Rationale: Anthracycline-induced cardiotoxicity (AIC) is a major side effect that limits the use of anthracyclines as effective chemotherapeutics. No mechanism-based therapy is available when cardiac function deteriorates. Objective: We aim to elucidate the dynamic autophagic defects in AIC and to identify a mechanism-based therapy via both genetic and pharmacological studies. Method and Results: Through phenotyping an adult AIC zebrafish model, we detected a biphasic response in autophagy: activation in the early stage and suppression in the later phase that is characterized by a decline in cardiac function. We conducted conditional genetic studies with atg7 , which encodes a rate-limiting autophagy core protein, and found that atg7 overexpression leads to therapeutic effects in the late phase but deleterious effects in the early phase of adult AIC. We then assessed the therapeutic effects of 37 Food and Drug Administration–approved autophagy activators using an embryonic AIC zebrafish model and identified spironolactone, pravastatin, and minoxidil as top-ranking drugs. We demonstrated the therapeutic efficacy of these Food and Drug Administration–approved autophagy activators in the adult AIC model and confirmed that these drugs exert therapeutic effects in the late phase but not in the early AIC phase. Finally, we demonstrated that the time-dependent therapeutic effects are conserved in a mouse AIC model and that spironolactone and rapamycin activated autophagy in an Atg7-dependent fashion. Conclusions: Our findings suggest that atg7-based autophagy activation is an effective therapeutic avenue to reversing the decline in cardiac function in AIC, highlighting the time-dependent nature of autophagy-based therapy.

Mutagenesis screening is a powerful forward genetic approach that has been successfully applied in lower-model organisms to discover genetic factors for biological processes. This phenotype-based approach has yet to be established in vertebrates for probing major human diseases, largely because of the complexity of colony management. Herein, we report a rapid strategy for identifying genetic modifiers of cardiomyopathy (CM). Based on the application of doxorubicin stress to zebrafish insertional cardiac (ZIC) mutants, we identified 4 candidate CM-modifying genes, of which 3 have been linked previously to CM. The long isoform of DnaJ (Hsp40) homolog, subfamily B, member 6b ( dnajb6b(L) ) was identified as a CM susceptibility gene, supported by identification of rare variants in its human ortholog DNAJB6 from CM patients. Mechanistic studies indicated that the deleterious, loss-of-function modifying effects of dnajb6b(L) can be ameliorated by inhibition of ER stress. In contrast, overexpression of dnajb6(L) exerts cardioprotective effects on both fish and mouse CM models. Together, our findings establish a mutagenesis screening strategy that is scalable for systematic identification of genetic modifiers of CM, feasible to suggest therapeutic targets, and expandable to other major human diseases.

RXRA is an endothelial- and early stage–predominant therapeutic target for treating anthracycline-induced cardiotoxicity.

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.

Ding-kun-dan (DKD), as one of well-known traditional Chinese medicine (TCM), is considered as an effective prescription to regulate menstruation, benefit Qi and nourish the blood. Previous studies had showed that DKD could improve sex hormone levels, insulin resistance, metabolism abnormalities and regulate immunity in animal models with polycystic ovary syndrome or endometriosis, however, little study conducted to reveal its clinical efficacy in Primary Dysmenorrhea (PD). To compare the effect of Ding-kun-dan (DKD) with Marvelon on relief of symptoms and change of serum pain-related factors in patients with primary dysmenorrhea. 136 patients with primary dysmenorrhea were randomly assigned to the DKD group (n = 73, take one tablet per day from 5th day of the menstrual cycle for 10 days every 28 days) and the Marvelon group (n = 63, take one tablet per day from 5th day of the menstrual cycle for 21 days every 28 days), the therapeutic effects were analyzed through evaluating the change of VAS scores, CMSS scores and the level of PGF_2a, PGE₂, PGF_2a/PGE₂ and NO during the 12 weeks intervention. Both DKD and Marvelon could effectively relief pain and other associated symptoms at each visit (Baseline, 4^th week, 8^th week and 12^th week). Although Marvelon was significantly superior to DKD in reducing VAS scores, the total CMSS, CMSS severity and duration scores at each posttreatment follow-up (P < 0.01), VAS scores in the DKD group decreased significantly over time while scores in the COC group only dropped rapidly after the first two months of treatment. Efficacy gap between two interventions continuously narrowed over time and the efficacy of DKD became non-inferior at the 12^th week compared to that of Marvelon (the difference between groups, - 0.78%; 95% confidence interval (CI), -13.67%-12.75%; non-inferiority margin, 15%). DKD group had better efficacy on mild pain compared to that of the COC group with no statistical difference (75% VS 61.9%, P > 0.05). DKD and Marvelon could effectively reduce PGF_2a, PGE₂ and higher PGF_2a/PGE₂ in patients with PD. There was no statistical difference in the level of PGF_2a, PGE₂, PGF_2a/PGE₂ and NO between DKD and Marvelon group at each follow-up. No serious adverse effect was observed. Ding-kun-dan is another available, effective and safe method for patients with primary dysmenorrhea to choose, especially for those who are suffered from mild pain and/or contraindicated to hormonal agents.