of PA pressure in mice using radio-telemetry.9
In conclusion, we described a new, easier-to-perform, mildly invasive trans-diaphragm-based
Chapter 2
pressure continued to rise. Detailed studies of these observations are planned for the near future.
RV pressure-telemetry approach for long-term monitoring of PA and RV pressures in the rat
model of monocrotaline-induced PH. Our findings may be applied to improve our understanding
of the disease processes involved in PH and develop better treatment strategies for the disease.
ACKNOWLEDGEMENT
We thank prof.dr. N. Westerhof, dr. C. Boer and F.S. de Man (VUmc, the Netherlands), A.
Brumagim and H. Michelau (DSI, USA), M. Hardziyenka (Academic Medical Center, Amsterdam, the Netherlands), and prof.dr. R. Remie (Solvay Pharmaceuticals, Weesp, the Netherlands)
for their expert advice and discussion.
Sources of funding
M.L. Handoko was supported by the Mosaic (‘Mozaïek’) program / the Netherlands Organization
for Scientific Research (NWO), The Hague, The Netherlands.
Disclosures
None
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McLaughlin VV, McGoon MD. Pulmonary arterial hypertension. Circulation 2006;114:1417-1431.
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and failure: report of a National Heart, Lung, and Blood Institute working group on cellular and
molecular mechanisms of right heart failure. Circulation 2006;114:1883-1891.
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Hess P, Clozel M, Clozel JP. Telemetry monitoring of pulmonary arterial pressure in freely moving
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5. Kramer K, Kinter LB. Evaluation and applications of radiotelemetry in small laboratory animals.
Physiol Genomics 2003;13:197-205.
6. Sebkhi A, Strange JW, Phillips SC, Wharton J, Wilkins MR. Phosphodiesterase type 5 as a target for
the treatment of hypoxia-induced pulmonary hypertension. Circulation 2003;107:3230-3235.
7. McMurtry MS, Bonnet S, Wu X, Dyck JR, Haromy A, Hashimoto K, Michelakis ED. Dichloroacetate
prevents and reverses pulmonary hypertension by inducing pulmonary artery smooth muscle cell
apoptosis. Circ Res 2004;95:830-840.
8. Schermuly RT, Dony E, Ghofrani HA, Pullamsetti S, Savai R, Roth M, Sydykov A, Lai YJ, Weissmann
N, Seeger W, Grimminger F. Reversal of experimental pulmonary hypertension by PDGF inhibition.
J Clin Invest 2005;115:2811-2821.
9. Schwenke DO, Pearson JT, Mori H, Shirai M. Long-term monitoring of pulmonary arterial pressure
in conscious, unrestrained mice. J Pharmacol Toxicol Methods 2006;53:277-283.
10. Zagorski J, Debelak J, Gellar M, Watts JA, Kline JA. Chemokines accumulate in the lungs of rats with
severe pulmonary embolism induced by polystyrene microspheres. J Immunol 2003;171:5529-5536.
11. Wilson DW, Segall HJ, Pan LC, Lamé MW, Estep JE, Morin D. Mechanisms and pathology of monocrotaline pulmonary toxicity. Crit Rev Toxicol 1992;22:307-325.
12. Lang RM, Bierig M, Devereux RB, Flachskampf FA, Foster E, Pellikka PA, Picard MH, Roman MJ,
Seward J, Shanewise JS, Solomon SD, Spencer KT, Sutton MS, Stewart WJ. Recommendations for
chamber quantification: a report from the American Society of Echocardiography’s Guidelines and
Standards Committee and the Chamber Quantification Writing Group, developed in conjunction
with the European Association of Echocardiography, a branch of the European Society of Cardiology.
J Am Soc Echocardiogr 2005;18:1440-1463.
13. Quinones MA, Otto CM, Stoddard M, Waggoner A, Zoghbi WA. Recommendations for quantification of Doppler echocardiography: a report from the Doppler Quantification Task Force of the
Nomenclature and Standards Committee of the American Society of Echocardiography. J Am Soc
Echocardiogr 2002;15:167-184.
14. Plante E, Lachance D, Roussel E, Drolet MC, Arsenault M, Couet J. Impact of anesthesia on echocardiographic evaluation of systolic and diastolic function in rats. J Am Soc Echocardiogr 2006;19:15201525.
15. Hardziyenka M, Campian ME, de Bruin-Bon HA, Michel MC, Tan HL. Sequence of echocardiographic changes during development of right ventricular failure in rat. J Am Soc Echocardiogr
2006;19:1272-1279.
16. Westerhof N, Stergiopulos N, Noble MIM. Snapshots of Hemdynamics: An aid for Clinical Research
and Graduate Education. New York (NY): Springer; 2005.
17. Chemla D, Castelain V, Humbert M, Hebert JL, Simonneau G, Lecarpentier Y, Herve P. New formula for predicting mean pulmonary artery pressure using systolic pulmonary artery pressure. Chest
2004;126:1313-1317.
18. Suga H. Ventricular energetics. Physiol Rev 1990;70:247-277.
19. Kozar RA, Kaplan LJ, Cipolla J, Meija J, Haber MM. Laparoscopic repair of traumatic diaphragmatic
injuries. J Surg Res 2001;97:164-171.
20. Gamblin TC, Wall CE, Jr., Morgan JH, III, Erickson DJ, Dalton ML, Ashley DW. The natural history
of untreated penetrating diaphragm injury: an animal model. J Trauma 2004;57:989-992.
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Zierold D, Perlstein J, Weidman ER, Wiedeman JE. Penetrating trauma to the diaphragm: natural history and ultrasonographic characteristics of untreated injury in a pig model. Arch Surg 2001;136:3237.
Kramer K, Grimbergen JA, van der Gracht L, van Iperen DJ, Jonker RJ, Bast A. The use of telemetry to
record electrocardiogram and heart rate in freely swimming rats. Methods Find Exp Clin Pharmacol
1995;17:107-112.
Schierok H, Markert M, Pairet M, Guth B. Continuous assessment of multiple vital physiological
functions in conscious freely moving rats using telemetry and a plethysmography system. J Pharmacol Toxicol Methods 2000;43:211-217.
Stephenson R, Liao KS, Hamrahi H, Horner RL. Circadian rhythms and sleep have additive effects on
respiration in the rat. J Physiol 2001;536:225-235.
Lewanowitsch T, White JM, Irvine RJ. Use of radiotelemetry to evaluate respiratory depression
produced by chronic methadone administration. Eur J Pharmacol 2004;484:303-310.
Hessel MH, Steendijk P, den Adel B, Schutte CI, van der Laarse A. Characterization of right ventricular function after monocrotaline-induced pulmonary hypertension in the intact rat. Am J Physiol
Heart Circ Physiol 2006.
Chapter 2
21.
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Louis BW.indd 34
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3
Opposite Effects
of Training in Rats
with Stable and
Progressive Pulmonary
Hypertension
M.L. Handoko1,2 * & F.S. de Man1,2 *, C.M.
Happé2, I. Schalij2, R.J.P. Musters1, N.
Westerhof1,2, P.E. Postmus2, W.J. Paulus1, W.J.
van der Laarse1, A. Vonk-Noordegraaf2
1
Departments of Physiology and 2Pulmonology,
VU University Medical Center / Institute for
Cardiovascular Research, Amsterdam, The
Netherlands
Circulation 2009; 120: 42-49
Louis BW.indd 35
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Exercise training
ABSTRACT
Exercise training in pulmonary arterial hypertension (PH) is a promising adjunct to medical
treatment. However, it is still unclear whether training is beneficial for all PH-patients. We
hypothesized that right ventricular adaptation plays a pivotal role in the response to training.
Two different dosages of monocrotaline were used in rats to model stable PH with preserved
cardiac output, and progressive PH developing right heart failure. Two weeks after injection, PH
was confirmed by echocardiography, and treadmill training was initiated. Rats were trained for
four weeks, unless manifest right heart failure developed earlier. At end of study protocol, all rats
were functionally assessed by endurance testing, echocardiography and invasive pressure measurements. Lungs and hearts were further analyzed for quantitative histomorphologic analyses.
In stable PH, exercise training was well-tolerated and markedly increased exercise endurance
(from 25 ±3.9 to 62 ±3.9 minutes; p < 0.001). Moreover, capillary density increased significantly
(from 1.21 ±0.12 to 1.51 ±0.07 capillaries per cardiomyocyte; p < 0.05). However, in progressive PH exercise training worsened survival (hazard ratio 2.7, 95%CI: 1.1-14.2) and increased
pulmonary vascular remodeling. In addition, training induced widespread leukocyte infiltration
into the right ventricle (from 135 ±14 to 276 ±18 leukocytes per mm2; p < 0.001).
In our rat model, exercise training was found to be beneficial in stable PH, but detrimental in
progressive PH. Future studies are necessary to address the clinical implications of our findings.
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INTRODUCTION
Pulmonary arterial hypertension (PH) is characterized by progressive pulmonary vascular
remodeling, which importantly increases right ventricular (RV) afterload, eventually leading to
right heart failure and premature death.1 Traditionally, PH-patients were advised to limit physical activity, because of risk of fatal cardiovascular compromise.2
Recent developments have however challenged this view.3 Firstly, prognosis of PH-patients improved by introduction of various potent PH-specific medications in the last decades.4 Secondly,
several studies have demonstrated beneficial effects of training in patients with COPD and with
congestive heart failure, and training was beneficial even for the most severely affected patients
(GOLD IV, NYHA IV) often suffering from secondary pulmonary hypertension.5,6 Finally, in a
al. reported marked improvement in exercise capacity and quality of life after exercise training.7
Although training might be a promising adjunct to medical treatment in PH, it remains to be
elucidated whether exercise training is beneficial for all PH-patients, and what its effect is on RV
Chapter 3
recent clinical trial with 30 stable PH-patients under optimized medical treatment, Mereles et
function and remodeling. RV adaptation might be a discriminating factor for responsiveness
to training. During exercise, pulmonary artery pressures and RV afterload increase,8 resulting
in a transient elevation of RV wall stress. Although this is unknown for right heart failure, for
left heart failure it has been demonstrated that even a temporary elevation of wall stress can upregulate local pro-inflammatory factors, leading to leukocyte infiltration into the myocardium.9
We hypothesized that such a pro-inflammatory reaction in the right ventricle might outbalance
the positive effects of exercise training, especially in the presence of RV maladaptation to pressure overload.
We therefore conducted an experimental study and assessed the effects of exercise training in
two phenotypes of PH; namely stable PH with a preserved cardiac output at rest, and progressive
PH developing right heart failure. Using a comprehensive set of physiologic and pathologic endpoints, we documented beneficial effects in stable PH, but detrimental effects in progressive PH.
METHODS
All experiments were approved by the Institutional Animal Care and Use Committee at the VU
University.
Experimental pulmonary hypertension
Male Wistar rats were used (56 in total, 150-175g; Harlan, Horst, the Netherlands). PH was
induced by a single subcutaneous injection of monocrotaline (Sigma-Aldrich, Zwijndrecht, The
Netherlands) dissolved in sterile saline.
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Exercise training
Monocrotaline 60 mg/kg body mass was used to model progressive PH developing right heart
failure (n = 18); with a dose of 40 mg/kg monocrotaline, stable PH with a preserved cardiac
output was mimicked (n = 18).10,11 The control group was injected with saline only (n = 20).
Study design and training program
The exercise program was adopted from a validated exercise program for Wistar rats, described
by Fenning and Harrison, et al.12
In the first week, all rats were accustomed to treadmill running: mild electrical stimulation was
used to encourage the rats to run. Then, rats were randomly assigned to any of the three experimental groups (control, stable or progressive PH) and injected accordingly with monocrotaline
or saline (Figure 1). In the following two weeks, all rats were placed on the treadmill for one
minute a day (5 times a week, at a constant speed of 13.3 m/min, no slope). After these two weeks,
the rats were again randomly assigned to an exercise training program (control-Ex, stable PH-Ex,
progressive PH-Ex; 5x /week; 30 min; 13.3m/min; no slope) or the sedentary group (control-Sed,
stable PH-Sed, progressive PH-Sed; 5x /week; 1 min; 13.3m/min; no slope). The level of training
Figure 1
Injection
Exercise period
Sedentary
Control
Training
Stable PH
(MCT40)
(n=10)
Control-Ex
(n=10)
Stable PH-Sed
(n=9)
Stable PH-Ex
(n=9)
Progressive PH-Sed (n=9)
Progressive PH
(MCT60)
Day 0
Echo
Control-Sed
Progressive PH-Ex (n=9)
14: Start
Echo
42 (max.): End
Echo / ET / Cath
Study design. The effect of exercise training was studied in two distinct phenotypes of established pulmonary
hypertension (stable and progressive PH).
Abbreviations: Start, start of exercise period (14 days after injection); End, end of study protocol (when manifest signs of
right heart failure developed, or 42 days after MCT-injection). Echo, echocardiographic evaluation; ET,endurance testing;
Cath, RV catheterization.
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represented moderate exercise intensity (≈50% VO2max).13 Animals were trained for maximally
four weeks (from day 14 until day 42 after monocrotaline-injection). Rats that developed manifest clinical signs of right heart failure (defined as: >5% loss of body mass /day and/or respiratory
distress, cyanosis, lethargy) were euthanized early, in keeping with the protocol approved by the
institutional animal care committee. Manifest right heart failure was the survival endpoint and
recorded as an event in the survival analysis.14
Endurance test
Only on rats that completed the four-week exercise program, endurance testing was performed.12
The treadmill was set at a constant speed of 15 m/min, and a slope of 20 degrees. The time from
start-until-exhaustion was used as a measure for exercise endurance of the rats. Exhaustion was
established when the rats accepted the electric stimulus three consecutive times as opposed to
Chapter 3
running. The maximal running time was 90 minutes, which was achieved by all control rats.
Hemodynamic evaluation
Echocardiography
The rats were evaluated by echocardiography at baseline (just before they received their injection), at start of training, and at the end of the study protocol (when manifest signs of right heart
failure developed, or 42 days after monocrotaline-injection). Transthoracic echocardiographic
measurements (ProSound SSD-4000 system equipped with a 13-MHz linear transducer (UST5542), Aloka, Tokyo, Japan) were performed on anaesthetized but spontaneously breathing rats
(isoflurane 2.0% in 1:1 O2/air mix; Pharmachemie, Haarlem, The Netherlands), as described
previously.15
Analyses were performed off-line (Image-Arena 2.9.1, TomTec Imaging Systems, Unterschleissheim / Munich, Germany). Measured parameters for cardiac and right ventricular function
were: Doppler-derived stroke volume, cardiac output, and tricuspid annular plane systolic
excursion (TAPSE). Parameters for RV remodeling were: RV end-diastolic diameter and RV wall
thickness. Parameters for pulmonary vascular remodeling were: pulmonary artery acceleration
time normalized for cycle length (PAAT/cl) and pulmonary vascular resistance (PVR).
Disease progression of PH during the period of exercise training was expressed as percentage
changes in hemodynamics over time, i.e. change in cardiac output:
ΔCO = [ (COEND OF PROTOCOL – COSTART OF TRANING) / COSTART OF TRANING ] * 100% / days-of-training .
Other parameters for disease progression (ΔSV, ΔTAPSE, and so on) were calculated similarly.
The non-invasive estimations of RV systolic pressures, PVR, and RV wall stress are described in
the Supplement.
39
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Exercise training
Invasive RV-pressure measurements
At the end of the study protocol, open-chest RV catheterization was performed under general
anesthesia in all animals (isoflurane 2.0% in 1:1 O2/air mix), as described before.16 Before the
procedure, the rats were intubated (16 G Teflon tube) and attached to a mechanical ventilator
(Micro-Ventilator, UNO, Zevenaar, The Netherlands; ventilator settings: breathing frequency 80/
min, pressures 9/0 cmH2O, inspiratory/expiratory ratio 1:1). The right ventricle was approached
via a lateral right thoracotomy through the fifth intercostal space. RV pressures were recorded
by the use of a high-fidelity catheter-tip transducer (Mikro-Tip SPR-671, Millar Instruments,
Houston TX). Analyses were performed when steady-state was reached over an interval of at
least 10 s and averaged.
Histology
After the final hemodynamic assessment, the rats were euthanized by exsanguination (under
isoflurane), and heart, lungs and other major organs were harvested. Lungs were weighed, the
airways of the left lobe subsequently filled with a 1:1 mix of saline and cryofixative (Tissue-Tek
O.C.T. compound, Sakura Fintek Europe, Zoeterwolde, The Netherlands), and snapfrozen in
liquid nitrogen. The right lobe was used to measure the ratio of dry to wet lung mass. The heart
was perfused, weighed, dissected and snapfrozen in liquid nitrogen.
Histomorphometric analysis of heart and lungs
The determination of cardiomyocyte cross-sectional area, cardiac fibrosis, and relative wall
thickness of pulmonary arterioles (PA) are described in detail in the Supplement.
Analysis of capillary density and cardiac inflammation was performed by using quantitative
immunofluorescence microscopy. Briefly, cardiac cryosections (5 μm) were incubated for 60
minutes with primary CD31- (1:35; sc-1506-R, Santa Cruz Biotechnology, Santa Cruz CA) and
CD45-antibodies (1:25; sc-53045, Santa Cruz) for capillary density and leukocyte infiltrations,
respectively, followed by appropriate secondary antibody staining as well as WGA (glycocalyx)
and DAPI (nuclei) counterstaining. Image acquisition was performed on a Marianas digital
imaging microscopy workstation (Intelligent Imaging Innovations (3i), Denver CO). SlideBook
imaging analysis software (SlideBook 4.2, 3i) was used to semi-automatically quantify the images. Capillary density was expressed as the number of capillaries per cardiomyocyte or number
of capillaries per section area, measured in at least three randomly chosen areas per ventricle,
where cardiomyocytes were transversally sectioned.17 Leukocyte infiltration was expressed as
the number of positive CD45-nuclei per section area, measured over minimally three randomly
chosen areas per ventricle.18
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Statistical analysis
All analyses were performed in a blinded fashion. All data were verified for normal distribution. Data are presented as mean ± SEM and analyses were performed on all rats, unless stated
otherwise. A p-value < 0.05 was considered significant.
Survival estimates were performed by Kaplan-Meier analyses, with post-hoc comparisons performed by log-rank test. Hazard ratios were calculated by the proportional hazards model. For
all other in vivo data, two-way analysis of variance was used; Interaction between PH-status and
training-status was tested, and subsequently Bonferroni post-hoc tests were performed (training
vs. sedentary in the three experimental groups). All reported p-values of post-hoc comparisons
are Bonferroni corrected (SPSS 16.0 for Windows, SPSS, Chicago IL).
For the histological data, multilevel analysis was used to correct for the non-independence
2.02.03, Center for Multilevel Modelling, Bristol, UK).19
The authors had full access to the data and take responsibility for its integrity. All authors have
read and agree to the manuscript as written.
Chapter 3
of successive measurements of cross sectional areas and PA wall thickness per animal (MLwiN
RESULTS
Established pulmonary hypertension at start of training
Estimated RV systolic pressure (using PAAT/cl) at start of training was elevated for stable PH
and progressive PH, compared to control (eRVSP, stable PH: 36 ±2.8 mmHg, progressive PH: 48
±3.5 mmHg, control: 26 ±2.3 mmHg, p < 0.001; Supplement Table S-1). In addition, compared
to control, PVR for stable and progressive PH was higher. Together with the rise in pulmonary
pressures, a modest increase of RV wall thickness was found, indicating mild RV hypertrophy
at start of training. At this time point, there were no signs of cardiac dysfunction or adverse
remodeling, measured by cardiac output, stroke volume, heart rate, TAPSE, or RV end-diastolic
diameter (Table S-1).
Induction of stable vs. progressive PH by different monocrotaline dosages
Serial echocardiographic measurements revealed different phenotype of PH induced by monocrotaline 40 or 60 mg/kg (Supplement: Figure S-1).
In stable PH-Sed (monocrotaline 40 mg/kg - untrained rats), from day 14 after monocrotalineinjection, there was no significantly further increase in PVR, and resting cardiac output was
preserved (ΔPVR: 7.5 ±5.0 %/day, ΔCO: -0.74 ±0.81 %/day, cardiac output at end: 109 ±10 ml/
min). Nevertheless, some signs of RV dysfunction and adverse remodeling were observed in
stable PH-Sed at end of study protocol (TAPSE, stable PH-Sed: 2.9 ±0.3 mm vs. control-Sed: 3.7
±0.1 mm; RV end-diastolic diameter, stable PH-Sed: 5.4 ±0.4 mm vs. control-Sed 3.5 ±0.1 mm;
all p < 0.05).
41
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Exercise training
In progressive PH-Sed (monocrotaline 60 mg/kg - untrained rats), a rapid increase in PVR and
a marked decline in cardiac output were seen (ΔPVR: 11 ±4.1 %/day, ΔCO: -3.1 ±0.76 %/day,
cardiac output at end: 69 ±9.7 ml/min; all p < 0.05 vs. stable PH-Sed).
Effects of training on disease progression in stable vs. progressive PH
Serial echocardiographic measurements were used to study the effect of training on disease
progression in control, stable and progressive PH. Figure 2 shows the evolution of the various
echocardiography-derived hemodynamic parameters over time for the different experimental
groups (for numeric data: Table S-2). The divergent effect of training on disease progression
is especially evident for cardiac output (Figure 2B). During the exercise period, a steeper fall
in cardiac output was observed for progressive PH-Ex vs. progressive PH-Sed, whereas cardiac
output was better preserved in stable PH-Ex than stable PH-Sed.
Figure 2
A
A.
PVR
PVR
1.0
1.0
B
B.
B.
p=0.001
p=0.001
14
14
21
28
35
21 Time28(days)35
Time (days)
C.
C.
RV EDD
RV EDD
4.0
4.0
21
28
35
21 Time28(days)35
Time (days)
Control-Sed
Control-Sed
Control-Ex
Control-Ex
42
42
21
28
35
21 Time28(days)35
Time (days)
TAPSE
TAPSE
4.0
4.0
p=0.03
p=0.03
6.0
6.0
14
14
14
14
D
D.
D.
(mm)
(mm)
8.0
8.0
50
50
42
42
C
(mm)
(mm)
p=0.01
p=0.01
100
100
0.5
0.5
0.0
0.0
Cardiac Output
Cardiac Output
150
150
(mL/min)
(mL/min)
(mmHg/ml/min)
(mmHg/ml/min)
A.
42
42
p=0.04
p=0.04
3.0
3.0
2.0
2.0
1.0
1.0
Stable PH-Sed
Stable PH-Sed
Stable PH-Ex
Stable PH-Ex
14
14
21
28
35
21 Time28(days)35
Time (days)
42
42
Progressive PH-Sed
Progressive PH-Sed
Progressive PH-Ex
Progressive PH-Ex
Opposite effects of training in stable vs. progressive PH were found for all important hemodynamic parameters for
disease progression (A-D), indicated by the slope of the connecting lines from ‘start of training’ to ‘end of study protocol’.
All data are presented as mean ±SEM. P-values represent the interactive effect per individual parameter. Each line
corresponds with an experimental group (see bottom). Numeric data at start of training are found in Supplement: Table
S-1, numeric data on disease progression are found in Table S-2. Abbreviations: PVR, pulmonary vascular resistance;
TAPSE, tricuspid annular plane systolic excursion; RVEDD, RV end-diastolic diameter.
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Effects of training on survival and endurance in stable vs. progressive PH
Loss in body mass and decline in cardiac output were closely correlated (r = 0.74, p < 0.001), which
verifies the clinical criteria that were used for right heart failure. From these criteria, we observed
that exercise training decreased survival significantly in progressive PH (hazard ratio, progressive PH-Ex vs. progressive PH-Sed: 2.7, 95%CI: 1.1-14.2); In stable PH-Sed, one rat prematurely
developed right heart failure, all rats in stable PH-Ex and the control groups survived (Figure 3A).
At four weeks, training significantly improved exercise endurance by twofold in stable PH (endurance, stable PH-Ex: 62 ±3.9 minutes vs. stable PH-Sed: 25 ±3.9 minutes, p < 0.001), whereas
an opposite trend was observed in progressive PH. A strong significant interaction between
training-status and PH-status was present (Figure 3B; p < 0.001).
The opposite effect of training on survival and endurance are in line with the previous findings
Figure 3
A
A.
B
B.
Survival
Endurance
100
Stable PH-Ex
Stable PH-Sed
1.0
p<0.001
Control
Ex
0.5
Progr. PH-Sed
(min)
(fraction)
Chapter 3
on disease progression.
50
Sed
p=0.04
0.0
Progr. PH-Ex
0 21
28
35
42
Days after MCT-injection
0
Stable PH
Progr. PH
A) Training had a detrimental effect on survival in progressive PH, whereas it did not affect survival in stable PH. All rats
from control groups survived, and were omitted here for clarity. B) Training improved endurance more than twofold
in stable PH, whereas an opposite trend was observed in progressive PH (interactive effect: p<0.001). All rats in the
control groups reached the predefined maximum endurance time (90 min, indicated by the horizontal line). All data are
presented as mean ±SEM. Endurance testing was only performed on surviving rats, therefore: n = 10 (control-Sed), n = 10
(control-Ex), n = 8 (stable PH-Sed), n = 9 (stable PH-Ex), n = 4 (progressive PH-Sed), n = 1 (progressive PH-Ex).
RV catheterization at end of study protocol
RV catheterization at end of study protocol confirmed the PH-status of monocrotaline-treated rats
(Figure S-2). There was a strong monocrotaline dose-dependent response at end of the study protocol
(p < 0.001): RV systolic pressure at rest doubled in stable PH and almost tripled in progressive PH
vs. control, and a similar pattern was seen for RV diastolic pressures. In addition, the intrinsic RV
contractility and relaxation were significantly changed in stable and progressive PH vs. control.
However, no (statistical) differences in RV dP/dtmax and RV dP/dtmin were found between the two PHphenotypes. RV catheterization at end of the study protocol revealed no (interactive) effect of training.
43
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Exercise training
Effects of training on pulmonary vascular remodeling in stable vs. progressive PH
Training increased (wet) lung weights in progressive PH (p < 0.001), whereas it had no effect
on lung weights in stable PH, independent of normalization (i.e. normalization by body mass
or tibia length; Figure 4A, Table S-3). Moreover, a strong interaction between training-status
and PH-status was present (p < 0.001). Ratios of wet to dry lung mass ratios were similar for
all experimental groups (wet/dry ratios between groups varied from 4.9 ±0.1 to 5.2 ±0.1, see
Table S-3). This suggests that training modulated pulmonary vascular remodeling, and that the
observed differences are not likely to be attributed to pulmonary edema. Measurements on PA
wall thickness confirmed the interactive effect of training on pulmonary vascular remodeling
(Figure 4B). Echo-derived PVR at end of the study protocol and PA wall thickness correlated well
(r = 0.82, p < 0.001), indicating that the pulmonary vascular remodeling must have had profound
hemodynamic effects in vivo.
Figure 4
(gram)
(gram)
4.0
4.0
Lung mass
Lung mass
B.
B
B.
p<0.001
p<0.001
2.0 Sed Ex
2.0 Sed Ex
0.0
0.0 Control Stable PH Progr. PH
Control Stable PH Progr. PH
C
C.
C.
60
60
(%)(%)
A.
A
A.
PA wall thickness
PA wall thickness
p<0.001
p<0.001
40
40
20
20 Sed Ex
Sed Ex
0
0 Control Stable PH Progr. PH
Control Stable PH Progr. PH
D
D.
D.
20 μm
20 μm
Training significantly worsened pulmonary vascular remodeling in progressive PH, demonstrated by elevated (wet) lung
mass (A) and increased PA wall thickness (B) in progressive PH-Ex, compared to progressive PH-Sed (p<0.001). Moreover,
significant interactions between training-status and PH-status were present (p<0.001 for lung mass, p<0.01 for PA wall
thickness). Lower panels show typical examples of pulmonary arterioles (Elastica von Giesson, 200x magnification) of
control-Sed (C) and progressive PH-Ex (D).
Data are presented as mean ±SEM.
44
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06-05-10 11:20
Effects of training on cardiac remodeling in stable vs. progressive PH
Histomorphology
In contrast with pulmonary vascular remodeling, the amount of RV hypertrophy was similar
among stable and progressive PH, whether it was expressed by RV mass (normalized or not)
or RV / (LV+S) (Table S-3), confirming echocardiographic observations. Measurements of RV
cardiomyocyte cross-sectional area also showed similar RV hypertrophy between stable and
progressive PH, compared to control (Figure 5A). RV fibrosis was significantly increased in
progressive PH only; no additional effect of training was found (Figure 5B). Similar levels of
RV hypertrophy but differences in RV diameters (Figure 5C) indicate concentric vs. eccentric
remodeling, which translated to severely elevated RV wall stress levels for progressive PH, but
only moderately elevated wall stress levels for stable PH; no interactive effect of training was
B. B
B.
800
### ###
800
### ###
600
600
Sed Ex
400 Sed Ex
400
200
200
0
0 Control Stable PH
Control Stable PH
CC.
C.
10.0
10.0
(mm)
(mm)
RV CSA
RV CSA
### ##
### ##
RV fibrosis
RV fibrosis
4.0
4.0
2.0
2.0
0.0
0.0
Progr. PH
Progr. PH
D. D
D.
RV EDD
RV EDD
5.0 Sed Ex
5.0 Sed Ex
0.0
0.0
### ###
### ###
(%)(%)
(µ m
(µ2m
) 2)
Figure 5
††† †††
††† †††
Control Stable PH Progr. PH
Control Stable PH Progr. PH
###
### ###
###
Sed Ex
Sed Ex
Control Stable PH Progr. PH
Control Stable PH Progr. PH
RV wall stress
RV wall stress
††† †††
††† †††
100
100
(mmHg)
(mmHg)
AA.
A.
Chapter 3
observed (Figure 5D).
50
50 Sed Ex
Sed Ex
0
0
### ###
### ###
Control Stable PH Progr. PH
Control Stable PH Progr. PH
The amount of RV hypertrophy indicated by RV CSA was similar in stable and progressive PH (A). RV fibrosis was only
seen in progressive PH, and no (interactive) effect of training was observed (B). The largest RV diameters were observed
in progressive PH (C), translating into the highest RV wall stress (D).
All data are presented as mean ±SEM. ##: p<0.01, ###: p<0.001 vs. control. †††: p<0.001 vs. stable PH (and p<0.001 vs.
control).
45
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Exercise training
Analyses of LV myocardium revealed atrophy (evident from lower LV mass and lower LV
cardiomyocyte cross-sectional area) and increased fibrosis in progressive PH only. No effect of
training was observed on LV myocardium in progressive PH (Figure S-3).
Cardiac capillarization and inflammation
Training improved RV capillarization in stable PH with approximately 25%, whereas a trend
for an opposite effect was seen in progressive PH, whether expressed as a ratio of capillaries per
cardiomyocyte or as capillaries per section area (Figure 6A,B). Moreover, a significant interaction between training-status and PH-status was present (p < 0.01). Training had no effect on
capillarization in LV (Figure S-3D).
In progressive PH, clusters of leukocytes were observed in various parts of the myocardium of
the right ventricle (Figure 7D). As a result, the number of leukocytes was significantly higher in
progressive PH, compared to control. Training dramatically increased RV leukocyte infiltration
A.
A
A.
RV capillaries per myocyte
Figure 6
RV capillaries
per myocyte
2.0
p<0.05
1.0
1.0
0.0
0.0
B.
p<0.05
Sed
Sed
2
(cap/mm
(cap/mm
) 2)
(Cp/Cm)
(Cp/Cm)
2.0
B.
B
Ex
Ex
Control
Stable PH Progr. PH
Control
Stable PH Progr. PH
C
C.
C.
RV capillary density
RV capillary
p<0.05density
Sed Ex
2000 Sed Ex
2000
p<0.05
1000
1000
0
0
Control
Stable PH Progr. PH
Control
Stable PH Progr. PH
D
D.
D.
20 μm
20 μm
Training improved RV capillarization in stable PH, whereas a trend for an opposite effect was observed in progressive PH,
whether it was expressed as capillary-to-cardiomyocyte ratio (A; Cp/Cm) or capillaries per area (B). Moreover, a significant
interaction between training-status and PH-status was present (p<0.01). Lower panels show typical examples of RV
capillarization (100x magnification) of control-Sed (C) and stable PH-Ex (D). Endothelin marker CD31 is stained green, cell
membranes red; capillaries appear as small yellow/orange dots (merging of red and green; arrows).
All data are presented as mean ±SEM.
46
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06-05-10 11:20
in progressive PH, whereas in stable PH it remained unchanged (Figure 7A). Moreover, a very
significant interaction between training-status and PH-status was present (p < 0.001). Leukocyte
infiltration was only observed in the right ventricle. Analysis of LV showed no differences between groups, their values were even slightly lower than RV control values (Figure 7B).
RV inflammation
RV inflammation
400
400
p<0.001
p<0.001
200
200
0
0
B.
BB.
400
400
LV inflammation
LV inflammation
200
200
Sed Ex
Sed Ex
Control Stable PH Progr. PH
Control Stable PH Progr. PH
C.
C
C.
0
0
Sed Ex
Sed Ex
Chapter 3
2) 2)
+-nuclei
+-nuclei
/ mm
/ mm
(CD45
(CD45
A.
A
2) 2)
+-nuclei
+-nuclei
/ mm
/ mm
(CD45
(CD45
A.
Figure 7
Control Stable PH Progr. PH
Control Stable PH Progr. PH
D.
D
D.
20 μm
20 μm
Training had a dramatic effect on RV leukocyte infiltration in progressive PH, whereas leukocyte infiltration remained
unchanged in stable PH (A). Moreover, there was a strong significant interaction between training-status and PH-status
(p<0.001). Inflammation was restricted to the right ventricle only, as values for leukocyte infiltration of LV did not differ
among the experimental groups and were even lower than RV control values (B). Lower panels show typical examples
of RV leukocyte infiltration (100x magnification) of control-Sed (C) and progressive PH-Ex (D). Lymphocyte-marker CD45
is stained: green, cell membranes: red, nuclei: blue; the clustering of aggregated leukocytes in progressive PH-Ex into the
myocardium of the right ventricle.
Data are presented as mean ±SEM.
DISCUSSION
To the best of our knowledge, this is the first study that investigated the effects of training in
stable and progressive PH, focusing on RV function and remodeling. Using a comprehensive set
of physiologic and pathologic endpoints, we have demonstrated that:
1) Exercise training was well tolerated and beneficial in PH with a preserved cardiac output. In
this group, training had no adverse effects on disease progression; it improved endurance,
and was associated with enhanced RV capillarization. However;
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Exercise training
2) Exercise training was detrimental in progressive PH developing right heart failure. Here,
exercise had adverse effects on hemodynamics and accelerated the progression to right heart
failure. Moreover, exercise was associated with adverse pulmonary vascular remodeling, and
massive RV inflammation.
Functional improvement after training, associated with enhanced capillarization
The only prospective clinical trial on exercise training in PH, by Mereles et al, showed that
functional capacity and quality of life of stable PH-patients could markedly be improved after
training.7 The general hemodynamic characteristics of the subjects in that trial were similar to
the stable PH -group in our study; comparable pulmonary artery pressures were found, together
with a mildly depressed cardiac index at rest, and moderate RV dilatation that remained stable
during the study period. In agreement with this clinical study we found a marked improvement
in exercise endurance in stable PH after training.
In addition, we found that in stable PH the functional improvement after training was associated with enhanced capillarization of the right ventricle. This phenomenon has been described
for ischemic heart failure,20 and recently two studies have evaluated the effect of training on
cardiac angiogenesis in systemic hypertension as well.21,22 In spontaneous hypertensive rats21 and
in rats with angiotensin II -induced hypertension,22 exercise was found to improve (LV) capillarization by about 40%. This is somewhat higher than the 25% increase in (RV) capillarization
observed in our present study, but the difference may be explained by the lower training intensity
and shorter exercise period in our study. A direct link between angiogenesis, hypertrophy and
cardiac function has been shown.23,24 Insufficient cardiac microvascular growth was recently
identified as an important underlying mechanism in the transition from compensatory hypertrophy to heart failure.23 Moreover, promotion of cardiac angiogenesis was found to normalize
the relative capillary deficit, to improve coronary flow reserve, and to restore cardiac dysfunction
under chronic pressure overload.24
It is likely that the improved endurance in stable PH is also partially attributable to other
beneficial effects of exercise that were not studied here. Especially, its effects on skeletal muscle
function in PH deserves further exploration in future studies, as this effect was shown to be
relevant in the rehabilitation of (left) heart failure- and COPD-patients.25,26
Worsened survival after training, associated with enhanced RV inflammation
Traditionally, PH-patients were encouraged to limit physical activity, a view that was mainly
based on theoretical arguments3. Here, we demonstrate that exercise training in progressive PH
can indeed be harmful in the case of a poorly adapted right ventricle, by augmenting of pressure
overload -associated RV inflammation.
It is unlikely that RV inflammation is primarily the result of a direct inflammatory effect of
monocrotaline on the heart.27 We found no evidence for LV inflammation, even in rats that
were treated with the highest monocrotaline dose. Furthermore, histology of the right ventricle
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06-05-10 11:20
revealed randomly distributed patches of infiltration, rather than a gradual pattern of inflammatory cells diffusing from the (sub)endocardium. For these reasons, RV inflammation in our
model is most likely ascribed to chronic RV pressure overload.
To the best of our knowledge, the link between RV inflammation and chronic RV pressure
overload has not been investigated yet, neither clinically nor experimentally. Nevertheless, in
patients suffering from acute pulmonary embolism, comparable observations of selective RV
inflammation were reported in a postmortem study,18 and similar findings were observed in
experimentally-induced acute pulmonary embolism.28 The mechanistic importance of RV inflammation was demonstrated, since suppression of the inflammatory response following acute
pulmonary embolism limited RV damage and prevented right heart failure.29 In these studies, it
was suggested that RV inflammation could have been triggered by ischemic injury of the right
High RV wall stress might be an alternative explanation, as recently shown in a model of
chronic LV pressure overload.9,30 We observed a similar amount of RV hypertrophy in all PHgroups. However, because of larger RV end-diastolic diameter, we found the highest RV wall
Chapter 3
ventricle or local and/or systemic over-production of catecholamines.
stress in progressive PH. At resting conditions, RV wall stress was similar in progressive PH-Ex
and progressive PH-Sed. Although we could not directly measure RV pressures during exercise,
RV afterload probably increased significantly during exercise, because of the elevated PVR.8
Therefore, it is likely that in progressive PH, RV wall stress was higher during exercise. These
episodes of elevated wall stress could have triggered RV inflammation, because short periods of
mechanical stretch (10 minutes) can induce myocardial over-expression of pro-inflammatory
cytokines (like TNF-α), which is followed by leukocyte infiltration.9,30
Finally, training might also directly aggravate pre-existing inflammation in the myocardium. In
viral myocarditis it is known that exercise augment the inflammatory reaction, enhances cardiac
dilatation, and increases its lethality.31,32
Our study suggests that RV inflammation in PH may be of pathophysiological importance.
Future studies should investigate its relevance for the different etiologies of clinical pulmonary
arterial hypertension.
Limitations
The model of PH that was caused by the use of monocrotaline, does not fully replicate the pathophysiology and resulting pulmonary and cardiovascular effects of clinical PH. Therefore, this
study should be viewed as a seminal analysis of exercise in stable and progressive PH from which
other (clinical) studies should arise. For example, validated clinical determinants that can predict
a favorable response to exercise training are currently absent.
The non-invasive estimation of PVR results from several measurements and is therefore susceptible to a large variability. Nevertheless, a close correlation was observed between echo-derived
PVR measurements and histological parameters for pulmonary vascular remodeling.
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Exercise training
Echo- or invasive hemodynamic measurements at end of study protocol failed to detect changes
that could explain the differences in survival and endurance between the trained and sedentary
groups. Echo- and invasive hemodynamic measurements were however obtained at rest and
therefore do not reflect exercise hemodynamics, which probably differed between both groups.
Moreover, in progressive PH, hemodynamic measurements at end of study protocol were obtained at a stage of terminal right heart failure, which was however reached earlier in the trained
than the sedentary group. Hence, the differences in survival between both groups are reflected
more by the time elapsed to reach right heart failure, than the hemodynamic findings at right
heart failure.
Conclusions
In our rat model, exercise training was found to be beneficial in stable PH, but detrimental in
progressive PH. The differential effect is probably due to enhanced RV myocardial capillarization
in stable PH, and RV myocardial inflammation in progressive PH. Future studies are necessary
to address the clinical implications of our findings.
ACKNOWLEDGEMENT
We thank dr. G. Harrison and prof.dr. A. de Haan for their introduction to exercise training of
rats, and prof.dr. J.W.M. Niessen & dr. K. Grünberg for their expert opinion and comments on
previous versions of the manuscript.
Sources of funding
M.L. Handoko (Mozaïek 017.002.122) and A. Vonk-Noordegraaf (Vidi 917.96.306) were supported by the Netherlands Organization for Scientific Research (NWO), The Hague, The Netherlands.
Disclosures
None.
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52
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Clinical relevance
The role of exercise training in current clinical management of patients with pulmonary arterial
hypertension (PH) remains controversial. Traditionally, these patients were advised to limit their
physical activity, as exercise was presumed to superimpose an additional right ventricular (RV)
load on an already compromised heart. Recently, exercise training was found to greatly improve
exercise capacity and quality of life in clinically stable PH-patients. It remains however uncertain
if exercise training is beneficial for all PH-patients, including the effects on the right ventricle.
In the present study, we therefore studied the effects of exercise training in a rodent model
inducing two distinct clinical phenotypes of PH. Training was found to be beneficial in stable
PH with preservation of cardiac function. However, training was detrimental in progressive PH,
inflammation.
These findings imply that disease progression and RV adaptation might be important clinical
determinants for favorable response to exercise training in PH. As clinical PH tends to be a
Chapter 3
as it accelerated the progression towards right heart failure and as it induced widespread RV
progressive disease, defining ‘stable’ PH might be challenging. In the absence of prospectively
validated criteria, PH-patients could be classified as stable, when there is constancy of clinical findings in repeated evaluations over a three-month period. These findings should include:
NYHA-classification, 6-minute-walk-distance, NT-proBNP levels, RV function and dimensions.
Prospective clinical studies should evaluate these criteria for stable PH in order to translate the
findings of the present study and to detect PH-patients, which will benefit from an exercise training program.
53
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Exercise training
SUPPLEMENT:
Expanded METHODS
Non-invasive estimation of RV systolic pressures, pulmonary vascular resistance, and
RV wall stress
The relationship between PAAT/cl and RV systolic pressure, measured at the end of the study
protocol, were used to non-invasively estimate RV systolic pressure (eRVSP) at baseline and at
the start of training:S1,S2
eRVSP ≈ 142 * e (-11*[PAAT/cl ]) .
Pulmonary vascular resistance (PVR) was estimated by Poiseuille’s law:S3-S6
PVR = (meanPAP – PCWP) / cardiac output ≈ (0.61 * RVSP + 2mmHg) / cardiac output .
RV wall stress was estimated using LaPlace’s law:S5
RV wall stress = (RVSP * RV end-diastolic diameter) / (4* RV wall thickness) .
Histomorphologic analyses of cardiomyocyte cross sectional area, cardiac fibrosis, and
relative wall thickness of pulmonary arterioles
Haematoxylin & eosin -stained cardiac cryosections (5 μm) were used to determine LV and RV
cardiomyocyte cross-sectional area.S7 ImageJ was used for image analysis (ImageJ for Windows
1.39a, National Institutes of Health, Bethesda MD), taking the pixel-to-aspect ratio into account.
Cardiomyocyte size for each ventricle was expressed as the average cross-sectional area of minimally twenty transversally cut cardiomyocytes at the level of the nucleus, randomly distributed
over the ventricles. Picrosirius red staining was used for analysis of cardiac fibrosis. By means
of an internally validated ImageJ-macro, cardiac fibrosis was automatically detected.S8 LV and
RV fibrosis were expressed as the percentage tissue area positive for collagen, measured over
minimally three randomly chosen areas per ventricle.
Pulmonary sections (5 μm) were stained with Elastica von Giesson for morphometric analysis
of vascular dimensions, as described before.S9,S10 Minimally 50 transversally cut pulmonary
arterioles, randomly distributed over the lungs with an outer diameter between 25 and 100 μm
were measured, using ImageJ. Relative wall thickness of pulmonary arterioles was calculated as:S9
PA wall thickness = 2 * (medial wall diameter / external diameter) * 100% .
54
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Supplemental FIGURES
Figure S-1
A.
A
B B.
PVR
150
Control
0.6
∆PVR = +11±4 %/day
0.4
∆PVR = +7.5±5 %/day
0.2
Stable PH
100
Progressive PH
50
0
7
14 21 28
Time (days)
35
0
42
0
7
14 21 28
Time (days)
35
42
Two distinct phenotypes of pulmonary hypertension (PH) were induced by the use of a low (40 mg/kg) and a high
dose of monocrotaline (60 mg/kg). In stable PH (MCT40 - untrained rats), from day 14 after MCT-injection, there was no
significant further increase in PVR (A) and resting cardiac output (B) was preserved (ΔPVR = 7.5 ±5.0 %/day, Δcardiac
output = -0.74 ±0.81 %/day, cardiac output at end = 109 ±10 ml/min). In progressive PH (MCT60 - untrained rats), a rapid
increase in PVR and a marked decline in cardiac output were seen (ΔPVR = 11 ±4.1 %/day, Δcardiac output = -3.1 ±0.76
%/day, cardiac output at end = 69 ±9.7 ml/min). ΔPVR and Δcardiac output are hemodynamic parameters for disease
progression and correspond with the slope indicated in the figure. They were calculated as stated in the methods section
of the main article, the numeric data can be found in Table S-2.
All data are presented as mean ±SEM.
Figure S-2
A.
A
††
80
40
††
###
Sed Ex
###
#
###
#
5.0
20
0
RV DP
10.0
(mmHg)
###
60
(mmHg)
B.
B
RV SP
Chapter 3
0.0
(ml/min)
(mmHg/ml/min)
0.8
cardiac output
Sed Ex
Control
0.0
Stable PH Progr. PH
Control
Stable PH Progr. PH
RV catheterization at the end of study confirmed the pulmonary hypertensive status of the sedentary and trained stable
and progressive PH rats (A-D). Although the intrinsic RV contractility and RV relaxation were significantly altered in
stable and progressive PH vs. control, rest-measurements did not reveal differences among the two PH phenotypes. No
(interactive) effect of training was observed.
All data are presented RV
as mean
±SEM. #:
p<0.05, ##: p<0.01, ###: p<0.001 vs. control; ††:RV
p<0.01
vs. stablemin
PH (and
dP/dt
max
dP/dt
p<0.001 vs. control). RV SP = RV systolic pressure; RV DP = RV diastolic pressure.
3.0
#
#
#
#
Ex
2.0 Sed
1.0
0.0
Louis BW.indd 55
D.
Control
Stable PH Progr. PH
0.0
(mmHg/s x1000)
(mmHg/s x1000)
C.
-1.0
-2.0
-3.0
55
Sed Ex
#
Control
#
#
#
Stable PH Progr. PH
06-05-10 11:20
Sed Ex
(m
(m
40
5.0
20
Sed Ex
0
Control
0.0
Stable PH Progr. PH
Control
Stable PH Progr. PH
Exercise training
Figure S-2 (Continued)
(mmHg/s x1000)
D.
D
RV dP/dt max
3.0
#
#
#
#
Ex
2.0 Sed
1.0
0.0
Control
RV dP/dt min
0.0
(mmHg/s x1000)
C.
C
-1.0
-2.0
-3.0
Stable PH Progr. PH
Sed Ex
#
Control
#
#
#
Stable PH Progr. PH
RV catheterization at the end of study confirmed the pulmonary hypertensive status of the sedentary and trained stable
and progressive PH rats (A-D). Although the intrinsic RV contractility and RV relaxation were significantly altered in
stable and progressive PH vs. control, rest-measurements did not reveal differences among the two PH phenotypes. No
(interactive) effect of training was observed.
All data are presented as mean ±SEM. #: p<0.05, ##: p<0.01, ###: p<0.001 vs. control; ††: p<0.01 vs. stable PH (and
p<0.001 vs. control). RV SP = RV systolic pressure; RV DP = RV diastolic pressure.
Figure S-3
### ###
0.5
0.0
CC.
Control
(%)
2.0
0.0
Sed Ex
Control
0
Stable PH Progr. PH
Control
Stable PH Progr. PH
LV capillary density
2.0 Sed Ex
###
###
200
DD.
##
###
400
Stable PH Progr. PH
LV fibrosis
4.0
LV CSA
600 Sed
Ex
Sed Ex
##
(Cp/Cm)
(gram)
1.0
BB.
LV mass
(µm2)
AA.
##
### ###
1.0
0.0
Control
Stable PH Progr. PH
LV atrophy (evident from lower LV mass and lower LV CSA; A,B) and LV fibrosis (C) was observed in progressive
pulmonary hypertension only. LV capillarization was decreased in both stable and progressive pulmonary hypertension
in comparison with control (D). Training had no effect on LV morphology in both stable and progressive pulmonary
hypertension.
All data are presented as mean ±SEM. ##: p<0.01; ###: p<0.001 vs. control. Cp/Cm = number of capillaries per
cardiomyocyte.
56
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Supplemental TABLES
Cardiac output (mL/min)
Stroke volume (mL)
Control
Stable PH
Progressive PH
(n = 20)
(n = 18)
(n = 18)
119 ±4
117 ±3
127 ±5
0.29 ±0.01
0.30 ±0.06
0.32 ±0.01
Heart rate (bpm)
405 ±5
394 ±5
399 ± 5
TAPSE (mm)
3.6 ±0.1
3.5 ±0.1
3.3 ±0.1
1.07 ±0.03 ##
RV wall thickness (mm)
0.96 ±0.01
1.08 ±0.02 ###
RVEDD (mm)
3.6 ±0.1
3.6 ±0.1
3.6 ±0.1
PAAT/cl (x100)
17 ±1
13 ±1 #
11 ±1 #### †
26 ±2
35 ±3 #
48 ±4 #### †
0.14 ±0.01
0.20 ±0.01 #
0.23 ±0.02 #
eRVSP (mmHg)
PVR (mmHg/ml/min)
Echocardiographic characteristics control vs. stable PH vs. progressive PH at the start of training confirmed the
pulmonary hypertensive status of MCT-treated rats. A strong MCT-dose dependent response was seen for PAAT/cl, eRVSP,
PVR and RV wall thickness (p<0.001).
All data are presented as mean ±SEM. #: p<0.05; ##: p<0.01; ###: p<0.001 vs. control; †: p <0.05 vs. stable PH.
Abbreviations: TAPSE, tricuspid annular plane systolic excursion; RVEDD, RV end-diastolic diameter; PAAT/cl , normalized
pulmonary artery acceleration time; eRVSP, estimated RV systolic pressure; PVR, pulmonary vascular resistance.
Chapter 3
Table S-1 Echocardiographic data at start of training
Table S-2 Effect of training on disease progression
Control
Stable PH
Progressive PH
Sed
Ex
Sed
Ex
Sed
Ex
(n = 10)
(n = 10)
(n = 9)
(n = 9)
(n = 9)
(n = 9)
Interaction
p-value
ΔCO (%/day)
0.4 ±0.2
0.2 ±0.3
-0.7 ±0.8
0.2 ±0.3
-3.1 ±0.8
-5.8 ±1.0 **
0.01
ΔSV (%/day)
0.6 ±0.2
0.4 ±0.2
-0.1 ±0.6
-0.7 ±0.2
-2.2 ±0.8
-4.9 ±1.0 **
0.02
ΔHR (%/day)
-0.2 ±0.1
-0.2 ±0.1
-0.8 ±0.4
-0.4 ±0.1
-0.9 ±0.2
-1.8 ±0.3 *
0.02
ΔTAPSE (%/day)
0.1 ±0.1
0.1 ±0.1
-1.1 ±0.8
-0.3 ±0.3
-2.5 ±0.6
-4.2 ±0.6 *
0.04
ΔRVWT (%/day)
0.0 ±0.1
-0.1 ±0.1
1.4 ±0.3
0.5 ±0.2
1.2 ±0.4
2.5 ±0.4 **
<0.001
ΔRVEDD (%/day)
-0.1 ±0.1
0.2 ±0.2
2.7 ±1.0
1.6 ±0.3
6.6 ±1.5
10.0 ±1.0 *
0.03
ΔRVSP (%/day)
0.7 ±0.5
0.4 ±0.5
2.8 ±1.0
2.7 ±0.6
2.0 ±0.7
6.6 ±2.1 *
0.04
ΔPVR (%/day)
0.8 ±0.7
0.3 ±0.6
7.5 ±5.0
2.7 ±0.9
11 ±4.1
35 ±9.0 **
0.001
Opposite effects of training on disease progression in stable vs. progressive PH were observed for all parameters. All data
are presented as mean ±SEM. *: p<0.05, **: p<0.01 progressive PH-Ex vs. pPH-Sed. Abbreviations: ΔCO (ΔSV, ΔHR, …),
daily percentage change of cardiac output, stroke volume, and so on, during exercise period.
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Exercise training
Table S-3 Autopsy data
Control
Stable PH
Progressive PH
Interaction
Sed
Ex
Sed
Ex
Sed
Ex
(n = 10)
(n = 10)
(n = 9)
(n = 9)
(n = 9)
(n = 9)
Body mass (g)
404 ±11
390 ±14
394 ±14
398 ±5
327 ±16
308 ±7
BM change (%/2d)
1.6 ±0.4
1.0 ±0.4
0.8 ±0.9
1.1 ±0.2
-4.2 ±2.1
-7.3 ±2.0
0.21
Lung mass (wet) (g)
1.47 ±0.05
1.42 ±0.07
1.95 ±0.08
1.68 ±0.09
2.23 ±0.14
2.88 ±0.15 ***
<0.001
Lung wet / dry mass ratio
Heart mass (g)
p-value
0.63
5.0 ±0.1
4.9 ±0.1
5.2 ± 0.2
5.1 ±0.1
5.2 ±0.1
5.0 ±0.2
0.98
1.42 ± 0.06
1.48 ±0.07
1.67 ±0.09
1.67 ±0.09
1.69 ±0.05
1.70 ±0.09
0.91
RV mass (g)
0.28 ±0.02
0.31 ±0.01
0.55 ±0.04
0.46 ±0.03
0.57 ±0.04
0.56 ±0.02
0.09
LV mass (+septum) (g)
0.98 ±0.05
1.01 ±0.05
0.93 ±0.04
0.96 ±0.03
0.79 ±0.03
0.76 ±0.04
0.69
RV / (LV + S) (g/g)
0.29 ±0.02
0.31 ±0.02
0.60 ±0.05
0.48 ±0.04
0.72 ±0.04
0.76 ±0.05
0.11
Liver (g)
14.5 ±0.6
13.9 ±0.8
15.0 ±0.6
15.2 ±0.6
11.3 ±1.0
10.2 ±0.4
0.65
Kidneys (g)
2.41 ±0.20
2.45 ±0.10
2.64 ±0.10
2.47 ±0.10
1.97 ±0.10
2.10 ±0.10
0.47
Spleen (g)
0.74 ±0.04
0.72 ±0.04
0.84 ±0.06
0.79 ±0.04
0.69 ±0.05
0.69 ±0.03
0.89
Brains (g)
2.02 ±0.03
1.96 ±0.05
1.99 ±0.02
1.96 ±0.04
1.88 ±0.05
1.93 ±0.04
0.31
37 ±0.3
37 ±0.3
37 ±0.3
38 ±0.4
36 ±0.3
36 ±0.3
0.34
Tibia length (mm)
A strong interactive effect Training x PH was observed for (wet) lungs mass only. This interactive effect remained strongly
significant independent of normalization (i.e. normalization by body mass or tibia length, and was not attributed to
edema (the lung wet/dry ratio did not differ between the experimental groups). A strong MCT-dose dependent response
was observed (p<0.001 for all parameters, except brain mass). All data are presented as mean ±SEM (wet weights). ***:
p<0.001 progressive PH-Ex vs. progressive PH-Sed. Abbreviations: BM change, relative body mass change over the last
two days; RV / (LV +S), RV over LV (including septum) mass ratio.
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