Reciprocal Interactions of STIM1 with Orai1 and L-Type Ca2+ Channels in Cardiac Myocytes

BACKGROUND: Pathological cardiac hypertrophy can be triggered by abnormal Ca2+ levels. It has been shown that the mechanisms governing context-dependent changes in Ca2+ influx are linked to stromal interaction molecule 1 (STIM1). STIM1 is a sarcoplasmic reticulum Ca2+ sensor that regulates Ca2+ influx by directly activating store operated calcium channels such as Orai1 in response to stress such as intracellular Ca2+ depletion. STIM1 is also known to regulate L-type Ca2+ channels in cardiomyocytes, though the mechanism has not been elucidated. HYPOTHESIS: The Ca2+-sensing protein, STIM1, molecularly interacts with Orai1 and L-type Ca2+ channels in cardiomyocytes to facilitate Ca2+ influx as a master regulator of Ca2+ channels. METHODS: To address this, we monitored STIM1 interactions with either Orai1 or L-type Ca2+ channels in neonatal rat ventricular cardiomyocytes (NRVMs) and adult rat ventricular cardiomyocytes (ARVMs) using the Duolink in situ fluorescence assay. This technique utilizes proximity ligation assay technology to directly monitor interactions between proteins within 40nm. These channel interactions were observed under endogenous culture conditions as well as thapsigargin-mediated calcium store depleted conditions. Differences in STIM1 interactions between control (untreated) and thapsigargin-treated NRVM and ARVM cells were observed. RESULTS: In NRVM cells, STIM1 interactions with Orai1 increased by 44.79 ± 2.68% when treated with thapsigargin as compared to the control population (n=73-83). Conversely, STIM1 interactions with L-type Ca2+ channels decreased by 52.31 ± 3.45% compared to the control (n=64-69). In ARVM cells, STIM1 interactions with Orai1 decreased by 59.19 ± 2.39% when treated with thapsigargin as compared to the control population (n=11-16). STIM1 interactions with L-type Ca2+ channels on the other hand increased by 74.13 ± 0.21% compared to the control (n=11-19). CONCLUSIONS: We observed an opposite trend in STIM1 interactions with Orai1 and L-type Ca2+ channels in NRVM and ARVM cells. When treated with thapsigargin, their degree of interaction changed significantly. This would suggest that STIM1 undergoes dynamic changes in response to calcium conditions in order to regulate Ca2+ influx. At a molecular level, it would seem that STIM1 is a versatile Ca2+ channel regulator as it can alternately interact with both channels. Further studies will aid in the development of novel therapeutic strategies for the treatment of cardiac hypertrophy and disease.