Abstract 240: Strategic Distribution of the Mitochondrial Ca2+ Uniporter in Cardiac Mitochondria

Propagation of SR-derived Ca2+ signals to the mitochondrial matrix is an important tuning tool of cardiac energy metabolism (excitation-bioenergetics coupling). Effective activation of the low-affinity mitochondrial Ca2+ uniporter (mtCU) is supported by high [Ca2+] nanodomains where SR cisternae are tethered to mitochondria. Cardiac mitochondria have large inner membrane (IMM) surface with extensive cristae folding. However, recent comparative patch clamp studies found cardiac mtCU current density extremely low. We hypothesized that efficient excitation-bioenergetics coupling would require strategic positioning of the mtCU. We thus analyzed the distribution of mtCU’s pore protein, MCU in rat and mouse cardiomyocytes and cardiac membrane fractions. Immunofluorescence (IF) using the commercial Sigma antibody showed significant non-mitochondrial (nuclear, cyto/sarcoplasmic) crossreactions as verified in MCU-KO MEFs and cardiomyocytes. To isolate mitochondrial labeling, we performed IF of isolated mitochondria (mtIF) and immunogold electron microscopy of freshly isolated cardiomyocytes. mtIF showed ~50% of MCU-positive structures colocalized with RyR2, in line with a location preference to SR-mitochondria associations. This distribution bias was also reflected in the immunogold TEM analysis. Moreover, WB showed both MCU and calsequestrin being enriched in the same sub-mitochondrial membrane fraction separated on sucrose gradient. The prevalence of MCU and even more the essential MCU regulator (EMRE) was higher in the mitochondria “contaminating” the heavy SR (SR-associated mito membranes) than in the crude mitochondrial fraction. [Ca2+] activation of mitochondrial Ca2+ uptake at similar IMM potentials was as effective in suspensions of SR-associated mito membranes as in the crude mitochondrial fraction. Collectively, our data suggest a location bias of the functionally mandatory mtCU channel components MCU and EMRE toward SR-mitochondrial associations. As the bias is much stronger for EMRE, the component that is required for MCU to function as a channel, it raises the possibility that EMRE availability may be a physiological limiting factor in regional mtCU activity in the cardiac muscle mitochondria.