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Cardiac pacemaking relies on the spontaneous electrical activity in the right atrium of sino-atrial myocytes (SANCs). Automaticity in SANCs results from a robust interplay of membrane ion channels activity and intracellular calcium dynamics. However, only a fraction of isolated SANCs exhibit rhythmic firing, whereas most SANCs show irregular (dysrhythmic) firing or remain dormant. To study the capability of L-type Cav1.3 calcium channels to initiate automaticity in dormant SANCs under β-adrenergic stimulation we used a knock-in mouse strain in which the sensitivity of Cav1.2 α1 subunits to dihydropyridines (DHP) was inactivated (Cav1.2DHP-/-). We performed current-clamp recordings on isolated SANCs under isoprenaline (ISO, 100 nM) and in the absence or presence of the DHP blocker Nifedipine (Nife, 3 μM). In rhythmic SANCs, Nife significantly reduced the spontaneous firing under ISO perfusion (ISO: 447 ± 12, ISO + Nife: 233 ± 25 bpm). In dysrhythmic SANCs, Nife significantly reduced the spontaneous rate under ISO in 60% of the cells (ISO: 386 ± 12, ISO + Nife: 188 ± 47 bpm) and completely stopped it in 40% (295 ± 29 bpm to 0). On 25 dormant SANCs, 50% started firing after ISO perfusion (0 to 320 ± 46 bpm). Strikingly, in 75% of them, Nife totally blocked this ISO-induced firing. Moreover, dormant SANCs showed a statistically significant increase in action potential (AP) threshold under ISO compared to dysrhythmic and rhythmic SANCs (dormant: −30.1 ± 2.5, dysrhythmic: −43.3 ± 2.3, rhythmic: −41.2 ± 2.1 mV). No significant difference was observed in the other AP parameters between dormant, dysrhythmic and rhythmic SANCs under ISO. Our results indicate that Cav1.3 channels can generate pacemaker activity autonomously, at least in a particular subpopulation of SANCs, while it is responsible of about half of the automaticity in the others. [ABSTRACT FROM AUTHOR]

Cardiac pacemaking relies on the spontaneous electrical activity of sino-atrial myocytes (SANCs) in the right atrium arising from a robust interplay of membrane ion channels activity (membrane clock) and intracellular calcium dynamics (calcium clock). Strikingly, most of isolated SANCs exhibit a dormant state (no apparent spontaneous activity) whereas only a small fraction shows regular firing. It has recently been shown that these non-firing SANCs are also present in intact SAN and can switch into a firing mode under beta-adrenergic stimulation. However, their functional relevance in vivo is unknown. To study the role of L-type Ca v 1.3 calcium channels in dormant SANCs using a knock-in mouse strain in which the sensitivity of Ca v 1.2 α1 subunits to dihydropyridines (DHP) was inactivated (Ca v 1.2DHP−/−). We performed voltage and current-clamp recordings and confocal live imaging on isolated SANCs under isoprenaline (ISO, 100 nM) in the absence or presence of the DHP blocker Nifedipine (Nife, 3 μM). Preliminary results seem to show less L-type calcium current I Ca,L in dormant SANCs (D) compared to rhythmic SANCs (R) (D: −6.3 ± 1.2, R: −15.6 pA/pF). Dormant SANCs also have less diastolic calcium (D: 96.9 ± 12, R: 218 ± 51.2 a.u, P = 0.14) but significant higher sarcoplasmic reticulum calcium load (D: 6.3 ± 0.6, R: 3.1 ± 0.3 a.u, P = 0.004). Interestingly, ISO perfusion could only switch half of dormant SANCs into a firing mode in both action potential (AP) and calcium transients (CT) recordings whereas the others remained dormant. Further Nife perfusion completely stopped this ISO-induced firing switching the cells back into a dormant state for the vast majority of them (75% in AP-recorded SANCs and 100% in CT-recorded SANCs). Taken together, these results indicate that Cav1.3 channels play a key role in sustaining the coupling of membrane and calcium clock in dormant SANCs under beta-adrenergic stimulation. [ABSTRACT FROM AUTHOR]

Transient Receptor Potential Vanilloid 4 (TRPV4) channel is a calcium permeable channel (P Ca /P Na ∼ 10). Its expression was 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 Ca v 1.2 was confirmed using western-blots biotinylation, immunoprecipitation and immunostaining experiments. Then, electrocardiograms (ECGs) and patch-clamp recordings showed shortened QTc and action potential (AP) duration in trpv4−/− compared to trpv4+/+ mice. Thus, TRPV4 activator GSK1016790A produced a transient and dose-dependent increase in AP duration at 90% of repolarization (APD 90) in trpv4+/+, but not in trpv4−/− myocytes or when combined with TRPV4 inhibitor GSK2193874 (100 nM). Hence, GSK1016790A increased 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 APD 90 in trpv4+/+ but not in trpv4−/− myocytes, suggesting that TRPV4 prolongs AP duration (APD) 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 accordance with what 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. [ABSTRACT FROM AUTHOR]

Introduction Sinoatrial cells generate cardiac automaticity through functional interactions between membrane ionic currents and intracellular Ca2+ dynamics. Mitochondrial Ca2+ plays a role in ATP synthesis regulation and intracellular Ca2+ levels. Interestingly, both could modify AMPc levels and possibly regulate cardiac automatism. We then hypothesized that mitochondrial Ca2+ homeostasis interact with β-adrenergic pathways and regulates cardiac automatism. Objective To determine whether pharmacological inhibition of the mitochondrial Ca2+ uniporter (MCU), which is responsible for Ca2+ influx in mitochondria, affects the sinoatrial node function and β-adrenergic responses. Method Basal level and amplitude of Ca2+ transients were recorded using a ratiometric fluorescent probe (Fura-2/AM) in mice isolated sinoatrial cells. Optical voltage mapping was also used to study the spontaneous frequency of action potentials generated by intact sinoatrial tissue isolated from C57Bl6 mice. Results In basal conditions, inhibition of mitochondrial Ca2+ influx with Ru360 in isolated sinoatrial cells did not affect automaticity. On the other hand, Ru360 prevents intracellular Ca2+ increase after stimulation of L-type Ca2+ channels. Similarly, MCU inhibition applied to intact sinoatrial tissues did not show significant effects under basal conditions, but induced a significant reduction of the spontaneous frequency (PA/min) under β-adrenergic stimulation. Conclusion We here demonstrated that mitochondrial Ca2+ regulates intracellular Ca2+ dynamics, and that mitochondrial Ca2+ is needed to support cAMP production, by stimulating ATP synthesis during a β-adrenergic stimulation. We propose that this mechanism is required to maintain a high spontaneous frequency, especially under physiological stress conditions. [ABSTRACT FROM AUTHOR]

Introduction Transient Receptor Potential Vanilloid 4 (TRPV4) channels, belonging to the TRP channels superfamily, are Ca2+-permeable channels activated by hypo-osmotic stress. In the heart, TRPV4 channels are expressed at the protein level in mouse and rat ventricular myocytes and fibroblasts. They are involved in valve development during embryogenesis, in ischemia-reperfusion injuries and in reactive fibrosis. Whether TRPV4 channel can participate in ventricular action potential (AP) is still unknown. Objective The aim of this study is to pharmacologically unmask the contribution of TRPV4 channels in left ventricular AP. Methods Left Ventricular myocytes were isolated from TRPV4 + / + and TRPV4 − / − mice and AP were recorded using the patch-clamp technique. Pharmacological activator (GSK1016790A) and inhibitors (HC-067047 and GSK2193874) were perfused during the recordings to evaluate the effect on AP parameters. Results TRPV4 activator GSK1016790A induces a transient and dose dependent increase in AP duration at 90% of repolarization (APD90) in TRPV4 + / + but not in TRPV4 − / − myocytes (+ 19.4 ± 3.8%; n = 11 vs − 0.6 ± 1.8%; n = 7 at 500 nM respectively). The activator mediated-APD increase is abolished by TRPV4 channels inhibitors and CamKII inhibitor KN93 at 1 μM. Conversely, TRPV4 channel inhibitor (GSK2193874) at 100 nM significantly decreases APD90 in TRPV4 + / + but not in TRPV4 − / − myocytes (− 7.8 ± 2.6%; n = 11 vs 1.0 ± 1.3%; n = 7 respectively). Similarly, HC-067047 at 300 nM significantly decreases APD90 in TRPV4 + / + but not in TRPV4 − / − myocytes (− 10.9 ± 5.0%; n = 6 vs − 1.2 ± 2.4%; n = 4 respectively). At the concentrations mentioned above, activator and inhibitors have no effect on the other AP parameters. Conclusion This study shows that TRPV4 channels exert a basal contribution in ventricular AP and could affect repolarization. It is thus a potential target for pharmacological approaches against ventricular arrhythmias. [ABSTRACT FROM AUTHOR]