Strategic Composition and Enrichment of the Mitochondrial Ca2+ Uniporter at Mitochondria-SR Associations Creates Hotspots for Mitochondrial Ca2+ Uptake in the Cardiac Muscle

Abstract

The bioenergetic fueling of cardiac muscle activity is tuned by Ca 2þ signals propagated from SR to the mitochondrial matrix (excitation-bioenergetics coupling). Effective activation of the low-affinity mitochondrial Ca 2þ uniporter (mtCU) is promoted by high [Ca 2þ ] nanodomains where SR is tethered to mitochondria. Cardiac mitochondria have very large inner membrane (IMM) surface with extensive cristae folding but their mtCU current density is reportedly extremely low. We hypothesized that efficient excitationbioenergetics coupling would require biased mtCU activity towards mitochondria-SR associations by mtCU being there more abundant and/or more efficient. To this end, we first analyzed the submitochondrial distribution of mtCU's pore protein, MCU in rat and mouse heart. Immunofluorescence (IF) in cells showed significant non-mitochondrial crossreactions; hence, we performed IF on mitochondria isolated from cardiac muscle (mtIF). mtIF showed 50% of punctate MCU positive structures colocalized with type-2 ryanodine receptor, suggesting a location bias to mitochondria-SR associations. Further, in submitochondrial membrane fractions MCU and the SR marker calsequestrin were both enriched with the inner-outer membrane contact points. Mitochondrial membranes in the SR fraction (MMSR) harbored as high concentrations of MCU as the crude mitochondria and much higher levels of the essential MCU regulator EMRE. Gel-filtration analysis showed size profiles of non-denaturing detergent-solubilized MCU-vs. EMRE-containing complexes largely overlapping in the SR fraction, while revealed MCU containing complexes without EMRE in the crude mitochondria. Finally, Ca 2þ flux assays revealed more efficient mtCU-mediated Ca 2þ uptake by the MMSR than the crude mitochondria. Our data thus suggest a distribution bias in the mtCU channel components over the cardiac IMM, turning mitochondria-SR interfaces to Ca 2þ uptake 'hotspots' harboring all-EMREsupported MCU while leaving other IMM areas less responsive to Ca 2þ owing to EMRE-less mtCUs.