S100A1's single cysteine is an indispensable redox switch for the protection against diastolic calcium waves in cardiomyocytes.

The EF-hand calcium (Ca2+) sensor protein S100A1 combines inotropic with antiarrhythmic potency in cardiomyocytes (CMs). Oxidative posttranslational modification (ox-PTM) of S100A1's conserved, single-cysteine residue (C85) via reactive nitrogen species (i.e., S-nitrosylation or S-glutathionylation) has been proposed to modulate conformational flexibility of intrinsically disordered sequence fragments and to increase the molecule's affinity toward Ca2+. Considering the unknown biological functional consequence, we aimed to determine the impact of the C85 moiety of S100A1 as a potential redox switch. We first uncovered that S100A1 is endogenously glutathionylated in the adult heart in vivo. To prevent glutathionylation of S100A1, we generated S100A1 variants that were unresponsive to ox-PTMs. Overexpression of wild-type (WT) and C85-deficient S100A1 protein variants in isolated CM demonstrated equal inotropic potency, as shown by equally augmented Ca2+ transient amplitudes under basal conditions and ß-adrenergic receptor (ßAR) stimulation. However, in contrast, ox-PTM defective S100A1 variants failed to protect against arrhythmogenic diastolic sarcoplasmic reticulum (SR) Ca2+ waves and ryanodine receptor 2 (RyR2) hypernitrosylation during ßAR stimulation. Despite diastolic performance failure, C85-deficient S100A1 protein variants exerted similar Ca2+-dependent interaction with the RyR2 than WT-S100A1. Dissecting S100A1's molecular structure-function relationship, our data indicate for the first time that the conserved C85 residue potentially acts as a redox switch that is indispensable for S100A1's antiarrhythmic but not its inotropic potency in CMs. We, therefore, propose a model where C85's ox-PTM determines S100A1's ability to beneficially control diastolic but not systolic RyR2 activity.NEW & NOTEWORTHY S100A1 is an emerging candidate for future gene-therapy treatment of human chronic heart failure. We aimed to study the significance of the conserved single-cysteine 85 (C85) residue in cardiomyocytes. We show that S100A1 is endogenously glutathionylated in the heart and demonstrate that this is dispensable to increase systolic Ca2+ transients, but indispensable for mediating S100A1's protection against sarcoplasmic reticulum (SR) Ca2+ waves, which was dependent on the ryanodine receptor 2 (RyR2) nitrosylation status.

Ligand-activated RXFP1 gene therapy ameliorates pressure overload-induced cardiac dysfunction.

Recurrent episodes of decompensated heart failure (HF) represent an emerging cause of hospitalizations in developed countries with an urgent need for effective therapies. Recently, the pregnancy-related hormone relaxin (RLN) was found to mediate cardio-protective effects and act as a positive inotrope in the cardiovascular system. RLN binds to the RLN family peptide receptor 1 (RXFP1), which is predominantly expressed in atrial cardiomyocytes. We therefore hypothesized that ventricular RXFP1 expression might exert potential therapeutic effects in an in vivo model of cardiac dysfunction. Thus, mice were exposed to pressure overload by transverse aortic constriction and treated with AAV9 to ectopically express RXFP1. To activate RXFP1 signaling, RLN was supplemented subcutaneously. Ventricular RXFP1 expression was well tolerated. Additional RLN administration not only abrogated HF progression but restored left ventricular systolic function. In accordance, upregulation of fetal genes and pathological remodeling markers were significantly reduced. In vitro, RLN stimulation of RXFP1-expressing cardiomyocytes induced downstream signaling, resulting in protein kinase A (PKA)-specific phosphorylation of phospholamban (PLB), which was distinguishable from ß-adrenergic activation. PLB phosphorylation corresponded to increased calcium amplitude and contractility. In conclusion, our results demonstrate that ligand-activated cardiac RXFP1 gene therapy represents a therapeutic approach to attenuate HF with the potential to adjust therapy by exogenous RLN supplementation.

Updated and enhanced pig cardiac transcriptome based on long-read RNA sequencing and proteomics.

Clinically translatable large animal models have become indispensable for cardiovascular research, clinically relevant proof of concept studies and for novel therapeutic interventions. In particular, the pig has emerged as an essential cardiovascular disease model, because its heart, circulatory system, and blood supply are anatomically and functionally similar to that of humans. Currently, molecular and omics-based studies in the pig are hampered by the incompleteness of the genome and the lack of diversity of the corresponding transcriptome annotation. Here, we employed Nanopore long-read sequencing and in-depth proteomics on top of Illumina RNA-seq to enhance the pig cardiac transcriptome annotation. We assembled 15,926 transcripts, stratified into coding and non-coding, and validated our results by complementary mass spectrometry. A manual review of several gene loci, which are associated with cardiac function, corroborated the utility of our enhanced annotation. All our data are available for download and are provided as tracks for integration in genome browsers. We deem this resource as highly valuable for molecular research in an increasingly relevant large animal model.

Development of an AAV9-RNAi-mediated silencing strategy to abrogate TRPM4 expression in the adult heart.

The cation channel transient receptor potential melastatin 4 (TRPM4) is a calcium-activated non-selective cation channel and acts in cardiomyocytes as a negative modulator of the L-type Ca2+ influx. Global deletion of TRPM4 in the mouse led to increased cardiac contractility under ß-adrenergic stimulation. Consequently, cardiomyocyte-specific inactivation of the TRPM4 function appears to be a promising strategy to improve cardiac contractility in heart failure patients. The aim of this study was to develop a gene therapy approach in mice that specifically silences the expression of TRPM4 in cardiomyocytes. First, short hairpin RNAmiR30 (shRNAmiR30) sequences against the TRPM4 mRNA were screened in vitro using lentiviral transduction for a stable expression of the shRNA cassettes. Western blot analysis identified three efficient shRNAmiR30 sequences out of six, which reduced the endogenous TRPM4 protein level by up to 90 ± 6%. Subsequently, the most efficient shRNAmiR30 sequences were delivered into cardiomyocytes of adult mice using adeno-associated virus serotype 9 (AAV9)-mediated gene transfer. Initially, the AAV9 vector particles were administered via the lateral tail vein, which resulted in a downregulation of TRPM4 by 46 ± 2%. Next, various optimization steps were carried out to improve knockdown efficiency in vivo. First, the design of the expression cassette was streamlined for integration in a self-complementary AAV vector backbone for a faster expression. Compared to the application via the lateral tail vein, intravenous application via the retro-orbital sinus has the advantage that the vector solution reaches the heart directly and in a high concentration, and eventually a TRPM4 knockdown efficiency of 90 ± 7% in the heart was accomplished by this approach. By optimization of the shRNAmiR30 constructs and expression cassette as well as the route of AAV9 vector application, a 90% reduction of TRPM4 expression was achieved in the adult mouse heart. In the future, AAV9-RNAi-mediated inactivation of TRPM4 could be a promising strategy to increase cardiac contractility in preclinical animal models of acute and chronic forms of cardiac contractile failure.

Epigenetic regulation of cardiac electrophysiology in atrial fibrillation: HDAC2 determines action potential duration and suppresses NRSF in cardiomyocytes.

Atrial fibrillation (AF) is associated with electrical remodeling, leading to cellular electrophysiological dysfunction and arrhythmia perpetuation. Emerging evidence suggests a key role for epigenetic mechanisms in the regulation of ion channel expression. Histone deacetylases (HDACs) control gene expression through deacetylation of histone proteins. We hypothesized that class I HDACs in complex with neuron-restrictive silencer factor (NRSF) determine atrial K+ channel expression. AF was characterized by reduced atrial HDAC2 mRNA levels and upregulation of NRSF in humans and in a pig model, with regional differences between right and left atrium. In vitro studies revealed inverse regulation of Hdac2 and Nrsf in HL-1 atrial myocytes. A direct association of HDAC2 with active regulatory elements of cardiac K+ channels was revealed by chromatin immunoprecipitation. Specific knock-down of Hdac2 and Nrsf induced alterations of K+ channel expression. Hdac2 knock-down resulted in prolongation of action potential duration (APD) in neonatal rat cardiomyocytes, whereas inactivation of Nrsf induced APD shortening. Potential AF-related triggers were recapitulated by experimental tachypacing and mechanical stretch, respectively, and exerted differential effects on the expression of class I HDACs and K+ channels in cardiomyocytes. In conclusion, HDAC2 and NRSF contribute to AF-associated remodeling of APD and K+ channel expression in cardiomyocytes via direct interaction with regulatory chromatin regions. Specific modulation of these factors may provide a starting point for the development of more individualized treatment options for atrial fibrillation.

From traditional pharmacological towards nucleic acid-based therapies for cardiovascular diseases.

Nucleic acid-based therapeutics are currently developed at large scale for prevention and management of cardiovascular diseases (CVDs), since: (i) genetic studies have highlighted novel therapeutic targets suggested to be causal for CVD; (ii) there is a substantial recent progress in delivery, efficacy, and safety of nucleic acid-based therapies; (iii) they enable effective modulation of therapeutic targets that cannot be sufficiently or optimally addressed using traditional small molecule drugs or antibodies. Nucleic acid-based therapeutics include (i) RNA-targeted therapeutics for gene silencing; (ii) microRNA-modulating and epigenetic therapies; (iii) gene therapies; and (iv) genome-editing approaches (e.g. CRISPR-Cas-based): (i) RNA-targeted therapeutics: several large-scale clinical development programmes, using antisense oligonucleotides (ASO) or short interfering RNA (siRNA) therapeutics for prevention and management of CVD have been initiated. These include ASO and/or siRNA molecules to lower apolipoprotein (a) [apo(a)], proprotein convertase subtilisin/kexin type 9 (PCSK9), apoCIII, ANGPTL3, or transthyretin (TTR) for prevention and treatment of patients with atherosclerotic CVD or TTR amyloidosis. (ii) MicroRNA-modulating and epigenetic therapies: novel potential therapeutic targets are continually arising from human non-coding genome and epigenetic research. First microRNA-based therapeutics or therapies targeting epigenetic regulatory pathways are in clinical studies. (iii) Gene therapies: EMA/FDA have approved gene therapies for non-cardiac monogenic diseases and LDL receptor gene therapy is currently being examined in patients with homozygous hypercholesterolaemia. In experimental studies, gene therapy has significantly improved cardiac function in heart failure animal models. (iv) Genome editing approaches: these technologies, such as using CRISPR-Cas, have proven powerful in stem cells, however, important challenges are remaining, e.g. low rates of homology-directed repair in somatic cells such as cardiomyocytes. In summary, RNA-targeted therapies (e.g. apo(a)-ASO and PCSK9-siRNA) are now in large-scale clinical outcome trials and will most likely become a novel effective and safe therapeutic option for CVD in the near future. MicroRNA-modulating, epigenetic, and gene therapies are tested in early clinical studies for CVD. CRISPR-Cas-mediated genome editing is highly effective in stem cells, but major challenges are remaining in somatic cells, however, this field is rapidly advancing.

Induction of cardiac dysfunction in developing and adult zebrafish by chronic isoproterenol stimulation.

Zebrafish is a widely used model to evaluate genetic variants and modifiers that can cause heart muscle diseases. Surprisingly, the ß-adrenergic receptor (ß-AR) pathway in zebrafish is not well characterized, although abnormal ß-AR signaling is a major contributor to human heart failure (HF). Chronic ß-AR activation in the attempt to normalize heart function in the failing heart results in a reduction of the ß-ARs expression and receptor desensitization, largely mediated through G-protein coupled receptor kinase 2 (GRK2) upregulation. This in turn leads to further deterioration of heart function and progression towards HF. This study seeks to systematically characterize the function of the ß-AR signaling in developing and adult zebrafish to ultimately assess the ability to induce HF through chronic ß-AR activation by isoproterenol (ISO) as established in the mouse model. Larval hearts first responded to ISO by 3dpf, in concordance with robust expression of key components of the ß-AR signaling pathway. Although ISO-induced ß1-AR and ß2-AR isoform upregulation persisted, chronic ISO stimulation for 5d caused systolic cardiac dysfunction concurrently with maximal expression of G-protein-coupled receptor kinase-2 (GRK2). More consistent to mammalians, adult zebrafish developed significant heart failure in concert with ß1-AR downregulation, and GRK2 and brain natriuretic peptide (BNP) upregulation in response to prolonged, 14d ISO-stimulation. This was accompanied by significant cell death and inflammation without detectable fibrosis. Our study unveils important characteristics of larvae and adult zebrafish hearts pertaining to ß-AR signaling. A lack of ß-AR responsiveness and atypical ß-AR/GRK2 ratios in larval zebrafish should be considered. Adult zebrafish resembled the mammalian situation on the functional and molecular level more closely, but also revealed differences to dysfunctional mammalian hearts, i.e. lack of fibrosis. Our study establishes the first ISO-inducible HF model in adult zebrafish and present critical characteristics of the zebrafish heart essential to be considered when utilizing the zebrafish as a human disease and future drug discovery model.

S100A1 Protein Does Not Compete with Calmodulin for Ryanodine Receptor Binding but Structurally Alters the Ryanodine Receptor·Calmodulin Complex.

S100A1 has been suggested as a therapeutic agent to enhance myocyte Ca(2+) cycling in heart failure, but its molecular mode of action is poorly understood. Using FRET, we tested the hypothesis that S100A1 directly competes with calmodulin (CaM) for binding to intact, functional ryanodine receptors type I (RyR1) and II (RyR2) from skeletal and cardiac muscle, respectively. Our FRET readout provides an index of acceptor-labeled CaM binding near donor-labeled FKBP (FK506-binding protein 12.6) on the cytoplasmic domain of RyR in isolated sarcoplasmic reticulum vesicles. S100A1 (0.01-400 µm) partially inhibited FRET (i.e. CaM binding), with Ki > 10 µm, for both RyR1 and RyR2. The high [S100A1] required for partial effects on FRET indicates a lack of competition by S100A1 on CaM/RyR binding under normal physiological conditions. High-resolution analysis of time-resolved FRET detects two structural states of RyR-bound CaM, which respond to [Ca(2+)] and are isoform-specific. The distribution of these structural states was perturbed only by high micromolar [S100A1], which promoted a shift of bound CaM to a lower FRET orientation (without altering the amount of CaM bound to RyR). Thus, high micromolar S100A1 does alter the CaM/RyR interaction, without involving competition. Nevertheless, submicromolar S100A1 can alter RyR function, an effect that is influenced by both [Ca(2+)] and [CaM]. We conclude that CaM and S100A1 can concurrently bind to and functionally modulate RyR1 and RyR2, but this does not involve direct competition at the RyR CaM binding site.

S100A6 Regulates Endothelial Cell Cycle Progression by Attenuating Antiproliferative Signal Transducers and Activators of Transcription 1 Signaling.

OBJECTIVE: S100A6, a member of the S100 protein family, has been described as relevant for cell cycle entry and progression in endothelial cells. The molecular mechanism conferring S100A6's proliferative actions, however, remained elusive. APPROACH AND RESULTS: Originating from the clinically relevant observation of enhanced S100A6 protein expression in proliferating endothelial cells in remodeling coronary and carotid arteries, our study unveiled S100A6 as a suppressor of antiproliferative signal transducers and activators of transcription 1 signaling. Discovery of the molecular liaison was enabled by combining gene expression time series analysis with bioinformatic pathway modeling in S100A6-silenced human endothelial cells stimulated with vascular endothelial growth factor A. This unbiased approach led to successful identification and experimental validation of interferon-inducible transmembrane protein 1 and protein inhibitors of activated signal transducers and activators of transcription as key components of the link between S100A6 and signal transducers and activators of transcription 1. CONCLUSIONS: Given the important role of coordinated endothelial cell cycle activity for integrity and reconstitution of the inner lining of arterial blood vessels in health and disease, signal transducers and activators of transcription 1 suppression by S100A6 may represent a promising therapeutic target to facilitate reendothelialization in damaged vessels.

Oral treatment with a zinc complex of acetylsalicylic acid prevents diabetic cardiomyopathy in a rat model of type-2 diabetes: activation of the Akt pathway.

BACKGROUND: Type-2 diabetics have an increased risk of cardiomyopathy, and heart failure is a major cause of death among these patients. Growing evidence indicates that proinflammatory cytokines may induce the development of insulin resistance, and that anti-inflammatory medications may reverse this process. We investigated the effects of the oral administration of zinc and acetylsalicylic acid, in the form of bis(aspirinato)zinc(II)-complex Zn(ASA)2, on different aspects of cardiac damage in Zucker diabetic fatty (ZDF) rats, an experimental model of type-2 diabetic cardiomyopathy. METHODS: Nondiabetic control (ZL) and ZDF rats were treated orally with vehicle or Zn(ASA)2 for 24 days. At the age of 29-30 weeks, the electrical activities, left-ventricular functional parameters and left-ventricular wall thicknesses were assessed. Nitrotyrosine immunohistochemistry, TUNEL-assay, and hematoxylin-eosin staining were performed. The protein expression of the insulin-receptor and PI3K/AKT pathway were quantified by Western blot. RESULTS: Zn(ASA)2-treatment significantly decreased plasma glucose concentration in ZDF rats (39.0 ± 3.6 vs 49.4 ± 2.8 mM, P < 0.05) while serum insulin-levels were similar among the groups. Data from cardiac catheterization showed that Zn(ASA)2 normalized the increased left-ventricular diastolic stiffness (end-diastolic pressure-volume relationship: 0.064 ± 0.008 vs 0.084 ± 0.014 mmHg/µl; end-diastolic pressure: 6.5 ± 0.6 vs 7.9 ± 0.7 mmHg, P < 0.05). Furthermore, ECG-recordings revealed a restoration of prolonged QT-intervals (63 ± 3 vs 83 ± 4 ms, P < 0.05) with Zn(ASA)2. Left-ventricular wall thickness, assessed by echocardiography, did not differ among the groups. However histological examination revealed an increase in the cardiomyocytes' transverse cross-section area in ZDF compared to the ZL rats, which was significantly decreased after Zn(ASA)2-treatment. Additionally, a significant fibrotic remodeling was observed in the diabetic rats compared to ZL rats, and Zn(ASA)2-administered ZDF rats showed a similar collagen content as ZL animals. In diabetic hearts Zn(ASA)2 significantly decreased DNA-fragmentation, and nitro-oxidative stress, and up-regulated myocardial phosphorylated-AKT/AKT protein expression. Zn(ASA)2 reduced cardiomyocyte death in a cellular model of oxidative stress. Zn(ASA)2 had no effects on altered myocardial CD36, GLUT-4, and PI3K protein expression. CONCLUSIONS: We demonstrated that treatment of type-2 diabetic rats with Zn(ASA)2 reduced plasma glucose-levels and prevented diabetic cardiomyopathy. The increased myocardial AKT activation could, in part, help to explain the cardioprotective effects of Zn(ASA)2. The oral administration of Zn(ASA)2 may have therapeutic potential, aiming to prevent/treat cardiac complications in type-2 diabetic patients.

S100A1 DNA-based Inotropic Therapy Protects Against Proarrhythmogenic Ryanodine Receptor 2 Dysfunction.

Restoring expression levels of the EF-hand calcium (Ca(2+)) sensor protein S100A1 has emerged as a key factor in reconstituting normal Ca(2+) handling in failing myocardium. Improved sarcoplasmic reticulum (SR) function with enhanced Ca(2+) resequestration appears critical for S100A1's cyclic adenosine monophosphate-independent inotropic effects but raises concerns about potential diastolic SR Ca(2+) leakage that might trigger fatal arrhythmias. This study shows for the first time a diminished interaction between S100A1 and ryanodine receptors (RyR2s) in experimental HF. Restoring this link in failing cardiomyocytes, engineered heart tissue and mouse hearts, respectively, by means of adenoviral and adeno-associated viral S100A1 cDNA delivery normalizes diastolic RyR2 function and protects against Ca(2+)- and ß-adrenergic receptor-triggered proarrhythmogenic SR Ca(2+) leakage in vitro and in vivo. S100A1 inhibits diastolic SR Ca(2+) leakage despite aberrant RyR2 phosphorylation via protein kinase A and calmodulin-dependent kinase II and stoichiometry with accessory modulators such as calmodulin, FKBP12.6 or sorcin. Our findings demonstrate that S100A1 is a regulator of diastolic RyR2 activity and beneficially modulates diastolic RyR2 dysfunction. S100A1 interaction with the RyR2 is sufficient to protect against basal and catecholamine-triggered arrhythmic SR Ca(2+) leak in HF, combining antiarrhythmic potency with chronic inotropic actions.

Heart failure gene therapy: the path to clinical practice.

Gene therapy, aimed at the correction of key pathologies being out of reach for conventional drugs, bears the potential to alter the treatment of cardiovascular diseases radically and thereby of heart failure. Heart failure gene therapy refers to a therapeutic system of targeted drug delivery to the heart that uses formulations of DNA and RNA, whose products determine the therapeutic classification through their biological actions. Among resident cardiac cells, cardiomyocytes have been the therapeutic target of numerous attempts to regenerate systolic and diastolic performance, to reverse remodeling and restore electric stability and metabolism. Although the concept to intervene directly within the genetic and molecular foundation of cardiac cells is simple and elegant, the path to clinical reality has been arduous because of the challenge on delivery technologies and vectors, expression regulation, and complex mechanisms of action of therapeutic gene products. Nonetheless, since the first demonstration of in vivo gene transfer into myocardium, there have been a series of advancements that have driven the evolution of heart failure gene therapy from an experimental tool to the threshold of becoming a viable clinical option. The objective of this review is to discuss the current state of the art in the field and point out inevitable innovations on which the future evolution of heart failure gene therapy into an effective and safe clinical treatment relies.

S100A1 deficiency impairs postischemic angiogenesis via compromised proangiogenic endothelial cell function and nitric oxide synthase regulation.

RATIONALE: Mice lacking the EF-hand Ca2+ sensor S100A1 display endothelial dysfunction because of distorted Ca2+ -activated nitric oxide (NO) generation. OBJECTIVE: To determine the pathophysiological role of S100A1 in endothelial cell (EC) function in experimental ischemic revascularization. METHODS AND RESULTS: Patients with chronic critical limb ischemia showed almost complete loss of S100A1 expression in hypoxic tissue. Ensuing studies in S100A1 knockout (SKO) mice subjected to femoral artery resection unveiled insufficient perfusion recovery and high rates of autoamputation. Defective in vivo angiogenesis prompted cellular studies in SKO ECs and human ECs, with small interfering RNA-mediated S100A1 knockdown demonstrating impaired in vitro and in vivo proangiogenic properties (proliferation, migration, tube formation) and attenuated vascular endothelial growth factor (VEGF)-stimulated and hypoxia-stimulated endothelial NO synthase (eNOS) activity. Mechanistically, S100A1 deficiency compromised eNOS activity in ECs by interrupted stimulatory S100A1/eNOS interaction and protein kinase C hyperactivation that resulted in inhibitory eNOS phosphorylation and enhanced VEGF receptor-2 degradation with attenuated VEGF signaling. Ischemic SKO tissue recapitulated the same molecular abnormalities with insufficient in vivo NO generation. Unresolved ischemia entailed excessive VEGF accumulation in SKO mice with aggravated VEGF receptor-2 degradation and blunted in vivo signaling through the proangiogenic phosphoinositide-3-kinase/Akt/eNOS cascade. The NO supplementation strategies rescued defective angiogenesis and salvaged limbs in SKO mice after femoral artery resection. CONCLUSIONS: Our study shows for the first time downregulation of S100A1 expression in patients with critical limb ischemia and identifies S100A1 as critical for EC function in postnatal ischemic angiogenesis. These findings link its pathological plasticity in critical limb ischemia to impaired neovascularization, prompting further studies to probe the microvascular therapeutic potential of S100A1.

IL-1α reversibly inhibits skeletal muscle ryanodine receptor. a novel mechanism for critical illness myopathy?

Critical illness myopathies in patients with sepsis or sustained mechanical ventilation prolong intensive care treatment and threaten both patients and health budgets; no specific therapy is available. Underlying pathophysiological mechanisms are still patchy. We characterized IL-1α action on muscle performance in "skinned" muscle fibers using force transducers and confocal Ca(2+) fluorescence microscopy for force/Ca(2+) transients and Ca(2+) sparks. Association of IL-1α with sarcoplasmic reticulum (SR) release channel, ryanodine receptor (RyR) 1, was investigated with coimmunoprecipitation and confocal immunofluorescence colocalization. Membrane integrity was studied in single, intact fibers challenged with IL-1α. IL-1α reversibly stabilized Mg(2+) inhibition of Ca(2+) release. Low Mg(2+)-induced force and Ca(2+) transients were reversibly abolished by IL-1α. At normal Mg(2+), IL-1α reversibly increased caffeine-induced force and Ca(2+) transients. IL-1α reduced SR Ca(2+) leak via RyR1, as judged by (1) increased SR Ca(2+) retention, (2) increased IL-1α force transients being reproduced by 25 µM tetracaine, and (3) reduced Ca(2+) spark frequencies by IL-1α or tetracaine. Coimmunoprecipitation confirmed RyR1/IL-1 association. RyR1/IL-1 immunofluorescence patterns perfectly colocalized. Long-term, 8-hour IL-1α challenge of intact muscle fibers compromised membrane integrity in approximately 50% of fibers, and confirmed intracellular IL-1α deposition. IL-1α exerts a novel, specific, and reversible interaction mechanism with the skeletal muscle RyR1 macromolecular release complex without the need to act via its membrane IL-1 receptor, as IL-1R membrane expression levels were not detectable in Western blots or immunostaining of single fibers. We present a potential explanation of how the inflammatory mediator, IL-1α, may contribute to muscle weakness in critical illness.

Orai1 deficiency leads to heart failure and skeletal myopathy in zebrafish.

Mutations in the store-operated Ca²âº entry pore protein ORAI1 have been reported to cause myopathies in human patients but the mechanism involved is not known. Cardiomyocytes express ORAI1 but its role in heart function is also unknown. Using reverse genetics in zebrafish, we demonstrated that inactivation of the highly conserved zebrafish orthologue of ORAI1 resulted in severe heart failure, reduced ventricular systolic function, bradycardia and skeletal muscle weakness. Electron microscopy of Orai1-deficient myocytes revealed progressive skeletal muscle instability with loss of myofiber integrity and ultrastructural abnormalities of the z-disc in both skeletal and cardiac muscle. Isolated Orai1-deficient cardiomyocytes showed loss of the calcineurin-associated protein calsarcin from the z-discs. Furthermore, we found mechanosignal transduction was affected in Orai1-depleted hearts, indicating an essential role for ORAI1 in establishing the cardiac signaling transduction machinery at the z-disc. Our findings identify ORAI1 as an important regulator of cardiac and skeletal muscle function and provide evidence linking ORAI1-mediated calcium signaling to sarcomere integrity and cardiomyocyte function.

AAV6.ßARKct cardiac gene therapy ameliorates cardiac function and normalizes the catecholaminergic axis in a clinically relevant large animal heart failure model.

AIMS: G protein-coupled receptor kinase 2 (GRK2), which is markedly upregulated in failing human myocardium, has been implicated as a contributing factor or consequence of heart failure (HF). Importantly, cardiac-specific GRK2 knockout mice have recently proved the pathological nature of GRK2 in HF. Targeted inhibition of GRK2 is possible using a peptide inhibitor known as the ßARKct, which has rescued several disparate small animal HF models. This study was designed to evaluate long-term ßARKct expression in a clinically relevant large animal HF model, using stable myocardial gene delivery with adeno-associated virus serotype 6 (AAV6). METHODS AND RESULTS: A porcine model of HF subsequent to left ventricular (LV) myocardial infarction (MI) was used to study the effects of retrograde injection into the anterior interventricular vein of either AAV6.ßARKct or AAV6.luciferase as a control 2 weeks after MI. Echocardiography and LV hemodynamics were performed before and 6 weeks after gene transfer. Robust and long-term ßARKct expression was found after AAV6-mediated delivery, leading to significant amelioration of LV haemodynamics and contractile function in HF pigs compared with AAV6.luciferase-treated control animals that showed a continued decline in cardiac function. Interestingly, the neurohormonal axis was virtually normalized in AVV6.ßARKct-treated HF animals, represented by reductions in plasma norepinephrine levels, whereas AAV6.luciferase-treated pigs showed further increases in plasma catecholamine levels. As a result, LV remodelling and foetal gene expression was reversed by AVV6.ßARKct gene therapy. CONCLUSION: These data--showing sustained amelioration of cardiac function in a post-MI pig HF model--demonstrate the therapeutic potential of ßARKct gene therapy for HF.

miR-222 inhibits pathological cardiac hypertrophy and heart failure.

AIMS: Physiological cardiac hypertrophy occurs in response to exercise and can protect against pathological stress. In contrast, pathological hypertrophy occurs in disease and often precedes heart failure. The cardiac pathways activated in physiological and pathological hypertrophy are largely distinct. Our prior work demonstrated that miR-222 increases in exercised hearts and is required for exercise-induced cardiac hypertrophy and cardiomyogenesis. Here, we sought to define the role of miR-222 in pathological hypertrophy. METHODS AND RESULTS: We found that miR-222 also increased in pathological hypertrophy induced by pressure overload. To assess its functional significance in this setting, we generated a miR-222 gain-of-function model through cardiac-specific constitutive transgenic miR-222 expression (TgC-miR-222) and used locked nucleic acid anti-miR specific for miR-222 to inhibit its effects. Both gain- and loss-of-function models manifested normal cardiac structure and function at baseline. However, after transverse aortic constriction (TAC), miR-222 inhibition accelerated the development of pathological hypertrophy, cardiac dysfunction, and heart failure. Conversely, miR-222-overexpressing mice had less pathological hypertrophy after TAC, as well as better cardiac function and survival. We identified p53-up-regulated modulator of apoptosis, a pro-apoptotic Bcl-2 family member, and the transcription factors, Hmbox1 and nuclear factor of activated T-cells 3, as direct miR-222 targets contributing to its roles in this context. CONCLUSION: While miR-222 is necessary for physiological cardiac growth, it inhibits cardiac growth in response to pressure overload and reduces adverse remodelling and cardiac dysfunction. These findings support the model that physiological and pathological hypertrophy are fundamentally different. Further, they suggest that miR-222 may hold promise as a therapeutic target in pathological cardiac hypertrophy and heart failure.

Cardiac G-protein-coupled receptor kinase 2 ablation induces a novel Ca2+ handling phenotype resistant to adverse alterations and remodeling after myocardial infarction.

BACKGROUND: G-protein-coupled receptor kinase 2 (GRK2) is a primary regulator of ß-adrenergic signaling in the heart. G-protein-coupled receptor kinase 2 ablation impedes heart failure development, but elucidation of the cellular mechanisms has not been achieved, and such elucidation is the aim of this study. METHODS AND RESULTS: Myocyte contractility, Ca(2+) handling and excitation-contraction coupling were studied in isolated cardiomyocytes from wild-type and GRK2 knockout (GRK2KO) mice without (sham) or with myocardial infarction (MI). In cardiac myocytes isolated from unstressed wild-type and GRK2KO hearts, myocyte contractions and Ca(2+) transients were similar, but GRK2KO myocytes had lower sarcoplasmic reticulum (SR) Ca(2+) content because of increased sodium-Ca(2+) exchanger activity and inhibited SR Ca(2+) ATPase by local protein kinase A-mediated activation of phosphodiesterase 4 resulting in hypophosphorylated phospholamban. This Ca(2+) handling phenotype is explained by a higher fractional SR Ca(2+) release induced by increased L-type Ca(2+) channel currents. After ß-adrenergic stimulation, GRK2KO myocytes revealed significant increases in contractility and Ca(2+) transients, which were not mediated through cardiac L-type Ca(2+) channels but through an increased SR Ca(2+). Interestingly, post-MI GRK2KO mice showed better cardiac function than post-MI control mice, which is explained by an improved Ca(2+) handling phenotype. The SR Ca(2+) content was better maintained in post-MI GRK2KO myocytes than in post-MI control myocytes because of better-maintained L-type Ca(2+) channel current density and no increase in sodium-Ca(2+) exchanger in GRK2KO myocytes. An L-type Ca(2+) channel blocker, verapamil, reversed some beneficial effects of GRK2KO. CONCLUSIONS: These data argue for novel differential regulation of L-type Ca(2+) channel currents and SR load by GRK2. G-protein-coupled receptor kinase 2 ablation represents a novel beneficial Ca(2+) handling phenotype resisting adverse remodeling after MI.

The inotropic peptide ßARKct improves ßAR responsiveness in normal and failing cardiomyocytes through G(ßγ)-mediated L-type calcium current disinhibition.

RATIONALE: The G(ßγ)-sequestering peptide ß-adrenergic receptor kinase (ßARK)ct derived from the G-protein-coupled receptor kinase (GRK)2 carboxyl terminus has emerged as a promising target for gene-based heart failure therapy. Enhanced downstream cAMP signaling has been proposed as the underlying mechanism for increased ß-adrenergic receptor (ßAR) responsiveness. However, molecular targets mediating improved cardiac contractile performance by ßARKct and its impact on G(ßγ)-mediated signaling have yet to be fully elucidated. OBJECTIVE: We sought to identify G(ßγ)-regulated targets and signaling mechanisms conveying ßARKct-mediated enhanced ßAR responsiveness in normal (NC) and failing (FC) adult rat ventricular cardiomyocytes. METHODS AND RESULTS: Assessing viral-based ßARKct gene delivery with electrophysiological techniques, analysis of contractile performance, subcellular Ca²(+) handling, and site-specific protein phosphorylation, we demonstrate that ßARKct enhances the cardiac L-type Ca²(+) channel (LCC) current (I(Ca)) both in NCs and FCs on ßAR stimulation. Mechanistically, ßARKct augments I(Ca) by preventing enhanced inhibitory interaction between the α1-LCC subunit (Cav1.2α) and liberated G(ßγ) subunits downstream of activated ßARs. Despite improved ßAR contractile responsiveness, ßARKct neither increased nor restored cAMP-dependent protein kinase (PKA) and calmodulin-dependent kinase II signaling including unchanged protein kinase (PK)Cε, extracellular signal-regulated kinase (ERK)1/2, Akt, ERK5, and p38 activation both in NCs and FCs. Accordingly, although ßARKct significantly increases I(Ca) and Ca²(+) transients, being susceptible to suppression by recombinant G(ßγ) protein and use-dependent LCC blocker, ßARKct-expressing cardiomyocytes exhibit equal basal and ßAR-stimulated sarcoplasmic reticulum Ca²(+) load, spontaneous diastolic Ca²(+) leakage, and survival rates and were less susceptible to field-stimulated Ca²(+) waves compared with controls. CONCLUSION: Our study identifies a G(ßγ)-dependent signaling pathway attenuating cardiomyocyte I(Ca) on ßAR as molecular target for the G(ßγ)-sequestering peptide ßARKct. Targeted interruption of this inhibitory signaling pathway by ßARKct confers improved ßAR contractile responsiveness through increased I(Ca) without enhancing regular or restoring abnormal cAMP-signaling. ßARKct-mediated improvement of I(Ca) rendered cardiomyocytes neither susceptible to ßAR-induced damage nor arrhythmogenic sarcoplasmic reticulum Ca²(+) leakage.

Level of G protein-coupled receptor kinase-2 determines myocardial ischemia/reperfusion injury via pro- and anti-apoptotic mechanisms.

RATIONALE: Activation of prosurvival kinases and subsequent nitric oxide (NO) production by certain G protein-coupled receptors (GPCRs) protects myocardium in ischemia/reperfusion injury (I/R) models. GPCR signaling pathways are regulated by GPCR kinases (GRKs), and GRK2 has been shown to be a critical molecule in normal and pathological cardiac function. OBJECTIVE: A loss of cardiac GRK2 activity is known to arrest progression of heart failure (HF), at least in part by normalization of cardiac ß-adrenergic receptor (ßAR) signaling. Chronic HF studies have been performed with GRK2 knockout mice, as well as expression of the ßARKct, a peptide inhibitor of GRK2 activity. This study was conducted to examine the role of GRK2 and its activity during acute myocardial ischemic injury using an I/R model. METHODS AND RESULTS: We demonstrate, using cardiac-specific GRK2 and ßARKct-expressing transgenic mice, a deleterious effect of GRK2 on in vivo myocardial I/R injury with ßARKct imparting cardioprotection. Post-I/R infarct size was greater in GRK2-overexpressing mice (45.0±2.8% versus 31.3±2.3% in controls) and significantly smaller in ßARKct mice (16.8±1.3%, P<0.05). Importantly, in vivo apoptosis was found to be consistent with these reciprocal effects on post-I/R myocardial injury when levels of GRK2 activity were altered. Moreover, these results were reflected by higher Akt activation and induction of NO production via ßARKct, and these antiapoptotic/survival effects could be recapitulated in vitro. Interestingly, selective antagonism of ß(2)ARs abolished ßARKct-mediated cardioprotection, suggesting that enhanced GRK2 activity on this GPCR is deleterious to cardiac myocyte survival. CONCLUSION: The novel effect of reducing acute ischemic myocardial injury via increased Akt activity and NO production adds significantly to the therapeutic potential of GRK2 inhibition with the ßARKct not only in chronic HF but also potentially in acute ischemic injury conditions.