Acute protein kinase C beta inhibition preserves coronary endothelial function after cardioplegic hypoxia/reoxygenation

Abstract

Objective: Protein kinase C (PKC) influences myocardial contractility and susceptibility to long-term cardiac dysfunction after ischemia-reperfusion injury. In diabetes, PKC inhibition has a protective effect in terms of microvascular dysfunction. SK-channel dysfunction also influences endothelial dysfunction in cardioplegic hypoxia-reoxygenation (CP-H/R). Here, we examine whether acute inhibition of PKC beta protects against CP-H/R-induced coronary endothelial and SK channel dysfunction.

Methods: Isolated mouse coronary arterioles, half pretreated with selective PKC inhibitor ruboxistaurin (RBX), were subjected to hyperkalemic, cardioplegic hypoxia (1 hour), and reoxygenation (1 hour) with Krebs buffer. Sham control vessels were continuously perfused with oxygenated Krebs buffer without CP-H/R. After 1 hour of reoxygenation, responses to the endothelium-dependent vasodilator adenosine-diphosphate (ADP) and the SK-channel activator NS309 were examined. Endothelial SK-specific potassium currents from mouse heart endothelial cells were examined using whole-cell path clamp configurations in response to NS309 and SK channel blockers apamin and TRAM34.

Results: CP-H/R significantly decreased coronary relaxation responses to ADP (P = .006) and NS309 (P = .0001) compared with the sham control group. Treatment with selective PKC beta inhibitor RBX significantly increased recovery of coronary relaxation responses to ADP (P = .031) and NS309 (P = .004) after CP-H/R. Treatment with RBX significantly increased NS309-mediated potassium currents following CP-H/R (P = .0415). Apamin and TRAM34 sensitive currents were significantly greater in CP-H/R + RBX versus CP-H/R mouse heart endothelial cells (P = .0027).

Conclusions: Acute inhibition of PKC beta significantly protected mouse coronary endothelial function after CP-H/R injury. This suggests that acute PKC beta inhibition may be a novel approach for preventing microvascular dysfunction during CP-H/R.

Keywords: cardioplegia; hypoxia–reoxygenation; protein kinase C; ruboxistaurin; vascular reactivity.

Graphical abstract

Increased PKC beta activity contributes to postcardiac surgery endothelial dysfunction.

Figure 1

Experimental design for microvascular reactivity and endothelial experiment protocols. A, Mouse coronary arterial microvessels (70-100 μm in internal diameter) from the left anterior descending artery dependent subepicardial region of the left ventricle were harvested and assigned to 3 groups: normoxia (sham controls), cardioplegic hypoxia–reoxygenation (1 hour), and cardioplegic hypoxia–reoxygenation (1 hour) with ruboxistaurin treatment. Following normoxia, cardioplegic hypoxia–reoxygenation, and/or ruboxistaurin treatment, microvascular responses to ADP, NS309, and SNP were measured. B, Mouse heart endothelial cells (MHECs) were harvested, and divided into the same 3 groups as were used for the microvascular reactivity study, with the key difference being 3 hours of cardioplegic hypoxia–reoxygenation. Following treatments, patch clamp methods were used to record whole cell potassium currents. ADP, Adenosine diphosphate; SNP, sodium nitroprusside.

Figure 2

Coronary endothelial responses of mouse microvessels to endothelium-dependent vasodilator NS309. A, Dose-dependent vasodilation of sham control mouse microvessels and vessels undergoing CP-H/R with or without RBX treatment in response to the SK channel activator NS309 (10^–9-10^–5 M). B, Bar graph shows vasodilation response to NS309 10^–5 M. CP-H/R, Microvessels undergoing hypoxia/reoxygenation; RBX + CP-H/R, microvessels pretreated with RBX before hypoxia and reoxygenation; RBX, ruboxistaurin.

Figure 3

Coronary endothelial responses of mouse microvessels to endothelium-dependent vasodilator ADP. A, Sham control (no CP-H/R) and CP-H/R vessels with or without RBX treatment in response to the endothelium-dependent vasodilator ADP (10^–9-10^–4 M). B, Bar graph shows vasodilatory response to ADP 10^–5 M. ADP, Adenosine diphosphate; CP-H/R, microvessels undergoing hypoxia/re-oxygenation; RBX + CP-H/R, microvessels pretreated with RBX before hypoxia and reoxygenation; RBX, ruboxistaurin.

Figure 4

Coronary endothelial responses of mouse microvessels to endothelium-independent vasodilator SNP. A, Sham control (no CP-H/R) and CP-H/R vessels with or without RBX treatment in response to the endothelium-independent vasodilator SNP (10^–9-10^–4 M). B, Bar graph shows vasodilatory response to SNP 10^–5 M. SNP, Sodium nitroprusside; CP-H/R, Microvessels undergoing hypoxia/reoxygenation; RBX + CP-H/R, microvessels pretreated with RBX before hypoxia and reoxygenation; RBX, ruboxistaurin.

Figure 5

RBX significantly increases SK-channel currents of mice heart endothelial cells (MHECs) in CP-H/R model. A, Representative traces of the whole cell currents of MHECs at holding potential of –50 mV and test potentials from –100 to +100 mV in 20-mV increments. B, Whole-cell I-V relationships sensitive to NS309 (1 μM) in MHECs of sham control and CP-H/R with or without RBX (50 nM) treatment. C, Box plots show NS309-sensitive component of potassium current at +100 mV in sham control MHECs and CP-H/R MHECs treated with or without RBX. ∗P = .0490, sham control (n = 6) versus CP-H/R (n = 7); P = .0415, CP-H/R (n = 7) versus CP-H/R + RBX (n = 6). D, whole-cell I-V relationships sensitive to TRAM34 (10 μM) + apamin (100 nM) in sham control MHECs and CP-H/R MHECs with or without RBX treatment. E, Box plots shows TRAM34 + apamin-sensitive component of potassium current at +100 mV in control MHECs and CP-H/R MHECs treated with or without RBX (n = 5/group). ∗∗P = .0032, control (n = 6) versus CP-H/R (n = 7); P = .0027, CP-H/R (n = 7) versus CP-H/R + RBX (n = 6). CP-H/R, MHECs undergoing hypoxia/reoxygenation; CP-H/R + RBX, MHECs pretreated with RBX before hypoxia and reoxygenation; RBX, ruboxistaurin.

Figure 6

Schematic of SK-channel–mediated vasodilation. A, CP-H/R increases PKC activity, which phosphorylates SK channels. This results in decreased SK-channel calcium sensitivity and reduces SK-channel opening, leading to reduced potassium currents and reduced endothelial hyperpolarization. This diminishes microvascular relaxation. B, RBX inhibits PKC, preventing PKC-mediated inhibition of SK-channel activity, thereby promoting endothelial hyperpolarization and coronary microvascular smooth muscle relaxation. SK channels are cyan on EC membranes. EC, Endothelial cell; CP-H/R, cardioplegic hypoxia–reoxygenation; PKC, protein kinase C; RBX, ruboxistaurin.

Figure 7

Depicts overview of premise, study, and key findings. RBX, Ruboxistaurin; PKC, protein kinase C; CP-H/R, cardioplegic hypoxia–reoxygenation; ADP, adenosine diphosphate.