Hypertrophy is the predominant method for the heart to increase its size and mass, because cardiomyocytes are terminally differentiated and have very limited capacity for hyperplastic growth. It is usually categorized as physiologic hypertrophy and pathologic hypertrophy.62,63 Physiologic hypertrophy occurs in response to growth, exercise, and pregnancy without apparent abnormalities in both systolic and diastolic function. In contrast, pathologic hypertrophy takes place in settings of chronically increased cardiac load represented by hypertension and valvular diseases. Pathologic hypertrophy is described as concentric (characterized by addition of sarcomeres in parallel, leading to increased thickness of the myocyte) and eccentric (characterized by the addition of sarcomeres in series, leading to increased length).62 If the load placed on the heart is not normalized, the heart may continue to hypertrophy, eventually leading to elevated filling pressures caused by increased ventricular stiffness. The hypertrophied heart may also begin to decompensate in the long term, leading to progressive dilatation and systolic dysfunction. Not surprisingly, cardiac hypertrophy is a significant risk factor for the development of heart failure and, in addition, for sudden death.64
Irrespective of the type of hypertrophy, the processes of hypertrophy require a dramatic reprogramming of gene expression to upregulate gene products necessary for growth of the cardiomyocytes. These include genes encoding contractile elements and proteins of the basic transcriptional and translational machinery that allow new protein production, and the genes encoding proteins that remodel the extracellular matrix, allowing growth to proceed. This process of reprogramming occurs in response to respective growth signals that are generated from a multitude of sources. In order to reprogram gene expression, these signals must be sensed at the cardiomyocyte membrane and transmitted into the interior of the cell, eventually into the nucleus, by a process called signal transduction. A large number of growth factors as well as signaling molecules have been reported to induce growth. Here, we will focus on the major arms for which strong evidence exists implicating components of the pathway in the hypertrophic response.
The Signal at the Cell Membrane
Two types of stimuli are believed to trigger the hypertrophic response: mechanical deformation of the membrane (cell stretch) and growth-promoting ligands binding to their cognate receptors in the myocyte cell membrane. Stretch of cardiomyocytes in culture leads to gene transcription and protein synthesis in a pattern that closely resembles the load-induced hypertrophic response in vivo.65 Stretch triggers responses both via the direct activation of signaling molecules and by inducing the release of humoral factors. Direct activation involves recruitment of integrin signaling and stretch-activated ion channels.66 For example, melusin, an integrin bound protein, is activated by mechanical stretch and promote adaptive hypertrophy via PI3-kinase/Akt and GRK3.67
The release of growth factors acts in an autocrine or paracrine fashion to amplify hypertrophic responses. Angiotensin II activates a number of prohypertrophic signaling pathways.68 The interleukin-6 family of cytokines, including cardiotrophin-1, is another group of factors released by mechanical stretch, which act via receptors specific to the cytokine and via the common receptor gp130.69 A wealth of data indicates that insulin-like growth factor-1 (IGF-1) signaling is also activated by a variety of stimuli that induce hypertrophy. Many of the prohypertrophic peptides bind to receptors that are linked to heterotrimeric G proteins of the Gq family. These G proteins convert receptor activation into mobilization of intracellular signaling pathways, and are the initial trigger for downstream events.70 Cardiac-specific overexpression of the α subunit of Gq led to cardiac hypertrophy.71 Later, it was reported that expressing a peptide that inhibits Gq-dependent signaling significantly limits the hypertrophic response to pressure overload in vivo.72 These studies, taken together, confirm a critical role of Gq in hypertrophic signaling and in the hypertrophic response to pressure overload. Activation of these proximal mediators of the stretch response results in increased cytosolic Ca2+, which plays a role in activating several signaling pathways, including calcineurin.
Signaling Within the Cell
There are two essential features of hypertrophic growth: reprogramming of gene expression and protein synthesis. Each is regulated by a series of intracellular pathways that are activated by events occurring at the membrane as described above. Signal transmission from the cell membrane to the nucleus is essential for gene reprogramming.46 This is generally accomplished by linear cascades of proteins, most commonly protein kinases and also by the protein phosphatase calcineurin, activating one another in sequence, culminating in the phosphorylation and activation of one or more transcription factors. The transcription factors then bind to promoter elements, promoting the gene transcription. Each transcription factor will usually target several genes, and the net result of the activation of the entire set of genes is the hypertrophic response. Characteristic of the response is a reestablishment of a gene program often described as “embryonic” or “fetal,” because some of the gene expression pattern resembles that normally expressed in utero. The genes induced by the hypertrophic response are often divided into three groups based on their time of expression: immediate early, intermediate, and late. Immediate early genes include the neurohormonal mediator, brain natriuretic peptide, and several stress-induced genes or genes involved in growth control. These include c-Fos, c-Jun, c-Myc, Egr-1, and heat shock protein 70 (HSP70). Intermediate response genes include atrial natriuretic peptide and angiotensinogen as well as several sarcomeric components, β-MHC (and corresponding downregulation of α-MHC), myosin light chain-2, and skeletal α-actin (replacing cardiac α-actin). Late response genes include ACE and the NCX.
In-Depth Evaluation of Calcium-Dependent Hypertrophic Signaling
The calcineurin pathway is the most consistently reported pathway that has been shown to regulate hypertrophic growth in a number of different models. Calcineurin, also known as protein phosphatase 2B (PP2B), is a Ca2+-calmodulin–activated protein phosphatase, and it is uniquely activated by sustained elevations in intracellular Ca2+35 (Fig. 6–4). Transgenic mice overexpressing an activated form of calcineurin in the hearts induced a profound hypertrophic response (two- to three-fold increase in heart size) that rapidly progressed to dilated heart failure within 2 to 3 months.57 Such data implicate calcineurin as a sufficient inducer of the hypertrophic response and as a potential causative factor associated with the transition to decompensation and heart failure. Calcineurin catalytic activity can be inhibited by the immunosuppressive drugs cyclosporine A (CsA) and FK506. Inhibition of calcineurin with either CsA or FK506 can antagonize cardiac hypertrophy and/or disease progression in pleiotropic rodent models.73 However, their potential usefulness in humans remains uncertain because both CsA and FK506 have a number of side effects in humans, including nephrogenic and neurogenic toxicity and immunosuppression.
Calcinuerin pathway in hypertrophy. Stimuli that induce hypertrophic signaling increases cytosolic Ca2+ levels. When the intracellular Ca2+ level rises to 10-5 M, four Ca2+ ions bind to calmodulin, and this Ca2+-calmodulin complex activates many calmodulin (CaM)-dependent enzymes, including the phosphatase calcineurin and the CaM-dependent kinases, Ca-CaM-dependent protein kinase II (CaMKII). Once activated, calcineurin directly dephosphorylates members of the nuclear factor of activated T-cells (NFAT) transcription factor family in the cytoplasm, promoting their translocation into the nucleus. In the nucleus, NFAT family members participate in the transcriptional induction of various immune response genes. CaMKII phosphorylates the histone deacetylases (HDACs) to induce their nuclear export and relieve the inhibition on the transcription factor myocyte enhancer factor-2 (MEF-2). These signals reprogram gene expression resulting in cardiac hypertrophy. Cyclosporin A (CsA) and FK506 are calcineurin inhibitors. Modified with permission from Passier R, Zeng H, Frey N, et al: CaM kinase signaling induces cardiac hypertrophy and activates the MEF2 transcription factor in vivo, J Clin Invest. 2000 May;105(10):1395-1406.
CaMKII is another target of Ca2+–calmodulin activation. CaMKII is involved in the regulation of intracellular Ca2+ handling, affecting contractility and relaxation of the cardiomyocytes.47 This molecule also modulates gene transcription by phosphorylating several transcription factors, including class II histone deacetylases, thereby enhancing the myocyte enhancer factor 2 activity. Increased CaMKII activity was found in hypertrophied and failing myocardium.47 CaMKII overexpression induced cardiac hypertrophy,74 whereas loss of CaMKII was associated with attenuated cardiac hypertrophy on hypertrophic stimulation.75
In summary, these pathways illustrate the paradigm of how a signal, increased cytosolic Ca2+, generated in response to either deformation of the membrane or to hypertrophic agonist binding to its receptor, activates key signaling factors (calcineurin/CaMKII), which then activate transcription factors that, in turn, reprogramming gene expression.
Regulation of Protein Synthesis
The second critical component of hypertrophic growth is the ability to dramatically upregulate protein synthesis capabilities. This is regulated by two very complex, interacting pathways. One is the PI3-kinase pathway that, in addition to its role in regulating protein synthesis, also plays a major role in reprogramming gene expression. The other is the mammalian target of rapamycin (mTOR) pathway. These pathways are essential in determining cell, organ, and body size (ie, normal growth) in species as diverse as Drosophila and human, but they are also recruited in, and regulate the response to, pathologic stress-induced hypertrophic growth. Both the PI3-K and mTOR pathways regulate protein synthesis by modulating the activity of various translation factors.
The Phosphoinositide 3-Kinase Pathway
This highly conserved pathway (Fig. 6–5) is remarkable in the fact that virtually every component of the pathway has been shown in animal models to regulate cell and organ growth, including growth of the heart. The pathway is activated by most (if not all) of the agonists implicated in inducing cardiac hypertrophy, including pressure overload.63 When activated, PI3-K phosphorylates the integral membrane phospholipid, phosphatidylinositol, leading to the recruitment of the protein kinase Akt (also known as protein kinase B, PKB) to the cell membrane. This brings PKB/Akt into proximity to its activator, the 3-phosphoinositide-dependent protein kinase-1 (PDK1), and results in activation of PKB/Akt.76 PKB/Akt then plays a role in activating mTOR and, consequently, p70S6K and the protein translation machinery.77 Consistent with this, all PI3-K, PDK1, and PKB/Akt have been shown to regulate cell and organ size, including size of the heart. Recently, inhibition of PI3-K p110γ was shown to normalize β-AR density and improve cardiac contractility in mouse heart failure,78 and its efficacy in humans is being explored in clinical trials.4
Mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways in hypertrophy. Left: MAPK pathway. MAPK signaling pathway transmits signals through a stream of several serial kinase reactions. Extracellular stimuli activate the MAPK pathways. Once MAPK kinase kinases (MAPKKKs), such as RAF, MAPK/ERK kinase kinase (MEKK) and TGF?-activated kinase (TAK), are activated, they phosphorylate MAPK kinases (MAPKK)s. The relayed signals eventually trigger activation of three major distal MAPKs including extracellular signal-regulated kinases (ERKs), p38, and c-Jun N-terminal kinases (JNKs). These activated MAPKs can translocate to the nucleus and phosphorylate numbers of transcription factors thereby altering gene expression. Reproduced with permission from Rose BA, Force T, Wang Y: Mitogen-activated protein kinase signaling in the heart: angels versus demons in a heart-breaking tale. Physiol Rev. 2010 Oct;90(4):1507-1546. Right: PI3-K pathway. PI3-K activation recruits Akt to the plasma membrane through activation by phosphoinositide dependent kinase 1 (PDK1). Akt promote complex assembly of mammalian target of rapamycin complex 1 (mTORC1) through a series of signaling pathways. On activation, mTORC1 phosphorylates its two major downstream targets p70 ribosome S6 kinase (p70S6K) and eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) and mediates cell growth, proliferation, and cell survival. mTORC1 consists of the regulatory-associated protein of mTOR (Raptor), the proline-rich Akt substrate 40 kDa (PRAS40), the mammalian lethal with Sec13 protein 8 (mLST8), and the DEP domain-containing mTOR interacting protein (Deptor). Reproduced with permission from Chong ZZ, Maiese K: Mammalian target of rapamycin signaling in diabetic cardiovascular disease, Cardiovasc Diabetol. 2012 Jul 16;11:45.
The importance of the protein kinase mTOR is illustrated by its conservation throughout evolution, from yeast to human. The critical importance of mTOR in regulating hypertrophic growth in vivo was demonstrated when its inhibitor, rapamycin, was found to attenuate the hypertrophic response to pressure overload in mice.79 Distinct functions of mTOR are reflected in its assembly as structurally distinct multiprotein complexes, mTORC1 and mTORC2 (see Fig. 6–5). Growth factors—including those leading to cardiac growth, such as insulin and IGF-1—activate mTORC1. Activation of mTORC1 leads to disinhibition of eukaryotic translation initiation factor 4E, resulting in enhanced protein synthesis.77 Because the mTORC pathway is also required for physiological hypertrophic response, complete inhibition of this pathway results in rapid ventricular dilatation and dysfunction upon pressure overload.80 In contrast, partial inhibition of mTORC1 seems to be able to reduce only pathological hypertrophy.77
Other Pathways Involved in Growth Regulation
MAPKs have been reported to be involved in a repertoire of biological processes including proliferation, differentiation, metabolism, motility, survival, and apoptosis.13 Stress-activated MAPKs, the c-Jun N-terminal kinases (JNKs), and p38 have been exhaustively studied (see Fig. 6–5); however, their exact role in hypertrophy remains obscure.13 Given the central role of these kinases in very basic responses of all cells to a wide range of cellular stressors (eg, oxidant stress, ionizing radiation, cytokine stimulation, osmolar stress, heat shock), compensatory adaptations are likely to be profound in various circumstances. Assessment of the present data suggest that the JNKs and p38 may be more involved in the progression of heart failure, including remodeling of the matrix, and may play much less of a role in hypertrophic growth.81 The extracellular signal-regulated kinase (ERK) family of MAPKs is also potently activated by hypertrophic stimuli. Interestingly, cardiac-specific overexpression of MEK1 (one of the immediate upstream activators of the ERKs) produced concentric cardiac hypertrophy, but unlike most other models, the MEK1 transgenic animals did not progress to contractile failure.82 These data raised the concept of “beneficial” hypertrophy versus “detrimental” hypertrophy by clearly demonstrating that hypertrophy per se need not inexorably progress to heart failure. This may have to do with the fact that the ERKs (in contrast to many other prohypertrophic signaling molecules, including calcineurin, the JNKs, and the p38) are, in many circumstances, cytoprotective.13
Glycogen synthase kinase-3 (GSK-3) is another target of PKB/Akt involved in hypertrophic signaling. GSK-3β is a negative regulator of cardiac growth. Transgenic animals expressing GSK-3β have dramatic reductions in normal cardiac growth and also have a markedly reduced hypertrophic response to pressure overload.83 Furthermore, inhibition of GSK-3β is necessary for the hypertrophic response to a number of agonists, likely mediated via several mechanisms. The GSK-3β targets include the nuclear factor of activated T cells (NFATs), and thus GSK-3β acts in opposition to the calcineurin pathway.84 Other known growth regulators negatively regulated by GSK-3β include c-Myc, GATA-4, β-catenin, and c-Jun.85
Alterations in Signaling in the Diseased Human Heart
Despite the continuous efforts to characterize molecular pathways in hypertrophy using transgenic and pressure overload animal models, analysis in human heart tissues exhibits somewhat inconsistent results. For example, in one study, various signaling factors (calcineurin, ERK1/2, JNK, p38, PKB/Akt, and GSK-3) were examined in the hypertrophic heart without heart failure, and calcineurin was the only factor that was consistently found to be activated.86 In contrast to the paucity of data on hypertrophied hearts, several studies have examined the signaling profile of hearts explanted from patients with advanced heart failure undergoing either transplant or left ventricular assist device placement. Where examined, calcineurin expression and activity were increased significantly.87,88 Activation of p38 was generally reported, although there seems to be a substantial variability.81 Meanwhile, no consistent results have been reported for JNKs and ERKs. PKB/Akt was found to be activated, irrespective of the etiology of the heart failure, and accordingly, its downstream target, GSK-3β, was inhibited.89 Variabilities among the studies could be accounted for by a number of factors, including different medical therapies, device therapies, status of the patient, and methods of preserving the tissue.
To summarize, current data are insufficient to conclude whether any signaling alterations are causal or simply a consequence of the hypertrophy and heart failure. The complexity of the heart failure signaling abnormalities and the changing activities of various pathways at various times in the progression of disease creates a “moving target” and leads to significant challenges to determine the key pathways. The patients with compensated hypertrophy versus advanced heart failure are obviously at different ends of the pathophysiologic spectrum of heart failure, and in between these points, including the transition to and early progression of heart failure, we have very little data on what pathways might be reasonable targets.