Journal
CIRCULATION
Volume 140, Issue 21, Pages 1720-1733Publisher
LIPPINCOTT WILLIAMS & WILKINS
DOI: 10.1161/CIRCULATIONAHA.118.037968
Keywords
calcium channels; calcium signaling; mitochondria; MCUB protein; human; MICU1 protein; human; oxidative phosphorylation; reperfusion injury
Funding
- National Institutes of Health [R01HL142271, R01HL136954, R01HL123966, P01HL134608-sub-5483]
- American Heart Association [17POST33660251, 15PRE25080299, 17PRE33460423]
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Background: The mitochondrial calcium uniporter (mtCU) is an approximate to 700-kD multisubunit channel residing in the inner mitochondrial membrane required for mitochondrial Ca2+ (Ca-m(2+)) uptake. Here, we detail the contribution of MCUB, a paralog of the pore-forming subunit MCU, in mtCU regulation and function and for the first time investigate the relevance of MCUB to cardiac physiology. Methods: We created a stable MCUB knockout cell line (MCUB-/-) using CRISPR-Cas9n technology and generated a cardiac-specific, tamoxifen-inducible MCUB mutant mouse (CAG-CAT-MCUB x MCM; MCUB-Tg) for in vivo assessment of cardiac physiology and response to ischemia/reperfusion injury. Live-cell imaging and high-resolution spectrofluorometery were used to determine intracellular Ca2+ exchange and size-exclusion chromatography; blue native page and immunoprecipitation studies were used to determine the molecular function and impact of MCUB on the high-molecular-weight mtCU complex. Results: Using genetic gain- and loss-of-function approaches, we show that MCUB expression displaces MCU from the functional mtCU complex and thereby decreases the association of mitochondrial calcium uptake 1 and 2 (MICU1/2) to alter channel gating. These molecular changes decrease MICU1/2-dependent cooperative activation of the mtCU, thereby decreasing Ca-m(2+) uptake. Furthermore, we show that MCUB incorporation into the mtCU is a stress-responsive mechanism to limit Ca-m(2+) overload during cardiac injury. Indeed, overexpression of MCUB is sufficient to decrease infarct size after ischemia/reperfusion injury. However, MCUB incorporation into the mtCU does come at a cost; acute decreases in Ca-m(2+) uptake impair mitochondrial energetics and contractile function. Conclusions: We detail a new regulatory mechanism to modulate mtCU function and Ca-m(2+) uptake. Our results suggest that MCUB-dependent changes in mtCU stoichiometry are a prominent regulatory mechanism to modulate Ca-m(2+) uptake and cellular physiology.
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