Search

Aims: Hyperactivity of Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) has emerged as a central cause of pathologic remodelling in heart failure. It has been suggested that CaMKII-induced hyperphosphorylation of the ryanodine receptor 2 (RyR2) and consequently increased diastolic Ca 2+ leak from the sarcoplasmic reticulum (SR) is a crucial mechanism by which increased CaMKII activity leads to contractile dysfunction. We aim to evaluate the relevance of CaMKII-dependent RyR2 phosphorylation for CaMKII-induced heart failure development in vivo., Methods and Results: We crossbred CaMKIIδC overexpressing [transgenic (TG)] mice with RyR2-S2814A knock-in mice that are resistant to CaMKII-dependent RyR2 phosphorylation. Ca 2+ -spark measurements on isolated ventricular myocytes confirmed the severe diastolic SR Ca 2+ leak previously reported in CaMKIIδC TG [4.65 ± 0.73 mF/F 0 vs. 1.88 ± 0.30 mF/F 0 in wild type (WT)]. Crossing in the S2814A mutation completely prevented SR Ca 2+ -leak induction in the CaMKIIδC TG, both regarding Ca 2+ -spark size and frequency, demonstrating that the CaMKIIδC-induced SR Ca 2+ leak entirely depends on the CaMKII-specific RyR2-S2814 phosphorylation. Yet, the RyR2-S2814A mutation did not affect the massive contractile dysfunction (ejection fraction = 12.17 ± 2.05% vs. 45.15 ± 3.46% in WT), cardiac hypertrophy (heart weight/tibia length = 24.84 ± 3.00 vs. 9.81 ± 0.50 mg/mm in WT), or severe premature mortality (median survival of 12 weeks) associated with cardiac CaMKIIδC overexpression. In the face of a prevented SR Ca 2+ leak, the phosphorylation status of other critical CaMKII downstream targets that can drive heart failure, including transcriptional regulator histone deacetylase 4, as well as markers of pathological gene expression including Xirp2, Il6, and Col1a1, was equally increased in hearts from CaMKIIδC TG on a RyR WT and S2814A background., Conclusions: S2814 phosphoresistance of RyR2 prevents the CaMKII-dependent SR Ca 2+ leak induction but does not prevent the cardiomyopathic phenotype caused by enhanced CaMKIIδC activity. Our data indicate that additional mechanisms-independent of SR Ca 2+ leak-are critical for the maladaptive effects of chronically increased CaMKIIδC activity with respect to heart failure., (© 2024 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of European Society of Cardiology.)

Mitophagy, a mitochondria-specific form of autophagy, removes dysfunctional mitochondria and is hence an essential process contributing to mitochondrial quality control. PTEN-induced kinase 1 (PINK1) and the E3 ubiquitin ligase Parkin are critical molecules involved in stress-induced mitophagy, but the intracellular signaling mechanisms by which this pathway is regulated are unclear. We tested the hypothesis that signaling through RhoA, a small GTPase, induces mitophagy via modulation of the PINK1/Parkin pathway as a protective mechanism against ischemic stress. We demonstrate that expression of constitutively active RhoA as well as sphingosine-1-phosphate induced activation of endogenous RhoA in cardiomyocytes result in an accumulation of PINK1 at mitochondria. This is accompanied by translocation of Parkin to mitochondria and ubiquitination of mitochondrial proteins leading to recognition of mitochondria by autophagosomes and their lysosomal degradation. Expression of RhoA in cardiomyocytes confers protection against ischemia, and this cardioprotection is attenuated by siRNA-mediated PINK1 knockdown. In vivo myocardial infarction elicits increases in mitochondrial PINK1, Parkin, and ubiquitinated mitochondrial proteins. AAV9-mediated RhoA expression potentiates these responses and a concurrent decrease in infarct size is observed. Interestingly, induction of mitochondrial PINK1 accumulation in response to RhoA signaling is neither mediated through its transcriptional upregulation nor dependent on depolarization of the mitochondrial membrane, the canonical mechanism for PINK1 accumulation. Instead, our results reveal that RhoA signaling inhibits PINK1 cleavage, thereby stabilizing PINK1 protein at mitochondria. We further show that active RhoA localizes at mitochondria and interacts with PINK1, and that the mitochondrial localization of RhoA is regulated by its downstream effector protein kinase D. These findings demonstrate that RhoA activation engages a unique mechanism to regulate PINK1 accumulation, induce mitophagy and protect against ischemic stress, and implicates regulation of RhoA signaling as a potential strategy to enhance mitophagy and confer protection under stress conditions., (© 2022. The Author(s).)

Rationale: CaMKII (Ca 2+ -Calmodulin dependent protein kinase) δC activation is implicated in pathological progression of heart failure (HF) and CaMKIIδC transgenic mice rapidly develop HF and arrhythmias. However, little is known about early spatio-temporal Ca 2+ handling and CaMKII activation in hypertrophy and HF., Objective: To measure time- and location-dependent activation of CaMKIIδC signaling in adult ventricular cardiomyocytes, during transaortic constriction (TAC) and in CaMKIIδC transgenic mice., Methods and Results: We used human tissue from nonfailing and HF hearts, 4 mouse lines: wild-type, KO (CaMKIIδ-knockout), CaMKIIδC transgenic in wild-type (TG), or KO background, and wild-type mice exposed to TAC. Confocal imaging and biochemistry revealed disproportional CaMKIIδC activation and accumulation in nuclear and perinuclear versus cytosolic regions at 5 days post-TAC. This CaMKIIδ activation caused a compensatory increase in sarcoplasmic reticulum Ca 2+ content, Ca 2+ transient amplitude, and [Ca 2+ ] decline rates, with reduced phospholamban expression, all of which were most prominent near and in the nucleus. These early adaptive effects in TAC were entirely mimicked in young CaMKIIδ TG mice (6-8 weeks) where no overt cardiac dysfunction was present. The (peri)nuclear CaMKII accumulation also correlated with enhanced HDAC4 (histone deacetylase) nuclear export, creating a microdomain for transcriptional regulation. At longer times both TAC and TG mice progressed to overt HF (at 45 days and 11-13 weeks, respectively), during which time the compensatory Ca 2+ transient effects reversed, but further increases in nuclear and time-averaged [Ca 2+ ] and CaMKII activation occurred. CaMKIIδ TG mice lacking δB exhibited more severe HF, eccentric myocyte growth, and nuclear changes. Patient HF samples also showed greatly increased CaMKIIδ expression, especially for CaMKIIδC in nuclear fractions., Conclusions: We conclude that in early TAC perinuclear CaMKIIδC activation promotes adaptive increases in myocyte Ca 2+ transients and nuclear transcriptional responses but that chronic progression of this nuclear Ca 2+ -CaMKIIδC axis contributes to eccentric hypertrophy and HF.

There is substantial evidence that chronic heart failure in humans and in animal models is associated with inflammation. Ischemic interventions such as myocardial infarction lead to necrotic cell death and release of damage associated molecular patterns, factors that signal cell damage and induce expression of proinflammatory chemokines and cytokines. It has recently become evident that nonischemic interventions are also associated with increases in inflammatory genes and immune cell accumulation in the heart and that these contribute to fibrosis and ventricular dysfunction. How proinflammatory responses are elicited in nonischemic heart disease which is not, at least initially, associated with cell death is a critical unanswered question. In this review we provide evidence supporting the hypothesis that cardiomyocytes are an initiating site of inflammatory gene expression in response to nonischemic stress. Furthermore we discuss the role of the multifunctional Ca 2+ /calmodulin-regulated kinase, CaMKIIδ, as a transducer of stress signals to nuclear factor-κB activation, expression of proinflammatory cytokines and chemokines, and priming and activation of the NOD-like pyrin domain-containing protein 3 (NLRP3) inflammasome in cardiomyocytes. We summarize recent evidence that subsequent macrophage recruitment, fibrosis and contractile dysfunction induced by angiotensin II infusion or transverse aortic constriction are ameliorated by blockade of CaMKII, of monocyte chemoattractant protein-1/C-C chemokine receptor type 2 signaling, or of NLRP3 inflammasome activation.

Cardiac ischemia/reperfusion, loss of blood flow and its subsequent restoration, causes damage to the heart. Oxidative stress from ischemia/reperfusion leads to dysfunction and death of cardiomyocytes, increasing the risk of progression to heart failure. Alterations in mitochondrial dynamics, in particular mitochondrial fission, have been suggested to play a role in cardioprotection from oxidative stress. We tested the hypothesis that activation of RhoA regulates mitochondrial fission in cardiomyocytes. Our studies show that expression of constitutively active RhoA in cardiomyocytes increases phosphorylation of Dynamin-related protein 1 (Drp1) at serine-616, and leads to localization of Drp1 at mitochondria. Both responses are blocked by inhibition of Rho-associated Protein Kinase (ROCK). Endogenous RhoA activation by the GPCR agonist sphingosine-1-phosphate (S1P) also increases Drp1 phosphorylation and its mitochondrial translocation in a RhoA and ROCK dependent manner. Consistent with the role of mitochondrial Drp1 in fission, RhoA activation in cardiomyocytes leads to formation of smaller mitochondria and this is attenuated by inhibition of ROCK, by siRNA knockdown of Drp1 or by expression of a phosphorylation-deficient Drp1 S616A mutant. In addition, activation of RhoA prevents cell death in cardiomyocytes challenged by oxidative stress and this protection is blocked by siRNA knockdown of Drp1 or by Drp1 S616A expression. Taken together our findings demonstrate that RhoA activation can regulate Drp1 to induce mitochondrial fission and subsequent cellular protection, implicating regulation of fission as a novel mechanism contributing to RhoA-mediated cardioprotection., (Copyright © 2018. Published by Elsevier Inc.)

Sphingosine-1-phosphate (S1P), a bioactive lysophospholipid, is generated and released at sites of tissue injury in the heart and can act on S1P 1 , S1P 2 , and S1P 3 receptor subtypes to affect cardiovascular responses. We established that S1P causes little phosphoinositide hydrolysis and does not induce hypertrophy indicating that it does not cause receptor coupling to G q . We previously demonstrated that S1P confers cardioprotection against ischemia/reperfusion by activating RhoA and its downstream effector PKD. The S1P receptor subtypes and G proteins that regulate RhoA activation and downstream responses in the heart have not been determined. Using siRNA or pertussis toxin to inhibit different G proteins in NRVMs we established that S1P regulates RhoA activation through Gα 13 but not Gα 12 , Gα q , or Gα i . Knockdown of the three major S1P receptors using siRNA demonstrated a requirement for S1P 3 in RhoA activation and subsequent phosphorylation of PKD, and this was confirmed in studies using isolated hearts from S1P 3 knockout (KO) mice. S1P treatment reduced infarct size induced by ischemia/reperfusion in Langendorff perfused wild-type (WT) hearts and this protection was abolished in the S1P 3 KO mouse heart. CYM-51736, an S1P 3 -specific agonist, also decreased infarct size after ischemia/reperfusion to a degree similar to that achieved by S1P. The finding that S1P 3 receptor- and Gα 13 -mediated RhoA activation is responsible for protection against ischemia/reperfusion suggests that selective targeting of S1P 3 receptors could provide therapeutic benefits in ischemic heart disease., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Several conditions of heart disease, including heart failure and diabetic cardiomyopathy, are associated with upregulation of cytosolic Ca(2+)/calmodulin-dependent protein kinase II (CaMKIIδC) activity. In the heart, CaMKIIδC isoform targets several proteins involved in intracellular Ca(2+) homeostasis. We hypothesized that high-intensity endurance training activates mechanisms that enable a rescue of dysfunctional cardiomyocyte Ca(2+) handling and thereby ameliorate cardiac dysfunction despite continuous and chronic elevated levels of CaMKIIδC CaMKIIδC transgenic (TG) and wild-type (WT) mice performed aerobic interval exercise training over 6 wk. Cardiac function was measured by echocardiography in vivo, and cardiomyocyte shortening and intracellular Ca(2+) handling were measured in vitro. TG mice had reduced global cardiac function, cardiomyocyte shortening (47% reduced compared with WT, P < 0.01), and impaired Ca(2+) homeostasis. Despite no change in the chronic elevated levels of CaMKIIδC, exercise improved global cardiac function, restored cardiomyocyte shortening, and reestablished Ca(2+) homeostasis to values not different from WT. The key features to explain restored Ca(2+) homeostasis after exercise training were increased L-type Ca(2+) current density and flux by 79 and 85%, respectively (P < 0.01), increased sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA2a) function by 50% (P < 0.01), and reduced diastolic SR Ca(2+) leak by 73% (P < 0.01), compared with sedentary TG mice. In conclusion, exercise training improves global cardiac function as well as cardiomyocyte function in the presence of a maintained high CaMKII activity. The main mechanisms of exercise-induced improvements in TG CaMKIIδC mice are mediated via increased L-type Ca(2+) channel currents and improved SR Ca(2+) handling by restoration of SERCA2a function in addition to reduced diastolic SR Ca(2+) leak., (Copyright © 2016 the American Physiological Society.)

The fast transient outward potassium current (Ito,f) plays a critical role in the electrical and contractile properties of the myocardium. Ito,f channels are formed by the co-assembly of the pore-forming α-subunits, Kv4.2 and Kv4.3, together with the accessory β-subunit KChIP2. Reductions of Ito,f are common in the diseased heart, which is also associated with enhanced stimulation of β-adrenergic receptors (β-ARs). We used cultured neonatal rat ventricular myocytes to examine how chronic β-AR stimulation decreases Ito,f. To determine which downstream pathways mediate these Ito,f changes, adenoviral infections were used to inhibit CaMKIIδc, CaMKIIδb, calcineurin, or nuclear factor κB (NF-κB). We observed that chronic β-AR stimulation with isoproterenol (ISO) for 48 h reduced Ito,f along with mRNA expression of all three of its subunits (Kv4.2, Kv4.3, and KChIP2). Inhibiting either CaMKIIδc nor CaMKIIδb did not prevent the ISO-mediated Ito,f reductions, even though CaMKIIδc and CaMKIIδb clearly regulated Ito,f and the mRNA expression of its subunits. Likewise, calcineurin inhibition did not prevent the Ito,f reductions induced by β-AR stimulation despite strongly modulating Ito,f and subunit mRNA expression. In contrast, NF-κB inhibition partly rescued the ISO-mediated Ito,f reductions in association with restoration of KChIP2 mRNA expression. Consistent with these observations, KChIP2 promoter activity was reduced by p65 as well as β-AR stimulation. In conclusion, NF-κB, and not CaMKIIδ or calcineurin, partly mediates the Ito,f reductions induced by chronic β-AR stimulation. Both mRNA and KChIP2 promoter data suggest that the ISO-induced Ito,f reductions are, in part, mediated through reduced KChIP2 transcription caused by NF-κB activation., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Ca(2+)-calmodulin kinase II (CaMKII) activation is deleterious in cardiac ischemia/reperfusion (I/R). Moreover, inhibition of CaMKII-dependent phosphorylations at the sarcoplasmic reticulum (SR) prevents CaMKII-induced I/R damage. However, the downstream targets of CaMKII at the SR level, responsible for this detrimental effect, remain unclear. In the present study we aimed to dissect the role of the two main substrates of CaMKII at the SR level, phospholamban (PLN) and ryanodine receptors (RyR2), in CaMKII-dependent I/R injury. In mouse hearts subjected to global I/R (45/120min), phosphorylation of the primary CaMKII sites, S2814 on cardiac RyR2 and of T17 on PLN, significantly increased at the onset of reperfusion whereas PKA-dependent phosphorylation of RyR2 and PLN did not change. Similar results were obtained in vivo, in mice subjected to regional myocardial I/R (1/24h). Knock-in mice with an inactivated serine 2814 phosphorylation site on RyR2 (S2814A) significantly improved post-ischemic mechanical recovery, reduced infarct size and decreased apoptosis. Conversely, knock-in mice, in which CaMKII site of RyR2 is constitutively activated (S2814D), significantly increased infarct size and exacerbated apoptosis. In S2814A and S2814D mice subjected to regional myocardial ischemia, infarct size was also decreased and increased respectively. Transgenic mice with double-mutant non-phosphorylatable PLN (S16A/T17A) in the PLN knockout background (PLNDM) also showed significantly increased post-ischemic cardiac damage. This effect cannot be attributed to PKA-dependent PLN phosphorylation and was not due to the enhanced L-type Ca(2+) current, present in these mice. Our results reveal a major role for the phosphorylation of S2814 site on RyR2 in CaMKII-dependent I/R cardiac damage. In contrast, they showed that CaMKII-dependent increase in PLN phosphorylation during reperfusion opposes rather than contributes to I/R damage., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The ATP-sensitive potassium (KATP) channels are crucial for stress adaptation in the heart. It has previously been suggested that the function of KATP channels is modulated by nitric oxide (NO), a gaseous messenger known to be cytoprotective; however, the underlying mechanism remains poorly understood. Here we sought to delineate the intracellular signalling mechanism responsible for NO modulation of sarcolemmal KATP (sarcKATP) channels in ventricular cardiomyocytes. Cell-attached patch recordings were performed in transfected human embryonic kidney (HEK) 293 cells and ventricular cardiomyocytes freshly isolated from adult rabbits or genetically modified mice, in combination with pharmacological and biochemical approaches. Bath application of the NO donor NOC-18 increased the single-channel activity of Kir6.2/SUR2A (i.e., the principal ventricular-type KATP) channels in HEK293 cells, whereas the increase was abated by KT5823 [a selective cGMP-dependent protein kinase (PKG) inhibitor], mercaptopropionyl glycine [MPG; a reactive oxygen species (ROS) scavenger], catalase (an H2O2-degrading enzyme), myristoylated autocamtide-2 related inhibitory peptide (mAIP) selective for Ca2+ / calmodulin-dependent protein kinase II (CaMKII) and U0126 [an extracellular signal-regulated protein kinase 1/2 (ERK1/2) inhibitor], respectively. The NO donors NOC-18 and N-(2-deoxy-α,β-d-glucopyranose-2-)-N2-acetyl-S-nitroso-d,l-penicillaminamide (glycol-SNAP-2) were also capable of stimulating native sarcKATP channels preactivated by the channel opener pinacidil in rabbit ventricular myocytes, through reducing the occurrence and the dwelling time of the long closed states whilst increasing the frequency of channel opening; in contrast, all these changes were reversed in the presence of inhibitors selective for soluble guanylyl cyclase (sGC), PKG, calmodulin, CaMKII or ERK1/2. Mimicking the action of NO donors, exogenous H2O2 potentiated pinacidil-preactivated sarcKATP channel activity in intact cardiomyocytes, but the H2O2-induced KATP channel stimulation was obliterated when ERK1/2 or CaMKII activity was suppressed, implying that H2O2 is positioned upstream of ERK1/2 and CaMKII for K(ATP) channel modulation. Furthermore, genetic ablation (i.e., knockout) of CaMKIIδ, the predominant cardiac CaMKII isoform, diminished ventricular sarcK(ATP) channel stimulation elicited by activation of PKG, unveiling CaMKIIδ as a crucial player. Additionally, evidence from kinase activity and Western blot analyses revealed that activation of NO-PKG signalling augmented CaMKII activity in rabbit ventricular myocytes and, importantly, CaMKII activation by PKG occurred in an ERK1/2-dependent manner, placing ERK1/2 upstream of CaMKII. Taken together, these findings suggest that NO modulates ventricular sarcK(ATP) channels via a novel sGC-cGMP-PKG-ROS(H2O2)-ERK1/2-calmodulin-CaMKII (δ isoform in particular) signalling cascade, which heightens K(ATP) channel activity by destabilizing the long closed states while facilitating closed-to-open state transitions. This pathway may contribute to regulation of cardiac excitability and cytoprotection against ischaemia-reperfusion injury, in part, by opening myocardial sarcK(ATP) channels.

Acute activation of calcium/calmodulin-dependent protein kinase (CaMKII) in permeabilized phospholamban knockout (PLN-KO) mouse myocytes phosphorylates ryanodine receptors (RyRs) and activates spontaneous local sarcoplasmic reticulum (SR) Ca release events (Ca sparks) even at constant SR Ca load. To assess how CaMKII regulates SR Ca release in intact myocytes (independent of SR Ca content changes or PLN effects), we compared Ca sparks in PLN-KO versus mice, which also have transgenic cardiac overexpression of CaMKIIδC in the PLN-KO background (KO/TG). Compared with PLN-KO mice, these KO/TG cardiomyocytes exhibited 1), increased twitch Ca transient and fractional release (both by ∼35%), but unaltered SR Ca load; 2), increased resting Ca spark frequency (300%) despite a lower diastolic [Ca]i, which also slowed twitch [Ca]i decline (suggesting CaMKII-dependent RyR Ca sensitization); 3), elevated Ca spark amplitude and rate of Ca release (which might indicate that more RyR channels participate in a single spark); 4), prolonged Ca spark rise time (which implies that CaMKII either delays RyR closure or prolongs the time when openings can occur); 5), more frequent repetitive sparks at single release sites. Analysis of repetitive sparks from individual Ca release sites indicates that CaMKII enhanced RyR Ca sensitivity, but did not change the time course of SR Ca refilling. These results demonstrate that there are dramatic CaMKII-mediated effects on RyR Ca release that occur via regulation of both RyR activation and termination processes., (Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Rationale: Differential effects of δ(B) and δ(C) subtypes of Ca²⁺/calmodulin-dependent protein kinase (CaMKII) on cardiomyocyte Ca²⁺ handling and survival have been suggested to result from their respective nuclear versus cytosolic localizations. CaMKIIδ subtype localization and its relationship to enzyme activation and target phosphorylation have not, however, been systematically evaluated., Objective: To determine whether CaMKIIδ subtypes are restricted to a particular subcellular location and assess the relationship of localization to enzyme activation and function., Methods and Results: CaMKIIδ is highly expressed in mouse heart and cardiomyocytes and concentrated in sarcoplasmic reticulum (SR)/membrane and nuclear fractions. CaMKIIδ(B) and δ(C) subtypes differ by a nuclear localization sequence, but both are present in nuclear and SR/membrane fractions. Nonselective subtype distribution is also seen in mice overexpressing CaMKIIδ(B) or δ(C), even in a CaMKIIδ null background. Fluorescently tagged CaMKIIδ(B) expressed in cardiomyocytes concentrates in nuclei whereas δ(C) concentrates in cytosol, but neither localization is exclusive. Mouse hearts exposed to phenylephrine show selective CaMKIIδ activation in the nuclear (versus SR) compartment, whereas caffeine selectively activates CaMKIIδ in SR (versus nuclei), independent of subtype. Compartmentalized activation extends to functional differences in target phosphorylation at CaMKII sites: phenylephrine increases histone deacetylase 5 phosphorylation (Ser498) but not phospholamban (Thr17), whereas the converse holds for caffeine., Conclusions: These studies demonstrate that CaMKIIδ(B) and δ(C) are not exclusively restricted to the nucleus and cytosol and that spatial and functional specificity in CaMKIIδ activation is elicited by mobilization of different Ca²⁺ stores rather than by compartmentalized subtype localization.

Mechanistic target of rapamycin (MTOR) plays a critical role in the regulation of cell growth and in the response to energy state changes. Drugs inhibiting MTOR are increasingly used in antineoplastic therapies. Myocardial MTOR activity changes during hypertrophy and heart failure (HF). However, whether MTOR exerts a positive or a negative effect on myocardial function remains to be fully elucidated. Here, we show that ablation of Mtor in the adult mouse myocardium results in a fatal, dilated cardiomyopathy that is characterized by apoptosis, autophagy, altered mitochondrial structure, and accumulation of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). 4E-BP1 is an MTOR-containing multiprotein complex-1 (MTORC1) substrate that inhibits translation initiation. When subjected to pressure overload, Mtor-ablated mice demonstrated an impaired hypertrophic response and accelerated HF progression. When the gene encoding 4E-BP1 was ablated together with Mtor, marked improvements were observed in apoptosis, heart function, and survival. Our results demonstrate a role for the MTORC1 signaling network in the myocardial response to stress. In particular, they highlight the role of 4E-BP1 in regulating cardiomyocyte viability and in HF. Because the effects of reduced MTOR activity were mediated through increased 4E-BP1 inhibitory activity, blunting this mechanism may represent a novel therapeutic strategy for improving cardiac function in clinical HF.

Rationale: Cardioprotective signaling mediates antiapoptotic actions through multiple mechanisms including maintenance of mitochondrial integrity. Pim-1 kinase is an essential downstream effector of AKT-mediated cardioprotection but the mechanistic basis for maintenance of mitochondrial integrity by Pim-1 remains unexplored. This study details antiapoptotic actions responsible for enhanced cell survival in cardiomyocytes with elevated Pim-1 activity., Objective: The purpose of this study is to demonstrate that the cardioprotective kinase Pim-1 acts to inhibit cell death by preserving mitochondrial integrity in cardiomyocytes., Methods and Results: A combination of biochemical, molecular, and microscopic analyses demonstrate beneficial effects of Pim-1 on mitochondrial integrity. Pim-1 protein level increases in the mitochondrial fraction with a corresponding decrease in the cytosolic fraction of myocardial lysates from hearts subjected to 30 minutes of ischemia followed by 30 minutes of reperfusion. Cardiac-specific overexpression of Pim-1 results in higher levels of antiapoptotic Bcl-X(L) and Bcl-2 compared to samples from normal hearts. In response to oxidative stress challenge, Pim-1 preserves the inner mitochondrial membrane potential. Ultrastructure of the mitochondria is maintained by Pim-1 activity, which prevents swelling induced by calcium overload. Finally, mitochondria isolated from hearts created with cardiac-specific overexpression of Pim-1 show inhibition of cytochrome c release triggered by a truncated form of proapoptotic Bid., Conclusion: Cardioprotective action of Pim-1 kinase includes preservation of mitochondrial integrity during cardiomyopathic challenge conditions, thereby raising the potential for Pim-1 kinase activation as a therapeutic interventional approach to inhibit cell death by antagonizing proapoptotic Bcl-2 family members that regulate the intrinsic apoptotic pathway.

The Na(+)-Ca(2+) exchanger gene (Ncx1) is upregulated in hypertrophy and is often found elevated in end-stage heart failure. Studies have shown that the change in its expression contributes to contractile dysfunction. beta-Adrenergic receptor (beta-AR) signaling plays an important role in the regulation of calcium homeostasis in the cardiomyocyte, but chronic activation in periods of cardiac stress contributes to heart failure by mechanisms which include Ncx1 upregulation. Here, using a Ca(2+)/calmodulin-dependent protein kinase II (CaMKIIdelta(c)) null mouse, we demonstrate that beta-AR-stimulated Ncx1 upregulation is dependent on CaMKII. beta-AR-stimulated Ncx1 expression is mediated by activator protein 1 (AP-1) factors and is independent of cAMP-response element-binding protein (CREB) activation. The MAP kinases (ERK1/2, JNK and p38) are not required for AP-1 factor activation. Chromatin immunoprecipitation demonstrates that beta-AR stimulation activates the ordered recruitment of JunB homodimers, which then are replaced by c-Jun homodimers binding to the proximal AP-1 elements of the endogenous Ncx1 promoter. In conclusion, this work has provided insight into the intracellular signaling pathways and transcription factors regulating Ncx1 gene expression in a chronically beta-AR-stimulated heart., (Copyright 2009 Elsevier Inc. All rights reserved.)

Background: Transgenic (TG) Ca/calmodulin-dependent protein kinase II (CaMKII)delta(C) mice have heart failure and isoproterenol (ISO)-inducible arrhythmias. We hypothesized that CaMKII contributes to arrhythmias and underlying cellular events and that inhibition of CaMKII reduces cardiac arrhythmogenesis in vitro and in vivo., Methods and Results: Under baseline conditions, isolated cardiac myocytes from TG mice showed an increased incidence of early afterdepolarizations compared with wild-type myocytes (P<0.05). CaMKII inhibition (AIP) completely abolished these afterdepolarizations in TG cells (P<0.05). Increasing intracellular Ca stores using ISO (10(-8) M) induced a larger amount of delayed afterdepolarizations and spontaneous action potentials in TG compared with wild-type cells (P<0.05). This seems to be due to an increased sarcoplasmic reticulum (SR) Ca leak because diastolic [Ca](i) rose clearly on ISO in TG but not in wild-type cells (+20+/-5% versus +3+/-4% at 10(-6) M ISO, P<0.05). In parallel, SR Ca leak assessed by spontaneous SR Ca release events showed an increased Ca spark frequency (3.9+/-0.5 versus 2.0+/-0.4 sparks per 100 microm(-1).s(-1), P<0.05). However, CaMKII inhibition (either pharmacologically using KN-93 or genetically using an isoform-specific CaMKIIdelta-knockout mouse model) significantly reduced SR Ca spark frequency, although this rather increased SR Ca content. In parallel, ISO increased the incidence of early (54% versus 4%, P<0.05) and late (86% versus 43%, P<0.05) nonstimulated events in TG versus wild-type myocytes, but CaMKII inhibition (KN-93 and KO) reduced these proarrhythmogenic events (P<0.05). In addition, CaMKII inhibition in TG mice (KN-93) clearly reduced ISO-induced arrhythmias in vivo (P<0.05)., Conclusions: We conclude that CaMKII contributes to cardiac arrhythmogenesis in TG CaMKIIdelta(C) mice having heart failure and suggest the increased SR Ca leak as an important mechanism. Moreover, CaMKII inhibition reduces cardiac arrhythmias in vitro and in vivo and may therefore indicate a potential role for future antiarrhythmic therapies warranting further studies.

The phosphoinositide 3-kinase (PI3K)/phosphoinositide dependent kinase 1 (PDK1) signaling pathway exerts cardioprotective effects in the myocardium through activation of key proteins including Akt. Activated Akt accumulates in nuclei of cardiomyocytes suggesting that biologically relevant targets are located in that subcellular compartment. Nuclear Akt activity could be potentiated in both intensity and duration by the presence of a nuclear-associated PI3K/PDK1 signaling cascade as has been described in other non-myocyte cell types. PI3K/PDK1 distribution was determined in vitro and in vivo by immunostaining and nuclear extraction of cultured rat neonatal cardiomyocytes or transgenic mouse hearts. Results show that PI3K and PDK1 are present at a basal level in cardiomyocytes nuclei and that cardioprotective stimulation with atrial natriuretic peptide (ANP) increases their nuclear localization. In comparison, overexpression of nuclear-targeted Akt does not mediate increased translocation of either PI3K or PDK1 indicating that accumulation of Akt does not drive PI3K or PDK1 into the nuclear compartment. Furthermore, PI3K and phospho-Akt(473) show parallel temporal accumulation in the nucleus following (MI) infarction challenge. These findings demonstrate the presence of a dynamically regulated nuclear-associated signaling cascade involving PI3K and PDK that presumably influences nuclear Akt activation.

Cardiomyocyte death is now recognized as a critical factor in the development of heart disease. Mitochondria are not only responsible for energy production to ensure that cardiac output meets the body's energy demands, but they serve as critical integrators of cell survival signals. Numerous stressors are known to induce cell death by necrosis and/or apoptosis mediated through mitochondrial dysregulation. Anti- and pro-apoptotic Bcl-2 family proteins regulate apoptosis by controlling mitochondrial outer membrane permeability, whereas opening of the mitochondrial permeability transition pore (PT-pore) induces large amplitude permeability of the inner membrane and consequent rupture of the outer membrane. Akt is one of the best described survival kinases activated by receptor ligands and its activation preserves mitochondrial integrity and protects cardiomyocytes against necrotic and apoptotic death. The mechanisms responsible for Akt-mediated mitochondrial protection have not been fully elucidated. There is, however, accumulating evidence that multiple Akt target molecules, recruited through both transcriptional and post-transcriptional mechanisms, directly impinge upon and protect mitochondria. In this review we discuss mechanisms by which Akt activation can effect changes at the mitochondria that protect cardiomyocytes and attenuate pathophysiological responses of the heart.

The insulin IGF-1-PI3K-Akt signaling pathway has been suggested to improve cardiac inotropism and increase Ca(2+) handling through the effects of the protein kinase Akt. However, the underlying molecular mechanisms remain largely unknown. In this study, we provide evidence for an unanticipated regulatory function of Akt controlling L-type Ca(2+) channel (LTCC) protein density. The pore-forming channel subunit Ca(v)alpha1 contains highly conserved PEST sequences (signals for rapid protein degradation), and in-frame deletion of these PEST sequences results in increased Ca(v)alpha1 protein levels. Our findings show that Akt-dependent phosphorylation of Ca(v)beta2, the LTCC chaperone for Ca(v)alpha1, antagonizes Ca(v)alpha1 protein degradation by preventing Ca(v)alpha1 PEST sequence recognition, leading to increased LTCC density and the consequent modulation of Ca(2+) channel function. This novel mechanism by which Akt modulates LTCC stability could profoundly influence cardiac myocyte Ca(2+) entry, Ca(2+) handling, and contractility.

RhoA a small G-protein that has an established role in cell growth and in regulation of the actin cytoskeleton. Far less is known about whether RhoA can modulate cell fate. We previously reported that sustained RhoA activation induces cardiomyocyte apoptosis (Del Re, D. P., Miyamoto, S., and Brown, J. H. (2007) J. Biol. Chem. 282, 8069-8078). Here we demonstrate that less chronic RhoA activation affords a survival advantage, protecting cardiomyocytes from apoptotic insult induced by either hydrogen peroxide treatment or glucose deprivation. Under conditions where RhoA is protective, we observe Rho kinase-dependent cytoskeletal rearrangement and activation of focal adhesion kinase (FAK). Activation of endogenous cardiomyocyte FAK leads to its increased association with the p85 regulatory subunit of phosphatidylinositol-3-kinase (PI3K) and to concomitant activation of Akt. Treatment of isolated perfused hearts with sphingosine 1-phosphate recapitulates this response. The pathway by which RhoA mediates cardiomyocyte Akt activation is demonstrated to require Rho kinase, FAK and PI3K, but not Src, based on studies with pharmacological inhibitors (Y-27632, LY294002, PF271 and PP2) and inhibitory protein expression (FAK-related nonkinase). Inhibition of RhoA-mediated Akt activation at any of these steps, including inhibition of FAK, prevents RhoA from protecting cardiomyocytes against apoptotic insult. We further demonstrate that stretch of cardiomyocytes, which activates endogenous RhoA, induces the aforementioned signaling pathway, providing a physiologic context in which RhoA-mediated FAK phosphorylation can activate PI3K and Akt. We suggest that RhoA-mediated effects on the cardiomyocyte cytoskeleton provide a novel mechanism for protection from apoptosis.

The response of cardiomyocytes to biomechanical stress can determine the pathophysiology of hypertrophic cardiac disease, and targeting the pathways regulating these responses is a therapeutic goal. However, little is known about how biomechanical stress is sensed by the cardiomyocyte sarcomere to transduce intracellular hypertrophic signals or how the dysfunction of these pathways may lead to disease. Here, we found that four-and-a-half LIM domains 1 (FHL1) is part of a complex within the cardiomyocyte sarcomere that senses the biomechanical stress-induced responses important for cardiac hypertrophy. Mice lacking Fhl1 displayed a blunted hypertrophic response and a beneficial functional response to pressure overload induced by transverse aortic constriction. A link to the Galphaq (Gq) signaling pathway was also observed, as Fhl1 deficiency prevented the cardiomyopathy observed in Gq transgenic mice. Mechanistic studies demonstrated that FHL1 plays an important role in the mechanism of pathological hypertrophy by sensing biomechanical stress responses via the N2B stretch sensor domain of titin and initiating changes in the titin- and MAPK-mediated responses important for sarcomere extensibility and intracellular signaling. These studies shed light on the physiological regulation of the sarcomere in response to hypertrophic stress.

Sphingosine 1-phosphate (S1P) is released at sites of tissue injury and effects cellular responses through activation of G protein-coupled receptors. The role of S1P in regulating cardiomyocyte survival following in vivo myocardial ischemia-reperfusion (I/R) injury was examined by using mice in which specific S1P receptor subtypes were deleted. Mice lacking either S1P(2) or S1P(3) receptors and subjected to 1-h coronary occlusion followed by 2 h of reperfusion developed infarcts equivalent to those of wild-type (WT) mice. However, in S1P(2,3) receptor double-knockout mice, infarct size following I/R was increased by >50%. I/R leads to activation of ERK, JNK, and p38 MAP kinases; however, these responses were not diminished in S1P(2,3) receptor knockout compared with WT mice. In contrast, activation of Akt in response to I/R was markedly attenuated in S1P(2,3) receptor knockout mouse hearts. Neither S1P(2) nor S1P(3) receptor deletion alone impaired I/R-induced Akt activation, which suggests redundant signaling through these receptors and is consistent with the finding that deletion of either receptor alone did not increase I/R injury. The involvement of cardiomyocytes in S1P(2) and S1P(3) receptor mediated activation of Akt was tested by using cells from WT and S1P receptor knockout hearts. Akt was activated by S1P, and this was modestly diminished in cardiomyocytes from S1P(2) or S1P(3) receptor knockout mice and completely abolished in the S1P(2,3) receptor double-knockout myocytes. Our data demonstrate that activation of S1P(2) and S1P(3) receptors plays a significant role in protecting cardiomyocytes from I/R damage in vivo and implicate the release of S1P and receptor-mediated Akt activation in this process.

The small G-protein RhoA regulates the actin cytoskeleton, and its involvement in cell proliferation has also been established. In contrast, little is known about whether RhoA participates in cell survival or apoptosis. In cardiomyocytes in vitro, RhoA induces hypertrophic cell growth and gene expression. In vivo, however, RhoA expression leads to development of heart failure (Sah, V. P., Minamisawa, S., Tam, S. P., Wu, T. H., Dorn, G. W., Ross, J. Jr., Chien, K. R., and Brown, J. H. (1999) J. Clin. Investig. 103, 1627-1634), a condition widely associated with cardiomyocyte apoptosis. We demonstrate here that adenoviral overexpression of activated RhoA in cardiomyocytes induces hypertrophy, which transitions over time to apoptosis, as evidenced by caspase activation and nucleosomal DNA fragmentation. The Rho kinase inhibitors Y-27632 and HA-1077 and expression of a dominant negative Rho kinase block these responses. Caspase-9, but not caspase-8, is activated, and its inhibition prevents DNA fragmentation, consistent with involvement of a mitochondrial death pathway. Interestingly, RhoA expression induces a 3-4-fold up-regulation of the proapoptotic Bcl-2 family protein Bax. RhoA also increases levels of activated Bax and the amount of Bax protein localized at mitochondria. Bax mRNA is increased by RhoA, indicating transcriptional regulation, and the ability of a dominant negative p53 mutant to block Bax up-regulation implicates p53 in this response. The involvement of Bax in RhoA-induced apoptosis was examined by treatment with a Bax-inhibitory peptide, which was found to significantly attenuate DNA fragmentation and caspase-9 and -3 activation. The dominant negative p53 also prevents RhoA-induced apoptosis. We conclude that RhoA/Rho kinase activation up-regulates Bax through p53 to induce a mitochondrial death pathway and cardiomyocyte apoptosis.

In heart failure (HF), Ca(2+)/calmodulin kinase II (CaMKII) expression is increased. Altered Na(+) channel gating is linked to and may promote ventricular tachyarrhythmias (VTs) in HF. Calmodulin regulates Na(+) channel gating, in part perhaps via CaMKII. We investigated effects of adenovirus-mediated (acute) and Tg (chronic) overexpression of cytosolic CaMKIIdelta(C) on Na(+) current (I(Na)) in rabbit and mouse ventricular myocytes, respectively (in whole-cell patch clamp). Both acute and chronic CaMKIIdelta(C) overexpression shifted voltage dependence of Na(+) channel availability by -6 mV (P < 0.05), and the shift was Ca(2+) dependent. CaMKII also enhanced intermediate inactivation and slowed recovery from inactivation (prevented by CaMKII inhibitors autocamtide 2-related inhibitory peptide [AIP] or KN93). CaMKIIdelta(C) markedly increased persistent (late) inward I(Na) and intracellular Na(+) concentration (as measured by the Na(+) indicator sodium-binding benzofuran isophthalate [SBFI]), which was prevented by CaMKII inhibition in the case of acute CaMKIIdelta(C) overexpression. CaMKII coimmunoprecipitates with and phosphorylates Na(+) channels. In vivo, transgenic CaMKIIdelta(C) overexpression prolonged QRS duration and repolarization (QT intervals), decreased effective refractory periods, and increased the propensity to develop VT. We conclude that CaMKII associates with and phosphorylates cardiac Na(+) channels. This alters I(Na) gating to reduce availability at high heart rate, while enhancing late I(Na) (which could prolong action potential duration). In mice, enhanced CaMKIIdelta(C) activity predisposed to VT. Thus, CaMKII-dependent regulation of Na(+) channel function may contribute to arrhythmogenesis in HF.

Our laboratory has previously shown that adenoviral-mediated overexpression of Galphaq in neonatal rat ventricular cardiomyocytes increases the phosphorylation of Akt, a well-established anti-apoptotic effector. As demonstrated here, Galphaq expression protects cardiomyocytes against apoptosis induced by treatment with 2-deoxyglucose (2DOG) and this protection is lost when Akt activation is prevented by treatment with LY294002 (an inhibitor of PI3K). Galphaq-induced Akt phosphorylation is not caused by increased Gbetagamma signaling and does not appear to involve PKC activation. Rather studies using the EGF receptor inhibitor AG1478 and the Src inhibitor PP2 implicate these tyrosine kinases in the pathway inducing Akt phosphorylation. EGFR phosphorylation is increased in cells expressing Galphaq and this effect is inhibited by PP2, placing Src upstream of EGFR phosphorylation. EGFR activation appears to be required for Galphaq-mediated protection since inhibition of Src or EGFR rendered cells susceptible to 2DOG-induced apoptosis. In contrast to the requirement for EGFR mediated Akt activation in cardioprotection, neither EGFR nor Akt activation are necessary for the hypertrophic increases in cell size or ANF content elicited by Galphaq overexpression. These data demonstrate that increased Galphaq activity can provide anti-apoptotic signals by eliciting EGFR phosphorylation and subsequent Akt activation, independent of the well-known ability of Galphaq signaling to elicit hypertrophy.

Previous work showed that calmodulin (CaM) and Ca2+-CaM-dependent protein kinase II (CaMKII) are somehow involved in cardiac hypertrophic signaling, that inositol 1,4,5-trisphosphate receptors (InsP3Rs) in ventricular myocytes are mainly in the nuclear envelope, where they associate with CaMKII, and that class II histone deacetylases (e.g., HDAC5) suppress hypertrophic gene transcription. Furthermore, HDAC phosphorylation in response to neurohumoral stimuli that induce hypertrophy, such as endothelin-1 (ET-1), activates HDAC nuclear export, thereby regulating cardiac myocyte transcription. Here we demonstrate a detailed mechanistic convergence of these 3 issues in adult ventricular myocytes. We show that ET-1, which activates plasmalemmal G protein-coupled receptors and InsP3 production, elicits local nuclear envelope Ca2+ release via InsP3R. This local Ca2+ release activates nuclear CaMKII, which triggers HDAC5 phosphorylation and nuclear export (derepressing transcription). Remarkably, this Ca2+-dependent pathway cannot be activated by the global Ca2+ transients that cause contraction at each heartbeat. This novel local Ca2+ signaling in excitation-transcription coupling is analogous to but separate (and insulated) from that involved in excitation-contraction coupling. Thus, myocytes can distinguish simultaneous local and global Ca2+ signals involved in contractile activation from those targeting gene expression.

The predominant cardiac Ca2+/calmodulin-dependent protein kinase (CaMK) is CaMKIIdelta. Here we acutely overexpress CaMKIIdeltaC using adenovirus-mediated gene transfer in adult rabbit ventricular myocytes. This circumvents confounding adaptive effects in CaMKIIdeltaC transgenic mice. CaMKIIdeltaC protein expression and activation state (autophosphorylation) were increased 5- to 6-fold. Basal twitch contraction amplitude and kinetics (1 Hz) were not changed in CaMKIIdeltaC versus LacZ expressing myocytes. However, the contraction-frequency relationship was more negative, frequency-dependent acceleration of relaxation was enhanced (tau(0.5Hz)/tau(3Hz)=2.14+/-0.10 versus 1.87+/-0.10), and peak Ca2+ current (ICa) was increased by 31% (-7.1+/-0.5 versus -5.4+/-0.5 pA/pF, P<0.05). Ca2+ transient amplitude was not significantly reduced (-27%, P=0.22), despite dramatically reduced sarcoplasmic reticulum (SR) Ca2+ content (41%; P<0.05). Thus fractional SR Ca2+ release was increased by 60% (P<0.05). Diastolic SR Ca2+ leak assessed by Ca2+ spark frequency (normalized to SR Ca2+ load) was increased by 88% in CaMKIIdeltaC versus LacZ myocytes (P<0.05; in an multiplicity-of-infection-dependent manner), an effect blocked by CaMKII inhibitors KN-93 and autocamtide-2-related inhibitory peptide. This enhanced SR Ca2+ leak may explain reduced SR Ca2+ content, despite measured levels of SR Ca2+-ATPase and Na+/Ca2+ exchange expression and function being unaltered. Ryanodine receptor (RyR) phosphorylation in CaMKIIdeltaC myocytes was increased at both Ser2809 and Ser2815, but FKBP12.6 coimmunoprecipitation with RyR was unaltered. This shows for the first time that acute CaMKIIdeltaC overexpression alters RyR function, leading to enhanced SR Ca2+ leak and reduced SR Ca2+ content but without reducing twitch contraction and Ca2+ transients. We conclude that this is attributable to concomitant enhancement of fractional SR Ca2+ release in CaMKIIdeltaC myocytes (ie, CaMKII-dependent enhancement of RyR Ca2+ sensitivity during diastole and systole) and increased ICa.

We previously reported that constitutively activated Galpha(q) (Q209L) expression in cardiomyocytes induces apoptosis through opening of the mitochondrial permeability transition pore. We assessed the hypothesis that disturbances in Ca(2+) handling linked Galpha(q) activity to apoptosis because resting Ca(2+) levels were significantly increased prior to development of apoptosis. Treating cells with EGTA lowered Ca(2+) and blocked both loss of mitochondrial membrane potential (an indicator of permeability transition pore opening) and apoptosis (assessed by DNA fragmentation). When cytosolic Ca(2+) and mitochondrial membrane potential were simultaneously measured by confocal microscopy, sarcoplasmic reticulum (SR)-driven slow Ca(2+) oscillations (time-to-peak approximately 4 s) were observed in Q209L-expressing cells. These oscillations were seen to transition into sustained increases in cytosolic Ca(2+), directly paralleled by loss of mitochondrial membrane potential. Ca(2+) transients generated by caffeine-induced release of SR Ca(2+) were greatly prolonged in Q209L-expressing cells, suggesting a decreased ability to extrude Ca(2+). Indeed, the Na(+)/Ca(2+) exchanger (NCX), which removes Ca(2+) from the cell, was markedly down-regulated at the mRNA and protein levels. Adenoviral NCX expression normalized cytosolic Ca(2+) levels and prevented DNA fragmentation in cells expressing Q209L. Interestingly, constitutively activated Akt, which rescues cells from Q209L-induced apoptosis, prevented the decrease in NCX expression, normalized cytosolic Ca(2+) levels and spontaneous Ca(2+) oscillations, shortened caffeine-induced Ca(2+) transients, and prevented loss of the mitochondrial membrane potential. Our findings demonstrate that NCX down-regulation and consequent increases in cytosolic and SR Ca(2+) can lead to Ca(2+) overloading-induced loss of mitochondrial membrane potential and suggest that recovery of Ca(2+) dysregulation is a target of Akt-mediated protection.

Background: Although the occurrence of cardiac myocyte apoptosis during heart failure has been documented, its importance in pathogenesis is unknown. Transgenic mice with cardiac-restricted overexpression of Galpha(q) exhibit a lethal, peripartum cardiomyopathy accompanied by apoptosis. To test whether apoptosis is causally linked to heart failure, we assessed whether inhibiting this cell death would improve left ventricular function and survival in the Galpha(q) peripartum cardiomyopathy model., Methods and Results: The potent polycaspase inhibitor IDN-1965 or vehicle was administered subcutaneously to Galpha(q) mice by osmotic minipump beginning on day 12 of pregnancy and continuing through euthanasia at day 14 postpartum. As expected, IDN-1965 markedly suppressed cardiac caspase-3-like activity (86.5%; P<0.01), accompanied by reduction in the frequency of cardiac myocyte apoptosis from 1.9+/-0.3% to 0.2+/-0.1% (P<0.01). Animals receiving IDN-1965 exhibited significant improvements in left ventricular end-diastolic dimension (vehicle, 4.7+/-0.1 mm; IDN-1965, 4.2+/-0.1 mm; P<0.01), fractional shortening (vehicle, 30.7+/-1.2%; IDN-1965, 38.9+/-1.0%; P<0.01), positive (vehicle, 3972+/-412; IDN-1965, 5870+/-295; P<0.01) and negative (vehicle, 2365+/-213; IDN-1965, 3413+/-201; P<0.01) dP/dt, and complete suppression of mortality (vehicle, 6 of 20 died; IDN-1965, 0 of 14 died; P<0.05)., Conclusions: Reduction in cardiac myocyte apoptosis by caspase inhibition improved left ventricular function and survival in pregnant Galpha(q) mice. These data indicate that cardiac myocyte apoptosis plays a causal role in the pathogenesis of cardiomyopathy in this model. Caspase inhibition may provide a novel therapeutic target for heart failure.

Expression of the wild type alpha subunit of Gq (GqWT) in cardiomyocytes induces hypertrophy, whereas a constitutively active G alpha q subunit (GqQ209L) induces apoptosis. Akt phosphorylation increases with GqWT expression but is markedly attenuated in cardiomyocytes expressing GqQ209L or in those expressing GqWT and treated with agonist. A membrane-targeted Akt rescues GqQ209L-expressing cardiomyocytes from apoptotic cell death. In contrast, leukemia inhibitory factor fails to activate Akt or promote cell survival in these cells. Association of Akt and PDK-1 with the membrane is also diminished in GqQ209L-expressing cardiomyocytes. Phosphatidylinositol 3,4,5-trisphosphate (PIP3), the primary regulator of Akt, increases significantly in GqWT-expressing cells but not in cardiomyocytes expressing GqQ209L. Levels of phosphatidylinositol 4,5-bisphosphate (PIP2), the immediate precursor of PIP3, are also markedly lower in GqQ209L-expressing compared to control cells. Expression of a GqQ209L mutant that has diminished capacity to activate phospholipase C does not decrease PIP2 or Akt or induce apoptosis. In transgenic mice with cardiac G alpha q overexpression, heart failure and increased cardiomyocyte apoptosis develop during the peripartal period. Akt phosphorylation and PIP2 levels decrease concomitantly. Our findings suggest that an Akt-mediated cell survival pathway is compromised by the diminished availability of PIP2 elicited by pathological levels of Gq activity.

The Rho family small GTPases play a crucial role in mediating cellular responses to stretch. However, it remains unclear how force is transduced to Rho signaling pathways. We investigated the effect of stretch on the activation and caveolar localization of RhoA and Rac1 in neonatal rat cardiomyocytes. In unstretched cardiomyocytes, RhoA and Rac1 were detected in both caveolar and non-caveolar fractions as assessed using detergent-free floatation analysis. Stretching myocytes for 4 min activated RhoA and Rac1. By 15 min of stretch, RhoA and Rac1 had dissociated from caveolae, and there was decreased coprecipitation of RhoA and Rac1 with caveolin-3. To determine whether compartmentation of RhoA and Rac1 within caveolae was necessary for stretch signaling, we disrupted caveolae with methyl beta-cyclodextrin (MbetaCD). Treatment with 5 mm MbetaCD for 1 h dissociated both RhoA and Rac1 from caveolae. Under this condition, stretch failed to activate RhoA or Rac1. Stretch-induced actin cytoskeletal organization was concomitantly impaired. Interestingly the ability of stretch to activate extracellular signal-regulated kinase (ERK) was unaffected by MbetaCD treatment, but ERK translocation to the nucleus was impaired. Stretch-induced hypertrophy was also inhibited. Actin cytoskeletal disruption with cytochalasin-D also prevented stretch from increasing nuclear ERK, whereas actin polymerization with jasplakinolide restored nuclear translocation of activated ERK in the presence of MbetaCD. We suggest that activation of RhoA or Rac1, localized in a caveolar compartment, is essential for sensing externally applied force and transducing this signal to the actin cytoskeleton and ERK translocation.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) delta is the predominant cardiac isoform, and the deltaC splice variant is cytoplasmic. We overexpressed CaMKIIdeltaC in mouse heart and observed dilated heart failure and altered myocyte Ca2+ regulation in 3-month-old CaMKIIdeltaC transgenic mice (TG) versus wild-type littermates (WT). Heart/body weight ratio and cardiomyocyte size were increased about 2-fold in TG versus WT. At 1 Hz, twitch shortening, [Ca2+]i transient amplitude, and diastolic [Ca2+]i were all reduced by approximately 50% in TG versus WT. This is explained by >50% reduction in SR Ca2+ content in TG versus WT. Peak Ca2+ current (ICa) was slightly increased, and action potential duration was prolonged in TG versus WT. Despite lower SR Ca2+ load and diastolic [Ca2+]i, fractional SR Ca2+ release was increased and resting spontaneous SR Ca2+ release events (Ca2+ sparks) were doubled in frequency in TG versus WT (with prolonged width and duration, but lower amplitude). Enhanced Ca2+ spark frequency was also seen in TG at 4 weeks (before heart failure onset). Acute CaMKII inhibition normalized Ca2+ spark frequency and ICa, consistent with direct CaMKII activation of ryanodine receptors (and ICa) in TG. The rate of [Ca2+]i decline during caffeine exposure was faster in TG, indicating enhanced Na+-Ca2+ exchange function (consistent with protein expression measurements). Enhanced diastolic SR Ca2+ leak (via sparks), reduced SR Ca2+-ATPase expression, and increased Na+-Ca2+ exchanger explain the reduced diastolic [Ca2+]i and SR Ca2+ content in TG. We conclude that CaMKIIdeltaC overexpression causes acute modulation of excitation-contraction coupling, which contributes to heart failure.

Addition of ATP to neonatal rat cardiomyocytes has been reported to inhibit hypertrophic growth responses, even though G(q)-coupled receptors are activated. In the current study, we investigated hypertrophic responses to activation of G(q)-coupled-purinergic receptors on cardiomyocytes using UTP as an alternative agonist to ATP. UTP (100 microM) activated phospholipase C via G(q) similarly to ATP, and responses to the two agonists were not additive. Similarly, UTP and ATP both induced phosphorylation of extracellular signal-regulated kinase (ERK1/2), while having little effect on p38 mitogen-activated protein kinase or c-Jun NH(2)-terminal kinase. However, addition of UTP (100 microM) to cardiomyocytes caused hypertrophic growth indicated by increased protein content without DNA synthesis. ATP (100 microM) caused no increase in protein. We conclude that activation of purinergic receptors on neonatal cardiomyocytes initiates hypertrophic signaling pathways, but that prolonged exposure to ATP, but not UTP, has growth-inhibitory effects.