Butyrate Feeding Reverses CypD-Related Mitoflash Phenotypes in Mouse Myofibers

Mitoflashes are spontaneous transients of the biosensor mt-cpYFP. In cardiomyocytes, mitoflashes are associated with the cyclophilin D (CypD) mediated opening of mitochondrial permeability transition pore (mPTP), while in skeletal muscle they are considered hallmarks of mitochondrial respiration burst under physiological conditions. Here, we evaluated the potential association between mitoflashes and the mPTP opening at different CypD levels and phosphorylation status by generating three CypD derived fusion constructs with a red shifted, pH stable Ca²⁺ sensor jRCaMP1b. We observed perinuclear mitochondrial Ca²⁺ efflux accompanying mitoflashes in CypD and CypDS42A (a phosphor-resistant mutation at Serine 42) overexpressed myofibers but not the control myofibers expressing the mitochondria-targeting sequence of CypD (CypDN30). Assisted by a newly developed analysis program, we identified shorter, more frequent mitoflash activities occurring over larger areas in CypD and CypDS42A overexpressed myofibers than the control CypDN30 myofibers. These observations provide an association between the elevated CypD expression and increased mitoflash activities in hindlimb muscles in an amyotrophic lateral sclerosis (ALS) mouse model previously observed. More importantly, feeding the mice with sodium butyrate reversed the CypD-associated mitoflash phenotypes and protected against ectopic upregulation of CypD, unveiling a novel molecular mechanism underlying butyrate mediated alleviation of ALS progression in the mouse model.