Download Heart failure with preserved ejection fraction: chronic low

J Appl Physiol 114: 131–147, 2013.
First published October 25, 2012; doi:10.1152/japplphysiol.01059.2012.
Heart failure with preserved ejection fraction: chronic low-intensity interval
exercise training preserves myocardial O2 balance and diastolic function
Kurt D. Marshall,1* Brittany N. Muller,1* Maike Krenz,2,3 Laurin M. Hanft,3 Kerry S. McDonald,3
Kevin C. Dellsperger,3,4,5 and Craig A. Emter1,3
1
Department of Biomedical Science, University of Missouri-Columbia, Columbia, Missouri; 2Dalton Cardiovascular Research
Center, University of Missouri-Columbia, Columbia, Missouri; 3Department of Medical Pharmacology and Physiology,
University of Missouri-Columbia, Columbia, Missouri; 4Department of Internal Medicine, University of Missouri-Columbia,
Columbia, Missouri; 5Center for Health Care Quality, University of Missouri-Columbia, Columbia, Missouri
Submitted 29 August 2012; accepted in final form 23 October 2012
diastolic heart failure; exercise; MV̇O2; fibrosis; hypertrophic signaling;
speckle tracking; coronary flow
PRIOR CLINICAL STUDIES, INCLUDING the recently completed HFACTION trial, indicates exercise training is a safe and effec-
* K. D. Marshall and B. N. Muller contributed equally to this work.
Address for reprint requests and other correspondence: C. A. Emter, Dept.
of Biomedical Science, Univ. of Missouri- Columbia, 1600 E. Rollins, E117
Veterinary Medicine, Columbia, MO 65211 (e-mail: [email protected]).
http://www.jappl.org
tive therapeutic modality in the treatment of patients with
stable heart failure (HF) due to left ventricular (LV) systolic
dysfunction (9, 25, 37, 58, 63). However, a significant limitation of these studies was the exclusion of patients with symptoms of HF who have preserved ejection fraction (HFpEF)
(58). Of an estimated 5 million people in the United States
afflicted with HF, ⬃50% are diagnosed as having HFpEF (4,
36, 47). The prevalence of HFpEF is increasing at a rate of
⬇1%/yr and will soon be the most prevalent HF phenotype (4).
Despite having preserved systolic function at rest, these patients display reduced cardiac functional reserve and morbidity
and mortality rates similar to values observed in HF patients
with reduced systolic function (4, 47, 55). Recent evidence
indicates patients diagnosed with HFpEF are a heterogeneous
group displaying multiple mechanisms that contribute to its
pathology, extending beyond only diastolic dysfunction (4,
47). Conventional HF therapies have failed to improve the
prognosis of this HF subgroup over the past 3 decades, illustrating the need for the development of novel treatment strategies (4, 6, 47, 61).
Although the benefits of exercise in HF are becoming
apparent, the mechanisms underlying these responses are
poorly understood. A significant gap in the literature exists
regarding the effects of chronic exercise training in treating
developing HF in large-animal models of cardiovascular disease. Recent data from our laboratory demonstrated chronic
low-intensity interval exercise training provides significant
benefits to the coronary vasculature and cardiac remodeling in
miniature swine hearts with LV hypertrophy generated from
pressure overload (20, 22). Specifically, we showed chronic
exercise limits LV fibrosis, collagen deposition, and mitochondrial dysfunction characterized by increased sensitivity to
Ca2⫹-induced mitochondrial permeability transition. Furthermore, we demonstrated chronic training prevents enhanced
coronary vascular sensitivity to endothelin-1 associated with a
decrease in smooth muscle Ca2⫹-sensitive composite K⫹ currents. The sum of these observations suggests chronic exercise
training could be used to effectively treat two critical factors
known to be involved in the pathogenesis of HF: 1) myocardial
oxygen supply/demand imbalance; and 2) impaired diastolic
function often associated with increased fibrotic remodeling of
the myocardium.
In the present study, we tested two hypotheses in treadmilltrained aortic-banded Yucatan miniature swine: 1) chronic
low-intensity interval training preserves normal myocardial
oxygen supply/demand balance; and 2) training-dependent
attenuation of LV fibrotic remodeling improves diastolic func-
8750-7587/13 Copyright © 2013 the American Physiological Society
131
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Marshall KD, Muller BN, Krenz M, Hanft LM, McDonald
KS, Dellsperger KC, Emter CA. Heart failure with preserved
ejection fraction: chronic low-intensity interval exercise training
preserves myocardial O2 balance and diastolic function. J Appl
Physiol 114: 131–147, 2013. First published October 25, 2012;
doi:10.1152/japplphysiol.01059.2012.—We have previously reported chronic low-intensity interval exercise training attenuates fibrosis,
impaired cardiac mitochondrial function, and coronary vascular dysfunction in miniature swine with left ventricular (LV) hypertrophy (Emter
CA, Baines CP. Am J Physiol Heart Circ Physiol 299: H1348 –H1356,
2010; Emter CA, et al. Am J Physiol Heart Circ Physiol 301: H1687–
H1694, 2011). The purpose of this study was to test two hypotheses:
1) chronic low-intensity interval training preserves normal myocardial
oxygen supply/demand balance; and 2) training-dependent attenuation of
LV fibrotic remodeling improves diastolic function in aortic-banded
sedentary, exercise-trained (HF-TR), and control sedentary male Yucatan
miniature swine displaying symptoms of heart failure with preserved
ejection fraction. Pressure-volume loops, coronary blood flow, and twodimensional speckle tracking ultrasound were utilized in vivo under
conditions of increasing peripheral mean arterial pressure and ␤-adrenergic stimulation 6 mo postsurgery to evaluate cardiac function. Normal
diastolic function in HF-TR animals was characterized by prevention of
increased time constant of isovolumic relaxation, normal LV untwisting
rate, and enhanced apical circumferential and radial strain rate. Reduced
fibrosis, normal matrix metalloproteinase-2 and tissue inhibitors of metalloproteinase-4 mRNA expression, and increased collagen III isoform
mRNA levels (P ⬍ 0.05) accompanied improved diastolic function
following chronic training. Exercise-dependent improvements in coronary blood flow for a given myocardial oxygen consumption (P ⬍ 0.05)
and cardiac efficiency (stroke work to myocardial oxygen consumption,
P ⬍ 0.05) were associated with preserved contractile reserve. LV hypertrophy in HF-TR animals was associated with increased activation of Akt
and preservation of activated JNK/SAPK. In conclusion, chronic lowintensity interval exercise training attenuates diastolic impairment by
promoting compliant extracellular matrix fibrotic components and preserving extracellular matrix regulatory mechanisms, preserves myocardial oxygen balance, and promotes a physiological molecular hypertrophic signaling phenotype in a large animal model resembling heart failure
with preserved ejection fraction.
132
Exercise Preserves Normal Cardiovascular Function in HFpEF
tion. Using pressure-volume (P-V) loops (a gold standard for
examining ventricular function) and two-dimensional (2D)
speckle tracking echocardiography (a clinical method of assessing LV function in humans), we provide a detailed characterization in vivo of LV systolic and diastolic function,
including its relationship to coronary blood flow and myocardial O2 consumption (MV̇O2), under conditions of increasing
peripheral mean arterial pressure (MAP) and ␤-adrenergic
stimulation. We also provide new mechanistic insight into
exercise-dependent improvements in fibrotic remodeling and
diastolic function in HFpEF by examining the expression of
several components of the extracellular matrix (ECM) and their
regulatory biomarkers, hypertrophic signaling pathways, and
passive elements of the cardiomyocyte contractile apparatus.
METHODS
Marshall KD et al.
artery (LAD) and near the apex for insertion of catheters and flow
probes. Great care was taken to leave the pericardium as intact as
possible. A 3PSB flow probe (Transonic Systems, Ithaca, NY) was
placed around the LAD to measure coronary blood flow. A custom
fluid-filled angiocatheter was inserted into the great cardiac vein for
coronary venous blood sampling. P-V loops were measured utilizing
a calibrated 7F admittance-based ADVantage catheter (SciSense,
London, Ontario, Canada) positioned in the LV via a small apical
incision. A 14F balloon occlusion catheter was advanced to the
inferior vena cava at the level of the apex of the heart via the deep
femoral vein. Peripheral systemic MAP was measured, and aortic
blood samples collected via a fluid-filled 6F LCB SH guide catheter
(Boston Scientific) introduced through a 7F sheath placed in the right
femoral artery and positioned in the aorta distal to the aortic band.
Catheter placement was visualized and confirmed using angiography
(Infimed software) and Visipaque contrast medium.
In vivo cardiovascular function. Following placement of the catheters and the flow probe, animals were brought to a peripheral MAP
of 80 mmHg using phenylephrine (1–5 ␮g·kg⫺1·min⫺1 iv) and allowed to stabilize until a stable coronary blood flow and HR were
observed for 5 min. This state of homeostasis was labeled “baseline”.
Swine lack coronary vascular ␣1-receptors (11, 68); therefore, the use
of phenylephrine is a valid method to induce systemic vasoconstriction and increase peripheral MAP without directly impacting coronary
function in swine. Coronary blood flow and arterial and venous blood
samples were collected simultaneously under conditions of increasing
pressure (peripheral MAP of 80, 90, 100, and 120 mmHg). Once the
target pressure was attained, coronary blood flow and HR were
allowed to stabilize until a new homeostasis was established. Following the pressure experiments, phenylephrine was reduced to the
baseline dose, and animals were allowed to return to baseline and
stabilize for 10 min. P-V loop measures were collected at baseline and
following one dose of dobutamine (a ␤1-adrenergic receptor agonist;
5 ␮g·kg⫺1·min⫺1 iv) administered for 5 min. Coronary blood flow
was averaged the last 30 s of each pressure/HR step. Pressure
experiments were utilized to examine myocardial oxygen demand/
supply balance and cardiac efficiency. ␤-Adrenergic stimulation was
utilized to examine myocardial contractile reserve.
MV̇O2 was determined as previously described (18). Briefly, blood
samples were analyzed immediately upon collection (ABL 700; Radiometer, Copenhagen, Denmark) for pH, PO2 (Torr), hemoglobin
count (Hb; g/dl), O2 saturation (SO2%), and lactate levels (Lactate
Scout; Sports Resource Group, Hawthorne, NY). Myocardial blood
O2 content [␮mol/ml; calculated using the following equation: (Hb ⫻
0.621 ⫻ SO2%) ⫹ (0.00131 ⫻ PO2)], O2 delivery (␮mol/ml; coronary
blood flow ⫻ arterial O2 content), and O2 extraction (%; ratio of
MV̇O2 to O2 delivery) were determined using methods published
previously (18). MV̇O2 was calculated as the product of coronary
arterial/venous O2 difference and coronary blood flow and then
normalized to the region of the heart perfused by the LAD. Feigl et al.
(24) previously estimated LAD perfusion area to be 30% of total heart
weight, and this standard has been utilized recently in swine (3).
P-V loops were recorded under conditions of reducing preload
achieved through transient occlusion of the inferior vena cava via
inflation of the balloon catheter. Indexes of LV function were generated using a minimum of 10 consecutive cardiac cycles with Lab
Scribe software (iWorx, Dover, NH), including HR, LV end-systolic
and diastolic volume (LVESV and LVEDV, respectively), LV endsystolic and diastolic pressure, ejection fraction (EF%), stroke volume, stroke work (SW), rate-pressure product, and cardiac output.
Other previously published indexes of LV function proposed to be
less sensitive to myocardial load and/or morphology were also determined using at least 15 consecutive cardiac cycles of constantly
reducing preload, including the end-systolic P-V relationship (ESPVR)
and preload recruitable SW (PRSW) (28, 66, 72). A quadratic fit
(which was judged to more accurately characterize this relationship)
was used to determine ESPVR and PRSW. Cardiac efficiency was
J Appl Physiol • doi:10.1152/japplphysiol.01059.2012 • www.jappl.org
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Aortic banding and exercise training. Before aortic banding, intact
male Yucatan miniature swine (27–30 kg; 8 mo old) were assigned
into three groups: nonsham sedentary control (Con; n ⫽ 4), banded
HF sedentary (HF; n ⫽ 5), and banded HF exercise trained (HF-TR;
n ⫽ 5). LV hypertrophy/HF was induced by aortic banding using
methods previously published by our laboratory (20) with modifications. These modifications included moving the aortic banding site to
the ascending aorta proximal to the brachiocephalic artery. A systolic
transstenotic gradient of ⬃70 mmHg [70 ⫾ 1 and 71 ⫾ 2 mmHg for
HF and HF-TR, respectively, P ⫽ nonsignificant (NS)] was achieved
while maintaining a distal peripheral vascular MAP of ⬃90 mmHg
(93 ⫾ 4 and 90 ⫾ 3 mmHg for HF and HF-TR, respectively, P ⫽ NS)
under anesthesia using phenylephrine (1–3 ␮g·kg⫺1·min⫺1 iv) at a
heart rate (HR) of 100 beats/min (101 ⫾ 4 and 97 ⫾ 4 beats/min for
HF and HF-TR, respectively, P ⫽ NS). Two months postsurgery,
transthoracic echocardiography was performed under inhaled isoflurane anesthesia (0.5%) to measure LV end-diastolic dimension and
LV diastolic wall thickness utilizing M-mode recordings using a
1.5– 4 MHz transducer on a GE Vividi Ultrasound system, as described previously (20). Aortic banding significantly increased LV
diastolic wall thickness in HF animals (P ⬍ 0.05, 5.6 ⫾ 0.3 and
7.7 ⫾ 0.5 mm for Con and HF, respectively) but did not alter LV
end-diastolic dimension (P ⫽ NS; 42.7 ⫾ 0.3 and 42.6 ⫾ 1.1 mm for
Con and HF, respectively), indicating concentric LV hypertrophy (an
observation commonly associated with LV pressure overload) was
present before the onset of exercise training. No differences in
echocardiographic measures of morphology existed between HF and
HF-TR groups at this time point; therefore, data from both aorticbanded groups were combined before the start of exercise training.
Following the development of LV hypertrophy, animals began lowintensity interval treadmill training consisting of treadmill running 3
days/wk, 55 min/day, for 15 wk with gradually increasing intensity as
tolerated until finally consisting of the following: 1) 5-min warm-up at
2 mph; 2) six 5-min sessions at 3 mph with five 3-min intervals at 4
mph in between; and 3) 5-min cool-down at 2 mph, as previously
published (20, 22). Animals were fed a standard diet averaging 15–20
g/kg once daily, and water was provided ad libitum. Dissection of
vital tissues and removal of skeletal muscle for analysis of citrate
synthase activity (70) occurred at the time of death. All animal
protocols were in accordance with the “Principles for the Utilization
and Care of Vertebrate Animals Used in Testing Research and
Training” and approved by the University of Missouri Animal Care
and Use Committee.
Terminal studies. Animals were initially anesthetized with a telazol
(5 mg/kg)/xylazine (2.25 mg/kg) mix and maintained on 100% oxygen using inhaled isoflurane (⬇1.75%). Heparin was given with an
initial loading dose of 300 U/kg iv, followed by maintenance of 100
U/kg each hour. A median sternotomy was performed, and the
pericardium was opened along the left anterior descending coronary
•
Exercise Preserves Normal Cardiovascular Function in HFpEF
133
Marshall KD et al.
Olympus Instrument), which was placed on a pneumatic vibration
isolation table with a cutoff frequency of ⬃1 Hz. Mechanical measurements were performed using a capacitance-gauge transducer
[model 403, sensitivity of 0.5 mN/V (plus a ⫻10 amplifier) and
resonant frequency of 600 Hz; Aurora Scientific, Aurora, ON, Canada]. Length changes were introduced using a DC torque motor
(model 308, Aurora Scientific) driven by voltage commands from a
personal computer via a 12-bit digital-to-analog converter (AT-MIO16E-1, National Instruments, Austin, TX). Passive force and length
signals were digitized at 1 kHz and stored on a personal computer
using LabView for Windows (National Instruments). Sarcomere
length was monitored simultaneous with force and length measurements using IonOptix SarcLen system (IonOptix, Milton, MA), which
used a fast Fourier transform algorithm of the video image of the
myocyte. Microscopy was done using a ⫻40 objective (Olympus
UWD 40) and a ⫻2.5 intermediate lens.
Passive tension measurements were performed at 14 ⫾ 1°C. For
sarcomere length-passive tension measurements, an experimental protocol was performed similar to that previously described (29). Following attachment of myocyte preparation to the apparatus, the
relaxed preparation was adjusted over a range of sarcomere lengths
from ⬃2.15 ␮m to ⬃2.65 ␮m at ⬃0.1-␮m increments by manual
manipulation of the length micrometer. After each sarcomere length
adjustment, ⬃20 s were provided to allow for development of steadystate passive force, and then the preparation was rapidly slackened
15–20% of initial length to yield zero force. Passive tension was
calculated as the difference in the force transducer signal before and
after the slack and divided by the myocyte’s cross-sectional area
determined from myocyte width measurements.
SDS-agarose gel electrophoresis and autoradiography. To determine baseline levels of PKA-mediated titin phosphorylation, 100 ␮g
of skinned cardiac myocytes were incubated with the catalytic subunit
of PKA (0.1 U/␮l) and 50 ␮Ci [␥-32P]ATP for 45 min. The reaction
was stopped by the addition of electrophoresis sample buffer and
heating at 95°C for 3 min. The samples were then separated by
SDS-agarose gel electrophoresis, silver stained, dried, and subsequently exposed to X-ray film (see Fig. 7, inset).
Quantitative RT-PCR. Quantitative real-time-PCR was performed
as previously described (21). Pulverized LV in TRIzol solution were
quick frozen in liquid nitrogen and stored at ⫺80°C until processed.
Total RNA was isolated according to the manufacturer’s published
protocol for TRIzol. cDNA was transcribed from total RNA using
High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) in a 20-␮l reaction. A minus RT reaction was also performed to
ensure no genomic DNA contamination. Quantitative RT-PCR was
performed on a Bio-Rad MyIQ2 cycler (model no. 170 –9790). Each
25-␮l reaction contained ⫻1 SYBER Green Master Mix (Bio-Rad),
0.8 ␮M forward and reverse primers, and 1 ␮g of cDNA. The reaction
conditions were optimized for each set of primers listed in Table 1.
Target gene expression was normalized to 18S ribosomal RNA using
the 2⫺⌬⌬ Ct method (45). Linearity and efficiency of each PCR
Table 1. Primer sequences (5= to 3=)
Target Gene
18S
BNP
Collagen I
Collagen III
MMP-2
MMP-9
TIMP-1
TIMP-4
Fibronectin
Elastin
Forward Primer
CGG
GCA
GCG
TCT
ATG
TCG
GTC
GAC
TGA
TGC
CTA
GCA
TCT
GAA
ACG
TGG
ATC
GGA
AGA
AGT
CCA
GCC
CTT
TTC
GAG
TTC
AGG
AAG
ACC
GGT
CAT
TCT
CCC
CCC
AGG
CGA
GCC
GTC
CTT
ACC
CCA
ATC
TCC
AGC
CTG
CCT
AAG
TTC
GCA
TCA
Reverse Primer
AGG
CTC
CTA
AAA
ACA
ACT
TTT
ATC
GTT
ACT
AA
TC
GT
AG
TC
TC
GT
CA
CC
CG
AGC
TCC
GTC
CCA
CCA
GTT
AAG
ACA
GCT
CTT
TGG
TGT
TCC
TTG
TAC
ACC
TAT
TAA
TAG
GGC
BNP, brain natriuretic peptide; MMP, matrix metalloproteinase; TIMP, tissue inhibitors of metalloproteinase.
J Appl Physiol • doi:10.1152/japplphysiol.01059.2012 • www.jappl.org
AAT
ATC
CTT
AGA
TTC
GTC
CCG
TGC
GCC
CTT
TAC
CCT
GGG
CAT
ACA
CCG
CAG
TGG
TTG
GAC
CGC
GGC
TCC
TTG
CGC
AGT
ACG
GCC
GTC
TCC
GGC
AGT
CTA
AAA
ACC
GAA
CTC
TGG
AAC
TGT
TC
TC
TTG G
AC
GA
TC
TA
AG
TC
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calculated as the ratio of SW to MV̇O2 (27, 71). The time constant of
isovolumic relaxation (␶) was calculated using the method of Raff and
Glantz (64).
2D speckle tracking echocardiography. Transthoracic echocardiography was performed under inhaled isoflurane anesthesia (0.5%) in
the supine/right lateral position 6 mo postbanding using a GE Vivid I
Ultrasound system, as previously described (20). Analysis was performed offline using GE EchoPac Software. Six segments of the LV
and septum were generated from apical four-chamber and short-axis
two-dimensional views (acquired at the mitral valve and apex levels)
and averaged to determine global strain, strain rate, and displacement
in the longitudinal, transverse, radial, and circumferential dimensions
over three cardiac cycles (53). Peak mitral annulus velocity at the
septum insertion was measured using tissue Doppler from an apical
four-chamber view. Torsion was calculated as the difference between
mitral and apical end-systolic rotation (degrees) and normalized to
both LV hypertrophy (wall thickness) and end-diastolic chamber
length, as previously described (65). Normalization to either of these
factors did not influence our results; thus only absolute torsion values
are reported.
Isolation of cardiac myocytes. Myocytes were isolated as previously described (33). Briefly, a section of LV free wall (⬃10 cm3)
near the LAD was removed, and half was rapidly frozen in liquid
nitrogen for biochemical analyses, and the other half was placed in
ice-cold relaxing solution for myocyte experiments. The piece in
relaxing solution was cut into smaller pieces (2–3 mm) and homogenized with a Waring blender. The resultant slurry was centrifuged for
75 s at 165 g, and the pellet was suspended for 3 min in 0.5% ultrapure
Triton X-100 (Pierce Chemical) in relaxing solution. The permeabilized myocytes were washed and centrifuged twice with cold relaxing
solution with the final suspension kept on ice during the day of the
experiment. Relaxing solution in which the ventricles were disrupted,
skinned, and suspended contained the following (in mmol/l): 2 EGTA,
5 MgCl2, 4 ATP, 10 imidazole, and 100 KCl at pH 7.0. The
compositions of relaxing solution used in passive tension measurements was as follows (mmol/l): 7 EGTA, 5 MgCl2, 20 imidazole, 4
ATP, 14.5 creatine phosphate, pH 7.0, Ca2⫹ concentrations of 10⫺9
M (relaxing solution), and sufficient KCl to adjust ionic strength to
180 mM.
Sarcomere length-passive tension measurements. The experimental
apparatus for physiological measurements of myocyte preparations
was similar to previous descriptions (49). Myocyte preparations were
attached between a force transducer and torque motor by placing the
ends of the myocyte preparation into stainless steel troughs (25
gauge). The ends of the myocyte preparations were secured by
overlaying a 0.5-mm length of 3– 0 monofilament nylon suture (Ethicon) onto each end of the myocyte and then tying the suture into the
troughs with two loops of 10 – 0 monofilament (Ethicon). The attachment procedure was performed under a stereomicroscope (⫻ ⬃100
magnification) using finely shaped forceps.
Before mechanical measurements, the experimental apparatus was
mounted on the stage of an inverted microscope (model IX-70,
•
Exercise Preserves Normal Cardiovascular Function in HFpEF
A
CON
HF
HF-TR
250
weight (g)
Marshall KD et al.
B
300
*
200
150
*
100
*
50
0
Wt (kg)
HW
LV+S
RV
CON
C
*
ATRIA
2.5
*
P=0.06
2
1.5
1
0.5
0
HF
p-ERK 1/2
akt
erk
HF-TR
jnk/sapk
kDa
44
42
ERK 1/2
44
42
p-SAPK/JNK
54
46
SAPK/JNK
46
p-Akt
60
Akt
60
condition were verified by creating a standard curve plotting the
critical threshold vs. log of the cDNA dilution.
Histology and immunohistochemistry. Cross sections of LV were
formalin fixed, embedded in paraffin, and immunohistochemistrystained for assessment of fibrosis. Briefly, total fibrosis was visualized
from 4-␮m-thick sections of LV using Masson’s trichrome stain with
methods previously established (20). Fibrosis was quantified from
four separate fields/animal using Image-Pro Plus analysis software
(MediaCybernetics, version 6.2, Bethesda, MD) and expressed as the
percent area stained.
Western blotting. LV tissue was lysed in buffer containing 150 mM
NaCl, 10 mM Tris (pH 7.4), 1 mM EDTA, and 1% Triton X-100, as
previously described (20). Proteins were resolved by SDS-PAGE
using 10 –15% acrylamide, transferred onto polyvinylidene difluoride
membranes, and blotted using the following commercially available
antibodies: all primary (1:500) and secondary (anti-rabbit, 1:1,000)
antibodies were from Cell Signaling Technologies. Membranes were
incubated with the appropriate alkaline phosphatase or horseradish
peroxidase-linked secondary antibody and visualized by enhanced
chemifluorescence and chemiluminescence (Amersham).
Scanning and transmission electron microscopy. LV tissue was
collected processed for scanning electron microscopy (SEM) and
transmission electron microscopy (TEM), as previously reported (81).
Briefly, all LV samples were fixed in a 2% paraformaldehyde, 2%
gluteraldehyde in 0.1 M sodium cadodylate solution. SEM samples
were rinsed in 0.1 M sodium cadodylate, then incubated at 4°C in a
secondary fixative solution of 2% OsO4 in 0.1 M citric acid complex
buffer, rinsed three times in 0.1 M Na cacodylate and Milli-Q water,
dehydrated in a series of ethanol washes, and dried. After fixation,
tissues were attached to Hitachi stubs with carbon adhesive, and silver
colloidal paint was applied around tissue edges (to maximize contact
with the carbon for optimum imaging) and sputter coated with
platinum before imaging on a Hitachi S4700 SEM microscope. TEM
samples were rinsed three times in 2-mercaptoethanol and then incubated at 4°C in a secondary fixative 1% osmium tetroxide solution,
washed three times in 2-mercaptoethanol and Milli-Q water, dehy-
drated in a series of acetone solutions and resin infiltration solutions,
and polymerized at 60°C. TEM samples were cut with a diamond
blade into 85-nm sections and then imaged on a JEOL 1400 Biological TEM microscope.
Three fields from each pig were randomly chosen to review and
obtain a minimum of three images from each LV sample at a
magnification of ⫻4,500 (SEM) or ⫻1,000 (TEM). To prevent sampling bias, the operator was blinded to the experimental condition of
each sample, and an independent investigator experienced in electron
micrographs analyzed our images postcollection to confirm our findings.
Zymography. LV was prepared for gelatin zymography, as described previously (76). Active human recombinant matrix metalloproteinase-2 (MMP-2) and MMP-9 (Calbiochem) were used as a
positive control. Briefly, 30 mg of LV were homogenized in a glass
homogenizer in 500 ␮l of cold lysis buffer (25 mM Tris·HCl, pH 7.5,
100 mM NaCl, 1% Nonident P-40) with protease inhibitors (10 ␮g/ml
aprotinin, 2 ␮g/ml leupeptin, 4 mM benzamidine). One hundred
micrograms of protein were subjected to electrophoresis on a 10%
polyacrylamide gel with gelatin (Bio-Rad) at 125 V for 90 min. Gels
were then incubated at room temperature in renaturing solution (2.5%
Triton X-100 in dH2O) for 30 min, washed with dH2O, and washed
for 30 min at room temperature in developing buffer (50 mM
Tris·HCl, pH 7.8, 0.2 M NaCl, 5 mM CaCl2, 0.02% Brij 35). Gels
were then incubated for 24 h in developing buffer at 37°C and stained
for 1 h with Coomassie blue R-250 (0.5% Coomassie blue R-250, 5%
methanol, 10% acetic acid), and then destained (10% methanol, 5%
acetic acid). Gels were imaged using a gel-doc system (Bio-Rad) and
quantified using ImageJ (National Institutes of Health) software.
Statistical analysis. All data analysis was performed using SPSS
version 19.0 or SigmaStat version 3.5. Group comparisons were made
using either one-way or repeated-measures ANOVA or independent
samples t-test, as appropriate. A mixed model incorporating linear
regression and analysis of covariance was used to compare response
variable [coronary blood flow, O2 extraction, coronary venous PO2
(cvPO2) and SO2% (cvSO2), change in lactate] slopes plotted vs.
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Fig. 1. Left ventricular (LV) remodeling and
expression of hypertrophic signaling proteins. A: aortic banding generates cardiac
hypertrophy, regardless of training status.
Global myocardial hypertrophy, as indicated
by increased postmortem heart (HW), LV ⫹
septum (LV⫹S), right ventricle (RV), and left ⫹
right atrial (Atria) weight, was present in all
aortic banded groups. Con, control; HF,
heart failure sedentary; HF-TR, heart failure
exercise trained. *P ⬍ 0.05, Con vs. HF-TR
and HF. B: expression of phosphorylated
(p)-Akt-to-total Akt protein ratio is increased in the LV of both HF-TR and HF
compared with Con animals. *P ⬍ 0.05, HF
vs. Con. A less significant decrease in pJNK/p-SAPK-to-total JNK/SAPK ratio was
observed only in the HF group. †P ⫽ 0.075,
HF vs. Con. Values are means ⫾ SE. C:
Western blots for all hypertrophic signaling
proteins.
•
Relative Protein ExpressionPhosphorylated:Total Protein
134
Exercise Preserves Normal Cardiovascular Function in HFpEF
Pressure (mmHg)
A
•
Marshall KD et al.
135
CON
120
100
80
60
40
20
0
0
20
40
60
80
100
120
Volume (mL)
HF
120
100
80
R
Fig. 2. Representative pressure-volume (P-V)
loops at baseline (mean arterial pressure ⫽ 80
mmHg) from individual Con (A), HF (B), and
HF-TR (C) animals. ESPVR, end-systolic
P-V relationship.
ESPV
60
40
20
0
0
20
40
60
80
100
120
100
120
Volume (mL)
HF-TR
120
100
VR
80
60
ESP
Pressure (mmHg)
C
40
20
0
0
20
40
60
80
Volume (mL)
MV̇O2, using group as the independent variable. Linear regression was
used to examine relationships between ultrasound variables (torsion,
early diastolic untwisting rate) and P-V loop measures (␶, LVESV,
EF%). Group differences revealed by ANOVA were found using
Student Newman-Keuls post hoc analysis. Within-group comparisons
were made using paired samples t-test. All data are means ⫾ SE, and
significance is reported at P ⬍ 0.10 and P ⬍ 0.05 levels (10, 83).
RESULTS
LV remodeling. Postmortem assessment of the heart indicated the presence of cardiac hypertrophy. Body weight was
not different between groups (P ⫽ NS; 36 ⫾ 2, 39 ⫾ 2, and
38 ⫾ 3 kg for HF, HF-TR, and Con, respectively); therefore,
absolute heart morphology measures are reported. Aortic banding significantly increased LV plus septum, atrial, right ventricle, and whole heart weights in HF and HF-TR groups
compared with Con (Fig. 1A). Myocardial hypertrophy occurred in all aortic-banded groups, regardless of training status,
similar to previous reports from our laboratory (20). LV
hypertrophy in the HF group was associated with an increase in
the ratio of phosphorylated Akt (p-Akt) to total Akt protein and
a less significant decrease in p-JNK/p-SAPK-to-total JNK/
SAPK protein ratio (Fig. 1B). The level of Akt phosphorylation
was also elevated to a lesser degree in HF-TR animals. Western blots for hypertrophic signaling proteins are show in
Fig. 1C.
LV brain natriuretic peptide mRNA expression. Aortic banding resulted in a 14-fold increase in LV (P ⬍ 0.05; 13.9 ⫾ 6.5,
0.5 ⫾ 0.2, 1.0 ⫾ 0.7 for HF, HF-TR, and Con, respectively)
brain natriuretic peptide mRNA expression in HF relative to
Con animals. This finding, indicative of the presence of compensatory HF, was attenuated by exercise training and suggests
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Pressure (mmHg)
B
136
Exercise Preserves Normal Cardiovascular Function in HFpEF
Table 2. Pressure-volume analysis of baseline systolic and
diastolic function
Con
Systolic function
LVESV, ml
LVESP, mmHg
SW, J
CO, l/min
RPP, beats 䡠 min⫺1 䡠
mmHg
SV, ml
EF, %
Diastolic function
LVEDV, ml
LVEDP, mmHg
␶ Glantz, ms
Cardiac Efficiency
SW/MV̇O2
HF
HF-TR
54 ⫾ 8
101 ⫾ 5
0.41 ⫾ 0.10
3.6 ⫾ 0.8
93 ⫾ 9*
112 ⫾ 13
0.69 ⫾ 0.05*
5.9 ⫾ 0.5
72 ⫾ 9
106 ⫾ 3
0.72 ⫾ 0.06*
5.9 ⫾ 1.0
8,912 ⫾ 591
40 ⫾ 9
42 ⫾ 3
11,764 ⫾ 1,669
57 ⫾ 3
38 ⫾ 3
9,019 ⫾ 689
68 ⫾ 10
49 ⫾ 6
94 ⫾ 16
11 ⫾ 1
53 ⫾ 3
150 ⫾ 8*
14 ⫾ 2
82 ⫾ 6*
140 ⫾ 8*
15 ⫾ 2
70 ⫾ 6
0.38 ⫾ 0.11
0.56 ⫾ 0.12
0.97 ⫾ 0.19*
our training protocol was effective in postponing the onset of
overt HF.
Citrate synthase activity. Citrate synthase activity was significantly elevated in the medial head of the triceps in HF-TR
animals, indicative of exercise-induced training adaptations to
our low-intensity exercise protocol (P ⬍ 0.05; 12.6 ⫾ 1.2,
18.0 ⫾ 1.0, and 14.4 ⫾ 1.4 ␮mol·g wet wt muscle⫺1·min⫺1 for
HF, HF-TR, and Con, respectively). These data, reproduced
separately from those previously published by our laboratory
(20), indicate our low-intensity interval training protocol is of
sufficient intensity to induce a classic metabolic marker of
training.
LV systolic function and contractile reserve. Representative
baseline P-V loops (Fig. 2) and measures (Table 2) are similar
to those previously reported in pigs (39). LVESV and SW were
increased in HF animals compared with Con. 2D speckle
tracking indicated torsion was increased in HF compared with
C
B
14
12
CON
HF
HF-TR
6
4
CON
HF
HF-TR
60
10
8
0
70
50
*
EF %
Torsion (degrees)
Con animals (Fig. 3A). Cumulative group data, indicated by the
regression line, showed torsion was positively correlated with
EF% (P ⬍ 0.05; Fig. 3B). Despite increased torsion, peak
systolic rotation rate at the apex was significantly reduced in
HF animals compared with Con (Fig. 3C). Global transverse
displacement (Fig. 4A) and strain (Fig. 4B) in the longitudinal
view and apical circumferential and radial end-systolic strain
rate (Fig. 4C) in the HF group were similar to Con. LV
contractility in response to ␤-adrenergic stimulation is presented in Table 3. Diminished contractile reserve was observed
in HF animals, as indicated by the reduced percent increase in
ESPVR and PRSW. The percent increase in HR in the HF
group was also reduced, suggesting ␤-adrenergic responsiveness was decreased in these animals.
Exercise training prevented the impairments in systolic function observed in HF animals. SW was increased in HF-TR
animals, but the parallel increase in LVESV observed in the
HF group was attenuated (Table 2). Torsion was also increased
in HF-TR animals (Fig. 3A), but, unlike the HF group, was
associated with maintenance of apical global peak systolic
rotation rate (Fig. 3C). Global transverse displacement (Fig.
4A) and strain (Fig. 4B; longitudinal view) were significantly
increased in HF-TR compared with Con and HF animals and
likely related to the improved EF%-torsion relationship. Increased torsion in the HF-TR group was also associated with
increased apical circumferential and radial end-systolic strain
rate (Fig. 4C). Despite similar reductions in ␤-adrenergic
responsiveness (reflected by percent increase in HR similar to
the HF group), chronic exercise training effectively preserved
contractile reserve, as indicated by the percent increase in
ESPVR and PRSW in relation to Con (Table 3). A significant
increase in PRSW was observed in HF-TR animals, further
suggesting LV contractility was enhanced following training.
Diastolic function. Diastolic function was impaired in HF
animals. P-V data showed increases in baseline LVEDV and ␶
in HF compared with Con animals (Table 2). 2D speckle
tracking indicated LV untwisting rate during early diastole was
reduced in HF animals (Fig. 5A). Cumulative data for all
groups (indicated by the regression line) showed untwisting
rate was negatively correlated to both ␶ and LVESV (Fig. 5, B
and C), reproducing findings from previous work (78). Group
Apical Global Peak Systolic
Rotation Rate (degrees/s)
A
Marshall KD et al.
40
30
20
2
10
0
0
y = 1.7467x + 25.892
R = 0.474
P < 0.05
-10
-20
-30
-40
-50
-60
*
-70
0
5
10
15
20
Torsion (degrees)
Fig. 3. LV torsion and apical systolic rotation rates. A: torsion is increased in aortic-banded animals independent of training status. B: linear regression plot
demonstrating a significant correlation between ejection fraction (EF%) and torsion. C: exercise training prevents a reduction in apical global peak systolic
rotation rate. Values are means ⫾ SE. *P ⬍ 0.05, HF vs. HF-TR and Con.
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Values are means ⫾ SE. Con, control; HF, heart failure sedentary; HF-TR,
heart failure exercise trained; LVESV, left ventricular end-systolic volume;
LVESP, left ventricular end-systolic pressure; SW, stroke work; CO, cardiac
output; RPP, rate-pressure product; SV, stroke volume; EF, ejection fraction;
LVEDV, left ventricular end-diastolic volume; LVEDP, left ventricular enddiastolic pressure; ␶, time constant of left ventricular pressure decay; MV̇O2,
myocardial O2 consumption; SW/MV̇O2, ratio of SW to MV̇O2. *Significance
is indicated at P ⬍ 0.05 vs. Con.
•
Exercise Preserves Normal Cardiovascular Function in HFpEF
B
6
CON
HF
HF-TR
C
50
*
5
4
3
2
1
0
137
Marshall KD et al.
40
35
30
25
20
15
*
2.5
*
45
2
Strain Rate (1/s)
7
Global Tranverse Strain (%)
Global Transverse Displacement
(mm)
A
•
1.5
1
0.5
0
-0.5
-1
10
-1.5
5
-2
*
Apical End Systolic
Circumferential Strain Rate
0
Apical End Systolic Radial
Strain Rate
Fig. 4. LV systolic transverse strain and apical circumferential and radial strain rates. A and B: exercise training increases global transverse displacement and
strain, respectively, in the longitudinal view. C: exercise training increases end-systolic circumferential and radial strain rate at the apex of the heart. Values are
means ⫾ SE. *P ⬍ 0.05, HF-TR vs. Con and HF.
Table 3. LV contractility in following ␤-adrenergic
stimulation
Baseline
HR, beats/min
Con
HF
HF-TR
ESPVR, mmHg/ml
Con
HF
HF-TR
PRSW, mmHg
Con
HF
HF-TR†
5 ␮g 䡠 kg⫺1 䡠 min⫺1
Dobutamine
Contractile Reserve,
%increase
92 ⫾ 4
112 ⫾ 5
98 ⫾ 8
138 ⫾ 6
151 ⫾ 7
135 ⫾ 4
50
35
38
7⫾3
12 ⫾ 3
11 ⫾ 4
22 ⫾ 3
20 ⫾ 4
31 ⫾ 6
210
65
176
48 ⫾ 7
71 ⫾ 10
74 ⫾ 7
99 ⫾ 7
129 ⫾ 7*
155 ⫾ 13*
107
82
109
Values are means ⫾ SE. HR, heart rate; ESPVR, end-systolic pressurevolume relationship; PRSW, preload recruitable stroke work. *Significance is
indicated at P ⬍ 0.05 vs. Con, same dose value. †P ⬍ 0.05 vs. Con
(repeated-measures ANOVA; group main effect).
stained) indicated general fibrosis was significantly elevated in
HF animals compared with Con (Fig. 7C). The approximate
20% increase in fibrosis is similar to previous reports from our
laboratory (20) and demonstrates the reproducibility of this
effect in our model. Increased fibrosis in the HF group was
associated with increases in MMP-2 and tissue inhibitors of
metalloproteinase-4 (TIMP-4) mRNA expression, along with
less significant increases in MMP-9 and TIMP-1 mRNA levels
relative to Con (Fig. 8A). MMP-2 and MMP-9 activity/abundance levels, determined using gel zymography, and a representative zymography gel are presented in Fig. 8, B and C,
respectively. TEM micrographs illustrated distinct collagen
bundles surrounding the capillary lumen in HF animals (Fig. 9B),
in addition to disruption of the structural organization of
mitochondria between sarcomeres and mitochondrial clustering (Fig. 10B) compared with Con (Fig. 10A).
Low-intensity interval exercise training prevented increased
myocardial fibrosis in response to pressure overload (Fig. 7C).
A reduction in fibrosis compared with Con animals was observed in the HF-TR group and associated with a significant
increase in collagen III mRNA expression (Fig. 7D). In contrast to the HF group, MMP-2, MPP-9, TIMP-1, and TIMP-4
mRNA levels were unchanged relative to Con (Fig. 8A). A
small decrease in MMP-2 activity/abundance (Fig. 8B) and less
significant increase in fibronectin mRNA expression relative to
Con (P ⫽ 0.068; data not shown) was also observed in HF-TR
animals. Analysis of relative elastin mRNA expression showed
no significant differences between groups (data not shown).
Chronic exercise training limited pericapillary fibrosis, mitochondrial clustering, and sarcomere/mitochondrial disorganization compared with HF animals (Figs. 9C and 10C).
Sarcomere length dependence of passive force and PKAmediated titin phosphorylation. Sarcomere length-passive
force relationships for permeabilized cardiac myocyte preparations are shown in Fig. 11. Passive force was greater for a
given sarcomere length in cardiac myocytes after aortic banding. Increased passive force was associated with greater PKAmediated back-phosphorylation of titin, indicative of lower
baseline levels of titin phosphorylation (autoradiogram inset).
Passive force and titin back-phosphorylation were elevated,
regardless of training status; consequently, HF and HF-TR data
were pooled for this measure.
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mean data from HF animals indicated a rightward shift of this
relationship compared with Con, demonstrating decreases in
LV untwisting was associated with increases in both ␶ and
LVESV. An increase in tissue Doppler A= (Fig. 6B) and increased
global longitudinal, mitral valve circumferential, and mitral
valve radial strain rates during late diastole (Fig. 6C) were
observed in the HF group, suggesting an enhanced role of atrial
systole compared with Con animals.
Exercise training prevented the diastolic dysfunction observed in the HF group. Baseline LVEDV was increased,
however, the significant increase in ␶ observed in HF animals
was prevented (Table 2). Early diastolic untwisting rate was
the same as Con animals (Fig. 5A), and rightward shifts in ␶
and LVESV in relation to early diastolic untwisting rate was
attenuated by exercise training (Fig. 5, B and C). Apical
circumferential and radial strain rate during early diastole were
also increased in HF-TR animals compared with both Con and
HF (Fig. 6A). Tissue Doppler A= (Fig. 6B) and late diastolic
strain rates (Fig. 6C) were not different than Con values,
indicating the enhanced atrial systole observed in HF animals
was not present in the HF-TR group.
LV fibrosis. Representative histological and SEM sections of
the LV from HF, Con, and HF-TR animals are shown in Fig. 7, A and
B. Trichrome staining (expressed as the percent area of LV
138
Exercise Preserves Normal Cardiovascular Function in HFpEF
Marshall KD et al.
100
90
80
*
70
60
CON
HF
HF-TR
50
40
30
20
10
0
C
120
Untwisting- Peak Global E Rotation
Rate (degree/s)
B
Untwisting- Peak Global E Rotation
Rate (degree/s)
Untwisting- Peak Global E Rotation
Rate (degree/s)
A
•
120
CON
HF
HF-TR
100
100
80
60
40
y = -0.3286x + 102.37
R = -0.45
P = 0.05
20
0
0
20
40
60
80
100
80
60
40
y = -0.3131x + 102.78
R = -0.61
P < 0.05
20
0
0
tau Glantz (msec)
50
100
150
LVESV (mL)
Myocardial oxygen supply/demand balance in response to
increasing peripheral pressure load. LV hemodynamic and
blood gas variables at baseline and during incremental increases in peripheral MAP are summarized in Table 4. Exercise training prevented the increase in HR observed in the HF
group. HR was significantly higher in HF compared with both
Con and HF-TR animals. Our MV̇O2 (50 –175 ␮mol/min before normalization to heart weight) and CBF (14 – 45 ml/min
before normalization to heart weight) values under sedation are
consistent with previously published studies in conscious resting pigs (MV̇O2: 125–225 ␮mol/min; CBF: 50 –75 ml/min)
(11–13, 50), verifying the validity of our hemodynamic and
blood-gas data. Exercise training improved the slope of the
coronary blood flow to MV̇O2 relationship, while coronary
blood flow increased at a slower rate in response to increasing
oxygen consumption (Fig. 12A) in HF animals. In the HF
group, decreased CBF for a given MV̇O2 was associated with
increased oxygen extraction (Fig. 12B) and decreased cvSO2
(Fig. 12C). The slope of the cvPO2 (Fig. 12D) to MV̇O2
relationship was also reduced in HF compared with HF-TR
animals. In total, these data indicate exercise training was able
to inhibit the development of myocardial oxygen supply/demand imbalance following pressure overload, despite the presence of significant LV hypertrophy.
Cardiac efficiency, expressed as the ratio of SW to MV̇O2
(SW/MV̇O2), was significantly increased under baseline condi-
A
B
4
8
*
3
CON
HF
HF-TR
1
C
6
(cm/s)
0
-1
-2
-4
4
5
4
3
-3
-5
2
*
Apical Early Diastolic Peak
Circumferential Strain Rate
Apical Early Diastolic Peak
Radial Strain Rate
1
0
*
3
Strain Rate (1/s)
Fig. 6. LV early-diastolic apical circumferential and radial strain rates, tissue Doppler, and
late-diastolic longitudinal and mitral circumferential and radial strain rates. A: exercise
training increases global circumferential and
radial strain rate during early diastole at the
apex of the heart. *P ⬍ 0.05, HF-TR vs. Con
and HF. †P ⬍ 0.05, HF vs. Con. B: movement of the mitral annulus at the insertion of
the septum during atrial systole increased in
sedentary HF animals (P ⫽ 0.055). C: exercise training prevented increases in global latediastolic longitudinal and circumferential and
radial strain rates at the mitral valve of the heart.
*P ⬍ 0.05, HF vs. Con and HF-TR. †P ⬍ 0.05,
HF-TR vs. Con. Values are means ⫾ SE.
Strain Rate (1/s)
2
*P=0.055
7
*
2
1
0
-1
-2
-3
*
-4
Late Diastolic Global Longitudinal
Strain Rate
Mitral Valve Late Diastolic Global
Circumferential Strain Rate
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Mitral Valve Late Diastolic Global
Radial Strain Rate
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Fig. 5. LV untwisting during early diastole. A: exercise training prevents a reduction in peak global LV rotation rate during early diastole. *P ⬍ 0.05, HF vs.
HF-TR and Con. B and C: linear regression plots demonstrating a significant correlation between untwisting and both the time constant of isovolumic relaxation
(␶) and LV end-systolic volume (LVESV). Exercise training attenuates the right downward shift in the relationship between untwisting during early diastole and
both ␶ and LVESV observed in sedentary HF animals. Values are means ⫾ SE.
Exercise Preserves Normal Cardiovascular Function in HFpEF
CON
•
139
Marshall KD et al.
HF
HF-TR
A
B
D
30
25
25
CON
HF
HF-TR
*
20
15
10
5
Relative mRNA Expression
FIBROSIS % AREA (LV)
35
*
20
15
10
5
0
0
Collagen I
Collagen III
Fig. 7. LV fibrosis and relative mRNA expression of collagen I and II isoforms. A: representative histological sections of trichrome-stained LV demonstrating
increased fibrosis in HF animals (magnification: ⫻40). B: representative scanning electron microscopy (SEM) micrographs showing increased quantity and
thickness of collagen fibers on the myocardium of the HF group (magnification: ⫻4,500). C: exercise training prevents increases in LV fibrosis, as indicated by
the percent area stained. *P ⬍ 0.05, HF vs. Con and HF-TR. †P ⬍ 0.05, HF-TR vs. Con. D: collagen III mRNA expression is increased in the LV of HF-TR
compared with Con and HF animals. *P ⬍ 0.05, HF vs. HF-TR and Con. Values are means ⫾ SE.
tions in HF-TR animals (Table 2) and associated with increased myocardial lactate uptake (indicated by the positive
slope, P ⫽ 0.088) compared with Con (Fig. 12E). Animals in
the HF group exhibited lactate uptake similar to Con animals,
suggesting, despite impaired myocardial oxygen supply/demand balance, the myocardium was not ischemic, and a metabolic shift to anaerobic glycolysis had not occurred under our
experimental conditions.
DISCUSSION
The results of this study illustrate several novel findings. In
the presence of existing hypertrophy and developing HF,
chronic low-intensity interval exercise training 1) prevents
diastolic dysfunction; 2) maintains normal myocardial oxygen
supply/demand balance; 3) improves cardiac efficiency; 4) encourages a physiological molecular hypertrophic signaling profile;
and 5) promotes expression of more compliant ECM components while preserving normal ECM regulatory mechanisms in
a large-animal model resembling HFpEF.
A clinically translational model of HFpEF. This investigation was primarily designed as a functional study with the
purpose of characterizing LV function following chronic exercise training in our swine model of pressure overload and
establishing it as a legitimate model of HFpEF using sophisticated in vivo techniques, including simultaneous coronary
blood flow, MV̇O2, and P-V measurements in addition to 2D
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C
Exercise Preserves Normal Cardiovascular Function in HFpEF
A
Relative mRNA expression
Marshall KD et al.
B
30
Fig. 8. Relative mRNA expression and activity of LV extracellular matrix regulating
biomarkers. A: exercise training prevents increases in the relative mRNA expression of
multiple metalloproteinase (MMP) and tissue inhibitors of metalloproteinase (TIMP)
isoforms. *P ⬍ 0.05, HF vs. HF-TR and
Con. B: quantification of MMP-2 and MMP-9
activity/abundance as determined by gel zymography. Values are means ⫾ SE. C: representative zymography gel of MMP-2 and
MMP-9 activity from all groups.
•
CON
HF
HF-TR
25
*
P=0.07
20
15
10
P=0.086
*
5
0
MMP-2
MMP-9
TIMP-1
C
Densitometry (arbitrary units)
140
3500
3000
2500
2000
1500
P=0.09
1000
500
0
TIMP-4
I
CON
MMP-2
I I
HF
MMP-9
I
I
HF-TR
MMP-9
75 kDa
MMP-2
speckle tracking echocardiography. Our previous work suggested diastolic dysfunction and myocardial oxygen supply/
demand imbalance were present in our model as a result of
pathological interactions between the coronary vasculature, LV
remodeling, and myocardial metabolism. Establishment of the
existence and nature of these cardiovascular functional deficits
was fundamental and substantiates our exploration of molecular mechanisms that we believe underlie exercise-dependent
improvements in cardiovascular function in our HFpEF model.
Results from the present study indicate our model exhibits
many characteristics of HFpEF, including LV hypertrophy,
early diastolic dysfunction, increased LV brain natriuretic
peptide mRNA levels, elevated HR, increased fibrosis, and
A
diminished LV contractile reserve. Combined with our laboratory’s previous work (20, 22), we believe our model accurately
reflects the pathophysiology of HFpEF and demonstrates the
relevance of our large-animal model for the study of this
patient population.
Exercise improves both systolic and diastolic cardiac
function. Patients with HFpEF demonstrate both systolic and
diastolic dysfunction, with diastolic impairment long thought
to be a key prognostic indicator for diagnosis. Our results show
chronic exercise alters mechanical properties of the LV that
may benefit systolic emptying and diastolic filling. 2D speckle
tracking echocardiography has become increasingly utilized
clinically as a diagnostic tool for assessing myocardial function
B
C
Mt
C
S
S
CL
Mt
CL
S
C
S
S
CL
C
S
Mt
Mt
Fig. 9. Representative transmission electron microscopy (TEM) micrographs showing pericapillary fibrosis in HF animals. Distinct collagen bundles indicating
interstitial pericapillary fibrosis surrounding the capillary lumen are more prominent in HF (B) compared with Con (A) or HF-TR (C) animals and demonstrate
coherence in relation to our histology results (magnification: ⫻4,500). C, collagen bundles; CL, capillary lumen; S, sarcomere; Mt, mitochondria.
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100 kDa
I
Exercise Preserves Normal Cardiovascular Function in HFpEF
A
B
•
141
Marshall KD et al.
C
S
S
Mt
S
Mt
S
S
Mt
S
in these patients. A key finding of the present study was
increased torsion following chronic exercise, which was associated with a commensurate increase in EF% that occurred in
a linear fashion predicted by our regression analysis. The
significant relationship between torsion and EF demonstrated
in our model has been shown previously in humans (79).
Central to this observation in the HF-TR group was prevention
of increased LVESV, maintenance of apical systolic rotation
rate, increased circumferential/radial strain rates, and enhanced
longitudinal transverse strain/displacement, findings not associated with increased torsion in HF animals. Previous studies
have demonstrated maintenance or enhancement of ventricular
torsion (or systolic twist) and circumferential, radial, and
longitudinal systolic strain in HFpEF patients at rest (60, 62,
79), although conflicting data reporting reductions in radial and
longitudinal strain exists (54, 75, 79). Data from the HF group
emulate these findings, demonstrating compensatory increases
HF & HF-TR Cardiac Myocytes
CON
Cardiac Myocytes
Passive Force (kN/m2)
14
12
10
Titin
8
6
4
2
0
2.0
2.1
2.2
2.3
2.4
2.5
2.6
2.7
Sarcomere Length (µm)
Fig. 11. Sarcomere length-passive force relationships for permeabilized cardiac myocyte preparations. Passive force was greater in cardiac myocytes after
aortic-banding (P ⬍ 0.05 vs. Con) and associated with lower baseline levels of
PKA-mediated titin phosphorylation, i.e., greater PKA-mediated back-phosphorylation in lane 1 samples from Con vs. lane 2 HF pigs in autoradiogram
inset. Exercise did not affect this finding; therefore, HF-TR and HF data were
pooled for this measure. Values are means ⫾ SE.
in torsion and preserved apical strain rates were associated with
maintenance (but not improvement) of EF at rest. Although
preservation of resting systolic function is a common feature of
HFpEF, decreased cardiac functional reserve is well established in this patient population (55, 75). Similarly, animals in
the HF group demonstrated reductions in contractile reserve in
response to ␤-adrenergic stimulation, implying an inability to
respond to increasing workloads, despite preserved resting
systolic function. Exercise-trained animals exhibited elevated
baseline contractility similar to that observed in the HF group.
However, in contrast to the HF group, contractile reserve
closely matched that seen in the Con group. These findings
suggest that diminished contractile reserve in HF animals was
not simply the result of less reserve available because of an
elevation in baseline contractility, but more so an impairment
in ␤-adrenergic sensitivity. Previous work demonstrated exercise training improves inotropic responsiveness to ␤-adrenergic activation in a rodent model of hypertension (46) and,
although speculative, may be a mechanism underlying exercise-dependent preservation of contractile reserve in response
to ␤-adrenergic stimulation in our study. Increased torsion and
strain rate following chronic exercise training were also associated with maintenance of contractile reserve, further demonstrating the utility of chronic exercise in preserving systolic
function and functional reserve in a setting of HFpEF.
Chronic exercise training also prevented the development of
diastolic impairment. A second key finding of our study was
the preservation of early diastolic untwisting rate, enhanced
early diastolic strain rates, and prevention of increased ␶
following 15 wk of chronic training. Subsequently, exercise
training was effective in preventing the rightward shift in the
relationship between LV untwisting rate and both LVESV and
␶ observed in Fig. 5, B and C. LV untwisting rate is used as a
diagnostic tool for assessment of diastolic function and temporally linked to ventricular relaxation and diastolic filling at
rest and in response to exercise (14, 56, 57). The untwisting
rate has been correlated with LVESV and indexes of LV
relaxation generated from P-V loops, such as ␶, in both health
and HF (14, 57, 78) and our data confirm these relationships.
Previous studies show ventricular untwisting rates during early
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Fig. 10. Representative TEM micrographs demonstrating structural disorganization or the myocardium in HF animals. Increased mitochondrial clustering
(outlined by white dashed line) and disrupted spatial orientation of mitochondria in relation to the sarcomere is evident in HF (B) compared with Con (A) animals
(magnification: ⫻1,000). Exercise training limits this structural remodeling to a lesser degree than that observed in the HF group (C).
142
Exercise Preserves Normal Cardiovascular Function in HFpEF
•
Marshall KD et al.
Table 4. Hemodynamic and blood-gas variables at baseline and during increasing pressure loads
90 mmHg
100 mmHg
120 mmHg
78 ⫾ 3
83 ⫾ 1
81 ⫾ 2
89 ⫾ 2
91 ⫾ 1
92 ⫾ 2
98 ⫾ 1
100 ⫾ 1
99 ⫾ 2
117 ⫾ 1
117 ⫾ 4
116 ⫾ 3
88 ⫾ 3
104 ⫾ 4*
85 ⫾ 6
80 ⫾ 6
98 ⫾ 6
86 ⫾ 6
71 ⫾ 4
99 ⫾ 11
81 ⫾ 6
71 ⫾ 8
109 ⫾ 16*
80 ⫾ 7
0.27 ⫾ 0.02
0.30 ⫾ 0.07
0.20 ⫾ 0.04
0.31 ⫾ 0.03
0.30 ⫾ 0.07
0.26 ⫾ 0.06
0.38 ⫾ 0.04
0.36 ⫾ 0.06
0.28 ⫾ 0.06
0.58 ⫾ 0.11
0.47 ⫾ 0.11
0.42 ⫾ 0.10
1.10 ⫾ 0.06
1.40 ⫾ 0.22
0.84 ⫾ 0.12
1.36 ⫾ 0.10
1.35 ⫾ 0.29
0.95 ⫾ 0.14
1.74 ⫾ 0.12
1.67 ⫾ 0.21
1.17 ⫾ 0.11
1.88 ⫾ 0.21
2.05 ⫾ 0.52
1.48 ⫾ 0.27
483 ⫾ 25
498 ⫾ 30
527 ⫾ 28
536 ⫾ 16
497 ⫾ 37
540 ⫾ 25
547 ⫾ 7
502 ⫾ 39
521 ⫾ 17
561 ⫾ 16
516 ⫾ 20
555 ⫾ 10
25 ⫾ 2
22 ⫾ 1
22 ⫾ 1
23 ⫾ 1
23 ⫾ 1
23 ⫾ 2
27 ⫾ 2
30 ⫾ 3
25 ⫾ 2
31 ⫾ 4
31 ⫾ 3
32 ⫾ 3
30 ⫾ 2
27 ⫾ 4
30 ⫾ 3
32 ⫾ 1
29 ⫾ 3
33 ⫾ 7
39 ⫾ 4
38 ⫾ 6
36 ⫾ 7
57 ⫾ 7
50 ⫾ 7
49 ⫾ 7
8.2 ⫾ 0.2
9.1 ⫾ 0.6
8.5 ⫾ 0.5
9.0 ⫾ 0.2
9.0 ⫾ 0.5
8.7 ⫾ 0.5
10.1 ⫾ 0.4
10.8 ⫾ 1.0
9.7 ⫾ 0.7
11.3 ⫾ 0.8
11.7 ⫾ 0.9
10.6 ⫾ 1.0
Values are means ⫾ SE. MAP, mean arterial pressure; CBF, coronary blood flow; PO2, O2 pressure; SO2, O2 saturation; Hb, hemoglobin. *Significance is
indicated at P ⬍ 0.05 vs. Con, same pressure load. †P ⬍ 0.05 vs. Con (repeated-measures ANOVA; group main effect).
diastole in HFpEF patients exhibit a great deal of variance,
including maintenance during the early stages of disease that
gradually declines as HF worsens (60, 62, 74, 75, 78). Consistent with these findings, reductions in early diastolic untwisting rate, increases in LVEDV and ␶, and the leftward shift
in untwisting rate in relation to both LVESV and ␶ indicate
significant diastolic impairment in the HF group.
A common compensatory mechanism of preserving diastolic
filling in HF patients is to augment atrial systole. Increased
mitral annulus movement and strain rates during late diastole
indicate this is indeed the case in our HF animals. Despite the
presence of atrial hypertrophy in both aortic-banded groups,
the HF-TR group did not demonstrate indicators of enhanced
atrial systole, as tissue Doppler and strain values during late
diastole were similar to Con values. In total, our results provide
new insight into utilizing 2D speckle tracking to differentiate
between pathological and physiological functional characteristics of the heart in HFpEF and elucidate positive adaptations to
chronic training in HF that could be utilized to assess the
effectiveness of exercise prescription.
Molecular mechanisms of exercise-dependent improvement
in diastolic function. Diastolic dysfunction resulting from increased myocardial stiffness has long been considered a hallmark feature of HFpEF (4). Central to our finding of preserved
diastolic function following chronic exercise is a reduction of
fibrosis in the HF-TR group. Our laboratory has previously
demonstrated this effect of training concurrent with a reduction
in collagen deposition (20). Results from the present study
suggest chronic exercise attenuates fibrotic LV remodeling via
two potential mechanisms: 1) maintenance of normal MMP/
TIMP expression; and 2) altering collagen isoform composition. In HF-TR animals, reduced fibrosis was associated with
normal mRNA levels of MMP-2, MMP-9, TIMP-1, and
TIMP-4. Relative to Con, expression of these regulatory biomarkers of the ECM was substantially increased in the HF
group. MMP-to-TIMP ratios are decreased in response to
pressure overload (31, 67, 69), and increased plasma expression of these MMP and TIMP isoforms has been proposed as
clinical predictors of LV hypertrophy and diastolic HF in
humans (86). Recent work by Yarbrough et al. (84) in swine
subject to acute and gradual pressure overload over 1 mo
demonstrated 1.5- to 2-fold increases in TIMP-1 and TIMP-4
mRNA expression relative to Con and proposed modulation of
these biomarkers could be therapeutically relevant. Our data
showed an ⬃15-fold increase in TIMP isoforms in HF animals,
suggesting under our chronically loaded conditions that TIMP
expression may continue to increase with the severity of HF.
The MMP/TIMP system is extremely complex, and regulation
of fibrotic remodeling in HF via manipulation of these molecular components has proven difficult in humans, as evident
from the equivocal results reported from studies such as the
PREMIER clinical trial (35). Our results demonstrate chronic
exercise may be a particularly powerful treatment option in this
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MAP, mmHg
Con
HF
HF-TR
HR, beats/min
Con
HF†
HF-TR
CBF, ml 䡠 min⫺1 䡠 g⫺1
Con
HF
HF-TR
MV̇O2, ␮mol
O2 䡠 min⫺1 䡠 g⫺1
Con
HF
HF-TR
Arterial PO2, Torr
Con
HF
HF-TR
Coronary venous PO2, Torr
Con
HF
HF-TR
Coronary venous SO2, %
Con
HF
HF-TR
Hb, g/100 ml
Con
HF
HF-TR
80 mmHg
Exercise Preserves Normal Cardiovascular Function in HFpEF
A
B
0.90
•
143
Marshall KD et al.
C
90
90
0.70
70
70
60
60
0.60
0.50
0.40
0.30
0.20
CON
0.10
HF
HF-TR
0.00
0.0
·
1.0
2.0
MVO2 (µmol O2
D
3.0
min-1 g-1)
30
10
20
15
10
5
P < 0.05
0
4.0
P = 0.05
0
0.0
·
1.0
2.0
MVO2 (µmol O2
3.0
min-1 g-1)
4.0
0.0
·
1.0
2.0
MVO2 (µmol O2
3.0
min-1 g-1)
4.0
0.50
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
P = 0.088
0.00
0.0
1.0
2.0
· (µmol O
MVO
2
2
3.0
min-1 g-1)
4.0
Fig. 12. Myocardial oxygen supply/demand balance in response to increasing peripheral mean arterial pressure loads. A: the slope of the relationship between
myocardial O2 consumption (MV̇O2) and coronary blood flow (CBF) was reduced in HF compared with HF-TR animals (P ⬍ 0.05). B: exercise training prevented
the increase in oxygen extraction observed in HF animals, as indicated by the significantly different slope of the O2 extraction- MV̇O2 relationship (P ⬍ 0.05).
C: increased oxygen extraction in the HF group was complemented by a concurrent decrease in coronary venous O2 saturation (cvSO2), apparent by the reduced
slope in the cvSO2- MV̇O2 relationship (P ⫽ 0.05). D: the slope for the relationship between coronary venous O2 pressure (cvPO2) and MV̇O2 was increased
following exercise training in HF-TR animals (P ⬍ 0.05). E: lactate consumption, evident by the positive slope of the relationship between coronary arterial vs.
venous lactate difference (lactate uptake) and MV̇O2, was increased in HF-TR animals (P ⫽ 0.088).
regard, enabling an integrated response in a diverse system that
has proven difficult to manipulate pharmacologically.
Improved diastolic function in HF-TR animals may be
linked to improved LV elasticity associated with increased
collagen III expression. Collagen type III is more compliant
than the type I isoform (80), and the ratio of type III to type I
is considered important to determining the elasticity/stiffness
of tissue with an increased III-to-I ratio representing increased
compliance (5). Burgess et al. previously examined collagen
type III-to-I ratio in response to hypertension or exercise in rats
(5). Their findings demonstrated a reduction in the type III-to-I
ratio that was associated with diminished diastolic function
following surgically induced hypertension. Conversely, exercise-trained animals maintained ratios similar to Con in the
presence of enhanced diastolic relaxation, demonstrating support for the association between collagen III-to-I ratio and
relaxation properties of the heart. Our results showed an
increase in collagen III mRNA with no significant change in
type I relative to Con in HF-TR animals, suggesting alteration
of collagen isoform ratios is a mechanism by which chronic
training preserves normal diastolic function and LV elasticity
in the presence of existing hypertrophy and developing HF.
Similar to previous findings in pressure-overloaded pigs (84),
collagen I and III mRNA expression was not altered in the HF
group relative to Con.
In addition to ECM remodeling, enhanced intrinsic cardiomyocyte stiffness is a potential factor contributing to diastolic
impairment in HFpEF (4). The enhanced passive stiffness in
cardiac myofibrils from aortic-banded animals in our study is
similar to that observed in human HFpEF patients by Borbely
et al. (2). Increased cardiomyocyte stiffness has been linked to
titin, a large elastic cytoskeletal protein that has been described
as a cardiac “spring”. Previous work has demonstrated an
increase in the N2BA (more compliant isoform)-to-N2B
(stiffer isoform) ratio in human hearts from patients with
HFpEF (2); however, shifts in titin isoform expression were
not observed in our study (data not shown). Changes in the
phosphorylation status of titin are known to alter passive
stiffness in human and pig cardiac myofibrils (2, 32, 40). Our
data showed a decrease in baseline PKA-mediated phosphorylation of titin (indicated by an increase in back-phosphorylation), which has been linked to increased passive force in
human HF (2) and confirmed in myocytes from our pig model.
Importantly, we provide evidence that diminished titin phosphorylation may precede shifting of titin isoforms during the
development of HF. Exercise training did not reduce passive
stiffness, implying modulation of the ECM plays a more
prominent role in exercise-dependent preservation of diastolic
function.
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cvPo2 (mmHg)
40
20
E
25
· (µmol O
MVO
2
2
20
4.0
30
3.0
min-1 g-1)
30
50
P < 0.05
35
2.0
40
0
40
1.0
50
10
45
0.0
cvSo2 (%)
80
O2 Extraction (%)
80
Lactate Uptake (µmol min-1 g-1)
CBF (mL min-1 g-1)
P < 0.05
0.80
144
Exercise Preserves Normal Cardiovascular Function in HFpEF
Marshall KD et al.
balance (3, 16, 17, 85), and imbalances in the relationship
between coronary blood flow and myocardial metabolism have
been observed in porcine and canine models of metabolic
syndrome (3, 85) and myocardial infarction (12, 13, 50, 51). To
the best of our knowledge, this is the first study to examine and
characterize LV function in a swine model of HF and chronic
exercise. Key to the interpretation of our results is the impact
of exercise on coronary oxygen balance and HR. An enhanced
rate of increase in coronary blood flow in response to increasing cardiac metabolic demands, coupled with preservation of
coronary oxygen extraction, cvPO2/cvSO2, and conservation of
HR similar to that observed in Con animals, demonstrates
maintenance of normal resistance vessel function and autonomic regulation are key mechanisms underlying traininginduced preservation of myocardial oxygen supply/demand
balance in HF. The coronary vasodilatory response to enhanced MV̇O2 stimulated by incrementally increasing peripheral MAP loads was diminished in HF animals and associated
with increased coronary oxygen extraction and decreased
cvSO2. Changes in oxygen extraction and cvSO2 are a reflection
of resistance vessel tone, supporting our assertion that the
impaired coronary vascular function our laboratory observed
previously (22) is associated with myocardial oxygen/supply
demand imbalance in our model approximating HFpEF. HR
was also significantly elevated across pressure loads in HF
animals compared with Con, potentially limiting coronary
blood flow primarily through increases in coronary back pressure brought about by increased extravascular compressive
forces (19) or reduced diastolic filling time. However, HR did
not change in response to increasing MAP load in HF animals,
demonstrating the impaired rate of coronary blood flow increase in response to increasing MV̇O2 was not merely a
consequence of elevated HR. The heart rate response in the HF
group also suggests the presence of baroreceptor dysfunction
as the normal response, a decline in heart rate in reaction to
increasing pressure observed in both CON and HF-TR groups,
was absent.
Our finding that preservation of oxygen supply/demand
balance following chronic exercise was associated with an
increase in overall LV efficiency is important. Efficiency in the
HF group was similar to that observed in Con; however, it was
sustained at a cost of increased myocardial oxygen extraction
resulting from the coronary blood flow/metabolic uncoupling
described previously. This represents a scenario that the heart
is unlikely to maintain long term. Although our data implied
the presence of resting myocardial ischemia in HF animals,
lactate consumption was similar to Con levels, suggesting
increased oxygen extraction was sufficient to prevent the onset
of myocardial anaerobic glycolysis. However, the change in
resistance vessel tone indicated by enhanced O2 extraction and
decreased cvSO2 would suggest that these animals are at a
perilous tipping point that, while not ischemic under our
experimental conditions of increasing peripheral MAP, may
certainly be underperfused and susceptible to ischemia under
other conditions of increasing myocardial metabolic demand,
such as exercise (which would also increase HR and LV
contractility).
Effect of anesthesia and Hb. A potential confounding variable of this study is the presence of incomplete coronary
autoregulation, likely due to the vasodilatory influence of
isoflurane. Elevated resting cvPO2 and cvSO2 values compared
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Exercise generates a unique hypertrophic signaling phenotype.
Considerable efforts have been made to identify the molecular
signaling components that differentiate between pathological
and physiological hypertrophy (15). Another central finding of
our study is that increases in activated Akt protein expression
was not associated with a commensurate decrease in activated
JNK/SAPK expression following chronic low-intensity interval training. In his 2007 review, Dorn discusses the “fuzzy
logic” of cardiac hypertrophy in the context that hypertrophy
typically exhibits both physiological and pathological characteristics (15). Activation of Akt has long been thought to play
a role in physiological hypertrophy, but studies in transgenic
mice have demonstrated chronic or excessive stimulation of the
Akt pathway can also lead to pathological adaptations in the
myocardium (48, 59). Exercise studies examining activation of
Akt under conditions of pressure overload were mostly done in
rodent models of hypertension and show equivocal findings
(26, 34, 38, 44, 52). The use of multiple rodent models and
varying modes, intensities, durations, frequencies, and overall
length of exercise training between these publications make
comparison difficult. Results from our large-animal model of
HFpEF clearly demonstrate the disparate action of Akt activation in the myocardium, and we speculate the physiological
adaptive action of Akt observed following chronic exercise in
our study might result from exercise-dependent activation of
growth factors such as insulin-like growth factor I (15, 59).
Exercise training also attenuated the decrease in activated
JNK/SAPK observed in HF animals. In 2004, Wilkins et al.
(82) demonstrated coordinated activation of calcineurin and
nuclear factor of activated T cell (NFAT) transcription factors
was increased in mice with pathological hypertrophy induced
by pressure overload, but not in mice with physiological
hypertrophy induced by two different types of exercise modes
(wheel running and swim training). Although this is not a
consensus finding (23), this study demonstrated calcineurinNFAT coupling likely plays a role in pathological, but not
physiological, growth of the myocardium. Supporting this
assertion are more recent studies demonstrating exercise training reduces calcineurin expression in hypertensive rats (26, 38,
44). The role of JNK activation in HF is controversial, as
conflicting results have been reported in human failing myocardium (7, 8, 30, 43, 73). However, Liang et al. (42) demonstrated JNK activation reduced cardiac hypertrophy via inhibition of the calcineurin-NFAT signaling pathway, suggesting,
under normal conditions, JNK signaling limits cardiac hypertrophy in response to pathological stimuli. Our data imply
exercise-dependent preservation of activated JNK/SAPK is a
potential mechanism of limiting calcium-mediated pathological remodeling of the myocardium. In total, our results demonstrate chronic exercise results in a unique molecular signaling profile that is associated with physiological hypertrophy, as
our laboratory has previously postulated (20).
Exercise preserves myocardial oxygen supply/demand balance and improves LV efficiency. A critical factor involved in
the pathogenesis of HF is development of myocardial oxygen
demand/supply imbalance. Another key finding in our study
was preservation of normal oxygen supply/demand balance,
despite the presence of antecedent pathological LV hypertrophy following chronic low-intensity interval training. Several
studies have examined the impact of acute exercise on coronary vascular function and myocardial oxygen supply/demand
•
Exercise Preserves Normal Cardiovascular Function in HFpEF
ACKNOWLEDGMENTS
The authors thank Cory Weimer, Melissa Cobb, Christine Schramm, and
Jan Ivey for considerable technical contributions, which were essential to the
successful completion of the study, and Dr. Heide Schatten for lending us
expertise in electron microscopy and critical independent evaluation of our
SEM and TEM images.
Marshall KD et al.
145
GRANTS
This study was supported by National Heart, Lung, and Blood Institute/
American Recovery and Reinvestment Act P30 HL101332 (written and
awarded to M. Harold Laughlin and managed by C.A. Emter).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: K.D.M., B.N.M., M.K., L.M.H., K.S.M., and C.A.E.
performed experiments; K.D.M., B.N.M., M.K., L.M.H., K.S.M., K.C.D., and
C.A.E. analyzed data; K.D.M., B.N.M., M.K., L.M.H., K.S.M., K.C.D., and
C.A.E. interpreted results of experiments; K.D.M., B.N.M., M.K., L.M.H.,
K.S.M., and C.A.E. prepared figures; K.D.M., B.N.M., L.M.H., and C.A.E.
drafted manuscript; K.D.M., B.N.M., M.K., L.M.H., K.S.M., K.C.D., and
C.A.E. approved final version of manuscript; M.K., L.M.H., K.S.M., K.C.D.,
and C.A.E. edited and revised manuscript; L.M.H., K.S.M., and C.A.E.
conception and design of research.
REFERENCES
1. Bender SB, van Houwelingen MJ, Merkus D, Duncker DJ, Laughlin
MH. Quantitative analysis of exercise-induced enhancement of early- and
late-systolic retrograde coronary blood flow. J Appl Physiol 108: 507–514,
2010.
2. Borbely A, Falcao-Pires I, van Heerebeek L, Hamdani N, Edes I,
Gavina C, Leite-Moreira AF, Bronzwaer JG, Papp Z, van der Velden
J, Stienen GJ, Paulus WJ. Hypophosphorylation of the stiff N2B titin
isoform raises cardiomyocyte resting tension in failing human myocardium. Circ Res 104: 780 –786, 2009.
3. Borbouse L, Dick GM, Payne GA, Payne BD, Svendsen MC, Neeb ZP,
Alloosh M, Bratz IN, Sturek M, Tune JD. Contribution of BK(Ca)
channels to local metabolic coronary vasodilation: effects of metabolic
syndrome. Am J Physiol Heart Circ Physiol 298: H966 –H973, 2010.
4. Borlaug BA, Paulus WJ. Heart failure with preserved ejection fraction:
pathophysiology, diagnosis, and treatment. Eur Heart J 32: 670 –679,
2011.
5. Burgess ML, Buggy J, Price RL, Abel FL, Terracio L, Samarel AM,
Borg TK. Exercise- and hypertension-induced collagen changes are related to left ventricular function in rat hearts. Am J Physiol Heart Circ
Physiol 270: H151–H159, 1996.
6. Burkhoff D. Mortality in heart failure with preserved ejection fraction: an
unacceptably high rate. Eur Heart J 33: 1718 –1720, 2012.
7. Communal C, Colucci WS, Remondino A, Sawyer DB, Port JD,
Wichman SE, Bristow MR, Singh K. Reciprocal modulation of mitogenactivated protein kinases and mitogen-activated protein kinase phosphatase 1 and 2 in failing human myocardium. J Card Fail 8: 86 –92, 2002.
8. Cook SA, Sugden PH, Clerk A. Activation of c-Jun N-terminal kinases
and p38-mitogen-activated protein kinases in human heart failure secondary to ischaemic heart disease. J Mol Cell Cardiol 31: 1429 –1434, 1999.
9. Crimi E, Ignarro LJ, Cacciatore F, Napoli C. Mechanisms by which
exercise training benefits patients with heart failure. Nat Rev Cardiol 6:
292–300, 2009.
10. Curran-Everett D, Benos DJ. Guidelines for reporting statistics in
journals published by the American Physiological Society. Am J Physiol
Regul Integr Comp Physiol 287: R247–R249, 2004.
11. de Beer VJ, Bender SB, Taverne YJ, Gao F, Duncker DJ, Laughlin
MH, Merkus D. Exercise limits the production of endothelin in the
coronary vasculature. Am J Physiol Heart Circ Physiol 300: H1950 –
H1959, 2011.
12. de Beer VJ, Sorop O, Pijnappels DA, Dekkers DH, Boomsma F,
Lamers JM, Duncker DJ, Merkus D. Integrative control of coronary
resistance vessel tone by endothelin and angiotensin II is altered in swine
with a recent myocardial infarction. Am J Physiol Heart Circ Physiol 294:
H2069 –H2077, 2008.
13. de Beer VJ, Taverne YJ, Kuster DW, Najafi A, Duncker DJ, Merkus
D. Prostanoids suppress the coronary vasoconstrictor influence of endothelin after myocardial infarction. Am J Physiol Heart Circ Physiol 301:
H1080 –H1089, 2011.
14. Dong SJ, Hees PS, Siu CO, Weiss JL, Shapiro EP. MRI assessment of
LV relaxation by untwisting rate: a new isovolumic phase measure of tau.
Am J Physiol Heart Circ Physiol 281: H2002–H2009, 2001.
J Appl Physiol • doi:10.1152/japplphysiol.01059.2012 • www.jappl.org
Downloaded from http://jap.physiology.org/ by 10.220.33.4 on June 14, 2017
with those previously observed in conscious resting Yucatan
mini-swine (1) suggest vasomotor tone was reduced slightly at
baseline. Anesthetic interruption of coronary autoregulation
conceivably resulted in modest overperfusion of the myocardium, particularly during pressure loading, leading to a gradual
increase in cvPO2 and cvSO2 over the course of our experimental protocol, ultimately resulting in blood-gas/ MV̇O2 relationships deviating from those normally observed in conscious
instrumented pigs (11–13, 50). We also explored the influence
that elevated and gradually increasing Hb levels over the
course of our protocol (likely the result of ␣-1 mediated splenic
contraction from the phenylephrine doses used to increase
pressure loads) may have had on our findings. However,
previous work by Van Woerkens et al. (77) demonstrated
increases in coronary venous oxygen concentrations in response to hemodilution, suggesting the increased Hb levels
probably did not contribute significantly to our results. Given
that our CBF and MV̇O2 values were consistent with those of
previously published studies in conscious resting pigs and all
animals were subject to the same conditions (likely affecting
the three groups similarly), we consider our results accurate
and valid as considered under our experimental conditions.
Limitations. We demonstrated our large-animal model accurately reflects the pathophysiology of HFpEF. However, there
are several limitations to our study. First, hypertension and
aortic stenosis are key contributors to the pathogenesis of
HFpEF. Although our aortic-banding procedure creates a pressure-overload scenario designed to mimic these disease states,
it does not accurately reflect the effects of hypertension systemically nor the comprehensive reduction in arterial distensibility observed along a large portion of the aorta in aortic
stenosis. Second, HFpEF is found more often in an aged
population, and our study was performed in young, sexually
mature swine and limited to only males. Finally, our study
design did not include a Con exercise-trained group, limiting
our understanding of the pressure overload independent aspect
of our training protocol. However, we consider this restriction
minimal, given that our training protocol is approximately
one-half the intensity of previously used swine training protocols (41) and may not be of sufficient intensity to elicit training
adaptations in a healthy animal.
In conclusion, chronic low-intensity interval exercise training attenuates diastolic impairment by promoting compliant
ECM fibrotic components and preserving ECM regulatory
mechanisms, preserves myocardial oxygen balance, and promotes a physiological molecular hypertrophic signaling phenotype. Our results demonstrate an integrated response in
multiple physiological systems following exercise during developing HF and provide novel mechanistic insight into the
benefits of training in a clinically translational large-animal
model of HFpEF.
•
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36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
Marshall KD et al.
loproteinase inhibitor (PG-116800) to prevent ventricular remodeling after
myocardial infarction: results of the PREMIER (Prevention of Myocardial
Infarction Early Remodeling) trial. J Am Coll Cardiol 48: 15–20, 2006.
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, Antman EM, Smith
SC Jr, Adams CD, Anderson JL, Faxon DP, Fuster V, Halperin JL,
Hiratzka LF, Jacobs AK, Nishimura R, Ornato JP, Page RL, Riegel B.
ACC/AHA 2005 Guideline Update for the Diagnosis and Management of
Chronic Heart Failure in the Adult: a report of the American College of
Cardiology/American Heart Association Task Force on Practice Guidelines (Writing Committee to Update the 2001 Guidelines for the Evaluation and Management of Heart Failure): developed in collaboration with
the American College of Chest Physicians and the International Society
for Heart and Lung Transplantation: endorsed by the Heart Rhythm
Society. Circulation 112: e154 –e235, 2005.
Keteyian SJ, Pina IL, Hibner BA, Fleg JL. Clinical role of exercise
training in the management of patients with chronic heart failure. J
Cardiopulm Rehabil Prev 30: 67–76, 2010.
Kolwicz SC, MacDonnell SM, Renna BF, Reger PO, Seqqat R, Rafiq
K, Kendrick ZV, Houser SR, Sabri A, Libonati JR. Left ventricular
remodeling with exercise in hypertension. Am J Physiol Heart Circ
Physiol 297: H1361–H1368, 2009.
Korvald C, Elvenes OP, Myrmel T. Myocardial substrate metabolism
influences left ventricular energetics in vivo. Am J Physiol Heart Circ
Physiol 278: H1345–H1351, 2000.
Kruger M, Kotter S, Grutzner A, Lang P, Andresen C, Redfield MM,
Butt E, dos Remedios CG, Linke WA. Protein kinase G modulates
human myocardial passive stiffness by phosphorylation of the titin
springs. Circ Res 104: 87–94, 2009.
Laughlin MH, Overholser KA, Bhatte MJ. Exercise training increases
coronary transport reserve in miniature swine. J Appl Physiol 67: 1140 –
1149, 1989.
Liang Q, Bueno OF, Wilkins BJ, Kuan CY, Xia Y, Molkentin JD.
c-Jun N-terminal kinases (JNK) antagonize cardiac growth through crosstalk with calcineurin-NFAT signaling. EMBO J 22: 5079 –5089, 2003.
Liang Q, Molkentin JD. Redefining the roles of p38 and JNK signaling
in cardiac hypertrophy: dichotomy between cultured myocytes and animal
models. J Mol Cell Cardiol 35: 1385–1394, 2003.
Libonati JR, Sabri A, Xiao C, Macdonnell SM, Renna BF. Exercise
training improves systolic function in hypertensive myocardium. J Appl
Physiol 111: 1637–1643, 2011.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data
using real-time quantitative PCR and the 2[-delta delta C(T)] method.
Methods 25: 402–408, 2001.
MacDonnell SM, Kubo H, Crabbe DL, Renna BF, Reger PO, Mohara
J, Smithwick LA, Koch WJ, Houser SR, Libonati JR. Improved
myocardial beta-adrenergic responsiveness and signaling with exercise
training in hypertension. Circulation 111: 3420 –3428, 2005.
Maeder MT, Kaye DM. Heart failure with normal left ventricular
ejection fraction. J Am Coll Cardiol 53: 905–918, 2009.
Matsui T, Li L, Wu JC, Cook SA, Nagoshi T, Picard MH, Liao R,
Rosenzweig A. Phenotypic spectrum caused by transgenic overexpression
of activated Akt in the heart. J Biol Chem 277: 22896 –22901, 2002.
McDonald KS. Ca2⫹ dependence of loaded shortening in rat skinned
cardiac myocytes and skeletal muscle fibers. J Physiol 525: 169 –181,
2000.
Merkus D, Houweling B, van den Meiracker AH, Boomsma F,
Duncker DJ. Contribution of endothelin to coronary vasomotor tone is
abolished after myocardial infarction. Am J Physiol Heart Circ Physiol
288: H871–H880, 2005.
Merkus D, Houweling B, van Vliet M, Duncker DJ. Contribution of
KATP⫹ channels to coronary vasomotor tone regulation is enhanced in
exercising swine with a recent myocardial infarction. Am J Physiol Heart
Circ Physiol 288: H1306 –H1313, 2005.
Miyachi M, Yazawa H, Furukawa M, Tsuboi K, Ohtake M, Nishizawa
T, Hashimoto K, Yokoi T, Kojima T, Murate T, Yokota M, Murohara
T, Koike Y, Nagata K. Exercise training alters left ventricular geometry
and attenuates heart failure in Dahl salt-sensitive hypertensive rats. Hypertension 53: 701–707, 2009.
Mondillo S, Galderisi M, Mele D, Cameli M, Lomoriello VS, Zaca V,
Ballo P, D’Andrea A, Muraru D, Losi M, Agricola E, D’Errico A,
Buralli S, Sciomer S, Nistri S, Badano L. Speckle-tracking echocardi-
J Appl Physiol • doi:10.1152/japplphysiol.01059.2012 • www.jappl.org
Downloaded from http://jap.physiology.org/ by 10.220.33.4 on June 14, 2017
15. Dorn GW 2nd. The fuzzy logic of physiological cardiac hypertrophy.
Hypertension 49: 962–970, 2007.
16. Duncker DJ, de Beer VJ, Merkus D. Alterations in vasomotor control of
coronary resistance vessels in remodelled myocardium of swine with a
recent myocardial infarction. Med Biol Eng Comput 46: 485–497, 2008.
17. Duncker DJ, Merkus D. Acute adaptations of the coronary circulation to
exercise. Cell Biochem Biophys 43: 17–35, 2005.
18. Duncker DJ, Stubenitsky R, Verdouw PD. Role of adenosine in the
regulation of coronary blood flow in swine at rest and during treadmill
exercise. Am J Physiol Heart Circ Physiol 275: H1663–H1672, 1998.
19. Duncker DJ, Van Zon NS, Crampton M, Herrlinger S, Homans DC,
Bache RJ. Coronary pressure-flow relationship and exercise: contributions of heart rate, contractility, and alpha 1-adrenergic tone. Am J Physiol
Heart Circ Physiol 266: H795–H810, 1994.
20. Emter CA, Baines CP. Low-intensity aerobic interval training attenuates
pathological left ventricular remodeling and mitochondrial dysfunction in
aortic-banded miniature swine. Am J Physiol Heart Circ Physiol 299:
H1348 –H1356, 2010.
21. Emter CA, Bowles DK. Store-operated Ca(2⫹) entry is not essential for
PDGF-BB induced phenotype modulation in rat aortic smooth muscle.
Cell Calcium 48: 10 –18, 2010.
22. Emter CA, Tharp DL, Ivey JR, Ganjam VK, Bowles DK. Lowintensity interval exercise training attenuates coronary vascular dysfunction and preserves Ca2⫹-sensitive K current in miniature swine with LV
hypertrophy. Am J Physiol Heart Circ Physiol 301: H1687–H1694, 2011.
23. Eto Y, Yonekura K, Sonoda M, Arai N, Sata M, Sugiura S, Takenaka
K, Gualberto A, Hixon ML, Wagner MW, Aoyagi T. Calcineurin is
activated in rat hearts with physiological left ventricular hypertrophy
induced by voluntary exercise training. Circulation 101: 2134 –2137,
2000.
24. Feigl EO, Neat GW, Huang AH. Interrelations between coronary artery
pressure, myocardial metabolism and coronary blood flow. J Mol Cell
Cardiol 22: 375–390, 1990.
25. Flynn KE, Pina IL, Whellan DJ, Lin L, Blumenthal JA, Ellis SJ, Fine
LJ, Howlett JG, Keteyian SJ, Kitzman DW, Kraus WE, Miller NH,
Schulman KA, Spertus JA, O’Connor CM, Weinfurt KP. Effects of
exercise training on health status in patients with chronic heart failure:
HF-ACTION randomized controlled trial. JAMA 301: 1451–1459, 2009.
26. Garciarena CD, Pinilla OA, Nolly MB, Laguens RP, Escudero EM,
Cingolani HE, Ennis IL. Endurance training in the spontaneously hypertensive rat: conversion of pathological into physiological cardiac hypertrophy. Hypertension 53: 708 –714, 2009.
27. Gibbs CL. Cardiac energetics. Physiol Rev 58: 174 –254, 1978.
28. Glower DD, Spratt JA, Snow ND, Kabas JS, Davis JW, Olsen CO,
Tyson GS, Sabiston DC Jr, Rankin JS. Linearity of the Frank-Starling
relationship in the intact heart: the concept of preload recruitable stroke
work. Circulation 71: 994 –1009, 1985.
29. Hanft LM, McDonald KS. Length dependence of force generation
exhibit similarities between rat cardiac myocytes and skeletal muscle
fibres. J Physiol 588: 2891–2903, 2010.
30. Haq S, Choukroun G, Lim H, Tymitz KM, del Monte F, Gwathmey J,
Grazette L, Michael A, Hajjar R, Force T, Molkentin JD. Differential
activation of signal transduction pathways in human hearts with hypertrophy versus advanced heart failure. Circulation 103: 670 –677, 2001.
31. Heymans S, Schroen B, Vermeersch P, Milting H, Gao F, Kassner A,
Gillijns H, Herijgers P, Flameng W, Carmeliet P, Van de Werf F,
Pinto YM, Janssens S. Increased cardiac expression of tissue inhibitor of
metalloproteinase-1 and tissue inhibitor of metalloproteinase-2 is related
to cardiac fibrosis and dysfunction in the chronic pressure-overloaded
human heart. Circulation 112: 1136 –1144, 2005.
32. Hidalgo C, Hudson B, Bogomolovas J, Zhu Y, Anderson B, Greaser
M, Labeit S, Granzier H. PKC phosphorylation of titin’s PEVK element:
a novel and conserved pathway for modulating myocardial stiffness. Circ
Res 105: 631– 638, 17 p following 638, 2009.
33. Hinken AC, Korte FS, McDonald KS. Porcine cardiac myocyte power
output is increased after chronic exercise training. J Appl Physiol 101:
40 –46, 2006.
34. Huang CY, Yang AL, Lin YM, Wu FN, Lin JA, Chan YS, Tsai FJ,
Tsai CH, Kuo CH, Lee SD. Anti-apoptotic and pro-survival effects of
exercise training on hypertensive hearts. J Appl Physiol 112: 883–891,
2012.
35. Hudson MP, Armstrong PW, Ruzyllo W, Brum J, Cusmano L,
Krzeski P, Lyon R, Quinones M, Theroux P, Sydlowski D, Kim HE,
Garcia MJ, Jaber WA, Weaver WD. Effects of selective matrix metal-
•
Exercise Preserves Normal Cardiovascular Function in HFpEF
54.
55.
56.
57.
58.
60.
61.
62.
63.
64.
65.
66.
67.
68.
Marshall KD et al.
147
69. Spinale FG. Myocardial matrix remodeling and the matrix metalloproteinases: influence on cardiac form and function. Physiol Rev 87: 1285–1342,
2007.
70. Srere PA. Citrate synthase. Methods Enzymol 13: 3–5, 1969.
71. Suga H. Ventricular energetics. Physiol Rev 70: 247–277, 1990.
72. Suga H, Sagawa K, Shoukas AA. Load independence of the instantaneous pressure-volume ratio of the canine left ventricle and effects of
epinephrine and heart rate on the ratio. Circ Res 32: 314 –322, 1973.
73. Takeishi Y, Huang Q, Abe J, Che W, Lee JD, Kawakatsu H, Hoit BD,
Berk BC, Walsh RA. Activation of mitogen-activated protein kinases and
p90 ribosomal S6 kinase in failing human hearts with dilated cardiomyopathy. Cardiovasc Res 53: 131–137, 2002.
74. Takeuchi M, Borden WB, Nakai H, Nishikage T, Kokumai M, Nagakura T, Otani S, Lang RM. Reduced and delayed untwisting of the left
ventricle in patients with hypertension and left ventricular hypertrophy: a
study using two-dimensional speckle tracking imaging. Eur Heart J 28:
2756 –2762, 2007.
75. Tan YT, Wenzelburger F, Lee E, Heatlie G, Leyva F, Patel K,
Frenneaux M, Sanderson JE. The pathophysiology of heart failure with
normal ejection fraction: exercise echocardiography reveals complex abnormalities of both systolic and diastolic ventricular function involving
torsion, untwist, and longitudinal motion. J Am Coll Cardiol 54: 36 –46,
2009.
76. Toth M, Fridman R. Assessment of gelatinases (MMP-2 and MMP-9) by
gelatin zymography. Methods Mol Med 57: 163–174, 2001.
77. Van Woerkens EC, Trouwborst A, Duncker DJ, Koning MM,
Boomsma F, Verdouw PD. Catecholamines and regional hemodynamics
during isovolemic hemodilution in anesthetized pigs. J Appl Physiol 72:
760 –769, 1992.
78. Wang J, Khoury DS, Yue Y, Torre-Amione G, Nagueh SF. Left
ventricular untwisting rate by speckle tracking echocardiography. Circulation 116: 2580 –2586, 2007.
79. Wang J, Khoury DS, Yue Y, Torre-Amione G, Nagueh SF. Preserved
left ventricular twist and circumferential deformation, but depressed longitudinal and radial deformation in patients with diastolic heart failure.
Eur Heart J 29: 1283–1289, 2008.
80. Weber KT, Brilla CG, Janicki JS. Myocardial fibrosis: functional
significance and regulatory factors. Cardiovasc Res 27: 341–348, 1993.
81. Whaley-Connell A, Govindarajan G, Habibi J, Hayden MR, Cooper
SA, Wei Y, Ma L, Qazi M, Link D, Karuparthi PR, Stump C, Ferrario
C, Sowers JR. Angiotensin II-mediated oxidative stress promotes myocardial tissue remodeling in the transgenic (mRen2) 27 Ren2 rat. Am J
Physiol Endocrinol Metab 293: E355–E363, 2007.
82. Wilkins BJ, Dai YS, Bueno OF, Parsons SA, Xu J, Plank DM, Jones
F, Kimball TR, Molkentin JD. Calcineurin/NFAT coupling participates
in pathological, but not physiological, cardiac hypertrophy. Circ Res 94:
110 –118, 2004.
83. Williams JL, Hathaway CA, Kloster KL, Layne BH. Low power, type
II errors, and other statistical problems in recent cardiovascular research.
Am J Physiol Heart Circ Physiol 273: H487–H493, 1997.
84. Yarbrough WM, Mukherjee R, Stroud RE, Rivers WT, Oelsen JM,
Dixon JA, Eckhouse SR, Ikonomidis JS, Zile MR, Spinale FG. Progressive induction of left ventricular pressure overload in a large animal
model elicits myocardial remodeling and a unique matrix signature. J
Thorac Cardiovasc Surg 143: 215–223, 2012.
85. Zhang C, Knudson JD, Setty S, Araiza A, Dincer UD, Kuo L, Tune
JD. Coronary arteriolar vasoconstriction to angiotensin II is augmented in
prediabetic metabolic syndrome via activation of AT1 receptors. Am J
Physiol Heart Circ Physiol 288: H2154 –H2162, 2005.
86. Zile MR, Desantis SM, Baicu CF, Stroud RE, Thompson SB, McClure
CD, Mehurg SM, Spinale FG. Plasma biomarkers that reflect determinants of matrix composition identify the presence of left ventricular
hypertrophy and diastolic heart failure. Circ Heart Fail 4: 246 –256, 2011.
J Appl Physiol • doi:10.1152/japplphysiol.01059.2012 • www.jappl.org
Downloaded from http://jap.physiology.org/ by 10.220.33.4 on June 14, 2017
59.
ography: a new technique for assessing myocardial function. J Ultrasound
Med 30: 71–83, 2011.
Morris DA, Perez AV, Blaschke F, Eichstadt H, Ozcelik C,
Haverkamp W. Myocardial systolic and diastolic consequences of left
ventricular mechanical dyssynchrony in heart failure with normal left
ventricular ejection fraction. Eur Heart J Cardiovasc Imaging 13: 556 –
567, 2012.
Norman HS, Oujiri J, Larue SJ, Chapman CB, Margulies KB,
Sweitzer NK. Decreased cardiac functional reserve in heart failure with
preserved systolic function. J Card Fail 17: 301–308, 2011.
Notomi Y, Martin-Miklovic MG, Oryszak SJ, Shiota T, Deserranno
D, Popovic ZB, Garcia MJ, Greenberg NL, Thomas JD. Enhanced
ventricular untwisting during exercise: a mechanistic manifestation of
elastic recoil described by Doppler tissue imaging. Circulation 113:
2524 –2533, 2006.
Notomi Y, Popovic ZB, Yamada H, Wallick DW, Martin MG,
Oryszak SJ, Shiota T, Greenberg NL, Thomas JD. Ventricular untwisting: a temporal link between left ventricular relaxation and suction. Am J
Physiol Heart Circ Physiol 294: H505–H513, 2008.
O’Connor CM, Whellan DJ, Lee KL, Keteyian SJ, Cooper LS, Ellis
SJ, Leifer ES, Kraus WE, Kitzman DW, Blumenthal JA, Rendall DS,
Miller NH, Fleg JL, Schulman KA, McKelvie RS, Zannad F, Pina IL.
Efficacy and safety of exercise training in patients with chronic heart
failure: HF-ACTION randomized controlled trial. JAMA 301: 1439 –1450,
2009.
O’Neill BT, Abel ED. Akt1 in the cardiovascular system: friend or foe?
J Clin Invest 115: 2059 –2064, 2005.
Park SJ, Miyazaki C, Bruce CJ, Ommen S, Miller FA, Oh JK. Left
ventricular torsion by two-dimensional speckle tracking echocardiography
in patients with diastolic dysfunction and normal ejection fraction. J Am
Soc Echocardiogr 21: 1129 –1137, 2008.
Paulus WJ, van Ballegoij JJ. Treatment of heart failure with normal
ejection fraction: an inconvenient truth! J Am Coll Cardiol 55: 526 –537,
2010.
Phan TT, Shivu GN, Abozguia K, Gnanadevan M, Ahmed I, Frenneaux M. Left ventricular torsion and strain patterns in heart failure with
normal ejection fraction are similar to age-related changes. Eur J Echocardiogr 10: 793–800, 2009.
Piepoli MF, Conraads V, Corra U, Dickstein K, Francis DP, Jaarsma
T, McMurray J, Pieske B, Piotrowicz E, Schmid JP, Anker SD, Solal
AC, Filippatos GS, Hoes AW, Gielen S, Giannuzzi P, Ponikowski PP.
Exercise training in heart failure: from theory to practice. A consensus
document of the Heart Failure Association and the European Association
for Cardiovascular Prevention and Rehabilitation. Eur J Heart Fail 13:
347–357, 2011.
Raff GL, Glantz SA. Volume loading slows left ventricular isovolumic
relaxation rate. Evidence of load-dependent relaxation in the intact dog
heart. Circ Res 48: 813–824, 1981.
Russel IK, Gotte MJ, Bronzwaer JG, Knaapen P, Paulus WJ, van
Rossum AC. Left ventricular torsion: an expanding role in the analysis of
myocardial dysfunction. JACC Cardiovasc Imaging 2: 648 –655, 2009.
Sagawa K, Suga H, Shoukas AA, Bakalar KM. End-systolic pressure/
volume ratio: a new index of ventricular contractility. Am J Cardiol 40:
748 –753, 1977.
Schubert A, Walther T, Falk V, Binner C, Loscher N, Kanev A,
Bleiziffer S, Rauch T, Autschbach R, Mohr FW. Extracellular matrix
gene expression correlates to left ventricular mass index after surgical
induction of left ventricular hypertrophy. Basic Res Cardiol 96: 381–387,
2001.
Schulz R, Oudiz RJ, Guth BD, Heusch G. Minimal alpha 1- and alpha
2-adrenoceptor-mediated coronary vasoconstriction in the anaesthetized
swine. Naunyn Schmiedebergs Arch Pharmacol 342: 422–428, 1990.
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