Combined subthreshold dose inhibition of myosin light chain phosphorylation and MMP-2 activity provides cardioprotection from ischaemic/reperfusion injury in isolated rat heart

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Abstract

BACKGROUND AND PURPOSE

Phosphorylation and degradation of myosin light chain 1 (MLC1) during myocardial ischaemia/reperfusion (I/R) injury is a well-established phenomenon. It has been established that MMP-2 is involved in MLC1 degradation and that this degradation is increased when MLC1 is phosphorylated. We hypothesized that simultaneous inhibition of MLC1 phosphorylation and MMP-2 activity will protect hearts from I/R injury. As phosphorylation of MLC1 and MMP-2 activity is important for normal heart function, we used a cocktail consisting combination of low (subthreshold for any protective effect alone) doses of MLC kinase, MMP-2 inhibitors and subthreshold dose of an MLC phosphatase activator.

EXPERIMENTAL APPROACH

Isolated rat hearts were subjected to 20 min of global, no-flow ischaemia and 30 min reperfusion in the absence and presence of inhibitors of MLC1 phosphorylation and degradation.

KEY RESULTS

The recovery of cardiac function was improved in a concentration-dependent manner by the MLC kinase inhibitor, ML-7 (1–5 μM), the MLC phosphatase activator, Y-27632 (0.05–1 μM) or the MMP inhibitor, doxycycline (Doxy, 1–30 μM). Co-administration of subthreshold doses of ML-7 (1 μM) and Y-27632 (0.05 μM) showed a potential synergistic effect in protecting cardiac contractility and MLC1 levels in I/R hearts. Further combination with a subthreshold concentration of Doxy (1 μM) showed additional protection that resulted in full recovery to control levels.

CONCLUSIONS AND IMPLICATIONS

The results of this study exemplify a novel low-dose multidrug approach to pharmacological prevention of reperfusion injury that will enable a reduction of unwanted side effects and/or cytotoxicity associated with currently available MMP-2 and kinase inhibiting drugs.

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