Exercise induces cardiac growth, protects against adverse remodeling, and may also induce a regenerative response. Since microRNAs (miRNA) play important roles in cardiovascular disease, we investigated their role in the cardiac exercise response.
We used the TaqMan rodent miRNA array to profile cardiac miRNA expressed at three weeks in two exercise models (swimming, running) compared to sedentary controls. Sixteen concordantly regulated miRNAs were identified and validated in both models, and examined for functional effects in neonatal rat ventricular cardiomyocytes (CMs). miR-222 was upregulated ~two-fold in both models and increased CM size (22%, p<0.01) and proliferation markers (EdU and Ki67, p<0.01). Bioinformatic and expression analyses identified four potential miR-222 targets (p27, Hipk1, Hipk2, and Hmbox-1) in CMs. These were confirmed as direct targets by luciferase assays, site-directed mutagenesis, and immunoblotting. siRNA knockdown (KD) of p27 or Hipk1 induced neonatal CM proliferation, while siRNA KD of Hmbox-1 increased CM size.
To examine miR-222’s role in vivo, LNA-antimiR-222 was injected via tail vein or subcutaneously and shown to reduce cardiac miR-222 levels to 1.5% (p<0.01). Untreated animals subjected to three weeks of swimming had the expected increase in heart size (15% in HW/TL, p<0.05), CM size (26%, p<0.05), and markers of CM proliferation (Ki67 and pHH3, p<0.05). Increases in heart and CM size were unaffected by control LNA-antimiR but completely blocked by LNA-antimiR-222, while CM proliferation markers decreased (60%, p<0.05). To see if miR-222 is sufficient to mediate the benefits of exercise, we made transgenic mice with cardiac-specific, regulated expression of miR-222 (Tg-miR-222). Tg-miR-222 have normal heart size and function at baseline. After ischemia-reperfusion injury (IRI), Tg-miR-222 had similar initial dysfunction but were protected against adverse remodeling over the next six weeks with better function (p<0.01), less cardiac fibrosis (68%, p<0.05), and increased CM proliferation markers (63%, p<0.05).
Conclusion: Cardiac miR-222 is upregulated by exercise, necessary for exercise-induced cardiac growth, and protects against pathological cardiac remodeling after IRI.