Creatine kinase overexpression improves ATP kinetics and contractile function in postischemic myocardium

Abstract

Reduced myofibrillar ATP availability during prolonged myocardial ischemia may limit post-ischemic mechanical function. Because creatine kinase (CK) is the prime energy reserve reaction of the heart and because it has been difficult to augment ATP synthesis during and after ischemia, we used mice that overexpress the myofibrillar isoform of creatine kinase (CKM) in cardiac-specific, conditional fashion to test the hypothesis that CKM overexpression increases ATP delivery in ischemic-reperfused hearts and improves functional recovery. Isolated, retrograde-perfused hearts from control and CKM mice were subjected to 25 min of global, no-flow ischemia and 40 min of reperfusion while cardiac function [rate pressure product (RPP)] was monitored. A combination of (31)P-nuclear magnetic resonance experiments at 11.7T and biochemical assays was used to measure the myocardial rate of ATP synthesis via CK (CK flux) and intracellular pH (pH(i)). Baseline CK flux was severalfold higher in CKM hearts (8.1 ± 1.0 vs. 32.9 ± 3.8, mM/s, control vs. CKM; P < 0.001) with no differences in phosphocreatine concentration [PCr] and RPP. End-ischemic pH(i) was higher in CKM hearts than in control hearts (6.04 ± 0.12 vs. 6.37 ± 0.04, control vs. CKM; P < 0.05) with no differences in [PCr] and [ATP] between the two groups. Post-ischemic PCr (66.2 ± 1.3 vs. 99.1 ± 8.0, %preischemic levels; P < 0.01), CK flux (3.2 ± 0.4 vs. 14.0 ± 1.2 mM/s; P < 0.001) and functional recovery (13.7 ± 3.4 vs. 64.9 ± 13.2%preischemic RPP; P < 0.01) were significantly higher and lactate dehydrogenase release was lower in CKM than in control hearts. Thus augmenting cardiac CKM expression attenuates ischemic acidosis, reduces injury, and improves not only high-energy phosphate content and the rate of CK ATP synthesis in postischemic myocardium but also recovery of contractile function.

Fig. 1.

Myofibrillar isoform of creatine kinase (CKM; A) and mitochondrial isoform of creatine kinase (CK-mito; B) protein expression as well as total CK activity (C) in control baseline, control end-ischemia, control reflow, CKM baseline, CKM end-ischemia, and CKM reflow hearts. Results expressed as means ± SE for n = 5 to 6 in each group. *P < 0.01 vs. control baseline.

Fig. 2.

Effect of ischemia and reperfusion on percent changes in ATP (A) and phosphocreatine (PCr; B) as well as changes in intracellular pH (pH_i; C) in control (gray) and CKM (black) mouse hearts. Results are means ± SE for n = 5 in each group. *P < 0.05 vs. control.

Fig. 3.

Representative ³¹P-magnetization transfer (MT) spectra from control (A, baseline; B, reflow) and CKM (C, baseline; D, reflow) hearts. The spectra were acquired with saturating irradiation (thick arrows) in the control position (left spectrum in each panel) and γ-ATP position (right spectrum in each panel). The decrease in the height of PCr peak between control and γ-ATP saturation (slope of the dotted lines) is directly related to the rate of ATP synthesis through the CK reaction. A greater decline in PCr signal with γ-ATP saturation observed in CKM vs. control hearts (both at baseline and during reperfusion) indicates higher ATP flux through CK in CKM hearts.

Fig. 4.

Effect of ischemia and reperfusion on levels of PCr and ATP (A), the CK pseudo-first-order rate constant (k_PCr→ATP), and rate of ATP synthesis via creatine kinase (CK flux; B) as well as P_i→ATP pseudo-first-order rate constant (k_Pi→ATP) and rate of ATP synthesis from P_i (C) in control (white) and CKM (gray) mouse hearts. Results are means ± SE for n = 5 in each group. *P < 0.01 vs. control (baseline); †P < 0.05 vs. CKM (baseline); #P < 0.01 vs. control (reflow).