Ca2+‐induced Complex I Inactivity: A Model for Early Mitochondrial Dysfunction Following Volumetric Muscle Loss Injury

Abstract

Calcium (Ca2+) is a secondary messenger, responsible for the regulation of a plethora of metabolic pathways that must be tightly regulated to avoid cytotoxicity 1. During early stage volumetric muscle loss (VML) injuries, damaged muscle fibers are open to the extracellular environment until the myofiber can be sealed, a process that may take hours 2– 3. This allows for free Ca2+ exchange between intra‐ and extracellular environments to concentrations beyond those appropriate for cell homeostasis. The purpose of this study was to model the changes in mitochondrial metabolism of early VML injuries as a response to a drastic increase of intracellular Ca2+.C57BL6 mice underwent unilateral surgery to create a VML defect in the gastrocnemius muscle, as approved by the University of Georgia’s Institutional Animal Care and Use Committee. Animals were sacrificed via CO2 inhalation, 18 hrs. following surgery. Total [Ca2+] per kg of tissue ([Ca2+]TWM) was measured using a BAPTA complexation assay. Gastrocnemius muscles from naïve mice were dissected into individual fiber bundles, permeabilized, and washed in either normal isotonic or Ca2+‐supplemented medium. Mitochondrial metabolic function and mitochondrial membrane potential were measured through oxygen consumption and Safranin‐O fluorescence rates, respectively, in a High‐Resolution Fluoro‐respirometer.[Ca2+]TWM was elevated in VML‐injured tissue, as expected (n=3, p< 0.05). Acute Ca2+ exposure negatively affected Complex I (C‐I) activity of the electron transport chain. Specifically, C‐I activity was targeted by the addition of glutamate and malate (Glu‐Mal) and confirmed by the sequential addition of C‐I inhibitor Rotenone (Rot). In normal fibers, respiration increased after Glu‐Mal addition and subsequently decreased significantly following Rot, while Ca2+‐incubated fibers exhibited no change in oxygen consumption rate. Succinate (Succ) was used to rescue respiratory function by bypassing C‐I. Succ addition significantly increased oxygen consumption in both normal and Ca2+‐incubated fibers (n=3, p<0.01), confirming a C‐I dysfunction in Ca2+‐treated fibers. Safranin fluorescence is sensitive to changes in mitochondrial membrane potential, decreasing with complex activity and increasing upon inhibition. In normal fibers, Safranin signal decreased with Glu‐Mal and Succ additions and increased with Rot, as expected. However, Ca2+‐treated fibers were only sensitive to Succ treatment, further supporting C‐I dysfunction (n=3, p< 0.01).Herein, we show that an acute increase in intracellular Ca2+ negatively affects the process of oxidative phosphorylation within the mitochondria by disturbing the electron potential and inactivating Complex I. This alteration in membrane potential and decrease in complex activity may be the first step in a systematic shut down of mitochondrial metabolic function that leaves the muscle remaining after injury beyond the hope of effective repair.Support or Funding InformationFunded through the Department of Defense Neuromusculoskeletal Injuries Rehabilitation Research Award W81XWH‐18‐1‐0710 to SMG (UMinn) and JAC (UGA)A‐ Quantification of internal [Ca2+] of VML‐injured and control gastrocnemius muscles using BAPTA absorbance (n=3, p < 0.05). B‐ Oxygen consumption rates of control and Ca2+‐treated muscle fibers after Complex I activation, inhibition, and bypass, respectively (n=3 in duplicate, p < 0.05). C‐ Safranin‐O signal fluctuation in control and Ca2+‐treated muscle fibers after Complex I activation, inhibition, and bypass, respectively (n=3, p< 0.05).Figure 1