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. 2022 Jun 22;11(13):e025021. doi: 10.1161/JAHA.121.025021

Figure 6. Inhibition of PI3K/AKT reversed Bmal1 knockdown‐mediated cardiomyocyte hypertrophy and fibroblost‐to‐myofibroblast differentiation.

Figure 6

A, H9c2 cells transfected with negative control (NC) or Bmal1 siRNA for 24 hour, and then treated with or without PE (200 µmol/L) for 30 minutes. The levels of phosphorylated‐AKT (P‐AKT) and total‐AKT (T‐AKT) were detected by western blot. n=3 per group. B and C, H9c2 cells were transfected with NC or Bmal1 siRNA for 24 hour, and then treated with or without PE (200 µmol/L, 6 hour) and MK2206 (10 nmol/L, 1 hour). Cells were stained with Phalloidin (red) to reflect cell size (B). Scale bar: 10 μm. Hypertrophic marker protein ANP was determined by western blot (C). n=5 per group. D, A schematic showing of an in‐direct co‐culture model: NRVMs were pre‐transfected with NC or Bma1 siRNA for 24 hour and then exposed with or without PE (200 µmol/L, 6 hour) and MK2206 (10 nmol/L, 1 hour). After PE and MK2206 stimulation, the supernatant was collected as conditional medium and added to NRCFs and further incubated for 24 hour. E through H. Western blot analysis and quantification of FN, Col1α1 and α‐SMA protein levels in NRCFs. n=5 to 7 per group. Ctrl, control group; PE, phenylephrine; CM, cardiac myocytes; CF, cardiac fibroblasts; NRVMs, neonatal rat ventricular myocytes; NRCFs, neonatal cardiac fibroblasts. FN: fibronectin; Col I: collagen 1a1; a‐SMA: alpha smooth muscle actin. Data were presented as mean±SEM, *P<0.05, **P<0.01.