Caveolin-3 Overexpression Attenuates Cardiac Hypertrophy via Inhibition of T-type Ca2+ Current Modulated by Protein Kinase C? in Cardiomyocytes.
ABSTRACT: Pathological cardiac hypertrophy is characterized by subcellular remodeling of the ventricular myocyte with a reduction in the scaffolding protein caveolin-3 (Cav-3), altered Ca(2+) cycling, increased protein kinase C expression, and hyperactivation of calcineurin/nuclear factor of activated T cell (NFAT) signaling. However, the precise role of Cav-3 in the regulation of local Ca(2+) signaling in pathological cardiac hypertrophy is unclear. We used cardiac-specific Cav-3-overexpressing mice and in vivo and in vitro cardiac hypertrophy models to determine the essential requirement for Cav-3 expression in protection against pharmacologically and pressure overload-induced cardiac hypertrophy. Transverse aortic constriction and angiotensin-II (Ang-II) infusion in wild type (WT) mice resulted in cardiac hypertrophy characterized by significant reduction in fractional shortening, ejection fraction, and a reduced expression of Cav-3. In addition, association of PKC? and angiotensin-II receptor, type 1, with Cav-3 was disrupted in the hypertrophic ventricular myocytes. Whole cell patch clamp analysis demonstrated increased expression of T-type Ca(2+) current (ICa, T) in hypertrophic ventricular myocytes. In contrast, the Cav-3-overexpressing mice demonstrated protection from transverse aortic constriction or Ang-II-induced pathological hypertrophy with inhibition of ICa, T and intact Cav-3-associated macromolecular signaling complexes. siRNA-mediated knockdown of Cav-3 in the neonatal cardiomyocytes resulted in enhanced Ang-II stimulation of ICa, T mediated by PKC?, which caused nuclear translocation of NFAT. Overexpression of Cav-3 in neonatal myocytes prevented a PKC?-mediated increase in ICa, T and nuclear translocation of NFAT. In conclusion, we show that stable Cav-3 expression is essential for protecting the signaling mechanisms in pharmacologically and pressure overload-induced cardiac hypertrophy.
Project description:Angiotensin (Ang) II participates in the pathogenesis of heart failure through induction of cardiac hypertrophy. Ang II-induced hypertrophic growth of cardiomyocytes is mediated by nuclear factor of activated T cells (NFAT), a Ca(2+)-responsive transcriptional factor. It is believed that phospholipase C (PLC)-mediated production of inositol-1,4,5-trisphosphate (IP(3)) is responsible for Ca(2+) increase that is necessary for NFAT activation. However, we demonstrate that PLC-mediated production of diacylglycerol (DAG) but not IP(3) is essential for Ang II-induced NFAT activation in rat cardiac myocytes. NFAT activation and hypertrophic responses by Ang II stimulation required the enhanced frequency of Ca(2+) oscillation triggered by membrane depolarization through activation of DAG-sensitive TRPC channels, which leads to activation of L-type Ca(2+) channel. Patch clamp recordings from single myocytes revealed that Ang II activated DAG-sensitive TRPC-like currents. Among DAG-activating TRPC channels (TRPC3, TRPC6, and TRPC7), the activities of TRPC3 and TRPC6 channels correlated with Ang II-induced NFAT activation and hypertrophic responses. These data suggest that DAG-induced Ca(2+) signaling pathway through TRPC3 and TRPC6 is essential for Ang II-induced NFAT activation and cardiac hypertrophy.
Project description:The RNA binding protein Human antigen R (HuR) interacts with specific AU-rich domains in target mRNAs and is highly expressed in many cell types, including cardiomyocytes. However, the role of HuR in cardiac physiology is largely unknown. Our results show that HuR undergoes cytoplasmic translocation, indicative of its activation, in hypertrophic cardiac myocytes. Specifically, HuR cytoplasmic translocation is significantly increased in NRVMs (neonatal rat ventricular myocytes) following treatment with phenylephrine or angiotensin II, agonists of two independent G?q-coupled GPCRs known to induce hypertrophy. This Gq-mediated HuR activation is dependent on p38 MAP kinase, but not canonical Gq-PKC signaling. Furthermore, we show that HuR activation is necessary for Gq-mediated hypertrophic growth of NRVMs as siRNA-mediated knockdown of HuR inhibits hypertrophy as measured by cell size and expression of ANF (atrial natriuretic factor). Additionally, HuR overexpression is sufficient to induce hypertrophic cell growth. To decipher the downstream mechanisms by which HuR translocation promotes cardiomyocyte hypertrophy, we assessed the role of HuR in the transcriptional activity of NFAT (nuclear factor of activated T cells), the activation of which is a hallmark of cardiac hypertrophy. Using an NFAT-luciferase reporter assay, we show an acute inhibition of NFAT transcriptional activity following pharmacological inhibition of HuR. In conclusion, our results identify HuR as a novel mediator of cardiac hypertrophy downstream of the Gq-p38 MAPK pathway, and suggest modulation of NFAT activity as a potential mechanism.
Project description:There is increasing evidence that angiotensin II (Ang II) is associated with the occurrence of ventricular arrhythmias. However, little is known about the electrophysiological effects of Ang II on ventricular repolarization. The rapid component of the delayed rectifier K(+) current (I(Kr)) plays a critical role in cardiac repolarization. Hence, the aim of this study was to assess the effect of Ang II on I(Kr) in guinea-pig ventricular myocytes.The whole-cell patch-clamp technique was used to record I(Kr) in native cardiocytes and in human embryonic kidney (HEK) 293 cells, co-transfected with human ether-a-go-go-related gene (hERG) encoding the alpha-subunit of I(Kr) and the human Ang II type 1 (AT(1)) receptor gene.Ang II decreased the amplitude of I(Kr) in a concentration-dependent manner with an IC(50) of 8.9 nM. Action potential durations at 50% (APD(50)) and 90% (APD(90)) repolarization were prolonged 20% and 16%, respectively by Ang II (100 nM). Ang II-induced inhibition of the I(Kr) was abolished by the AT(1) receptor blocker, losartan (1 muM). Ang II decreased hERG current in HEK293 cells and significantly delayed channel activation, deactivation and recovery from inactivation. Moreover, PKC inhibitors, stausporine and Bis-1, significantly attenuated Ang II-induced inhibition of I(Kr).Ang II produces an inhibitory effect on I(Kr)/hERG currents via AT(1) receptors linked to the PKC pathway in ventricular myocytes. This is a potential mechanism by which elevated levels of Ang II are involved in the occurrence of arrhythmias in cardiac hypertrophy and failure.
Project description:Previous studies have demonstrated that calcium-/calmodulin-dependent protein kinase II (CaMKII) and calcineurin A-nuclear factor of activated T-cell (CnA-NFAT) signaling pathways play key roles in cardiac hypertrophy (CH). However, the interaction between CaMKII and CnA-NFAT signaling remains unclear. H9c2 cells were cultured and treated with angiotensin II (Ang II) with or without silenced CaMKII? (siCaMKII) and cyclosporine A (CsA, a calcineurin inhibitor) and subsequently treated with Wenxin Keli (WXKL). Patch clamp recording was conducted to assess L-type Ca2+ current (ICa-L), and the expression of proteins involved in signaling pathways was measured by western blotting. Myocardial cytoskeletal protein and nuclear translocation of target proteins were assessed by immunofluorescence. The results indicated that siCaMKII suppressed Ang II-induced CH, as evidenced by reduced cell surface area and ICa-L. Notably, siCaMKII inhibited Ang II-induced activation of CnA and NFATc4 nuclear transfer. Inflammatory signaling was inhibited by siCaMKII and WXKL. Interestingly, CsA inhibited CnA-NFAT pathway expression but activated CaMKII signaling. In conclusion, siCaMKII may improve CH, possibly by blocking CnA-NFAT and MyD88 signaling, and WXKL has a similar effect. These data suggest that inhibiting CaMKII, but not CnA, may be a promising approach to attenuate CH and arrhythmia progression.
Project description:Recent studies have demonstrated that urotensin-II (U-II) plays important roles in cardiovascular actions including cardiac positive inotropic effects and increasing cardiac output. However, the mechanisms underlying these effects of U-II in cardiomyocytes still remain unknown. We show by electrophysiological studies that U-II dose-dependently potentiates L-type Ca(2+) currents (ICa,L) in adult rat ventricular myocytes. This effect was U-II receptor (U-IIR)-dependent and was associated with a depolarizing shift in the voltage dependence of inactivation. Intracellular application of guanosine-5'-O-(2-thiodiphosphate) and pertussis toxin pretreatment both abolished the stimulatory effects of U-II. Dialysis of cells with the QEHA peptide, but not scrambled peptide SKEE, blocked the U-II-induced response. The phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin as well as the class I PI3K antagonist CH132799 blocked the U-II-induced ICa,L response. Protein kinase C antagonists calphostin C and chelerythrine chloride as well as dialysis of cells with 1,2bis(2aminophenoxy)ethaneN,N,N',N'-tetraacetic acid abolished the U-II-induced responses, whereas PKC? inhibition or PKA blockade had no effect. Exposure of ventricular myocytes to U-II markedly increased membrane PKC?1 expression, whereas inhibition of PKC?1 pharmacologically or by shRNA targeting abolished the U-II-induced ICa,L response. Functionally, we observed a significant increase in the amplitude of sarcomere shortening induced by U-II; blockade of U-IIR as well as PKC? inhibition abolished this effect, whereas Bay K8644 mimicked the U-II response. Taken together, our results indicate that U-II potentiates ICa,L through the ?? subunits of Gi/o-protein and downstream activation of the class I PI3K-dependent PKC?1 isoform. This occurred via the activation of U-IIR and contributes to the positive inotropic effect on cardiomyocytes.
Project description:The increase in protein activity and upregulation of G-protein coupled receptor kinase 2 (GRK2) is a hallmark of cardiac stress and heart failure. Inhibition of GRK2 improved cardiac function and survival and diminished cardiac remodeling in various animal heart failure models. The aim of the present study was to investigate the effects of GRK2 on cardiac hypertrophy and dissect potential molecular mechanisms. In mice we observed increased GRK2 mRNA and protein levels following transverse aortic constriction (TAC). Conditional GRK2 knockout mice showed attenuated hypertrophic response with preserved ventricular geometry 6 weeks after TAC operation compared to wild-type animals. In isolated neonatal rat ventricular cardiac myocytes stimulation with angiotensin II and phenylephrine enhanced GRK2 expression leading to enhanced signaling via protein kinase B (PKB or Akt), consecutively inhibiting glycogen synthase kinase 3 beta (GSK3?), such promoting nuclear accumulation and activation of nuclear factor of activated T-cells (NFAT). Cardiac myocyte hypertrophy induced by in vitro GRK2 overexpression increased the cytosolic interaction of GRK2 and phosphoinositide 3-kinase ? (PI3K?). Moreover, inhibition of PI3K? as well as GRK2 knock down prevented Akt activation resulting in halted NFAT activity and reduced cardiac myocyte hypertrophy. Our data show that enhanced GRK2 expression triggers cardiac hypertrophy by GRK2-PI3K? mediated Akt phosphorylation and subsequent inactivation of GSK3?, resulting in enhanced NFAT activity.
Project description:Cyclic nucleotide phosphodiesterases (PDEs) through the degradation of cGMP play critical roles in maintaining cardiomyocyte homeostasis. Ca(2+)/calmodulin (CaM)-activated cGMP-hydrolyzing PDE1 family may play a pivotal role in balancing intracellular Ca(2+)/CaM and cGMP signaling; however, its function in cardiomyocytes is unknown.Herein, we investigate the role of Ca(2+)/CaM-stimulated PDE1 in regulating pathological cardiomyocyte hypertrophy in neonatal and adult rat ventricular myocytes and in the heart in vivo.Inhibition of PDE1 activity using a PDE1-selective inhibitor, IC86340, or downregulation of PDE1A using siRNA prevented phenylephrine induced pathological myocyte hypertrophy and hypertrophic marker expression in neonatal and adult rat ventricular myocytes. Importantly, administration of the PDE1 inhibitor IC86340 attenuated cardiac hypertrophy induced by chronic isoproterenol infusion in vivo. Both PDE1A and PDE1C mRNA and protein were detected in human hearts; however, PDE1A expression was conserved in rodent hearts. Moreover, PDE1A expression was significantly upregulated in vivo in the heart and myocytes from various pathological hypertrophy animal models and in vitro in isolated neonatal and adult rat ventricular myocytes treated with neurohumoral stimuli such as angiotensin II (Ang II) and isoproterenol. Furthermore, PDE1A plays a critical role in phenylephrine-induced reduction of intracellular cGMP- and cGMP-dependent protein kinase (PKG) activity and thereby cardiomyocyte hypertrophy in vitro.These results elucidate a novel role for Ca(2+)/CaM-stimulated PDE1, particularly PDE1A, in regulating pathological cardiomyocyte hypertrophy via a cGMP/PKG-dependent mechanism, thereby demonstrating Ca(2+) and cGMP signaling cross-talk during cardiac hypertrophy.
Project description:Although angiotensin II (Ang II) plays an important role in heart disease associated with pump dysfunction, its direct effects on cardiac pump function remain controversial. We found that after Ang II infusion, the developed pressure and +dP/dt(max) in isolated Langendorff-perfused mouse hearts showed a complex temporal response, with a rapid transient decrease followed by an increase above baseline. Similar time-dependent changes in cell shortening and L-type Ca(2+) currents were observed in isolated ventricular myocytes. Previous studies have established that Ang II signaling involves phosphoinositide 3-kinases (PI3K). Dominant-negative inhibition of PI3Kalpha in the myocardium selectively eliminated the rapid negative inotropic action of Ang II (inhibited by approximately 90%), whereas the loss of PI3Kgamma had no effect on the response to Ang II. Consistent with a link between PI3Kalpha and protein kinase C (PKC), PKC inhibition (with GF 109203X) reduced the negative inotropic effects of Ang II by approximately 50%. Although PI3Kalpha and PKC activities are associated with glycogen synthase kinase-3beta and NADPH oxidase, genetic ablation of either glycogen synthase kinase-3beta or p47(phox) (an essential subunit of NOX2-NADPH oxidase) had no effect on the inotropic actions of Ang II. Our results establish that Ang II has complex temporal effects on contractility and L-type Ca(2+) channels in normal mouse myocardium, with the negative inotropic effects requiring PI3Kalpha and PKC activities.
Project description:Angiotensin II (Ang II) is a major determinant of inward remodeling and hypertrophy in pial arterioles that may have an important role in stroke during chronic hypertension. Previously, we found that epidermal growth factor receptor is critical in Ang II-mediated hypertrophy that may involve caveolin-1 (Cav-1). In this study, we examined the effects of Cav-1 and matrix metalloproteinase-9 (MMP9) on Ang II-mediated structural changes in pial arterioles. Cav-1-deficient (Cav-1(-/-)), MMP9-deficient (MMP9(-/-)), and wild-type mice were infused with either Ang II (1000 ng/kg per minute) or saline via osmotic minipumps for 28 days (n=6-8 per group). Systolic arterial pressure was measured by a tail-cuff method. Pressure and diameter of pial arterioles were measured through an open cranial window in anesthetized mice. Cross-sectional area of the wall was determined histologically in pressurized fixed pial arterioles. Expression of Cav-1, MMP9, phosphorylated epidermal growth factor receptor, and Akt was determined by Western blotting and immunohistochemistry. Deficiency of Cav-1 or MMP9 did not affect Ang II-induced hypertension. Ang II increased the expression of Cav-1, phosphorylated epidermal growth factor receptor, and Akt in wild-type mice, which was attenuated in Cav-1(-/-) mice. Ang II-induced hypertrophy, inward remodeling, and increased MMP9 expression in pial arterioles were prevented in Cav-1(-/-) mice. Ang II-mediated increases in MMP9 expression and inward remodeling, but not hypertrophy, were prevented in MMP9(-/-) mice. In conclusion, Cav-1 is essential in Ang II-mediated inward remodeling and hypertrophy in pial arterioles. Cav-1-induced MMP9 is exclusively involved in inward remodeling, not hypertrophy. Further studies are needed to determine the role of Akt in Ang II-mediated hypertrophy.
Project description:Adiponectin and miR-133a are key regulators in cardiac hypertrophy. However, whether APN has a potential effect on miR-133a remains unclear. In this study, we aimed to investigate whether APN could regulate miR-133a expression in Angiotensin II (Ang II) induced cardiac hypertrophy in vivo and in vitro. Lentiviral-mediated adiponectin treatment attenuated cardiac hypertrophy induced by Ang II infusion in male wistar rats as determined by reduced cell surface area and mRNA levels of atrial natriuretic peptide (ANF) and brain natriuretic peptide (BNP), also the reduced left ventricular end-diastolic posterior wall thickness (LVPWd) and end-diastolic interventricular septal thickness (IVSd). Meanwhile, APN elevated miR-133a level which was downregulated by Ang II. To further investigate the underlying molecular mechanisms, we treated neonatal rat ventricular myocytes (NRVMs) with recombinant rat APN before Ang II stimulation. Pretreating cells with recombinant APN promoted AMP-activated protein kinase (AMPK) phosphorylation and inhibited ERK activation. By using the inhibitor of AMPK or a lentiviral vector expressing AMPK short hairpin RNA (shRNA) cancelled the positive effect of APN on miR-133a. The ERK inhibitor PD98059 reversed the downregulation of miR-133a induced by Ang II. These results indicated that the AMPK activation and ERK inhibition were responsible for the positive effect of APN on miR-133a. Furthermore, adiponectin receptor 1 (AdipoR1) mRNA expression was inhibited by Ang II stimulation. The positive effects of APN on AMPK activation and miR-133a, and the inhibitory effect on ERK phosphorylation were inhibited in NRVMs transfected with lentiviral AdipoR1shRNA. In addition, APN depressed the elevated expression of connective tissue growth factor (CTGF), a direct target of miR-133a, through the AMPK pathway. Taken together, our data indicated that APN reversed miR-133a levels through AMPK activation, reduced ERK1/2 phosphorylation in cardiomyocytes stimulated with Ang II, revealing a previously undemonstrated and important link between APN and miR-133a.