BIN1, Myotubularin, and Dynamin-2 Coordinate T-Tubule Growth in Cardiomyocytes.

BACKGROUND: Transverse tubules (t-tubules) form gradually in the developing heart, critically enabling maturation of cardiomyocyte Ca2+ homeostasis. The membrane bending and scaffolding protein BIN1 (bridging integrator 1) has been implicated in this process. However, it is unclear which of the various reported BIN1 isoforms are involved, and whether BIN1 function is regulated by its putative binding partners MTM1 (myotubularin), a phosphoinositide 3'-phosphatase, and DNM2 (dynamin-2), a GTPase believed to mediate membrane fission. METHODS: We investigated the roles of BIN1, MTM1, and DNM2 in t-tubule formation in developing mouse cardiomyocytes, and in gene-modified HL-1 and human-induced pluripotent stem cell-derived cardiomyocytes. T-tubules and proteins of interest were imaged by confocal and Airyscan microscopy, and expression patterns were examined by RT-qPCR and Western blotting. Ca2+ release was recorded using Fluo-4. RESULTS: We observed that in the postnatal mouse heart, BIN1 localizes along Z-lines from early developmental stages, consistent with roles in initial budding and scaffolding of t-tubules. T-tubule proliferation and organization were linked to a progressive and parallel increase in 4 detected BIN1 isoforms. All isoforms were observed to induce tubulation in cardiomyocytes but produced t-tubules with differing geometries. BIN1-induced tubulations contained the L-type Ca2+ channel, were colocalized with caveolin-3 and the ryanodine receptor, and effectively triggered Ca2+ release. BIN1 upregulation during development was paralleled by increasing expression of MTM1. Despite no direct binding between MTM1 and murine cardiac BIN1 isoforms, which lack exon 11, high MTM1 levels were necessary for BIN1-induced tubulation, indicating a central role of phosphoinositide homeostasis. In contrast, the developing heart exhibited declining levels of DNM2. Indeed, we observed that high levels of DNM2 are inhibitory for t-tubule formation, although this protein colocalizes with BIN1 along Z-lines, and binds all 4 isoforms. CONCLUSIONS: These findings indicate that BIN1, MTM1, and DNM2 have balanced and collaborative roles in controlling t-tubule growth in cardiomyocytes.

Membrane remodelling triggers maturation of excitation-contraction coupling in 3D-shaped human-induced pluripotent stem cell-derived cardiomyocytes.

The prospective use of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) for cardiac regenerative medicine strongly depends on the electro-mechanical properties of these cells, especially regarding the Ca2+-dependent excitation-contraction (EC) coupling mechanism. Currently, the immature structural and functional features of hiPSC-CM limit the progression towards clinical applications. Here, we show that a specific microarchitecture is essential for functional maturation of hiPSC-CM. Structural remodelling towards a cuboid cell shape and induction of BIN1, a facilitator of membrane invaginations, lead to transverse (t)-tubule-like structures. This transformation brings two Ca2+ channels critical for EC coupling in close proximity, the L-type Ca2+ channel at the sarcolemma and the ryanodine receptor at the sarcoplasmic reticulum. Consequently, the Ca2+-dependent functional interaction of these channels becomes more efficient, leading to improved spatio-temporal synchronisation of Ca2+ transients and higher EC coupling gain. Thus, functional maturation of hiPSC-cardiomyocytes by optimised cell microarchitecture needs to be considered for future cardiac regenerative approaches.

Distress-Mediated Remodeling of Cardiac Connexin-43 in a Novel Cell Model for Arrhythmogenic Heart Diseases.

Gap junctions and their expression pattern are essential to robust function of intercellular communication and electrical propagation in cardiomyocytes. In healthy myocytes, the main cardiac gap junction protein connexin-43 (Cx43) is located at the intercalated disc providing a clear direction of signal spreading across the cardiac tissue. Dislocation of Cx43 to lateral membranes has been detected in numerous cardiac diseases leading to slowed conduction and high propensity for the development of arrhythmias. At the cellular level, arrhythmogenic diseases are associated with elevated levels of oxidative distress and gap junction remodeling affecting especially the amount and sarcolemmal distribution of Cx43 expression. So far, a mechanistic link between sustained oxidative distress and altered Cx43 expression has not yet been identified. Here, we propose a novel cell model based on murine induced-pluripotent stem cell-derived cardiomyocytes to investigate subcellular signaling pathways linking cardiomyocyte distress with gap junction remodeling. We tested the new hypothesis that chronic distress, induced by rapid pacing, leads to increased reactive oxygen species, which promotes expression of a micro-RNA, miR-1, specific for the control of Cx43. Our data demonstrate that Cx43 expression is highly sensitive to oxidative distress, leading to reduced expression. This effect can be efficiently prevented by the glutathione peroxidase mimetic ebselen. Moreover, Cx43 expression is tightly regulated by miR-1, which is activated by tachypacing-induced oxidative distress. In light of the high arrhythmogenic potential of altered Cx43 expression, we propose miR-1 as a novel target for pharmacological interventions to prevent the maladaptive remodeling processes during chronic distress in the heart.

The Structural and the Functional Aspects of Intercellular Communication in iPSC-Cardiomyocytes.

Recent advances in the technology of producing novel cardiomyocytes from induced pluripotent stem cells (iPSC-cardiomyocytes) fuel new hope for future clinical applications. The use of iPSC-cardiomyocytes is particularly promising for the therapy of cardiac diseases such as myocardial infarction, where these cells could replace scar tissue and restore the functionality of the heart. Despite successful cardiogenic differentiation, medical applications of iPSC-cardiomyocytes are currently limited by their pronounced immature structural and functional phenotype. This review focuses on gap junction function in iPSC-cardiomyocytes and portrays our current understanding around the structural and the functional limitations of intercellular coupling and viable cardiac graft formation involving these novel cardiac muscle cells. We further highlight the role of the gap junction protein connexin 43 as a potential target for improving cell-cell communication and electrical signal propagation across cardiac tissue engineered from iPSC-cardiomyocytes. Better insight into the mechanisms that promote functional intercellular coupling is the foundation that will allow the development of novel strategies to combat the immaturity of iPSC-cardiomyocytes and pave the way toward cardiac tissue regeneration.

Improved Generation of Human Induced Pluripotent Stem Cell-Derived Cardiac Pacemaker Cells Using Novel Differentiation Protocols.

Current protocols for the differentiation of human-induced pluripotent stem cells (hiPSC) into cardiomyocytes only generate a small amount of cardiac pacemaker cells. In previous work, we reported the generation of high amounts of cardiac pacemaker cells by co-culturing hiPSC with mouse visceral endoderm-like (END2) cells. However, potential medical applications of cardiac pacemaker cells generated according to this protocol, comprise an incalculable xenogeneic risk. We thus aimed to establish novel protocols maintaining the differentiation efficiency of the END2 cell-based protocol, yet eliminating the use of END2 cells. Three protocols were based on the activation and inhibition of the Wingless/Integrated (Wnt) signaling pathway, supplemented either with retinoic acid and the Wnt activator CHIR99021 (protocol B) or with the NODAL inhibitor SB431542 (protocol C) or with a combination of all three components (protocol D). An additional fourth protocol (protocol E) was used, which was originally developed by the manufacturer STEMCELL Technologies for the differentiation of hiPSC or hESC into atrial cardiomyocytes. All protocols (B, C, D, E) were compared to the END2 cell-based protocol A, serving as reference, in terms of their ability to differentiate hiPSC into cardiac pacemaker cells. Our analysis revealed that protocol E induced upregulation of 12 out of 15 cardiac pacemaker-specific genes. For comparison, reference protocol A upregulated 11, while protocols B, C and D upregulated 9, 10 and 8 cardiac pacemaker-specific genes, respectively. Cells differentiated according to protocol E displayed intense fluorescence signals of cardiac pacemaker-specific markers and showed excellent rate responsiveness to adrenergic and cholinergic stimulation. In conclusion, we characterized four novel and END2 cell-independent protocols for the differentiation of hiPSC into cardiac pacemaker cells, of which protocol E was the most efficient.

AAV-mediated expression of NFAT decoy oligonucleotides protects from cardiac hypertrophy and heart failure.

Previous studies have underlined the substantial role of nuclear factor of activated T cells (NFAT) in hypertension-induced myocardial hypertrophy ultimately leading to heart failure. Here, we aimed at neutralizing four members of the NFAT family of transcription factors as a therapeutic strategy for myocardial hypertrophy transiting to heart failure through AAV-mediated cardiac expression of a RNA-based decoy oligonucleotide (dON) targeting NFATc1-c4. AAV-mediated dON expression markedly decreased endothelin-1 induced cardiomyocyte hypertrophy in vitro and resulted in efficient expression of these dONs in the heart of adult mice as evidenced by fluorescent in situ hybridization. Cardiomyocyte-specific dON expression both before and after induction of transverse aortic constriction protected mice from development of cardiac hypertrophy, cardiac remodeling, and heart failure. Singular systemic administration of AAVs enabling a cell-specific expression of dONs for selective neutralization of a given transcription factor may thus represent a novel and powerful therapeutic approach.

Endothelial cell modulation of cardiomyocyte gene expression.

The anatomic arrangement of microvascular endothelial cells and cardiomyocytes in vivo enables close interactions among these cells. In our in vitro co-culture system, ANP and BNP expression in the mouse atrial cardiomyocyte cell line HL-1 and subsequent ANP release were significantly upregulated when co-cultured with mouse cardiac microvascular endothelial cells or exposed to endothelial cell-conditioned medium. Endothelin-1 (ET-1) activation of endothelial cells remarkably enhanced their paracrine effect on cardiomyocyte gene expression, suggesting that ET-1 stimulation of endothelial cells affects expression of fetal genes such as ANP and BNP in adult cardiomyocytes through paracrine signalling. Exposure of HL-1 cells and murine induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to authentic angiopoietin-2 (Ang2) caused a concentration-dependent decrease in ANP expression while ET-1-induced ANP expression was augmented by low but inhibited by high concentrations of Ang2. FK506-mediated inhibition of the calcineurin-NFAT pathway in the HL-1 cells selectively inhibited the stimulatory effect of the conditioned medium derived from ET-1-pre-stimulated endothelial cells on cardiomyocyte fetal gene expression. Combined with previous results indicating a crucial role for ANP and BNP in cardiac homeostasis, our findings provide further evidence that paracrine signalling by cardiac microvascular endothelial cells modulates cardiomyocyte function.

Somatic mutations and promotor methylation of the ryanodine receptor 2 is a common event in the pathogenesis of head and neck cancer.

Genomic sequencing projects unraveled the mutational landscape of head and neck squamous cell carcinoma (HNSCC) and provided a comprehensive catalog of somatic mutations. However, the limited number of significant cancer-related genes obtained so far only partially explains the biological complexity of HNSCC and hampers the development of novel diagnostic biomarkers and therapeutic targets. We pursued a multiscale omics approach based on whole-exome sequencing, global DNA methylation and gene expression profiling data derived from tumor samples of the HIPO-HNC cohort (n = 87), and confirmed new findings with datasets from The Cancer Genome Atlas (TCGA). Promoter methylation was confirmed by MassARRAY analysis and protein expression was assessed by immunohistochemistry and immunofluorescence staining. We discovered a set of cancer-related genes with frequent somatic mutations and high frequency of promoter methylation. This included the ryanodine receptor 2 (RYR2), which showed variable promoter methylation and expression in both tumor samples and cell lines. Immunohistochemical staining of tissue sections unraveled a gradual loss of RYR2 expression from normal mucosa via dysplastic lesion to invasive cancer and indicated that reduced RYR2 expression in adjacent tissue and precancerous lesions might serve as risk factor for unfavorable prognosis and upcoming malignant conversion. In summary, our data indicate that impaired RYR2 function by either somatic mutation or epigenetic silencing is a common event in HNSCC pathogenesis. Detection of RYR2 expression and/or promoter methylation might enable risk assessment for malignant conversion of dysplastic lesions.

The VAMP-associated protein VAPB is required for cardiac and neuronal pacemaker channel function.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels encode neuronal and cardiac pacemaker currents. The composition of pacemaker channel complexes in different tissues is poorly understood, and the presence of additional HCN modulating subunits was speculated. Here we show that vesicle-associated membrane protein-associated protein B (VAPB), previously associated with a familial form of amyotrophic lateral sclerosis 8, is an essential HCN1 and HCN2 modulator. VAPB significantly increases HCN2 currents and surface expression and has a major influence on the dendritic neuronal distribution of HCN2. Severe cardiac bradycardias in VAPB-deficient zebrafish and VAPB-/- mice highlight that VAPB physiologically serves to increase cardiac pacemaker currents. An altered T-wave morphology observed in the ECGs of VAPB-/- mice supports the recently proposed role of HCN channels for ventricular repolarization. The critical function of VAPB in native pacemaker channel complexes will be relevant for our understanding of cardiac arrhythmias and epilepsies, and provides an unexpected link between these diseases and amyotrophic lateral sclerosis.-Silbernagel, N., Walecki, M., Schäfer, M.-K. H., Kessler, M., Zobeiri, M., Rinné, S., Kiper, A. K., Komadowski, M. A., Vowinkel, K. S., Wemhöner, K., Fortmüller, L., Schewe, M., Dolga, A. M., Scekic-Zahirovic, J., Matschke, L. A., Culmsee, C., Baukrowitz, T., Monassier, L., Ullrich, N. D., Dupuis, L., Just, S., Budde, T., Fabritz, L., Decher, N. The VAMP-associated protein VAPB is required for cardiac and neuronal pacemaker channel function.

Improving electrical properties of iPSC-cardiomyocytes by enhancing Cx43 expression.

The therapeutic potential of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) is limited by immature functional features including low impulse propagation and reduced cell excitability. Key players regulating electrical activity are voltage-gated Na+ channels (Nav1.5) and gap junctions built from connexin-43 (Cx43). Here we tested the hypothesis that enhanced Cx43 expression increases intercellular coupling and influences excitability by modulating Nav1.5. Using transgenic approaches, Cx43 and Nav1.5 localization and cell coupling were studied by confocal imaging. Nav1.5 currents and action potentials (APs) were measured using the patch-clamp technique. Enhanced sarcolemmal Cx43 expression significantly improved intercellular coupling and accelerated dye transfer kinetics. Furthermore, Cx43 modulated Nav1.5 function leading to significantly higher current and enhanced AP upstroke velocities, thereby improving electrical activity as measured by microelectrode arrays. These findings suggest a mechanistic link between cell coupling and excitability controlled by Cx43 expression in iPSC-CMs. Therefore, we propose Cx43 as novel molecular target for improving electrical properties of iPSC-CMs to match the functional properties of native myocytes.

Bacopa monnieri extract increases rat coronary flow and protects against myocardial ischemia/reperfusion injury.

BACKGROUND: This study explored Bacopa monnieri, a medicinal Ayurvedic herb, as a cardioprotectant against ischemia/reperfusion injury using cardiac function and coronary flow as end-points. METHODS: In normal isolated rat hearts, coronary flow, left ventricular developed pressure, heart rate, and functional recovery were measured using the Langendorff preparation. Hearts were perfused with either (i) Krebs-Henseleit (normal) solution, (control), or with 30, 100 µg/ml B. monnieri ethanolic extract (30 min), or (ii) with normal solution or extract for 10 min preceding no-perfusion ischemia (30 min) followed by reperfusion (30 min) with normal solution. Infarct volumes were measured by triphenyltetrazolium staining. L-type Ca2+-currents (ICa, L) were measured by whole-cell patching in HL-1 cells, a mouse atrial cardiomyocyte cell line. Cytotoxicity of B. monnieri was assessed in rat isolated ventricular myocytes by trypan blue exclusion. RESULTS: In normally perfused hearts, B. monnieri increased coronary flow by 63 ± 13% (30 µg/ml) and 216 ± 21% (100 µg/ml), compared to control (5 ± 3%) (n = 8-10, p < 0.001). B. monnieri treatment preceding ischemia/reperfusion improved left ventricular developed pressure by 84 ± 10% (30 µg/ml), 82 ± 10% (100 µg/ml) and 52 ± 6% (control) compared to pre- ischemia/reperfusion. Similarly, functional recovery showed a sustained increase. Moreover, B. monnieri (100 µg/ml) reduced the percentage of infarct size from 51 ± 2% (control) to 25 ± 2% (n = 6-8, p < 0.0001). B. monnieri (100 µg/ml) reduced ICa, L by 63 ± 4% in HL-1 cells. Ventricular myocyte survival decreased at higher concentrations (50-1000 µg/ml) B. monnieri. CONCLUSIONS: B. monnieri improves myocardial function following ischemia/reperfusion injury through recovery of coronary blood flow, contractile force and decrease in infarct size. Thus this may lead to a novel cardioprotectant strategy.

Functional Characterization and Comparison of Intercellular Communication in Stem Cell-Derived Cardiomyocytes.

One novel treatment strategy for the diseased heart focuses on the use of pluripotent stem cell-derived cardiomyocytes (SC-CMs) to overcome the heart's innate deficiency for self-repair. However, targeted application of SC-CMs requires in-depth characterization of their true cardiogenic potential in terms of excitability and intercellular coupling at cellular level and in multicellular preparations. In this study, we elucidated the electrical characteristics of single SC-CMs and intercellular coupling quality of cell pairs, and concomitantly compared them with well-characterized murine native neonatal and immortalized HL-1 cardiomyocytes. Firstly, we investigated the electrical properties and Ca(2+) signaling mechanisms specific to cardiac contraction in single SC-CMs. Despite heterogeneity of the new cardiac cell population, their electrophysiological activity and Ca(2+) handling were similar to native cells. Secondly, we investigated the capability of paired SC-CMs to form an adequate subunit of a functional syncytium and analyzed gap junctions and signal transmission by dye transfer in cell pairs. We discovered significantly diminished coupling in SC-CMs compared with native cells, which could not be enhanced by a coculture approach combining SC-CMs and primary CMs. Moreover, quantitative and structural analysis of gap junctions presented significantly reduced connexin expression levels compared with native CMs. Strong dependence of intercellular coupling on gap junction density was further confirmed by computational simulations. These novel findings demonstrate that despite the cardiogenic electrophysiological profile, SC-CMs present significant limitations in intercellular communication. Inadequate coupling may severely impair functional integration and signal transmission, which needs to be carefully considered for the prospective use of SC-CMs in cardiac repair. Stem Cells 2015;33:2208-2218.

Posttranslational modifications of cardiac ryanodine receptors: Ca(2+) signaling and EC-coupling.

In cardiac muscle, a number of posttranslational protein modifications can alter the function of the Ca(2+) release channel of the sarcoplasmic reticulum (SR), also known as the ryanodine receptor (RyR). During every heartbeat RyRs are activated by the Ca(2+)-induced Ca(2+) release mechanism and contribute a large fraction of the Ca(2+) required for contraction. Some of the posttranslational modifications of the RyR are known to affect its gating and Ca(2+) sensitivity. Presently, research in a number of laboratories is focused on RyR phosphorylation, both by PKA and CaMKII, or on RyR modifications caused by reactive oxygen and nitrogen species (ROS/RNS). Both classes of posttranslational modifications are thought to play important roles in the physiological regulation of channel activity, but are also known to provoke abnormal alterations during various diseases. Only recently it was realized that several types of posttranslational modifications are tightly connected and form synergistic (or antagonistic) feed-back loops resulting in additive and potentially detrimental downstream effects. This review summarizes recent findings on such posttranslational modifications, attempts to bridge molecular with cellular findings, and opens a perspective for future work trying to understand the ramifications of crosstalk in these multiple signaling pathways. Clarifying these complex interactions will be important in the development of novel therapeutic approaches, since this may form the foundation for the implementation of multi-pronged treatment regimes in the future. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.

Establishment of a human skeletal muscle-derived cell line: biochemical, cellular and electrophysiological characterization.

Excitation-contraction coupling is the physiological mechanism occurring in muscle cells whereby an electrical signal sensed by the dihydropyridine receptor located on the transverse tubules is transformed into a chemical gradient (Ca2+ increase) by activation of the ryanodine receptor located on the sarcoplasmic reticulum membrane. In the present study, we characterized for the first time the excitation-contraction coupling machinery of an immortalized human skeletal muscle cell line. Intracellular Ca2+ measurements showed a normal response to pharmacological activation of the ryanodine receptor, whereas 3D-SIM (super-resolution structured illumination microscopy) revealed a low level of structural organization of ryanodine receptors and dihydropyridine receptors. Interestingly, the expression levels of several transcripts of proteins involved in Ca2+ homoeostasis and differentiation indicate that the cell line has a phenotype closer to that of slow-twitch than fast-twitch muscles. These results point to the potential application of such human muscle-derived cell lines to the study of neuromuscular disorders; in addition, they may serve as a platform for the development of therapeutic strategies aimed at correcting defects in Ca2+ homoeostasis due to mutations in genes involved in Ca2+ regulation.

Cardiac stereotactic body radiotherapy to treat malignant ventricular arrhythmias directly affects the cardiomyocyte electrophysiology.

BACKGROUND: Promising as a treatment option for life-threatening ventricular arrhythmias, cardiac stereotactic body radiotherapy (cSBRT) has demonstrated early antiarrhythmic effects within days of treatment. The mechanisms underlying the immediate and short-term antiarrhythmic effects are poorly understood. OBJECTIVE: We hypothesize that cSBRT has a direct antiarrhythmic effect on cellular electrophysiology through reprogramming of ion channel and gap junction protein expression. METHODS: After exposure to 20 Gy of x-rays in a single fraction, neonatal rat ventricular cardiomyocytes were analyzed 24 and 96 hours postradiation to determine changes in conduction velocity, beating frequency, calcium transients, and action potential duration in both monolayers and single cells. In addition, the expression of gap junction proteins, ion channels, and calcium handling proteins was evaluated at protein and messenger RNA levels. RESULTS: After irradiation with 20 Gy, neonatal rat ventricular cardiomyocytes exhibited increased beat rate and conduction velocity 24 and 96 hours after treatment. Messenger RNA and protein levels of ion channels were altered, with the most significant changes observed at the 96-hour mark. Upregulation of Cacna1c (Cav1.2), Kcnd3 (Kv4.3), Kcnh2 (Kv11.1), Kcnq1 (Kv7.1), Kcnk2 (K2P2.1), Kcnj2 (Kir2.1), and Gja1 (Cx43) was noted, along with improved gap junctional coupling. Calcium handling was affected, with increased Ryr2 ryanodin-rezeptor 2 and Slc8a1 Na+/Ca2+ exchanger expression and altered properties 96 hours posttreatment. Fibroblast and myofibroblast levels remained unchanged. CONCLUSION: cSBRT modulates the expression of various ion channels, calcium handling proteins, and gap junction proteins. The described alterations in cellular electrophysiology may be the underlying cause of the immediate antiarrhythmic effects observed after cSBRT.

'Eventless' InsP3-dependent SR-Ca2+ release affecting atrial Ca2+ sparks.

Augmented inositol 1,4,5-trisphosphate receptor (InsP3R) function has been linked to a variety of cardiac pathologies, including cardiac arrhythmia. The contribution of inositol 1,4,5-trisphosphate-induced Ca(2+) release (IP3ICR) in excitation-contraction coupling (ECC) under physiological conditions, as well as under cellular remodelling, remains controversial. Here we test the hypothesis that local IP3ICR directly affects ryanodine receptor (RyR) function and subsequent Ca(2+)-induced Ca(2+) release in atrial myocytes. IP3ICR was evoked by UV-flash photolysis of caged InsP3 under whole-cell configuration of the voltage-clamp technique in atrial myocytes isolated from C57/BL6 mice. Photolytic release of InsP3 was accompanied by a significant increase in the Ca(2+) release event frequency (4.14 ± 0.72 vs. 6.20 ± 0.76 events (100 µm)(-1) s(-1)). These individual photolytically triggered Ca(2+) release events were identified as Ca(2+) sparks, which originated from RyR openings. This was verified by Ca(2+) spark analysis and pharmacological separation between RyR and InsP3R-dependent sarcoplasmic reticulum (SR)-Ca(2+) release (2-aminoethoxydiphenyl borate, xestospongin C, tetracaine). Significant SR-Ca(2+) flux but eventless SR-Ca(2+) release through InsP3R were characterized using SR-Ca(2+) leak/SR-Ca(2+) load measurements. These results strongly support the idea that IP3ICR can effectively modulate RyR openings and Ca(2+) spark probability. We conclude that eventless and highly efficient InsP3-dependent SR-Ca(2+) flux is the main mechanism of functional cross-talk between InsP3Rs and RyRs, which may be an important factor in the modulation of ECC sensitivity.

PKA phosphorylation of cardiac ryanodine receptor modulates SR luminal Ca2+ sensitivity.

During physical exercise and stress, the sympathetic system stimulates cardiac contractility via ß-adrenergic receptor activation, resulting in protein kinase A (PKA)-mediated phosphorylation of the cardiac ryanodine receptor, RyR2, at Ser2808. Hyperphosphorylation of RyR2-S2808 has been proposed as a mechanism contributing to arrhythmogenesis and heart failure. However, the role of RyR2 phosphorylation during ß-adrenergic stimulation remains controversial. We examined the contribution of RyR2-S2808 phosphorylation to altered excitation-contraction coupling and Ca(2+) signaling using an experimental approach at the interface of molecular and cellular levels and a transgenic mouse with ablation of the RyR2-S2808 phosphorylation site (RyR2-S2808A). Experimentally challenging the communication between L-type Ca(2+) channels and RyR2 led to a spatiotemporal de-synchronization of RyR2 openings, as visualized using confocal Ca(2+) imaging. ß-Adrenergic stimulation re-synchronized RyR2s, but less efficiently in RyR2-S2808A than in control cardiomyocytes, as indicated by comprehensive analysis of RyR2 activation. In addition, spontaneous Ca(2+) waves in RyR2-S2808A myocytes showed significantly slowed propagation and complete absence of acceleration during ß-adrenergic stress, unlike wild type cells. Single channel recordings revealed an attenuation of luminal Ca(2+) sensitivity in RyR2-S2808A channels upon addition of PKA. This suggests that phosphorylation of RyR2-S2808 may be involved in RyR2 modulation by luminal (intra-SR) Ca(2+) ([Ca(2+)](SR)). We show here by three independent experimental approaches that PKA-dependent RyR2-S2808 phosphorylation plays significant functional roles at the subcellular level, namely, Ca(2+) release synchronization, Ca(2+) wave propagation and functional adaptation of RyR2 to variable [Ca(2+)](SR). These results indicate a direct mechanistic link between RyR2 phosphorylation and SR luminal Ca(2+) sensing.

Alterations of excitation-contraction coupling and excitation coupled Ca(2+) entry in human myotubes carrying CAV3 mutations linked to rippling muscle.

Rippling muscle disease is caused by mutations in the gene encoding caveolin-3 (CAV3), the muscle-specific isoform of the scaffolding protein caveolin, a protein involved in the formation of caveolae. In healthy muscle, caveolin-3 is responsible for the formation of caveolae, which are highly organized sarcolemmal clusters influencing early muscle differentiation, signalling and Ca(2+) homeostasis. In the present study we examined Ca(2+) homeostasis and excitation-contraction (E-C) coupling in cultured myotubes derived from two patients with Rippling muscle disease with severe reduction in caveolin-3 expression; one patient harboured the heterozygous c.84C>A mutation while the other patient harbored a homozygous splice-site mutation (c.102+ 2T>C) affecting the splice donor site of intron 1 of the CAV3 gene. Our results show that cells from control and rippling muscle disease patients had similar resting [Ca(2+) ](i) and 4-chloro-m-cresol-induced Ca(2+) release but reduced KCl-induced Ca(2+) influx. Detailed analysis of the voltage-dependence of Ca(2+) transients revealed a significant shift of Ca(2+) release activation to higher depolarization levels in CAV3 mutated cells. High resolution immunofluorescence analysis by Total Internal Fluorescence microscopy supports the hypothesis that loss of caveolin-3 leads to microscopic disarrays in the colocalization of the voltage-sensing dihydropyridine receptor and the ryanodine receptor, thereby reducing the efficiency of excitation-contraction coupling.

Inhibition of cAMP degradation improves regulatory T cell-mediated suppression.

Naturally occurring regulatory T cells (nTreg cells) are crucial for the maintenance of peripheral tolerance. We have previously shown that a key mechanism of their suppressive action is based on a contact-dependent transfer of cAMP from nTreg cells to responder T cells. Herein, we further elucidate the important role of cAMP for the suppressive properties of nTreg cells. Prevention of cAMP degradation by application of the phosphodiesterase 4 inhibitor rolipram led to strongly increased suppressive potency of nTreg cells for Th2 cells in vitro and in vivo. Detailed analyses revealed that rolipram caused, in the presence of nTreg cells, a synergistic increase of cAMP in responder Th2 cells. In vivo, the application of nTreg cells in a strictly Th2-dependent preclinical model of asthma had only a marginal effect. However, the additional treatment with rolipram led to a considerable reduction of airway hyperresponsiveness and inflammation in a prophylactic as well as in a therapeutic model. This amelioration was correlated with enhanced cAMP-levels in lung Th2 cells in vivo. Collectively, these data support our observation that cAMP has a key function for nTreg cell-based suppression and they clearly demonstrate that the effect of cAMP on T responder cells can be greatly enhanced upon application of phosphodiesterase 4 inhibitors.

Substrate Stiffness Influences Structural and Functional Remodeling in Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Novel treatment strategies for cardiac tissue regeneration are heading for the use of engineered cardiac tissue made from induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Despite the proven cardiogenic phenotype of these cells, a significant lack of structural and functional properties of mature myocytes prevents safe integration into the diseased heart. To date, maturation processes of cardiomyocytes remain largely unknown but may comprise biophysical cues from the immediate cell environment. Mechanosensing is one critical ability of cells to react to environmental changes. Accordingly, the surrounding substrate stiffness, comprised of extracellular matrix (ECM), cells, and growth surface, critically influences the myocyte's physiology, as known from deleterious remodeling processes in fibrotic hearts. Conversely, the mechanical properties during culture of iPSC-CMs may impact on their structural and functional maturation. Here, we tested the hypothesis that the environmental stiffness influences structural and functional properties of iPSC-CMs and investigated the effect of different substrate stiffnesses on cell contractility, excitation-contraction (EC) coupling, and intercellular coupling. Culture surfaces with defined stiffnesses ranging from rigid glass with 25GPa to PDMS of physiological softness were coated with ECM proteins and seeded with murine iPSC-CMs. Using confocal imaging, cardiac protein expression was assessed. Ca2+ handling and contractile properties were analyzed on different substrate stiffnesses. Intercellular coupling via gap junctions was investigated by fluorescence recovery after photobleaching (FRAP). Our data revealed greater organization of L-type Ca2+ channels and ryanodine receptors and increased EC-coupling gain, demonstrating structural and functional maturation in cells grown on soft surfaces. In addition, increased shortening and altered contraction dynamics revealed increased myofilament Ca2+ sensitivity in phase-plane loops. Moreover, connexin 43 expression was significantly increased in iPSC-CMs grown on soft surfaces leading to improved intercellular coupling. Taken together, our results demonstrate that soft surfaces with stiffnesses in the physiological range improve the expression pattern and interaction of cardiac proteins relevant for EC-coupling. In parallel, soft substrates influence contractile properties and improve intercellular coupling in iPSC-CMs. We conclude that the mechanical stiffness of the cell environment plays an important role in driving iPSC-CMs toward further maturation by inducing adaptive responses.