Exploration Of TRPC3/6-mediated Store-Operated Ca²⁺ Entry In Cardiomyocytes

Ca²⁺ is an important second messenger in cells. Intracellular Ca²⁺ maintaines a certain dynamic balance under normal circumstances. Once the calcium homeostasis is broken, it will cause cell dysfunction, such as the abnormity of cytomorphology, movement, fission and proliferation, metabolism etc. In non-excitable cells, store-operated Ca²⁺ entry（SOCE） or capacitative Ca²⁺ entry（CCE） is generally accepted to function as a main mediator of Ca²⁺ entry pathway. The activation of SOCE mainly depends on the G protein coupled receptors. The coupling of these Gq PCRs to the phospholipase C（PLC） pathway produces inositol trisphosphate（IP3）, and involves the activation of calcium channels by IP3-induced depletion of endoplasmic reticulum calcium stores. However, a number of recent studies have provided evidences that SOCE also is functional in excitable cells, such as neurocytes, skeletal muscle cel s and myocardial cel s.During the process of studying the molecular components of SOCE, a team of scientists discovered mammalian transient receptor potential canonical（TRPC） family. There are seven members of TRPC family（TRPC1-7）, TRPC2 is a pseudogene in humans. Functional TRPC channels are divided into TRPC1/4/5 and TRPC3/6/7 subfamily members based on the biochemical and functional similarities. Studies reported by a large number of investigators showed that some TRPC channels contribute to SOCE, but others were regulated by store-independent mechanism.So far, the role of TRPC channels in SOCE in cardiac myocytes is not well defined. In order to find out the relationship between TRPC and SOCE, we used TRPC3/6/7 gene knockout mice, H9C2 cells and TRPC inhibitors. By calcium imaging, this study explored the idea that TRPC was shown to induce SOCE, which was inhibited partially with TRPC3/6/7 gene knockout cells. This showed that TRPC3/6/7 might be implicated in SOCE. Meanwhile, we couldn’t detect the m RNA expression of TRPC7 by RT-PCR in heart tissue. It indicated that TRPC3/6 tended to be involved in SOCE.In addition, cells show an increase of intracellular Ca²⁺ after the release of calcium store. However, we couldn’t find this phenomenon in cardiomyocytes. The Plasma membrane Ca²⁺-ATPase（PMCA）,which mediate Ca²⁺-efflux across the sarcolemma, are known to regulate signaling in the heart. There are four PMCA subtypes（PMCA1-4）.PMCA4 is one of the predominant PMCA isoforms in the heart. To investigate whether PMCA4 related to this phenomenon, we used Caloxin 1b1 to selectively inhibit PMCA4 then detected SOCE in myocardial cell. Result showed that after adding Caloxin 1b1 still did not detect the increase of intracellular Ca2 + after the release of calcium store. So we speculated that this phenomenon had nothing to do with PMCA4.H9C2 cells showed a robust SOCEIn this experiment, we used the conventional protocol to validate the presence of endogenous SOCE in the cardiomyocytes. We treated H9C2 cells with 2μmol/L thapsigargin alone for seven minutes in calcium- free solution, leading to passive diffusion of ER calcium into the cytoplasm, and mimicking the physiological process of store-depletion and the activation of SOCE. Then we observed the change of [Ca²⁺]i by Ca²⁺-sensitive dye Fura-2 and calcium imaging.Under these conditions, H9C2 cells showed a robust and signif icant Ca²⁺ influx that could be blocked with2-APB（50μmol /L）, an inhibitor of SOCE. This showed that SOCE existed in myocardial cel s.TRPC channels participated in primary cardiomyocytes SOCETo determine if TRPC channels play a role in SOCE of cardiomyocytes, we first used the pan TRPC antagonist SKF-96365 in H9C2 cells and primary cardiomyocytes. The inhibition of SKF96365 on TRPC channels is non-selective, so it is always used for TRPC studies. The results showed that SKF-96365 completely reversed the sustained Ca²⁺ increases. And cardiomyocytes displayed SOCE operated by TRPC.SOCE in TRPC3/6/7-/- knockout mice was decreasing.It is currently believed that SOCE regulates myocardial apoptosis, hypertrophy, ischemia and reperfusion which are often caused by intracellular calcium overload. Meanwhile, numerous studies have demonstrated that TRPC is involved in heart pathological process, especially TRPC3 and TRPC6 in pathologic cardiac hypertrophy. In addition, among many subtypes, TRPC3, TRPC6 are ⁷5% identical, have relatively low selectivity for Ca²⁺, and are sensitive to intracellular Ca²⁺. Their relatively high expression levels in smooth muscle and heart cells make them promising candidates for the as yet unidentified non-selective cationic channels in these muscle cells. To further characterize the properties of TRPC channels in SOCE in cardiac myocytes, we used TRPC3/6/7 gene knockout mice. The results showed that the peak amplitude of the Ca²⁺ transient was inhibited in TRPC3/6/7-/-cardiomyocytes compared with wildtype cardiomyocytes. These data indicated that TRPC3/6/7 might take part in SOCE. However, I couldn’t detecte the expression of TRPC7 in heart tissue, so TRPC3/6 became the best candidate molecules.PMCA4 had nothing to do with the invariability of intracellular Ca²⁺ after the release of calcium storePMCA is a kind of P2 type Ca²⁺-ATPase, it has high affinity with Ca2 + and can driver Ca²⁺ from inside cell to the extracellular by ATP. PMCA4 is one of the predominant PMCA isoforms in the heart which mediate Ca²⁺-efflux across the sarcolemma. We did not detect the increase of intracellular Ca²⁺ after the release of calcium store in cardiomyocytes. To investigate whether PMCA4 associated with this phenomenon, we use Caloxin1b1（including 10μmol/L, 30μmol/L, 100 μmol/L） to selectively inhibit PMC A4 then doing calcium imaging tests again. Results showed that after joining Caloxin 1b1 still did not detect the increase of intracellular Ca²⁺ after the release of calcium store in cardiomyocytes. So we speculated that this phenomenon had nothing to do with PMCA4.Collectively these results suggested that SOCE operated by TRPC exhibited in cardiac myocytes and TRPC3/6 were the most likely candidates. Additionally, PMCA4 had nothing to do with the invariability of intracellular Ca²⁺ after the release of calcium store. Therefore, this present study provides a new experimental basis to reveal TRPC mediated SOCE in myocardial cells, and to study the physiological characters of myocardial cel s.