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) and/or TMEM65. 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. slc8b1 knockouts had no major phenotype but showed attenuated, although not abolished sodium-dependent mitochondrial calcium efflux. tmbim5/slc8b1 double knockouts showed altered 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 brain tissue and decreased calcium levels in muscle. These findings suggest that TMBIM5 functions as an auxiliary calcium efflux pathway cooperating with NCLX in a tissue-specific manner.