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Background: Functional disorders of mitochondria are closely related to muscle diseases. Many studies have also shown that oxidative stress can stimulate the production of a large number of reactive oxygen species (ROS), which have various adverse effects on mitochondria and can damage muscle cells. Purpose: In this study, based on our previous research, we focused on the PINK1/Parkin pathway to explore the mechanism by which AS-IV alleviates muscle injury by inhibiting excessive mitophagy. Methods: L6 myoblasts were treated with AS-IV after stimulation with hydrogen peroxide (H2O2) and carbonyl cyanide m-chlorophenylhydrazone (CCCP). Then, we detected the related indices of oxidative stress and mitophagy by different methods. A PINK1 knockdown cell line was established by lentiviral infection to obtain further evidence that AS-IV reduces mitochondrial damage through PINK1/Parkin. Results: After mitochondrial damage, the expression of malondialdehyde (MDA) and intracellular ROS in L6 myoblasts significantly increased, while the expression of superoxide dismutase (SOD) and ATP decreased. The mRNA and protein expression levels of Tom20 and Tim23 were decreased, while those of VDAC1 were increased. PINK1, Parkin, and LC3 II mRNA and protein expression increased, and P62 mRNA and protein expression decreased·H2O2 combined with CCCP strongly activated the mitophagy pathway and impaired mitochondrial function. However, abnormal expression of these factors could be reversed after treatment with AS-IV, and excessive mitochondrial autophagy could also be reversed, thus restoring the regulatory function of mitochondria. However, AS-IV-adjusted function was resisted after PINK1 knockdown. Conclusion: AS-IV is a potential drug for myasthenia gravis (MG), and its treatment mechanism is related to mediating mitophagy and restoring mitochondrial function through the PINK1/Parkin pathway.

Triiodothyronine (T3), the biologically active form of thyroid hormone, was reported to protect myocardium from ischemia/reperfusion (I/R) injury when given before sustained ischemia, but its cardioprotective effects when given at the onset of reperfusion (postconditioning), a protocol with more clinical impact is unknown. Therefore, the present study was designed to determine whether T3 postconditioning (THPostC) is able to protect the heart from reperfusion injury and its underlying mechanisms. Isolated Sprague-Dawley rat hearts were subjected to 30 min ischemia/45 min reperfusion, triiodothyronine was delivered at the first 5 min of reperfusion. Our data shown that T3 from 1 to 10 μM during the first 5-min of reperfusion concentration-dependently improved post-ischemic myocardial function. A similar protection was observed in isolated rat cardiomyocytes characterized by the alleviation of I/R-induced loss of mitochondrial membrane potential and exacerbated cell death. Moreover, mitophagy (selectively recognize and remove damaged mitochondria) was significantly stimulated by myocardial I/R, which was enhanced with THPostC. Meanwhile, we found that THPostC stimulated PINK1/Parkin pathway, a critical regulator for mitophagy. Then, adenoviral knockdown of PINK1 and Parkin conformed its roles in the THPostC-mediated cardioprotection. Our results suggest that THPostC confers cardioprotection against I/R injury at least in part by reinforcing PINK1-dependent mitophagy. These findings reveal new roles and mechanisms of triiodothyronine in the cardioprotection against I/R injury.

Functional disorders of mitochondria are closely related to muscle diseases. Many studies have also shown that oxidative stress can stimulate the production of a large number of reactive oxygen species (ROS), which have various adverse effects on mitochondria and can damage muscle cells. In this study, based on our previous research, we focused on the PINK1/Parkin pathway to explore the mechanism by which AS-IV alleviates muscle injury by inhibiting excessive mitophagy. L6 myoblasts were treated with AS-IV after stimulation with hydrogen peroxide (H 2 O 2) and carbonyl cyanide m-chlorophenylhydrazone (CCCP). Then, we detected the related indices of oxidative stress and mitophagy by different methods. A PINK1 knockdown cell line was established by lentiviral infection to obtain further evidence that AS-IV reduces mitochondrial damage through PINK1/Parkin. After mitochondrial damage, the expression of malondialdehyde (MDA) and intracellular ROS in L6 myoblasts significantly increased, while the expression of superoxide dismutase (SOD) and ATP decreased. The mRNA and protein expression levels of Tom20 and Tim23 were decreased, while those of VDAC1 were increased. PINK1, Parkin, and LC3 II mRNA and protein expression increased, and P62 mRNA and protein expression decreased·H 2 O 2 combined with CCCP strongly activated the mitophagy pathway and impaired mitochondrial function. However, abnormal expression of these factors could be reversed after treatment with AS-IV, and excessive mitochondrial autophagy could also be reversed, thus restoring the regulatory function of mitochondria. However, AS-IV-adjusted function was resisted after PINK1 knockdown. AS-IV is a potential drug for myasthenia gravis (MG), and its treatment mechanism is related to mediating mitophagy and restoring mitochondrial function through the PINK1/Parkin pathway. [Display omitted] • The combination of CCCP and H 2 O 2 can induce excessive mitophagy. • AS-IV can reduce excessive mitophagy through the PINK1/Parkin pathway. • AS-IV can improve L6 cells damage and restore mitochondrial function. • The regulatory effect of AS-IV on damaged L6 cells is offset by PINK1 knockdown. [ABSTRACT FROM AUTHOR]

• THPostC concentration-dependently improved post-ischemic contractile function and reduces the cell death during I/R. • THPostC alleviates mitochondrial dysfunction of cardiomyocytes during simulated I/R. • THPostC activates I/R-induced mitophagy and suppresses mitophagy blocks protective effects of THPostC. • PINK1-Parkin pathway is responsible for THPostC-afforded cardioprotective effects. Triiodothyronine (T 3), the biologically active form of thyroid hormone, was reported to protect myocardium from ischemia/reperfusion (I/R) injury when given before sustained ischemia, but its cardioprotective effects when given at the onset of reperfusion (postconditioning), a protocol with more clinical impact is unknown. Therefore, the present study was designed to determine whether T 3 postconditioning (THPostC) is able to protect the heart from reperfusion injury and its underlying mechanisms. Isolated Sprague-Dawley rat hearts were subjected to 30 min ischemia/45 min reperfusion, triiodothyronine was delivered at the first 5 min of reperfusion. Our data shown that T 3 from 1 to 10 μM during the first 5-min of reperfusion concentration-dependently improved post-ischemic myocardial function. A similar protection was observed in isolated rat cardiomyocytes characterized by the alleviation of I/R-induced loss of mitochondrial membrane potential and exacerbated cell death. Moreover, mitophagy (selectively recognize and remove damaged mitochondria) was significantly stimulated by myocardial I/R, which was enhanced with THPostC. Meanwhile, we found that THPostC stimulated PINK1/Parkin pathway, a critical regulator for mitophagy. Then, adenoviral knockdown of PINK1 and Parkin conformed its roles in the THPostC-mediated cardioprotection. Our results suggest that THPostC confers cardioprotection against I/R injury at least in part by reinforcing PINK1-dependent mitophagy. These findings reveal new roles and mechanisms of triiodothyronine in the cardioprotection against I/R injury. [ABSTRACT FROM AUTHOR]