Mitochondrial Calcium Uptake and Matrix Calcium Buffering in Skeletal Muscle

Abstract

Many clues suggest that mitochondrial calcium uptake plays an essential role in muscle function. Mitochondria can contribute to shaping of the sarcoplasmic [Ca2+] transients by their Ca2+ handling and are providing energy for muscle contraction and relaxation by their ATP production. Mitochondrial calcium handling involves uptake, release and Ca2+ chelation in the matrix, which allows storage of vast amounts of Ca2+. Recent identification of the molecular machinery of the Ca2+ uniporter (mtCU) created an opportunity to target specific mechanisms of Ca2+ handling and address their function in Ca2+ homeostasis and contractile function. To elucidate the role of mitochondrial Ca2+ uptake in skeletal muscle (SM) function we ablated MICU1, the Ca2+ sensing regulator of mtCU in mouse. MICU1 ablation in SM resulted in impaired gatekeeping and attenuated Ca2+ uptake and resulted in less endurance exercise performance when challenged with fatigue protocols. However, in this mouse and in cell lines, upon MICU1 ablation protein levels of other components of the mtCU and Ca2+ chelation in the matrix were also altered. To overcome the compensatory effects we decided to apply two strategies. First, we induce acute MICU1 knock out using a tamoxifen-inducible CRE-system. Secondly, we also targeted the mitochondrial phosphate (Pi) carrier (PiC) in a similar manner. Pi transport into mitochondria is crucial for both mitochondrial ATP synthesis and Ca2+ chelation in the matrix. To quantitatively and simultaneously measure Ca2+ uptake and matrix buffering, mitochondria are isolated from SM for fluorometry are loaded with furaFF/AM and are incubated in the presence of rhodFF. SM mitochondria acutely depleted for PiC show a decreased Ca2+ chelation capacity in the matrix and unexpectedly, take up more Ca2+ than the control. When challenged in an incremental exercise test, PiC-ablated mice could only run for 8.5±1.7 min, whereas control mice ran for the full 20 min, showing impaired performance. The effects of acute MICU1 depletion in SM on mtCU composition, mitochondrial Ca2+ handling and SM function are currently evaluated. Thus, our results provide genetic evidence for the contribution of mitochondrial Ca2+ homeostasis to SM function and are expected to determine the function and mechanism dependent on specifically on MICU1 and PiC.