Localization of myoglobin in mitochondria: implication in regulation of mitochondrial respiration in rat skeletal muscle

Abstract

Mitochondria play a principal role in metabolism, and mitochondrial respiration is an important process for producing adenosine triphosphate. Recently, we showed the possibility that the muscle‐specific protein myoglobin (Mb) interacts with mitochondrial complex IV to augment the respiration capacity in skeletal muscles. However, the precise mechanism for the Mb‐mediated upregulation remains under debate. The aim of this study was to ascertain whether Mb is truly integrated into the mitochondria of skeletal muscle and to investigate the submitochondrial localization. Isolated mitochondria from rat gastrocnemius muscle were subjected to different proteinase K (PK) concentrations to digest proteins interacting with the outer membrane. Western blotting analysis revealed that the PK digested translocase of outer mitochondrial membrane 20 (Tom20), and the immunoreactivity of Tom20 decreased with the amount of PK used. However, the immunoreactivity of Mb with PK treatment was better preserved, indicating that Mb is integrated into the mitochondria of skeletal muscle. The mitochondrial protease protection assay experiments suggested that Mb localizes within the mitochondria in the inner membrane from the intermembrane space side. These results strongly suggest that Mb inside muscle mitochondria could be implicated in the regulation of mitochondrial respiration via complex IV.

Myoglobin‐mediated lipid shuttling increases adrenergic activation of brown and white adipocyte metabolism and is as a marker of thermogenic adipocytes in humans

“…Previous studies have demonstrated mitochondrial localization of MB in skeletal muscle 18,19 and also mesenchymal stem cells 33 . We first analyzed whether MB is also localized in isolated mitochondria from imBA_hMB and controls.…”

“…Mitochondria from differentiated imBA_Ctrl adipocytes and imBA_hMB clones were isolated using a mitochondria isolation kit (#89874, Thermo Fisher). A mitochondrial protease protection assay using protease K to investigate mitochondrial integration of MB in differentiated imBA adipocytes was done as previously described 18 with minor modifications. Mitochondria from differentiated imBA_Ctrl and imBA_hMB adipocytes were isolated in solution A, followed by two rounds of ultrasound sonification for 15 s each.…”

“…MB further regulates mitochondrial activity and capacity via modulation of cellular nitric oxide (NO) levels 15 or as a scavenger of reactive oxygen species (ROS) 16 . In myocardial cells, MB is localized in mitochondria, 17,18 where it may directly interact with respiratory chain complex IV 19 . Recent studies also demonstrated fatty acid (FA) and acyl carnitine binding especially to oxygenated MB suggesting a potential role in lipid metabolism 20–22 .…”

TOM complex‐independent transport pathway of myoglobin into mitochondria in C2C12 myotubes

“…Mb plays a principal role in O 2 storage, buffering intracellular O 2 concentrations and facilitating O 2 diffusion. Although Mb is generally recognized as a cytoplasmic protein (Kanatous & Mammen, 2010 ; Ordway & Garry, 2004 ; Postnikova & Shekhovtsova, 2018 ), we previously demonstrated that Mb is also localized within mitochondria in rat skeletal muscle by using a proteinase K (PK) protection assay, a technique to digest proteins interacting with the outer mitochondrial membrane (OMM; Koma et al, 2021 ).…”

“…Non‐mitochondrial proteins are also translocated into mitochondria via the TOM complex pathway (Budas et al, 2010 ; Fu et al, 2012 ; Rodriguez‐Sinovas et al, 2006 ). Given that Mb inside mitochondria is localized in the IMS (Koma et al, 2021 ), it is a possible that Mb uses the TOM complex to transport into mitochondria. The purpose of the present study was to examine whether Mb import into mitochondria is mediated by the TOM complex.…”