Overexpression of microRNA-99a Attenuates Cardiac Hypertrophy

Abstract

Pathological cardiomyocyte hypertrophy is associated with significantly increased risk of heart failure, one of the leading medical causes of mortality worldwide. MicroRNAs are known to be involved in pathological cardiac remodeling. However, whether miR-99a participates in the signaling cascade leading to cardiac hypertrophy is unknown. To evaluate the role of miR-99a in cardiac hypertrophy, we assessed the expression of miR-99a in hypertrophic cardiomyocytes induced by isoprenaline (ISO)/angiotensin-II (Ang II) and in mice model of cardiac hypertrophy induced by transverse aortic constriction (TAC). Expression of miR-99a was evaluated in these hypertrophic cells and hearts. We also found that miR-99a expression was highly correlated with cardiac function of mice with heart failure (8 weeks after TAC surgery). Overexpression of miR-99a attenuated cardiac hypertrophy in TAC mice and cellular hypertrophy in stimuli treated cardiomyocytes through down-regulation of expression of mammalian target of rapamycin (mTOR). These results indicate that miR-99a negatively regulates physiological hypertrophy through mTOR signaling pathway, which may provide a new therapeutic approach for pressure-overload heart failure.

Fig 1. The expression of miR-99a and mTOR in heart with hypertrophy and failure induced by TAC in mice.

A. Representative images of hearts. B-D. UCG measurement after TAC surgery. There was no significant difference in EF% and LVID;d between sham and TAC group 1 week after surgery (concentric hypertrophy). Decreased EF% and enlarged LVID;d were observed in TAC group 8 weeks after surgery (eccentric hypertrophy). IVS;d was increased both 1 week and 8 weeks after surgery. E-F. mTOR expression was increased 1 week and 8 weeks after TAC. G-H. MiR-99a expression was increased 1 week after surgery, but low correlated with cardiac function. I-J. MiR-99a expression was increased 8 weeks after surgery, and showed a high correlated with cardiac function. (1 week after TAC: n = 11; 8 weeks after TAC: n = 16; Sham group: n = 6). *, p<0.05.

Fig 2. The expression of miR-99a and mTOR in NMVMs upon hypertrophic stimuli.

A. Expression of mTOR and ANP in NMVMs under Ang II stimulation assessed by western blotting analysis. B. ANP expression was up-regulated at least 2.68-fold upon Ang II stimulation. C. Increased mTOR expression was observed as early as 1 hour after Ang II stimulation (increased about 1.60-fold), and lasted for at least 24 hours. D. Expression of mTOR and ANP in NMVMs under ISO stimulation assessed by western blotting analysis. E. mTOR expression was increased under ISO stimulation. F. ANP expression was up-regulated upon ISO stimulation. G. MiR-99a expression was increased about 2.00-fold after ISO stimulation. H. MiR-99a was increased at least 2.00-fold upon Ang II stimulation. Each experiment was repeated more than 3 times. *, p<0.05.

Fig 3. The anti-hypertrophic role of MiR-99a in NMVMs under hypertrophic stimuli.

A-B. NMVMs transfected with lenti-GFP and lenti-99a-GFP without ISO or Ang II stimulation. C-F. NMVMs transfected with lenti-GFP or lenti-99a-GFP were treated with 10 μmol/L Ang II (C, D) for 6 hours or 20 μmol/L ISO (E, F) for 6 hours. G-I. Inhibition of mTOR and ANP expression by miR-99a in NMVMs under ISO or Ang II stimulation assessed by western blotting analysis. J-M. Decreased cell apoptosis in MiR-99a overexpressing NMVMs under ISO or Ang II stimulation assessed by western blotting analysis and TUNEL assay. *, p<0.05.

Fig 4. MiR-99a gene therapy attenuates cardiac hypertrophy in a mice model with TAC.

A, Immunofluorescence for GFP tag, indicating successful expression of exogenous miR-99a in heart after lenti-99a-GFP delivery. (α-actin: red; GFP: green; DAPI: cyan). B. Increased miR-99a expression in heart 1 week and 7 weeks after lentiviral delivery assessed by TaqMan RT-PCR. C More spherical shape of hearts from lenti-GFP mice (right column) comparing to lenti-99a-GFP (middle column) and sham mice (left column). Bigger size of lungs from lenti-GFP mice (right column) relative to lenti-99a-GFP (middle column) and sham mice (left column). The bottom panel shows paraffin-embedded sections of the hearts from the top panel. Bars = 3mm. D. Heart-to-body-weight ratio (mg/g) was increased in TAC mice, but attenuated in miR-99a overexpressing heart (n = 8 in lenti-99a-GFP group; n = 19 in lenti-GFP group). E-G. Western blotting analysis showed ANP (F) and α-SMA (G) were both down-regulated in miR-99a treated group compared to lenti-GFP group. H. BNP, β-MHC, PPAR-α, ACTA1, Hif-α, GLUT1 and HK2 levels were strongly increased in lenti-GFP treated hearts, but attenuated in lenti-99a treated hearts. Serca2a was decreased after surgery, but there was no significant difference in Serca2a expression among these three groups. I-J. Analysis of cardiomyocytes size in HE—stained sections (n = 3–5 per each group). K-L. Analysis of cardiac fibrosis in Masson—stained sections (n = 3–5 per each group). *, p<0.05.

Fig 5. MiR-99a suppressed hypertrophy and improved cardiac function in response to pressure overload.

A. Interventricular septum thickness was decreased as early as 1 week after MiR-99a lentiviral delivery, and this effect lasted for at least 7 weeks. B. Hypertrophy of LV posterior wall was attenuated in TAC mice with treatment of miR-99a as early as 1 week after lentiviral delivery. C-D. LVID;s (C) and LVID;d (D) were enlarged in response to pressure overload. MiR-99a treated group showed preserved LVID;s and LVID;d at 5 weeks and 7 weeks after lentiviral delivery. E-F. EF% and FS% were improved in TAC mice with miR-99a treatment at 5 weeks and 7 weeks after infection. IVS, interventricular septum; LVID;d, LV end-diastolic diameter; LVID;s, LV end-systolic diameter; LVPW, LV posterior wall; %EF, Ejection fraction; %FS, percent fractional shortening. *, p<0.05.

Fig 6. MiR-99a regulated mTOR/P70/S6K signaling pathway and decreased cell apoptosis in heart of TAC mice.

A-B. The expression of mTOR, SMARCA5, SMARCD1 and FGFR3 in heart of TAC mice. C-D. Phosphorylation of p70/S6-kinase was significantly decreased in lenti-99a-GFP group compared to lenti-GFP group. E-F. Decreased cleavage of caspase 3 at 17 kD in lenti-99a-GFP group compared to lenti-GFP group. n = 5 per group; *, p<0.05.