Abstract-Myocardial G protein-coupled receptor kinase (GRK)2 is a critical regulator of cardiac -adrenergic receptor (AR) signaling and cardiac function. Its upregulation in heart failure may further depress cardiac function and contribute to mortality in this syndrome. Preventing GRK2 translocation to activated AR with a GRK2-derived peptide that binds G  ␥ (ARKct) has benefited some models of heart failure, but the precise mechanism is uncertain, because GRK2 is still present and ARKct has other potential effects. We generated mice in which cardiac myocyte GRK2 expression was normal during embryonic development but was ablated after birth (␣MHC-CreϫGRK2 fl/fl) or only after administration of tamoxifen (␣MHC-MerCreMerϫGRK2 fl/fl) and examined the consequences of GRK2 ablation before and after surgical coronary artery ligation on cardiac adaptation after myocardial infarction. Absence of GRK2 before coronary artery ligation prevented maladaptive postinfarction remodeling and preserved AR responsiveness. Strikingly, GRK2 ablation initiated 10 days after infarction increased survival, enhanced cardiac contractile performance, and halted ventricular remodeling. These results demonstrate a specific causal role for GRK2 in postinfarction cardiac remodeling and heart failure and support therapeutic approaches of targeting GRK2 or restoring AR signaling by other means to improve outcomes in heart failure.
Cardiac-restricted overexpression of the Ca 2+ -binding protein S100A1 has been shown to lead to increased myocardial contractile performance in vitro and in vivo. Since decreased cardiac expression of S100A1 is a characteristic of heart failure, we tested the hypothesis that S100A1 gene transfer could restore contractile function of failing myocardium. Adenoviral S100A1 gene delivery normalized S100A1 protein expression in a postinfarction rat heart failure model and reversed contractile dysfunction of failing myocardium in vivo and in vitro. S100A1 gene transfer to failing cardiomyocytes restored diminished intracellular Ca 2+ transients and sarcoplasmic reticulum (SR) Ca 2+ load mechanistically due to increased SR Ca 2+ uptake and reduced SR Ca 2+ leak. Moreover, S100A1 gene transfer decreased elevated intracellular Na + concentrations to levels detected in nonfailing cardiomyocytes, reversed reactivated fetal gene expression, and restored energy supply in failing cardiomyocytes. Intracoronary adenovirus-mediated S100A1 gene delivery in vivo to the postinfarcted failing rat heart normalized myocardial contractile function and Ca 2+ handling, which provided support in a physiological context for results found in myocytes. Thus, the present study demonstrates that restoration of S100A1 protein levels in failing myocardium by gene transfer may be a novel therapeutic strategy for the treatment of heart failure.
Background— Diminished cardiac S100A1 protein levels are characteristic of ischemic and dilated human cardiomyopathy. Because S100A1 has recently been identified as a Ca 2+ -dependent inotropic factor in the heart, this study sought to explore the pathophysiological relevance of S100A1 levels in development and progression of postischemic heart failure (HF). Methods and Results— S100A1-transgenic (STG) and S100A1-knockout (SKO) mice were subjected to myocardial infarction (MI) by surgical left anterior descending coronary artery ligation, and survival, cardiac function, and remodeling were compared with nontransgenic littermate control (NLC) and wild-type (WT) animals up to 4 weeks. Although MI size was similar in all groups, infarcted S100A1-deficient hearts (SKO-MI) responded with acute contractile decompensation and accelerated transition to HF, rapid onset of cardiac remodeling with augmented apoptosis, and excessive mortality. NLC/WT-MI mice, displaying a progressive decrease in cardiac S100A1 expression, showed a later onset of cardiac remodeling and progression to HF. Infarcted S100A1-overexpressing hearts (STG-MI), however, showed preserved global contractile performance, abrogated apoptosis, and prevention from cardiac hypertrophy and HF with superior survival compared with NLC/WT-MI and SKO-MI. Both Gq-protein–dependent signaling and protein kinase C activation resulted in decreased cardiac S100A1 mRNA and protein levels, whereas Gs-protein–related signaling exerted opposite effects on cardiac S100A1 abundance. Mechanistically, sarcoplasmic reticulum Ca 2+ cycling and β-adrenergic signaling were severely impaired in SKO-MI myocardium but preserved in STG-MI. Conclusions— Our novel proof-of-concept study provides evidence that downregulation of S100A1 protein critically contributes to contractile dysfunction of the diseased heart, which is potentially responsible for driving the progressive downhill clinical course of patients with HF.
S100A1, a Ca 2؉ binding protein of the EF-hand type, is preferentially expressed in myocardial tissue and has been found to colocalize with the sarcoplasmic reticulum (SR) and the contractile filaments in cardiac tissue. Because S100A1 is known to modulate SR Ca 2؉ handling in skeletal muscle, we sought to investigate the specific role of S100A1 in the regulation of myocardial contractility. To address this issue, we investigated contractile properties of adult cardiomyocytes as well as of engineered heart tissue after S100A1 adenoviral gene transfer. S100A1 gene transfer resulted in a significant increase of unloaded shortening and isometric contraction in isolated cardiomyocytes and engineered heart tissues, respectively. Analysis of intracellular Ca 2؉ cycling in S100A1-overexpressing cardiomyocytes revealed a significant increase in cytosolic Ca 2؉ transients, whereas in functional studies on saponinpermeabilized adult cardiomyocytes, the addition of S100A1 protein significantly enhanced SR Ca 2؉ uptake. Moreover, in Triton-skinned ventricular trabeculae, S100A1 protein significantly decreased myofibrillar Ca 2؉ sensitivity ([EC50%]) and Ca 2؉ cooperativity, whereas maximal isometric force remained unchanged. Our data suggest that S100A1 effects are cAMP independent because cellular cAMP levels and protein kinase A-dependent phosphorylation of phospholamban were not altered, and carbachol failed to suppress S100A1 actions. These results show that S100A1 overexpression enhances cardiac contractile performance and establish the concept of S100A1 as a regulator of myocardial contractility. S100A1 thus improves cardiac contractile performance both by regulating SR Ca 2؉ handling and myofibrillar Ca 2؉ responsiveness.
Background-The incidence of heart failure is ever-growing, and it is urgent to develop improved treatments. An attractive approach is gene therapy; however, the clinical barrier has yet to be broken because of several issues, including the lack of an ideal vector supporting safe and long-term myocardial transgene expression. Methods and Results-Here, we show that the use of a recombinant adeno-associated viral (rAAV6) vector containing a novel cardiac-selective enhancer/promoter element can direct stable cardiac expression of a therapeutic transgene, the calcium (Ca 2ϩ )-sensing S100A1, in a rat model of heart failure. The chronic heart failure-rescuing properties of myocardial S100A1 expression, the result of improved sarcoplasmic reticulum Ca 2ϩ handling, included improved contractile function and left ventricular remodeling. Adding to the clinical relevance, long-term S100A1 therapy had unique and additive beneficial effects over -adrenergic receptor blockade, a current pharmacological heart failure treatment.Conclusions-These findings demonstrate that stable increased expression of S100A1 in the failing heart can be used for long-term reversal of LV dysfunction and remodeling. Thus, long-term, cardiac-targeted rAAV6-S100A1 gene therapy may be of potential clinical utility in human heart failure.
As a prerequisite to clinical application, we determined the long-term therapeutic effectiveness and safety of adeno-associated viral (AAV) S100A1 gene therapy in a preclinical, large animal model of heart failure. S100A1, a positive inotropic regulator of myocardial contractility, becomes depleted in failing cardiomyocytes in humans and various animal models, and myocardial-targeted S100A1 gene transfer rescues cardiac contractile function by restoring sarcoplasmic reticulum calcium Ca2+ handling in acutely and chronically failing hearts in small animal models. We induced heart failure in domestic pigs by balloon-occlusion of the left circumflex coronary artery, resulting in myocardial infarction. After 2 weeks, when the pigs displayed significant left ventricular contractile dysfunction, we administered through retrograde coronary venous delivery, AAV9-S100A1 to the left ventricular non-infarcted myocardium. AAV9-luciferase and saline treatment served as control. At 14 weeks, both control groups showed significantly decreased myocardial S100A1 protein expression along with progressive deterioration of cardiac performance and left ventricular remodeling. AAV9-S100A1 treatment prevented and reversed this phenotype by restoring cardiac S100A1 protein levels. S100A1 treatment normalized cardiomyocyte Ca2+ cycling, sarcoplasmic reticulum calcium handling and energy homeostasis. Transgene expression was restricted to cardiac tissue and extra-cardiac organ function was uncompromised indicating a favorable safety profile. This translational study shows the pre-clinical feasibility, long-term therapeutic effectiveness and a favorable safety profile of cardiac AAV9-S100A1 gene therapy in a preclinical model of heart failure. Our study presents a strong rational for a clinical trial of S100A1 gene therapy for human heart failure that could potentially complement current strategies to treat end-stage heart failure.
Cardiac hypertrophy is an independent risk for heart failure (HF) and sudden death. Deciphering signalling pathways dependent on extracellular calcium (Ca2+) influx that control normal and pathological cardiac growth may enable identification of novel therapeutic targets. The objective of the present study is to determine the role of the Ca2+ release-activated Ca2+ (CRAC) channel Orai1 and stromal interaction molecule 1 (Stim1) in postnatal cardiomycoyte store-operated Ca2+ entry (SOCE) and impact on normal and hypertrophic postnatal cardiomyocyte growth. Employing a combination of siRNA-mediated gene silencing, cultured neonatal rat ventricular cardiomyocytes together with indirect immunofluorescence, epifluorescent Ca2+ imaging and site-specific protein phosphorylation and real-time mRNA expression analysis, we show for the first time that both Orai1 and Stim1 are present in cardiomyocytes and required for SOCE due to intracellular Ca2+ store depletion by thapsigargin. Stim1-KD but not Orai1-KD significantly decreased diastolic Ca2+ levels and caffeine-releasable Ca2+ from the sarcoplasmic reticulum (SR). Conversely, Orai1-KD but not Stim1-KD significantly diminished basal NRCM cell size, anp and bnp mRNA levels and activity of the calcineurin (CnA) signaling pathway although diminishing both Orai1 and Stim1 protein similarly attenuated calmodulin kinase II (CamKII) and ERK1/2 activity under basal conditions. Both Orai1- and Stim1-KD completely abrogated phenylephrine (PE) mediated hypertrophic NRCM growth and enhanced natriuretic factor expression by inhibiting Gq-protein conveyed activation of the CaMKII and ERK1/2 signaling pathway. Interestingly, only Orai1-KD but not Stim1-KD prevented Gq-mediated CaN-dependent prohypertrophic signalling. This study shows for the first time that both Orai1 and Stim1 have a key role in cardiomyocyte SOCE regulating both normal and hypertrophic postnatal cardiac growth in vitro.
S100A1, a Ca2؉ -sensing protein of the EF-hand family, is most highly expressed in myocardial tissue, and cardiac S100A1 overexpression in vitro has been shown to enhance myocyte contractile properties. To study the physiological consequences of S100A1 in vivo, transgenic mice were developed with cardiac-restricted overexpression of S100A1. Characterization of two independent transgenic mouse lines with ϳ4-fold overexpression of S100A1 in the myocardium revealed a marked augmentation of in vivo basal cardiac function that remained elevated after -adrenergic receptor stimulation. Contractile function and Ca 2؉ handling properties were increased in ventricular cardiomyocytes isolated from S100A1 transgenic mice. Enhanced cellular Ca 2؉ cycling by S100A1 was associated both with increased sarcoplasmic reticulum Ca 2؉ content and enhanced sarcoplasmic reticulum Ca 2؉ -induced Ca 2؉ release, and S100A1 was shown to associate with the cardiac ryanodine receptor. No alterations in -adrenergic signal transduction or major cardiac Ca 2؉ -cycling proteins occurred, and there were no signs of hypertrophy with chronic cardiac S100A1 overexpression. Our findings suggest that S100A1 plays an important in vivo role in the regulation of cardiac function perhaps through interacting with the ryanodine receptor. Because S100A1 protein expression is downregulated in heart failure, increasing S100A1 expression in the heart may represent a novel means to augment contractility.
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