Kun Zhang,Kehui Yang,Lingyuan Zeng,Yang Xiao,JinGui Yu
Myocardial cell injury resulting from myocardial ischemia and reperfusion is one of the primary drivers behind the onset and progression of heart disease. Penehyclidine hydrochloride (PHC), a novel selective anti-cholinergic agent, exerts a protective effect against myocardial ischemia-reperfusion injury (MIRI). Rab32 belongs to the family of small GTPase proteins. The present study aimed to investigate whether PHC improved MIRI by regulating Rab32 and to explore the underlying mechanisms. Oxygen-glucose deprivation/reoxygenation (OGD/R) and left anterior descending coronary artery ligation were used to establish MIRI models in vitro and in vivo. Here, we showed that PHC upregulated the expression of Rab32 in OGD/R treated HL-1 cells. PHC alleviated OGD/R-induced cell apoptosis and intracellular and mitochondrial ROS levels, while the downregulation of Rab32 exacerbated cell injury. Rab32 was upregulated in MIRI mice and downregulated in OGD/R-induced HL-1 cells. Rab32 overexpression improved cardiac function, reduced the myocardial infarct size, and inhibited cell apoptosis and mitochondrial dysfunction, either in MIRI mice or OGD/R-induced HL-1 cells. On the mechanism, Rab32 interacted with Fancd2 in HL-1 cells. Rab32 downregulation decreased Fancd2 protein expression in mitochondria. Rab32 anchored Fancd2 to mitochondria in OGD/R treated HL-1 cells. Fancd2 knockdown reversed the protective effect of Rab32 on OGD/R-induced HL-1 cells and aggravated cardiomyocyte injury. Finally, the protective role of PHC on MIRI-caused cardiomyocyte injury was confirmed in MIRI mice. Overall, we demonstrated that Rab32 protects mitochondrial function by anchoring Fancd2 and mediates the protective effect of PHC against MIRI.
Regulator of G‑protein signaling 12 (RGS12) is a regulatory factor that is involved in various physiological processes. However, the role of RGS12 in myocardial ischemia/reperfusion injury (MIRI) currently remains unclear. The present study established a mouse model of MIRI by ligating the left main coronary artery followed by reperfusion. In addition, mouse HL‑1 cells were cultured in a hypoxic and serum‑free medium, followed by reoxygenation to establish an in vitro cell model of hypoxia/reoxygenation (H/R). Adenoviruses targeting RGS12 were subsequently used to either overexpress or silence RGS12 expression. RGS12 was highly expressed in both the myocardial tissues of mice with MIRI and HL‑1 cells subjected to H/R. The results from the in vitro experiments demonstrated that the knockdown of RGS12 reduced oxidative stress under a pathological environment, as indicated by decreased reactive oxygen species (ROS) levels and malondialdehyde activity and increased activities of superoxide dismutase and catalase. Furthermore, mice with MIRI and HL‑1 cells that underwent H/R stimulation exhibited increased ferroptosis, whereas RGS12 knockdown reversed these changes. These results showed that post‑RGS12 silencing the levels of Fe2+ and lipid ROS were decreased, the expression levels of glutathione peroxidase 4 and cystine transporter solute carrier family 7 member 11 were increased and mitochondrial structure was improved by preventing the loss of the mitochondrial crest. Mechanistically, the nuclear factor erythroid 2‑related factor 2 (Nrf2) pathway with anti‑ferroptosis and anti‑oxidative stress capacities was activated by RGS12 knockdown. Conversely, RGS12 overexpression exerted the opposite effects both in vivo and in vitro. Notably, it was demonstrated that penehyclidine hydrochloride (PHC), known to block the MIRI process, decreased RGS12 expression levels both in vivo and in vitro, and RGS12 overexpression inhibited the therapeutic effects of PHC on MIRI. In conclusion, the present study demonstrated that RGS12, a target of PHC, potentially enhanced the progression of MIRI by promoting oxidative stress and ferroptosis, and this effect may involve the regulation of the Nrf2 pathway.
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