Non-RyR Calcium Leak of the Sarcoplasmic Reticulum is Governed by TRPC1 in Cardiomyocytes

Abstract

Sarcoplasmic calcium leak in cardiomyocytes has been explained by spontaneous sparks from ryanodine receptors (RyRs), non-spark-mediated RyR leak and non-RyR leak. Ion channels underlying non-RyR leak are currently ill defined. Here, we test the hypothesis that transient receptor potential cation (TRPC) 1 channels contribute to non-RyR leak. We investigated neonatal rat ventricular myocytes (NRVMs) infected with adenoviral eGFP, TRPC1-eGFP, shRNA-TRPC1-eGFP and scrambled RNA-eGFP constructs. NRVMs were studied 4-5 days after infection. NRVMs were fixed and labeled for TRPC1 and sarco/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2). Three-dimensional image stacks of NRVMs were acquired using a confocal microscope (Leica SP8 TCS). Protein colocalization was measured using the Pearson correlation coefficient (Rr). We also investigated SR calcium load in Rhod-3 loaded NRVMs. In short, NRVMs were paced and then superfused with 0 sodium/0 calcium solution for 2 min. Subsequently caffeine (20 mM) was applied to trigger sarcoplasmic calcium release. Fluorescence signals were acquired using a Leica SP8 TCS microscope and analysed after noise reduction and self-ratioing. We found a mid-level degree of colocalization of SERCA2 and TRPC1 for NRVMs infected with with eGFP (Rr=0.46±0.03), TRPC1-eGFP (0.41±0.03) and shRNA-TRPC1-eGFP (0.47±0.03). Colocalization of eGFP with SERCA2 was higher for TRPC1-eGFP (0.41±0.04) than for eGFP (0.26±0.02) and shRNA-TRPC1-eGFP infected NRVMs (0.22±0.04). We measured decreased and increased sarcoplasmic calcium content in NRVMs infected with TRPC1-eGFP (1.64±0.21) and shRNA-TRPC1-eGFP (4.30±0.49), respectively, versus eGFP (3.18±0.23). Differences between calcium content in eGFP and scrambled RNA-eGFP infected NRVMs were not significant. The investigations support our hypothesis that TRPC1 channels contribute to non-RyR sarcoplasmic calcium leak in cardiomyocytes. Structural data indicate that TRPC1 is located in the sarcoplasmic membrane. We suggest that expression and activity of TRPC1 channels modulate the concentration of sarcoplasmic and cytosolic calcium in cardiac myocytes.