Potential Arrhythmogenic Role of TRPC Channels and Store-Operated Calcium Entry Mechanism in Mouse Ventricular Myocytes

Abstract

Background and Purpose: Store-operated calcium entry (SOCE) is an important physiological phenomenon that extensively mediates intracellular calcium ion (Ca2+) load. It has been previously found in myocytes isolated from neonatal or diseased hearts. We aimed to determine its existence, molecular nature in undiseased hearts and its potential arrhythmogenic implications under hyperactive conditions.Experimental Approach: Ventricular myocytes isolated from adult FVB mice were studied by using Ca2+ imaging and whole-cell perforated patch-clamp recording. In addition, lead II ECGs were recorded in isolated Langendorff-perfused mice hearts. Functional TRPC channel antibodies and inhibitors, and TRPC6 activator hyperforin were used.Key Results: In this study, we demonstrate the existence and contribution of SOCE in normal adult mouse cardiac myocytes. For an apparent SOCE activation, complete depletion of sarcoplasmic reticulum (SR) Ca2+ by employing both caffeine (10 mM) and thapsigargin (1 μM) or cyclopiazonic acid (10 μM) was required. Consistent with the notion that SOCE may be mediated by heteromultimeric TRPC channels, SOCEs observed from those myocytes were significantly reduced by the pretreatment with anti-TRPC1, 3, and 6 antibodies as well as by gadolinium, a non-selective TRPC channel blocker. In addition, we showed that SOCE may regulate spontaneous SR Ca2+ release, Ca2+ waves, and triggered activities which may manifest cardiac arrhythmias. Since the spontaneous depolarization in membrane potential preceded the elevation of intracellular Ca2+, an inward membrane current presumably via TRPC channels was considered as the predominant cause of cellular arrhythmias. The selective TRPC6 activator hyperforin (0.1–10 μM) significantly facilitated the SOCE, SOCE-mediated inward current, and calcium load in the ventricular myocytes. ECG recording further demonstrated the proarrhythmic effects of hyperforin in ex vivo mouse hearts.Conclusion and Implications: We suggest that SOCE, which is at least partially mediated by TRPC channels, exists in adult mouse ventricular myocytes. TRPC channels and SOCE mechanism may be involved in cardiac arrhythmogenesis via promotion of spontaneous Ca2+ waves and triggered activities under hyperactivated conditions.

Specific Upregulation of TRPC1 and TRPC5 Channels by Mineralocorticoid Pathway in Adult Rat Ventricular Cardiomyocytes

“…For example, the traditional protocol using only thapsigargin or cyclopiazonic acid (CPA) to block the SR Ca 2+ pumps indicates that there is no or limited SOCE in normal adult ventricular cells [9,17,35,45]. However, these conditions might not sufficiently deplete the SR Ca 2+ stores of quiescent adult ventricular cells [15,46], even if one might think that, in the presence of an SERCA pump inhibitor, the SR Ca 2+ leak would deplete the SR [45]. Thus, when a clear SR Ca 2+ depletion is observed (with ryanodine receptor activation through either stimulation or caffeine/ryanodine application), a moderated SOCE activity might be observed [11,14,[47][48][49], as we show in the present study.…”

“…A number of studies, conducted in various cell models, implicated members of TRPC channels in SOC activity [59][60][61][62]. Notably, TRPC1 channels are believed to mediate the non-selective cation current and to form SOC channels as a component in human atria [63], in the mouse sinoatrial node [64], in human cardiac c-kit+ progenitor cells [65], in NRVMs [66], and in adult ventricular cardiomyocytes [36,46,67]. Similarly, TRPC5, which was found to contribute to the formation of SOCE in smooth muscle cells isolated from rabbit pial arterioles [68], contributes to SOCE in NRVMs [11,69].…”

Role of Transient Receptor Potential Canonical Channels in Heart Physiology and Pathophysiology

“…Importantly, SOCE has been reported in myocytes isolated from fetal, neonatal, and hypertrophic hearts (29,30), however, its existence, molecular nature and pathological/physiological relevance in normal adult human hearts has not drawn much attention. Recent studies, including ours, have revealed TRPC channels play an important role in the regulation of electromechanical activity of the developing heart (8), Ca 2+ paradox injury (31), pathological remodeling after myocardial infarction (32), as well as has a proarrhythmic effect under hyperactive conditions (by activators) (33). Hence, TRPC channels appear to be another important way by which Ca 2+ can enter cardiomyocytes and regulate myocardial contractility in addition to voltage-gated Ca 2+ channels (such as L-type or Ttype) and the Na + /Ca 2+ exchanger.…”

“…Besides genetic approaches, pharmacological TRPC modulators have been widely used as a tool to define the functional roles of TRPCs. For example, Gd 3+ , 2aminoethoxydiphenyl borate (2-APB), and SKF 96365 are generally used as non-selective TRPC blockers (8,33,52,53). Some relatively selective blockers include Ethyl-1-(4-(2,3,3-trichloroacrylamide)phenyl)-5-(trifluoromethyl)-1Hpyrazole-4-carboxylate (Pyr3, a TRPC3 blocker) (54), GsMTx4 (a TRPC1 and TRPC6 blocker) (55).…”

“…For example, (-) Englerin A is an agonist for TRPC5 and TRPC4 (17). Hyperforin has been used as an agonist for TRPC6 (33). The identification of these new selective TRPC modulators provides possibilities for pharmacological therapy for TRPC-related diseases.…”