Testosterone antagonizes paraquat-induced cardiomyocyte senescence via the mIGF-1/SIRT1 signaling pathway

Abstract

Testosterone has been demonstrated to antagonize doxorubicin-induced cardiomyocyte senescence. However, whether testosterone prevents the paraquat-induced cardiomyocyte senescence is largely unknown. The detection of SA-β-gal activity was performed using senescence β-gal staining kit and the reactive oxygen species levels were determined by reactive oxygen species assay kit. The plasmids for insulin-like growth factor 1 shRNA (sh-mIGF-1), sirtuin-1 shRNA (sh-SIRT1), scramble shRNA (sh-NC), overexpressing mIGF-1 (mIGF-1), overexpressing SIRT1 (SIRT1), and negative controls (NC) were obtained for this study. The expression of target genes was detected using quantitative real-time PCR, immunolabeling, and western blot. We found that testosterone significantly delayed the paraquat-induced HL-1 cardiomyocyte senescence as evidenced by decreasing senescence-associated β-galactosidase activity and reactive oxygen species generation, which were accompanied by the up-regulated expression of mIGF-1 and SIRT1. RNA interference to reduce mIGF-1 and SIRT1 expression showed that testosterone prevented paraquat-induced HL-1 senescence via the mIGF-1/SIRT1 signaling pathway. Furthermore, myocardial contraction was evaluated by expression of genes of the contractile proteins/enzymes, such as α-myosin heavy chain 6 (MHC6), α-myosin heavy chain 7 (MHC7), α-skeletal actin (ACTA-1), and sarco/endoplasmic reticulum calcium ATPase-2 (SERCA2). Testosterone adjusted the above four gene expressions and the adjustment was blocked by mIGF-1 or SIRT1 inhibition. Our findings suggested that the mIGF-1/SIRT1 signaling pathway mediated the protective function of testosterone against the HL-1 cardiomyocyte senescence by paraquat, which provided new clues for the mechanisms underlying the anti-aging role of testosterone in cardiomyocytes.

Figure 1. Testosterone (T) prevents paraquat (PQ)-induced cell senescence in HL-1 cardiomyocytes. A, Percentages of senescence-associated β-galactosidase (SA-β-gal)-positive HL-1 cells after no treatment or exposure to PQ for 1 h, and with or without incubation with testosterone for 72 h at the indicated concentrations. B, Effect of testosterone on reactive oxygen species (ROS) production of HL-1 cardiomyocytes after the same treatments as in panel A. C, Protein expression of mIGF-1 and SIRT1 in HL-1 cardiomyocytes was measured by western blotting after no treatment or exposure to PQ for 1 h post-incubated with or without testosterone (1.0 μM) for 72 h. D, Detection of SIRT1 expression in HL-1 cardiomyocytes by immunofluorescence after the same treatment as in panel C. Scale bar indicates 50 μm. Data are reported as means±SE of four independent experiments (ANOVA and Dunnett's multiple comparisons test).

Figure 2. Testosterone (T) delayed paraquat (PQ)-induced cardiomyocyte senescence via the mIGF-1/SIRT1 pathway. A, Representative western blotting and band densitometry for mIGF-1 and SIRT1 in HL-1 cardiomyocytes transfected with or without sh-mIGF-1 and mIGF-1 in the presence of PQ and T. B, Percentages of senescence-associated β-galactosidase (SA-β-gal)-positive HL-1 cardiomyocytes and reactive oxygen species (ROS) production of cardiomyocytes after the same treatments as in panel A. C, Detection of SIRT1 expression in HL-1 cardiomyocytes by immunofluorescence and the statistical results after the same treatment as in panel A. Scale bar indicates 50 μm. D, Detection of SIRT1 expression in HL-1 cardiomyocytes by immunofluorescence and the statistical results after transfected with or without sh-SIRT1 and SIRT1 in the presence of PQ and T. Scale bar indicates 50 μm. E, Representative western blotting and band densitometry for SIRT1 in HL-1 cardiomyocytes after the same treatment as in panel 2D. F, Percentages of SA-β-gal-positive HL-1 cardiomyocytes and the ROS production of cardiomyocytes after the same treatments as in panel 2D. Data are reported as means±SE of four independent experiments (ANOVA and Dunnett's multiple comparisons test). CK: control.

Figure 3. Testosterone (T) changed the gene expression of the contractile protein/enzyme via mediating mIGF-1/SIRT1 pathway in HL-1 cardiomyocytes. The expressions of MHC7 mRNAs (A), ACTA-1 mRNAs (B), MHC6 mRNAs (C), and SERCA2 mRNAs (D) were examined by qRT-PCR. Data are reported as means±SE of four independent experiments (ANOVA and Dunnett's multiple comparisons test). PQ: paraquat; CK: control; NC: negative control.