SUMMARYIn the developing chicken embryo yolk sac vasculature, the expression of arterial identity genes requires arterial hemodynamic conditions. We hypothesize that arterial flow must provide a unique signal that is relevant for supporting arterial identity gene expression and is absent in veins. We analyzed factors related to flow, pressure and oxygenation in the chicken embryo vitelline vasculature in vivo. The best discrimination between arteries and veins was obtained by calculating the maximal pulsatile increase in shear rate relative to the time-averaged shear rate in the same vessel: the relative pulse slope index (RPSI). RPSI was significantly higher in arteries than veins. Arterial endothelial cells exposed to pulsatile shear in vitro augmented arterial marker expression as compared with exposure to constant shear. The expression of Gja5 correlated with arterial flow patterns: the redistribution of arterial flow provoked by vitelline artery ligation resulted in flow-driven collateral arterial network formation and was associated with increased expression of Gja5. In situ hybridization in normal and ligation embryos confirmed that Gja5 expression is confined to arteries and regulated by flow. In mice, Gja5 (connexin 40) was also expressed in arteries. In the adult, increased flow drives arteriogenesis and the formation of collateral arterial networks in peripheral occlusive diseases. Genetic ablation of Gja5 function in mice resulted in reduced arteriogenesis in two occlusion models. We conclude that pulsatile shear patterns may be central for supporting arterial identity, and that arterial Gja5 expression plays a functional role in flow-driven arteriogenesis.
Rationale: Monitoring and controlling cardiac myocyte activity with optogenetic tools offer exciting possibilities for fundamental and translational cardiovascular research. Genetically encoded voltage indicators may be particularly attractive for minimal invasive and repeated assessments of cardiac excitation from the cellular to the whole heart level. Objective: To test the hypothesis that cardiac myocyte–targeted voltage-sensitive fluorescence protein 2.3 (VSFP2.3) can be exploited as optogenetic tool for the monitoring of electric activity in isolated cardiac myocytes and the whole heart as well as function and maturity in induced pluripotent stem cell–derived cardiac myocytes. Methods and Results: We first generated mice with cardiac myocyte–restricted expression of VSFP2.3 and demonstrated distinct localization of VSFP2.3 at the t-tubulus/junctional sarcoplasmic reticulum microdomain without any signs for associated pathologies (assessed by echocardiography, RNA-sequencing, and patch clamping). Optically recorded VSFP2.3 signals correlated well with membrane voltage measured simultaneously by patch clamping. The use of VSFP2.3 for human action potential recordings was confirmed by simulation of immature and mature action potentials in murine VSFP2.3 cardiac myocytes. Optical cardiograms could be monitored in whole hearts ex vivo and minimally invasively in vivo via fiber optics at physiological heart rate (10 Hz) and under pacing-induced arrhythmia. Finally, we reprogrammed tail-tip fibroblasts from transgenic mice and used the VSFP2.3 sensor for benchmarking functional and structural maturation in induced pluripotent stem cell–derived cardiac myocytes. Conclusions: We introduce a novel transgenic voltage-sensor model as a new method in cardiovascular research and provide proof of concept for its use in optogenetic sensing of physiological and pathological excitation in mature and immature cardiac myocytes in vitro and in vivo.
Background-The basis for the unique effectiveness of long-term amiodarone treatment on cardiac arrhythmias is incompletely understood. The present study investigated the pharmacogenomic profile of amiodarone on genes encoding ion-channel subunits. Methods and Results-Adult male mice were treated for 6 weeks with vehicle or oral amiodarone at 30, 90, or 180 mg · kg Ϫ1 · d Ϫ1. Plasma and myocardial levels of amiodarone and N-desethylamiodarone increased dose-dependently, reaching therapeutic ranges observed in human. Plasma triiodothyronine levels decreased, whereas reverse triiodothyronine levels increased in amiodarone-treated animals. In ECG recordings, amiodarone dose-dependently prolonged the RR, PR, QRS, and corrected QT intervals. Specific microarrays containing probes for the complete ion-channel repertoire (IonChips) and real-time reverse transcription-polymerase chain reaction experiments demonstrated that amiodarone induced a dose-dependent remodeling in multiple ion-channel subunits. Genes encoding Na ϩ (SCN4A, SCN5A, SCN1B), connexin (GJA1), Ca 2ϩ (CaCNA1C), and K ϩ channels (KCNA5, KCNB1, KCND2) were downregulated. In patch-clamp experiments, lower expression of K ϩ and Na ϩ channel genes was associated with decreased I to,f , I K,slow , and I Na currents. Inversely, other K ϩ channel ␣-and -subunits, such as KCNA4, KCNK1, KCNAB1, and KCNE3, were upregulated. Conclusions-Long-term amiodarone treatment induces a dose-dependent remodeling of ion-channel expression that is correlated with the cardiac electrophysiologic effects of the drug. This profile cannot be attributed solely to the amiodarone-induced cardiac hypothyroidism syndrome. Thus, in addition to the direct effect of the drug on membrane proteins, part of the therapeutic action of long-term amiodarone treatment is likely related to its effect on ion-channel transcripts. Key Words: antiarrhythmic agents Ⅲ ion channels Ⅲ molecular biology Ⅲ electrophysiology A miodarone, a widely used antiarrhythmic drug, has remarkable efficacy for the treatment of ventricular tachyarrhythmias and atrial fibrillation. However, the basis for its effectiveness is still poorly understood. The pharmacologic profile of this drug is complex, and much remains to be clarified about both short-and long-term actions. Amiodarone has been referred to as a class III antiarrhythmic agent, 1 but it also possesses electrophysiologic characteristics of class I and IV agents and minor class II effects. 2 The drug is also known to modify thyroid function extensively because of its iodinated nature. 3 The question arose as to whether the long-term effects of amiodarone might stem from its molecular interaction with thyroid hormone receptors or other mechanisms. In particular, it has been hypothesized that the effects of amiodarone could depend on modulation of transcript expression in addition to its direct effect on cell membrane channels. 4 Genomic techniques now bring gene expression studies to a genome scale, allowing investigators to examine simultaneous changes in th...
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