Butyrate Feeding Reverses CypD-Related Mitoflash Phenotypes in Mouse Myofibers.

Ang Li,Jianjie Ma,Xuejun Li,Jingsong Zhou,Jianxun Yi

doi:10.3390/ijms22147412

Abstract

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.

Highlights

Mitoflash events are spontaneous fluorescent transients of the mitochondrial targeted, circularly permuted yellow fluorescent protein [1]
The jRCaMP1b intensity notably increased in response to external Ca2+ stimulation (0 mM to 2 mM) for all three fusion constructs (Figure 2 and Videos S1–S3), confirming that the fusion setup did not undermine the functionality of jRCaMP1b
Butyrate is a short-chain fatty acid derived from microbial fermentation of dietary fibers in the colon [63]

Summary

Introduction

Mitoflash events are spontaneous fluorescent transients of the mitochondrial targeted, circularly permuted yellow fluorescent protein (mt-cpYFP) [1]. 1.9-fold of normoxia levels 5 min after reoxygenation, implying an association between these events and a reperfusion induced burst of reactive oxygen species (ROS) [1]. Mitoflash events have been associated with ROS production, mitochondrial respiration acceleration under tetanic electrical field stimulation, and heat-induced hypermetabolism [2,3]. New evidence emerged to link mitoflash events with pathological elevations of mitochondrial ROS levels in myofibers after denervation or during the progression of neuromuscular degeneration of amyotrophic lateral sclerosis (ALS) [4,5]. The connections between mitoflash events and ROS production have been reported in multiple studies, it is debated whether these events are authentic indicators of superoxide burst or merely representing mitochondrial matrix alkalization [1,6,7,8]. There may exist a common ground that can be substantiated by both sides, as both matrix

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Discussion

Conclusion