Subthreshold nitric oxide synthase inhibition improves synergistic effects of subthreshold MMP‐2/MLCK‐mediated cardiomyocyte protection from hypoxic injury

Abstract

Injury of myocardium during ischaemia/reperfusion (I/R) is a complex and multifactorial process involving uncontrolled protein phosphorylation, nitration/nitrosylation by increased production of nitric oxide and accelerated contractile protein degradation by matrix metalloproteinase‐2 (MMP‐2). It has been shown that simultaneous inhibition of MMP‐2 with doxycycline (Doxy) and myosin light chain kinase (MLCK) with ML‐7 at subthreshold concentrations protects the heart from contractile dysfunction triggered by I/R in a synergistic manner. In this study, we showed that additional co‐administration of nitric oxide synthase (NOS) inhibitor (1400W or L‐NAME) in subthreshold concentrations improves this synergistic protection in the model of hypoxia–reoxygenation (H‐R)‐induced contractile dysfunction of cardiomyocytes. Isolated cardiomyocytes were subjected to 3 min. of hypoxia and 20 min. of reoxygenation in the presence or absence of the inhibitor cocktails. Contractility of cardiomyocytes was expressed as myocyte peak shortening. Inhibition of MMP‐2 by Doxy (25–100 μM), MLCK by ML‐7 (0.5–5 μM) and NOS by L‐NAME (25–100 μM) or 1400W (25–100 μM) protected myocyte contractility after H‐R in a concentration‐dependent manner. Inhibition of these activities resulted in full recovery of cardiomyocyte contractility after H‐R at the level of highest single‐drug concentration. The combination of subthreshold concentrations of NOS, MMP‐2 and MLCK inhibitors fully protected cardiomyocyte contractility and MLC1 from degradation by MMP‐2. The observed protection with addition of L‐NAME or 1400W was better than previously reported combination of ML‐7 and Doxy. The results of this study suggest that addition of NOS inhibitor to the mixture of inhibitors is better strategy for protecting cardiomyocyte contractility.

Mixture of MMP-2, MLC, and NOS Inhibitors Affects NO Metabolism and Protects Heart from Cardiac I/R Injury

“…In conclusion, this study confirmed our previous results [3,16] that showed that coadministration of subthreshold doses of Doxy, L-NAME, and ML-7 serves cardioprotective. Additionally, this study provided an important insight into understanding the interaction of iNOS, eNOS, and ADMA, which is crucial for the development of the therapy beneficial for patients after myocardial infarction.…”

“…However, subsequently, it leads to ischemia/reperfusion injury (IRI), resulting in a cascade of processes detrimental to cardiac tissue. Impaired nitric oxide (NO) metabolism along with oxidative stress is known to be an important factor contributing to the pathophysiology of cardiovascular disorders, including myocardial infarction [2][3][4][5].…”

“…•− ) instead of NO [10]. ere is evidence that the L-arginine/NO/ADMA pathway plays a role in the development of CVD [3,4,8].…”

L-NAME improves doxycycline and ML-7 cardioprotection from oxidative stress

“…We have previously shown that co-administration of low concentrations of drugs that prevent both phosphorylation and degradation of MLC1 improves cardiac mechanical function after I/R (27). Additionally, in a model of hypoxia-reoxygenation with use of isolated cardiomiocytes we showed that the combination of a subthreshold concentration of a NOS inhibitor with subthreshold concentrations of MLCK and MMP-2 inhibitors resulted in full recovery of cardiomyocyte contractility (28). Thus, we propose that an alternative to monotherapy at full therapeutic dose (often limited by cytotoxicity) is the use of combination therapy at lower therapeutic concentrations without loss of protective effects with the added advantage of limiting adverse physiological side effects.…”

“…It is well accepted, that one of the main factors contributing to the pathogenesis of I/R injury is an increased activity of matrix metalloproteinases (MMPs) (1,2) and degradation of contractile proteins (3-6). Recently we showed that the co-administration of a combination of low, subthreshold concentrations of MMP-2 and MLCK inhibitors is as effective and protective against I/R as the same inhibitors given alone at full protective doses (27,28). In addition, protective doses of single drug often exhibit undesired effects.…”