Cardiac surgery or transplant using cardioplegic arrest and cardiopulmonary bypass (CP/CPB) subjects the heart to mild reversible ischemic injury that results in pronounced myocardial stunning. Mitochondrial large conductance calcium-activated K⁺ channels (BK_Ca) in cardiomyocytes are potently cardioprotective and beneficially modulate respiration and ROS post-I/R injury. However, how BK_Ca-dependent K⁺ flux regulates ROS and respiration is unclear. We evaluated the BK_Ca activator rottlerin in a preclinical large animal pig CP/CPB model of myocardial stunning and evaluated BK_Ca-dependent cardioprotective mechanisms in vitro and in vivo. BK_Ca activation with the small molecule rottlerin significantly improved LV cardiac functional recovery and contractility at 1 h reperfusion. TEM showed larger mitochondria in the rottlerin group consistent with K⁺ influx. We also found that pigs with rottlerin treatment had increased electron transport chain supercomplexes (mSCs), which are thought to improve respiratory efficiency and reduce ROS. Consistent with increased mSC, rottlerin reduced ROS-dependent protein modification in CP/CPB pigs. In complementary studies, rottlerin and/or adenoviral overexpression of a mutant mito-BK_Ca increased respiration, altered mitochondrial morphology, increased mSC assembly, and significantly reduced ROS following in vitro CP/reoxygenation, similar to CP/CPB pigs. In summary, mito-BK_Ca activation increases mSCs and respiration while reducing ROS, suggesting that mSC-dependent improved respiratory efficiency promotes robust cardioprotection.

Mitochondrial dysfunction, including impaired respiration and increased reactive oxygen species (ROS), is an early feature of Alzheimer’s disease (AD) models. Electron transport chain supercomplexes (mSCs) regulate respiratory efficiency and ROS generation, yet genetic determinants of mSC organization in AD models remain underappreciated. Cox7a2l is known to promote CIII₂/CIV association and CIV incorporation into mSCs. We examined the commonly used mixed-background B6;129S 3xTg-AD mice and B6129SF2/J controls aged 12–15 months, as well as specific rat AD models for changes in mSC organization using blue and clear native-PAGE and DIA-MS. BN and CN-PAGE revealed a marked shift toward larger mSC assemblies in 3xTg-AD mice and a distinct ~650 kDa assembly of complex III absent from controls. Gene sequencing showed that 3xTg-AD mice retained the full-length Cox7a2l variant similar to 129S strains, whereas controls predominantly carried a shortened C57BL/6-associated variant. DIA-MS identified greatly increased Cox7a2l in differential mSC bands. Similar changes were found in 3xTg-AD heart tissue. In contrast, AD rat models and their controls, all expressing full-length Cox7a2l, did not exhibit comparable mSC differences. These findings suggest Cox7a2l genotype as a determinant of mSC organization and highlight the need to account for genetic background when interpreting mSC-dependent mitochondrial phenotypes in mixed-background 3xTg-AD studies. This distinction is essential for accurately separating strain-dependent effects from AD-associated mitochondrial changes.