Figure 5.

FUNDC1 promotes endoplasmic reticulum (ER) Ca²⁺ transfer to mitochondria and cytosol. A, Rhod-2 measurements of [Ca²⁺]_m in response to ATP (0.1 mM) in Fundc1^wt/Y/Cre^αMyHC+/− or Fundc1^f/Y/Cre^αMyHC+/− neonatal cardiomyocytes. B, Mean ± SD of [Ca²⁺]_m from A (n = 5 independent experiments; ^*P < 0.05 versus Fundc1^wt/Y/Cre^αMyHC+/−). C, Fura-2 measurements of cytosolic [Ca²⁺]_i in response to ATP (0.1 mM) in Fundc1^wt/Y/Cre^αMyHC+/− or Fundc1^f/Y/Cre^αMyHC+/− neonatal cardiomyocytes. D, Mean ± SD of [Ca²⁺]_i from C (n = 5 independent experiments; ^*P < 0.05 versus Fundc1^wt/Y/Cre^αMyHC+/−). E, Representative recordings of ER Ca²⁺ levels [Ca²⁺]_ER during store filling initiated by addition of 100 nM Ca²⁺ and 1.5 mM ATP in Fundc1^wt/Y/Cre^αMyHC+/− or Fundc1^f/Y/Cre^αMyHC+/− neonatal cardiomyocytes. F, Mean ± SD of [Ca²⁺]_ER from E (n = 5 independent experiments; ^*P < 0.05 versus Fundc1^wt/Y/Cre^αMyHC+/−). G-J, The neonatal cardiomyocytes overexpressing Fundc1 by infected with FUNDC1 adenovirus (ad-Fundc1) were transfected with three types of inositol 1,4,5-trisphosphate receptors (IP₃Rs) siRNAs (siIp₃r1, siIp₃r2, and siIp₃r3) for 48 h. A scramble siRNA (siControl) was used as gene silence control. Beta-galactosidase adenovirus (ad-β-Gal) was used as gene overexpression control. Flow cytometry (FCM) analysis were applied to detect resting [Ca²⁺]_m and [Ca²⁺]_i levels labeled with a florescence probe Rhod-2 and CaG5N, respectively in the cells. Representative FCM blots (G) and histogram (H) of the percentage of positive population of Rhod-2 cells in the indicated groups (mean ± SD, n = 5 independent experiments; ^*P < 0.05, ^**P < 0.01 versus siControl & ad-β-Gal). Representative FCM blots (I) and histogram (J) of the percentage of positive population of CaG5N cells in the indicated groups (mean ± SD, n = 5 independent experiments; ^*P < 0.05, ^**P < 0.01 versus siControl & ad-β-Gal). K-M, Effects of FUNDC1 on [Ca²⁺]_ER. Representative recordings of [Ca²⁺]_ER during store filling initiated by addition of 100 nM Ca²⁺ and 1.5 mM ATP in permeabilized cardiomyocytes infected with ad-β-Gal or ad-Fundc1 in the presence (K) or absence (L) of IP₃Rs. Each trace represents mean of 15–20 cells in a single image field. M, Summary data showing the steady-state [Ca²⁺]_ER after store filling (mean ± SD) for 40–100 cells pooled from at least 3 independent trials (mean ± SD, n = 5 independent experiments; ^*P < 0.05 versus siControl & ad-β-Gal).