N-Methyl-D-Aspartate Receptor-Driven Calcium Influx Potentiates the Adverse Effects of Myocardial Ischemia-Reperfusion Injury Ex Vivo

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Abstract

Background:

Despite the adverse effects of N-methyl-D-aspartate receptor (NMDAR) activity in cardiomyocytes, no study has yet examined the effects of NMDAR activity under ex vivo ischemic-reperfusion (I/R) conditions. Therefore, our aim was to comprehensively evaluate the effects of NMDAR activity through an ex vivo myocardial I/R rat model.

Methods:

Isolated rat hearts were randomly segregated into 6 groups (n = 20 in each group): (1) an untreated control group; (2) a NMDA-treated control group; (3) an untreated I/R group; (4) an I/R+NMDA group treated with NMDA; (5) an I/R+NMDA+MK-801 group treated with NMDA and the NMDAR inhibitor MK-801; and (6) an I/R+NMDA+[Ca2+]-free group treated with NMDA and [Ca2+]-free buffer. The 4 I/R groups underwent 30 minutes of ischemia followed by 50 minutes of reperfusion. Left ventricular pressure signals were analyzed to assess cardiac performance. Myocardial intracellular calcium levels ([Ca2+]i) were assessed in isolated ventricular cardiomyocytes. Creatine kinase, creatine kinase isoenzyme MB, lactate dehydrogenase, cardiac troponin I, and cardiac troponin T were assayed from coronary effluents. TTC and TUNEL staining were used to measure generalized myocardial necrosis and apoptosis levels, respectively. Western blotting was applied to assess the phosphorylation of PKC-δ, PKC-ε, Akt, and extracellular signal-regulated kinase.

Results:

Enhanced NMDAR activity under control conditions had no significant effects on the foregoing variables. In contrast, enhanced NMDAR activity under I/R conditions produced significant increases in [Ca2+]i levels (∼1.2% increase), significant losses in left ventricular function (∼5.4% decrease), significant multi-fold increases in creatine kinase, creatine kinase isoenzyme MB, lactate dehydrogenase, cardiac troponin I, and cardiac troponin T, significant increases in generalized myocardial necrosis (∼36% increase) and apoptosis (∼150% increase), and significant multi-fold increases in PKC-δ, PKC-ε, Akt, and extracellular signal-regulated kinase phosphorylation (all P < 0.05). These adverse effects were rescued by the NMDAR inhibitor MK-801 or [Ca2+]-free buffer (all P < 0.05).

Conclusions:

NMDAR-driven calcium influx potentiates the adverse effects of myocardial I/R injury ex vivo.

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