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Chapter
10
General Discussion
Summary
Future Perspectives
General discussion
Heart failure with preserved ejection fraction (HFpEF) is widely prevalent and has a
cumbersome prognosis, while its pathophysiological mechanisms are incompletely
understood and specific therapeutic strategies remain uncertain1-3. It is still debated whether
HFpEF and HF with reduced EF (HFrEF) represent distinct HF phenotypes4. However,
prominent differences in patient demographics, risk factor profile, hemodynamic
characteristics and macroscopic and myocellular remodeling coupled to disparate responses to
similar HF pharmacotherapy would all seem to justify that HFpEF and HFrEF indeed
represent distinct HF phenotypes2,5-7. Elevated diastolic left ventricular (LV) stiffness is an
important feature in HFpEF8-10. Numerous studies, of which several are included in the
present thesis, demonstrated that raised intrinsic cardiomyocyte stiffness (Fpassive) importantly
contributes to high diastolic LV stiffness in HFpEF5,11-14. Cardiomyocyte Fpassive is mainly
determined by the giant elastic sarcomeric protein titin, which regulates myocardial passive
tension, stiffness and biomechanical stress/stretch signaling15-17. Titin spans a half sarcomere,
running from the Z-disk to the M-band and functions as a bidirectional spring responsible for
early diastolic recoil and late diastolic resistance to stretch15-17. Cardiomyocyte titin-based
elasticity can be adjusted through transcriptional and posttranslational modifications. At the
transcriptional level, titin modulates stiffness through shifts in expression of its compliant
N2BA (3.2-3.7 MDa) and stiff N2B (3.0MDa) isoforms, which are co-expressed in the
sarcomere at a N2BA:N2B ratio of approximately 35:65 in the normal heart15-17. In
eccentrically remodeled explanted hearts from dilated18,19 or ischemic cardiomyopathy20
patients, as well as in LV endomyocardial biopsies from HFrEF patients5,13, the N2BA:N2B
expression ratio was increased, resulting in reduced myocardial stiffness. In contrast, the
N2BA:N2B expression ratio was decreased in LV endomyocardial biopsies from HFpEF
patients compared to HFrEF5,13, whereas mixed results were reported in LV biopsies from
patients with severe AS, ranging from either reduced21 or similar22 N2BA:N2B expression
ratios compared to donor hearts. While titin-based stiffness may be altered by titin isoform
switching within days or weeks, titin-based stiffness can also be modulated acutely through
posttranslational modifications, such as titin isoform phosphorylation16.
Both protein kinases A and G (PKA and PKG) phosphorylate the stiff titin N2B isoform
thereby acutely lowering titin-based cardiomyocyte stiffness, which has been demonstrated in
skinned human LV muscle strips23,24, in isolated human LV myofibrils24 and in isolated
cardiomyocytes from HFpEF and HFrEF patients2,11,13,14. Two additional posttranslational
modifications of titin, both increasing titin-based stiffness, include protein kinase C α (PKCα) mediated phosphorylation of titin’s PEVK segment (rich in Proline, Glutamic acid, Valine
and Lysine)25 and oxidative stress-induced formation of disulfide bridges in the stiff N2B
segment of titin26. Contribution by other myofilamentary proteins, such as myosin heavy
chain, desmin, actin, troponin T, troponin I (TnI), myosin light chain 1 and -2 was recently
ruled out11,13, suggesting that phosphorylation-mediated posttranslational modification of titin
indeed represents the most likely mechanism for acute modulation of cardiomyocyte Fpassive.
Intrinsic cardiomyocyte stiffness is significantly higher in patients with HFpEF than in
controls, in patients with HFrEF and in patients operated for severe aortic stenosis (AS)5,11-14.
Because the fall in cardiomyocyte resting tension following in-vitro administration of PKA
and PKG is significantly larger in HFpEF than in HFrEF and AS5,12-14, a larger
phosphorylation deficit of presumably titin could be present in HFpEF as recently
suggested13. Indeed, stimulation of PKG activity through inhibited breakdown of cyclic
guanosine monophosphate (cGMP) by phosphodiesterase type 5A (PDE5A)27 by the PDE5A
inhibitor sildenafil, was shown to ameliorate diastolic dysfunction in various experimental
and clinical studies. Sildenafil restored LV relaxation kinetics in mice exposed to transverse
aortic constriction (TAC)28 and reduced diastolic LV stiffness in an old hypertensive dog
model29, in patients with HFrEF30 and in HFpEF patients with pulmonary hypertension31.
Administration of sildenafil to old hypertensive dogs lowered diastolic LV stiffness through
restored phosphorylation of the titin N2B segment29. Moreover, PDE5A inhibitors have also
been shown to attenuate adrenergic stimulation32, reduce ventricular-vascular stiffening33,
improve endothelial function34, reduce pulmonary vascular resistance35 and enhance exercise
tolerance35,36 in HFrEF. Recently, we measured PKG activity, upstream control of PKG
activity and downstream effects of PKG activity in LV myocardial biopsies of HFpEF
patients14. To discern altered control by PKG of the myocardial remodeling process in
HFpEF, measurements were compared to measurements obtained from LV myocardium
remodeled concentrically by severe AS or eccentrically by nonischemic HFrEF. Our study
showed that relative to both AS and HFrEF, HFpEF patients had reduced myocardial PKG
activity and lower cGMP concentration, which related to higher cardiomyocyte Fpassive and
increased myocardial nitrotyrosine levels, indicative of raised nitrosative/oxidative stress. In
all groups, cardiomyocyte Fpassive was acutely lowered by in vitro administration of PKG,
whereas cardiomyocytes from HFpEF patients demonstrated the largest PKG-induced fall in
Fpassive14. Reduced PKG activity and lower myocardial cGMP concentration in HFpEF did not
result from altered myocardial soluble guanylate cyclase (sGC) or PDE5A expression, which
were similar in all groups, or from unequal brain type natriuretic peptide (BNP) expression,
which was comparable in HFpEF and AS. Because of comparable proBNP-108 expression in
HFpEF and AS, BNP is unlikely to account for the widely different PKG activities and cGMP
concentrations observed in both conditions14. Lower proBNP-108 expression in HFpEF than
in HFrEF also explains the low positive predictive value of BNP for the diagnosis of
HFpEF37. Downregulation of cGMP-PKG signaling in HFpEF was therefore most likely
related to low myocardial NO bioavailability because of high nitrosative/oxidative stress,
which was almost fourfold higher in HFpEF than in both HFrEF and AS14. Oxidative stress is
known to lower NO bioavailability and downregulate cGMP-PKG signaling38-41.
Importance of NO for diastolic function
LV distensibility
Endothelial dysfunction plays a prominent role in the pathophysiology and prognosis of HF42.
Impaired endothelium-dependent vasodilation in HF patients has been attributed to reduced
activity of the L-arginine-NO synthetic pathway, increased degradation of NO by reactive
oxygen species (ROS) and hyporesponsiveness in vascular smooth muscle43,44.
NO is crucially important for diastolic function, since it potently enhances LV relaxation and
distensibility through cGMP-PKG dependent and independent mechanisms, including
reduction of myofilament calcium (Ca2+) sensitivity by TnI phosphorylation and enhancing
phospolamban-mediated sarcoplasmic reticular (SR) Ca2+ reuptake, respectively45-47. NO is an
ubiquitous intra- and intercellular signaling molecule generated in a temporally and spatially
restricted manner by a family of NO synthases (NOSs), including endothelial NOS (eNOS),
neuronal NOS (nNOS) and inducible NOS (iNOS), which vary in their subcellular
localization46,47. The myocardial autocrine and paracrine effects of NO depend on where and
by which NOS isoform NO is produced46. Myocardial eNOS-derived NO was demonstrated
to enhance LV relaxation and distensibility48-50 and to modulate cardiac β-adrenergic
responsiveness51. Stimulation of eNOS activity by intracoronary administration of NO donors
instantaneously hastened LV relaxation, accelerated LV pressure decline and increased
diastolic LV distensibility in the normal human heart48 as well as in patients with dilated
cardiomyopathy49 and severe AS50. The importance of myocardial NO bioavailability for
diastolic LV function was recently reappraised because of close correlations between
asymmetric dimethylarginine and diastolic LV dysfunction in patients with ischemic and nonischemic dilated cardiomyopathy52,53.
In addition, myocardial endothelial eNOS-derived NO regulates cardiac metabolism through
modulation of myocardial substrate utilization and by reduction of myocardial oxygen
consumption through inhibition of complexes I and II of the mitochondrial electron transport
chain46,54. Interestingly, mitochondrial biogenesis is stimulated in response to increased
myocardial NO bioavailability and cGMP levels55. Because cross-bridge detachment is an
energy consuming process, slow LV relaxation in HFpEF could therefore also result from a
myocardial energy deficit because of reduced myocardial NO bioavailability due to increased
nitrosative/oxidative stress burden. Recent 31P-magnetic resonance spectroscopy studies
indeed demonstrated reduced myocardial energy reserve in patients with HFpEF56-58, HFrEF59
and in both diabetic60 and obese61 patients, in whom impaired myocardial bioenergetics
correlated with diastolic LV dysfunction60,61.
NO-mediated activation of cGMP-PKG signaling
NO activates sGC, which is present predominantly in the cytosol and in smaller amounts at
the plasma membrane, via a complex interplay between binding of NO to both heme and nonheme sites of sGC47,62,63. Stimulated sGC subsequently enhances cGMP generation, which
activates its primary effector kinase PKG27,47. In turn, PKG phosphorylates a vast number of
target proteins, exerting a wide range of downstream effects like enhanced SR Ca2+ reuptake,
inhibition of Ca2+ influx and suppression of pro-hypertrophic and fibrotic signaling as well as
stimulation of relaxation and LV distensiblity by phosphorylation of TnI and the stiff titin
N2B segment16,24,27,64. Because NO diffusion distances in cardiomyocytes are limited, NOsGC-cGMP signaling is compartmentalized into functional signalosomes, coupling NO
synthesis to downstream sGC-cGMP signals47. For instance, in cardiomyocytes and
endothelial cells, eNOS, sGC and PKG colocalize with caveolin at plasma membrane
invaginations, called caveolae, which may serve as microdomains for optimized NO-sGCcGMP signaling47,63. Furthermore, besides being subject to intricate intracellular
compartmentalization, signaling through the eNOS-NO-cGMP-PKG pathway is also
characterized by considerable crosstalk with β-adrenergic mediated cAMP signaling,
providing the heart with a complex and highly differentially regulated machinery to modulate
and integrate divergent cardiovascular (patho)physiological conditions47,65-67.
Importance of cGMP-PKG signaling in myocardial remodelling
Cardiac remodeling in HFpEF is characterized by concentric LV and cardiomyocyte
hypertrophy and interstitial fibrosis5,9,68-70. The Framingham Heart Study determined that LV
hypertrophy follows aging as independent risk factor for cardiovascular morbidity and
mortality71. Stimuli for LV hypertrophy activate an array of membrane-bound receptors
coupled to multiple intracellular signaling cascades, which ultimately stimulate prohypertrophic gene expression72,73. Important upstream triggers for cardiac hypertrophy
include mechanical stretch, pressure overload, cytokines, activation of RAAS, endothelin and
catecholamines, whereas cGMP-PKG signaling exerts prominent anti-hypertrophic and antifibrotic effects27,72,73. Indeed, modulation of the sGC-PKG-PDE5A axis affected myocardial
remodeling with less cardiomyocyte hypertrophy and interstitial fibrosis in TAC mice
exposed to sildenafil28. Despite similar levels of LV and cardiomyocyte hypertrophy in
patients with HFpEF and AS, HFpEF patients demonstrated higher cardiomyocyte Fpassive and
lower myocardial PKG activity than AS patients14. The finding in AS, of higher PKG activity
corresponding with low Fpassive but not with cardiomyocyte diameter, suggests cardiomyocyte
diastolic stiffness to evolve independently from cardiomyocyte hypertrophy. This concept
also emerged from the VALIDD trial, in which only 3% of hypertensives had significant LV
hypertrophy despite all having diastolic LV dysfunction74. External myocardial force signals
imposed during hemodynamic load are transmitted to ECM components and the
cardiomyocytes15. Mechanical stimuli sensed at the outer cardiomyocyte cell membrane can
activate cytoskeletal and sarcomeric signaling cascades, which subsequently trigger prohypertrophic factors15. In addition to its prominent role in regulating cardiomyocyte stiffness,
titin also acts as a dominant signaling protein, which integrates both internally generated
sarcomeric forces and externally sensed biomechanical stretch/stress signals to hypertrophy
regulation75. Thus, it could be speculated that increased cardiomyocyte stiffness sensed by
titin precedes titin-mediated stimulation of pro-hypertrophic signaling.
Nitrosative/oxidative stress lowers NO bioavailability and NO signalling in
HFpEF
Reduced NO bioavailability, dysregulation of NO-mediated signaling and increased
nitrosative/oxidative stress are firmly implicated in the pathogenesis of HF40,41,76-78. Although
generation of ROS (i.e. superoxide, hydroxyl radical and non-radical species such as
hydrogen peroxide) is a normal component of cellular redox-based signaling, excessive ROS
production causes nitrosative/oxidative stress40,41,76-79.
Nitrosative/oxidative stress impairs NO bioavailability and NO-mediated signaling through a
number of mechanisms. First, superoxide rapidly reacts with NO to form peroxynitrite, which
triggers eNOS uncoupling with subsequent generation of superoxide instead of NO, thereby
augmenting oxidative stress. Second, by scavenging NO, superoxide reduces NO
bioavailability and prevents NO from stimulating sGC-mediated activation of cGMP-PKG
signaling37,40,41. Finally, sGC activity is adversely affected by nitrosative/oxidative stress
through several mechanisms, including superoxide and peroxynitrite mediated decrease in
sGC activity and sGC heme group oxidation converting sGC to its NO-insensitive state40,63.
The lower myocardial PKG activity and reduced cGMP concentration in HFpEF patients,
which was associated with increased myocardial nitrotyrosine levels14, could therefore have
resulted from upstream nitrosative/oxidative stress-induced impairments of NO bioavailability
and reduced NO-mediated activation of sGC-induced cGMP/PKG signaling. Accordingly, a
number of recent studies also suggested oxidative stress to be involved in the pathogenesis of
diastolic LV dysfunction in HFpEF. In HFpEF rodent models, oxidative stress uncoupled
cardiac NOS and induced diastolic LV dysfunction, whereas stimulation of eNOS activity
reversed oxidative stress and improved diastolic LV function80,81. Furthermore, high plasma
levels of methylated L-arginine metabolites, indicative of increased oxidative stress, were
strongly related to diastolic LV dysfunction in patients with HFrEF52. Moreover, in patients
with HFpEF, myocardial fibrosis and inflammation were associated with diastolic LV
dysfunction and with increased oxidative stress in endomyocardial biopsies compared to
controls82. Abundant evidence demonstrates that ROS potently activate pro-hypertrophic and
pro-fibrotic signaling, and by interfering with NO-sGC-cGMP-PKG signalling, ROS inhibit
anti-hypertrophic and anti-fibrotic signalling cascades41,72.
In addition, nitrosative/oxidative stress induces a state of NO/redox disequilibrium, which
impairs S-nitrosylation signaling (a posttranslational protein modification process through
coupling of an NO moiety to a reactive cysteine thiol). S-nitrosylation serves as a major
effector of NO bioactivity and an important mode of cellular signal transduction, regulating
the activity of numerous metabolic enzymes, kinases, phosphatases and respiratory proteins as
well as cytoskeletal and structural components and transcription factors41,76,83.
Possible mechanisms for enhanced nitrosative/oxidative stress in HFpEF
It was recently suggested that the particularly high prevalence of metabolic risk factors in
HFpEF could be associated with increased inflammation and nitrosative/oxidative stress in
this condition14. HFpEF patients demonstrate a high prevalence of obesity and DMII84-86.
Obesity and DMII are strongly related to insulin resistance (IR) and the metabolic syndrome
(a constellation of cardiovascular risk factors, including obesity, hypertension, IR,
hyperglycemia, dyslipidemia, microalbuminuria and hypercoagulability)87 and the frequent
clustering of these metabolic risk factors causes synergistic adverse effects on myocardial
structure and function88. The prevalence of obesity, DMII, IR and metabolic syndrome
increases rapidly and is expected to reach pandemic proportions in the next few decades88-92,
while these metabolic risk factors have all been prospectively identified as precursors of
incident HF93-95 and are independently associated with early development of diastolic LV
dysfunction96-99. Diastolic LV dysfunction is recognized as the earliest manifestation of
DMII-induced LV dysfunction96,100-104, while the excessive diastolic LV stiffness of the
diabetic heart has been related to myocardial fibrosis105 and to circulating advanced glycation
endproducts (AGEs)106. In a recent study, DM increased diastolic LV stiffness in both HFpEF
and HFrEF patients through distinct mechanisms12. In diabetic HFrEF, raised diastolic
stiffness was associated with both increased CVF and myocardial deposition of AGEs.
Conversely, in diabetic HFpEF, myocardial CVF was unchanged and elevated diastolic
stiffness related to increased cardiomyocyte Fpassive12. Diabetes also increases LV diastolic
and cardiomyocyte stiffness in patients with severe AS102 and elevated cardiomyocyte
stiffness in diabetic AS patients related to hypophosphorylation of the titin N2B segment,
which could be corrected by phosphorylation with PKA22.
Obesity, DMII and IR can have direct adverse effects on myocardial structure and function
independently from common confounders as hypertension or CAD, which has been referred
to as “obesity”107, “diabetic”102, or “insulin-resistant”108 cardiomyopathy. Moreover,
metabolic risk factors are strongly associated with endothelial dysfunction, inflammation,
oxidative stress, impaired myocardial energetics, abnormal cardiomyocyte Ca2+ handling,
reduced NO bioavailability and maladaptive cardiac remodelling102,107,108.
Metabolic risk factors interfere with NO-cGMP-PKG signaling
Oxidative stress-mediated downregulation of NO-cGMP-PKG signaling has been
demonstrated in various experimental models of obesity, diabetes, IR and metabolic
syndrome109,110. Cultured rodent aortic smooth muscle cells subjected to hyperglycaemia
demonstrated significantly reduced PKG expression and activity, which occurred through a
PKC-dependent activation of NADPH oxidase-derived superoxide production109. In this
model, high glucose-mediated lowering of PKG levels was inhibited by a superoxide
scavenger or NADPH oxidase inhibitors109. In addition, in human diabetic cardiomyopathy,
sildenafil improved measurements of LV geometry, strain and torsion determined by
magnetic resonance imaging111. Furthermore, in a rodent model of diet-induced obesity and
IR, signaling through the NO-cGMP pathway was reduced, while vascular inflammation and
IR were completely prevented by administration of sildenafil110.
Physiologically, insulin phosphorylates titin through activation of the phosphoinositol-3kinase (PI3K)/Akt/eNOS pathway, while IR, FFAs and adipokines inhibit insulin-dependent
PI3K/Akt signaling, resulting in reduced eNOS activity and impaired NO generation112,113.
Inflammation and oxidative stress associated with metabolic risk factors can reduce eNOS
activity and NO bioavailability through a number of ways, including RAAS-mediated
activation of ROS producing enzymes, increased flux through the hexosamine biosynthesis
pathway, mitochondrial uncoupling, activation of the polyol pathway, lipotoxicity, glucose
auto-oxidation and formation of AGEs113. Moreover, obesity, DM and IR induce activation of
multiple PKC isoforms, with PKCs being prominently involved in numerous cellular
functions and signal transduction pathways114. PKC can hamper myocardial eNOS activity
and NO generation by inhibition of PI3K/Akt signaling and stimulation of NADPH oxidase
activity114, whereas PKCα was recently shown to increase titin-based cardiomyocyte stiffness
through phosphorylation of the PEVK segment25. Interestingly, in a rodent diabetic HFpEF
model, PKCβ inhibition improved diastolic distensiblity, contractility and maladaptive cardiac
remodelling115. Recent evidence demonstrates that in addition to downregulation of NOcGMP-PKG signaling, metabolic conditions such as IR, metabolic syndrome and DM also
induce a state of myocardial energy deficiency with decreased mitochondrial oxidative
phosphorylation and diminished mitochondrial biogenesis, which predisposes to a metabolic
cardiomyopathy characterized by early development of diastolic LV dysfunction116,117.
In summary, metabolic risk factors could contribute to diastolic LV dysfunction and elevated
myocardial stiffness in HFpEF through provoking myocardial inflammation and
nitrosative/oxidative stress, which on its turn results in decreased NO bioavailability,
downregulation of cGMP-PKG signaling, impaired myocardial bioenergetics and maladaptive
remodeling. Furthermore, downregulation of cGMP-PKG signaling could subsequently result
in hypophosphorylation of titin, which elevates cardiomyocyte stiffness and possibly triggers
cardiomyocyte hypertrophy.
Novel treatment strategies for HFpEF
In HFpEF, the reduced myocardial PKG activity, increased nitrosative/oxidative stress and the
presence of myocardial fibrosis, impaired myocardial energetics and the high prevalence of
metabolic risk factors supports use of NO-donors, PDE5A inhibitors, statins, anti-fibrotic
agents, such as aldosterone antagonists and optimized treatment of metabolic risk.
Stimulation of NO-cGMP-PKG signaling
NOS uncoupling has emerged as an important contributor to pathologic chamber remodeling,
endothelial dysfunction and diastolic dysfunction in animal models of HF80,81. Oxidative
depletion of the essential NOS cofactor tetrahydrobiopterin (BH4), results in NOS
destabilization and uncoupling subsequently causing NOS to generate superoxide instead of
NO118. Administration of BH4 in animal models reduces pressure-overload hypertrophy,
fibrosis, NOS uncoupling and oxidative stress, while at the same time systolic and diastolic
function are improved80,81,119, suggesting that targeting NOS uncoupling could improve
diastolic LV dysfunction in HFpEF. Exogenous administration of BH4 has also been shown
to improve endothelial dysfunction in patients with hypercholesterolemia120, DM121 and
hypertension122 through improvement of NOS function and enhanced NO bioactivity coupled
with BH4 treatment.
Acute administration of NO donors is known to improve diastolic LV function with an earlier
onset of LV relaxation, lower LV peak systolic, end-systolic and end-diastolic pressures, with
rightward displacement of the diastolic LV pressure-volume relation45,48.
Current ESC HF guidelines accord a class IIa indication for administration of NO donors in
patients admitted for acute HF with pulmonary edema without concomitant cardiogenic
shock123. Unfortunately, longterm use of NO donors is frequently hampered by development
of NO resistance124. NO resistance largely results from a combination of scavenging of NO by
superoxide and of (reversible) inactivation of sGC125. Conversely, chronic use of isosorbide
dinitrate combined with the antioxidant hydralazine improved outcome in V-HeFT I and AHeFT trials126,127. Hydralazine reduces superoxide generation by xanthine oxidase and
NADPH oxidase128. The clinical characteristics of the A-HeFT HFrEF patients revealed a
high prevalence of obesity and DM129, conditions which are also highly prevalent in HFpEF.
Combined use of isosorbide dinitrate and the antioxidant hydralazine could therefore be
potentially favourable in HFpEF.
An exciting promising new inroad for HFpEF treatment is represented by agents that enhance
cellular cGMP signaling27. Upstream activation of sGC by NO stimulates cGMP-PKG
activation in the cytosolic and subsarcolemmal compartments, while upstream activation of
particulate GC (pGC) by natriuretic peptides stimulates cGMP-PKG signaling at the plasma
membrane63,65. Several key transcription factors and sarcomeric proteins involved in
hypertrophy signaling, diastolic relaxation and stiffness and vasorelaxation are favourably
modified by PKG-dependent phosphorylation, suggesting that these agents may be beneficial
in HFpEF13,24,27,28,48. PDE5A inhibitors, such as sildenafil, increase cGMP levels by blocking
their catabolism. PDE5A inhibitors attenuate adrenergic stimulation32, reduce ventricularvascular stiffening32, antagonize maladaptive chamber remodeling28,30, improve endothelial
function34, reduce pulmonary vascular resistance30,31,35 and lower diastolic LV stiffness in
patients with HFrEF30 and in HFpEF patients with pulmonary hypertension31.
An alternative approach to enhance cGMP-PKG signaling is through direct stimulation of
sGC activity. Recently, two classes of drugs have been discovered, the sGC activators and
sGC stimulators, which target two different redox states of sGC: the NO-sensitive reduced
(ferrous) sGC and NO-insensitive oxidized (ferric) sGC respectively130. sGC consists of an
α/β-heterodimeric protein with a prosthetic ferrous heme group. The presence of a reduced
Fe2+ (ferrous) heme group is crucial for NO-sensing and NO-dependent sGC stimulation130.
The heme group can exist in different redox states, which may enable sGC to modulate
intracellular redox homeostasis in addition to its NO-sensing capability130. Oxidative stress
favours heme-free sGC, which is unresponsive to NO. Hence, oxidative stress synergistically
hampers NO-cGMP signaling through sGC oxidation and through ROS-mediated scavenging
of NO38,40 thereby compromising NO-sGC-cGMP mediated signaling130-132. The sGC
stimulators have a dual mode of action; they sensitize sGC to low levels of NO and can
stimulate sGC directly in the absence of any endogenous NO. Conversely, sGC activators
specifically activate the NO-unresponsive, heme-free form of the enzyme irrespective of NO
bioavailability130-133.
In a nonrandomized proof of concept study, the direct NO- and heme-independent sGC
activator cinaciguat was shown to acutely reduce pulmonary capillary and artery pressures,
lower pulmonary and systemic vascular resistance, while improving cardiac output in patients
admitted for acute decompensated HF134. A number of phase IIb studies (COMPOSE
program) evaluated the safety and efficacy of fixed low doses of intravenous cinaciguat as
add-on therapy in patients hospitalized for acute HF and demonstrated cinaciguat to be
associated with an excess of non-fatal hypotensive episodes without improvements in dyspnea
or cardiac index135. Recently, the sustained sGC stimulator (BAY 41-8543) was found to
offset NOS inhibition and to restore acute cardiac modulation by sildenafil in adult mice,
which suggests that direct sGC stimulators could be used to enhance the action of PDE5A
inhibitors in settings where critical sGC-generated cGMP is inadequate136.
Stimulation of cGMP-PKG signaling can also be achieved through NP-mediated activation of
pGC. The synthetic NP nesiritide was shown to acutely reduce pulmonary capillary wedge
pressure and systemic vascular resistance, while increasing cardiac index and stroke volume
index in HFrEF patients137. Acute NP administration was recently reported to lower diastolic
LV stiffness and to increase myocardial titin phosphorylation in an old hypertensive HFpEF
dog model29 but failed to improve clinical endpoints in a large, randomized clinical trial of
acutely decompensated HF patients with either LVEF <40% and LVEF ≥40%138. Conversely,
the ubiquitous enzyme dipeptidyl peptidase IV (DPP IV) cleaves BNP1-32 into BNP3-32, which
has reduced biological activity compared to BNP1-32139,140 and was found increased in patients
with chronic HF141. Interestingly, sitagliptin, a specific DPP IV inhibitor, preserved renal
function, modulated stroke volume and heart rate and potentiated the positive inotropic effect
of exogenous BNP in a pacing-induced pig HF model142.
Modulation of myocardial remodeling and nitrosative/oxidative stress
Prominent features of HFpEF, DM, IR and obesity are cardiac hypertrophy and fibrosis.
Hypertrophic stimuli, such as mechanical stretch, neurohumoral activation and inflammatory
markers activate various downstream pro-hypertrophic signaling pathways, whereas NO-PKG
mediated signaling exerts anti-hypertrophic and anti-fibrotic effects72,143. Furthermore,
abundant evidence demonstrates that ROS potently activate pro-hypertrophic and pro-fibrotic
pathways, and by interfering with both NO-sGC-cGMP-PKG signalling and NO-mediated Snitrosylation, ROS inhibit anti-hypertrophic and anti-fibrotic signalling cascades72,76,78,79,143.
Hence, in HFpEF, the combination of low myocardial PKG activity and nitrosative/oxidative
stress could accelerate myocardial maladaptive remodeling, because of unopposed activation
of pro-hypertrophic and pro-fibrotic signaling.
Angiotensin converting enzyme inhibitors and angiotensin receptor blockers
Angiotensin converting enzyme inhibitors (ACE-I) and angiotensin receptor blockers (ARBs)
prevent new onset DMII, ameliorate IR and inhibit formation of AGEs144-148. Moreover,
ACE-I and ARBs improve maladaptive remodeling in HFrEF149,150, reduce myocardial
fibrosis and LV mass in patients with hypertensive heart disease151,152, inhibit inflammation
and NADPH oxidase-mediated ROS production153,154 and stimulate NO production40,155,156.
Although ACE-I and ARBs improved diastolic LV dysfunction in hypertensive heart
disease157-159, their importance for HFpEF treatment has been seriously challenged, because of
the neutral outcomes of large HFpEF trials investigating ACE-I160 and ARBs161,162. A recent
large meta-analysis demonstrated divergent stimulatory effects of ACE-I on endothelial
function163, with failure of ACE-I to improve brachial flow mediated dilation in diabetic164
and obese patients165. Furthermore, in the ALLHAT study (Antihypertensive and LipidLowering Treatment to Prevent Heart Attack Trial), lisinopril was inferior to chlorthalidone in
preventing new-onset HFpEF166, while enalapril failed to improve exercise capacity or aortic
distensibility in HFpEF patients167. The reason why ACE-I and ARBs fail to improve
outcome in HFpEF patients, despite their ability to improve endothelial function remains
elusive. However, other mechanisms than RAAS activation can impair NO bioavailability and
PKG signaling in the setting of metabolic risk factors. Insulin resistance, FFA and adipokines
inhibit insulin-dependent PI3K/Akt signaling, resulting in reduced eNOS activity and
impaired NO generation113. In addition, hyperglycemia and adipokines can hamper eNOS
activity through various mechanisms, including increased flux through the hexosamine
biosynthesis pathway113. Oxidative stress in DMII, obesity and IR can be generated from
other sources than RAAS-associated ROS-producing enzymes, including mitochondrial
uncoupling, activation of the polyol pathway, lipotoxicity, glucose auto-oxidation and
formation of AGEs113,168. Furthermore, DM, obesity and IR induce activation of multiple PKC
isoforms, with PKCs being prominently involved in numerous cellular functions and signal
transduction pathways169. PKC can hamper myocardial eNOS activity and NO generation by
inhibition of PI3K/Akt signaling and stimulation of NADPH oxidase activity113,169, whereas
PKCα was recently shown to increase titin-based cardiomyocyte stiffness through
phosphorylation of the PEVK segment25. Interestingly, in a rodent diabetic HFpEF model,
PKCβ inhibition improved diastolic distensiblity, contractility and maladaptive cardiac
remodeling170. Thus, in HFpEF, increased prevalence of (frequently co-existing) metabolic
risk factors will exert complex and profound adverse effects on endothelial function and NO
bioavailability, which are mediated by synergistic contributions of inflammation, oxidative
stress, metabolic disturbances and deranged intracellular endothelial and cardiomyocyte
signaling and which could explain why isolated treatment of ACE-I and ARBs resulted in
neutral clinical outcome in the large HFpEF trials.
Aldosterone antagonists
Aldosterone plays an important role in the pathogenesis of vascular stiffening and endothelial
dysfunction, and increased plasma aldosterone levels are associated with inflammation,
oxidative stress, IR and the metabolic syndrome171-173. In addition, elevated aldosterone levels
are also related to cardiac hypertrophy, fibrosis and diastolic LV dysfunction171-173. In HFpEF
clinical trials, aldosterone antagonists improved exercise capacity and diastolic LV
stiffness174, as well as LV long axis function and cardiac geometry175. In a recent multicenter,
prospective, randomized, double-blind, placebo controlled trial, spironolactone improved
diastolic LV stiffness, but failed to improve exercise capacity176.
AGE breaking compounds
In small scale clinical HFpEF177 and HFrEF178 trials, the AGE breaking compound
alagebrium was shown to improve diastolic function and LV remodeling, whereas it enhanced
arterial compliance in aged hypertensives with vascular stiffening179. In contrast, in a recent
randomized study, alagebrium did not improve exercise tolerance, nor systolic or diastolic
function in HFrEF patients180.
Statins
Statins have been shown to improve endothelial function by stimulating activity and
expression of eNOS and by enhancing NO production through both cholesterol-dependent and
cholesterol-independent mechanisms181-183. The “pleiotropic effects” of statins also include
suppression of inflammation and oxidative stress, inhibition of neurohormonal activation,
inhibition of small GTP-binding protein (Rho, Rac and Ras) signaling and attenuation of
cardiac hypertrophy and fibrosis181-183. In a prospective, observational study, statin treatment
substantially improved survival in HFpEF, in contrast to ACE-I, ARBs, beta blockers or
calcium channel blockers which had no effect on survival184. More recently, two additional
prospective clinical studies demonstrated statins to be associated with improved survival in
HFpEF patients followed over a 5 year period185,186.
Life style changes and exercise training
Standard lifestyle recommendations (exercise, smoking cessation, weight loss) improve
insulin sensitivity and protect against DM development in obese, non-diabetic
individuals108,187. Obesity induces cardiac structural and functional changes characterized by
concentric LV remodeling188, altered myocardial substrate metabolism189,190 and diastolic LV
dysfunction, which is independently related to the extent of visceral adiposity191. Conversely,
weight loss in obesity ameliorates cardiac hypertrophy and diastolic dysfunction and improves
myocardial bioenergetics and vascular stiffness independent of changes in blood pressure and
other obesity-related co-morbidities192-199.
Physical exercise was shown to improve both clinical outcome and insulin sensitivity in nonischemic HFrEF patients200. The ESC HF guidelines firmly recommend regular physical
activity and structured exercise training. This recommendation is based on the fact that
exercise training improves exercise capacity and quality of life, does not adversely affect LV
remodeling and may reduce mortality and hospitalization in patients with mild-to-moderate
chronic HFrEF123,201-203. Exercise training could also be beneficial for improving outcome in
patients with HFpEF as exercise training was shown to improve endothelial dysfunction,
systemic inflammation and metabolic syndrome204-206. Moreover, exercise training was
suggested to attenuate the age-dependent decline in diastolic function207. Recently, the
randomized Exercise training in Diastolic Heart Failure Pilot Study (Ex-DHF) indeed
demonstrated in HFpEF patients that exercise training improved exercise capacity and quality
of life, which was associated with reverse atrial remodeling and enhanced diastolic LV
function208. Conversely, in a more recent study, HFpEF patients were allocated to a 16 weeks
exercise training program and although exercise training improved peak VO2 max, no
significant changes were observed in systolic and diastolic function209.
Improved metabolic control
Under physiological conditions, myocardial energy production is derived from a balanced
metabolism of glucose utilization and FFAs. Due to a decrease in glucose transport through
the sarcolemmal membrane of cardiomyocytes in diabetic patients with depletion of glucose
transporters 1 and 4, energy production is shifted to the beta-oxidation of FFAs210,211. The
oxidation of FFAs is less favourable in the energy balance due to the higher oxygen
demand212, while the increase in cardiac triglyceride accumulation is associated with diastolic
dysfunction213. Partial inhibitors of myocardial fatty acid oxidation, such as trimetazidine,
improved myocardial ischemia and exercise capacity in diabetic patients214. Trimetazidine,
increases myocardial efficiency by enhancing glucose metabolism and decreasing FFA
metabolism through inhibiting FFA beta-oxidation108,215,216. Trimetazidine exerts
cardioprotective effects by reducing oxidative damage, inhibiting inflammation and apoptosis
and improving endothelial function217-219. Indeed, in HFrEF, trimetazidine improved
myocardial function, remodeling, bioenergetics and functional class, while trimetazidine
decreased HF hospitalization215,220.
Metformin is a widely prescribed drug in DMII that improves insulin sensitivity and clinical
outcome in diabetic HF patients221, while metformin also exhibits anti-oxidant effects222.
Concerns about metformin being associated with lactic acidosis restricted its use in HF,
although clinical experience suggests that this risk is low223. Indeed, observational data
suggest that metformin may actually be beneficial for congestive HF224,225 and metformin
improved insulin sensitivity and minute ventilation, while it induced weight loss in nondiabetic IR HFrEF patients226. Since metformin is not a hypoglycemic agent, metformin could
represent a suitable metabolic therapy for nondiabetic HF patients.
Previously, it was shown that each 1% increase in HbA1c was associated with an 8%
increased risk of HF in diabetic patients even after adjusting for other factors, including
CAD227. In a small observational study, insulin-based metabolic control in DMII patients,
improved diastolic LV function and myocardial perfusion reserve228. Conversely, a recent
large prospective randomized trial demonstrated that long-term intensive insulin-based
glycemic control aiming at a HbA1c below 6% was associated with increased 5-year mortality
in DMII patients229. In addition, the DADD (Diabetes mellitus And Diastolic Dysfunction)
trial also failed to confirm improvement of diastolic dysfunction with tight insulin-based
glycemic control in DMII patients230. A statistical type II error could have contributed to the
negative outcome in DADD, because of strict DADD exclusion criteria, which resulted in
patients only having subtle evidence of diastolic dysfuntion with slow relaxation but normal
LV distensibility231. In addition to a type II error, this negative outcome could also suggest
that in DMII, myocardial damage not only results from hyperglycemia but also from
hyperinsulinemia, inflammation and oxidative stress100-102. Indeed, a recent study observed
improvement of diastolic LV function in DMII patients treated with rosiglitazone, to relate
more closely to the fall in malondialdehyde, a plasma marker of oxidative stress, than to the
fall in HbA1c, insulin or IL6232.
Signaling through NO-cGMP-PKG is downregulated in metabolic conditions such as obesity,
metabolic syndrome, IR and DM109,110, while in human diabetic cardiomyopathy, sildenafil
improved measurements of LV geometry, strain and torsion determined by magnetic
resonance imaging111.
Conclusion
In HFpEF, pathophysiological mechanisms, diagnostic and therapeutic strategies remain
uncertain. This is reflected in the lack of improvement of prognosis in HFpEF, over the last
decennia despite the steady increase of HFpEF prevalence. Advances in therapeutic strategies
are only to be expected when more comprehensive insight has been gained into underlying
pathophysiological mechanisms. Recent studies have contributed to improved understanding
of HFpEF pathophysiology demonstrating the importance of elevated cardiomyocyte stiffness
due to reduced myocardial PKG activity and high nitrosative/oxidative stress, which could be
related to the particularly high prevalence of metabolic risk factors in HFpEF.
Pharmacological agents that enhance myocardial NO-cGMP-PKG signaling and reduce
nitrosative/oxidative stress, in conjunction with strategies that improve overall metabolic risk
could therefore have therapeutic potential in HFpEF treatment.
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Summary
This thesis investigates several aspects of structural, functional and biochemical remodeling at
the myocardial and cardiomyocyte level in patients with heart failure with preserved ejection
fraction (HFpEF) and compared these measurements to measurements obtained in patients
with HF with reduced EF (HFrEF) as well as in patients operated for severe aortic stenosis
(AS). We used a clinical to basic translational “bed-to-bench” approach, relating clinical,
hemodynamic and echocardiographic parameters to structural, functional and biochemical
characteristics of the myocardium and cardiomyocytes through procurement of left ventricular
(LV) endomyocardial biopsies. In addition, the importance of metabolic risk factors, such as
diabetes mellitus type II, for diastolic LV dysfunction is investigated. According to the results
of this thesis, raised diastolic LV stiffness in the different patient groups results from distinct
pathophysiological mechanisms, which provides evidence that HFpEF and HFrEF actually
represent distinct HF phenotypes.
Heart failure with preserved ejection fraction (HFpEF) is widely prevalent and is associated
with a cumbersome prognosis, while its pathophysiological mechanisms are incompletely
understood and specific therapeutic strategies remain uncertain. It is however still debated
whether HFpEF and HFrEF are indeed distinct HF phenotypes despite prominent differences
in myocardial remodeling and clinical characteristics. To support the clinical distinction
between HFpEF and HFrEF, LV myocardial structure and function were compared in LV
endomyocardial biopsy samples of patients with HFpEF and HFrEF, which is described in
Chapter 2. Patients hospitalized for worsening HF were classified as having HFpEF (n=22;
LVEF 62 ± 2%) or HFrEF (n=22; LVEF 34 ± 2%). None of the patients had significant
coronary artery disease and transvascularly procured LV endomyocardial biopsies did not
demonstrate evidence of infiltrative or inflammatory myocardial disease. Biopsy samples
were analyzed with histomorphometry and electron microscopy. Single, permeabilized
cardiomyocytes were isolated from the biopsies, stretched to a sarcomere length of 2.2 μm to
measure intrinsic stiffness (passive force (Fpassive)), and activated with calcium (Ca2+)containing solutions to measure total force. HFpEF and HFrEF patients demonstrated
important differences in clinical, hemodynamic and echocardiographic characteristics. HFpEF
patients were more often hypertensive and obese and tended to be older than HFrEF patients.
LV peak systolic pressure, EF, LV wall thickness and myocardial stiffness were higher in
HFpEF. Furthermore, HFpEF patients demonstrated concentric remodeling with increased LV
mass index/LV end-diastolic volume index ratio (LVMI/LVEDVI), whereas HFrEF patients
had eccentric remodeling with lowered LVMI/LVEDVI ratio. HFpEF and HFrEF patients
also demonstrated important differences in myocellular structural and functional
characteristics. In HFpEF, cardiomyocyte diameter, myofibrillar density, myofilament Ca2+
sensitivity and cardiomyocyte Fpassive were higher than in HFrEF, while myocardial collagen
volume fraction (CVF) and total cardiomyocyte force were comparable. Following in-vitro
administration of protein kinase A (PKA), cardiomyocyte Fpassive was reduced in both HF
groups, but the drop in cardiomyocyte Fpassive was significantly larger in HFpEF, suggesting a
larger myofilament phosphorylation deficit in HFpEF. The giant sarcomeric protein titin
importantly determines cardiomyocyte Fpassive through shifts in expression of its stiff N2B and
compliant N2BA isoforms and through shifts in isoform phosphorylation status. In HFpEF,
the titin N2BA/N2B isoform expression ratio was lower than in HFrEF. We concluded that
important differences exist in both macroscopic and microscopic myocardial structural and
functional properties in LV myocardium of HFpEF and HFrEF patients, which suggests
HFpEF and HFrEF to be associated with phenotypically distinct myocardial and
cardiomyocyte abnormalities. These differences support the clinical discrimination of HF
patients into HFpEF and HFrEF groups.
Chapter 3. Excessive diastolic LV stiffness is an important contributor to HF in patients with
diabetes mellitus (DM). Diabetes is presumed to increase stiffness through myocardial
deposition of collagen and advanced glycation end products (AGEs). Cardiomyocyte Fpassive
also elevates stiffness, especially in HFpEF. We assessed the contribution to diastolic stiffness
of fibrosis, AGEs and cardiomyocyte Fpassive in diabetic HF patients with preserved or reduced
LVEF. In this study, LV endomyocardial biopsies were procured in both HFpEF and HFrEF
patients without significant coronary disease, who were hospitalized for worsening HF.
Biopsy samples were used for quantification of CVF and AGEs and for isolation of
cardiomyocytes to measure Fpassive. Diabetic HF patients had higher diastolic LV stiffness
irrespective of LVEF. The underlying mechanisms for increased diastolic LV stiffness in
diabetic HFrEF consisted predominantly of increased myocardial CVF and AGEs deposition.
In contrast, increased cardiomyocyte Fpassive was the most important contributor to raised
diastolic LV stiffness in diabetic HFpEF, with similar CVF and only a trend towards
increased myocardial AGEs deposition in diabetic HFpEF compared to non-diabetic HFpEF
patients. Furthermore, the higher Fpassive in diabetic HFpEF than in non-diabetic HFpEF
cardiomyocytes was paralleled by widening of the sarcomeric Z-disc, which was significantly
larger in diabetic HFpEF than in non-diabetic HFpEF cardiomyocytes. This study was the
first to report Z-disc widening in humans and because of the simultaneous elevation of Fpassive,
it suggests that Z-disc widening results from altered elastic properties of cytoskeletal proteins,
which pull at and open up adjacent Z-discs. Raised cardiomyocyte Fpassive was acutely lowered
with in-vitro administration of PKA with the decrement of Fpassive lowering being greatest in
diabetic HFpEF cardiomyocytes, suggesting the largest phosphorylation deficit in this
condition.
In this study, we concluded that the mechanisms responsible for the increased diastolic
stiffness of the diabetic heart differ in HFpEF and HFrEF. In diabetic HFpEF patients,
increased cardiomyocyte Fpassive more importantly underlies raised diastolic LV stiffness,
whereas in diabetic HFrEF patients, elevated fibrosis and myocardial AGEs deposition are
more important.
Chapter 4. In this editorial comment, the implications of transcriptional and posttranslational
modifications of the giant sarcomeric protein titin for diastolic function are discussed. Titin
importantly determines myocardial stiffness through modulation of cardiomyocyte Fpassive.
Titin spans a half sarcomere, running from the Z-disc to the M-band and functions as a
bidirectional spring responsible for early diastolic recoil and late diastolic resistance to
stretch. Cardiomyocyte titin-based elasticity can be adjusted through transcriptional and
posttranslational modifications. At the transcriptional level, titin modulates stiffness through
shifts in expression of its compliant N2BA (3.2-3.7 MDa) and stiff N2B (3.0MDa) isoforms,
which are co-expressed in the sarcomere. Titin-based stiffness is posttranslationally modified
by PKA and protein kinase G (PKG) mediated phosphorylation of the stiff N2B segment of
titin, which acutely lowers cardiomyocyte Fpassive in skinned human LV muscle strips, in
isolated human LV myofibrils and in isolated cardiomyocytes from HFpEF and HFrEF
patients. This acute adjustment of titin-based stiffness by PKA and PKG mediated
phosphorylation provides the cardiomyocyte with an “adaptive suspension”, regulating short
term modulation of myocardial stiffness, while extracellular matrix changes provide long term
modulation of myocardial stiffness.
Chapter 5. Transcriptional and posttranslational changes in the giant sarcomeric protein titin
underlie increased cardiomyocyte Fpassive, which importantly contributes to high diastolic
stiffness of failing myocardium. Shifts in titin isoform expression ratio and isoform
phosphorylation can alter cardiomyocyte Fpassive. In LV biopsies of HF patients, AS patients
and controls, we therefore related Fpassive of isolated cardiomyocytes to expression of titin
isoforms and to phosphorylation of titin and titin isoforms. Biopsies were procured by
transvascular technique in HF patients and perioperatively in AS patients or from explanted
hearts. All patients were free of significant coronary disease. Isolated, permeabilized
cardiomyocytes were stretched to 2.2 μm sarcomere length to measure Fpassive. Expression and
phosphorylation of titin isoforms were analyzed using gel electrophoresis with ProQ Diamond
and SYPRO Ruby stains. Cardiomyocyte Fpassive was higher in HF than in AS or controls,
while in-vitro administration of PKA acutely lowered Fpassive only in HF cardiomyocytes.
High Fpassive of HF cardiomyocytes also fell on administration of PKG and remained unaltered
on subsequent PKA treatment. The titin N2BA/N2B isoform expression ratio was higher in
HF than in controls and comparable in HF and AS patients, while a significant relation was
observed between N2BA/N2B ratio and LV end-diastolic wall stress. Overall titin
phosphorylation was comparable in HF and AS, but relative phosphorylation of the stiff N2B
titin isoform was significantly lower in HF than in AS. Because of comparable expression of
titin isoforms and similar overall phosphorylation of titin, the higher Fpassive of HF
cardiomyocytes compared to AS cardiomyocytes was attributed to a shift in titin isoform
phosphorylation with relative hypophosphorylation of the stiff N2B titin isoform.
Contribution to Fpassive of the thin filament and crossbridge interaction were ruled out because
of unaltered Fpassive after exposure to gelsolin (which removes the thin filament) and 2,3butanedione monoxine (which prevents cross bridge interactions). Cardiomyocyte Fpassive,
PKA-induced decrease in Fpassive and N2B phosphorylation deficit rose from AS to HFrEF to
HFpEF. A larger N2B titin isoform phosphorylation deficit in HFpEF explains both their
higher Fpassive and larger PKA-induced fall of Fpassive. We concluded that relative
hypophosphorylation of the stiff N2B titin isoform represents a novel mechanism responsible
for raised Fpassive of human HF cardiomyocytes. In HF, phosphorylation of the stiff N2B titin
isoform acutely lowers cardiomyocyte Fpassive, which could therefore have important
therapeutic implications, as stimulation of titin phosphorylation may represent a means to
relax the stiffened heart.
Chapter 6. In this review pathophysiological mechanisms for increased diastolic LV stiffness
in diabetic heart failure are discussed. Increased diastolic LV stiffness is recognized as the
earliest manifestation of DM-induced LV dysfunction. Mechanisms responsible for increased
diastolic LV stiffness in diabetic HF include myocardial fibrosis, AGEs, metabolic
disturbances, impaired Ca2+ homeostasis, endothelial dysfunction, myocardial oxidative
stress, impaired nitric oxide (NO) bioavailability, downregulated myocardial cyclic guanosine
monophosphate (cGMP) signaling and altered titin regulation. Furthermore, mechanisms
responsible for the elevated diastolic LV stiffness differ in diabetic HFpEF and diabetic
HFrEF patients. Myocardial fibrosis and deposition of AGEs are important determinants of
increased diastolic LV stiffness in diabetic HFrEF, whereas increased Fpassive of hypertrophied
cardiomyocytes is the main determinant of increased diastolic LV stiffness in diabetic HFpEF.
Downregulation of NO-mediated cGMP-PKG signaling, probably induced by vascular
inflammation and oxidative stress, could account for the high Fpassive of hypertrophied
cardiomyocytes observed in diabetic HFpEF patients.
Chapter 7. Diabetes mellitus (DM)-induced diastolic LV dysfunction is recognized as an
important determinant of morbidity and mortality in HF. In this chapter, the effects of DM on
diastolic LV function and its underlying mechanisms were identified in patients with severe
AS with or without DM who were referred for aortic valve replacement in the absence of
significant coronary artery disease. Preoperative Doppler echocardiography and
hemodynamics were implemented with perioperative LV biopsies. Myocardial CVF and
AGEs deposition were quantified by histomorphometry and immunohistochemistry, whereas
Fpassive was determined in isolated, permeabilized cardiomyocytes stretched to a sarcomere
length of 2.2 μm. Expression and phosphorylation of titin isoforms were analyzed with gel
electrophoresis with ProQ Diamond and SYPRO Ruby stains. Diabetic AS patients had
reduced LV end-diastolic distensibility as evident from higher LV end-diastolic pressure at
comparable LV end-diastolic volume index and attributed to higher myocardial CVF, more
AGEs deposition and higher cardiomyocyte Fpassive. The higher cardiomyocyte Fpassive in
diabetic AS patients was related to significant hypophosphorylation of the stiff N2B titin
isoform, while in-vitro administration of PKA normalized cardiomyocyte Fpassive. We
concluded that an additional impairment of LV end-diastolic distensibility is observed when
severe, symptomatic AS was associated with DM. This additional impairment related to both
structural and functional alterations of LV myocardium consisting of increased interstitial
collagen deposition, augmented intramyocardial AGE accumulation and higher intrinsic
cardiomyocyte stiffness. The contribution of increased cardiomyocyte Fpassive to raised
diastolic LV stiffness could be amenable to novel therapeutic strategies modulating
posttranslational titin-based stiffness through stimulation of titin phosphorylation.
Chapter 8. Prominent features of myocardial remodeling in HFpEF are high cardiomyocyte
Fpassive and cardiomyocyte hypertrophy. In experimental models, both reacted favourably to
raised PKG activity. In this chapter, we assessed myocardial PKG activity, its downstream
effects on cardiomyocyte Fpassive and cardiomyocyte diameter, and its upstream control by
cGMP, nitrosative/oxidative stress, and brain natriuretic peptide (BNP). To discern altered
control of myocardial remodeling by PKG, HFpEF was compared with HFrEF and AS. All
patients were free of significant coronary disease. More HFpEF patients were obese or had
DM. LV myocardial biopsies were procured transvascularly in HFpEF and HFrEF and
perioperatively in AS. Fpassive was measured in isolated, permeabilized cardiomyocytes before
and after PKG administration. Myocardial homogenates were used for assessment of PKG
activity, cGMP concentration and expression of proBNP-108 and nitrotyrosine. Nitrotyrosine
represents an indirect measure of nitrosative/oxidative stress. Additional quantitative
immunohistochemical analysis was performed for PKG activity, nitrotyrosine expression and
expression of soluble guanylate cyclase (sGC) and phosphodiesterase type 5A (PDE5A),
which breaks down cGMP, thereby terminating cGMP-PKG signaling. Our study showed that
relative to both AS and HFrEF, HFpEF patients had reduced myocardial PKG activity and
lower cGMP concentration, which related to higher cardiomyocyte Fpassive and increased
myocardial nitrotyrosine levels, indicative of raised nitrosative/oxidative stress. In all groups,
cardiomyocyte Fpassive was acutely lowered by in vitro administration of PKG, whereas
cardiomyocytes from HFpEF patients demonstrated the largest PKG-induced fall in Fpassive.
Reduced PKG activity and lower myocardial cGMP concentration in HFpEF did not result
from altered myocardial sGC or PDE5A expression, which were similar in all groups, or from
unequal natriuretic peptide (NP) expression, which was comparable in HFpEF and AS.
Downregulation of cGMP-PKG signaling in HFpEF was therefore most likely related to low
myocardial NO bioavailability because of high nitrosative/oxidative stress, which was almost
fourfold higher in HFpEF than in both HFrEF and AS. Oxidative stress is indeed known to
lower NO bioavailability and downregulate cGMP-PKG signaling. Increased
nitrosative/oxidative stress in HFpEF could have resulted from the high prevalence in HFpEF
of metabolic comorbidities. We concluded that HFpEF myocardium is characterized by
downregulated NO-cGMP-PKG signaling probably resulting from high nitrosative/oxidative
stress. Low PKG activity raises cardiomyocyte intrinsic stiffness and could be a potential
target for a specific HFpEF treatment strategy.
Chapter 9. In this review underlying molecular and cellular mechanisms for diastolic LV
dysfunction are discussed. HFpEF is widely prevalent, associated with a dismal prognosis and
incompletely understood pathophysiology, which precludes specific therapy. Prominent
features of HFpEF are impaired relaxation, increased diastolic LV stiffness and distinct
cardiac remodeling processes at the macroscopic, microscopic and ultrastructural level. In
HFpEF, increased diastolic LV stiffness can be explained by maladaptive changes in the
extracellular matrix and by elevated cardiomyocyte stiffness, which seem to be related to
myocardial inflammation, nitrosative/oxidative stress, endothelial dysfunction and
downregulation of NO-cGMP-PKG signaling. NO is crucially important for diastolic
function, since it potently enhances LV relaxation and distensibility through a variety of
mechanisms including cGMP-PKG dependent and independent mechanisms and modulation
of β-adrenergic responsiveness, S-nitrosylation signaling and myocardial bioenergetics.
Patients with HFpEF demonstrate a high prevalence of metabolic disorders, which may
contribute to enhanced myocardial inflammation, nitrosative/oxidative stress, downregulation
of NO bioavailability and NO-mediated cGMP-PKG signaling, thereby triggering diastolic
LV dysfunction. We concluded that targeting metabolic risk and improving endothelial
function, NO bioactivity and cGMP-PKG signaling can be promising strategies for specific
HFpEF treatment.
Future perspectives
Heart failure with preserved ejection fraction (HFpEF) demonstrates an increasing prevalence,
and currently approximately 50% of HF patients present with this type of HF. Although
clinical outcome is slightly better for HFpEF than for HFrEF, mortality associated with
HFpEF is considerable and most patients with HFpEF die from cardiovascular causes.
Unfortunately, in HFpEF, pathophysiological mechanisms, diagnostic and therapeutic
strategies remain uncertain and this is reflected in the lack of improvement of prognosis in
HFpEF over the last decennia. Advances in therapeutic strategies are only to be expected
when more comprehensive insight has been gained into underlying pathophysiological
mechanisms responsible for development and progression of HFpEF. HFpEF appears a
complex syndrome characterized by maladaptive changes in myocardial and cardiomyocyte
structural, functional and biochemical characteristics, probably induced in a multifactorial
fashion by a combination of adverse effects of myocardial inflammation, nitrosative/oxidative
stress, endothelial dysfunction, downregulation of NO bioavailability and NO-mediated
signaling, impaired myocardial bioenergetics, disturbed calcium handling and concentric
hypertrophy. Our recent translational studies have contributed to improved understanding of
HFpEF pathophysiology and exciting insights with potential direct applications for
therapeutic strategies have emerged. For instance, pharmacological agents that enhance
myocardial NO-cGMP-PKG signaling and reduce nitrosative/oxidative stress, in conjunction
with strategies that improve overall metabolic risk could have therapeutic potential in HFpEF
treatment. Nevertheless, because of the high complexity of HFpEF pathophysiology,
continuing research efforts, based on an integrative translational approach are undisputably
valuable to keep on moving this exciting field forward. This importance of integrative
physiology to gain insight into clinical pathophysiology in order to improve HFpEF outcome
has recently been acknowledged and awarded a significant grant from the European
Committee, to launch a large collaborative project, including 20 centers across Europe,
coordinated by Prof Dr. W.J. Paulus. This large scale integrating project includes both
experimental models of diastolic dysfunction as well as HFpEF patients and focuses on
pathophysiological mechanisms, improved diagnostic modalities and potential specific
HFpEF therapeutic strategies.