Therapeutic delivery of cyclin-A2 via recombinant adeno-associated virus serotype 9 restarts the myocardial cell cycle: An in vitro study

Abstract

Cyclin-A2, which is downregulated following birth, has previously been established as a key regulator of the cell cycle. The present study aimed to detect the effects of cyclin-A2 on myocardial cells by using recombinant adeno-associated virus 9 (rAAV9). Sixty mice were selected and randomly divided into two groups (n=30). The control group were injected with saline and the experimental group were transfected with the rAAV9-cyclinA2-CMV vector by intravenous injection into the tail vein. Tissues were harvested at two and four weeks following injection. Cyclin-A2 expression levels and localization were evaluated using western blot and immunohistochemical analyses. DNA synthesis and mitosis in the myocardium were confirmed by analyzing proliferating cell nuclear antigen (PCNA) and phospho-histone H3 (H3P) expression levels. Expression of Cyclin-A2 in the myocardium commenced two weeks following tail vein injection in the cyclin-A2-treated group, while no expression was observed in the control group. Four weeks following injection, expression levels of cyclin-A2 were higher than those observed at two weeks following injection into the myocardium (two weeks: 0.146±0.013 vs. 27.1±3.33%, P<0.001; four weeks: 0.142±0.107 vs. 74.4±3.36%, P<0.001). PCNA displayed increased expression levels in the cyclin-A2-treated group (two weeks: 13.1±0.54 vs. 65.8±3.44%, P<0.001; four weeks: 13.2±0.55 vs. 71.2±1.58%, P<0.001); however, no change was observed in those of the control group. By contrast, no significant difference was observed in mitosis marker H3P expression levels between the two groups. Immunohistochemical analysis of cyclin-A2 indicated cytoplasmic, but not nuclear, localization. cyclin-A2 and PCNA expression levels in the liver, lung and kidney showed no significant difference between the two groups (P>0.05). It was therefore concluded that the delivery of cyclin-A2 via rAAV9 to the mouse myocardium restarted the myocardial cell cycle, thereby establishing steady and specific expression in the myocardium. Furthermore, the effect of Cyclin-A2 on the myocardium may provide a novel method for achieving cardiac regeneration following cardiac injury.