Abstract 20427: Activation of Large Conductance Ca++ Activated K+ Channels (BK) During Ischemic Cardioplegic Arrest (CP) Promotes Formation of Mitochondrial Supercomplexes and Enhanced Mitochondrial Respiration
Introduction: Activation of mitochondrial BK channels is cardioprotective in response to ischemic insults. Activation of BK channels enhances cardiac function and reduces ROS generation in cell and rodent models of ischemic cardioplegic arrest. Recent advances demonstrate that components of the mitochondrial electron transport chain can organize into respiratory supercomplexes yielding more efficient respiration and reduced ROS generation.
Hypothesis: BK channel activation during ischemic CP may enhance cardioprotection through mitochondrial supercomplex formation and respiration.
Methods: H9c2 cells were subjected to in vitro CP (3 hrs) under hypoxic, hypothermic (20C), conditions with or without the BK channel activators rottlerin (500 nM) or NS11021 (100 nM), followed by 30 minute reoxygenation. Cell respiration was measured using a Seahorse XFe analyzer and mitochondria were isolated to assess supercomplex formation using Blue-Native electrophoresis (BN-PAGE). Complexes were identified via mass spectrometry.
Results: Treatment with rottlerin caused a robust increase (>2 fold) in large mitochondrial supercomplexes (~1.2 mD) in both control and CP cells (n=3, p<.05). MS analysis of the large complex bands verified constituents of complex I,III, and IV. Following CP, basal respiration (oxygen consumption rate) was reduced compared to control cells (41.9 +/-4.1 % control, n=5)). BK activators (rott and NS11021) enhanced basal respiration in both control and CP/R groups (con+ rott:130.6 +/-8.7 % control , (p<.03 vs con), CP +rott: 63.2 +/- 2.3 % control, (p<.01 vs CP alone)) . BK activation also enhanced peak respiration (FCCP-induced) following CP/R. Rottlerin modulated effects on respiration were largely blunted by the BK blocker paxilline. In addition, preliminary BN-PAGE experiments in mouse isolated hearts following CP/R demonstrated that BK activation also promoted robust supercomplex formation in situ.
Conclusions: Activation of BK channels during ischemic insults promotes enhanced mitochondrial respiration which associates with enhanced mitochondrial supercomplex formation. To our knowledge this is the first report of therapeutically enhanced formation of respiratory supercomplexes following ischemic insults.
Author Disclosures: B. Cordeiro: None. R.T. Clements: None.
This research has received full or partial funding support from the American Heart Association.
- © 2014 by American Heart Association, Inc.