Figure 1.

Stim1 and Orai1 are expressed and are required for SOCE in neonatal and cardiac myocytes A, Immunoblotting for Stim1 and Orai1 in cultured neonatal and Adult cardiomyocytes revealed sustained expression. B, Immunolocalization of Orai1 and Stim1 in NCVM. Endogenous Orai1 (left panel) and Stim1 (right panel) are detected in NCVM and showed perinuclear enrichment. Cardiomyocytes are stained with actinin. Nuclear are stained with Sytox-Blue. C RT-PCR showing decreased expression of Orai1 mRNA in silenced cells compared to control cell (scrambled siRNA), 96h after transfection (n=6 experiments, * p<0.01), D, Representative Western Blots and densitometric analysis showing significant downregulation of Orai1 after 96h after siRNA transfection (n=6 experiments, *p<0.01) E RT-PCR showing decreased expression of Stim1 mRNA in silenced cells compared to control cell (scrambled siRNA), 96h after transfection (n=6 experiments, * p<0.01). F, Representative Western Blots and densitometric analysis showing significant downregulation of Stim1 after 96h after siRNA transfection (n=6 experiments, *p<0.01). G, Representative records and quantitative analyses of SOCE in neonatal cardiac myocytes. Blue Curve represents control cells treated with PE. Orai1 KD reduced SOCE (−75% decrease vs control group, Stim1 KD −62%, p<0.05, n>30 cells in each condition.). H Representative original traces and quanttitative analyses of global Ca²⁺ transients in electrically stimulated control, Stim1 siRNA treated and Orai1 siiRNA treated cardiomyocytes and representative original traces and quanttitative analyses of SR Ca²⁺ load assessed by caffeine-induced cytosolic Ca²⁺ rise. Stim1 knockdown significantly decrease SR Ca2+ load (p<0.05, n>40 cells for each conditions)