The protein kinase C (PKC) family of serine/threonine kinases functions downstream of nearly all membrane-associated signal transduction pathways. Here we identify PKC-alpha as a fundamental regulator of cardiac contractility and Ca(2+) handling in myocytes. Hearts of Prkca-deficient mice are hypercontractile, whereas those of transgenic mice overexpressing Prkca are hypocontractile. Adenoviral gene transfer of dominant-negative or wild-type PKC-alpha into cardiac myocytes enhances or reduces contractility, respectively. Mechanistically, modulation of PKC-alpha activity affects dephosphorylation of the sarcoplasmic reticulum Ca(2+) ATPase-2 (SERCA-2) pump inhibitory protein phospholamban (PLB), and alters sarcoplasmic reticulum Ca(2+) loading and the Ca(2+) transient. PKC-alpha directly phosphorylates protein phosphatase inhibitor-1 (I-1), altering the activity of protein phosphatase-1 (PP-1), which may account for the effects of PKC-alpha on PLB phosphorylation. Hypercontractility caused by Prkca deletion protects against heart failure induced by pressure overload, and against dilated cardiomyopathy induced by deleting the gene encoding muscle LIM protein (Csrp3). Deletion of Prkca also rescues cardiomyopathy associated with overexpression of PP-1. Thus, PKC-alpha functions as a nodal integrator of cardiac contractility by sensing intracellular Ca(2+) and signal transduction events, which can profoundly affect propensity toward heart failure.

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A calcineurin-nuclear factor of activated T cells (NFAT) regulatory pathway has been implicated in the control of cardiac hypertrophy, suggesting one mechanism whereby alterations in intracellular calcium handling are linked to the expression of hypertrophy-associated genes. Although recent studies have demonstrated a necessary role for calcineurin as a mediator of cardiac hypertrophy, the potential involvement of NFAT transcription factors as downstream effectors of calcineurin signaling has not been evaluated. Accordingly, mice with targeted disruptions in NFATc3 and NFATc4 genes were characterized. Whereas the loss of NFATc4 did not compromise the ability of the myocardium to undergo hypertrophic growth, NFATc3-null mice demonstrated a significant reduction in calcineurin transgene-induced cardiac hypertrophy at 19 days, 26 days, 6 weeks, 8 weeks, and 10 weeks of age. NFATc3-null mice also demonstrated attenuated pressure overload-and angiotensin II-induced cardiac hypertrophy. These results provide genetic evidence that calcineurin-regulated responses require NFAT effectors in vivo.Cardiac hypertrophy is defined by an increase in ventricular wall thickness accompanied by an increase in cardiomyocyte cell volume. Hypertrophic enlargement is precipitated by increased workload or by decreased efficiency within the heart, conditions that are associated with hypertension, ischemic heart disease, valvular insufficiency, neuroendocrine disruptions, or intrinsic defects in contractile proteins (reviewed in reference 30). Although initially compensatory, sustained cardiac hypertrophy predisposes an individual to sudden death, arrhythmias, functional decompensation, and overt heart failure (30).Numerous regulatory pathways have been implicated in the transduction of hypertrophic signaling, linking neuroendocrine and mechanical stress stimuli to altered cardiac gene expression (reviewed in reference 40). Although numerous hypertrophic regulatory pathways have been identified, the recent characterization of the calcium-regulated phosphatase calcineurin as an important signaling factor in the heart has generated considerable interest. Transgenic mice expressing an activated form of calcineurin in the heart developed robust hypertrophy that quickly transitioned to dilation and failure (41). Subsequently, the calcineurin inhibitory drugs cyclosporine (Cs) and FK506 were shown to inhibit or attenuate cardiac hypertrophy or cardiomyopathy in most, but not all, rodent models of heart disease, suggesting a necessary regulatory role for this signaling pathway in the heart (reviewed in reference 39). More recently, transgenic mice expressing either the calcineurin inhibitory domains of Cain, AKAP79, MCIP1, or dominant-negative calcineurin were shown to have attenuated cardiac hypertrophy in response to pathophysiologic stimulation (7,52,72).Perhaps the best-characterized target of calcineurin is the nuclear factor of activated T cells (NFAT) transcription factor family. Calcineurin directly dephosphorylates NFAT transcription fa...

Members of the protein kinase C (PKC) isozyme family are important signal transducers in virtually every mammalian cell type. Within the heart, PKC isozymes are thought to participate in a signaling network that programs developmental and pathological cardiomyocyte hypertrophic growth. To investigate the function of PKC signaling in regulating cardiomyocyte growth, adenoviral-mediated gene transfer of wild-type and dominant negative mutants of PKCα, βII, δ, and ɛ (only wild-type ζ) was performed in cultured neonatal rat cardiomyocytes. Overexpression of wild-type PKCα, βII, δ, and ɛ revealed distinct subcellular localizations upon activation suggesting unique functions of each isozyme in cardiomyocytes. Indeed, overexpression of wild-type PKCα, but not βII, δ, ɛ, or ζ induced hypertrophic growth of cardiomyocytes characterized by increased cell surface area, increased [3H]-leucine incorporation, and increased expression of the hypertrophic marker gene atrial natriuretic factor. In contrast, expression of dominant negative PKCα, βII, δ, and ɛ revealed a necessary role for PKCα as a mediator of agonist-induced cardiomyocyte hypertrophy, whereas dominant negative PKCɛ reduced cellular viability. A mechanism whereby PKCα might regulate hypertrophy was suggested by the observations that wild-type PKCα induced extracellular signal–regulated kinase1/2 (ERK1/2), that dominant negative PKCα inhibited PMA-induced ERK1/2 activation, and that dominant negative MEK1 (up-stream of ERK1/2) inhibited wild-type PKCα–induced hypertrophic growth. These results implicate PKCα as a necessary mediator of cardiomyocyte hypertrophic growth, in part, through a ERK1/2-dependent signaling pathway.

The Ca 2؉ -calmodulin-activated Ser͞Thr protein phosphatase calcineurin and the downstream transcriptional effectors of calcineurin, nuclear factor of activated T cells, have been implicated in the hypertrophic response of the myocardium. Recently, the calcineurin inhibitory agents cyclosporine A and FK506 have been extensively used to evaluate the importance of this signaling pathway in rodent models of cardiac hypertrophy. However, pharmacologic approaches have rendered equivocal results necessitating more specific or genetic-based inhibitory strategies. In this regard, we have generated Tg mice expressing the calcineurin inhibitory domains of Cain͞Cabin-1 and A-kinase anchoring protein 79 specifically in the heart. ⌬Cain and ⌬A-kinase-anchoring protein Tg mice demonstrated reduced cardiac calcineurin activity and reduced hypertrophy in response to catecholamine infusion or pressure overload. In a second approach, adenoviral-mediated gene transfer of ⌬Cain was performed in the adult rat myocardium to evaluate the effectiveness of an acute intervention and any potential species dependency. ⌬Cain adenoviral gene transfer inhibited cardiac calcineurin activity and reduced hypertrophy in response to pressure overload without reducing aortic pressure. These results provide genetic evidence implicating calcineurin as an important mediator of the cardiac hypertrophic response in vivo.C ardiac hypertrophy is broadly defined as an adaptive enlargement of the myocardium characterized by the growth of individual cardiac myocytes rather than an increase in cell number. Whereas cardiac hypertrophy is a beneficial response that temporarily augments output, sustained hypertrophy often becomes maladaptive and is a leading predictor of future heart failure (1, 2). To understand the molecular mechanisms that underlie adaptive and maladaptive cardiac hypertrophy, investigation has centered around a characterization of the intracellular signal transduction pathways that promote cardiac myocyte growth (3, 4).One such intracellular signaling pathway involves the calciumcalmodulin, Ser͞Thr protein phosphatase calcineurin (PP2B). Sustained elevations in intracellular calcium concentration, in association with calmodulin, directly activate calcineurin phosphatase activity leading to the dephosphorylation and nuclear translocation of a family of transcription factors known as nuclear factor of activated T cells (5, 6).A role for calcineurin and nuclear factor of activated T cells as regulators of cardiac hypertrophy was recently identified (7). Transgenic (Tg) mice expressing an activated calcineurin or a constitutively nuclear nuclear factor of activated T cells c4 factor in the heart demonstrated profound hypertrophy that rapidly progressed to heart failure (7). In vitro, adenoviral-mediated gene transfer of activated calcineurin also promoted hypertrophic growth of neonatal cardiac myocytes (8). Treatment of cultured cardiomyocytes with the calcineurin inhibitory agents cyclosporine A (CsA) or FK506 blocked agonist-induced hypertrop...

Recent studies identify conventional protein kinase C (PKC) isoform phosphorylations at conserved residues in the activation loop and C terminus as maturational events that influence enzyme activity and targeting but are not dynamically regulated by second messengers. In contrast, this study identifies phorbol 12-myristoyl 13-acetate (PMA)-and norepinephrine-induced phosphorylations of PKC⑀ (at the C-terminal hydrophobic motif) and PKC␦ (at the activation loop) as events that accompany endogenous novel PKC (nPKC) isoform activation in neonatal rat cardiomyocytes. Agonist-induced nPKC phosphorylations are prevented (and the kinetics of PMAdependent PKC down-regulation are slowed) by pharmacologic inhibitors of nPKC kinase activity. PKC␦ is recovered from PMA-treated cultures with increased in vitro lipid-independent kinase activity (and altered substrate specificity); the PMA-dependent increase in PKC␦ kinase activity is attenuated when PKC␦ activation loop phosphorylation is prevented. To distinguish roles of individual nPKC isoforms in nPKC phosphorylations, wild-type (WT) and dominant negative (DN) PKC␦ and PKC⑀ mutants were introduced into cardiomyocyte cultures using adenovirus-mediated gene transfer. WT-PKC␦ and WT-PKC⑀ are highly phosphorylated at activation loop and hydrophobic motif sites, even in the absence of allosteric activators. DN-PKC␦ is phosphorylated at the activation loop but not the hydrophobic motif; DN-PKC⑀ is phosphorylated at the hydrophobic motif but not the activation loop. Collectively, these results identify a role for PKC⑀ in nPKC activation loop phosphorylations and PKC␦ in nPKC hydrophobic motif phosphorylations. Agonist-induced nPKC isoform phosphorylations that accompany activation/translocation of the enzyme contribute to the regulation of PKC␦ kinase activity, may influence nPKC isoform trafficking/down-regulation, and introduce functionally important cross-talk for nPKC signaling pathways in cardiomyocytes. Protein kinase C (PKC)1 constitutes a family of serine/threonine kinases that play key roles in signal transduction by receptors coupled to phosphoinositide hydrolysis (1). The basic primary structure of all PKC isozymes consists of a single polypeptide chain with N-terminal regulatory and C-terminal catalytic domains. PKC isoforms are subdivided into three distinct subfamilies based upon structural differences in their N-terminal regulatory domains. Conventional PKC isoforms (cPKCs) contain an autoinhibitory pseudosubstrate domain followed by two membrane-targeting modules, the C1 domain that binds diacylglycerol and phosphatidylserine (PS) and the C2 domain that binds anionic lipids in a calcium-dependent manner. Novel PKC (nPKC) isoforms lack the C2 domain and are maximally activated by diacylglycerol/phorbol ester in the absence of calcium; atypical PKCs are regulated by lipids but are not activated by calcium and diacylglycerol. Current models of PKC isoform activation focus largely on the allosteric changes that are induced by cofactor binding to N-terminal membrane-t...

The MAPKs are important transducers of growth and stress stimuli in virtually all eukaryotic cell types. In the mammalian heart, MAPK signaling pathways have been hypothesized to regulate myocyte growth in response to developmental signals or physiologic and pathologic stimuli. Here we generated cardiac-specific transgenic mice expressing dominant-negative mutants of p38α, MKK3, or MKK6. Remarkably, attenuation of cardiac p38 activity produced a progressive growth response and myopathy in the heart that correlated with the degree of enzymatic inhibition. Moreover, dominant-negative p38α, MKK3, and MKK6 transgenic mice each showed enhanced cardiac hypertrophy following aortic banding, Ang II infusion, isoproterenol infusion, or phenylephrine infusion for 14 days. A mechanism underlying this enhanced-growth profile was suggested by the observation that dominant-negative p38α directly augmented nuclear factor of activated T cells (NFAT) transcriptional activity and its nuclear translocation. In vivo, NFAT-dependent luciferase reporter transgenic mice showed enhanced activation in the presence of the dominant-negative p38α transgene before and after the onset of cardiac hypertrophy. More significantly, genetic disruption of the calcineurin Aβ gene rescued hypertrophic cardiomyopathy and depressed functional capacity observed in p38-inhibited mice. Collectively, these observations indicate that reduced p38 signaling in the heart promotes myocyte growth through a mechanism involving enhanced calcineurin-NFAT signaling

Background-Phospholamban (PLN) is an inhibitor of the Ca 2ϩ affinity of sarcoplasmic reticulum (SR) Ca 2ϩ -ATPase (SERCA2). The amino acid sequence of PLN is highly conserved, and although all species contain asparagine (Asn), human PLN is unique in containing lysine (Lys) at amino acid 27. Methods and Results-Human PLN was introduced in the null background. Expression of human PLN, at similar levels to mouse wild-type PLN, resulted in significant decreases in the affinity of SERCA2 for Ca