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Cardiac hypertrophy is a complex adaptive response to mechanical and neurohumoral stimuli and under continual stressor, it contributes to maladaptive responses, heart failure and death. Protein kinase C (PKC) and several other kinases play a role in the maladaptative cardiac responses, including cardiomyocyte hypertrophy, myocardial fibrosis and inflammation. Identifying specific therapies that regulate these kinases is a major focus of current research. PKC, a family of serine/threonine kinases, has emerged as potential mediators of hypertrophic stimuli associated with neurohumoral hyperactivity in heart failure. In this review, we describe the role of PKC isozymes that are involved in cardiac hypertrophy and heart failure.
Cardiac remodeling is a target organ response to cardiovascular diseases and is an independent risk factor for coronary heart disease, stroke, arrhythmias, heart failure, and cardiovascular morbidity and mortality [1–3]. The process of cardiac hypertrophy involves multiple progressive alterations of heart geometry mediated by neurohumoral stimulating stress (i.e. epinephrine, norepinephrine, angiotensin II and aldosterone). Various kinases have been described as candidate mediators of the cardiac biochemical stress and trophic response induced by activation of neurohormone receptors . Subcellular changes associated with cardiac hypertrophy and remodeling may be beneficial in the short term, but are often maladaptative and lead to functional decompensation in the long term. Along with this, the potential molecular events underlying the transition from compensated cardiac hypertrophy to failure are still under investigation. Thus a number of studies have focused on identifying intracellular distal strategic nodes where signals converge and/or serve as multi-effector brakes to suppress or reverse hypertrophy inside the cardiac muscle cell, which would become attractive targets for heart failure pharmacological therapy. In this review, we focus on βIIPKC and εPKC isozymes as potential intracellular nodes that play critical roles in cardiac hypertrophy and failure.
Identified in 1977 by Nishizuka and coworkers , protein kinase C (PKC) is a group of closely related phospholipid-dependent serine-threonine protein kinases, which are activated as a result of receptor-dependent activation of phospholipase C and the hydrolysis of membrane phosphoinositides . The physiological importance of PKC is underscored by the existence of ten different isozymes. These enzymes are classified according to their structure and activation requirements into the following groups: classical or conventional PKCs (α, βI, βII, and γ), which are Ca2+-dependent and activated by binding to diacylglycerol (DAG) and phosphatidylserine (PS); novel PKCs (δ, ε, θ, and η), which are Ca2+-independent but are activated by DAG and PS; and the atypical PKCs (ζ, ι/λ), which are Ca2+ and DAG independent but are PS sensitive. For all PKC isozymes, translocation to membrane structures provides a mechanism to regulate access to substrate and has been taken as the hallmark of activation . At least for some PKC isozymes, auto-phosphorylation on particular sites can also be used as a hallmark of PKC activation .
PKC is ubiquitously expressed in all tissues, whereas its distribution is tissue, species and time dependent. PKC isozymes α, δ, ε, η, and ζ have been identified in cultured cardiomyocytes [9, 10]. While the mouse myocardium expresses low levels of both βI and βIIPKC , abundant expression of these β PKCs in human and rat cardiomyocytes has been reported [12–14]. Immunoblotting analyses demonstrated the presence of PKC isozymes α, βI, βII, δ, ε, γ, μ, η, λ, and θ in human heart tissue . Further species-specific differences in the expression of cardiac η, θ and εPKC were also reported . Thus, caution must be taken when translating PKC findings from animal models, since expression patterns of specific PKC isozymes differs among species. Further, semi-quantitative assessment of PKC isozymes levels using specific antibodies might provide misleading information on the relative abundance of the PKC isozymes . Using recombinant proteins as a standard, to provide absolute amounts of each PKC isozyme is essential, because of differences in relative immunoreactivity of the isozymes. Finally, temporal expression and activation of different PKC isozymes during ageing and their contribution to disease progression should be considered . For example, during heart failure progression, εPKC and βIIPKC isozymes levels are increased at early (left ventricular hypertrophy) and late (cardiac dysfunction) stages, respectively .
Prolonged increase of workload triggers cardiac hypertrophy, an adaptative response to normalize wall stress and compensate for the increased neurohormonal stimuli and hemodynamic load. At a cellular level, cardiac myocytes assume a hypertrophic phenotype associated with reactivation of fetal gene programs and quantitative/qualitative changes in the contractile machinery, subcellular organelles, cellular signaling and myocardial metabolism [19, 20]. Several studies have addressed the intracellular mechanisms underlying cardiac hypertrophy and PKC isozymes emerged as potential mediators of hypertrophic stimuli [21, 22]. In fact, PKC activation with PMA (a non-selective PKC activator) causes cardiac myocyte hypertrophy, whereas inhibitory peptides of the cpKCs display an anti-hypertrophic effect . Neurohormones- and mechanical stress-induced cardiac hypertrophic stimuli converge to PKC activation with increased PKC expression and activity in various in vivo models of cardiac hypertrophy [18, 24–26]. Therefore, experimental approaches using rat cultured myocytes, transgenic mice overexpressing PKC isozymes, and isozyme-selective agonist and antagonist peptides have been used to address the relative contribution of PKC isozymes to cardiac hypertrophy and heart failure. Among different PKC isozymes, εPKC and βIIPKC have been considered the main effectors. αPKC has also been involved in rat cardiomyocyte growth and cardiac dysfunction [27, 28].
εPKC is activated in response to hypertrophic stimuli in rat cultured cardiac myocyte and in vivo, and its overexpression in mice leads to cardiac hypertrophy associated with concentric remodeling and preserved cardiac contractility [18, 29]. Treatment with anti-sense εPKC decreased myotrophin-induced stimulation of protein synthesis in neonatal rat cardiomyocytes . In fact, cardiac myocyte-restricted εPKC activation in transgenic mice expressing the εPKC-specific activator (ψεRACK) induces non-pathological hypertrophy, and inhibition of εPKC by a selective inhibitor, εV1 fragment, expressed in low levels in transgenic mice, results in a thin ventricular wall and myocyte hypertrophy [31, 32]. Additionally, high levels of εV1 expression lead to a lethal form of heart failure from dilated cardiomyopathy . We have also demonstrated that εPKC activation during transition from compensated cardiac hypertrophy to heart failure increased mast cell degranulation-induced inflammatory responses, induced cardiac fibrosis and ventricular dysfunction, and significantly reduced animal survival, whereas sustained εPKC inhibition abrogated this pathological phenotype in a rat model [33, 34].
βPKC isozymes also play an important role in cardiac hypertrophy and failure. Even though βPKC is restricted to embryonic and neonatal cardiac myocytes, its expression is upregulated in adult cardiac myocytes under hypertrophic stimuli and in human heart failure [12, 14, 18, 24, 35]. Of interest, increased βIIPKC levels in cultured mouse cardiac myocytes are paralleled by increased ANF and βMHC, genes involved in the transition from cardiac maladaptative hypertrophy to heart failure in cardiac fetal reprogramming .
Targeted overexpression of βIIPKC in mouse cardiomyocytes results in left ventricular hypertrophy and fibrosis and oral treatment of the transgenic animal with LY333531, a βPKC inhibitor, prevents cardiac hypertrophy , supporting a direct relationship between βIIPKC and the pathological response. In contrast to these findings, another study using βPKC knockout mice showed no role for βPKC in heart failure progression. Thus, the role of βPKC in cardiac hypertrophy in genetically modified mice is controversial. More recently, hypertensive Dahl salt-sensitive rats treated with the βIIPKC-specific inhibitor peptide, but not the βIPKC specific inhibitor, greatly delayed the development of heart failure and improved survival . Similar results were observed in myocardial infarction-induced heart failure in rats treated with βIIPKC-specific inhibitor peptide . Finally, studies characterizing the level and activity of PKC isozymes in human heart failure found a significant increase in level and activation of βPKC [12, 14]. Together, these studies indicate that changes in βIIPKC correlate with human heart failure, suggesting that focusing on this PKC isozyme in considering therapeutic intervention is advisable. A summary of several studies using genetic and pharmacological approaches to determine the role of βIIPKC and εPKC isozymes in cardiac hypertrophy and heart failure is provided in Table 1.
As discussed above, βIIPKC and εPKC isozymes are suited to operate as molecular switches at nodal points in signaling pathways leading to cardiac hypertrophy and heart failure, and downstream mediators of PKC effects have been identified. Pro-hypertrophic stimulation of adult cardiomyocyte cultures with endothelin-1 (ET-1), angiotensin II or phorbol myristate acetate (PMA) resulted in PKC-mediated phosphorylation with further activation of several pro-survival kinases, including mTOR and S6K1 (Fig. 1). Indeed, expression of dominant negative εPKC abrogated ET-1 stimulated mTOR and S6K1 phosphorylation, suggesting that εPKC activates mTOR and S6K1 leading to cardiomyocyte hypertrophy (Fig. 1).
Another kinase that has been implicated in PKC-mediated hypertrophic signaling is ERK, a MAPK involved in growth and cell survival (Fig. 1). Under hypertrophic stimuli, εPKC acts as an upstream regulator of the Ras/Raf/-ERK cascade  and mediates GPCR-dependent mobilization of transcription factors in cardiomyocyte cultures . Indeed, using a constitutively-activated εPKC mutant, Heidkamp et al.  demonstrated that εPKC selectively activates ERK, resulting in cardiomyocyte remodeling. Of interest, using the same approach for δPKC, they observed that δPKC preferentially activates JNK and p38, which are implicated in stress-activated protein kinase cascades. Taken together these studies support the involvement of both εPKC and δPKC isozymes in MAPK cascade activation with distinctly different MAPK pathway downstream targets.
As described above, increased βPKC levels in rat primary cardiac myocytes under hypertrophic stimuli are paralleled by increased ANF expression, and the mechanism underlying this response seems to be related to GATA-4, a transcription factor that mediates βPKC activation of the ANF promoter in response to pro-hypertrophic Ang II, ultimately resulting in enhanced DNA binding activity . In another study, Lim et al. showed that PKC-dependent TAK1 phosphorylation and ATF2 (activating transcription factor 2) activation is involved in TGF-β1-induced cardiac hypertrophy . Indeed, non-selective PKC inhibition completely blocked TGF-β1-induced TAK1 kinase activity and subsequent downstream signaling, such as inhibition of β-MHC gene induction and ANF promoter activity. Lastly, protein kinase D, another downstream effector of PKC, directly phosphorylates HDAC5 and stimulates its nuclear export and cardiac hypertrophy, whereas a non-selective PKC inhibitor (GF 109203X) prevents nuclear export of HDAC5 in response to hypertrophic agonists . According to these studies, neurohumoral activation of GPCR engages classical and novel PKC pathways towards cardiac hypertrophy-related gene reprogramming (Fig. 1). Therefore, reduced myofilament responsiveness to Ca2+ associated with a significant increase in troponin I phosphorylation levels have been seen in the myocardium of transgenic mice overexpressing βIIPKC. The depressed cardiomyocyte function improved after sequential perfusion of LY333531, a βPKC inhibitor. This study shows that βIIPKC-mediated phosphorylation of troponin I in vivo may decrease the Ca2+ responsiveness of myofilaments, and thus lead to cardiac myocyte dysfunction . Depressed myofilament contractility-associated myocyte dysfunction was also observed in αPKC transgenic mice .
In addition to the effects of βIIPKC on cardiac hypertrophy-related gene reprogramming proteins and contractile myofilaments, our laboratory has recently identified new βIIPKC targets associated with protein quality control disruption in heart failure . The cellular protein quality control (PQC) is set to detect, repair and dispose of cytotoxic damaged proteins by multilayered control mechanisms including chaperones, the ubiquitin-proteasome system and autophagy. We found that increased βIIPKC activity leads to proteasome dysfunction, the main effector of the ubiquitin-proteasome system, and contributes to heart failure development  (Ferreira et al in preparation). Therefore, blocking βIIPKC and proteasome interaction using a rationally designed peptide inhibitor for βIIPKC improves cardiac function and survival in both ischemic and hypertensive-induced heart failure in rats. These phenomena were mediated by increasing proteasomal activity and re-established cardiac protein quality control. A summary of βIIPKC and εPKC isozyme-mediated cellular responses in cardiac hypertrophy and heart failure is provided in Figure 2.
Because specific PKC isozymes contribute to a wide variety of human diseases and sometimes exert even opposing effects in the same disease, the need to produce highly selective pharmacological PKC inhibitors is highlighted. A group of bisindolyl malelmide (BIM) compounds that are based on the scaffold of the nonspecific kinase inhibitor staurosporine have been a focus of active research. These compounds were reported to function as βPKC selective inhibitors and reverse cardiac remodeling and ventricular dysfunction [44, 46]. However, several staurosporine-originated PKC inhibitors have been tested in animal models and clinical trial and were found to have undesirable toxicity. Moreover, specific PKC inhibitors such as LV333531, BIM1and BMI2 inhibit the activity of several PKC isozymes as well as the activity of other kinases . For example, the βPKC-selective inhibitor (LV333531) that is currently in clinical trial for diabetic retinopathy and diabetic macular edema has been reported to affect other PKC isozymes .
Our lab has taken another approach to generate PKC isozyme-selective pharmacological tools. We used a rational design to identity the interaction site between each PKC isozyme and its selective anchoring protein (RACK) and identified peptides corresponding to these sites that selectively interfere with the interaction. Importantly, we showed that inhibition of interaction of a particular isozyme with its RACK causes a selective loss of the function mediated by that isozyme without affecting the function of other PKC isozymes that are present in the same cells (reviewed elsewhere ). The peptides are delivered into cells and in vivo by cross-linking them to cell-permeating peptides, such as TAT47–57. These peptide inhibitors are highly selective and efficacious in treating animals used as models for different human diseases where specific PKC isozyme is activated [50–54]. Relevant to the topic of this review, selective inhibition of βIIPKC and εPKC with these peptide inhibitors significantly improved cardiac function and prolonged survival in different heart failure animal models [22, 33, 45, 53]. Therefore, although intermolecular proteins interactions are considered to be difficult to target for therapeutic, short peptides corresponding to sequences that mediate protein-protein interactions may offer a new class of drugs for treating a number of diseases including cardiovascular diseases.
Taken together, studies using cultured cardiomyocytes, transgenic animals and selective pharmacological tools suggest that both βIIPKC and εPKC are key molecules involved in cardiac hypertrophy and heart failure. Avoiding pathological cardiac remodeling is a goal of heart failure therapy . In this review, we describe a continuum of responses emanating from βIIPKC and εPKC isozymes that contribute to decompensated hypertrophy and heart failure (see Table 1 and Figure 1). Because specific modulators of PKC isozymes are already in clinical trials for a variety of diseases [56–59], it may now be possible to consider using βIIPKC and εPKC isozyme-specific inhibitors to treat human heart failure. Therefore, recent clinical trials suggest that systemic delivery of inhibitors of PKC isozymes is well tolerated [57, 58, 60]. Further, advances in drug delivery suggest that organ-selective delivery may also be another possibility in the near future, for example by delivering slow drug-releasing molecules to specific organs . Therefore, clinical trials with βIIPKC and εPKC inhibitors to treat heart failure should be considered using either systemic or cardiac-specific drug delivery. Further in vivo studies using larger animal models will help to point out the relevance of βIIPKC and εPKC as therapeutic targets for the treatment of maladaptative cardiac remodeling and heart failure in humans.
This study was supported by National Institute of Health Grant HL076675 and HL52141 to DMR. JCF holds a post-doctoral fellowship from Fundação de Amparo a Pesquisa do Estado de São Paulo - Brasil (FAPESP 2009/03143-1). PCB holds a scholarship from Conselho Nacional de Pesquisa e Desenvolvimento, Brasil (CNPq BPQ 301519/2008-0).
DM-R is the founder of KAI Pharmaceuticals, Inc, a company that plans to bring PKC regulators to the clinic. However, none of the work described in this study is based on or supported by the company. Other authors have no disclosure.
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