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-b-gal activity was performed using senescence b-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 b-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 a-myosin heavy chain 6 (MHC6), a-myosin heavy chain 7 (MHC7), a-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 antiaging role of testosterone in cardiomyocytes.

Secreted Cytokines within the Urine of AKI Patients Modulate TP53 and SIRT1 Levels in a Human Podocyte Cell Model

“…Since we only had access to a limited sample pool for this study, we aimed at excluding individual variability by pooling the samples. Gender-specific analyses were carried out to avoid variations due to the influence of sex hormones, such as testosterone and oestradiol, in the modulation of SIRT1 expression [36][37][38]. While the male urine samples 24 h post-surgery revealed only seven cytokines as significantly altered, the female urine samples contained a plethora of significantly altered cytokines.…”

Pharmacological senolysis reduces doxorubicin-induced cardiotoxicity and improves cardiac function in mice

“…As adult mammalian cardiomyocytes are predominantly post-mitotic and cannot be expanded in vitro, we selected the proliferating HL-1 cardiac myocyte cell line to validate different therapeutical approaches [28]. These cells, even immortalized, maintain a cardiac-specific phenotype and have been previously used as experimental model for cardiomyocyte-induced senescence [29].…”

Pharmacological Senolysis as a New Therapeutic Approach for the Prevention of Doxorubicin-Induced Cardiotoxicity

“…To investigate whether the selective elimination of senescent cells would prevent long-term associated cardiac pathologies to DOX, we first established an in vitro model using the HL-1 cardiac myocyte cell line (Figure 18A). HL-1 cells represent a cardiac myocyte cell line that can be repeatedly passaged, and have been previously used to induce a cardiomyocyte senescence model (Yu et al, 2020), as even immortalized, these cells maintain a cardiac-specific phenotype and allow to obtain reproducible experiments (Claycomb et al, 1998). We first performed a doseresponse curve of DOX for 72 h and found that 100 nM treatment was enough to induce senescence in HL-1 cells without causing significant cell death, keeping cell viability up to 80% (Figure 18B).…”

“…An alternative for in vitro drug evaluation is the HL-1 cardiomyocyte cell line (Claycomb et al, 1998). These cells, even immortalized, maintain a cardiac-specific phenotype and have been previously used as an experimental model for cardiomyocyte-induced senescence (Yu et al, 2020). We treated HL-1 cells with low-dose DOX (100 nM) since it has been shown that cardiac cells undergo a senescence program rather than apoptotic damage under these conditions (Piegari et al, 2013;Spallarossa et al, 2009).…”