Objective: The aim of this study was to determine whether uncoupling of gap junctions (GJ) prior to ischaemia would modify the antiarrhythmic effect of ischaemic preconditioning (PC) in a canine model of ischaemia/reperfusion.
A cannula was aseptically implanted in the lateral, third or fourth ventricle of dogs and, after recovery, Hydergine or mechlorethamine was injected into these cerebral ventricles to induce emesis. With Hydergine, the ED5O was 0.55 µg for the lateral, 0.15 µg for the third and 0.02 µg for the fourth ventricle. The corresponding geometric mean latencies were 19.0, 12.0 and 7.7 min respectively. Statistical analyses of these values showed that both the dosage and latency for one ventricular route were significantly different from those for another. Furthermore, in four dogs with chemoceptive trigger-zone ablation, emesis was not obtained with as much as 20 to 100 times the ED50 for the fourth ventricle route. With mechlorethamine, the ED509s could not be accurately determined. They were approximately 750, 320 and 113 µg for the lateral, third and fourth ventricles respectively. In three dogs with chemoceptive trigger-zone ablation, emesis was not observed at doses 9 or more times the estimated ED50 for the fourth ventricle route. Perphenazine, 0.1 mg/kg i.v., was found to be effective, while chlorpromazine, 2.0 mg/kg i.v., was not effective in preventing emesis induced by moderately large doses of mechlorethamine injected into the fourth ventricle.
Nitric oxide (NO) donors provide a preconditioning-like anti-arrhythmic protection in the anaesthetized dog. As NO may modulate gap junction (GJ) function, the present study investigated whether this anti-arrhythmic effect is due to a modification of GJs by NO, derived from the NO donor sodium nitroprusside (SNP).In chloralose-urethane-anaesthetized, open-chest dogs, either saline (controls; n= 11) or SNP (0.2 microg x kg(-1) x min(-1); n= 10) was infused at a rate of 0.5 mL x min(-1) by the intracoronary route. The infusions were started 20 min prior to and maintained throughout the entire 60 min occlusion period of the left anterior descending coronary artery. The severity of ischaemia and of arrhythmias, tissue electrical impedance and permeability, as well as the phosphorylation of connexin43, were assessed.Compared with the controls, SNP infusion markedly suppressed the total number of ventricular premature beats (666 +/- 202 vs. 49 +/- 18; P < 0.05), and the number of ventricular tachycardiac episodes (8.1 +/- 2.3 vs. 0.2 +/- 0.1; P < 0.05) without significantly modifying the incidence of ventricular tachycardia or ventricular fibrillation. The severity of ischaemia (epicardial ST-segment changes, inhomogeneity of electrical activation) and tissue electrical impedance changes were significantly less in the SNP-treated dogs. SNP improved GJ permeability and preserved the phosphorylated form of connexin43.The anti-arrhythmic protection resulting from SNP infusion in the anaesthethized dog may, in part, be associated with the modulation of gap junctional function by NO.
Introduction: Bradycardia is known to prolong APD and DOR and is a factor that promotes arrhythmia in long QT type 2. In numerous conditions, spontaneous Ca 2+ release from sarcoplasmic reticulum has been shown to trigger early afterdepolarizations, ectopic activity that initiate arrhythmias, but the interplay between secondary Ca 2+ elevation (SCE) and membrane excitability has not been demonstrated in bradycardia. Objectives: To correlate voltage-depolarization during the AP plateau to SCE during bradycardia. Methods: Dual optical mapping of intracellular calcium transient (CaT) and AP was performed in Langendorff perfused rabbit hearts. After AV node ablation, CaT and AP dynamics were investigated at physiological (120 beats per minute (bpm)) and slow heart rate (50 bpm). Results: Upon changing HR from 120 to 50 bpm, APD gradually increased with a time-constant of 53.8±8.9 s, consistent with clinical QT measurements. The shift from 120 to 50 bpm elicited SCE during the AP plateau that was a) regionally heterogeneous, b) associated with enhanced depolarization of the AP plateau and was reversed by pacing at 120 bpm. Regional differences of SCE at 50 bpm were significantly increased (P<0.01, n=7) and were correlated with dispersion of APD (r=0.9277±0.03, n=7). SCE and APD prolongation were more pronounced at the base of right ventricles than the apex of left ventricles (P<0.01, n=7) (Figure). Suppression of SCE with K201 (1 µM/L) (to stabilize RyR2) reduced APD (P<0.01, n=5) and DOR (P<0.02, n=5). The molecular basis of the spatial distribution of SCE is currently being correlated to the intrinsic distributions of Ca 2+ handling channels and transporters (Cav1.2α, RyR2, NCX and SERCA2a) Conclusion: These data show for the first time that bradycardia elicits SCE which contributes to AP prolongation and its spatial heterogeneity increases DOR. These changes explain why bradycardia is a critical factor to trigger Torsade de Pointes in LQT2.
Women have a higher risk of lethal arrhythmias than men in long QT syndrome type 2 (LQTS2), but the mechanisms remain uncertain due to the limited availability of healthy control human tissue. We have previously reported that in female rabbits, estrogen increases arrhythmia risk in drug-induced LQTS2 by upregulating L-type Ca2+ (ICa,L) and sodium-calcium exchange (INCX) currents at the base of the epicardium by a genomic mechanism. This study investigates if the effects of estrogen on rabbit ICa,L and INCX apply to human hearts.Postmortem human left ventricular tissue samples were probed with selective antibodies for regional heterogeneities of ion channel protein expression and compared to rabbit myocardium. Functionally, ICa,L and INCX were measured from female and male cardiomyocytes derived from human induced pluripotent stem cells (iPS-CMs) with the voltage-clamp technique from control and estrogen-treated iPS-CMs.In women (n = 12), Cav1.2α (primary subunit of the L-type calcium channel protein 1) and NCX1 (sodium-calcium exchange protein) levels were higher at the base than apex of the epicardium (40 ± 14 and 81 ± 30%, respectively, P < 0.05), but not in men (n = 6) or postmenopausal women (n = 6). Similarly, in cardiomyocytes derived from female human iPS-CMs, estrogen (1 nM, 1-2 days) increased ICa,L (31%, P < 0.05) and INCX (7.5-fold, - 90 mV, P < 0.01) and their mRNA levels (P < 0.05). Moreover, in male human iPS-CMs, estrogen failed to alter ICa,L and INCX.The results show that estrogen upregulates cardiac ICa,L and INCX in women through genomic mechanisms that account for sex differences in Ca2+ handling and spatial heterogeneities of repolarization due to base-apex heterogeneities of Cav1.2α and NCX1. By analogy with rabbit studies, these effects account for human sex-difference in arrhythmia risk.
Introduction: Depolarization of pulmonary arterial smooth muscle cells (PASMCs) and subsequent increase of intracellular calcium concentration are suggested to be crucial events in the pathomechanism of pulmonary hypertension. In animal models of PH the calcium activated chloride channel TMEM16A has been recently identified as a novel ion channel contributing to sustained pulmonary vasoconstriction.
Hypothesis: We hypothetised that TMEM16A is also upregulated in human pulmonary arteries obtained from patients with idiopathic pulmonary arterial hypertension (IPAH) and it is responsible for a relevant depolarizing current.
Methods: Laser capture microdissection (LCM) of the intima and media layers of pulmonary arteries from healthy donors (n=7) and IPAH patients (n=7) was performed in order to investigate the expression of several chloride channels by QPCR. These findings were confirmed on cultured PASMCs isolated from healthy donor and IPAH lungs. The membrane potential of PASMCs from both groups was measured with the patch-clamp technique and the effect of the specific TMEM16A inhibitor T16A-inh01 (10 μM) was studied.
Results: Our QPCR studies have shown marked upregulation of TMEM16A both in the LCM material and in the PASMCs isolated from IPAH patients. Measurement of the resting membrane potential showed that the PASMCs from IPAH patients were more depolarized compared to the PASMCs of healthy donors (-34 ± 2 mV vs -47 ± 2 mV, p< 0.05). Interestingly, treatment of the IPAH PASMCs with T16A-inh01 resulted in a significant hyperpolarization of the membrane potential (-41 ± 2 mV, p< 0.05 compared to untreated IPAH).
Conclusions: Our data suggest that TMEM16A is upregulated in the PASMCs of human IPAH patients and the resulting increase in chloride efflux indeed contributes to PASMC depolarization, which may lead to excess vasoconstriction and remodeling of pulmonary arteries.
