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.

Protection of the heart by remote preconditioning using IOA is as powerful as classical preconditioning. Both protection methods share protein kinase C as a common element in their signal transduction pathways. Since hexamethonium could not block the protection from IOA and a reperfusion period has to be necessarily interspaced between the IOA and the infarct inducing ischemia of the heart, a neuronal signal transmission from the remote area to the heart can be excluded with certainty. A humoral factor must be responsible for the remote protection. Interestingly the production of the protecting factor is dependent on the duration of the ischemia of the lower limb. The protecting substance, which must be upstream of protein kinase C, remains to be identified.

Objectives This study investigated the hypothesis whether S100A1 gene therapy can improve pathological key features in human failing ventricular cardiomyocytes (HFCMs). Background Depletion of the Ca2+-sensor protein S100A1 drives deterioration of cardiac performance toward heart failure (HF) in experimental animal models. Targeted repair of this molecular defect by cardiac-specific S100A1 gene therapy rescued cardiac performance, raising the immanent question of its effects in human failing myocardium. Methods Enzymatically isolated HFCMs from hearts with severe systolic HF were subjected to S100A1 and control adenoviral gene transfer and contractile performance, calcium handling, signaling, and energy homeostasis were analyzed by video-edge-detection, FURA2-based epifluorescent microscopy, phosphorylation site-specific antibodies, and mitochondrial assays, respectively. Results Genetically targeted therapy employing the human S100A1 cDNA normalized decreased S100A1 protein levels in HFCMs, reversed both contractile dysfunction and negative force-frequency relationship, and improved contractile reserve under beta-adrenergic receptor (β-AR) stimulation independent of cAMP-dependent (PKA) and calmodulin-dependent (CaMKII) kinase activity. S100A1 reversed underlying Ca2+ handling abnormalities basally and under β-AR stimulation shown by improved SR Ca2+ handling, intracellular Ca2+ transients, diastolic Ca2+ overload, and diminished susceptibility to arrhythmogenic SR Ca2+ leak, respectively. Moreover, S100A1 ameliorated compromised mitochondrial function and restored the phosphocreatine/adenosine-triphosphate ratio. Conclusions Our results demonstrate for the first time the therapeutic efficacy of genetically reconstituted S100A1 protein levels in HFCMs by reversing pathophysiological features that characterize human failing myocardium. Our findings close a gap in our understanding of S100A1’s effects in human cardiomyocytes and strengthen the rationale for future molecular-guided therapy of human HF.

Members of the S100 protein family have been reported to function as endogenous danger signals (alarmins) playing an active role in tissue inflammation and repair when released from necrotic cells. Here, we investigated the role of S100A1, the S100 isoform with highest abundance in cardiomyocytes, when released from damaged cardiomyocytes during myocardial infarction (MI). Patients with acute MI showed significantly increased S100A1 serum levels. Experimental MI in mice induced comparable S100A1 release. S100A1 internalization was observed in cardiac fibroblasts (CFs) adjacent to damaged cardiomyocytes. In vitro analyses revealed exclusive S100A1 endocytosis by CFs, followed by Toll-like receptor 4 (TLR4)-dependent activation of MAP kinases and NF-κB. CFs exposed to S100A1 assumed an immunomodulatory and anti-fibrotic phenotype characterized i.e. by enhanced intercellular adhesion molecule-1 (ICAM1) and decreased collagen levels. In mice, intracardiac S100A1 injection recapitulated these transcriptional changes. Moreover, antibody-mediated neutralization of S100A1 enlarged infarct size and worsened left ventricular functional performance post-MI. Our study demonstrates alarmin properties for S100A1 from necrotic cardiomyocytes. However, the potentially beneficial role of extracellular S100A1 in MI-related inflammation and repair warrants further investigation.

Neuron-astrocyte communication is an important regulatory mechanism in various brain functions but its complexity and role are yet to be fully understood. In particular, the temporal pattern of astrocyte response to neuronal firing has not been fully characterized. Here, we used neuron-astrocyte cultures on multi-electrode arrays coupled to Ca2+ imaging and explored the range of neuronal stimulation frequencies while keeping constant the amount of stimulation. Our results reveal that astrocytes specifically respond to the frequency of neuronal stimulation by intracellular Ca2+ transients, with a clear onset of astrocytic activation at neuron firing rates around 3-5 Hz. The cell-to-cell heterogeneity of the astrocyte Ca2+ response was however large and increasing with stimulation frequency. Astrocytic activation by neurons was abolished with antagonists of type I metabotropic glutamate receptor, validating the glutamate-dependence of this neuron-to-astrocyte pathway. Using a realistic biophysical model of glutamate-based intracellular calcium signaling in astrocytes, we suggest that the stepwise response is due to the supralinear dynamics of intracellular IP3 and that the heterogeneity of the responses may be due to the heterogeneity of the astrocyte-to-astrocyte couplings via gap junction channels. Therefore our results present astrocyte intracellular Ca2+ activity as a nonlinear integrator of glutamate-dependent neuronal activity.

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.

Rationale: Genome editing by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is evolving rapidly. Recently, second-generation CRISPR/Cas9 activation systems based on nuclease inactive dead (d)Cas9 fused to transcriptional transactivation domains were developed for directing specific guide (g)RNAs to regulatory regions of any gene of interest, to enhance transcription. The application of dCas9 to activate cardiomyocyte transcription in targeted genomic loci in vivo has not been demonstrated so far. Objective: We aimed to develop a mouse model for cardiomyocyte-specific, CRISPR-mediated transcriptional modulation, and to demonstrate its versatility by targeting Mef2d and Klf15 loci (2 well-characterized genes implicated in cardiac hypertrophy and homeostasis) for enhanced transcription. Methods and Results: A mouse model expressing dCas9 with the VPR transcriptional transactivation domains under the control of the Myh (myosin heavy chain) 6 promoter was generated. These mice innocuously expressed dCas9 exclusively in cardiomyocytes. For initial proof-of-concept, we selected Mef2d , which when overexpressed, led to hypertrophy and heart failure, and Klf15 , which is lowly expressed in the neonatal heart. The most effective gRNAs were first identified in fibroblast (C3H/10T1/2) and myoblast (C2C12) cell lines. Using an improved triple gRNA expression system (TRISPR [triple gRNA expression construct]), up to 3 different gRNAs were transduced simultaneously to identify optimal conditions for transcriptional activation. For in vivo delivery of the validated gRNA combinations, we employed systemic administration via adeno-associated virus serotype 9. On gRNA delivery targeting Mef2d expression, we recapitulated the anticipated cardiac hypertrophy phenotype. Using gRNA targeting Klf15 , we could enhance its transcription significantly, although Klf15 is physiologically silenced at that time point. We further confirmed specific and robust dCas9VPR on-target effects. Conclusions: The developed mouse model permits enhancement of gene expression by using endogenous regulatory genomic elements. Proof-of-concept in 2 independent genomic loci suggests versatile applications in controlling transcription in cardiomyocytes of the postnatal heart.

Simultaneous calcium imaging and extra-cellular recordings from cultured cortical rat neurons were performed to directly map the efficacy of extra-cellular recordings with microelectrodes. For the first time, we can associate extra-cellular recordings with neuronal activity of specific neurons in the vicinity of the electrode. We demonstrate that recorded cells can be identified by correlating the electrical signals and the calcium response. Our data demonstrate that in sparse cultures, microelectrodes record exclusively from cells which reside at very close proximity to the recording electrode. Moreover, we show that recording appears to be limited to only a partial subset of the cells residing in this range. We further show that even in cases of strong neuron-electrode coupling, extra-cellular signals recorded from single, well-identified neurons vary in shape over time rendering spike sorting and network activity rate analysis incongruous. As multi-electrode array technology is becoming increasingly widespread, the visualization technique we report here will help users better understand the limits of this versatile and useful method.