Download Molecular and Cellular Basis for Diastolic Dysfunction

Curr Heart Fail Rep
DOI 10.1007/s11897-012-0109-5
PREVENTION OF HEART FAILURE AFTER MYOCARDIAL INFARCTION (M ST. JOHN SUTTON, SECTION EDITOR)
Molecular and Cellular Basis for Diastolic Dysfunction
Loek van Heerebeek & Constantijn P. M. Franssen &
Nazha Hamdani & Freek W. A. Verheugt &
G. Aernout Somsen & Walter J. Paulus
# Springer Science+Business Media, LLC 2012
Abstract Heart failure with preserved ejection fraction
(HFpEF) is highly prevalent and is frequently associated
with metabolic risk factors. Patients with HFpEF have only
a slightly lower mortality than patients with HF and reduced
EF. The pathophysiology of HFpEF is currently incompletely understood, which precludes specific therapy. Both HF
phenotypes demonstrate distinct cardiac remodeling processes at the macroscopic, microscopic, and ultrastructural
levels. Increased diastolic left-ventricular (LV) stiffness and
impaired LV relaxation are important features of HFpEF,
which can be explained by changes in the extracellular
matrix and the cardiomyocytes. In HFpEF, elevated intrinsic
cardiomyocyte stiffness contributes to high diastolic LV
stiffness. Posttranslational changes in the sarcomeric protein
titin, affecting titin isoform expression and phosphorylation,
contribute to elevated cardiomyocyte stiffness. Increased
nitrosative/oxidative stress, impaired nitric oxide bioavailability, and down-regulation of myocardial cyclic guanosine
monophosphate and protein kinase G signaling could trigger
posttranslational modifications of titin, thereby augmenting
cardiomyocyte and LV diastolic stiffness.
L. van Heerebeek : C. P. M. Franssen : N. Hamdani : W. J. Paulus
Department of Physiology, Institute for Cardiovascular Research,
VU University Medical Center,
Amsterdam, The Netherlands
L. van Heerebeek : F. W. A. Verheugt : G. A. Somsen
Department of Cardiology, Onze Lieve Vrouwe Gasthuis,
Oosterpark 9,
1091 AC Amsterdam, The Netherlands
W. J. Paulus (*)
ICaR-VU, VU University Medical Center,
Van der Boechorststraat 7,
1081 BT Amsterdam, The Netherlands
e-mail: [email protected]
Keywords Cardiomyocytes . Cyclic guanosine
monophosphate . Diabetes mellitus . Diastole . Diastolic
dysfunction . Extracellular matrix . Fibrosis . Guanylate
cyclase . Heart failure . Hypertrophy . Inflammation .
Metabolic risk factors . Metabolic syndrome . Nitric oxide .
Natriuretic peptides . Obesity . Oxidative stress . Protein
Kinase G . Phosphodiesterase . Titin
Introduction
Heart failure with preserved ejection fraction (HFpEF) currently accounts for 50 % of all HF patients, and its prevalence relative to HF with reduced EF (HFrEF) is rising at a
rate of approximately 1 % per year [1]. Patients with HFpEF
have a dismal prognosis, with only slightly lower mortality
rates than patients with HFrEF [1, 2]. As a result of
evidence-based HF therapy, the prognosis of patients with
HFrEF improved progressively over the past 3 decades.
Conversely, despite frequent use of similar pharmacological
agents, the prognosis of patients with HFpEF remained
unaltered over the same time period [1]. Indeed, most trials
testing modern HF therapy in patients with HFpEF had a
neutral outcome, in contrast to the positive outcome observed with similar pharmacotherapy in patients with HFrEF
[3]. The main reason for this discrepancy is failure of
modern HF therapy to sufficiently address pathophysiological mechanisms driving left-ventricular (LV) remodeling in
HFpEF [3, 4•]. LV remodeling substantially differs between
HFpEF and HFrEF. In HFpEF, the left ventricle is concentrically remodeled with normal LV cavity size, increased
wall thickness, and increased LV mass/volume ratio, whereas in HFrEF, the left ventricle is eccentrically remodeled
with LV dilatation, normal or decreased wall thickness, and
low LV mass/volume ratio [5, 6]. Similar findings are also
observed at the microscopic and ultrastructural levels, with
concentrically thickened, stiff cardiomyocytes in HFpEF
Curr Heart Fail Rep
and eccentrically enlarged, more compliant cardiomyocytes
in HFrEF [5, 6]. In addition to disparate responses to similar
pharmacotherapy and different patterns of chamber and
myocellular remodeling in HFpEF and HFrEF, distinct differences are also observed in demographic and cardiovascular risk factor profiles [1, 2, 7, 8], supporting HFpEF and
HFrEF as two separate HF phenotypes. The pathophysiology of HFpEF is incompletely understood, which precludes
development of specific therapeutic strategies.
LV Diastolic Dysfunction in HFpEF
According to current criteria, HFpEF is diagnosed in the
presence of signs and/or symptoms of HF, preserved systolic
LV function with an EF of >50 %, and LV end-diastolic
volume index of <97 ml/m2, as well as evidence of diastolic
LV dysfunction [9]. The importance of diastolic LV dysfunction for HFpEF was recently reappraised by invasive
studies that showed slow LV relaxation and elevated diastolic LV stiffness at rest [10, 11]. In addition, elevated
diastolic LV stiffness limited cardiac performance during
atrial pacing and exercise [11, 12]. Reduced LV distensibility and increased diastolic stiffness in HFpEF can be
explained by the left and upward shift of the LV enddiastolic pressure–volume relation (LV EDPVR), with an
increased slope of the LV EDPV curve [6, 10]. Hence, each
LV end-diastolic volume is associated with a high enddiastolic pressure. In HFpEF, the steep LV EDPVR seems
the most important determinant for impaired exercise tolerance, with deficient early diastolic LV recoil, blunted LV
lusitropic or chronotropic response, vasodilator incompetence, and deranged ventriculovascular coupling serving
contributory roles [13]. In the absence of endocardial or
pericardial disease, the steep LV EDPVR results from increased myocardial stiffness, which is regulated by the
extracellular matrix (ECM) and the cardiomyocytes [14].
Regulation of Diastolic Stiffness by the ECM
The ECM contributes to passive stiffness in diastole and
prevents overstretch, myocyte slippage, and tissue deformation during ventricular filling, while ECM components also
serve as modulators of growth and tissue differentiation
[15]. Chronic pressure overload, as occurs in hypertensive
heart disease, is associated with excessive forms of collagen
deposition, which increases myocardial stiffness [16]. Collagen importantly determines ECM-based stiffness, through
regulation of its total amount, expression of collagen type I,
and degree of collagen cross-linking [15, 16], which are
increased and linked to diastolic LV dysfunction in patients
with HFpEF [17, 18•]. In a recent substudy from the
landmark HFpEF I-PRESERVE (The Irbesartan in Heart
Failure with Preserved Systolic Function) trial, which had
a neutral overall outcome [19], increased baseline plasma
levels of collagen markers were not independently associated with adverse outcome on multivariable analysis but were
on single-variable analysis [20]. Hence, this study partially
supports the hypothesis that pathological fibrosis contributes to adverse prognosis in patients with HFpEF [20].
Myocardial collagen turnover is dynamically modulated by
the local renin-angiotensin-aldosterone system (RAAS) and
transforming growth factor β(TGF-β) [15, 16] and through
modulation of collagen synthesis and degradation by matrix
metalloproteinases (MMPs) and tissue inhibitors of MMPs
(TIMPs) [21]. In HFpEF, matrix degradation is decreased
because of altered expression of MMPs and up-regulation of
TIMPs [22–25]. Previously, serological markers of collagen
turnover significantly predicted diastolic dysfunction and
HFpEF, with MMP2 representing the most sensitive and
specific biomarker for the identification of HFpEF [23].
Plasma levels of TIMP-1 also predicted diastolic LV dysfunction [24] and have recently been proposed as a potential
biomarker of HFpEF in hypertensive patients [25]. In
patients with dilated cardiomyopathy, matrix degradation
is increased because of up-regulation of MMPs [26]. Distinct expression profiles of MMPs and TIMPs also correspond with unequal patterns of myocardial collagen
deposition, with mainly interstitial fibrosis in HFpEF
and both replacement and interstitial fibrosis in dilated
cardiomyopathy [4•].
Importance of Inflammation in HFpEF
Myocardial inflammation was shown to contribute to ECM
changes and diastolic dysfunction in HFpEF [27•]. In an
endomyocardial biopsy study, when compared with controls,
HFpEF patients had increased inflammatory cell TGF-β expression, which induces transdifferentiation of fibroblasts into
myofibroblasts, with high production of collagen and low
expression of MMP1, whereas both myocardial collagen and
the amount of inflammatory cells correlated with diastolic LV
dysfunction [27•]. As compared with asymptomatic hypertensives, HFpEF patients had increased circulating biomarkers of inflammation (interleukin 6 [IL6], IL8, monocyte
chemoattractant protein 1), of collagen metabolism (aminoterminal propeptide of collagen III, carboxy-terminal telopeptide of collagen I), and of ECM turnover (MMP2 and MMP9)
[28]. In addition, MMP9, TIMP1, and the ratio of MMP9/
TIMP1 correctly identified patients with a high left atrial
volume index, which reflects chronic diastolic LV dysfunction
[28]. Moreover, the Health ABC study reported inflammatory
biomarkers such as IL6 and tumour necrosis factor α to be
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strongly associated with the propability of HFpEF development in elderly patients [29].
Interestingly, LV endomyocardial biopsy studies demonstrated that one third of HFpEF patients had a normal myocardial collagen volume fraction (CVF), despite similarly
elevated LVend-systolic wall stress and LV stiffness modulus,
as compared with HFpEF patients with raised CVF [30].
Numerous subsequent studies unequivocally attributed high
diastolic LV stiffness to elevated intrinsic cardiomyocyte stiffness (Fpassive) in HFpEF patients [4•, 30, 31, 32••].
Regulation of Diastolic Stiffness by the Cardiomyocytes
Cardiomyocyte Fpassive is mainly determined by the giant
elastic sarcomeric protein titin, which regulates myocardial
passive tension, stiffness, and biomechanical stress/stretch
signaling [33•]. 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 stretch [33•]. Within physiological volumes at a
sarcomere length (SL) range of 1.8–2.2 μm, titin was identified as the dominant determinant of LV passive pressure,
contributing approximately 80 % to LV passive stiffness,
while the contribution of the ECM appeared more important
at an SL higher than 2.2 μm [34]. 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 (3.0 MDa) isoforms, which are co-expressed in the sarcomere at a N2BA:
N2B ratio of approximately 35:65 in the normal heart [33•].
In eccentrically remodeled hearts from dilated [4•, 35, 36] or
ischemic cardiomyopathy [37] patients, 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, as compared with patients with HFrEF [4•].
Titin-based stiffness is posttranslationally modified by a
variety of mechanisms. Protein kinase A (PKA) and protein
kinase G (PKG) mediated phosphorylation of the stiff N2B
segment of titin acutely lowers Fpassive in skinned human LV
muscle strips [38, 39••], in isolated human LV myofibrils
[39••], and in isolated cardiomyocytes from HFpEF and
HFrEF patients [4•, 30, 31, 32••]. 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) [40] and oxidative stressinduced formation of disulfide bridges in the N2B segment
[41]. Contribution by other myofilamentary proteins, such as
myosin heavy chain, desmin, actin, troponin T, troponin I, and
myosin light chain 1 and 2 was recently ruled out [30, 32••],
suggesting that phosphorylation-mediated posttranslational
modification of titin represents the most likely mechanism
for acute modulation of cardiomyocyte Fpassive. Acute lowering of cardiomyocyte Fpassive by PKA and PKG suggests a
myofilamentary protein phosphorylation deficit in HF. In
HFpEF, slow LV relaxation and high diastolic LV stiffness
have been shown to react favorably to raised PKG activity
following in vivo administration of sildenafil, which raises
myocardial PKG activity through inhibited breakdown of
cyclic guanosine monophosphate (cGMP) by phosphodiesterase type 5A (PDE5A) [42•]. Sildenafil restored LV relaxation
kinetics in mice exposed to transverse aortic constriction
(TAC) [43] and reduced diastolic LV stiffness in an old
hypertensive dog model [44••], in patients with HFrEF
[45•], and in HFpEF patients with pulmonary hypertension
[46••]. Administration of sildenafil to elderly hypertensive
dogs lowered diastolic LV stiffness through restored phosphorylation of the titin N2B segment [44••], which was recently shown to be hypophosphorylated in HF patients [32••]
and to have PKG phosphorylation sites [39••]. We recently
measured PKG activity, upstream control of PKG activity, and
downstream effects of PKG activity in LV myocardial biopsies of HFpEF patients [47••]. To discern altered control by
PKG of the myocardial remodeling process in HFpEF, measurements were compared with measurements obtained from
LV myocardium remodeled concentrically by severe aortic
stenosis (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 F passive was acutely lowered by in vitro
administration of PKG, whereas cardiomyocytes from HFpEF
patients demonstrated the largest PKG-induced fall in Fpassive
[47••]. 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 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 nitric oxide
(NO) bioavailability because of high nitrosative/oxidative
stress, which was almost fourfold higher in HFpEF than in
both HFrEF and AS [47••]. Oxidative stress is known to lower
NO bioavailability and down-regulate cGMP-PKG signaling
[48, 49].
Importance of Myocardial NO for Diastolic LV Function
NO is crucially important for diastolic function, since it potently enhances LV relaxation and distensibility through
Curr Heart Fail Rep
cGMP-PKG dependent and independent mechanisms, including reduction of myofilament calcium (Ca2+) sensitivity by
troponin I phosphorylation and enhancing phospholambanmediated sarcoplasmic reticular (SR) Ca2+ reuptake, respectively [50]. 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 localization [42•, 51, 52•]. The myocardial autocrine and paracrine
effects of NO depend on where and by which NOS isoform
NO is produced [51]. Myocardial eNOS-derived NO was
demonstrated to enhance LV relaxation and distensibility
[53–55] and to modulate cardiac β-adrenergic responsiveness
[56]. 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 heart [53], as well as in
patients with dilated cardiomyopathy [54] and severe AS [55].
NO- and NP-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 sGC [42•, 52•]. Stimulated sGC subsequently enhances cGMP generation, which modulates
phosphodiesterase (PDE) enzyme activity and activates its
primary effector kinase PKG [42•, 52•]. In turn, PKG phosphorylates a vast number of target proteins, exerting a wide
range of downstream effects like enhanced reuptake of Ca2+
into the SR, inhibition of Ca2+ influx, and suppression of
hypertrophic and fibrotic signaling, as well as stimulation of
LV relaxation and LV distensibility by phosphorylation of
troponin I and the stiff titin N2B segment [39••, 42•, 52•].
Because NO diffusion distances in cardiomyocytes are
limited, NO-sGC-cGMP signaling is compartmentalized into
functional signalosomes, coupling NO synthesis to downstream sGC-cGMP signals [52•]. For instance, in cardiomyoyctes and endothelial cells, eNOS, sGC, and PKG
colocalize with caveolin at plasma membrane invaginations,
called caveolae, which may serve as microdomains for optimized NO-sGC-cGMP signaling [52•, 57].
In addition, NP-mediated activation of cGMP-PKG signaling is spatially confined to a distinct subcellular compartment. Natriuretic peptides stimulate the NP receptor at the
plasma membrane, resulting in activation of particulate GC
(pGC). Activated pGC subsequently stimulates cGMP generation and PKG activation confined to a subsarcoplasmic
membrane compartment [52•, 57].
Atrial natriuretic peptide (ANP) and brain natriuretic
peptide (BNP) are released by atrial and ventricular cardiomyocytes, respectively, in response to increased myocardial
wall stress due to volume- or pressure-overload conditions
[52•, 58]. Both NPs stimulate natriuresis, vasorelaxation,
and myocardial relaxation, while they inhibit neurohumoral
activation, fibrosis, and hypertrophy [58, 59]. We recently
observed myocardial proBNP-108 expression to be similar
in HFpEF and AS and higher in HFrEF than in both HFpEF
and AS [47••]. These observations are consistent with BNP
expression being predominantly regulated by diastolic LV
wall stress, which is higher in HFrEF because of eccentric
LV remodeling and similar in HFpEF and AS because of
concentric LV remodeling [60]. 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 conditions, which were
therefore attributed to higher nitrosative/oxidative stress in
HFpEF than in AS [47••]. Lower proBNP-108 expression in
HFpEF than in HFrEF also explains the low positive predictive value of BNP for the diagnosis of HFpEF [61].
Acute BNP administration was recently reported to lower
diastolic LV stiffness and to increase myocardial titin phosphorylation in an old hypertensive HFpEF dog model [44••]
but failed to improve clinical endpoints in acutely decompensated HF patients with LVEF ≥ 40 % [62].
Compartmentalization of cGMP-PKG Signaling
and Cross Talk With cAMP Signaling
Particulate GC and sGC generate spatially and functionally
distinct cellular pools of cGMP, with NP-pGC-cGMP signals regulated by PDE type 2 (PDE2) and NO-sGC-cGMP
signals regulated by PDE5A [63•]. Functionally distinct
compartmentalization of NP-pGC-cGMP and NO-sGCcGMP pathways was shown in rodent cardiomyocytes, in
which the PDE5A inhibitor sildenafil suppressed cardiac βadrenergic receptor (β-AR) responsiveness, which was abrogated by NOS inhibition, whereas activation of pGC by
ANP had no effect, despite higher myocardial cGMP elevations [56]. Importantly, considerable cross talk exists between both NO- and NP-mediated cGMP-PKG signaling
and the β-AR-cAMP-PKA signaling pathway, which importantly regulates excitation–contraction coupling, relaxation,
and myofilament Ca2+ sensitivity [64].
Recently, it was demonstrated that activation of a signalosome consisting of the beta3-adrenergic receptor (β3-AR)
and eNOS-NO-sGC-cGMP counteracts myocardial β1-ARand β2-AR-mediated cardiac stimulation. This inhibition
occurred both through cGMP-induced activation of PDE2,
which subsequently inhibited cAMP, and through PKGmediated phosphorylation of troponin I [65, 66]. In rat
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cardiomyocytes, it was demonstrated that NP-pGCmediated cGMP generation elicits a strong feed-forward
mechanism that further enhances cGMP production in the
subsarcolemmal pool [63•]. In contrast, increased cGMP
production by NO-sGC activates a negative feedback regulation through PKG-mediated activation of PDE5A, which
subsequently inhibits further cGMP generation [63•].
The beneficial clinical and experimental effects of
PDE5A inhibitors on diastolic LV stiffness and cardiac
remodeling [43, 44••, 45•, 46••] might be explained by
PDE5A inhibitors interfering with the negative feedback
regulation mediated by PKG, finally resulting in enhanced
PKG signaling.
Importance of cGMP-PKG Signaling in Myocardial
Remodeling
Cardiac remodeling in HFpEF is characterized by concentric
LV and cardiomyocyte hypertrophy and interstitial fibrosis
[4•, 5, 17, 18•]. The Framingham Heart Study determined
that LV hypertrophy follows aging as an independent risk
factor for cardiovascular morbidity and mortality [67]. Stimuli for LV hypertrophy activate an array of membranebound receptors coupled to multiple intracellular signaling
cascades, which ultimately stimulate prohypertrophic gene
expression [59]. Important upstream triggers for cardiac
hypertrophy include mechanical stretch, pressure overload,
cytokines, activation of RAAS, endothelin, and catecholamines, whereas cGMP-PKG signaling exerts prominent
antihypertrophic and antifibrotic effects [42•, 59]. Indeed,
modulation of the sGC-PKG-PDE5A axis affected myocardial remodeling with less cardiomyocyte hypertrophy and
interstitial fibrosis in TAC mice exposed to sildenafil [43].
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 did AS patients [47••]. The finding in AS,
of higher PKG activity corresponding with low Fpassive but
not with cardiomyocyte diameter, suggests that cardiomyocyte diastolic stiffness evolves independently of 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
dysfunction [68]. External myocardial force signals imposed
during hemodynamic load are transmitted to ECM components and the cardiomyocytes [69]. Mechanical stimuli
sensed at the outer cardiomyocyte cell membrane can activate cytoskeletal and sarcomeric signaling cascades, which
subsequently trigger prohypertrophic factors [69]. 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 regulation [70•]. Thus, one could speculate that
increased cardiomyocyte stiffness sensed by titin precedes
titin-mediated stimulation of prohypertrophic signaling.
Importance of NO-cGMP Signaling in Myocardial
Energetics
Another paracrine action of endothelial eNOS-derived NO
is regulation of myocardial 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
chain [51, 71]. Interestingly, mitochondrial biogenesis is
stimulated in response to increased myocardial NO bioavailability and cGMP levels [72]. Because cross-bridge detachment is an energy-consuming process, slow LV relaxation in
HFpEF could therefore also result from a myocardial energy
deficit. Indeed, recent 31P-magnetic resonance spectroscopy
studies demonstrated reduced myocardial energy reserve
both in patients with HFpEF [73] and in asymptomatic,
normotensive patients with diabetes mellitus type II
(DMII), in whom impaired myocardial bioenergetics
correlated with diastolic LV dysfunction [74]. Furthermore, cardiac energetic deficiency relating to diastolic
dysfunction was also recently demonstrated in obese, as
compared with normal weight, subjects both at rest and
during inotropic stress with high-dose intravenous
dobutamine administration [75].
Nitrosative/Oxidative Stress Lowers NO Bioavailability
and NO Signaling in HFpEF
Reduced NO bioavailability, dysregulation of NO-mediated
signaling, and increased nitrosative/oxidative stress are
firmly implicated in the pathogenesis of HF [49, 76]. Enhanced nitrosative/oxidative stress impairs NO bioavailability and NO-mediated signaling through a number of
mechanisms. First, superoxide rapidly reacts with NO to
form peroxynitrite. Peroxynitrite oxidizes the essential
eNOS cofactor tetrahydrobiopterin (BH4) and 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 signaling [48, 49, 76]. Finally, sGC activity
is adversely affected by nitrosative/oxidative stress through
several mechanisms, including superoxide- and peroxynitritemediated decrease in sGC activity and sGC heme group
oxidation converting sGC to its NO-insensitive state [49,
57]. In a TAC mouse model, pressure overload decreased
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sGC responsiveness to NO through sGC heme oxidation
and through a shift of sGC out of caveolae-enriched
microdomains [57].
The lower myocardial PKG activity and reduced cGMP
concentration in HFpEF patients, which was associated with
increased myocardial nitrotyrosine levels [47••], 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 that oxidative stress is 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 function [77, 78]. Furthermore, high plasma levels
of methylated L-arginine metabolites, indicative of increased oxidative stress, were strongly related to diastolic
LV dysfunction in patients with HFrEF [79]. Moreover, in
patients with HFpEF, myocardial fibrosis and inflammation
were associated with diastolic LV dysfunction and with
increased oxidative stress in endomyocardial biopsies, as
compared with controls [27•].
Abundant evidence demonstrates that reactive oxygen
species (ROS) potently activate prohypertrophic and profibrotic signaling, and by interfering with NO-sGC-cGMPPKG signaling, ROS inhibit antihypertrophic and antifibrotic signaling cascades [59, 76].
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 factors [76].
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 condition [47••]. HFpEF patients demonstrate a high
prevalence of obesity and DMII [7, 8, 80]. 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),
and the frequent clustering of these metabolic risk factors
causes synergistic adverse effects on myocardial structure
and function [81]. The prevalence of obesity, DMII, IR, and
metabolic syndrome increases rapidly and is expected to
reach pandemic proportions in the next few decades [81],
while these metabolic risk factors have all been prospectively identified as precursors of incident HF [82–85] and are
independently associated with early development of diastolic LV dysfunction [86•, 87, 88•, 89]. Diabetes increases LV
diastolic and cardiomyocyte stiffness in both HFpEF [31]
and AS [90] patients. Elevated cardiomyocyte stiffness in
diabetic AS patients related to hypophosphorylation of the
titin N2B segment, which could be corrected by phosphorylation with PKA [90].
Obesity, DMII, and IR can have direct adverse effects
on myocardial structure and function independently of
common confounders as hypertension or CAD, which
has been referred to as “obesity” [91], “diabetic” [92],
or “insulin-resistant” [93] 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 remodeling [91–93].
Metabolic Risk Factors Interfere with NO-cGMP-PKG
Signaling
Oxidative stress-mediated down-regulation of NO-cGMPPKG signaling has been demonstrated in various experimental models of obesity, diabetes, IR, and metabolic syndrome [94, 96•]. 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 production [94]. In this model, high glucosemediated lowering of PKG levels was inhibited by a superoxide scavenger or NADPH oxidase inhibitors [94]. In
addition, in human diabetic cardiomyopathy, sildenafil improved measurements of LV geometry, strain, and torsion
determined by magnetic resonance imaging [95]. 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 sildenafil [96•].
Physiologically, insulin phosphorylates titin through
activation of the phosphoinositol-3-kinase (PI3K)/Akt/
eNOS pathway, while IR, free fatty acids, and adipokines inhibit insulin-dependent PI3K/Akt signaling,
resulting in reduced eNOS activity and impaired NO
generation [97, 98]. Inflammation and oxidative stress
associated with metabolic risk factors can reduce eNOS
activity and NO bioavailability through a number of
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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 autooxidation, and formation of advanced glycation end
products [98]. Moreover, obesity, DM, and IR induce
activation of multiple PKC isoforms, with PKCs being
prominently involved in numerous cellular functions and
signal transduction pathways [99]. PKC can hamper
myocardial eNOS activity and NO generation by inhibition of PI3K/Akt signaling and stimulation of NADPH
oxidase activity [99], whereas PKCα was recently
shown to increase titin-based cardiomyocyte stiffness
through phosphorylation of the PEVK segment [40].
Interestingly, in a rodent diabetic HFpEF model, PKCβ
inhibition improved diastolic distensiblity, contractility,
and maladaptive cardiac remodeling [100].
In summary, metabolic risk factors could contribute to
diastolic LV dysfunction and elevated myocardial stiffness
in HFpEF by provoking myocardial inflammation and nitrosative/oxidative stress, which in turn results in decreased
NO bioavailability, down-regulation of cGMP-PKG signaling, impaired myocardial bioenergetics, and maladaptive
remodeling. Furthermore, down-regulation of cGMP-PKG
signaling could subsequently result in hypophosphorylation
of titin, which elevates cardiomyocyte stiffness and possibly
triggers cardiomyocyte hypertrophy.
Conclusion
HFpEF is widely prevalent and is associated with a poor
prognosis because of the absence of effective therapeutic
strategies due to an incomplete understanding of its pathophysiology. In HFpEF, increased diastolic LV stiffness can be
explained by maladaptive changes in the ECM and by elevated cardiomyocyte stiffness, which seem to be related to myocardial inflammation and nitrosative/oxidative stress. Patients
with HFpEF demonstrate a high prevalence of metabolic
disorders, which may contribute to enhanced myocardial inflammation, nitrosative/oxidative stress, and down-regulation
of NO bioavailability and NO-mediated cGMP-PKG signaling, thereby triggering diastolic LV dysfunction. Therefore,
targeting metabolic risk and improving endothelial function,
NO bioactivity and cGMP-PKG signaling can be promising
strategies for specific HFpEF treatment.
Acknowledgments L. van Heerebeek, C.P.M. Franssen, N. Hamdani,
and W.J. Paulus are funded through a grant from European Commission
FP7 Health 2010, Large Collaborative Research Project on Diastolic
Heart Failure MEDIA (261409).
Disclosure No potential conflicts of interest relevant to this article
were reported.
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