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Background: The mitochondrial calcium uniporter (mtCU) is an ≈700-kD multisubunit channel residing in the inner mitochondrial membrane required for mitochondrial Ca2+ (mCa2+) 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 mCa2+ uptake. Furthermore, we show that MCUB incorporation into the mtCU is a stress-responsive mechanism to limit mCa2+ 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 mCa2+ uptake impair mitochondrial energetics and contractile function.Conclusions: We detail a new regulatory mechanism to modulate mtCU function and mCa2+ uptake. Our results suggest that MCUB-dependent changes in mtCU stoichiometry are a prominent regulatory mechanism to modulate mCa2+ uptake and cellular physiology. [ABSTRACT FROM AUTHOR]

Introduction: Recent studies demonstrated that plasma exosomes from diabetic patients and animals lose their ability to maintain angiogenetic properties of endothelial progenitor cells (EPCs)/endothelial cells. However, the underlying mechanisms remain incompletely understood. Hypothesis: Systemic blocking of exosome synthesis/release improves ischemic hindlimb repair in diabetes. Methods: Unilateral hindlimb ischemia (HLI) surgery was conducted by ligation of left femoral artery in 12-week male db/db and db/+ mice which were administrated with either exosome synthesis inhibitor GW4869 (GW, 2 mg/kg body weight, i.p) or vehicle for 4 weeks starting from one week before HLI. Blood perfusion in ischemic hindlimbs was examined by Laser Doppler at pre-HLI, and 0, 3, 7, 14 and 21 days post-HLI. Plasma exosomes were isolated and analyzed by NanoSight. Results: Administration of GW increased blood concentration of CD31+/Sca-1+ cell, decreased cell death in the muscles of ischemic hindlimbs of db/db mice 3-day post-HLI. GW also decreased necrosis and loss of toe/toenail; improved blood flow; enhanced capillary/arterial density determined by CD31 and SMA-α staining and decreased fibrosis in the muscles of ischemic hindlimbs in db/db mice 21-day post-HLI. Plasma exosomes from db/db mice significantly impaired human cardiac microvascular endothelial cells (HCMVECs) tube formation and migration. Mechanistically, diabetic plasma exosomes increased mitochondria proton leak and mitochondrial uncoupling protein-2 (UCP-2) which allows a more rapid electron flux thus reducing membrane potential resulting in reduced reactive oxygen species (ROS) production creating proton leaks across the inner mitochondria membrane to 3.34-fold in HCMVECs. Diabetic plasma exosomes also decreased superoxide dismutase 1 (SOD1), a major antioxidase levels in mitochondria, and mitoROS in HCMVECs. Finally, RNA-seq studies identified highly enriched exosomal microRNA-17 which may directly target SOD1. Conclusions: Systemic inhibition of exosome synthesis/release by GW improved ischemic limb repair in diabetic db/db mice. Mechanistically, although preliminary data, increased mitochondria proton leak/exosome-curried miR-17-induced downregulation of SOD1/mitochondria oxidative stress cascade, at least partially, is involved in plasma exosomes-impaired angiogenic property in diabetes. [ABSTRACT FROM AUTHOR]