Structural and Functional Impact of Mitochondrial Ca2+ Signaling in Skeletal Muscle

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pharmacology and Physiology, 2016.Skeletal muscle is an extremely energy demanding tissue. ATP in skeletal muscle is derived either via anaerobic pathways or through oxidative phosphorylation in mitochondria. ATP generation rate in mitochondria depends on the level of Ca2+ ions in mitochondrial matrix. The balance of mitochondrial matrix Ca2+ level is achieved through tight regulation of mitochondrial membrane potential (ΔΨm), mitochondrial localization with regard to the Ca2+ release channels as well as fine tuning of mitochondrial Ca2+ uptake and efflux. In adult skeletal muscle, mitochondria are primarily located at the Iband of the sarcomere and are connected to the SR by proteinaceous tethers. Mitofusin 2 (Mfn2) is a transmembrane GTPase that participates in mitochondrial fusion and is thought to constitute a part of the SR-mitochondrial tether. Here the structural and functional role of Mfn2 in SR-mitochondrial crosstalk in skeletal muscle was evaluated. Reduction of Mfn2 protein expression (Mfn2 KD) resulted in mitochondrial fragmentation and mislocalization from the I-band. Mitochondrial Ca2+ uptake in Mfn2 KD FDB fibers was reduced along with a reduction in mitochondrial membrane potential (ΔѰm), suggesting that the reduction in activity-dependent mitochondrial Ca2+ accumulation results in part from a reduction in the electrochemical driving force for Ca2+ uptake. Molecular mechanisms involved in the regulation of activity dependent mitochondrial Ca2+ uptake and efflux were evaluated. Mitochondrial Ca2+ uniporter (MCU) and Mitochondrial Leucine zipper-EF-hand containing transmembrane protein 1 (LETM1) were studied as Ca2+ uptake mechanisms whereas mitochondrial Ca2+/Na+ exchanger (NCLX) and the mitochondrial permeability pore (mPTP) were evaluated as potential Ca2+ efflux pathways. MCU is a mitochondrial transmembrane protein that forms homomultimers to form a Ca2+ permeable channel to allow Ca2+ entry upon Ca2+ release from the sarcoplasmic reticulum. LETM1 is EF hand containing transmembrane ion exchanger, that is thought to be involved in Ca2+/H+ or K+/H+ exchange across inner mitochondrial membrane (IMM). In vivo knockdown of 50% of endogenous MCU expression (MCU KD) resulted in a significant reduction in activity-dependent mitochondrial Ca2+ uptake and expression of the dominant negative form of MCU (DNMCU) in FDB fibers resulted in almost complete inhibition of mitochondrial Ca2+ uptake following electrical stimulations. Mitochondrial Ca2+ uptake was not altered in FDB fibers where LETM1 expression was reduced by 50% (LETM1 KD). Mitochondrial Ca2+ efflux rate was significantly reduced following blockage of NCLX by application of 10 μM CGP- 37157, a specific NCLX inhibitor. The role of mPTP in mitochondrial Ca2+ efflux was studied in FDB fibers isolated from cyclophilin D knockout mice (CypD KO), in which mPTP is desensitized for Ca2+ activation. Mitochondrial Ca2+ efflux rate was not altered in CypD KO FDB fibers; however, the extent of mitochondrial Ca2+ uptake in CypD KO fibers was significantly increased compared to WT control. These results indicate that MCU is required for achieving optimal activity-dependent mitochondrial Ca2+ uptake, whereas LETM1 is less likely to play a role in mitochondrial Ca2+ uptake under conditions applied. Also, NCLX is a major mitochondrial Ca2+ efflux channel, whereas mPTP plays a role in the fast mode of mitochondrial Ca2+ efflux, preventing mitochondrial Ca2+ overload. Oxidative phosphorylation in mitochondria is one of the major ATP producing pathways and it depends on matrix Ca2+ levels; therefore the effects of in vivo inhibition of mitochondrial Ca2+ uptake in skeletal muscle on muscle performance and fatigability were studied. For this purpose, a transgenic mouse model with an inducible, musclespecific expression of dominant negative form of MCU (DN-MCU: dnMCU+/-rtTA+/- and dnMCU+/-rtTA+/+) was generated. Following a series of doxycycline (DOX) injections the DN-MCU transgene was expressed specifically in skeletal muscle fibers in a transgene DNA copy dependent manner. Activity-dependent mitochondrial Ca2+ uptake in FDB fibers isolated from DN-MCU mice was reduced by 50% compared to control, non DOX injected DN-MCU mice. Ex vivo muscle fatigability of oxidative muscles was enhanced and in vivo running performance of dnMCU+/-rtTA+/+ mice was reduced. Together these results suggest that proper mitochondrial Ca2+ uptake is important for maintenance of sufficient power output, especially in more oxidative muscles