Calcium (Ca 2+ ) signaling plays a major role in a wide range of physiological functions including control and regulation of cardiac and skeletal muscle performance and vascular tone [1,2]. As all Ca 2+ signals require proteins to relay intracellular Ca 2+ oscillations downstream to different signaling networks, a specific toolkit of Ca 2+ -sensor proteins involving members of the EF-hand S100 Ca 2+ binding protein superfamily maintains the integrity of the Ca 2+ signaling in a variety of cardiac and vascular cells, transmitting the message with great precision and in a temporally and spatially coordinated manner [3][4][5][6]. Indeed, the possibility that S100 proteins might contribute to heart and vascular diseases was first suggested by the discovery of distinctive patterns of S100 expression in healthy and diseased hearts and vasculature from humans and animal heart failure (HF) models [7][8][9][10][11][12][13][14][15][16][17][18]. Based on more elaborate genetic studies in mice and strategies to manipulate S100 protein expression in human cardiac, skeletal muscle and vascular cells, it is now apparent that the integrity of distinct S100 protein isoforms in striated muscle and vascular cells such as S100A1, S100A4, S100A6, S100A8/A9 or S100B is a basic requirement for normal cardiovascular and muscular development and function; loss of integrity would naturally lead to profound deregulation of the implicated Ca 2+ signaling systems with detrimental consequences to cardiac, skeletal muscle, and vascular function [7][8][9][10][11][12][13][14][15][16][17][18][19][20]. The brief debate and discussion here are confined by design to the biological actions and pathophysiological relevance of the EF-hand Ca 2+ -sensor protein S100A1 in the heart, vasculature and skeletal muscle with a particular focus on current translational therapeutic strategies [4,21,22]. By virtue of its ability to modulate the activity of numerous key effector proteins that are essentially involved in the control of Ca 2+ -and NO-homeostasis in cardiac, sketelal muscle and vascular cells, S100A1 has been proven to play a critical role both in cardiac performance, blood pressure regulation and skeletal muscle function [4,21,23]. Given that deregulated S100A1 expression in cardiomyocytes and endothelial cells has recently been linked to heart failure and Corresponding author: Patrick Most,

Rationale The Gβγ-sequestering peptide βARKct derived from the G-protein coupled receptor kinase 2 (GRK2) carboxy-terminus has emerged as a promising target for gene-based heart failure (HF) therapy. Enhanced downstream cAMP signaling has been proposed as the underlying mechanism for increased β-adrenergic receptor (βAR) responsiveness. However, molecular targets mediating improved cardiac contractile performance by βARKct and its impact on Gβγ-mediated signaling have yet to be fully elucidated. Objective We sought to identify Gβγ-regulated targets and signaling mechanisms conveying βARKct-mediated enhanced βAR responsiveness in normal (NC) and failing (FC) adult rat ventricular cardiomyocytes. Methods and Results Assessing viral-based βARKct gene delivery with electrophysiological techniques, analysis of contractile performance, subcellular Ca2+ handling and site-specific protein phosphorylation, we demonstrate that βARKct enhances the cardiac L-type Ca2+ channel (LCC) current (Ica) both in NCs and FCs upon βAR stimulation. Mechanistically, βARKct augments Ica by preventing enhanced inhibitory interaction between the α1-LCC subunit (Cav1.2α) and liberated Gβγ subunits downstream of activated βARs. Despite improved βAR contractile responsiveness, βARKct neither increased nor restored cAMP-dependent protein kinase A (PKA) and calmodulin-dependent kinase II (CaMKII) signaling including unchanged protein kinase Cε (PKCε), ERK1/2, Akt, ERK5 and p38 activation both in NCs and FCs. Accordingly, though βARKct significantly increases Ica and Ca2+ transients being susceptible to suppression by recombinant Gβγ protein and use-dependent LCC blocker, βARKct-expressing cardiomyocytes exhibit equal basal and βAR-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 Gβγ-dependent signaling pathway attenuating cardiomyocyte Ica upon βAR as molecular target for the Gβγ-sequestering peptide βARKct. Targeted interruption of this inhibitory signaling pathway by βARKct confers improved βAR contractile responsiveness through increased Ica without enhancing regular or restoring abnormal cAMP-signaling. βARKct-mediated improvement of Ica rendered cardiomyocytes neither susceptible to βAR-induced damage nor arrhythmogenic SR Ca2+ leakage.

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Background: S100A1 is an inotropic calcium (Ca) sensor protein in cardiomyocytes interacting with the cardiac ryanodine receptor (RyR2). Previous studies of our group have shown that S100A1 protein can decrease Ca spark frequency in permeabilized ventricular cardiomyocytes (CMs). We therefore hypothesized that S100A1 might prevent arrhythmogenic sarcoplasmic reticulum (SR) Ca leak in CMs. Methods and Results: Ventricular rat cardiomyocytes were enzymatically isolated and transfected either with 10 MOI of a CMV-GFP control or CMV-S100A1/CMV-GFP adenovirus resulting in GFP fluorescence in >99% of cultured GFP-CMs and S100A1-CMs as assessed by epifluorescent microscopy after 24-hours. Expression analysis revealed a 3-fold increase in S100A1 protein in S100A1-CM levels compared to GFP-CM. Confocal line-scan microscopy yielded a 50% reduction in Ca spark frequency in rhodamin2-AM loaded quiescent S100A1-CMs and epifluorescent imaging showed a 20% and 50% increase in the SR Ca content and systolic Ca transient amplitude, respectively, in FURA2-AM loaded 2Hz-stimulated S100A1-CM (37°C, 1.8 mM Ca) compared with GFP-CMs. Pathophysiological diastolic SR Ca leak was induced subjecting 2Hz-stimulated FURA2-AM loaded CMs to 0.5 mM caffeine and 10–7 M isoproterenol (caff/iso) as previously published by Isner and co-workers. As expected, this protocol resulted in permanent diastolic Ca waves in 100% (n=60/60) of 2Hz-stimulated iso/caff-treated GFP-CMs and was accompanied by significantly increased diastolic Ca levels and reduced systolic Ca transient amplitudes compared with iso-treated GFP-CMs. However, S100A1 efficiently protected more than 80% of iso/caff treated S100A1-CMs (n=49/60 cells, P<0.05 vs. iso/caff GFP-CMs) from diastolic Ca waves and further alterations of CM Ca handling. Importantly, similar results were obtained in caff/iso-treated failing rat GFP- and S100A1-CMs (data not shown). Conclusions: Here we show for the first time that S100A1 can both diminish the physiological SR Ca leak and protect from arrhythmogenic Ca waves in CMs. Given its beneficial effects in the context of experimental HF animal models, S100A1 therapeutic inotropic actions might be complemented by an antiarrhythmic effect targeting dysfunctional RyR2.