A Platform for Generation of Chamber-Specific Cardiac Tissues and Disease Modeling.

Tissue engineering using cardiomyocytes derived from human pluripotent stem cells holds a promise to revolutionize drug discovery, but only if limitations related to cardiac chamber specification and platform versatility can be overcome. We describe here a scalable tissue-cultivation platform that is cell source agnostic and enables drug testing under electrical pacing. The plastic platform enabled on-line noninvasive recording of passive tension, active force, contractile dynamics, and Ca2+ transients, as well as endpoint assessments of action potentials and conduction velocity. By combining directed cell differentiation with electrical field conditioning, we engineered electrophysiologically distinct atrial and ventricular tissues with chamber-specific drug responses and gene expression. We report, for the first time, engineering of heteropolar cardiac tissues containing distinct atrial and ventricular ends, and we demonstrate their spatially confined responses to serotonin and ranolazine. Uniquely, electrical conditioning for up to 8 months enabled modeling of polygenic left ventricular hypertrophy starting from patient cells.

Dexmedetomidine inhibited arrhythmia susceptibility to adrenergic stress in RyR2R2474S mice through regulating the coupling of membrane potential and intracellular calcium.

BACKGROUND: Dexmedetomidine (DEX), a highly selective α2-adrenoceptor agonist, can decrease the incidence of arrhythmias, such as catecholaminergic polymorphic ventricular tachycardia (CPVT). However, the underlying mechanisms by which DEX affects cardiac electrophysiological function remain unclear. METHODS: Ryanodine receptor (RyR2) heterozygous R2474S mice were used as a model for CPVT. WT and RyR2R2474S/+ mice were treated with isoproterenol (ISO) and DEX, and electrocardiograms were continuously monitored during both in vivo and ex vivo experiments. Dual-dye optical mapping was used to explore the anti-arrhythmic mechanism of DEX. RESULTS: DEX significantly reduced the occurrence and duration of ISO-induced of VT/VF in RyR2R2474S/+ mice in vivo and ex vivo. DEX remarkably prolonged action potential duration (APD80) and calcium transient duration (CaTD80) in both RyR2R2474S/+ and WT hearts, whereas it reduced APD heterogeneity and CaT alternans in RyR2R2474S/+ hearts. DEX inhibited ectopy and reentry formation, and stabilized voltage-calcium latency. CONCLUSION: DEX exhibited an antiarrhythmic effect through stabilizing membrane voltage and intracellular Ca2+. DEX can be used as a beneficial perioperative anesthetic for patients with CPVT or other tachy-arrhythmias.

Activation of G Protein-Coupled Estrogen Receptor (GPER) Negatively Modulates Cardiac Excitation-Contraction Coupling (ECC) through the PI3K/NOS/NO Pathway.

The G-protein-coupled estrogen receptor (GPER) has been described to exert several cardioprotective effects. However, the exact mechanism involved in cardiac protection remains unclear. The aim of this study is to investigate the role of GPER activation on excitation-contraction coupling (ECC) and the possibility that such effect participates in cardioprotection. The cardiac myocytes of male Wistar rats were isolated with a digestive buffer and loaded with Fura-2-AM for the measurement of intracellular calcium transient (CaT). Sarcomere shortening (SS) and L-type calcium current (ICaL) were also registered. The confocal technique was used to measure nitric oxide (NO) production in cells loaded with DAF-FM-diacetate. Cardiac myocytes exposed to 17-ß-estradiol (E2, 10 nM) or G-1 (1 µM) for fifteen minutes decreased CaT, SS, and ICaL. These effects were prevented using G-36 (antagonist of GPER, 1 µM), L-Name (NO synthase -NOS- inhibitor, 100 nM), or wortmannin (phosphoinositide-3-kinase -PI3K- inhibitor, 100 nM). Moreover, G1 increased NO production, and this effect was abolished in the presence of wortmannin. We concluded that the selective activation of GPER with E2 or G1 in the isolated cardiac myocytes of male rats induced a negative inotropic effect due to the reduction in ICaL and the decrease in CaT. Finally, the pathway that we proposed to be implicated in these effects is PI3K-NOS-NO.

Stretch-induced reactive oxygen species contribute to the Frank-Starling mechanism.

Myocardial stretch physiologically activates NADPH oxidase 2 (NOX2) to increase reactive oxygen species (ROS) production. Although physiological low-level ROS are known to be important as signalling molecules, the role of stretch-induced ROS in the intact myocardium remains unclear. To address this, we investigated the effects of stretch-induced ROS on myocardial cellular contractility and calcium transients in C57BL/6J and NOX2-/- mice. Axial stretch was applied to the isolated cardiomyocytes using a pair of carbon fibres attached to both cell ends to evaluate stretch-induced modulation in the time course of the contraction curve and calcium transient, as well as to evaluate maximum cellular elastance, an index of cellular contractility, which is obtained from the end-systolic force-length relationship. In NOX2-/- mice, the peak calcium transient was not altered by stretch, as that in wild-type mice, but the lack of stretch-induced ROS delayed the rise of calcium transients and reduced contractility. Our mathematical modelling studies suggest that the augmented activation of ryanodine receptors by stretch-induced ROS causes a rapid and large increase in the calcium release flux, resulting in a faster rise in the calcium transient. The slight increase in the magnitude of calcium transients is offset by a decrease in sarcoplasmic reticulum calcium content as a result of ROS-induced calcium leakage, but the faster rise in calcium transients still maintains higher contractility. In conclusion, a physiological role of stretch-induced ROS is to increase contractility to counteract a given preload, that is, it contributes to the Frank-Starling law of the heart. KEY POINTS: Myocardial stretch increases the production of reactive oxygen species by NADPH oxidase 2. We used NADPH oxidase 2 knockout mice to elucidate the physiological role of stretch-induced reactive oxygen species in the heart. We showed that stretch-induced reactive oxygen species modulate the rising phase of calcium transients and increase myocardial contractility. A mathematical model simulation study demonstrated that rapid activation of ryanodine receptors by reactive oxygen species is important for increased contractility. This response is advantageous for the myocardium, which must contract against a given preload.

Extracellular Calcium-Induced Calcium Transient Regulating the Proliferation of Osteoblasts through Glycolysis Metabolism Pathways.

During bone remodeling, high extracellular calcium levels accumulated around the resorbing bone tissue as soon as the activation of osteoclasts. However, if and how calcium is involved in the regulation of bone remodeling remains unclear. In this study, the effect of high extracellular calcium concentrations on osteoblast proliferation and differentiation, intracellular calcium ([Ca2+]i) levels, metabolomics, and the expression of proteins related to energy metabolism were investigated. Our results showed that high extracellular calcium levels initiated a [Ca2+]i transient via the calcium-sensing receptor (CaSR) and promoted the proliferation of MC3T3-E1 cells. Metabolomics analysis showed that the proliferation of MC3T3-E1 cells was dependent on aerobic glycolysis, but not the tricarboxylic acid cycle. Moreover, the proliferation and glycolysis of MC3T3-E1 cells were suppressed following the inhibition of AKT. These results indicate that calcium transient triggered by high extracellular calcium levels activated glycolysis via AKT-related signaling pathways and ultimately promoted the proliferation of osteoblasts.

Early Alterations in Structural and Functional Properties in the Neuromuscular Junctions of Mutant FUS Mice.

Amyotrophic lateral sclerosis (ALS) is manifested as skeletal muscle denervation, loss of motor neurons and finally severe respiratory failure. Mutations of RNA-binding protein FUS are one of the common genetic reasons of ALS accompanied by a 'dying back' type of degeneration. Using fluorescent approaches and microelectrode recordings, the early structural and functional alterations in diaphragm neuromuscular junctions (NMJs) were studied in mutant FUS mice at the pre-onset stage. Lipid peroxidation and decreased staining with a lipid raft marker were found in the mutant mice. Despite the preservation of the end-plate structure, immunolabeling revealed an increase in levels of presynaptic proteins, SNAP-25 and synapsin 1. The latter can restrain Ca2+-dependent synaptic vesicle mobilization. Indeed, neurotransmitter release upon intense nerve stimulation and its recovery after tetanus and compensatory synaptic vesicle endocytosis were markedly depressed in FUS mice. There was a trend to attenuation of axonal [Ca2+]in increase upon nerve stimulation at 20 Hz. However, no changes in neurotransmitter release and the intraterminal Ca2+ transient in response to low frequency stimulation or in quantal content and the synchrony of neurotransmitter release at low levels of external Ca2+ were detected. At a later stage, shrinking and fragmentation of end plates together with a decrease in presynaptic protein expression and disturbance of the neurotransmitter release timing occurred. Overall, suppression of synaptic vesicle exo-endocytosis upon intense activity probably due to alterations in membrane properties, synapsin 1 levels and Ca2+ kinetics could be an early sign of nascent NMJ pathology, which leads to neuromuscular contact disorganization.

Region-specific distribution of transversal-axial tubule system organization underlies heterogeneity of calcium dynamics in the right atrium.

The atrial myocardium demonstrates the highly heterogeneous organization of the transversal-axial tubule system (TATS), although its anatomical distribution and region-specific impact on Ca2+ dynamics remain unknown. Here, we developed a novel method for high-resolution confocal imaging of TATS in intact live mouse atrial myocardium and applied a custom-developed MATLAB-based computational algorithm for the automated analysis of TATS integrity. We observed a twofold higher (P < 0.01) TATS density in the right atrial appendage (RAA) than in the intercaval regions (ICR, the anatomical region between the superior vena cava and atrioventricular junction and between the crista terminalis and interatrial septum). Whereas RAA predominantly consisted of well-tubulated myocytes, ICR showed partially tubulated/untubulated cells. Similar TATS distribution was also observed in healthy human atrial myocardium sections. In both mouse atrial preparations and isolated mouse atrial myocytes, we observed a strong anatomical correlation between TATS distribution and Ca2+ transient synchronization and rise-up time. This region-specific difference in Ca2+ transient morphology disappeared after formamide-induced detubulation. ICR myocytes showed a prolonged action potential duration at 80% of repolarization as well as a significantly lower expression of RyR2 and Cav1.2 proteins but similar levels of NCX1 and Cav1.3 compared with RAA tissue. Our findings provide a detailed characterization of the region-specific distribution of TATS in mouse and human atrial myocardium, highlighting the structural foundation for anatomical heterogeneity of Ca2+ dynamics and contractility in the atria. These results could indicate different roles of TATS in Ca2+ signaling at distinct anatomical regions of the atria and provide mechanistic insight into pathological atrial remodeling.NEW & NOTEWORTHY Mouse and human atrial myocardium demonstrate high variability in the organization of the transversal-axial tubule system (TATS), with more organized TATS expressed in the right atrial appendage. TATS distribution governs anatomical heterogeneity of Ca2+ dynamics and thus could contribute to integral atrial contractility, mechanics, and arrhythmogenicity.

TRPV1 channels regulate the automaticity of embryonic stem cell-derived cardiomyocytes through stimulating the Na+ /Ca2+ exchanger current.

Calcium controls the excitation-contraction coupling in cardiomyocytes. Embryonic stem cell-derived cardiomyocytes (ESC-CMs) are an important cardiomyocyte source for regenerative medicine and drug screening. Transient receptor potential vanilloid 1 (TRPV1) channels are nonselective cation channels that permeate sodium and calcium. This study aimed to investigate whether TRPV1 channels regulate the electrophysiological characteristics of ESC-CMs. If yes, what is the mechanism behind? By immunostaining and subcellular fractionation, followed by western blotting, TRPV1 was found to locate intracellularly. The staining pattern of TRPV1 was found to largely overlap with that of the sarco/endoplasmic reticulum Ca2+ -ATPase, the sarcoplasmic reticulum (SR) marker. By electrophysiology and calcium imaging, pharmacological blocker of TRPV1 and the molecular tool TRPV1ß (which could functionally knockdown TRPV1) were found to decrease the rate and diastolic depolarization slope of spontaneous action potentials, and the amplitude and frequency of global calcium transients. By calcium imaging, in the absence of external calcium, TRPV1-specific opener increased intracellular calcium; this increase was abolished by preincubation with caffeine, which could deplete SR calcium store. The results suggest that TRPV1 controls calcium release from the SR. By electrophysiology, TRPV1 blockade and functional knockdown of TRPV1 decreased the Na+ /Ca2+ exchanger (NCX) currents from both the forward and reverse modes, suggesting that sodium and calcium through TRPV1 stimulate the NCX activity. Our novel findings suggest that TRPV1 activity is important for regulating the spontaneous activity of ESC-CMs and reveal a novel interplay between TRPV1 and NCX in regulating the physiological functions of ESC-CMs.

Transient receptor potential vanilloid 4 channel participates in mouse ventricular electrical activity.

The TRPV4 channel is a calcium-permeable channel (PCa/PNa ∼ 10). Its expression has been reported in ventricular myocytes, where it is involved in several cardiac pathological mechanisms. In this study, we investigated the implication of TRPV4 in ventricular electrical activity. Left ventricular myocytes were isolated from trpv4+/+ and trpv4-/- mice. TRPV4 membrane expression and its colocalization with L-type calcium channels (Cav1.2) was confirmed using Western blot biotinylation, immunoprecipitation, and immunostaining experiments. Then, electrocardiograms (ECGs) and patch-clamp recordings showed shortened QTc and action potential (AP) duration in trpv4-/- compared with trpv4+/+ mice. Thus, TRPV4 activator GSK1016790A produced a transient and dose-dependent increase in AP duration at 90% of repolarization (APD90) in trpv4+/+ but not in trpv4-/- myocytes or when combined with TRPV4 inhibitor GSK2193874 (100 nM). Hence, GSK1016790A increased calcium transient (CaT) amplitude in trpv4+/+ but not in trpv4-/- myocytes, suggesting that TRPV4 carries an inward Ca2+ current in myocytes. Conversely, TRPV4 inhibitor GSK2193874 (100 nM) alone reduced APD90 in trpv4+/+ but not in trpv4-/- myocytes, suggesting that TRPV4 prolongs AP duration in basal condition. Finally, introducing TRPV4 parameters in a mathematical model predicted the development of an inward TRPV4 current during repolarization that increases AP duration and CaT amplitude, in accord with what was found experimentally. This study shows for the first time that TRPV4 modulates AP and QTc durations. It would be interesting to evaluate whether TRPV4 could be involved in long QT-mediated ventricular arrhythmias.NEW & NOTEWORTHY Transient receptor potential vanilloid 4 (TRPV4) is expressed at the membrane of mouse ventricular myocytes and colocalizes with non-T-tubular L-type calcium channels. Deletion of trpv4 gene in mice results in shortened QT interval on electrocardiogram and reduced action potential duration of ventricular myocytes. Pharmacological activation of TRPV4 channel leads to increased action potential duration and increased calcium transient amplitude in trpv4-/- but not in trpv4-/- ventricular myocytes. To the contrary, TRPV4 channel pharmacological inhibition reduces action potential duration in trpv4+/+ but not in trpv4-/- myocytes. Integration of TRPV4 channel in a computational model of mouse action potential shows that the channel carries an inward current contributing to slowing down action potential repolarization and to increase calcium transient amplitude, similarly to what is observed experimentally. This study highlights for the first time the involvement of TRPV4 channel in ventricular electrical activity.

Cardiomyocyte depolarization triggers NOS-dependent NO transient after calcium release, reducing the subsequent calcium transient.

Cardiac excitation-contraction coupling and metabolic and signaling activities are centrally modulated by nitric oxide (NO), which is produced by one of three NO synthases (NOSs). Despite the significant role of NO in cardiac Ca2+ homeostasis regulation under different pathophysiological conditions, such as Duchenne muscular dystrophy (DMD), no precise method describes the production, source or effect of NO through two NO signaling pathways: soluble guanylate cyclase-protein kinase G (NO-sGC-PKG) and S-nitrosylation (SNO). Using a novel strategy involving isolated murine cardiomyocytes loaded with a copper-based dye highly specific for NO, we observed a single transient NO production signal after each electrical stimulation event. The NO transient signal started 67.5 ms after the beginning of Rhod-2 Ca2+ transient signal and lasted for approximately 430 ms. Specific NOS isoform blockers or NO scavengers significantly inhibited the NO transient, suggesting that wild-type (WT) cardiomyocytes produce nNOS-dependent NO transients. Conversely, NO transient in mdx cardiomyocyte, a mouse model of DMD, was dependent on inducible NOS (iNOS) and endothelial (eNOS). In a consecutive stimulation protocol, the nNOS-dependent NO transient in WT cardiomyocytes significantly reduced the next Ca2+ transient via NO-sGC-PKG. In mdx cardiomyocytes, this inhibitory effect was iNOS- and eNOS-dependent and occurred through the SNO pathway. Basal NO production was nNOS- and iNOS-dependent in WT cardiomyocytes and eNOS- and iNOS-dependent in mdx cardiomyocytes. These results showed cardiomyocyte produces NO isoform-dependent transients upon membrane depolarization at the millisecond time scale activating a specific signaling pathway to negatively modulate the subsequent Ca2+ transient.