Heart failure and PAH - Chest

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The right ventricle under pressure;

Cellular and molecular mechanisms of right heart failure in pulmonary hypertension

Harm J. Bogaard MD, PhD1,2 hjbogaard@vcu.edu

Kohtaro Abe MD, PhD2,3 kabe@vcu.edu2

Anton Vonk Noordegraaf, MD PhD1 a.vonk@vumc.nl

Norbert F. Voelkel, MD2

1Dept of Pulmonary Medicine, VU University Medical Center, Amsterdam, The Netherlands; 2Dept of Pulmonary Medicine and Critical Care, Virginia Commonwealth University, Richmond, Virginia; 3Dept of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan

First Author: Harm J Bogaard

Contact Address: 1101 E Marshall Street

Sanger Hall room 7-020

Richmond VA 23298

Fax: 001-804-628-0325

Tel:  001-804-628-9618

Correspondence to: Norbert Voelkel nvoelkel@mcvh-vcu.edu

Conflict of interest statement: There are no conflicts of interest for any of the authors


AC adenylate cyclase

ACE angiotensin converting enzyme

ADAM-12 a disintegrin and metalloprotease 12

AM adrenomedullin

ANP atrial natriuretic peptide

ATII angiotensin II

AT1R angiotensin type 1 receptor

β-AR β-adrenergic receptor

β-ARK β-AR kinase

BNP brain natriuretic peptide

CaMKII Ca2+-calmodulin dependent protein kinase II

cAMP cyclic adenosine monophosphate

cGMP cyclic guanosine monophosphate

CREB cAMP-response element binding protein

CSC cardiac stem cell

CT-1 cardiotropin-1

Cu copper

DAG diacylglycerol

Dvl disheveled protein

E-C excitation-contraction

ECM extracellular matrix

EGF epidermal growth factor

eNOS endothelial NO synthase (also NOS3)

EPO erythropoietin

ET-1 endothelin-1

ETA,B ET-1 receptor A and B

Fz Frizzled

GEF guanine nucleotide exchange factor

GH growth hormone

G protein guanine nucleotide binding protein

GPCR G protein coupled receptor

GSK-3 glycogen synthase kinase-3

HAT histone acetyltransferase

HDAC histone deacetylase

HIF-1α hypoxia inducible factor 1α

HO-1 heme oxygenase 1

IGF-1 insulin-like growth hormone 1

IL interleukin

IP3 inositol-1,4,5-triphosphate

JAK Janus kinase

JNK c-Jun-N-terminal kinase

LIF Leukemia inhibitory factor

LIMP-2 lysosomal integral membrane protein 2

LRP LDL receptor–related protein

LV left ventricle

LTCC L-type Ca2+ channel

MAPK mitogen activated protein kinase

MCIP myocyte-enriched calcineurin-interacting protein

MCP-1 monocyte chemoattractant protein-1

MCT monocrotaline

2-ME methoxyestradiol

MHC myosin heavy chain

miRNA microRNA

MKK MAPK kinase

MKKK MKK kinase

MMP matrix metalloproteinase

MOMP mitochondrial outer membrane permeabilization

MR mineralocorticoid receptor

mTOR mammalian target of rapamycin

NCX Na/Ca2+ exchanger

NEP neutral endopeptidase

NFAT nuclear factor of activated T cells

NGF neuronal growth factor

NO nitric oxide

NPR-A, B, C natriuretic peptide receptors A, B and C

PAB pulmonary artery banding

PAH pulmonary arterial hypertension

PDE-5 phosphodiesterase type 5

PDGF platelet-derived growth factor

PG prostaglandin

pGC particulate guanylate cyclase

PGI2 prostacyclin

PI3K phosphatidylinositol-3 kinase

PIP2 phosphatidylinositol-4,5-biphosphate

PIP3 phosphatylinositol-3,4,5-triphosphate

PKA protein kinase A

PKC protein kinase C

PKG protein kinase G

PLC phospholipase C

PPARα peroxisome proliferator-activated receptor α

PTEN phosphatase and tensin homolog on chromosome 10

RAS renin-angiotensin system

RNS reactive nitrogen species

ROCK Rho kinase

ROS reactive oxygen species

RTK receptor tyrosine kinase

RV right ventricle

RyR Ryanodine receptor

SERCA sarcoplasmic Ca2+ ATPase

sGC soluble guanylate cyclase

Sir2α silent information regulator 2 α

SNO NO-modified cysteine thiols

SOD superoxide dismutase

SR sarcoplasmic reticulum

SRF serum response factor

STARS striated muscle activator of rho signaling

STAT-3 signal transducer and activator of transcription 3

TAC transverse aortic constriction

TAK1 TGF-β-activated kinase 1

Tcf/Lef T-cell factor/Lymphocyte enhancer factor

TGF-β1 transforming growth factor β1

TNF tumor necrosis factor

TRF-1,2 telomeric binding proteins 1 and 2

Trx thioredoxin

VEGF vascular endothelial growth factor

VHL von Hippel-Lindau protein

XO xanthine oxidase

1. Animal models for the study of pulmonary hypertension and right heart failure

The degree of RV adaptation and failure varies substantially in current PAH animal models (see table e1). Presently used animal models to study the vascular changes in pulmonary hypertension all have their limitations in the study of PAH associated right heart failure. Chronic hypoxia is associated with increased RV afterload due to hypoxic pulmonary vasoconstriction and pulmonary vascular smooth muscle cell hyperplasia 1-4. However, the impossibility to differentiate between the effects of pressure overload and the direct effects of hypoxia limits extrapolation from this model to right heart failure in PAH. The toxic effects of monocrotaline (MCT), a pyrrolizidine alkaloid that causes pulmonary vasculitis and subsequently vascular remodeling, are generally assumed to be pulmonary specific 2;5-12. However, MCT is also used to generate liver damage and hepatic veno-occlusive disease 13. It is possible that the pro-inflammatory and pro-coagulant responses elicited by MCT-induced pulmonary vaculitis have systemic effects and contribute to heart failure. In fact, Akhavein et al recently demonstrated that shortly after MCT administration (even before pulmonary hypertension develops), extensive inflammatory changes can be seen in both ventricles and that these changes are associated with depressed contractile function, especially in the LV 14. When MCT is combined with aortocaval shunting, the developing pulmonary vascular changes more closely resemble those of human severe PAH 15. Since right heart failure in this model of flow-associated PAH comes about by a combination of pressure and volume overload, this model may reflect failure in congenital heart disease, but not RV failure in most types of human PAH. More recently a model of severe angioproliferative pulmonary hypertension has been developed based on a single administration of the vascular endothelial growth factor (VEGF) receptor blocker SU5416 16-18. This drug induces pulmonary endothelial cell apoptosis and secondary vascular remodeling, but the specificity of SU5416 for the pulmonary endothelium has not been determined. Both SU5416 and MCT may affect the myocardial microcirculation directly.

Pulmonary artery banding (PAB) has no other direct effects than increasing afterload, but RV adaptation in this model is very dose and species dependent. Cat and dog PAB models have been used incidentally to study the RV response to acute and chronic increases in afterload 19-22. Although rabbit and rodent PAB models are useful to study acute increases in RV afterload, the high short-term mortality rates in some of these models (e.g. 50% after one week of PAB in rabbits) questions their suitability to study the development of right heart failure in PAH 23-31. The rodent RV subjected to PAB displays many changes that have been originally described in the pressure overloaded LV: fetal gene re-expression, β-adrenergic receptor (β-AR) dysregulation, altered expression of sarcoplasmic reticulum (SR) proteins, myocardial fibrosis and increased apoptosis 23-27;29-31. State-of-the-art research on LV pressure overload has moved forward to address the relative importance of these changes in the transition from compensated hypertrophy to heart failure, making use of transgenic knock-outs and constitutive activation of signaling pathways, with or without additional stressors like transverse aortic constriction (TAC)32-60 and agonist infusion (e.g. catecholamines and angiotensin II (ATII), see table e2) 34-37;39;49;51;58;61;62. Transgenic approaches have not been applied to specifically study the transition from RV hypertrophy to failure. Other important left heart failure models are based on coronary ligation (myocardial infarction) or ischemia/reperfusion 63-68 The relevance of these models to right heart failure associated with PAH is unknown, since it is undetermined whether ischemia plays a role in severe RV pressure overload.

2. Mechanisms of contractile dysfunction in heart failure

Myocyte excitation-contraction coupling

Myocyte excitation-contraction (E-C) coupling involves cytosolic Ca2+ entry through L-type Ca2+ channels (LTCCs); the resultant increase in intracellular Ca2+ triggers further Ca2+ release from the SR through the ryanodine receptor (RyR, see fig. e1)69. Intracellular Ca2+ binds to troponin C within the myofilaments, which initiates contraction. Subsequent relaxation depends on dissociation of Ca2+ from troponin C and Ca2+ reuptake by the SR through a Ca2+-ATPase (SERCA), interacting with phospholamban 70. Ca2+ is than removed trans-sarcolemmally through the Na/Ca2+ exchanger (NCX) in its forward mode. Unphosphorylated phospholamban inhibits SERCA, and by phosphorylating phospholamban protein kinase A (PKA) enhances SERCA-mediated Ca2+ re-entry into the SR during diastole 70. The efficiency of the trigger (the size of the inward Ca2+ current) needed to cause Ca2+ release from the SR (i.e. the E-C coupling gain, a determinant of contraction velocity) has been shown to be reduced in human heart failure. This can be the result of either functional defects in LTCCs, an increased distance between LTCCs and RyRs, decreased SR Ca2+ stores or functional abnormalities of the RyR 71. It has also been shown that heart failure is associated with a sustained increase in intracellular Ca2+ concentration, interfering with normal E-C coupling and diastolic relaxation (in addition to inducing maladaptive hypertrophic pathways). Proposed mechanisms are hyperphosphorylation of the RyRs by PKA (while PKA initially improves Ca2+ handling, long-term PKA signaling makes RyRs leaky), decreased SERCA expression/activity and enhanced SERCA inhibition through phospholamban 71-73. Heart failure is also associated with an increase in the intracellular Na+ concentration, putting the NCX in its reverse mode and contributing to a further increase in intracellular Ca2+ concentration 71. The described abnormalities are well established in left heart failure and some of these (decreased expression of mRNAs of SERCA, phospholamban and RyR) have also been shown after PAB in rabbits and rats, without clarification of their consequences for RV systolic function and protein expression 25;26.

Mitochondria, ATP and high energy phosphates

Mitochondria of cardiomyocytes in the failing left heart (there are no data on RV failure in this respect) have structural abnormalities and display reduced activities of electron transport-chain complexes, reduced ATPase synthase capacities and increased levels of uncoupling proteins that cause them to produce heat rather than ATP 74. In advanced heart failure, myocardial ATP levels decrease by 30-40%, but these levels are still well above those required for ATP consuming reactions. A more profound decrease is seen in levels of the high-energy phosphate metabolites creatine and phosphocreatin, contributing to contractile dysfunction when the heart is stressed, such as during the increased sympathetic drive of exercise 74. Concomitantly increased levels of free intracellular ADP further reduce the inotropic reserve. Since there are no known interventions that can directly address these abnormalities, improving myofibrillar efficiency of ATP utilization with calcium-sensitizing drugs is thus far the only possible intervention in this regard 75.

Myocardial substrate use

It has been postulated that modification of myocardial substrate use (from fatty acids to glucose) could be used as a strategy to increase the heart’s efficiency and lower the oxygen cost of energy generation 76. This is still a matter of debate, however, since there are inconsistencies in reports on glucose uptake and utilization in heart failure 74. Using [18F]fluorodeoxyglucose positron emission tomography, increased RV glucose utilization was shown in PAH, which was reversed by PGI2 treatment 77. End-stage heart failure, however, is associated with insulin resistance and decreased glucose uptake and utilization 74. The nuclear receptor peroxisome proliferator-activated receptor (PPAR)α plays an important role in the balance between lipid and glucose metabolism. PPARα regulates the expression of genes that encode proteins involved in the uptake and β-oxidation of free fatty acids and cellular cholesterol trafficking. PPARα is downregulated in human heart failure, but the consequences of this are controversial. On the one hand, a switch to glucose as a substrate yields more ATP per molecule of oxygen, which could be beneficial in a hypoxic heart 78. On the other hand, downregulation of PPARα is associated with a dysbalance between reactive oxygen species (ROS) and antioxidants via decreased superoxide dismutase (SOD) expression 79.

3. Causes and consequences of neurohormonal activation and autocrine/paracrine signaling

Reduced tissue perfusion due to a decreased cardiac output activates neurohormonal pathways that are first beneficial (maintenance of blood pressure and renal perfusion), but will eventually decrease cardiac function. Heart failure is associated with upregulation of the renin-angiotensin system (RAS, with ATII as the most important factor involved in cardiac remodeling), adrenergic overstimulation and increased expression of several counter regulating peptides (e.g. natriuretic peptides), all of which have been shown to influence cardiac myocytes, fibroblasts, immune cells and the extracellular matrix. Many of the neurohormones that reach the heart through the systemic circulation are also secreted locally by resident cardiac cells (myocytes, endothelial cells and fibroblasts) and, together with factors that are secreted locally only, affect cardiomyocyte growth, proliferation and survival.

Angiotensin II: primary example of a maladaptive hypertrophic signal

Activation of the RAS involves secretion of renin by juxtaglomerular cells in the kidney in response to reduced perfusion, subsequent renin-induced cleavage of hepatogenic angiotensinogen and production of angiotensin I, and finally conversion of angiotensin I by angiotensin coverting enzyme (ACE) to ATII. The plasma-localized RAS is important in the regulation of salt/water homeostasis and vasoconstriction, regulating blood pressure. ATII can also be produced locally in tissues under different forms of stress, not only by circulating renin and ACE, but also by other enzymes which cleave angiotensinogen and convert angiotensin I 80. Locally produced ATII is involved in tissue remodeling by promoting hyperplasia and hypertrophy of vascular smooth muscle cells, hypertrophic cardiac remodeling and myocardial fibrosis 81. Whereas most evidence concerning the role of ATII signaling in pressure overload related heart failure comes from studies on the LV, PAB in rabbits has been shown to cause RV hypertrophy and systolic dysfunction due to signaling defects downstream of ATII (in fact, the density of its receptor, AT1R, was increased) 28. Genetic variation in ACE expression has been implicated in the differences in survival between PAH patients 82.

Most effects of ATII on the heart are mediated by the AT1R, which is a G protein coupled receptor (GPCR). GPCRs are transmembrane receptors with seven domains linked to a guanine nucleotide binding protein (G protein) 83. GPCR binding with ligand (ATII, catecholamines, ET-1 and others) activates the G protein. Depending on the stimulating ligand and receptor, different downstream effectors are activated, such as phospholipases, adenylate cyclase (AC) and various kinases (see fig. e2 and fig. 3). This results in the release of the second messenger molecules such as inositol-1,4,5-triphosphate (IP3), diacylglycerol (DAG) and cyclic adenosine monophosphate (cAMP) 83. Stimulation of the AT1R is also associated with production of arachidonic acid, linking ATII to inflammatory pathways. Moreover, the AT1R participates in several G-protein independent pathways, including those involving receptor tyrosine kinases (RTKs, e.g. receptors for insulin, epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) ) and non-receptor tyrosine kinases (Src family kinases, Janus kinase (JAK) ), providing a link with signaling through transforming growth factor (TGF)-β1, mitogen activated protein kinases (MAPKs) and aldosterone. Many of ATII’s effects on the heart and vasculature are potentiated by interactions with TGF-β1 and aldosterone, although the underlying mechanisms are still ill defined. Other ATII mediated pathologic effects in the vasculature occur via activation of small GTP binding proteins, NAD(P)H oxidases and subsequent generation of ROS 81.

Phospholipase C, protein kinase C, calcineurin

Binding of ATII to the AT1R (and similarly, ET-1 or catecholamines to their GPCR) leads to phospholipase (PLC)-β activation. PLC-β releases IP3 and DAG from the plasma membrane; IP3 subsequently activates Ca2+ channels in the SR and the resulting Ca2+ release into the cytoplasm, in conjunction with upregulation of transient receptor potentials (TRCPs), leads to a sustained increase in the intracellular Ca2+ concentration which activates calmodulin and calcineurin phosphatase 49;72;84. DAG activates kinases of the PKC family (see below). PLC-β/PKC signaling-related hypertrophy is mainly eccentric and is associated with reduced contractility, adrenergic dysfunction and apoptosis 84.

In a thus far undetermined way, cardiac myocytes are able to distinguish between the Ca2+ pools involved in contraction and pools involved in transcription-dependent remodeling. It is assumed that Ca2+ compartmentalization and distinct patterns of Ca2+ concentration waveforms trigger specific signal transduction pathways that are otherwise insensitive to the moment-to-moment fluctuations in Ca2+ concentration that are associated with myocyte contraction 72. After activation by the increase in intracellular Ca2+ concentration, calmodulin activates calcium/calmodulin-dependent protein kinase, which promotes the expression of the transcription factor MEF2 (via nucleo-cytoplasmic transfer of repressing histone deacetylases (HDACs, see below) 85. MEF2, mediated by the action of the cytoskeletal protein STARS (striated muscle activator of rho signaling) upregulates the activity of the pro-hypertophic transcription factor SRF (serum response factor) 48. STARS expression is upregulated in human heart failure, and transgenic overexpression of STARS in mice enhances the development of maladaptive hypertrophy and heart failure after TAC 48;86. Overexpression of SRF without external stimuli is sufficient to cause eccentric hypertrophy and heart failure. In addition to its direct effects on the expression of transcription factors, calmodulin regulates Ca2+ handling and E-C coupling via LTCCs, RyR and SERCA, with a recent report demonstrating early cardiac hypertrophy in mice with impaired calmodulin regulation of RyR2 87.

Calcineurin activity is increased in human hearts with compensated hypertrophy, while constitutive activation of calcineurin in transgenic mouse hearts is sufficient to induce massive cardiac enlargement and eventually heart failure 88;89. Active calcineurin dephosphorylates the NFAT (nuclear factor of activated T cell) transcription factor, and dephosphorylated NFAT translocates into the nucleus (see fig. 3). In the nucleus NFAT activates transcription in cooperation with other transcription factors, including MEF2 and GATA-4 90. Dephosphorylation of NFAT by calcineurin is inhibited by the transcription factor glycogen synthase kinase-3 (GSK-3, see below) 91. Recently, a family of calcineurin inhibitory proteins termed MCIPs (myocyte-enriched calcineurin-interacting proteins) were identified that seem to function as endogenous modulators of calcineurin activation in the heart 92. Suppression of calcineurin signaling by either overexpression of MCIP or GSK-3 aborts the hypertrophic response to TAC without affecting LV systolic function 37;38. These findings may not be extrapolated to the pressure overloaded RV in PAH without caution. Normal RV afterload and RV wall thickness are only one fifth of that of the LV; the stress imposed by PAH on the RV (doubling or tripling of afterload in comparison with an approximate 50% increase in TAC models) might still require a considerable degree of hypertrophy. There is limited published data available on PKC and calcineurin signaling in the pressure overloaded RV. Braun et al showed that whereas PKC activity was enhanced after PAB in rats, there was no change in expression of calcineurin subunits 31. Moreover, ACE inhibition did not affect the degree of hypertrophy or PKC upregulation. In another report, PAB was shown to be associated with increased expression of MEF2 and GATA-4 in the RV 30.

MAPK cascades

Signaling cascades involving MAPKs are recognized as important determinants of the cardiac response to stress. In these cascades, MAPKs are phosphorylated and activated by upstream MAPK kinases (MKKs) which are, in turn, phosphorylated and activated by MKK kinases (MKKKs) 93. In the heart, extracellular signal-regulated kinase ERK1/2 is activated by pro-hypertrophic signals (partly mediated by GPCRs and small GTP binding proteins) 94, whereas the c-Jun-N-terminal kinases (JNKs) and p38 MAPKs are activated by cellular stress (hypoxia, stretch, oxidative injury) and are associated with cardiac myocyte apoptosis, inflammation and fibrosis (see fig. e2) 95-97. Overexpression of MAPK phosphatase 1, which inhibits ERK1/2, JNK and p38, prevents both agonist-induced hypertrophy in vitro and pressure overload-associated hypertrophy in vivo, thus demonstrating a significant role for these pathways in hypertrophic signaling 34. Stretched cardiac fibroblasts show integrin-dependent activation of ERK1/2 and JNK 98. How cell receptors or stress link to the activation of MAPK cascades is not well understood. It is largely undefined which transcription factors MAPKs phosphorylate and what genes are expressed or suppressed as a result of MAPK signaling in the heart, although NFAT and MEF2 have been implicated 92;99.

Small GTP binding proteins

This family consists of multiple members, regulating diverse cellular processes such as cell growth, division and survival, organization of the cytoskeleton, membrane trafficking, and cellular motility. Activation of various receptors (GPCRs, RTKs, receptor-independent tyrosine kinases) is associated with the activation of guanine nucleotide exchange factors (GEFs). GEFs mediate substitution of the GDP bound to small GTP binding proteins for GTP. The small GTP binding proteins subsequently acquire GTPase activity and hydrolyze GTP, using the energy to activate various other signaling processes. Five families of small GTP binding proteins have been described (ras, rho, ARFs, rab, ran), each consisting of several members 100. Ras signaling is coupled to multiple downstream effectors involved in the hypertrophic response, including phosphatidylinositol 3-Kinase (PI3K) and MAPKs 92;100. Moreover, activated ras promotes nuclear localization of NFAT, whereas a dominant-negative ras-mutant (N17ras) has been shown to abrogate phenylephrine induced increase in NFAT activity 101. In the heart, the rho family of small GTP binding proteins (RhoA, Rac, and Cdc42 subfamilies) regulates the cytoskeletal organization of non-muscle cells as well as cardiomyocytes 102;103. Rho signaling is in many different ways involved in the hypertrophic cardiac response and there are excellent reviews with detailed information 104. To give a few examples, activation of Rho kinase (ROCK) by RhoA is involved in adrenergic and ET-1 induced upregulation of MEF2, SRF and GATA-4 92;105-108. ROCK activation may contribute to hypertrophic sarcomere organization 92. Two recent reports showed that ROCK and caspase-3 activation are tightly intertwined in causing cardiomyocyte apoptosis, with ROCK acting both up- and downstream of caspase-3 46;109. A recent study that used targeted deletion of the ROCK-1 isotype in the mouse heart contrasted with many previous studies that relied on pharmacological inhibitors of ROCK. ROCK-1 knock-out in a TAC mouse model did not prevent the development of hypertrophy but rather attenuated the development of cardiac fibrosis 44. There is strong evidence that ROCK activation also plays a role in the initiation and/or propagation of pulmonary vasoconstriction and vascular remodeling in PAH 3;4;7;110-112.


As an important mediator of the RAS, aldosterone is best known for its effects on extracellular fluid and potassium homeostasis. The neurohormone is increasingly recognized, however, for its role in the development of heart failure associated with pressure overload and myocardial infarction. Aldosterone has been shown to promote endothelial dysfunction, induce vascular inflammation and myocardial ischemia, increase collagen synthesis in cardiac fibroblasts, increase oxidative stress via NADPH oxidase, and stimulate cardiomyocyte apoptosis 113. Likewise, mineralocorticoid receptor (MR) blockade (despite concomitant use of ACE inhibitors or AT1R blockers) is associated with increased nitric oxide (NO) bioavailability, reduced cardiac fibrosis and LV mass, improved LV ejection fraction and diastolic function, and reduced mortality 113-115. The underlying signaling mechanisms are not yet fully elucidated. Aldosterone is produced by the adrenals in response to ATII; whether local production in the heart occurs in humans is still a matter of debate 113. MR binding with aldosterone is associated with ERK1/2 activation, which seems to be mediated by MR induced transactivation of the EGF receptor 116. The AT1R is capable of a similar transactivation of the EGF receptor (as are other GPCRs), which effect is potentiated by aldosterone 117.

Transforming growth factor Beta 1 (TGF-β1)

TGF-β1 is upregulated in the heart in response to chronic pressure overload, predominantly mediated by ATII 118;119. In fact, many of ATII’s effects on ventricular mass, cardiomyocyte size, and contractility are mediated by TGF-β1 120. Most cardiac TGF-β1 mRNA can be found in cardiac fibroblasts, which is reflected in the association of TGF-β1 activation with increased myocardial fibrosis and the progression to systolic and diastolic heart failure during chronic pressure overload 118. After excretion, TGF-β1 binds to a dimerized complex of two serine-threonine kinase receptors (TGF-β1 receptors 1 and 2) and subsequent signaling is realized via two different pathways: phosphorylation of Smad proteins and activation of TGF-β-activated kinase (TAK)1 121. The TGF-β1–Smad pathway appears to be involved in the activation of collagen-gene promoter sites, primarily enhancing DNA translation of collagen type I 121. TAK1 is a MKKK family member and links TGF-β1 to the MAPKs p38 and JNK122. TAK1 is expressed at low levels in the normal adult heart, but transgenic constitutive activation of TAK1 in the myocardium without overload is sufficient to reproduce the full histological picture of heart failure, including hypertrophied cardiomyocytes, fetal gene re-expression, cardiomyocyte drop-out (with high rates of apoptosis) and interstitial fibrosis 122. TAK1 can also be activated by other cytokines than TGF-β1, including tumor necrosis factor (TNF)α and interleukin (IL)-1 123.


The effect of interaction of adrenergic agonists (adrenalin and noradrenalin) with their GPCR is dependent on the adrenergic receptor subtype. Upon receptor binding, AC is activated and cAMP is produced (β1 and β2 receptors), PI3K is activated (β2 receptors), MAPKs are activated (β2 and α1 receptors) and IP3 and DAG are produced (α1 receptor) 83. There are a number of ways in which adrenergic stimulation contributes to hypertrophy and, after prolonged simulation, to cardiac dysfunction. Production of cAMP leads to the activation of PKA, which initially improves Ca2+ handling (phosphorylation of phospholamban increases Ca2+ reuptake by the SR through SERCA). Long-term PKA signaling, however, leads to hyperphosphorylation of RyRs and a sustained increase in intracellular Ca2+ and maladaptive hypertrophy. PKA phosphorylates the transcription factor CREB (cAMP-response element binding protein), which has also been suggested to contribute to the development of ventricular dilatation and failure 124. Phosphorylated CREB interacts with CREB binding protein and p300, a histone acetyltransferase (HAT), to induce relaxation of the chromatin structure and promote gene activation (see below) 125. It was recently shown that cAMP may also affect cardiomyocyte calcium handling through PKA independent mechanisms, such as activation of epac (exchange protein activated by cAMP) and, subsequently, Ca2+-calmodulin dependent protein kinase II (CaMKII) 126.

Chronically increased levels of circulating catecholamines, such as in human heart failure, are associated with a decrease in β-AR density 83 and a PI3K (subtype p110γ) mediated increase in the expression of β-AR kinase (β-ARK), which is a kinase that phosphorylates the cytoplasmic tail of the receptor and decreases its sensitivity 43. Expression of a dominant negative β-ARK mutant prevents pathological hypertrophy and heart failure in mice chronically infused with isoproterenol 61 and in rabbits subjected to PAB 29. This seems paradoxal in the light of the clinical finding that blocking the β-AR in patients with heart failure improves survival and prevents pathological remodeling 127. The consequences of PLC-β and MAPKs activation by chronic adrenergic stimulation were discussed above. Finally, pressure overload in the MCT model is associated with RV specific anatomical sympathetic hyperinnervation, which is due to upregulation of cardiomyocyte-derived neuronal growth factor (NGF) 12. The newly developed neurons have embryonic characteristics and are functionally inferior to mature neurons. Upregulation of NGF in the MCT model likely results from ET-1 signaling 128.


In addition to its vasoactive effects, ET-1 regulates a variety of biological processes in non-vascular tissues. ET-1 augments cardiomyocyte contractility and plays a role in the development of pressure overload induced cardiac hypertrophy 129;130. ET-1 can induce mast cell degranulation and subsequent activation of matrix metalloproteinases (MMPs) 131. In PAH and heart failure, ET-1 serum concentrations are elevated, due to increased production by endothelial cells and cardiomyocytes in response to various stimuli (e.g. vasoactive hormones, growth factors, shear stress, hypoxia, ROS) 130;132;133. ET-1 production is inhibited by cGMP, either produced in response to NO or natriuretic peptides 130.

ET-1 exerts its effects through two GPCR-receptor subtypes, ETA and ETB; the former predominates in the rat myocardium 134. Heart failure in rats leads to an increased ETA receptor density 133. Upon activation of the receptor, PLC-β is activated (with downstream activation of the calcineurin/NFAT pathway), IP3 and DAG are released from the cell membrane (with subsequent activation of PKC) and the small GTP binding proteins RhoA and Ras are activated 129;135. Mediated by calcineurin-NFAT signaling, ET-1 transactivates the pro-survival transcription factor bcl-2 in cardiomyocytes, protecting the heart from apoptosis 136. In addition, ETA receptor activation (as AT1R and MR activation) is associated with activation of PI3K subtype p110γ and transactivation of the EGF receptor. This transactivation is mediated by a disintegrin and metalloprotease 12 (ADAM-12) 36. Blocking ADAM-12 signaling prevents the development of hypertrophy in mice subjected to TAC, improving systolic function at the same time 36. Finally, ET-1 signaling leads to activation of the MAPK cascade involving ERK1/2 129. In summary, ET-1 is associated with a myriad of signaling pathways, but the relative importance and interdependence of these pathways in ET-1’s pro-hypertrophic action are not yet fully clear. In patients with PAH associated heart failure, the direct effects of ET-1 signaling on the heart are mixed with its stimulation of pulmonary vasoconstriction and vascular remodeling.


For more than a decade, prostaglandins (PGs) have been the cornerstone of PAH treatment 137. However, little is still known about their mode of action in the heart. The effects of PGI2 and 4 related, naturally occurring, cyclooxygenase metabolites (prostanoids) PGD2, PGE1, PGE2, and PGF2α are cell type specific and depend on the activation of a specific PG receptor subtype (GPCRs coupled to inhibiting or stimulating G proteins) 138. Through either inhibition or stimulation of AC, PGs affect platelet aggregation, vascular tone and growth and proliferation of endothelial cells, smooth muscle cells and fibroblasts. It is generally assumed that the therapeutic effect of PGs in PAH comes about by induction of pulmonary vasodilatation and inhibition of vascular remodeling 139. It has to be recognized, however, that PGs have important direct effects on the heart. In patients with severe heart failure, i.v. administration of epoprostenol (synthetic PGI2) results in an immediate and substantial increase in cardiac output and a reduction in cardiac filling pressures 140. Whereas this could follow reflex tachycardia and pulmonary vasodilation with improved right ventriculoarterial coupling 21;140, molecular effects on cardiac cells and signaling pathways are also possible. In a model of flow-associated PAH, the synthetic PGI2 analog iloprost improved RV contractility and capillary-to-myocyte ratio (but not density), independently from a change in RV afterload 15. PGI2 has been reported to suppress pressure overload–induced cardiac hypertrophy via the inhibition of both cardiomyocyte hypertrophy and cardiac fibrosis. Both effects are considered to originate from the action on non-cardiomyocytes, but the underlying mechanisms are undetermined 40.

Atrial and brain natriuretic peptides (ANP and BNP)

The expression of natriuretic peptides is increased both in PAH and in heart failure; the primary stimulus is increased ventricular stretch, but this response is modulated by many other factors, such as ATII, ET-1, circulating catecholamines, α1 and β2 stimulation and hypoxia 141. Integrins are important for linking stress to increased atrial natriuretic peptide (ANP) gene expression 142. ANP and brain natriuretic peptide (BNP) bind to the natriuretic peptide receptors NPR-A, NPR-B and NPR-C. Upon binding of the natriuretic peptides to NPR-A, the particulate guanylate cyclase (pGC) that is linked to the receptor produces cyclic guanosine monophosphate (cGMP), which in turn activates protein kinase G (PKG, see fig. e3 and below) 143. Natriuretic peptides are primarily involved in vasodilation and fluid balance. By inhibiting RAS and the sympathetic system 141, they indirectly suppress cardiac hypertrophy and fetal gene expression. Moreover, there is evidence that ANP/BNP induced cGMP signaling directly attenuates cardiomyocyte hypertrophy in response to TAC 39, inhibits cardiomyocyte apoptosis via nuclear accumulation of zyxin and Akt1144 and prevents myocardial fibrosis through inhibition of cardiac fibroblasts 33;145. Binding of ANP and BNP to NPR-C is followed by endocytosis and lysomal degradation 146, but more important for peptide clearance is inactivation by neutral endopeptidase (NEP) 147.

Nitric oxide, cyclic guanosine monophosphate and protein kinase G

cGMP is a ubiquitous intracellular secondary messenger in the cardiovascular system. Whereas the natriuretic peptides activate pGC, NO induces the formation of cGMP through activation of soluble guanylate cyclase (sGC, see fig. e3) 143. cGMP is degraded by the action of PDEs; some PDE subtypes hydrolyze cGMP only (PDE5, PDE6, PDE9), whereas others degrade cAMP (PDE3, PDE4, PDE7, PDE8) or both cGMP and cAMP (PDE1, PDE2).148 Whereas a role for cGMP in cardiac contractility, lusitropy and ion channel responsivity is well established, the extent to which natriuretic peptides versus NO mediate these effects is less clear 149. Part of the divergent actions of natriuretic peptides and NO seem to result from the fact that they are involved in the generation of cGMP in different subcelllular locations: at the plasma membrane in case of the former and in the cytosol in case of the latter. This compartimentalization is enhanced by the fact that PDE5 controls the soluble but not the particulate cGMP pool 150.

The effect of cGMP on myocardial contractility depends on its interaction with the PDEs and cAMP. Theoretically, cGMP can decrease contractility by decreasing cAMP concentrations through inhibition of AC and induction of PDE2 (see fig. e3) 151;152. In addition, phosphorylation of troponin I by a cGMP-dependent protein kinase can decrease the sensitivity of the contractile apparatus to Ca2+ and accelerate myocardial relaxation 151. On the other hand, it was recently shown in PAH patients and MCT induced RV hypertrophy that cGMP can in fact increase contractility by increasing cAMP concentrations 153. The authors explained this apparent paradox by cGMP related inhibition of the cGMP sensitive PDE3. They also showed that compared to the normal RV, RV hypertrophy (both human and experimentally induced) is associated with a considerable decrease in PKG activity. At the same time, PDE5 was only expressed in the hypertrophic RV and not in the normal RV 153.

cGMP/PKG signaling protects the heart from apoptosis 154;155 and blunts the hypertrophic response to pressure overload and isoproterenol, which is associated with inhibition of the calcineurin/NFAT pathway, PI3K/Akt1 signaling and ERK1/2 cascades 39;42;156. PKG interacts in a complex way with RhoA signaling. Whereas PKG can phosphorylate RhoA, forcing its cytosolic location and thereby preventing downstream activation (a potentially additional way of counteracting pro-hypertrophic signals), a basal level of PKG is necessary for transcription and protein stabilization of this small GTP binding protein 157.

It should be noted that NO plays many roles in the cardiovascular system, some of which are independent from its induction of sGC. Whereas some issues concerning nitrosative stress will be dealt with below, we refer to other reviews for a thorough discussion on how NO affects excitation-contraction coupling and myocardial relaxation, heart rate, myocardial energetics and myocardial substrate utilization, and on how NO can exert both beneficial and deleterious effects in pathological situations (ischemia-reperfusion, left ventricular hypertrophy, heart failure, transplant vasculopathy and rejection, myocarditis) 158.


Adrenomedullin (AM) is another peptide that is upregulated in heart failure and could have important cardio-protective effects 159. Circulating levels are also elevated in PAH and correlate strongly with right atrial pressure 160. AM gene expression is promoted by various stimuli, including inflammation, hypoxia, oxidative stress, mechanical stress and activation of RAS and the sympathetic nervous system 161. Its signaling compares to natriuretic peptides in many aspects: AM induces systemic and pulmonary vasodilatation and natriuresis, AM inhibits ET-1 signaling, AM inhibits RAS and the sympathetic nervous systems, AM inhibits fibroblast proliferation and AM is cleared by NEP. Surprisingly, binding of AM to its receptor results in activation of AC instead of pGC, which could be responsible for a possible positive inotropic effect of AM (although this is still controversial) 161. As discussed above, chronic stimulation of AC may be detrimental for cardiac function. The fact that AM signaling mimics natriuretic peptide signaling could be due to its stimulating effect on endothelial NO synthase expression. Finally, AM stimulates Akt1 mediated angiogenesis (see below for details on the complex relation between Akt1 and angiogenesis) and inhibits endothelial and cardiomyocyte apoptosis 161;162.


Apelin is a recently discovered neurohormone that is upregulated in heart failure and ischemia (via hypoxia inducible factor, HIF-1α) and seems to have natriuretic, vasodilating, anti-proliferative and positive inotropic effects 163. High levels of mRNA of both apelin and its GPCR APJ are found in cardiac myocytes, vascular smooth muscle cells and endothelial cells. It has been proposed as a new therapeutic target, but more research is warranted on its exact mode of action 163.

Growth hormones and the PI3K/Akt1 pathway

Growth hormone (GH) and insulin-like growth hormone (IGF-1, secreted by the liver in response to GH) play a role in cardiac development and the maintenance of its structure and function. There is a high incidence of concentric cardiomyopathy in acromegalic patients 164 and cardiac function improves when patients with GH deficiency are treated with GH 165. IGF-I directly causes cardiomyocyte hypertrophy in rats, 166 is involved in myofilament calcium sensitization167 and inhibits apoptosis 168.

An important signaling pathway of IGF-1 is the PI3K/Akt1 pathway (see fig. 3). The same pathway is also used by insulin, cardiotropin-1 (CT-1) and PDGF. Binding of these ligands to their membrane RTK activates PI3K (subtype p110α). PI3K phosphorylates the membrane phospholipid phosphatidylinositol-4,5-biphosphate (PIP2), which leads to the formation of phosphatylinositol-3,4,5-triphosphate (PIP3) and recruitment of the protein kinase Akt1 (also known as PKB) to the cell membrane together with its activator PDK1. After activation of Akt1, signaling events are induced that are associated with normal myocardial growth, physiological hypertrophy and prevention of cellular senescence 84;169. The PI3K/Akt1 pathway is inhibited by PTEN (phosphatase and tensin homolog on chromosome 10), which is a tumor-suppressor phosphatase that dephosphorylates PIP3 and therefore prevents Akt1 activation 170.

After activation of Akt1 the mammalian target of rapamycin (mTOR) is activated and GSK-3 is inhibited. mTOR is a central signaling molecule for hypertrophy-associated protein synthesis. mTOR can be up-regulated by a second -Akt1 independent- way through the activation of the ERK1/2 pathway by GPCRs. GSK-3 is a negative regulator of both normal and pathologic stress-induced growth 37;171. Induction of the PI3K/Akt1 pathway releases the cellular protein synthesis machinery from its tonic inhibition by GSK-3. Examples of hypertrophic growth regulating transcription factors that are normally inhibited by GSK-3 are c-Myc, GATA-4 and NFAT (see above for the link with calcineurin signaling) 84. Transgenic mice that express a constitutively active form of GSK-3 under control of a cardiac-specific promoter are physiologically normal under nonstressed conditions, but have a diminished hypertrophic response to chronic β-adrenergic stimulation and pressure overload. Remarkably, systolic function in these circumstances was unaffected despite the absence of hypertrophy 37. It should be noted that GATA-4 provides a substrate for the anti-apoptotic aspects of Akt1 signaling, since this transcription factor upregulates the survival factor Bcl3 172. There are, however, many other ways in which Akt1 acts as a pro-survival factor (activation of Bad, IKKβ, Foxo3a and procaspase-9) 173.

Binding of ATII, catecholamines and ET-1 to their GPCR is associated with a similar pathway, involving Akt1 and another PI3K subtype (p110γ). While activation of Akt1 through p110α is considered a beneficial response, activation through p110γ is associated with maladaptive hypertrophy. Since this type of Akt1 activation is associated with the same induction of mTOR and inhibition of GSK-3, it is likely that other detrimental effects of p110γ signaling such as the activation of β-ARK and a decreased capillary density are responsible 43;84;174. Not only the trigger, but also the duration of Akt1 stimulation seems to determine whether adaptive or maladaptive hypertrophy (with reduced capillarization) follows 174.

p53 and Sir2α

LV and RV pressure overload are associated with accumulation of the tumor suppressor gene p53 and subsequent suppression of HIF-1α and angiogenic growth factors 27;50. p53 induces apoptosis of cells with DNA damage via activation of Bax and via direct, transcription independent, induction of the mitochondrial death pathway 175;176. p53 dependent apoptosis is suppressed by silent information regulator 2 (Sir2)α, which also functions as a HDAC 177. Sir2α expression is increased in heart failure and has been shown to inhibit apoptosis in cultured cardiomyocytes, thereby providing some endogenous counterbalancing effect 178. The effect of p53 upregulation on myocardial capillary density in hypertrophic hearts will be discussed below.

Platelet derived growth factor

PDGF has been implicated in the pathobiology of pulmonary vascular remodeling in PAH 9. The same molecule has been attributed with cardio-protective effects in models of myocardial infarction 64;65. The proposed explanations are enhanced cardiomyocyte survival in the early period after acute infarction, anti-inflammatory effects and potential activation of progenitor cells (either resident or bone-marrow derived), which may differentiate into cardiomyocytes and coronary vessels 179-181. Active PDGF is built up by polypeptides (A and B chain) that form homo- or heterodimers and stimulate α and β cell surface receptors 182. PDGF receptors belong to a family of transmembrane RTKs. When the RTK binds with PDGF, it is autophosphorylated and subsequently activates different signaling pathways, e.g. PI3K, MAPK and signal transducer and activator of transcription 3 (STAT-3) 182.

gp130 signaling cytokines

Leukemia inhibitory factor (LIF) and CT-1 are cytokines that induce hypertrophic growth and suppress apoptosis via gp130 receptors 32;183. Whereas stimulation of GPCRs generally induces hypertrophy through parallel assembly of additional sarcomeres, stimulation of gp130 receptors results in assembly of sarcomeres in series. The former results in an increase in myocyte width, the latter in an increase in myocyte length 184. It remains to be determined whether this implies that gp130 signaling cytokines are predominantly involved in remodeling after volume overload (which is known to be associated with serial sarcomere assembly, in contrast to pressure overload), or whether the activation of these cytokines signifies maladaptive remodeling, i.e. pathologic dilatation 32. LIF and CT-1 are also involved in cardiac remodeling after ischemia and have been shown to stimulate angiogenesis, fibroblast migration and collagen synthesis in this context 185. It is unclear whether the net result of gp130 signaling in the overloaded human heart is beneficial or detrimental. The anti-apoptotic effect of gp130 signaling involve both activation of ERK1/2 and activation of Akt1 186;187.

Upon stimulation of the gp130 receptor, JAKs are phosphorylated; this is associated with the activation of multiple intracellular signaling pathways (PI3K, MAPKs, STAT-3, and Src) 185. STAT-3 is a transcription factor that directs a wide variety of biologic processes, such as cell survival and apoptosis, inflammation, angiogenesis, and cardiac hypertrophy 188. In the heart, its activation primarily follows that of phosphorylation at tyrosine 705 by JAK-1, which occurs upon binding of ligand to gp130 receptors. In fact, the JAK-STAT-3 signaling pathway has been shown to mediate most hypertrophic and cyto-protective effects of gp130 activation in cardiomyocytes subjected to different kinds of stress (doxorubicin, ischemia/reperfusion) 189-191. Phosphorylation of STAT-3 at serine 727 by MAPKs and the PDGF receptor could provide an alternative pathway for activation 192. STAT-3 affects capillary density and the composition of the extracellular matrix through maintaining a balance between anti-angiogenic (connective tissue growth factor, thrombospondin-1, tissue inhibitor of metalloproteinase 1) and pro-angiogenic factors (VEGF) 188.

The Wnt pathway and β-catenin

A recently discovered pathway associated with cardiac hypertrophy is the Wnt/Frizzled (Fz) pathway (see fig. e4). The putative sequence of signaling events is as follows: Wnt ligands (a large family of glycoproteins, secreted in a paracrine fashion) bind to a membrane-bound complex consisting of a member of the Fz receptor family and a LDL receptor–related protein (LRP); activation of a member of the disheveled (Dvl) protein family follows; GSK-3 is subsequently inhibited; inhibition of GSK-3 as a result of Wnt signaling releases pro-hypertrophic transcription factors (e.g. GATA-4 and NFAT) and additionally contributes to hypertrophy by decreasing phosphorylation of β-catenin and allowing it to accumulate in the cytoplasm 193. It was recently questioned whether β-catenin accumulation is indeed necessary for the hypertrophic response. Rather, it was suggested that adapative cardiac remodeling after GPCR stimulation requires β-catenin downregulation 62. β-catenin is a master switch involved in a myriad of cell functions and is co-activated by members of the T-cell factor/Lymphocyte enhancer factor (Tcf/Lef) family of transcription factors. β-catenin is not only of importance to the cardiac hypertrophic response to pressure overload (together with Lef-1), but also to cell proliferation (e.g. cardiac progenitor cells) and survival under conditions of oxidative stress (by inducing cell cycle arrest and quiescence) 47;194;195.

Interruption of Wnt signaling in mice lacking the Dvl-1 gene attenuated the onset of TAC-induced cardiac hypertrophy 56. In these mice, the amount of β-catenin protein was reduced and natriuretic pepide upregulation was prevented. Unfortunately, no LV functional data were provided. It was suggested that Wnt inhibited GSK-3 directly and indirectly via activation of Akt1, the latter being of minor importance after 7 days of TAC (Akt1 presumably being upregulated in the first days of TAC only) 56. Similarly, cardiac specific disruption of β-catenin/Lef-1 signaling prevented TAC-induced myocardial hypertrophy 47. β-catenin is a key component of adherens junctions, which are structures that hold epithelial cells together, as well as cardiomyocytes in the intercalated disc 57;194. The latter fact could be essential for β-catenin’s role in myocardial hypertrophy, since it has been shown that interruption of the binding of β-catenin to cadherin in the intercalated disc (by transgenic loss of lysosomal integral membrane protein 2 (LIMP-2) expression), prevents TAC-induced hypertrophy and leads to heart failure 57. In contrast with these findings, another study showed that ATII-induced cardiac hypertrophy was prevented by transgenic cardiac specific stabilization of β-catenin, which was accompanied by a decreased systolic function and unrelated to apoptosis 62. In this study, cardiac specific depletion of β-catenin resulted in mild cardiac hypertrophy under baseline conditions and enhanced the hypertrophic response to ATII 62. These divergent responses may have resulted from differences in stimuli (TAC vs ATII), differences in transgenic approaches and different intervals between genetic targeting of the Wnt pathway and subsequent induction of hypertrophy. Cancer research has shown that HIF-1α and β-catenin interact in the cellular response to hypoxia (see below)196.

The female factor

One of the intriguing aspects of PAH epidemiology is the gender difference in prevalence. Over the human life span, there are two female incidence peaks: one in early adulthood and one after the menopause 197. While the first peak could be related to the use of anorexigens, a known risk factor for PAH, the second peak is also present in connective tissue diseases and could be due to female sex hormone deficiency. Ovariectomized rats exposed to chronic hypoxia or MCT develop more severe pulmonary hypertension than animals with intact ovaries 1;6. Obviously, by limiting the progression of pulmonary vascular remodeling in these models, estrogens delayed right heart failure. However, there are also a number of direct effects of estrogens on the heart that could be cardio-protective. Most cardiovascular research has focused on the potential of 2-methoxyestradiol (2-ME) as a possible drug. Among possible protective effects of 2-ME are inhibition of ET-1 synthesis, reduction of mast cell related MMP activation and stimulation of PGI2 synthesis 198-200. In contrast, some ME-2 related mechanisms that have been demonstrated in cancer tissue could contribute to the development of right heart failure: post-transcriptional inhibition of HIF-1α, generation of ROS and activation of pro-apoptotic pathways 201;202. 2-ME activates different MAPK pathways (ERK1/2, p38 and JNK) in the lung 203, if the same would hold true in the heart the end result would be hard to predict. Many effects of 2-ME are independent from estrogen receptor binding, which may explain why other estrogens exert different effects. High dose 17β estradiol has been recently shown to prevent the transition from hypertrophy to failure in a genetic model for spontaneous heart failure, which was associated with antioxidant mechanisms (inhibition of NADPH oxidase and upregulation of thioredoxin, Trx) and reduced apoptosis (inhibition of apoptosis signal-regulating kinase 1(ASK-1) and its downstream MAPKs p38 and JNK) 204.

Control of gene expression by histone acetylation/deacetylation

A central mechanism for gene regulation in eukaryotes is histone-dependent packaging of genomic DNA. When there is no transcription, DNA is wrapped around histone octameres in nucleosomes, which are the basic units of chromatin. The highly compact structure that is formed by interacting nucleosomes limits access of transcriptional enzymes to genomic DNA, thereby repressing gene expression 205. Acetylation of histones by HATs (e.g. p300, when co-activated by CREB) relaxes the nucleosomal structures, thereby facilitating gene expression. The opposite effect is established by class II HDACs, which repress transcription and constitutively inhibit hypertrophic pathways 125. One strategy to override HDAC activity is by exporting it out of the nucleus (nucleocytoplasmic shuttling). The latter mechanism has been shown to be involved in the regulation of the activity of the MEF2 transcription factor in cardiac hypertrophy 85;206. Nucleocytoplasmic transfer of HDACs can be established by PKC signaling (with activation of PKD as an intermediate step), and this could be one of the ways that GPCR agonists use to induce transcription of hypertrophic factors 207. Surprisingly, HDAC inhibitors do not increase hypertrophy but strongly suppress agonist-dependent cardiac hypertrophy and increase α-MHC levels 208. Recent findings may explain this paradox, since it was demonstrated that class I HDACs (e.g. Hdac2) constitutively repress anti-hypertrophic pathways such as GSK-3 51.

Translational repression by microRNAs (miRNAs)

miRNAs are small RNA molecules that negatively modulate gene expression through base paring to mRNAs, thereby inducing their cleavage and/or translational repression. miRNAs are involved in a variety of biological processes, including apoptosis, cell proliferation, tumor suppression and stress responses 209. In human heart failure, there seems to be reactivation of a fetal microRNA program that may contribute to alterations of gene expression 210. miRNA-133 has been recently shown to control cardiac hypertrophy in mice 52. Exercise, TAC and selective cardiac overexpression of Akt1 were associated with reduced expression of miRNA-133 and suppression of miRNA-133 by decoy sequences induced hypertrophy in the absence of a stimulus. Some pro-hypertrophic target proteins normally inhibited by miRNA-133 were identified: RhoA, Cdc42 and Nelf-A/WHSC2. Moreover, in dilated atria of humans with mitral stenosis a 50% reduction in miRNA-133 expression was observed 52. In another study, miRNA-208 has been shown to mediate myocardial fibrosis and the switch from αMHC to βMHC in response to TAC 53.

4. Reactive oxygen species and reactive nitrogen species in heart failuire

Excessive production of ROS in heart failure can result from upregulation of xanthine oxidase (XO), NAD(P)H oxidases, cytochrome P450 and auto-oxidation of catecholamines 212;226;227. Constitutively expressed endothelial NO synthase (eNOS or NOS3) and hemoglobin are the principal sources of reactive nitrogen species (RNS) in the heart, including NO and SNOs (NO-modified cysteine thiols in amino acids, peptides, and proteins). Sustained desaturation of hemoglobin contributes to a NO/redox disequilibrium 149. The resulting decreased levels of S-nitrosylated hemoglobin (SNO-Hb) impair red blood cell induced vasodilatation and contribute to reduced tissue perfusion in heart failure 228, with subsequent RAS activation. Uncoupling of NOS3 further contributes to ROS generation 229. NOS3 is normally present as a homodimer, but chronic pressure overload is associated with uncoupling of NOS3 and monomeric NOS3 generates ROS rather than NO 41. The cellular signal transduction pathways of ROS and RNS are tightly intertwined and complex, and beyond the scope of this review. Heart failure is not only associated with excessive ROS production, but also with a failing defense against ROS through downregulation of PPARα and subsequent decreased superoxide dismutase expression 79.

ROS reduce myocyte contractility through suppression of enzymes involved in excitation-contraction coupling (LTCCs 230 and SERCA 231, see online supplement). Polynitrosylation of the Ryanodine receptor (RyR; see online supplement for a comparable mechanism involving hyperphosphorylation of the RyR by protein kinase A, PKA) can further contribute to contractile dysfunction 232. Many signaling molecules (e.g. ATII, TGFβ1, PDGF, TNFα and ET-1) use ROS formation to induce hypertrophic pathways (involving MAPKs, PKC, calcineurin, Akt1, Src), while ROS formation is accompanied by “side-effects” of inflammation, cell damage and enzyme inactivation 212. ATII induces ROS via NAD(P)H oxidases, an effect that seems to be mediated by rho activation since it can be blocked by HMG-CoA reductase inhibitors (statins) 35. ROS have been implicated in cardiac remodeling after ischemia through activation of matrix metalloproteinases (MMPs) 233. In the overloaded heart, ROS can induce both adaptive hypertrophy and apoptosis; the level of ROS produced seems to determine the direction of the response, since relatively low levels are associated with ERK1/2 activation related protein synthesis, whereas higher levels activate pro-apoptotic pathways (via p38 and JNK, see online supplement for details on MAPK signaling) 234. It is unclear whether S-nitrosylation exerts mainly anti-apoptotic effects (e.g. by S-nitrosylating and therefore inhibiting caspases 3 and 9, JNK and ASK-1; see online supplement) or pro-apoptotic effects (e.g. by S-nitrosylating and therefore inhibiting NF-κB) 149.

5. Heart failure and Immune Cells

In conditions of ischemia/reperfusion, macrophages recruit neutrophils through the secretion of IL-6, and neutrophils contribute to tissue injury 235. Various cell types exert a direct or indirect influence on the composition of the extracellular matrix. Macrophages, present in greater numbers in failing than in normal human hearts 236, are recruited by monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule-1, and contribute to remodeling through secretion of TGF-β 237. Mast cell density is increased in pressure overloaded ventricles and these cells can activate fibroblasts, MMPs and proteases 238. T-helper (CD4+) lymphocytes interact with cardiac fibroblasts and are essential components in the cardiac remodeling process 239;240. Finally, B-cells may contribute to the development of heart failure by the secretion of auto-antibodies against mitochondrial proteins, contractile proteins, cardiac β1-receptors and muscarinergic receptors; such antibodies have been demonstrated in dilated cardiomyopathy 241-246. It is unclear whether these antibodies play a role in the initiation or propagation of the disorder, whether they are formed in response to tissue injury, and whether they and the cytokines discussed above could play a role in right heart failure.

6. Cardiac hypoxia contributing to the transition from compensated hypertrophy to dilatation and failure

Hypoxia-inducible factor 1α

Expression of HIF-1α in cardiomyocytes is required to maintain normal myocardial metabolism, vascularity, calcium handling and contractile function 211. Myocardial HIF-1α levels are upregulated in patients with ischemic cardiomyopathy and although most effects are obviously beneficial (induction of angiogenesis, facilitation of glucose uptake and metabolism), it has been speculated that chronic activation of HIF-1α could be deleterious due to induction of oxidative stress, 212 but data are lacking.

Mismatch between myocardial oxygen delivery and demand results in enhanced signaling through the basic helix-loop-helix transcription factor HIF-1α (see fig. e4 and fig. e5). HIF-1α is constitutively transcribed and translated, but under conditions in which oxygen is abundant it undergoes proteosomal degradation 213. Under such conditions, cellular prolyl-hydroxylases hydroxylate HIF-1α, producing a binding site on the HIF-1α molecule for the von Hippel-Lindau protein (VHL). VHL is part of a ubiquitin ligase complex that polyubiquitinates HIF-1α and targets it for rapid destruction by the proteosome 213. Under hypoxic circumstances, HIF-1α is not hydroxylated and regulates the transcription of an extensive repertoire of genes involved in angiogenesis (VEGF, AM), vascular remodeling, erythropoiesis (erythropoietin, EPO), metabolism, apoptosis, reactive oxygen species (ROS) formation, vascular tone and inflammation 212. Additional non-oxygen dependent regulation of HIF-1α expression occurs via the tumor-suppressor protein p53, intracellular concentrations of copper (Cu),50;54 and, as recently shown in cancer research, via competion with Tcf/Lef for binding with β-catenin within the nucleus (see fig. e4). In hypoxic conditions, binding to HIF-1α is more prevalent and induction of cell cycle arrest follows, together with transcription of angiogenic growth factors. In normoxic conditions and when Wnt/Fz signaling is active, β-catenin binding to Tcf/Lef prevails and hypertrophic and proliferative growth factors are activated 196. It would be very interesting to know whether this mechanism plays a role in the cardiac hypertrophic response as well. It is likely that the role of the Wnt pathway in cardiac hypertrophy will become clearer in the near future.

Vascular endothelial growth factor

VEGF signaling is initially upregulated in the LV exposed to TAC, but after two weeks insufficient VEGF signaling contributes to decreased cardiac microvascular density and systolic dysfunction. Restoring VEGF signaling leads to an increase in capillary density and an improvement in systolic function 50. The explanation for this biphasic response of VEGF protein expression may reside in an Akt1 - p53 - HIF-1α signaling axis (see fig. e5). Short-term activation of Akt1 leads to adaptive cardiac hypertrophy together with increased cardiac myocyte VEGF secretion and angiogenesis, while chronic Akt1 activation is associated with cardiac dilatation, a decreased secretion of VEGF and a reduced capillary density 174. Similarly, chronic pressure overload leads to accumulation of the tumor suppressor p53 and, as a consequence, downregulation of HIF-1α and VEGF. Either preventing p53 accumulation or introducing adenoviral vectors encoding VEGF directly into the heart enhances the number of microvessels, facilitates myocardial hypertrophic growth and restores myocardial function 50. This seems not to be a coincidence, since Akt1 and p53 signaling are highly interdependent 214. The importance of VEGF signaling in the hypertrophic response to pressure overload is further demonstrated by a study in mice exposed to TAC in which disruption of VEGF signaling led to the development of thin-walled, dilated and hypovascular hearts displaying contractile dysfunction 45. The exact mechanism by which VEGF influences cardiomyocyte hypertrophy is not known. Both indirect effects through secretion of paracrine factors (NO, ET-1, PGI2) by newly formed endothelial cells and direct activation of hypertrophic pathways (e.g. involving MAPKs) in the cardiomyocyte have been proposed 215;216. VEGF not only affects cardiomyocyte growth but also apoptosis, which can be prevented in rabbits exposed to pressure overload by intra-pericardial installation of VEGF 217. Another regulator of VEGF signaling is EPO. Recently, EPO receptors were demonstrated in the heart and disruption of cardiac EPO signaling accelerates the development of heart failure in mice subjected to TAC, which is associated with decreased VEGF signaling and capillary density 60.

7. Cardiac cell loss, regeneration and cellular senescence in heart failure


There are two pathways leading to apoptosis. The first pathway, in which caspases are activated via “death receptors” (e.g. Fas and TNF receptor) may be important in immune mediated heart failure, but does not seem to be important in more common forms of heart failure such as ischemic and dilated cardiomyopathy 218. The second more important pathway in heart failure is the mitochondrial pathway, involving mitochondrial outer membrane permeabilization (MOMP) 218. MOMP leads to cytosolic release of cytochrome c and other proteins that are normally found in between the mitochondrial outer and inner membrane. Subsequent to the release of cytochrome c, a self-amplifying caspase cascade is activated that ultimately leads to activation of caspase 3. Activated caspase 3 induces nuclear protein cleavage and DNA fragmentation. One of the proteins that are cleaved is β-catenin; cleavage is likely necessary to dismantle cell-cell contacts during apoptosis 219. The mitochondrial pathway is tightly regulated by the balance between anti-apoptotic proteins, such as Bcl-2, and pro-apoptotic proteins, such as Bax.

Caspase 3 induction of cardiomyocyte apoptosis is tightly related to rho signaling, with caspase 3 acting both up- and downstream of Rho kinase (ROCK, see online supplement). Chang et al. demonstrated that in the pathway leading to cardiomyocyte apoptosis, either induced by toxins or TAC, caspase 3 cleaves and activates ROCK, which generates a pro-apoptotic amplifying loop through activation of PTEN and subsequent inhibition of Akt1-mediated survival pathways 46. Del Re et al. confirmed this interaction and provided evidence for even more interaction between caspase 3 and ROCK. Activation of ROCK is associated with p53 mediated upregulation and activation of Bax, which subsequently translocates to the mitochondrial membrane to permeabilize it, releasing caspases 109.

Cardiomyocyte proliferation and senescence

It was calculated that the entire normal heart is replaced every 4.5 years; that is about 18 times over the human life span 220. The degree of cell loss is severely increased in heart failure (see above) and although the rate of myocyte formation is also increased, the pace of renewal is insufficient to prevent net cell loss 220. Since cardiac stem cells (CSCs) rarely divide, it is their progeny that actually replicates. High rates of cell turn-over do require repeated CSC division, however, which inevitably implies telomeric shortening and dysfunction, replicative senescence, cell death, reduction of the stem cell pool and exhaustion of the myocardial growth reserve 221;222. Telomeres are chromatin structures capping the ends of chromosomes that prevent the recognition of chromosomal ends as double-stranded DNA breaks, protecting these regions from recombination and degradation and avoiding a DNA damage cellular response. Telomeric DNA is composed of noncoding double-stranded G-rich tandem repeats that are extended several thousand base pairs. In human dividing cells including CSCs telomerase protects the integrity of the telomeric structure 223. Upregulated telomerase activity in heart failure cannot prevent telomeric attrition, however, which fact may also be related to alterations in telomeric binding proteins (TRF-1 and 2, polymerase and many others) 222;224. These events are accompanied by increased expression of p14ARF, p16 INK4a, p53 and phospho-p53, which together block the cell cycle, prohibit proliferation and activate the death program 220. Other consequences of cellular senescence that also affect mature cardiomyocytes are a reduced ability to synthesize hypertrophic proteins, decreased secretion of autocrine or paracrine factors and impaired antioxidant defense mechanisms 225. IGF-1 seems to protect aging CSCs and cardiomyocytes from senescence by phosphorylation of Akt1 which is associated with an increase in telomerase activity 169. The roles of apoptosis and senescence in PAH related right heart failure have not been investigated.

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|Table e1. Main animal models for research on pulmonary hypertension and right heart failure. |

|Model |Species |Notes |

|Chronic Hypoxia 1-4 |Rodents and |Effects of hypoxia mixed with those of increased afterload due to hypoxic |

| |larger animals|pulmonary vascular constriction |

|Monocrotaline 2;5-12;14;15 |Rat |Possible systemic activation of pro-inflammatory and pro-coagulant pathways |

| | |contributing to heart failure |

| | | |

|SU5416/Hypoxia 16-18 |Rat |effects of VEGF receptor blockade on myocardial microcirculation are unknown |

|Pulmonary artery banding 19-31 |Rat, Rabbit, |High mortality rates in rodents, only suitable to study chronic pressure |

| |Dog, Cat |overload with moderate constriction |

| | |re-expression of fetal genes (e.g. ANP, GATA-4 MEF2C) 23;30, β-AR |

| | |dysregulation 29, altered expression of SR proteins 25;26, increased apoptosis|

| | |27;31, myocardial fibrosis 24 as in left ventricular overload |

| | |no studies on the relative importance of different signaling pathways in the |

| | |transition from hypertrophy to failure |

VEGF = vascular endothelial growth factor; ANP = atrial natriuretic peptide; β-AR = β-adrenergic receptor.

Table e2. Important animal models for research on left heart failure, with relevance to the study of right heart failure in pulmonary hypertension.

|Model |Species |Notes |

|TAC 32-60 |Rodents and |frequently combined with transgenic approaches and/or agonist infusion (ATII, |

| |larger animals|Iso, PE, ET-1) |

| | |suppression of hypertrophy can be associated with enhanced systolic function |

| | |36-38;42;58;59 |

| | |neurohormonal activation due to decreased renal perfusion) is superimposed on |

| | |increased afterload |

| | | |

|Ischemia ± |Rodents and |may enhance understanding of pathobiology of PAH associated right heart failure |

|Reperfusion 63-68 |larger animals| |

TAC = transverse aortic constriction; ATII = angiotensin II; Iso = isoproterenol; PE = phenylepinephrine; ET-1 = endothelin-1; PAH = pulmonary arterial hypertension.

Legends to the figures

Figure e1. Excitation-contraction coupling. Cytosolic Ca2+ enters the sarcoplasimic reticulum (SR) through L-type Ca2+ channels (LTCCs). The resultant increase in intracellular Ca2+ triggers further Ca2+ release from the SR through the ryanodine receptor (RyR). Intracellular Ca2+ binds to troponin C within the myofilaments, which initiates contraction (not shown). Subsequent relaxation depends on dissociation of Ca2+ from troponin C and Ca2+ reuptake by the SR through a Ca2+-ATPase (SERCA), interacting with phospholamban. Ca2+ is removed trans-sarcolemmally through the Na/Ca2+ exchanger (not shown). Unphosphorylated phospholamban inhibits SERCA, and by phosphorylating phospholamban, protein kinase A (PKA) enhances SERCA-mediated Ca2+ re-entry into the SR during diastole.

Figure e2. G protein coupled receptors (GPCRs) involved in the myocardial hypertrophic response. Different receptor types use different secondary messengers. One of the consequences of angiotensin II (ATII) binding to the angiotensin type 1 receptor (AT1R) is activation of phospholipase C (PLCβ), which is followed by an increase in intracellular Ca2+ and protein kinase C (PKC) activation. After binding of catecholamines to the β-adrenergic receptor (β-AR), adenylate cyclase is activated, cAMP is produced and protein kinase A is activated. Other consequences of GPCR activation are activation of small GTP binding proteins (GTPases) and MAPK cascades. See text for more details.

Figure e3. The central role of cGMP/PKG signaling in the attenuation of myocardial hypertrophy. Activation of particulate gyanylate cyclase (pGC) by natriuretic peptides and soluble gyanylate cyclase (sGC) by nitric oxide is followed by production of cGMP and activation of protein kinase G (PKG). PKG affects myocardial contractility by reducing cAMP concentrations through inhibition of adenylate cyclase and the induction of phosphodiesterase PDE2. The production of pro-hypertrophic transcription factors that ultimately follows AT1R and β-AR activation is counterbalanced by cGMP/PKG signaling. cGMP/PKG signaling blunts the hypertrophic response through inhibition of the calcineurin/NFAT pathway, PI3K/Akt1 signaling (not shown), MAPK cascades and Rho signaling.

Figure e4. Wnt signaling and the balance between growth and angiogenesis. After Wnt ligands bind to a membrane-bound complex consisting of a member of the Fz receptor family and a LDL receptor–related protein (LRP), a member of the disheveled (Dvl) protein family is activated. This prevents inhibition of pro-hypertrophic transcription factors and decreases phosphorylation of β-catenin by GSK-3. β-catenin accumulates in the cytoplasm and is transferred to the nucleus, where it combines with Tcf/Lef to induce growth (proliferation or hypertrophy). However, in hypoxic conditions β-catenin combines with hypoxia inducible factor (HIF)-1α to induce cell cycle arrest and angiogenesis. Hypoxia prevents ubiquitination of HIF-1α and occurs when angiogenesis is insufficient in comparison to the degree of growth. In normoxic conditions, HIF-1α is hydroxylated by prolyl hydroxylases which allows binding of von Hippel-Lindau protein (VHL). HIF-1α is subsequently ubiquitinated in the proteosome.

Figure e5. The possible role of Akt1 signaling in the transition from compensated hypertrophy to dilatation and failure of the pressure overloaded right ventricle. An increased right ventricular afterload is associated with Akt1 signaling (e.g. induced by G protein coupled receptors or receptor tyrosine kinases) and subsequent activation of hypertrophic transcription factors. Hypertrophy must be met with angiogenesis to prevent ischemia, and this is realized by hypoxic prevention of hypoxia inducible factor (HIF)-1α ubiquitination and subsequent vascular endothelial growth factor (VEGF) transcription. However, prolonged Akt1 signaling leads to upregulation of the tumor suppressor gene p53, which inhibits HIF-1α independently from oxygen concentrations. Copper is another oxygen independent regulator of HIF-1α and the copper deficiency that may occur in heart failure contributes to insufficient angiogenesis.


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