Introduction: BKCa channels are large conductance Ca++-activated K+ channels, which reside in the inner mitochondrial membrane in cardiomyocytes. We have demonstrated that the BKCa channel activator rottlerin (a.k.a mallotoxin) is cardioprotective during cardioplegic arrest in isolated rodent hearts and in vitro models. Here we used a clinically relevant in vivo swine model of cardioplegic arrest and cardiopulmonary bypass (CP/CPB) with and without rottlerin supplemented CP solution. Mitochondrial signaling alterations were identified by TMT-MS analysis of phosphorylation enriched mitochondrial protein fractions. Methods: Yorkshire swine (50 Kg) of both sexes were subjected to hypothermic CP/CPB (1hour) followed by 1 hour reperfusion. Groups included sham (n = 5), CP/CPB alone (n = 8) and CP/CPB with rottlerin supplemented cardioplegia (1uM) (CP/CPB+R) (n = 7). Mitochondria-enriched fractions were isolated from the left ventricular free wall and subjected to Tandem Mass Tag (TMT) labeling coupled mass spectrometry analysis. Results: CP/CPB pigs had reduced myocardial function indicative of myocardial stunning as shown by decreased LVDevP, and ±dP/dt vs sham (P<.05). BKCa activation significantly restored left ventricular function compared to CP/CPB alone (LVDevP: 74.0 ± 2.8 vs . 52.5±5.0 mmHg, P < .01, +dP/dt: 1482±116 vs . 923±126 mmHg/s, P < .01, and - dP/dt: 1120±116 vs . 629±82 mmHg/s, P < .01). TMT-MS analysis identified 3363 proteins. 410 unique as well as numerous novel phosphorylation sites were significantly regulated in CP/CPB vs sham. Rottlerin treatment generally normalized phosphorylation changes with 175 and 63 different vs CP/CPB and sham, respectively. Enrichment analysis demonstrated significant changes in pathways associated with adrenergic and PKA signaling, calcium handling, mitochondrial dynamics, cristae organization, and oxidative phosphorylation. Conclusions: BKCa-activator supplemented cardioplegia significantly improves myocardial functional recovery following CP/CPB in a clinically relevant large animal model. Furthermore, mitochondrial BKCa-channel activation during cardioplegic arrest normalizes numerous signaling alterations associated with myocardial stunning and cardiac surgery.

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Background: Inactivation of small-conductance calcium-activated potassium (SK) channels contributes to coronary microvascular dysfunction in patients after cardioplegic arrest, cardiopulmonary bypass (CP/CPB) and cardiac surgery. SK-channel activation is cardioprotective in rodent isolated heart model of myocardial infarction and ischemia/reperfusion. In the current study, we investigated the impact of SK activation on myocardial protection in a clinically relevant large animal model of pigs subjected to cardioplegic ischemia/reperfusion and CPB. Methods and Results: Ten Yorkshire pigs were subjected to 1 hour of CP/CPB followed by 1 hour of reperfusion. Pigs received either cold blood hyperkalemic CP alone (CP control) or CP containing the selective SK channel activator NS309 (10 -6 M, n = 5/group). Left ventricular (LV) function was assessed by using pressure-volume loop analysis. Coronary arteriolar relaxation response (in-vitro) to the endothelium-dependent vasodilators adenosine diphosphate (ADP, 10 -9 -10 -4 M) and substance P (10 -12 -10 -7 M) and independent vasodilator sodium nitroprusside (SNP) was evaluated with video-microscopy. Inclusion of NS309 in cold blood CP tends to improve the recovery of LV function compared with the CP controls. CP/CPB and reperfusion significantly reduced endothelium-dependent relaxation response to ADP, 10 -5 M) and substance P (10 -8 M) as compared to control (p<0.05). In contrast, CP-containing NS309 significant improved the recovery of endothelium-dependent relaxation response to ADP and substance P as compared with the control group (p<0.05, respectively). Conclusion: In pig model of CP/CPB, inclusion of the selective SK channel activator in the cold blood cardioplegia improves recovery of coronary arteriolar endothelial function and microvascular relaxation, which may contribute to increased myocardial perfusion and improved LV function.

Introduction: Activation of mitochondrial large conductance Ca++-activated K+ channels (BKCa) is cardioprotective in cell and rodent isolated heart models of cardioplegic arrest and reperfusion. We previously demonstrated cardioprotection is associated with reduced mitochondrial ROS generation and reorganization of individual electron transport chain complexes into physically associated respiratory supercomplexes (SC) made of ETC complex I/III/IV. In this study, we examined the efficacy of BKCa activation during cardioplegic arrest in a clinically relevant large animal study using pigs subjected to CP/CPB and reperfusion. Methods: 50 kg Yorkshire pigs of either sex were subjected to CP/CPB (hyperkalemic cold blood CP, 1 hr) with or without the BKCa activator mallotoxin (MTX (aka rottlerin)) supplied in the CP solution, followed by reperfusion (1hr). Pigs were instrumented with a PV catheter and IVC occlusions performed at baseline and 30 and 60 mins after weening from bypass. Following surgery, mitochondria were isolated and subjected to Blue-Native PAGE and immunoblotting of ETC complexes to visualize large (~100-1500 kD) mitochondrial SC. Results: Baseline hemodynamics were similar between groups. CP/CPB resulted in significant myocardial stunning in pigs as assessed by LVDP (pre 64.8 mmHg vs post 42.0, P<.05 ), +/-dP/dt (pre 971.4 and -911.8 vs post:588.0 and -541.0, P<.05 ), and end-systolic elastance (Ees: pre 1.28 vs post .63, P=0.1 ) at 60 min reperfusion. However, pigs arrested with CP containing MTX (500 nM) exhibited greatly reduced myocardial stunning with significant improvements in LVDP (pre:65.7 post:67.8 mmHg, P<.01 vs post CP/CPB group) +/- dP/dt (pre:1026.0 and -911.8 vs post:1441.7 and -984.8 mmHg/s, P=.02 and .04 vs CP/CPB) and Ees (pre 1.1, post 1.35, P=.02 vs CP/CPB). LV mitochondria isolated from both groups had two major I/III/IV mitochondrial SC’s of ~1300 (SC1) and ~1000 kD (SC2) as assessed by BN-PAGE and immunoblot. The CP/CPB + MTX group had significant increases in both SC1 (6.0 % vs 18.0%, P=.01) and SC2 (19.7% vs 52.3%, P<.01) expressed as percent of total assembled complex IV. Conclusion: Addition of a BKCa activator to CP solutions ameliorates myocardial stunning in large animals subjected to CP/CPB and reperfusion.