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  • Results are expressed as mean s e m Paired or

    2019-11-07

    Results are expressed as mean±s.e.m. Paired or unpaired Student\'s t test was used for comparisons between 2 groups and one-way ANOVA followed by Bonferroni\'s post-test for more groups. Statistical significance was assigned if p<0.05.
    Results and discussion Variations in [Ca2+]i in cardiomyocytes loaded with Fluo-3 were recorded by confocal fluorescence. Cells were first exposed to the SERCA inhibitor thapsigargin (TG, 5μM) and stimulated 2min to empty the sarcoplasmic reticulum (SR) and then exposed to 0 Ca solution with LTCC blocker nifedipine (Nif, 10μM) and KB-R7943 (KB-R, 5μM) as NCX inhibitor. The efficient Ca2+ store depletion was confirmed by the disappearance of [Ca2+]i transient and cell contraction. Changes in [Ca2+]i of entire myocyte were then monitored following reperfusion with 1.8mM [Ca2+]o in the continued presence of TG, Nif and KB-R (Fig. 1A). In control Verapamil HCl synthesis incubated 4 to 6h without 8-pCPT, when Ca2+ was added back to the external solution there was a weak increase in the Ca2+ fluorescence level (Fig. 1A and B) consistent with a low functional SOCE mechanism in adult cardiomyocytes. However, ~80% of cardiomyocytes incubated with 8-pCPT displayed a significant increase in Ca2+ fluorescence (Fig. 1A and B). This EPAC-enhanced SOCE-like activity was abolished in the presence of the non-specific TRPC channel blocker SKF-96365 (30μM) and was prevented in cardiomyocytes co-incubated with the specific EPAC inhibitors CE3F4 (EPAC 1 inhibitor, 10μM) or ESI-05 (EPAC 2 inhibitor, 10μM). No significant difference was seen between both EPAC inhibitors that have no effect by themselves (data not shown), suggesting that sustained activation of both EPAC isoforms are involved. Moreover, acute EPAC activation with 10μM 8-pCPT for 2–5min resulted in a weak SOCE-like activity (peak F/F0=1.15±0.02, n=28) comparable to control conditions (peak F/F0=1.15±0.03, n=53). It is thus clear that sustained EPAC activation leads to the emergence of a robust SOCE-like mechanism of little significance in cardiomyocytes under control conditions. To further investigate the nature of the store-operated channel we recorded the background current elicited by a 3750ms voltage ramp from +70 to −120mV (holding potential −80mV), following SR depletion (2min stimulation with 300ms voltage-clamp pulses from −80 to 0mV at 4s intervals in the presence of Nif, KB-R and TG, and 0 [Ca2+]o). After stabilization for at least 3min, when Ca2+ was added back to the bathing media (2mM in the continued presence of Nif, KB-R and TG) the current–voltage relationship was almost linear in the control cell incubated without 8-pCPT (Fig.1C). On average (14 cells), the current densities were −0.6±0.1 and 1.0±0.2 pA/pF at −100 and +50mV, respectively. However, in 13 of 17 cells incubated with 8-pCPT (Fig. 1D) we observe robust increases in both inward and outward currents (−2.4±0.4 and 2.9±0.4 pA/pF at −100 and +50mV, respectively, p<0.05). Perfusion with 30μM SKF-96365 reduced both inward and outward currents in the cell incubated with 8-pCPT but had no significant effect on the cell incubated without 8-pCPT (Fig. 1C and D). As a result, the SKF-sensitive current was virtually absent in control cells (−0.12±0.06 and 0.16±0.07 pA/pF at −100 and +50mV, respectively) but was significantly increased in cells incubated with 8-pCPT (−0.9±0.1 and 0.7±0.1 pA/pF at −100 and +50mV, respectively, p<0.05). It might be pointed out that even if SKF-96365 has been shown to inhibit other cardiac ionic channels [13], the present experiments were conducted in conditions where the major Ca2+ flux pathways were blocked thus minimizing unspecific effect of SKF-96365. This limited pharmacological approach suggested that sustained EPAC activation unveils a TRP-like current related to the SOCE mechanism. We analyzed the expression levels of 7 possible molecular candidates, namely TRPC1, C3, C4, C5, C6, Stim1 and Orai1 [2] in 8-pCPT treated and untreated cells. Whereas no significant changes in TRPC1, C5, C6, Orai1 and Stim1 were observed (Supplementary Fig.S1), cardiomyocytes incubated with 8-pCPT showed increased expression of TRPC3 and C4 protein levels (Fig. 1E). Moreover, immunocytochemical staining showed higher labeling of TRPC3 and C4 in 8-pCPT-incubated cells compared with control-incubated cells or cells co-incubated with EPAC inhibitors (Fig. 1F). These results are consistent with TRPC3 immunoreactivity localized to the sarcolemma and the T-tubular network of adult myocytes and with TRPC4 showing a tendency to be more expressed at the intercalated discs [10], [14]. These effects were not associated with concomitant upregulation of mRNA levels of TRPC3 and C4 evaluated by qRT-PCR (Fig. 1G), suggesting a post-transcriptional modulation. Although this subject deserves further investigation, among the diverse pathways that regulate TRPC channel trafficking, a strong candidate that could help to explain our findings is phospholipase C (PLC). It has been previously shown that EPAC activates PLC in cardiomyocytes [7], [15]. Now, PLC activation leads to the production of DAG and TRPC3 activation [16], as well as TRPC4 channel trafficking in hippocampal neurons [17]. Another factor that could influence TRPC channel trafficking is Ca2+. Rise in [Ca2+]i could increase intracellular trafficking of TRPC1/C4 channels [18]. One may hypothesize that the demonstrated increases in [Ca2+]i by EPAC activation [7], [8] could also participate to the TRPC4 post-transcriptional regulation by EPAC.