The PTEN/PI3K signaling pathway regulates a vast array of fundamental cellular responses. We show that cardiomyocyte-specific inactivation of tumor suppressor PTEN results in hypertrophy, and unexpectedly, a dramatic decrease in cardiac contractility. Analysis of double-mutant mice revealed that the cardiac hypertrophy and the contractility defects could be genetically uncoupled. PI3Kalpha mediates the alteration in cell size while PI3Kgamma acts as a negative regulator of cardiac contractility. Mechanistically, PI3Kgamma inhibits cAMP production and hypercontractility can be reverted by blocking cAMP function. These data show that PTEN has an important in vivo role in cardiomyocyte hypertrophy and GPCR signaling and identify a function for the PTEN-PI3Kgamma pathway in the modulation of heart muscle contractility.

Differential modes for ␤ 1 -and ␤ 2 -adrenergic receptor (AR) regulation of adenylyl cyclase in cardiomyocytes is most consistent with spatial regulation in microdomains of the plasma membrane. This study examines whether caveolae represent specialized subdomains that concentrate and organize these moieties in cardiomyocytes. Caveolae from quiescent rat ventricular cardiomyocytes are highly enriched in ␤ 2 -ARs, G␣ i , protein kinase A RII␣ subunits, caveolin-3, and flotillins (caveolin functional homologues); ␤ 1 -ARs, m 2 -muscarinic cholinergic receptors, G␣ s , and cardiac types V/VI adenylyl cyclase distribute between caveolae and other cell fractions, whereas protein kinase A RI␣ subunits, G protein-coupled receptor kinase-2, and clathrin are largely excluded from caveolae. Cell surface ␤ 2 -ARs localize to caveolae in cardiomyocytes and cardiac fibroblasts (with markedly different ␤ 2 -AR expression levels), indicating that the fidelity of ␤ 2 -AR targeting to caveolae is maintained over a physiologic range of ␤ 2 -AR expression. In cardiomyocytes, agonist stimulation leads to a marked decline in the abundance of ␤ 2 -ARs (but not ␤ 1 -ARs) in caveolae. Other studies show co-immunoprecipitation of cardiomyocytes adenylyl cyclase V/VI and caveolin-3, suggesting their in vivo association. However, caveolin is not required for adenylyl cyclase targeting to low density membranes, since adenylyl cyclase targets to low buoyant density membrane fractions of HEK cells that lack prototypical caveolins. Nevertheless, cholesterol depletion with cyclodextrin augments agonist-stimulated cAMP accumulation, indicating that caveolae function as negative regulators of cAMP accumulation. The inhibitory interaction between caveolae and the cAMP signaling pathway as well as domainspecific differences in the stoichiometry of individual elements in the ␤-AR signaling cascade represent important modifiers of cAMP-dependent signaling in the heart.Catecholamines act through cardiac ␤-adrenergic receptors (␤-ARs) 1 to influence the contractile state of the heart. The direct inotropic and chronotropic support provided by cardiac ␤-ARs represents a critical compensatory mechanism to preserve cardiac function during stress and/or states associated with circulatory compromise. In the hearts of most mammalian species, the physiologic effects of catecholamines are mediated by the predominant ␤ 1 -AR subtype (75-80% of the total ␤-ARs), which activates a signaling pathway involving the G sdependent stimulation of adenylyl cyclase leading to the accumulation of cAMP and protein kinase A-dependent phosphorylation of key target proteins. Cardiomyocytes also express ␤ 2 -ARs that support contractile function. Until quite recently, most studies of ␤ 2 -AR signaling in cardiomyocytes were wedded to the concept that ␤ 2 -ARs signal to the G s /cAMP pathway in a manner that is essentially equivalent to the pathway activated by ␤ 1 -ARs. However, there is evidence that ␤ 2 -ARs are not functionally redundant, including the findings that ␤ 2 -ARs ...

There is a growing body of evidence that G proteincoupled receptors function in the context of plasma membrane signaling compartments. These compartments may facilitate interaction between receptors and specific downstream signaling components while restricting access to other signaling molecules. We recently reported that ␤ 1 -and ␤ 2 -adrenergic receptors (AR) regulate the intrinsic contraction rate in neonatal mouse myocytes through distinct signaling pathways. By studying neonatal myocytes isolated from ␤ 1 AR and ␤ 2 AR knockout mice, we found that stimulation of the ␤ 1 AR leads to a protein kinase A-dependent increase in the contraction rate. In contrast, stimulation of the ␤ 2 AR has a biphasic effect on the contraction rate. The biphasic effect includes an initial protein kinase A-independent increase in the contraction rate followed by a sustained decrease in the contraction rate that can be blocked by pertussis toxin. Here we present evidence that caveolar localization is required for physiologic signaling by the ␤ 2 AR but not the ␤ 1 AR in neonatal cardiac myocytes. Evidence for ␤ 2 AR localization to caveolae includes co-localization by confocal imaging, co-immunoprecipitation of the ␤ 2 AR and caveolin 3, and co-migration of the ␤ 2 AR with a caveolin-3-enriched membrane fraction. The ␤ 2 AR-stimulated increase in the myocyte contraction rate is increased by ϳ2-fold and markedly prolonged by filipin, an agent that disrupts lipid rafts such as caveolae and significantly reduces co-immunoprecipitation of ␤ 2 AR and caveolin 3 and comigration of ␤ 2 AR and caveolin-3 enriched membranes. In contrast, filipin has no effect on ␤ 1 AR signaling. These observations suggest that ␤ 2 ARs are normally restricted to caveolae in myocyte membranes and that this localization is essential for physiologic signaling of this receptor subtype.Catecholamines act through cardiac ␤-adrenergic receptors (␤ARs) 1 to modulate heart rate and contractility. Three ␤AR subtypes have been cloned (␤ 1 AR, ␤ 2 AR, and ␤ 3 AR). ␤ 1 AR and ␤ 2 AR are the primary subtypes responsible for cardiac response to catecholamines. ␤ 1 AR and ␤ 2 AR are also pharmacologically more similar to each other than they are to the ␤ 3 AR. The close structural and functional properties of ␤ 1 AR and ␤ 2 AR are paradigmatic of many other G protein-coupled receptor families in which two or more receptor subtypes respond to the same hormone or neurotransmitter and couple to the same effector systems. Although ␤ 1 AR and ␤ 2 AR have very similar signaling properties when expressed in undifferentiated cell lines (1), there is a growing body of experimental evidence that suggests that they have different signaling properties in regulating cardiac function. The ␤ 1 AR knockout (␤ 1 AR-KO) mice lack the normal chronotropic and inotropic responses to the non-selective agonist isoproterenol (2). Thus, in the murine heart, ␤ 2 ARs play no significant role in controlling heart rate and contractility. ␤ 2 AR knockout (␤ 2 AR-KO) mice have normal inotropic and chr...

To determine whether age-dependent differences in cardiac responses to autonomic agonists could result from developmental changes in protein kinase C (PKC) isoform expression, we probed extracts from the fetal, neonatal, and adult heart as well as cultured neonatal and isolated adult ventricular myocytes with specific antisera to calcium-dependent (alpha and beta) and calcium-independent (delta, epsilon and zeta) isoforms of the enzyme. Although PKC-beta immunoreactivity could not be detected in cultured neonatal or isolated adult ventricular myocytes, adult and neonatal myocytes expressed multiple other isoforms of PKC. Our studies revealed an age-dependent decline in the immunoreactivity for three PKC isoforms. PKC-alpha was detected in extracts from the fetal and 2-day-old neonatal heart as well as cultured neonatal rat ventricular myocytes. Only faint PKC-alpha immunoreactivity was detected in extracts from the adult heart, and PKC-alpha was not detected in extracts from isolated adult ventricular myocytes, suggesting that PKC-alpha resides in nonmyocyte elements in the adult heart. PKC-delta also was detected in greater abundance in fetal and neonatal than in adult myocardial extracts. The decline in PKC-alpha and PKC-delta expression occurred during the first 2 postnatal weeks. PKC-zeta was detected in greatest abundance in extracts from the fetal heart. PKC-zeta expression declined markedly by the second postnatal day, and only faint PKC-zeta immunoreactivity was detected in extracts from adult myocardium. Failure to detect PKC-zeta in extracts from isolated adult ventricular myocytes suggests that PKC-zeta resides primarily in nonmyocyte elements in the adult heart. PKC-epsilon was detected in all preparations, but it was detected in greatest abundance in extracts from neonatal hearts. In vitro sympathetic innervation of previously noninnervated neonatal ventricular myocytes or in vivo chemical sympathectomy of the neonatal heart did not modulate PKC isoform expression, suggesting that sympathetic innervation does not significantly regulate PKC isoform expression. PKC-alpha partitioned to the soluble fraction of unstimulated myocytes and was selectively translocated to the particulate fraction by Ca2+. In contrast, a major portion of the novel PKC isoforms partitioned to the particulate fraction of unstimulated myocytes. The subcellular distribution of novel PKC isoforms was not influenced by Ca2+. 12-O-Tetradecanoylphorbol 13-acetate (TPA, 300 nmol/L) induced translocation of soluble PKC-alpha, PKC-delta, and PKC-epsilon to the particulate fraction at 30 minutes and complete (PKC-alpha and PKC-delta) or 80% (PKC-epsilon) downregulation at 24 hours. PKC-zeta was not affected by TPA.(ABSTRACT TRUNCATED AT 400 WORDS)

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Abstract-Protein kinase C (PKC) isoforms constitute an important component of the signal transduction pathway used by cardiomyocytes to respond to a variety of extracellular stimuli. Translocation to distinct intracellular sites represents an essential step in the activation of PKC isoforms, presumably as a prerequisite for stable access to substrate. Caveolae are specialized subdomains of the plasma membrane that are reported to concentrate key signaling proteins and may represent a locus for PKC action, given that PKC activators have been reported to dramatically alter caveolae morphology. Accordingly, this study examines whether PKC isoforms initiate signaling in cardiomyocyte caveolae. Phorbol ester-sensitive PKC isoforms were detected at very low levels in caveolae fractions prepared from unstimulated cardiomyocytes; phorbol 12-myristate 13-acetate (PMA) (but not 4␣-PMA, which does not activate PKC) recruited calcium-sensitive PKC␣ and novel PKC␦ and PKC⑀ to this compartment. The subcellular localization of the phorbol ester-insensitive PKC isoform was not influenced by PMA. Endothelin also induced the selective translocation of PKC␣ and PKC⑀ (but not PKC␦ or PKC) to caveolae. Multiple components of the extracellular signal-regulated protein kinase (ERK) cascade, including A-Raf, c-Raf-1, mitogen-activated protein kinase kinase, and ERK, were detected in caveolae under resting conditions. Although levels of these proteins were not altered by PMA, translocation of phorbol ester-sensitive PKC isoforms to caveolae was associated with the activation of a local ERK cascade as well as the phosphorylation of a Ϸ36-kDa substrate protein in this fraction. Finally, a minor fraction of a protein that has been designated as a receptor for activated protein kinase C resides in caveolae and (along with caveolin-3) could represent a mechanism to target PKC isoforms to cardiomyocyte caveolae. These studies identify cardiomyocyte caveolae as a meeting place for activated PKC isoforms and their downstream target substrates. (Circ Res. 1999;84:980-988.)

Protein kinase C (PKC) isoforms play key roles in the regulation of cardiac contraction, ischemic preconditioning, and hypertrophy/failure. Models of PKC activation generally focus on lipid cofactor-induced PKC translocation to membranes. This study identifies tyrosine phosphorylation as an additional mechanism that regulates PKC␦ actions in cardiomyocytes. Rather, tyrosine phosphorylation regulates PKC␦ kinase activity. PKC␦ is recovered from the soluble fraction of H 2 O 2 -treated cardiomyocytes as a tyrosine-phosphorylated, lipid-independent enzyme with altered substrate specificity. In vitro PKC␦ phosphorylation by Src also increases lipid-independent kinase activity. The magnitude of this effect varies, depending upon the substrate, suggesting that tyrosine phosphorylation fine-tunes PKC␦ substrate specificity. The stimulus-specific modes for PKC␦ signaling identified in this study allow for distinct PKC␦-mediated phosphorylation events and responses during growth factor stimulation and oxidant stress in cardiomyocytes. Protein kinase C (PKC)1 comprises a multigene family of at least 10 structurally distinct phospholipid-dependent serinethreonine kinases that regulate cardiac contraction, play a role in ischemic preconditioning, and contribute to the pathogenesis of cardiac hypertrophy and heart failure (1, 2). PKC isoforms are single polypeptide chains with structurally homologous C-terminal catalytic domains and more variable N-terminal regulatory domains. This diverse group of enzymes is subdivided into three distinct subfamilies based upon structural differences in their N-terminal regulatory domain that confer distinct patterns of cofactor activation. Conventional PKC isoforms (cPKCs; ␣, ␤I, ␤II, ␥) contain an autoinhibitory pseudosubstrate domain followed by membrane-targeting C1 and C2 domains that are regulated by diacylglycerol (DAG) and calcium, respectively. Novel PKCs (nPKCs; ␦, ⑀, , and ) lack a calcium-binding C2 domain and are maximally activated by DAG and phorbol ester, in the absence of calcium. Atypical PKCs (aPKCs; and /) are regulated by lipids, but are not activated by second messengers such as calcium and DAG. Current models of PKC isoform activation in the heart have focused largely on the conformational changes induced by cofactor interactions with N-terminal membrane-targeting modules that anchor the enzyme to membranes, expel the autoinhibitory pseudosubstrate domain from the substrate-binding pocket, and thereby relieve autoinhibition. According to this model, individual PKC isoforms elicit distinct (and occasionally functionally opposing) cellular responses as a result of cofactorinduced compartmentation to distinct membrane subdomains, in close proximity to their unique sets of target protein substrates (1).Recent studies identify an additional mechanism for PKC regulation via sequential phosphorylations on a conserved threonine in the activation loop and two conserved serine/threonines in turn and hydrophobic motifs in the C terminus (3). For cPKCs, these phosphorylation even...

PKC␦ leads to depressed maximum tension and cross-bridge kinetics, attributable to a dominant effect of cTnI-Thr144 phosphorylation. Our data implicate PKC␦-Tyr 311 /Thr 505 phosphorylation as dynamically regulated modifications that alter PKC␦ enzymology and allow for stimulus-specific control of cardiac mechanics during growth factor stimulation and oxidative stress. Protein kinase C␦ (PKC␦)2 is a ubiquitous serine/threonine kinase implicated in a wide range of cellular responses (1, 2). PKC␦ is conventionally viewed as a lipid-dependent enzyme that is anchored to membranes in close proximity to target substrates through interactions with lipid cofactors. However, there is recent evidence that PKC␦ also is dynamically regulated through activation loop (Thr 505 ) phosphorylation (3, 4). For other PKC isoforms, activation loop phosphorylation is a stable "priming" phosphorylation completed during de novo enzyme synthesis (5). In the case of cPKCs, activation loop phosphorylations are mediated by phosphoinositide-dependent kinase-1 and are essential to generate a catalytically competent enzyme. Although newly synthesized PKC␦ also undergoes maturational phosphoinositide-dependent kinase-1-dependent Thr 505 phosphorylation, PKC␦ differs from other PKC isoforms in that 1) PKC␦ is a catalytically active enzyme even without Thr 505 phosphorylation and 2) PKC␦-Thr 505 phosphorylation is dynamically regulated through an autocatalytic mechanism (4). Although there are hints that Thr 505 phosphorylation might contribute to the control of PKC␦ enzymology, a PKC␦-Thr 505 autophosphorylation mechanism that regulates the actions of PKC␦ toward a physiologically relevant substrate in a differentiated cell has never been reported.PKC␦ also is regulated through tyrosine phosphorylation. However, the consequences of PKC␦ tyrosine phosphorylation remain disputed, because PKC␦ tyrosine phosphorylation is variably linked to increased, decreased, or unchanged PKC␦ activity (1). Inconsistencies in the literature have been attributed to the presence of multiple tyrosine residues throughout the structure of PKC␦ (including