Role In Cardiac Physiology Research Articles

Cardiac diffusion-weighted and tensor imaging: a Society for Cardiovascular Magnetic Resonance (SCMR) special interest group consensus statement

Thanks to recent developments in Cardiovascular magnetic resonance (CMR), cardiac diffusion-weighted magnetic resonance is fast emerging in a range of clinical applications. Cardiac diffusion-weighted imaging (cDWI) and diffusion tensor imaging (cDTI) now enable investigators and clinicians to assess and quantify the 3D microstructure of the heart. Free-contrast DWI is uniquely sensitized to the presence and displacement of water molecules within the myocardial tissue, including the intra-cellular, extra-cellular and intra-vascular spaces. CMR can determine changes in microstructure by quantifying: a) mean diffusivity (MD) –measuring the magnitude of diffusion; b) fractional anisotropy (FA) – specifying the directionality of diffusion; c) helix angle (HA) and transverse angle (TA) –indicating the orientation of the cardiomyocytes; d) E2A and E2A mobility – measuring the alignment and systolic-diastolic mobility of the sheetlets, respectively.This document provides recommendations for both clinical and research cDWI and cDTI, based on published evidence when available and expert consensus when not. It introduces the cardiac microstructure focusing on the cardiomyocytes and their role in cardiac physiology and pathophysiology. It highlights methods, observations and recommendations in terminology, acquisition schemes, post-processing pipelines, data analysis and interpretation of the different biomarkers. Despite the ongoing challenges discussed in the document and the need for ongoing technical improvements, it is clear that cDTI is indeed feasible, can be accurately and reproducibly performed and, most importantly, can provide unique insights into myocardial pathophysiology.

MiR-6721-5p as a natural regulator of Meta-VCL is upregulated in the serum of patients with coronary artery disease

BackgroundCoronary artery disease (CAD), the leading cause of mortality globally, arises from atherosclerotic blockage of the coronary arteries. Meta-vinculin (meta-VCL), a large spliced isoform of VCL, co-localizes in muscular adhesive structures and plays significant roles in cardiac physiology and pathophysiology. This study aimed to identify microRNAs (miRNAs) regulating meta-VCL expression and investigate the expression alterations of the miRNAs of interest and meta-VCL as potential biomarkers in the serum of CAD patients. MethodsBioinformatics tools were employed to select miRNAs targeting meta-VCL. Cell-based ectopic expression analysis and a dual-luciferase assay were used to examine the interactions between miRNAs and meta-VCL. An ELISA assessed the concentrations of interleukin-6 (IL-6), IL-10, and tumor necrosis factor-α (TNF-α). MiRNA and meta-VCL expression patterns and biomarker suitability were evaluated in serum samples from CAD and non-CAD individuals using real-time PCR. A cardiac cell-line data set and CAD blood exosome samples were analyzed using bioinformatics and ROC curve analyses, respectively. ResultsmiR-6721-5p directly interacted with the putative target sites at the 3′-UTR of meta-VCL and regulated its expression. IL-10 and TNF-α concentrations, which may act as anti-inflammatory factors, decreased following miR-6721-5p upregulation and meta-VCL downregulation. Bioinformatics and experimental expression analyses confirmed downregulated meta-VCL expression and upregulated miR-6721-5p expression in CAD samples. ROC curve analysis yielded an AUC score of 0.705 (P = 0.018), indicating the potential suitability of miR-6721-5p as a biomarker for CAD. ConclusionsmiR-6721-5p plays a regulatory role in meta-VCL expression and may contribute to CAD development by reducing anti-inflammatory factors. These findings suggest that miR-6721-5p could serve as a novel biomarker in the pathogenesis of CAD.

Neural control of ventricular electrophysiology: the role of the posterior descending ganglionated plexus

Abstract Background The autonomic nervous system plays an integral role in cardiac physiology. While the atrially located ganglionated plexus have been studied in several species, there is limited knowledge regarding the functional characterization of ventricular ganglionated plexus. Purpose The aim of the present study was to assess the role of the posterior descending ganglionated plexus (PD-GP) for neural control of ventricular electrophysiology. Methods First, to investigate whether the mouse may be useful as an animal model, a review of whole-mount immunohistological stained murine hearts (n=43) was conducted. Second, functional studies were performed in an ex-vivo retrograde-perfused porcine model (n=3) at baseline (pacing with a cycle length of 600 ms), during PD-GP high-frequency and local nicotine stimulation (Figure 1A). Wave propagation characteristics were determined by epicardial activation mapping. Activation recovery intervals (ARIs) were analyzed with a multi-electrode sock placed around the epicardium (Figure 1B). Finally, morphological analysis of explanted human hearts was conducted to determine the potential of translation into clinical practice. Results In murine hearts, ventricular ganglionated plexus were present in only 10% of hearts. In porcine hearts, dispersion of conduction velocity was increased during PD-GP high-frequency (8.52±2.24 radian vs. 2.79±0.89 radian; p=0.018) and nicotine stimulation (19.79±6.49 radian vs. 2.79±0.89 radian; p=0.044) compared to paced rhythm (Figure 1C). High-frequency stimulation prolonged ARIs in the posterior (257.8±6.7 ms vs. 244.8±1.9 ms; p=0.044) and basal (258.1±4.2 ms vs. 244.8±1.9 ms; p=0.039) right ventricle compared to the posterior left ventricle, while nicotine did not affect ARIs (right ventricle: 255.7±29.0 ms vs. left ventricle: 245.0±29.3 ms; p=0.677) (Figure 1D). Analysis of explanted human hearts confirmed the presence of the PD-GP in close relationship to the posterior descending coronary artery within epicardial adipose tissue (Figure 2). Conclusions While we demonstrate functional relevance of the PD-GP in explanted porcine hearts, important species-related differences might exist.Ex-vivo retrograde-perfused swine modelAnalysis of explanted human hearts

Mitochondrial calcium uniporter channel gatekeeping in cardiovascular disease.

The mitochondrial calcium (mCa2+) uniporter channel (mtCU) resides at the inner mitochondrial membrane and is required for Ca2+ to enter the mitochondrial matrix. The mtCU is essential for cellular function, as mCa2+ regulates metabolism, bioenergetics, signaling pathways and cell death. mCa2+ uptake is primarily regulated by the MICU family (MICU1, MICU2, MICU3), EF-hand-containing Ca2+-sensing proteins, which respond to cytosolic Ca2+ concentrations to modulate mtCU activity. Considering that mitochondrial function and Ca2+ signaling are ubiquitously disrupted in cardiovascular disease, mtCU function has been a hot area of investigation for the last decade. Here we provide an in-depth review of MICU-mediated regulation of mtCU structure and function, as well as potential mtCU-independent functions of these proteins. We detail their role in cardiac physiology and cardiovascular disease by highlighting the phenotypes of different mutant animal models, with an emphasis on therapeutic potential and targets of interest in this pathway.

Stress-Induced Proteasome Sub-Cellular Translocation in Cardiomyocytes Causes Altered Intracellular Calcium Handling and Arrhythmias.

The ubiquitin-proteasome system (UPS) is an essential mechanism responsible for the selective degradation of substrate proteins via their conjugation with ubiquitin. Since cardiomyocytes have very limited self-renewal capacity, as they are prone to protein damage due to constant mechanical and metabolic stress, the UPS has a key role in cardiac physiology and pathophysiology. While altered proteasomal activity contributes to a variety of cardiac pathologies, such as heart failure and ischemia/reperfusion injury (IRI), the environmental cues affecting its activity are still unknown, and they are the focus of this work. Following a recent study by Ciechanover's group showing that amino acid (AA) starvation in cultured cancer cell lines modulates proteasome intracellular localization and activity, we tested two hypotheses in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs, CMs): (i) AA starvation causes proteasome translocation in CMs, similarly to the observation in cultured cancer cell lines; (ii) manipulation of subcellular proteasomal compartmentalization is associated with electrophysiological abnormalities in the form of arrhythmias, mediated via altered intracellular Ca2+ handling. The major findings are: (i) starving CMs to AAs results in proteasome translocation from the nucleus to the cytoplasm, while supplementation with the aromatic amino acids tyrosine (Y), tryptophan (W) and phenylalanine (F) (YWF) inhibits the proteasome recruitment; (ii) AA-deficient treatments cause arrhythmias; (iii) the arrhythmias observed upon nuclear proteasome sequestration(-AA+YWF) are blocked by KB-R7943, an inhibitor of the reverse mode of the sodium-calcium exchanger NCX; (iv) the retrograde perfusion of isolated rat hearts with AA starvation media is associated with arrhythmias. Collectively, our novel findings describe a newly identified mechanism linking the UPS to arrhythmia generation in CMs and whole hearts.

Effects of Gap 26, a Connexin 43 Inhibitor, on Cirrhotic Cardiomyopathy in Rats.

Introduction Cirrhotic cardiomyopathy (CCM) is recognized by impaired cardiac responsiveness to stress, prolonged QT interval, and systolic and diastolic dysfunctions. Connexins are a family of transmembrane proteins that play a key role in cardiac physiology. Connexin 43 (Cx43) inhibition showed cardio-protective effects. Peptide drug Cx43 inhibitor, Gap 26, could inhibit gap junction 43. This study was designed to evaluate the effects of a connexin mimetic peptide, Gap 26, in the CCM model in rats. Methods The cirrhosis was induced through carbon tetrachloride (CCl4). On day 56, electrocardiography (ECG) was recorded, spleen weight was measured, and tissue and serum samples were collected. Further, Cx43 mRNA expression in heart tissue was checked. Results The chronotropic responses decreased in the CCl4/saline and increased in the CCl4/Gap. The spleen weight, QTc interval, and brain natriuretic peptide (BNP), tumor necrosis factor-alpha (TNF-α), aspartate aminotransferase (AST), alanine transaminase (ALT), and malondialdehyde (MDA) levels elevated in the CCl4/saline, and the spleen weight, QTc interval, and MDA and ALT levels were reduced by Gap 26 treatment. The level of nuclear factor (erythroid-derived 2) factor 2 (Nrf2) decreased in the CCl4/saline. The Cx43 expression was downregulated in the CCl4/saline and upregulated with the Gap 26 treatment. Conclusion Gap 26 not only alleviated the chronotropic hyporesponsiveness and the severity of liver damage and upregulated the atrial Cx43 expression, but it also had an antioxidant effect on the heart.

The dispensability of 14-3-3 proteins for the regulation of human cardiac sodium channel Nav1.5.

14-3-3 proteins are ubiquitous proteins that play a role in cardiac physiology (e.g., metabolism, development, and cell cycle). Furthermore, 14-3-3 proteins were proposed to regulate the electrical function of the heart by interacting with several cardiac ion channels, including the voltage-gated sodium channel Nav1.5. Given the many cardiac arrhythmias associated with Nav1.5 dysfunction, understanding its regulation by the protein partners is crucial. In this study, we aimed to investigate the role of 14-3-3 proteins in the regulation of the human cardiac sodium channel Nav1.5. Amongst the seven 14-3-3 isoforms, only 14-3-3η (encoded by YWHAH gene) weakly co-immunoprecipitated with Nav1.5 when heterologously co-expressed in tsA201 cells. Total and cell surface expression of Nav1.5 was however not modified by 14-3-3η overexpression or inhibition with difopein, and 14-3-3η did not affect physical interaction between Nav1.5 α-α subunits. The current-voltage relationship and the amplitude of Nav1.5-mediated sodium peak current density were also not changed. Our findings illustrate that the direct implication of 14-3-3 proteins in regulating Nav1.5 is not evident in a transformed human kidney cell line tsA201.

The Role of Nucleoporins in Cardiac Tissue Development and Disease.

Nuclear pore complexes (NPCs) are intricate intracellular structures composed of approximately 30 nuclear pore proteins (NUPs) that regulate the transport of materials between the nucleus and cytoplasm in eukaryotic cells. The heart is a crucial organ for sustaining the vital functions of the body, pumping blood rich in nutrients and energy to all organs and tissues. Recent studies have shown that NPCs play pivotal roles not only in normal cardiac physiological processes such as myocardial cell proliferation and differentiation but also in various pathological processes such as ischemic and hypoxic myocardial injury. Due to their mass and complicated nature, the structures of NPCs have been challenging to identify by the scientific community. With the development of cryo-electron microscopy and advanced sampling techniques, researchers have made significant progress in understanding the structures of NPCs. This review aims to summarize the latest research on the structural aspects of NPCs and their roles in cardiac physiology and pathology, increase the understanding of the intricate mechanisms of NPC actions, provide valuable insights into the pathogenesis of heart diseases and describe the development of potential novel therapeutic strategies.

Fluorofenidone enhances cardiac contractility by stimulating CICR and CaV1.2

Fluorofenidone (AKF-PD) is a novel pyridone derivative that inhibits fibrosis and inflammation in many tissues. Accordingly, it has been effective in disease models, such as liver failure, nephropathy, and pulmonary fibrosis. However, its potential role in cardiac physiology and pathology has yet to be elucidated. Thus, this paper investigated a possible functional impact of AKF-PD on adult rat cardiac myocytes. Cells were kept in culture for 1–2 days under either control conditions or the presence of AKF-PD (500 μM). They were next examined concerning cell contractility, intracellular Ca2+ homeostasis, and activity of voltage-gated Ca2+ channels. Remarkably, AKF-PD enhanced the percentage of cell shortening and rates of both contraction and relaxation by nearly 100%. A stimulus in Ca2+-induced Ca2+ release (CICR) most likely accounts for these effects because AKF-PD also increased the magnitude of electrically evoked Ca2+ transients. Of note, the compound did not alter the peak value of caffeine-elicited Ca2+ transients, indicating stimulation of CICR at constant sarcoplasmic reticulum Ca2+ load. Since CICR is triggered by the entry of Ca2+ through CaV1.2 (ICa), a possible effect on these Ca2+ channels was also investigated. AKF-PD increased the magnitude of both ICa and maximal macroscopic Ca2+ conductance (Gmax) by about 50%. However, no differences were found in either voltage dependence of inactivation or the amount of maximal immobilization-resistant charge movement (Qmax). Thus, the effect on ICa could be explained by a higher channel's open probability (Po) rather than a greater abundance of channel proteins. Additional data indicate that AKF-PD reduces the rate of Ca2+ extrusion in the presence of caffeine, suggesting inhibition of the Na/Ca exchanger. Overall, these results indicate that AKF-PD upregulates the Po of CaV1.2 and then sequentially enhances ICa, CICR, and contractility. Therefore, the novel compound is also a candidate to be tested in cardiac disease models.

Acute Modulation of Left Ventricular Control by Selective Intracardiac Sympathetic Denervation.

The sympathetic nervous system plays an integral role in cardiac physiology. Nerve fibers innervating the left ventricle are amenable to transvenous catheter stimulation along the coronary sinus (CS). The aim of the present study was to modulate left ventricular control by selective intracardiac sympathetic denervation. First, the impact of epicardial CS ablation on cardiac electrophysiology was studied in a Langendorff model of decentralized murine hearts (n=10 each, ablation and control groups). Second, the impact of transvenous, anatomically driven axotomy by catheter-based radiofrequency ablation via the CS was evaluated in healthy sheep (n=8) before and during stellate ganglion stimulation. CS ablation prolonged epicardial ventricular refractory period without (41.8 ± 8.4ms vs 53.0 ± 13.5ms; P=0.049) and with β1-2-adrenergic receptor blockade (47.8 ± 7.8ms vs 73.1 ± 13.2ms; P<0.001) in mice. Supported by neuromorphological studies illustrating a circumferential CS neural network, intracardiac axotomy by catheter ablation via the CS in healthy sheep diminished the blood pressure increase during stellate ganglion stimulation (Δ systolic bloodpressure 21.9 ± 10.9mmHg vs 10.5 ± 12.0mmHg; P=0.023; Δ diastolic blood pressure 9.0 ± 5.5mmHg vs 3.0± 3.5mmHg; P=0.039). Transvenous, anatomically driven axotomy targeting nerve fibers along the CS enables acute modulation of left ventricular control by selective intracardiac sympathetic denervation.

The dual role of the hexosamine biosynthetic pathway in cardiac physiology and pathophysiology

The heart is a highly metabolic organ with extensive energy demands and hence relies on numerous fuel substrates including fatty acids and glucose. However, oxidative stress is a natural by-product of metabolism that, in excess, can contribute towards DNA damage and poly-ADP-ribose polymerase activation. This activation inhibits key glycolytic enzymes, subsequently shunting glycolytic intermediates into non-oxidative glucose pathways such as the hexosamine biosynthetic pathway (HBP). In this review we provide evidence supporting the dual role of the HBP, i.e. playing a unique role in cardiac physiology and pathophysiology where acute upregulation confers cardioprotection while chronic activation contributes to the onset and progression of cardio-metabolic diseases such as diabetes, hypertrophy, ischemic heart disease, and heart failure. Thus although the HBP has emerged as a novel therapeutic target for such conditions, proposed interventions need to be applied in a context- and pathology-specific manner to avoid any potential drawbacks of relatively low cardiac HBP activity.

Molecular and functional characterization of the mouse intrinsic cardiac nervous system.

The intrinsic cardiac nervous system (ICNS) refers to clusters of neurons, located within the heart, that participate in the neuronal regulation of cardiac functions and that are involved in the initiation of cardiac arrhythmias. Therefore, deciphering its role in cardiac physiology and physiopathology is mandatory. The aim of this study was to provide a phenotypic, electrophysiological, and pharmacological characterization of the mouse ICNS, which is still poorly characterized. Global cardiac innervation and phenotypic diversity were investigated using immunohistochemistry on cleared murine hearts and on tissue sections. The patch clamp technique was used for the electrophysiological and pharmacological characterization of isolated mouse intracardiac neurons. We have identified the expression of 7 distinct neuronal markers within the mouse ICNS, thus proving the neurochemical diversity of this network. Of note, it was the first time that the existenceof neurons expressing the calcium-binding protein calbindin, neuropeptide Y, and cocaine and amphetamine regulated transcript peptide was described in the mouse. Electrophysiology studies also revealed the existence of 4 different neuronal populations on the basis of their electrical behavior. Finally, we showed that these neurons can be modulated by several neuromodulators. This study showed that the mouse ICNS presents a molecular and functional complexity similar to other species and is therefore a suitable model to decipher the role of individual neuronal subtypes regarding the modulation of cardiac function and the initiation of cardiac arrhythmias.

Selective intracardiac sympathetic denervation acutely modulates left ventricular control

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): German Centre for Cardiovascular Research (DZHK) Background The sympathetic nervous system plays an integral role in cardiac physiology. Neuromodulation is emerging as a treatment option for ventricular arrhythmias, but selective intracardiac approaches are rare. Sympathetic nerve fibers innervating the left ventricle have been demonstrated to be amenable to transvenous catheter stimulation along the coronary sinus (CS). Purpose The aim of the present study was to modulate left ventricular control by selective sympathetic denervation using epicardial or standard catheter ablation for intracardiac axotomy at the level of the CS. Methods First, the impact of epicardial CS ablation on cardiac electrophysiology was studied in a Langendorff model of murine hearts (n=10 each, ablation and control). Second, the impact of transvenous, anatomically-driven axotomy by catheter-based radiofrequency ablation along the CS was evaluated in a healthy ovine in vivo model (n=8) before and during left stellate ganglion stimulation (LSGS). Results CS ablation for intracardiac sympathetic axotomy prolonged epicardial ventricular refractory period (VRP) without (41.8±8.4 ms vs. 53.0±13.5 ms; P=0.0487) and with beta1-2-adrenergic receptor blockade (47.8±2.8 ms vs. 73.1±5.0 ms; P=0.0009) and enhanced the increasing effect of beta1-2-adrenergic receptor blockade on epicardial VRP (∆VRP 6.3±7.0 ms vs. 20.0±7.5 ms; P=0.0045) in mice (Figure, A). Mean epicardial wave propagation velocity in the left ventricle was faster in ablated hearts than in controls (1.13±0.05 m/s vs. 1.00±0.02 m/s; P=0.0463), but did not differ in the right ventricle (1.15±0.05 m/s vs. 1.20±0.08 m/s; P=0.7938). Transvenous catheter ablation of the CS reduced systolic (SBP, 57.7±5.0 mmHg vs. 46.9±3.6 mmHg; P=0.0428) and diastolic blood pressure (DBP, 35.5±3.0 mmHg vs. 26.7±1.8 mmHg; P=0.0106) and diminished the blood pressure increase during LSGS in sheep (∆SBP 21.9±3.8 mmHg vs. 10.5±4.2 mmHg; P=0.0234; ∆DBP 9.0±1.9 mmHg vs. 3.0±1.2 mmHg; P=0.0391) (Figure, B, C). Cycle length remained unchanged by LSGS, both before (baseline 653.2±20.6 ms vs. LSGS 627.8±27.5 ms; P=0.2309) and after CS ablation (baseline 734.8±24.2 ms vs. LSGS 746.2±37.3 ms; P=0.7145). Conclusions Anatomically-driven axotomy targeting nerve fibers along the CS enables selective intracardiac sympathetic denervation resulting in acute modulation of left ventricular control.

Cardiac expression of microRNA-7 is associated with adverse cardiac remodeling

Although microRNA-7 (miRNA-7) is known to regulate proliferation of cancer cells by targeting Epidermal growth factor receptor (EGFR/ERBB) family, less is known about its role in cardiac physiology. Transgenic (Tg) mouse with cardiomyocyte-specific overexpression of miRNA-7 was generated to determine its role in cardiac physiology and pathology. Echocardiography on the miRNA-7 Tg mice showed cardiac dilation instead of age-associated physiological cardiac hypertrophy observed in non-Tg control mice. Subjecting miRNA-7 Tg mice to transverse aortic constriction (TAC) resulted in cardiac dilation associated with increased fibrosis bypassing the adaptive cardiac hypertrophic response to TAC. miRNA-7 expression in cardiomyocytes resulted in significant loss of ERBB2 expression with no changes in ERBB1 (EGFR). Cardiac proteomics in the miRNA-7 Tg mice showed significant reduction in mitochondrial membrane structural proteins compared to NTg reflecting role of miRNA-7 beyond the regulation of EGFR/ERRB in mediating cardiac dilation. Consistently, electron microscopy showed that miRNA-7 Tg hearts had disorganized rounded mitochondria that was associated with mitochondrial dysfunction. These findings show that expression of miRNA-7 in the cardiomyocytes results in cardiac dilation instead of adaptive hypertrophic response during aging or to TAC providing insights on yet to be understood role of miRNA-7 in cardiac function.

Contribution of K2P Potassium Channels to Cardiac Physiology and Pathophysiology.

Years before the first two-pore domain potassium channel (K2P) was cloned, certain ion channels had already been demonstrated to be present in the heart with characteristics and properties usually attributed to the TREK channels (a subfamily of K2P channels). K2P channels were later detected in cardiac tissue by RT-PCR, although the distribution of the different K2P subfamilies in the heart seems to depend on the species analyzed. In order to collect relevant information in this regard, we focus here on the TWIK, TASK and TREK cardiac channels, their putative roles in cardiac physiology and their implication in coronary pathologies. Most of the RNA expression data and electrophysiological recordings available to date support the presence of these different K2P subfamilies in distinct cardiac cells. Likewise, we show how these channels may be involved in certain pathologies, such as atrial fibrillation, long QT syndrome and Brugada syndrome.

OPN5 a new optogenetic receptor to study Gq signalling in the heart with light

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft (DFG) Deutsche Zentrum für Herz-Kreislauf-Forschung (DZHK) Background Gq signalling plays an imperative role in cardiac physiology as well as pathophysiologies such as arrhythmias, hypertrophy, and heart failure. However, the underlying kinetics and the transition from physiological to pathological signalling is yet to be investigated. Optogenetics provides an unprecedented spatial and temporal precision as well as cell specificity, but up to now, selective control of Gq signalling in the intact heart with light has not been achieved. Aim To prove the potential of human Neuropsin (OPN5) to selectively control Gq signalling in the adult heart. Methods The biophysical behaviour of OPN5 in HEK293 cells was characterized by measuring IP1 accumulation, Ca2+ imaging, and patch-clamp analysis of GIRK current activity. We generated a new transgenic mouse model expressing OPN5 in fusion with eYFP under the control of the chicken-β-actin promotor and analysed the expression pattern in the whole heart after tissue clearing with light sheet microscopy and the expression rate after single cell dissociation. Adult isolated cardiomyocytes were examined to measure the effect of OPN5 activation on contractility with a novel custom-made software for on-line contraction analysis by pixel tracking. In Langendorff-perfused hearts, illumination of the sinus node region with UV light (385 nm, 10 s, 1 mW/mm2) was used to measure the influence on the heart rate in electrogram recordings. The light-induced effects and basal ECG parameters as well as the heart-weight-to-femur-length ratio of OPN5 hearts were compared to hearts from non-expressing siblings as well as from CD1 wild-type. Results We prove the selective activation of the Gq proteins by UV light-induced increase in IP3 production and induction of Ca2+ transients in HEK293 cells because both effects were abolished after applying the Gq protein specific blocker FR900359. Promiscuous activation of Gi proteins was excluded by light-induced inhibition of GIRK channels instead of activation. Within two different mouse lines, we found an OPN5/eYFP expression rate of ∼70% within the cardiomyocytes targeted to the plasmamembrane. UV light induced a significant increase in contraction velocity in ventricular cardiomyocytes, and this effect could be abolished by blocking Gq proteins. In the right atrium HCN4 positive sinus nodal cells expressed OPN5/ eYFP and illumination of this region resulted in OPN5 expressing hearts in an average increase of 5.9 ± 0.97% (n= 20, in founder line 1) in the heart rate, which was significant lager than control hearts (all groups &amp;lt;2.4% increase, n≥17). Importantly we could not find any differences in basal ECG parameters or the heart weight-to-femur-length between OPN5 expressing and control. Conclusion We herein prove the specificity of OPN5 for Gq protein activation and demonstrate its use in cardiomyocytes as well as the whole heart. Thus OPN5 is the first optogenetic tool allowing to control Gq signalling in the whole heart precisely.

Proteome characterisation of extracellular vesicles isolated from heart.

Cardiac intercellular communication is critical for heart function and often dysregulated in cardiovascular diseases. While cardiac extracellular vesicles (cEVs) are emerging mediators of signalling, their isolation remains a technical challenge hindering our understanding of cEV protein composition. Here, we utilised Langendorff-collagenase-based enzymatic perfusion and differential centrifugation to isolate cEVs from mouse heart (yield 3-6 μg/heart). cEVs are ∼200 nm, express classical EV markers (Cd63/81/9+ , Tsg101+ , Pdcd6ip/Alix+ ), and are depleted of blood (Alb/Fga/Hba) and cardiac damage markers (Mb, Tnnt2, Ldhb). Comparison with mechanically-derived EVs revealed greater detection of EV markers and decreased cardiac damage contaminants. Mass spectrometry-based proteomic profiling revealed 1721 proteins in cEVs, implicated in proteasomal and autophagic proteostasis, glycolysis, and fatty acid metabolism; essential functions often disrupted in cardiac pathologies. There was striking enrichment of 942 proteins in cEVs compared to mouse heart tissue - implicated in EV biogenesis, antioxidant activity, and lipid transport, suggesting active cargo selection and specialised function. Interestingly, cEVs contain marker proteins for cardiomyocytes, cardiac progenitors, B-cells, T-cells, macrophages, smooth muscle cells, endothelial cells, and cardiac fibroblasts, suggesting diverse cellular origin. We present a method of cEV isolation and provide insight into potential functions, enabling future studies into EV roles in cardiac physiology and disease.

No functional TRPA1 in cardiomyocytes.

There is mounting evidence that TRPA1 has a role in cardiac physiology and pathophysiology. We aim to clarify the site of TRPA1 expression in the heart and in particular whether the channel is expressed in cardiomyocytes. Due to the high calcium conductance of TRPA1, and marginal calcium changes being detectable, microfluorimetry in primary mouse cardiomyocytes, and in the cardiomyocyte cell lines H9c2 and HL-1, was applied. TRPA1 mRNA in mouse and human hearts, primary cardiomyocytes, and the cardiac cell lines were quantified. Dorsal root ganglia served as control for both methods. In addition to AITC, the more potent and specific TRPA1 agonists JT010 and PF-4840154 failed to elicit a TRPA1-mediated response in native and electrically paced primary cardiomyocytes, and the cardiomyocyte cell lines H9c2 and HL-1. There were only marginal levels of TRPA1 mRNA in cardiomyocytes and cardiac cell lines, also in conditions of cell differentiation or inflammation, which might occur in pathophysiological conditions. Similarly, TRPV1 agonist capsaicin did not activate primary mouse cardiomyocytes, did not alter electrically paced activity in these, and did not activate H9c2 cells or alter spontaneous activity of HL-1 cells. Human pluripotent stem cells differentiated to cardiomyocytes had no relevant TRPA1 mRNA levels. Also in human post-mortem heart samples, TRPA1 mRNA levels were substantially lower compared with the respective dorsal root ganglion. The results do not question a role of TRPA1 in the heart but exclude a direct effect in cardiomyocytes.

Connexins in the Heart: Regulation, Function and Involvement in Cardiac Disease.

Connexins are a family of transmembrane proteins that play a key role in cardiac physiology. Gap junctional channels put into contact the cytoplasms of connected cardiomyocytes, allowing the existence of electrical coupling. However, in addition to this fundamental role, connexins are also involved in cardiomyocyte death and survival. Thus, chemical coupling through gap junctions plays a key role in the spreading of injury between connected cells. Moreover, in addition to their involvement in cell-to-cell communication, mounting evidence indicates that connexins have additional gap junction-independent functions. Opening of unopposed hemichannels, located at the lateral surface of cardiomyocytes, may compromise cell homeostasis and may be involved in ischemia/reperfusion injury. In addition, connexins located at non-canonical cell structures, including mitochondria and the nucleus, have been demonstrated to be involved in cardioprotection and in regulation of cell growth and differentiation. In this review, we will provide, first, an overview on connexin biology, including their synthesis and degradation, their regulation and their interactions. Then, we will conduct an in-depth examination of the role of connexins in cardiac pathophysiology, including new findings regarding their involvement in myocardial ischemia/reperfusion injury, cardiac fibrosis, gene transcription or signaling regulation.

Cardiac L-Type Channel Modulation by LRRC10 Proteins

L-type calcium channels (LTCC) play a central role in cardiac physiology by conveying Ca2+ influx that initiates cardiac muscle contraction following membrane depolarization. LTCC activity is fine-tuned by diverse regulatory subunits, and alterations in channel regulation cause debilitating cardiac diseases. Importantly, leucine-rich repeat containing protein 10 (LRRC10) was recently identified as a novel regulatory subunit of cardiac LTCC (Woon et al (2018) JAHA 7:e006528). Mutations in LRRC10 have been identified in patients with dilated cardiomyopathy (DCM) and sudden unexplained nocturnal death syndrome (SUNDS).