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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