Sathyamangla V. Naga Prasad

β-Arrestin–mediated β1-adrenergic receptor transactivation of the EGFR confers cardioprotection

Deleterious effects on the heart from chronic stimulation of β-adrenergic receptors (βARs), members of the 7 transmembrane receptor family, have classically been shown to result from Gs-dependent adenylyl cyclase activation. Here, we identify a new signaling mechanism using both in vitro and in vivo systems whereby β-arrestins mediate β1AR signaling to the EGFR. This β-arrestin–dependent transactivation of the EGFR, which is independent of G protein activation, requires the G protein–coupled receptor kinases 5 and 6. In mice undergoing chronic sympathetic stimulation, this novel signaling pathway is shown to promote activation of cardioprotective pathways that counteract the effects of catecholamine toxicity. These findings suggest that drugs that act as classical antagonists for G protein signaling, but also stimulate signaling via β-arrestin–mediated cytoprotective pathways, would represent a novel class of agents that could be developed for multiple members of the 7 transmembrane receptor family.

Intermittent pressure overload triggers hypertrophy-independent cardiac dysfunction and vascular rarefaction

For over a century, there has been intense debate as to the reason why some cardiac stresses are pathological and others are physiological. One long-standing theory is that physiological overloads such as exercise are intermittent, while pathological overloads such as hypertension are chronic. In this study, we hypothesized that the nature of the stress on the heart, rather than its duration, is the key determinant of the maladaptive phenotype. To test this, we applied intermittent pressure overload on the hearts of mice and tested the roles of duration and nature of the stress on the development of cardiac failure. Despite a mild hypertrophic response, preserved systolic function, and a favorable fetal gene expression profile, hearts exposed to intermittent pressure overload displayed pathological features. Importantly, intermittent pressure overload caused diastolic dysfunction, altered beta-adrenergic receptor (betaAR) function, and vascular rarefaction before the development of cardiac hypertrophy, which were largely normalized by preventing the recruitment of PI3K by betaAR kinase 1 to ligand-activated receptors. Thus stress-induced activation of pathogenic signaling pathways, not the duration of stress or the hypertrophic growth per se, is the molecular trigger of cardiac dysfunction.

Phosphoinositide 3-kinase regulates β2-adrenergic receptor endocytosis by AP-2 recruitment to the receptor/β-arrestin complex

Internalization of β-adrenergic receptors (βARs) occurs by the sequential binding of β-arrestin, the clathrin adaptor AP-2, and clathrin. D-3 phosphoinositides, generated by the action of phosphoinositide 3-kinase (PI3K) may regulate the endocytic process; however, the precise molecular mechanism is unknown. Here we demonstrate that βARKinase1 directly interacts with the PIK domain of PI3K to form a cytosolic complex. Overexpression of the PIK domain displaces endogenous PI3K from βARK1 and prevents βARK1-mediated translocation of PI3K to activated β2ARs. Furthermore, disruption of the βARK1/PI3K interaction inhibits agonist-stimulated AP-2 adaptor protein recruitment to the β2AR and receptor endocytosis without affecting the internalization of other clathrin dependent processes such as internalization of the transferrin receptor. In contrast, AP-2 recruitment is enhanced in the presence of D-3 phospholipids, and receptor internalization is blocked in presence of the specific phosphatidylinositol-3,4,5-trisphosphate lipid phosphatase PTEN. These findings provide a molecular mechanism for the agonist-dependent recruitment of PI3K to βARs, and support a role for the localized generation of D-3 phosphoinositides in regulating the recruitment of the receptor/cargo to clathrin-coated pits.

Cardiac Overexpression of a Gq Inhibitor Blocks Induction of Extracellular Signal–Regulated Kinase and c-Jun NH2-Terminal Kinase Activity in In Vivo Pressure Overload

Background —Understanding the cellular signals that initiate cardiac hypertrophy is of critical importance in identifying the pathways that mediate heart failure. The family of mitogen-activated protein kinases (MAPKs), including the extracellular signal–regulated kinases (ERKs), c-Jun NH2-terminal kinase (JNK), and p38 MAPKs, may play specific roles in myocardial growth and function. Methods and Results —To determine the mechanism of activation of MAPK pathways during the development of cardiac hypertrophy, we evaluated the induction of MAPK activity after aortic constriction in wild-type and in 2 types of cardiac gene-targeted mice: one overexpressing a carboxyl-terminal peptide of G&agr;q that inhibits Gq-mediated signaling (TG GqI mouse) and another overexpressing a carboxyl-terminal peptide of &bgr;-adrenergic receptor kinase-1 that inhibits G&bgr;&ggr; signaling (TG &bgr;ARKct mouse). Wild-type mice with pressure overload showed an acute induction of JNK, followed by the induction of p38/p38&bgr; at 3 days and ERK at 7 days. Both JNK and p38 activity remained elevated at 7 days after banding. In TG GqI mice, hypertrophy was significantly attenuated, and induction of ERK and JNK activity was abolished, whereas the induction of p38 and p38&bgr; was robust, but delayed. By contrast, all 3 MAPK pathways were activated by aortic constriction in the TG &bgr;ARKct hearts, suggesting a role for G&agr;q, but not G&bgr;&ggr;. Conclusions —Taken together, these data show that the induction of ERK and JNK activity in in vivo pressure-overload hypertrophy is mediated through the stimulation of Gq-coupled receptors and that non–Gq-mediated pathways are recruited to activate p38 and p38&bgr;.

Protein kinase activity of phosphoinositide 3-kinase regulates β-adrenergic receptor endocytosis

Phosphoinositide 3-kinase (PI(3)K) is a unique enzyme characterized by both lipid and protein kinase activities. Here, we demonstrate a requirement for the protein kinase activity of PI(3)K in agonist-dependent β-adrenergic receptor (βAR) internalization. Using PI(3)K mutants with either protein or lipid phosphorylation activity, we identify the cytoskeletal protein non-muscle tropomyosin as a substrate of PI(3)K, which is phosphorylated in a wortmannin-sensitive manner on residue Ser 61. A constitutively dephosphorylated (S61A) tropomyosin mutant blocks agonist-dependent βAR internalization, whereas a tropomyosin mutant that mimics constitutive phosphorylation (S61D) compliments the PI(3)K mutant, with only lipid phosphorylation activity reversing the defective βAR internalization. Notably, knocking down endogenous tropomyosin expression using siRNAs that target different regions of tropomyosin resulted in complete inhibition of βAR endocytosis, showing that non-muscle tropomyosin is essential for agonist-mediated receptor internalization. These studies demonstrate a previously unknown role for the protein phosphorylation activity of PI(3)K in βAR internalization and identify non-muscle tropomyosin as a cellular substrate for protein kinase activity of PI(3)K.

Restoration of β-Adrenergic Receptor Signaling and Contractile Function in Heart Failure by Disruption of the βARK1/Phosphoinositide 3-Kinase Complex

Background—Desensitization and downregulation of myocardial &bgr;-adrenergic receptors (&bgr;ARs) are initiated by the increase in &bgr;AR kinase 1 (&bgr;ARK1) levels. By interacting with &bgr;ARK1 through the phosphoinositide kinase (PIK) domain, phosphoinositide 3-kinase (PI3K) is targeted to agonist-stimulated &bgr;ARs, where it regulates endocytosis. We tested the hypothesis that inhibition of receptor-targeted PI3K activity would alter receptor trafficking and ameliorate &bgr;AR signaling, ultimately improving contractility of failing cardiomyocytes. Methods and Results—To competitively displace PI3K from &bgr;ARK1, we generated mice with cardiac-specific overexpression of the PIK domain. Seven-day isoproterenol administration in wild-type mice induced desensitization of &bgr;ARs and their redistribution from the plasma membrane to early and late endosomes. In contrast, transgenic PIK overexpression prevented the redistribution of &bgr;ARs away from the plasma membrane and preserved their responsiveness to agonist. We further tested whether PIK overexpression could normalize already established &bgr;AR abnormalities and ameliorate contractile dysfunction in a large animal model of heart failure induced by rapid ventricular pacing in pigs. Failing porcine hearts showed increased &bgr;ARK1-associated PI3K activity and marked desensitization and redistribution of &bgr;ARs to endosomal compartments. Importantly, adenoviral gene transfer of the PIK domain in failing pig myocytes resulted in reduced receptor-localized PI3K activity and restored to nearly normal agonist-stimulated cardiomyocyte contractility. Conclusions—These data indicate that the heart failure state is associated with a maladaptive redistribution of &bgr;ARs away from the plasma membrane that can be counteracted through a strategy that targets the &bgr;ARK1/PI3K complex.

Hyperammonemia in cirrhosis induces transcriptional regulation of myostatin by an NF-κB–mediated mechanism

Significance Loss of skeletal muscle mass, or sarcopenia, is nearly universal in cirrhosis and adversely affects the outcome of these patients. There are no established therapies to prevent or reverse sarcopenia because the mechanisms are not known. We show that the expression of myostatin, a negative regulator of skeletal muscle mass, is increased in the cirrhotic muscle and is mediated by increased ammonia concentration. Skeletal muscle ammonia concentrations are significantly increased in cirrhosis, resulting in activation of the transcription factor NF-κB, which in turn increases the expression of myostatin. Given the high prevalence of cirrhosis, these studies are of broad general interest because ammonia-lowering strategies, NF-κB antagonists, and myostatin blocker are potential therapies to reverse sarcopenia of cirrhosis. Loss of muscle mass, or sarcopenia, is nearly universal in cirrhosis and adversely affects patient outcome. The underlying cross-talk between the liver and skeletal muscle mediating sarcopenia is not well understood. Hyperammonemia is a consistent abnormality in cirrhosis due to impaired hepatic detoxification to urea. We observed elevated levels of ammonia in both plasma samples and skeletal muscle biopsies from cirrhotic patients compared with healthy controls. Furthermore, skeletal muscle from cirrhotics had increased expression of myostatin, a known inhibitor of skeletal muscle accretion and growth. In vivo studies in mice showed that hyperammonemia reduced muscle mass and strength and increased myostatin expression in wild-type compared with postdevelopmental myostatin knockout mice. We postulated that hyperammonemia is an underlying link between hepatic dysfunction in cirrhosis and skeletal muscle loss. Therefore, murine C2C12 myotubes were treated with ammonium acetate resulting in intracellular concentrations similar to those in cirrhotic muscle. In this system, we demonstrate that hyperammonemia stimulated myostatin expression in a NF-κB–dependent manner. This finding was also observed in primary murine muscle cell cultures. Hyperammonemia triggered activation of IκB kinase, NF-κB nuclear translocation, binding of the NF-κB p65 subunit to specific sites within the myostatin promoter, and stimulation of myostatin gene transcription. Pharmacologic inhibition or gene silencing of NF-κB abolished myostatin up-regulation under conditions of hyperammonemia. Our work provides unique insights into hyperammonemia-induced myostatin expression and suggests a mechanism by which sarcopenia develops in cirrhotic patients.

Dynamic Regulation of Phosphoinositide 3-Kinase-γ Activity and β-Adrenergic Receptor Trafficking in End-Stage Human Heart Failure

Background— Downregulation of &bgr;-adrenergic receptors (&bgr;ARs) under conditions of heart failure requires receptor targeting of phosphoinositide 3-kinase (PI3K)–&ggr; and redistribution of &bgr;ARs into endosomal compartments. Because support with a left ventricular assist device (LVAD) results in significant improvement of cardiac function in humans, we investigated the effects of mechanical unloading on regulation of PI3K&ggr; activity and intracellular distribution of &bgr;ARs. Additionally, we tested whether displacement of PI3K&ggr; from activated &bgr;ARs would restore agonist responsiveness in failing human cardiomyocytes. Methods and Results— To test the role of PI3K on &bgr;AR endocytosis in failing human hearts, we assayed for PI3K activity in human left ventricular samples before and after mechanical unloading (LVAD). Before LVAD, failing human hearts displayed a marked increase in &bgr;AR kinase 1 (&bgr;ARK1)–associated PI3K activity that was attributed exclusively to enhanced activity of the PI3K&ggr; isoform. Increased &bgr;ARK1-coupled PI3K activity in the failing hearts was associated with downregulation of &bgr;ARs from the plasma membrane and enhanced sequestration into early and late endosomes compared with unmatched nonfailing controls. Importantly, LVAD support reversed PI3K&ggr; activation, normalized the levels of agonist-responsive &bgr;ARs at the plasma membrane, and depleted the &bgr;ARs from the endosomal compartments without changing the total number of receptors (sum of plasma membrane and early and late endosome receptors). To test whether the competitive displacement of PI3K from the &bgr;AR complex restored receptor responsiveness, we overexpressed the phosphoinositide kinase domain of PI3K (which disrupts &bgr;ARK1/PI3K interaction) in primary cultures of failing human cardiomyocytes. Adenoviral-mediated phosphoinositide kinase overexpression significantly increased basal contractility and rapidly reconstituted responsiveness to &bgr;-agonist. Conclusions— These results suggest a novel paradigm in which human &bgr;ARs undergo a process of intracellular sequestration that is dynamically reversed after LVAD support. Importantly, mechanical unloading leads to complete reversal in PI3K&ggr; and &bgr;ARK1-associated PI3K activation. Furthermore, displacement of active PI3K from &bgr;ARK1 restores &bgr;AR responsiveness in failing myocytes.

β-Adrenergic axis and heart disease

Beta-adrenergic receptors (beta-ARs) belong to a large family of G-protein-coupled receptors (GPCRs) that form the interface between the sympathetic nervous system and the cardiovascular system. The beta-AR signal system is one of the most powerful regulators of cardiac function, mediated by the effects of the sympathetic transmitters epinephrine and norepinephrine. In a number of cardiac diseases, however, the biology of beta-AR signaling pathways is altered dramatically. Here we discuss the role of beta-AR signaling in the normal and abnormal heart and how the use of genetically engineered mouse models has helped in our understanding of the pathophysiology of cardiac disease.

Agonist-stimulated β-Adrenergic Receptor Internalization Requires Dynamic Cytoskeletal Actin Turnover

Stimulation of β-adrenergic receptors (βARs) leads to sequential recruitment of β-arrestin, AP-2 adaptor protein, clathrin, and dynamin to the receptor complex, resulting in endocytosis. Whether a dynamic actin cytoskeleton is required for βAR endocytosis is not known. In this study, we have used β1- and β2 ARs, two ubiquitously expressed members of the βAR family, to comprehensively evaluate the requirement of the actin cytoskeleton in receptor internalization. The integrity of the actin cytoskeleton was manipulated with the agent latrunculin B (LB) and mutants of cofilin to depolymerize actin filaments. Treatment of cells with LB resulted in dose-dependent depolymerization of the cortical actin cytoskeleton that was associated with significant attenuation in internalization of β2ARs, β1ARs, and mutants of β1ARs that internalize via either clathrin- or caveolin-dependent pathways. Importantly, LB treatment did not inhibit β-arrestin translocation or dynamin recruitment to the agonist-stimulated receptor. To unequivocally demonstrate the requirement of the actin cytoskeleton for β2AR endocytosis, we used an actin-binding protein cofilin that biochemically depolymerizes and severs actin filaments. Isoproterenol-mediated internalization of β2AR was completely blocked in the presence of wild type cofilin, which could be rescued by a mutant of cofilin that mimics a constitutive phosphorylated state and leads to normal agonist-stimulated β2AR endocytosis. Finally, treatment with jasplakinolide, an inhibitor of actin turnover, resulted in dose-dependent inhibition of β2AR internalization, suggesting that turnover of actin filaments at the receptor complex is required for endocytosis. Taken together, these data demonstrate that intact and functional dynamic actin cytoskeleton is required for normal βAR internalization.