Large scale, unbiased analysis of elementary calcium signaling events in cardiac myocytes.

The identification of spatiotemporally restricted Ca2+ signals, Ca2+ sparks, was instrumental for our understanding of cardiac Ca2+ homeostasis. High-speed 2D confocal imaging enables acquisition of such Ca2+ sparks with high-content information but their full appreciation is constrained by the lack of unbiased and easy-to-use analysis tools. We developed a software toolset for unbiased and automatic Ca2+ spark analysis for huge data sets of subcellular Ca2+ signals. iSpark was developed to be scanner and detector independent. In myocytes from hearts subjected to various degrees of hypertrophy we acquired >5.000.000 Ca2+ sparks from 14 mice. The iSpark-enabled analysis of this large Ca2+ spark data set showed that the highly organized distribution of Ca2+ sparks present in healthy cells disarrayed concomitant with the development of aberrant transverse tubules and disease severity. Thus, iSpark represents a versatile and universal tool for analyzing local Ca2+ signaling in healthy as well as diseased, aberrant local Ca2+ signal transduction. The results from the unbiased analysis of large data sets provide a deeper insight into possible mechanisms contributing to the onset and progression of cardiac diseases such as hypertrophy.

Hyperaldosteronism induces left atrial systolic and diastolic dysfunction.

Patients with hypertension and hyperaldosteronism show an increased risk of stroke compared with patients with essential hypertension. Aim of the study was to assess the effects of aldosterone on left atrial function in rats as a potential contributor to thromboembolism. Osmotic mini-pumps delivering 1.5 µg aldosterone/h were implanted in rats subcutaneously (Aldo, n = 39; controls, n = 38). After 8 wk, left ventricular pressure-volume analysis of isolated working hearts was performed, and left atrial systolic and diastolic function was also assessed by atrial pressure-diameter loops. Moreover, left atrial myocytes were isolated to investigate their global and local Ca2+ handling and contractility. At similar heart rates, pressure-volume analysis of isolated hearts and in vivo hemodynamic measurements revealed neither systolic nor diastolic left ventricular dysfunction in Aldo. In particular, atrial filling pressures and atrial size were not increased in Aldo. Aldo rats showed a significant reduction of atrial late diastolic A wave, atrial active work index, and increased V waves. Consistently, in Aldo rats, sarcomere shortening and the amplitude of electrically evoked global Ca2+ transients were substantially reduced. Sarcoplasmic reticulum-Ca2+ content and fractional Ca2+ release were decreased, substantiated by a reduced sarcoplasmic reticulum calcium ATPase activity, resulting from a reduced CAMKII-evoked phosphorylation of phospholamban. Hyperaldosteronism induced atrial systolic and diastolic dysfunction, while atrial size and left ventricular hemodynamics, including filling pressures, were unaffected in rats. The described model suggests a direct causal link between hyperaldosteronism and decreased atrial contractility and diastolic compliance.

Direct nkx2-5 transcriptional repression of isl1 controls cardiomyocyte subtype identity.

During cardiogenesis, most myocytes arise from cardiac progenitors expressing the transcription factors Isl1 and Nkx2-5. Here, we show that a direct repression of Isl1 by Nkx2-5 is necessary for proper development of the ventricular myocardial lineage. Overexpression of Nkx2-5 in mouse embryonic stem cells (ESCs) delayed specification of cardiac progenitors and inhibited expression of Isl1 and its downstream targets in Isl1(+) precursors. Embryos deficient for Nkx2-5 in the Isl1(+) lineage failed to downregulate Isl1 protein in cardiomyocytes of the heart tube. We demonstrated that Nkx2-5 directly binds to an Isl1 enhancer and represses Isl1 transcriptional activity. Furthermore, we showed that overexpression of Isl1 does not prevent cardiac differentiation of ESCs and in Xenopus laevis embryos. Instead, it leads to enhanced specification of cardiac progenitors, earlier cardiac differentiation, and increased cardiomyocyte number. Functional and molecular characterization of Isl1-overexpressing cardiomyocytes revealed higher beating frequencies in both ESC-derived contracting areas and Xenopus Isl1-gain-of-function hearts, which associated with upregulation of nodal-specific genes and downregulation of transcripts of working myocardium. Immunocytochemistry of cardiomyocyte lineage-specific markers demonstrated a reduction of ventricular cells and an increase of cells expressing the pacemaker channel Hcn4. Finally, optical action potential imaging of single cardiomyocytes combined with pharmacological approaches proved that Isl1 overexpression in ESCs resulted in normally electrophysiologically functional cells, highly enriched in the nodal subtype at the expense of the ventricular lineage. Our findings provide an Isl1/Nkx2-5-mediated mechanism that coordinately regulates the specification of cardiac progenitors toward the different myocardial lineages and ensures proper acquisition of myocyte subtype identity.

Genetically encoded Ca2+ indicators in cardiac myocytes.

Genetically encoded Ca(2+) indicators constitute a powerful set of tools to investigate functional aspects of Ca(2+) signaling in isolated cardiomyocytes, cardiac tissue, and whole hearts. Here, we provide an overview of the concepts, experiences, state of the art, and ongoing developments in the use of genetically encoded Ca(2+) indicators for cardiac cells and heart tissue. This review is supplemented with in vivo viral gene transfer experiments and comparisons of available genetically encoded Ca(2+) indicators with each other and with the small molecule dye Fura-2. In the context of cardiac myocytes, we provide guidelines for selecting a genetically encoded Ca(2+) indicator. For future developments, we discuss improvements of a broad range of properties, including photophysical properties such as spectral spread and biocompatibility, as well as cellular and in vivo applications.

Cardiac N-methyl D-aspartate Receptors as a Pharmacological Target.

This study focuses on characterization of the cardiac N-methyl D-aspartate receptors (NMDARs) as a target for endogenous and synthetic agonists and antagonists. Using isolated perfused rat hearts, we have shown that intracoronary administration of the NMDAR agonists and antagonists has a pronounced effect on autonomous heart function. Perfusion of rat hearts with autologous blood supplemented with NMDAR agonists was associated with induction of tachycardia, sinus arrhythmia, and ischemia occurring within physiological plasma concentration range for glutamate and glycine. Intracoronary administration of the NMDAR antagonists exerted an antiarrhythmic effect and resulted in bradycardia and improvement of capillary perfusion. Action of antagonists eliprodil, Ro25-6981, memantine, ketamine, and MK-801 on autonomous heart function diverged strikingly from that of L-type Ca channel blockers. Cardiac NMDAR subunit composition differed from that of neuronal receptors and was age specific and chamber specific. Transcripts of the GluN3A and GluN2D were found in all heart chambers, whereas expression of GluN1 and GluN2A and 2C was restricted to the atria. Expression of the GluN2B protein in ventricles increased markedly with age of the animals. The obtained data reveal that NMDARs are expressed in rat heart contributing to the autonomic heart rate regulation and the function of the cardiac conduction system.

A background Ca2+ entry pathway mediated by TRPC1/TRPC4 is critical for development of pathological cardiac remodelling.

AIMS: Pathological cardiac hypertrophy is a major predictor for the development of cardiac diseases. It is associated with chronic neurohumoral stimulation and with altered cardiac Ca(2+) signalling in cardiomyocytes. TRPC proteins form agonist-induced cation channels, but their functional role for Ca(2+) homeostasis in cardiomyocytes during fast cytosolic Ca(2+) cycling and neurohumoral stimulation leading to hypertrophy is unknown. METHODS AND RESULTS: In a systematic analysis of multiple knockout mice using fluorescence imaging of electrically paced adult ventricular cardiomyocytes and Mn(2+)-quench microfluorimetry, we identified a background Ca(2+) entry (BGCE) pathway that critically depends on TRPC1/C4 proteins but not others such as TRPC3/C6. Reduction of BGCE in TRPC1/C4-deficient cardiomyocytes lowers diastolic and systolic Ca(2+) concentrations both, under basal conditions and under neurohumoral stimulation without affecting cardiac contractility measured in isolated hearts and in vivo. Neurohumoral-induced cardiac hypertrophy as well as the expression of foetal genes (ANP, BNP) and genes regulated by Ca(2+)-dependent signalling (RCAN1-4, myomaxin) was reduced in TRPC1/C4 knockout (DKO), but not in TRPC1- or TRPC4-single knockout mice. Pressure overload-induced hypertrophy and interstitial fibrosis were both ameliorated in TRPC1/C4-DKO mice, whereas they did not show alterations in other cardiovascular parameters contributing to systemic neurohumoral-induced hypertrophy such as renin secretion and blood pressure. CONCLUSIONS: The constitutively active TRPC1/C4-dependent BGCE fine-tunes Ca(2+) cycling in beating adult cardiomyocytes. TRPC1/C4-gene inactivation protects against development of maladaptive cardiac remodelling without altering cardiac or extracardiac functions contributing to this pathogenesis.

Arrhythmia causes lipid accumulation and reduced glucose uptake.

Atrial fibrillation (AF) is characterized by irregular contractions of atrial cardiomyocytes and increased energy demand. The aim of this study was to characterize the influence of arrhythmia on glucose and fatty acid (FA) metabolism in cardiomyocytes, mice and human left atrial myocardium. Compared to regular pacing, irregular (pseudo-random variation at the same number of contractions/min) pacing of neonatal rat cardiomyocytes induced shorter action potential durations and effective refractory periods and increased diastolic [Ca(2+)]c. This was associated with the activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and AMP-activated protein kinase (AMPK). Membrane expression of fatty acid translocase (FAT/CD36) and (14)C-palmitic acid uptake were augmented while membrane expression of glucose transporter subtype 4 (GLUT-4) as well as (3)H-glucose uptake were reduced. Inhibition of AMPK and CaMKII prevented these arrhythmia-induced metabolic changes. Similar alterations of FA metabolism were observed in a transgenic mouse model (RacET) for spontaneous AF. Consistent with these findings samples of left atrial myocardium of patients with AF compared to matched samples of patients with sinus rhythm showed up-regulation of CaMKII and AMPK and increased membrane expression of FAT/CD36, resulting in lipid accumulation. These changes of FA metabolism were accompanied by decreased membrane expression of GLUT-4, increased glycogen content and increased expression of the pro-apoptotic protein bax. Irregular pacing of cardiomyocytes increases diastolic [Ca(2+)]c and activation of CaMKII and AMPK resulting in lipid accumulation, reduced glucose uptake and increased glycogen synthesis. These metabolic changes are accompanied by an activation of pro-apoptotic signalling pathways.

Genetically Encoded Voltage Indicators in Circulation Research.

Membrane potentials display the cellular status of non-excitable cells and mediate communication between excitable cells via action potentials. The use of genetically encoded biosensors employing fluorescent proteins allows a non-invasive biocompatible way to read out the membrane potential in cardiac myocytes and other cells of the circulation system. Although the approaches to design such biosensors date back to the time when the first fluorescent-protein based Förster Resonance Energy Transfer (FRET) sensors were constructed, it took 15 years before reliable sensors became readily available. Here, we review different developments of genetically encoded membrane potential sensors. Furthermore, it is shown how such sensors can be used in pharmacological screening applications as well as in circulation related basic biomedical research. Potentials and limitations will be discussed and perspectives of possible future developments will be provided.

Noise-free visualization of microscopic calcium signaling by pixel-wise fitting.

RATIONALE: Our insights into physiological and pathophysiological cardiac excitation-contraction coupling has greatly benefited from significant advancement in optical technologies such as high-speed confocal microscopy. This has pushed pixel dwell times into the time domain of nanoseconds, resulting in low signal-to-noise ratios, which have limited data analysis and interpretation. OBJECTIVE: Line scan imaging has been and still is dominant in high speed confocal recording. It allows analysis only of a small fraction of a cell's cross section (1.5%), but the appreciation of spatiotemporal fine details of excitation-contraction coupling is instrumental for the further understanding of pathological mechanisms. We aim to provide a novel analysis tool to extract otherwise hidden fine details in cardiac excitation-contraction coupling from high-speed 2-dimensional confocal image series. METHODS AND RESULTS: We demonstrate that high-speed 2-dimensional confocal data (150 frames/s) can be analyzed quantitatively by a pixel-wise fitting approach, using a mathematical formalism to phenomenologically describe local calcium transients. Such an approach produces virtually noise-free fluorescence data originating from minute volumes (0.025 femtoliter) and allows extraction of detailed and most importantly quantitative and mechanistically novel information on microscopic calcium signaling and excitation-contraction coupling in a robust manner. CONCLUSIONS: Pixel-wise fitting provides novel insights into cardiac excitation-contraction coupling. Specifically, it revealed microscopic calcium alternans on the level of individual coupling sites. Microscopic calcium alternans is an early precursor of cellular alternans and as such will shed more light onto this mechanism leading to cardiac arrhythmia.

Mutation of the calmodulin binding motif IQ of the L-type Ca(v)1.2 Ca2+ channel to EQ induces dilated cardiomyopathy and death.

Cardiac excitation-contraction coupling (EC coupling) links the electrical excitation of the cell membrane to the mechanical contractile machinery of the heart. Calcium channels are major players of EC coupling and are regulated by voltage and Ca(2+)/calmodulin (CaM). CaM binds to the IQ motif located in the C terminus of the Ca(v)1.2 channel and induces Ca(2+)-dependent inactivation (CDI) and facilitation (CDF). Mutation of Ile to Glu (Ile1624Glu) in the IQ motif abolished regulation of the channel by CDI and CDF. Here, we addressed the physiological consequences of such a mutation in the heart. Murine hearts expressing the Ca(v)1.2(I1624E) mutation were generated in adult heterozygous mice through inactivation of the floxed WT Ca(v)1.2(L2) allele by tamoxifen-induced cardiac-specific activation of the MerCreMer Cre recombinase. Within 10 days after the first tamoxifen injection these mice developed dilated cardiomyopathy (DCM) accompanied by apoptosis of cardiac myocytes (CM) and fibrosis. In Ca(v)1.2(I1624E) hearts, the activity of phospho-CaM kinase II and phospho-MAPK was increased. CMs expressed reduced levels of Ca(v)1.2(I1624E) channel protein and I(Ca). The Ca(v)1.2(I1624E) channel showed "CDI" kinetics. Despite a lower sarcoplasmic reticulum Ca(2+) content, cellular contractility and global Ca(2+) transients remained unchanged because the EC coupling gain was up-regulated by an increased neuroendocrine activity. Treatment of mice with metoprolol and captopril reduced DCM in Ca(v)1.2(I1624E) hearts at day 10. We conclude that mutation of the IQ motif to IE leads to dilated cardiomyopathy and death.

Epicardioid single-cell genomics uncovers principles of human epicardium biology in heart development and disease.

The epicardium, the mesothelial envelope of the vertebrate heart, is the source of multiple cardiac cell lineages during embryonic development and provides signals that are essential to myocardial growth and repair. Here we generate self-organizing human pluripotent stem cell-derived epicardioids that display retinoic acid-dependent morphological, molecular and functional patterning of the epicardium and myocardium typical of the left ventricular wall. By combining lineage tracing, single-cell transcriptomics and chromatin accessibility profiling, we describe the specification and differentiation process of different cell lineages in epicardioids and draw comparisons to human fetal development at the transcriptional and morphological levels. We then use epicardioids to investigate the functional cross-talk between cardiac cell types, gaining new insights into the role of IGF2/IGF1R and NRP2 signaling in human cardiogenesis. Finally, we show that epicardioids mimic the multicellular pathogenesis of congenital or stress-induced hypertrophy and fibrotic remodeling. As such, epicardioids offer a unique testing ground of epicardial activity in heart development, disease and regeneration.

Intra-pituitary follicle-stimulating hormone signaling regulates hepatic lipid metabolism in mice.

Inter-organ communication is a major hallmark of health and is often orchestrated by hormones released by the anterior pituitary gland. Pituitary gonadotropes secrete follicle-stimulating hormone (FSH) and luteinizing hormone (LH) to regulate gonadal function and control fertility. Whether FSH and LH also act on organs other than the gonads is debated. Here, we find that gonadotrope depletion in adult female mice triggers profound hypogonadism, obesity, glucose intolerance, fatty liver, and bone loss. The absence of sex steroids precipitates these phenotypes, with the notable exception of fatty liver, which results from ovary-independent actions of FSH. We uncover paracrine FSH action on pituitary corticotropes as a mechanism to restrain the production of corticosterone and prevent hepatic steatosis. Our data demonstrate that functional communication of two distinct hormone-secreting cell populations in the pituitary regulates hepatic lipid metabolism.

Functional and morphological preservation of adult ventricular myocytes in culture by sub-micromolar cytochalasin D supplement.

In cardiac myocytes, cytochalasin D (CytoD) was reported to act as an actin disruptor and mechanical uncoupler. Using confocal and super-resolution STED microscopy, we show that CytoD preserves the actin filament architecture of adult rat ventricular myocytes in culture. Five hundred nanomolar CytoD was the optimal concentration to achieve both preservation of the T-tubular structure during culture periods of 3 days and conservation of major functional characteristics such as action potentials, calcium transients and, importantly, the contractile properties of single myocytes. Therefore, we conclude that the addition of CytoD to the culture of adult cardiac myocytes can indeed be used to generate a solid single-cell model that preserves both morphology and function of freshly isolated cells. Moreover, we reveal a putative link between cytoskeletal and T-tubular remodeling. In the absence of CytoD, we observed a loss of T-tubules that led to significant dyssynchronous Ca(2+)-induced Ca(2+) release (CICR), while in the presence of 0.5 µM CytoD, T-tubules and homogeneous CICR were majorly preserved. Such data suggested a possible link between the actin cytoskeleton, T-tubules and synchronous, reliable excitation-contraction-coupling. Thus, T-tubular re-organization in cell culture sheds some additional light onto similar processes found during many cardiac diseases and might link cytoskeletal alterations to changes in subcellular Ca(2+) signaling revealed under such pathophysiological conditions.

High-throughput optical action potential recordings in hiPSC-derived cardiomyocytes with a genetically encoded voltage indicator in the AAVS1 locus.

Cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs) represent an excellent in vitro model in cardiovascular research. Changes in their action potential (AP) dynamics convey information that is essential for disease modeling, drug screening and toxicity evaluation. High-throughput optical AP recordings utilizing intramolecular Förster resonance energy transfer (FRET) of the voltage-sensitive fluorescent protein (VSFP) have emerged as a substitute or complement to the resource-intensive patch clamp technique. Here, we functionally validated our recently generated voltage indicator hiPSC lines stably expressing CAG-promoter-driven VSFP in the AAVS1 safe harbor locus. By combining subtype-specific cardiomyocyte differentiation protocols, we established optical AP recordings in ventricular, atrial, and nodal CMs in 2D monolayers using fluorescence microscopy. Moreover, we achieved high-throughput optical AP measurements in single hiPSC-derived CMs in a 3D context. Overall, this system greatly expands the spectrum of possibilities for high-throughput, non-invasive and long-term AP analyses in cardiovascular research and drug discovery.

Human BIN1 isoforms grow, maintain, and regenerate excitation-contraction couplons in adult rat and human stem cell-derived cardiomyocytes.

AIMS: In ventricular myocytes, transverse-tubules (T-tubules) are instrumental for excitation-contraction (EC)coupling and their disarray is a hallmark of cardiac diseases. BIN1 is a key contributor to their biogenesis. Our study set out to investigate the role of human BIN1 splice variants in the maintenance and regeneration of EC-coupling in rat adult ventricular myocytes and human-induced pluripotent stem cell-derived cardiac myocytes (hiPS-CMs). METHODS AND RESULTS: In heart samples from healthy human donors expression patterns of five BIN1 splice variants were identified. Following viral transduction of human BIN1 splice variants in cellular models of T-tubular disarray, we employed high-speed confocal calcium imaging and CaCLEAN analysis to identify functional EC-coupling sites (couplons) and T-tubular architecture. Adult rat ventricular myocytes were used to investigate the regeneration after loss and maintenance of EC-coupling while we studied the enhancement of EC-coupling in hiPS-CMs. All five human BIN1 splice variants induced de-novo generation of T-tubules in both cell types. Isoforms with the phosphoinositide-binding motif (PI) were most potent in maintenance and regeneration of T-tubules and functional EC-coupling in adult rat myocytes. In hiPSC-CMs, BIN1 variants with PI-motif-induced de novo generation of T-tubules, functional couplons and enhanced calcium handling. CONCLUSION: BIN1 is essential for the maintenance, regeneration, and de novo generation of functional T-tubules. Isoforms with PI-motifs appeared as particulalrly potent. These T-tubules trigger the development of functional couplons resulting in enhanced calcium handling.

Optical action potential screening on adult ventricular myocytes as an alternative QT-screen.

BACKGROUND/AIMS: QT-interval screens are increasingly important for cardiac safety on all new medications. So far, investigations rely on animal experiments or cell-based screens solely probing for conductance alterations in heterologously expressed hERG-channels in cell lines allowing for a high degree of automation. Adult cardiomyocytes can not be handled by automated patch-clamp setups. Therefore optical screening of primary isolated ventricular myocytes is regarded as an alternative. Several optical voltage sensors have been reported for ratiometric measurements, but they all influenced the naïve action potential. The aim of the present study was to explore the recording conditions and define settings that allow optical QT-interval screens. METHODS: Based on an improved optical design, individual action potentials could be recorded with an exceptional signal-to-noise-ratio. The sensors were validated using the patch-clamp technique, confocal microscopy and fluorescence lifetime imaging in combination with global unmixing procedures. RESULTS: We show that the small molecule dye di-8-ANEPPS and the novel genetically encoded sensor Mermaid provide quantitative action potential information. When applying such sensors we identified distinctly different pharmacological profiles of action potentials for adult and neonatal rat cardiomyocytes. CONCLUSION: Optical methods can be used for QT-interval investigations based on cellular action potentials using either the small molecule dye di-8-ANEPPS or the genetically encoded sensor Mermaid. Adult cardiomyocytes are superior to neonatal cardiomyocytes for such pharmacological investigations. Optical QT-screens may replace intricate animal experiments.

Apparent calcium spark properties and fast-scanning 2D confocal imaging modalities.

Ca2+ sparks are instrumental to understand physiological and pathological Ca2+ signaling in the heart. High-speed two spatially dimensional (2D) confocal imaging (>120 Hz) enables acquisition of sparks with high-content information, however, owing to a wide variety of different acquisition modalities the question arises: how much they reflect the "true" Ca2+ spark properties. To address this issue, we compared a fast point and a 2D-array scanner equipped with a range of different detectors. As a quasi-standard biological sample, we employed Ca2+ sparks in permeabilized and intact mouse ventricular myocytes and utilized an unbiased, automatic Ca2+ spark analysis tool, iSpark. Data from the point scanner suffered from low pixel photon fluxes (PPF) concomitant with high Poissonian noise. Images from the 2D-array scanner displayed substantially increased PPF, lower Poissonian noise and almost 3-fold increased sign-to-noise ratios. Noteworthy, data from the 2D scanner suffered from considerable inter-pinhole crosstalk evident for the permeabilized cells. Spark properties, such as frequency, amplitude, decay time and spatial spread were distinctly different for any scanner/detector combination. Our study reveals that the apparent Ca2+ spark properties differ dependent on the particular recording modality and set-up employed, quantitatively.

Transcriptional signatures regulated by TRPC1/C4-mediated Background Ca2+ entry after pressure-overload induced cardiac remodelling.

AIMS: After summarizing current concepts for the role of TRPC cation channels in cardiac cells and in processes triggered by mechanical stimuli arising e.g. during pressure overload, we analysed the role of TRPC1 and TRPC4 for background Ca2+ entry (BGCE) and for cardiac pressure overload induced transcriptional remodelling. METHODS AND RESULTS: Mn2+-quench analysis in cardiomyocytes from several Trpc-deficient mice revealed that both TRPC1 and TRPC4 are required for BGCE. Electrically-evoked cell shortening of cardiomyocytes from TRPC1/C4-DKO mice was reduced, whereas parameters of cardiac contractility and relaxation assessed in vivo were unaltered. As pathological cardiac remodelling in mice depends on their genetic background, and the development of cardiac remodelling was found to be reduced in TRPC1/C4-DKO mice on a mixed genetic background, we studied TRPC1/C4-DKO mice on a C57BL6/N genetic background. Cardiac hypertrophy was reduced in those mice after chronic isoproterenol infusion (-51.4%) or after one week of transverse aortic constriction (TAC; -73.0%). This last manoeuvre was preceded by changes in the pressure overload induced transcriptional program as analysed by RNA sequencing. Genes encoding specific collagens, the Mef2 target myomaxin and the gene encoding the mechanosensitive channel Piezo2 were up-regulated after TAC in wild type but not in TRPC1/C4-DKO hearts. CONCLUSIONS: Deletion of the TRPC1 and TRPC4 channel proteins protects against development of pathological cardiac hypertrophy independently of the genetic background. To determine if the TRPC1/C4-dependent changes in the pressure overload induced alterations in the transcriptional program causally contribute to cardio-protection needs to be elaborated in future studies.

DREADD technology reveals major impact of Gq signalling on cardiac electrophysiology.

AIMS: Signalling via Gq-coupled receptors is of profound importance in many cardiac diseases such as hypertrophy and arrhythmia. Nevertheless, owing to their widespread expression and the inability to selectively stimulate such receptors in vivo, their relevance for cardiac function is not well understood. We here use DREADD technology to understand the role of Gq-coupled signalling in vivo in cardiac function. METHODS AND RESULTS: We generated a novel transgenic mouse line that expresses a Gq-coupled DREADD (Dq) in striated muscle under the control of the muscle creatine kinase promotor. In vivo injection of the DREADD agonist clozapine-N-oxide (CNO) resulted in a dose-dependent, rapid mortality of the animals. In vivo electrocardiogram data revealed severe cardiac arrhythmias including lack of P waves, atrioventricular block, and ventricular tachycardia. Following Dq activation, electrophysiological malfunction of the heart could be recapitulated in the isolated heart ex vivo. Individual ventricular and atrial myocytes displayed a positive inotropic response and arrhythmogenic events in the absence of altered action potentials. Ventricular tissue sections revealed a strong co-localization of Dq with the principal cardiac connexin CX43. Western blot analysis with phosphor-specific antibodies revealed strong phosphorylation of a PKC-dependent CX43 phosphorylation site following CNO application in vivo. CONCLUSION: Activation of Gq-coupled signalling has a major impact on impulse generation, impulse propagation, and coordinated impulse delivery in the heart. Thus, Gq-coupled signalling does not only modulate the myocytes' Ca2+ handling but also directly alters the heart's electrophysiological properties such as intercellular communication. This study greatly advances our understanding of the plethora of modulatory influences of Gq signalling on the heart in vivo.