Mitoregulin Microprotein Influences Mitochondrial Membrane Integrity And Myocardial Ischemia-Reperfusion Injury

Microproteins have emerged as a class of proteins often found to be encoded by misannotated long noncoding RNAs (lncRNAs). We and others discovered that a ubiquitously-expressed “lncRNA” ( LINC00116 ) encodes for a highly-conserved mitochondrial transmembrane microprotein that regulates beta-oxidation; we named this protein Mitoregulin (Mtln). In cells and mice, we found that Mtln increases mitochondrial respiratory efficiencies and Ca 2+ retention capacities, while reducing ROS. To date, the molecular function of Mtln remains unclear, with instances of conflicting data across papers. Recently, we found that mitochondria from Mtln-knockout (KO) mice are susceptible to membrane freeze-damage, leading to matrix protein leak during mitochondrial isolation. This appears not to be a distal downstream consequence of chronic Mtln loss, as we have confirmed these findings in cells after acute Mtln knockdown and in a mouse model of inducible cardiac-specific KO of Mtln (AAV-based CRISPR in aMHC-Cas9 mice; ~90% reduction in Mtln). Furthermore, acute re-expression of Mtln using AAV in Mtln-KO mice rescues the membrane deficit. Transmission electron microscopy revealed that acute Mtln knockdown in cells disrupts mitochondrial cristae integrity / density. Given that we previously found that Mtln strongly binds cardiolipin, we assessed the effects of synthetic Mtln peptide on membrane dynamics in mitochondrial-simulated lipid monolayers and found that Mtln modulates lipid packing and mixing and increases monolayer elasticity. Whether these potential direct effects mediate Mltn’s broad positive impact on mitochondria remain unknown. To further explore a role for Mtln in cardiac disease, we subjected Mtln-KO and AAV-Mtln overexpression mice to myocardial ischemia-reperfusion (IR), given Mtln’s influence on mitochondrial Ca 2+ buffering and ROS. Mtln-KO mice showed significantly worse outcomes (increased infarct size, cardiac dilation, and decreased ejection fraction) at 72 h post-IR, whereas AAV-Mtln mice were protected (decreased infarct size and increased ejection fraction), each compared to control littermates. Overall, this work points to new molecular functions for Mtln and highlights its protective (therapeutic) role in IR injury.