Cardiotoxicity is one of the main side effects of doxorubicin (Dox) treatment. Dox could induce oxidative stress, leading to an opening of the mitochondrial permeability transition pore (mPTP) and apoptosis in cardiomyocytes. Previous studies have shown that Cryptotanshinone (Cts) has potential cardioprotective effects, but its role in Dox-induced cardiotoxicity (DIC) remains unknown. A Dox-stimulated H9C2 cell model was established. The effects of Cts on cell viability, reactive oxygen species (ROS), superoxide ion accumulation, apoptosis and mitochondrial membrane potential (MMP) were evaluated. Expressions of proteins in Akt-GSK-3β pathway were detected by Western blot. An Akt inhibitor was applied to investigate the effects of Cts on the Akt-GSK-3β pathway. The effects of Cts on the binding of p-GSK-3β to ANT and the formation of the ANT-CypD complex were explored by immunoprecipitation assay. The results showed that Cts could increase cell viability, reduce ROS levels, inhibit apoptosis and protect mitochondrial membrane integrity. Cts increased phosphorylated levels of Akt and GSK-3β. After cells were co-treated with an Akt inhibitor, the effects of Cts were abolished. An immunoprecipitation assay showed that Cts significantly increased GSK-3β-ANT interaction and attenuated Dox-induced formation of the ANT-CypD complex, thereby inhibiting opening of the mPTP. In conclusion, Cts could ameliorate oxidative stress and apoptosis via the Akt-GSK-3β-mPTP pathway.

Mitophagy can regulate mitochondrial homeostasis, preserve energy metabolism and cardiomyocytes survival effectively to restrain the development of heart failure (HF). Danqi Pill (DQP), composed of the dry roots of Salvia miltiorrhiza Bunge and Panax notoginseng, is included in the 2015 national pharmacopeia and effective in the clinical treatment of coronary heart diseases. Our previous studies have approved that DQP exerted remarkable cardioprotective effects on HF. However, the effect and mechanism of DQP on mitophagy have not been proved yet. We aim to explore whether DQP regulates mitophagy to protect against HF and to elucidate the in-depth mechanism. The HF rat model for evaluating DQP's efficacy was established with left anterior descending coronary artery ligation. The oxygen-glucose deprivation-reperfusion-induced cardiomyocyte model was conducted to clarify the potential mechanism of DQP. The mitochondria-targeted fluorescent protein Keima (mt-Keima) was applied for detecting mitophagy flux. Co-immunofluorescence and co-immunoprecipitation were performed to detect protein co-localization. Flow cytometry for JC-1 and Annexin-FITC/PI staining was utilized for assessing mitochondrial activity and function. In vivo, medium dose of DQP (1.5 g/kg) notably improved cardiac function and inhibited cardiac apoptosis in HF rats. Co-immunofluorescent staining of LC3B and TOM20 showed that DQP restored mitophagy. Further co-immunoprecipitation demonstrated that DQP increased the co-localization of FUNDC1 with either ULK1 or PGAM5. In vitro, DQP markedly protected mitochondrial membrane potential damage, reduced cardiomyocytes apoptosis, decreased the level of mitochondrial ROS, and increased the ATP level. Parallel with the in vitro results, DQP increased the interaction of FUNDC1 and LC3B, while knockdown of FUNDC1 diminished the interaction. Besides, Mt-Keima signaling detection further confirmed that DQP significantly promoted mitophagy. Intriguingly, knockdown of ULK1 or PGAM5 separately weakened rather than eliminated these effects of DQP on FUNDC1-mediated mitophagy, mitochondrial homeostasis and energy metabolism. Our results demonstrated that DQP protected against HF by improving FUNDC1-mediated mitophagy to perverse energy metabolism through the coordinated regulation of ULK1 and PGAM5.

A de novo missense variant in Rag GTPase protein C (RagC^S75Y) was recently identified in a syndromic dilated cardiomyopathy (DCM) patient. However, its pathogenicity and the related therapeutic strategy remain unclear. We generated a zebrafish Rragc^S56Y (corresponding to human RagC^S75Y) knock-in (KI) line via TALEN technology. The KI fish manifested cardiomyopathy-like phenotypes and poor survival. Overexpression of RagC^S75Y via adenovirus infection also led to increased cell size and fetal gene reprogramming in neonatal rat ventricle cardiomyocytes (NRVCMs), indicating a conserved mechanism. Further characterization identified aberrant mammalian target of rapamycin complex 1 (mTORC1) and transcription factor EB (TFEB) signaling, as well as metabolic abnormalities including dysregulated autophagy. However, mTOR inhibition failed to ameliorate cardiac phenotypes in the RagC^S75Y cardiomyopathy models, concomitant with a failure to promote TFEB nuclear translocation. This observation was at least partially explained by increased and mTOR-independent physical interaction between RagC^S75Y and TFEB in the cytosol. Importantly, TFEB overexpression resulted in more nuclear TFEB and rescued cardiomyopathy phenotypes. These findings suggest that S75Y is a pathogenic gain-of-function mutation in RagC that leads to cardiomyopathy. A primary pathological step of RagC^S75Y cardiomyopathy is defective mTOR–TFEB signaling, which can be corrected by TFEB overexpression, but not mTOR inhibition.