Tmbim5 and Slc8b1 cooperate in tissue-specific mitochondrial calcium regulation in zebrafish

Mitochondrial calcium homeostasis involves coordinated uptake via the mitochondrial calcium uniporter (MCU) and efflux through sodium-dependent NCLX (encoded by SLC8B1). Mild Mcu knockout phenotypes suggest additional transport mechanisms. We investigated TMBIM5, a proposed bidirectional mitochondrial calcium/proton transporter, by generating zebrafish lacking tmbim5, slc8b1, plus tmbim5/mcu and tmbim5/slc8b1 double knockouts. Tmbim5-deficient fish exhibited growth impairment, muscle atrophy, and increased brain cell death. tmbim5/mcu double knockouts showed no additive effects, arguing against Tmbim5 functioning as an independent calcium uptake pathway. However, tmbim5/slc8b1 double knockouts showed disrupted mitochondrial calcium handling with reduced uptake and efflux. Remarkably, brain phenotypes were rescued while muscle dysfunction was exacerbated in double mutants, corresponding to restored mitochondrial membrane potential in Tmbim5-deficient brain tissue and exacerbated decreased calcium levels in double knockout muscle. We also found broad downregulation of mitochondrial calcium transport proteins and decreased mitochondrial DNA content in double knockout brain tissue, indicating reduced mitochondrial mass and a potential beneficial glycolytic shift in energy metabolism. These findings demonstrate that TMBIM5 functions as a calcium efflux pathway cooperating with NCLX in a tissue-specific manner.