NMMHC IIA Inhibition Ameliorates Cerebral Ischemic/Reperfusion-Induced Neuronal Apoptosis Through Caspase-3/ROCK1/MLC Pathway.

PURPOSE

Our previous studies have indicated that non-muscle myosin heavy chain IIA (NMMHC IIA) is involved in HO-induced neuronal apoptosis, which is associated with the positive feedback loop of caspase-3/ROCK1/MLC pathway. However, the neuroprotective effect of NMMHC IIA inhibition with an adeno-associated virus (AAV) vector after transient middle cerebral artery occlusion (MCAO) and its role in caspases-3/ROCK1/MLC pathway remain blurred.

METHODS

Green fluorescent protein (GFP) and a small hairpin RNA targeting Myh9 (encoding NMMHC IIA) were cloned and packaged into the AAV9 vector. AAV-shMyh9 or control vector were injected into C57BL/6J mice four weeks prior to 60 min MCAO. Twenty-four hours after reperfusion, functional and histological analyses of the mice were performed.

RESULTS

In this study, AAV-shMyh9 was used to down-regulate NMMHC IIA expression in mice. We found that down-regulation of NMMHC IIA could improve neurological scores and histological injury in ischemic mice. Ischemic attack also activated neuronal apoptosis, and this effect was partially attenuated when NMMHC IIA was inhibited by AAV-shMyh9. In addition, AAV-shMyh9 significantly reduced cerebral ischemic/reperfusion (I/R)-induced NMMHC IIA-actin interaction, caspase-3 cleavage, Rho-associated kinase1 (ROCK1) activation and myosin light-chains (MLC) phosphorylation.

CONCLUSION

Consequently, we showed that AAV-shMyh9 inhibits I/R-induced neuronal apoptosis linked with caspase-3/ROCK1/MLC/NMMHC IIA-actin cascade, which has also been confirmed to be a positive feedback loop. These findings put some insights into the neuroprotective effect of AAV-shMyh9 associated with the regulation of NMMHC IIA-related pathway under ischemic attack and provide a therapeutic strategy for ischemic stroke.