YY1 transcriptionally represses TGFB1 in mice and may be a potential drug target for treating diabetic nephropathy.

Background: Diabetic heart dysfunction is a common complication of diabetes. Cell death is a core event that leads to diabetic heart dysfunction. However, the time sequence of cell death pathways and the precise time to intervene of particular cell death type remain largely unknown in the diabetic heart. This study aims to identify the particular cell death type that is responsible for diabetic heart dysfunction and to propose a promising therapeutic strategy by intervening in the cell death pathway. Methods: Type 2 diabetes models were established using db/db leptin receptor–deficient mice and high-fat diet/streptozotocin–induced mice. The type 1 diabetes model was established in streptozotocin-induced mice. Apoptosis and programmed cell necrosis (necroptosis) were detected in diabetic mouse hearts at different ages. G protein–coupled receptor–targeted drug library was searched to identify potential receptors regulating the key cell death pathway. Pharmacological and genetic approaches that modulate the expression of targets were used. Stable cell lines and a homemade phosphorylation antibody were prepared to conduct mechanistic studies. Results: Necroptosis was activated after apoptosis at later stages of diabetes and was functionally responsible for cardiac dysfunction. Cannabinoid receptor 2 (CB2R) was a key regulator of necroptosis. Mechanically, during normal glucose levels, CB2R inhibited S6 kinase–mediated phosphorylation of BACH2 at serine 520, thereby leading to BACH2 translocation to the nucleus, where BACH2 transcriptionally repressed the necroptosis genes Rip1 , Rip3 , and Mlkl . Under hyperglycemic conditions, high glucose induced CB2R internalization in a β-arrestin 2–dependent manner; thereafter, MLKL (mixed lineage kinase domain-like), but not receptor-interacting protein kinase 1 or 3, phosphorylated CB2R at serine 352 and promoted CB2R degradation by ubiquitin modification. Cardiac re-expression of CB2R rescued diabetes-induced cardiomyocyte necroptosis and heart dysfunction, whereas cardiac knockout of Bach2 diminished CB2R-mediated beneficial effects. In human diabetic hearts, both CB2R and BACH2 were negatively associated with diabetes-induced myocardial injuries. Conclusions: CB2R transcriptionally repressed necroptosis through interaction with BACH2; in turn, MLKL formed a negative feedback to phosphorylate CB2R. Our study provides the integrative view of a novel molecular mechanism loop for regulation of necroptosis centered by CB2R, which represents a promising alternative strategy for controlling diabetic heart dysfunction.

BACKGROUND: GRKs (GPCR [G protein–coupled receptor] kinases) are key regulators of GPCR signaling. Because GPCRs are targeted by approximately 40% of clinical drugs, GRKs also represent therapeutic targets for combating cardiovascular diseases. However, the predominant GRK that responds to diabetic heart injury and the underlying mechanisms remain largely unknown. METHODS: Human and mouse diabetic heart tissues were used to assess the expression pattern of the predominant GRK. Cardiac conditional knockout mice, heart-restricted adeno-associated virus 9, CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9)-mediated gene editing, and pharmacological modulators were used for functional investigation. Heart histopathology and function were evaluated by hematoxylin-eosin staining, wheat germ agglutinin staining, Sirius red staining, Masson trichrome staining, and echocardiography. Immunoprecipitation, electrophoretic mobility shift assays, site-specific mutation, autodocking, and molecular dynamics simulation were conducted for mechanism studies. Computer-aided virtual screening was conducted to identify inhibitors of the predominant GRK. RESULTS: GRK3 was the predominant GRK that was significantly upregulated in cardiomyocytes from both human and mouse diabetic hearts. Expression of GRK3 positively correlated with cardiac fibrosis and hypertrophy in human diabetic hearts. Heart-restricted overexpression of GRK3 aggravated, whereas conditional knockout of GRK3 in cardiomyocytes significantly relieved, heart injuries elicited by hyperglycemia. Mechanistically, hyperglycemia upregulated GRK3 expression by inhibiting the transcriptional repressor YY1 (Yin-Yang 1). GRK3 then essentially targeted CB2R (cannabinoid receptor 2) to phosphorylate CB2R at serine 335 (S335) and promote β-arrestin 2–mediated internalization and ubiquitin-dependent degradation of CB2R. Modulation of either YY1 or CB2R perturbed GRK3 functions in the diabetic hearts. Virtual screening identified a small molecule, isochlorogenic acid A, as an efficient GRK3 inhibitor that protects the heart from diabetic injury without causing additional side effects. CONCLUSIONS: GRK3 is the predominant GRK that aggravates diabetic heart injury. YY1-dependent upregulation of GRK3 functions through direct phosphorylation of CB2R. Small molecules targeting GRK3 may represent a promising avenue for curing diabetic heart injury in the clinic.

Pathological hypertrophic myocardium under consistent adverse stimuli eventually can cause heart failure. This study aims to explore the role of BACH2, a member of the basic region leucine zipper transcription factor family, in cardiac hypertrophy and failure. Transverse aortic constriction surgery was operated to induce cardiac hypertrophy and failure in mice. BACH2 was overexpressed in mice through tail vein injection of AAV9-Bach2. Mice with systemic or cardiac-specific knockdown of Bach2 were adopted. Neonatal rat ventricular myocytes (NRVMs) were isolated and infected with lentivirus to overexpress Bach2 or transfected with siRNA to knock down Bach2. Our data showed that overexpression of BACH2 ameliorated TAC-induced cardiac hypertrophy and failure in mice and decreased isoproterenol (ISO)-triggered myocyte hypertrophy in NRVMs. Systemic or cardiac-specific knockdown of Bach2 worsened the cardiac hypertrophy and failure phenotype in mice. Further assays showed that BACH2 bound to the promotor region of Akap6 at the -600 to -587 site and repressed its expression, which functioned as a crucial scaffold for cardiac hypertrophy and failure signaling pathways. Small molecular natural product library screening suggested that myricetin could up-regulate expression of Bach2 and simultaneously suppress the transcriptional levels of hypertrophic marker genes Bnp and Myh7. Further studies showed that myricetin exerted a BACH2-dependent protective effect against cardiac hypertrophy in vivo and in vitro. Taken together, our findings demonstrated that BACH2 plays a crucial role in the regulation of cardiac hypertrophy and failure and can be a potential therapeutic target in the future.

Background: Immunotherapy has revolutionized the treatment of malignant tumors; however, it may lead to fatal cardiotoxicity. Herein, we explored the mechanisms underlying cardiac side‐effects induced by immune checkpoint inhibitors (ICIs) and proposed a promising therapeutic target. Methods: Serum samples were collected from 168 patients with advanced non‐small cell lung cancer (NSCLC) receiving ICIs treatment or not. Representative ICI (IBI308) was intraperitoneally injected into normal C57BL/6 and congenital immune deficient nude mice. NOD‐like receptor family, pyrin domain containing 3 ( Nlrp3 ) globally knockout mice and gasdermin D ( Gsdmd ) globally knockout mice were involved in this study. Mice with cardiac‐specific BTB domain and CNC homolog 2 (Bach2) knock‐in and knock‐out were also included. The Cleavage Under Targets and Tagmentation (CUT&Tag) experiment was conducted to identify downstream molecules of BACH2, which was further validated with dual‐luciferase and electrophoretic mobility shift assays (EMSA). A library of small‐molecule products was screened to identify a specific agonist of BACH2, followed by in vivo and in vitro verification. Results: Patients treated with ICIs had significantly higher cardiac troponin T (cTNT) and interleukin 18 (IL‐18) levels. IBI308 significantly reduced cardiac function, increased cardiac fibrosis, and induced myocyte pyroptosis in wild type mice and T‐cell deficient nude mice. IBI308‐elicited toxicity was reversed by depleting pyroptotic genes Nlrp3 or Gsdmd . Furthermore, cardiac‐specific knock‐in of Bach2 rescued, whereas cardiac‐specific knock‐out of Bach2 exacerbated IBI308‐induced cardiotoxicity and pyroptosis. BACH2 directly bound to the promoter of G‐Rich RNA sequence binding factor 1 ( GRSF1 ) and promoted its transcription, which then activated the nuclear factor κB (NF‐κB) signaling cascade. The protective effect of BACH2 was dismissed after knockdown of GRSF1 or inhibition of the NF‐κB pathway. Lipoic acid was identified as an activator of BACH2 and reversed IBI308‐induced pyroptosis in a BACH2‐dependent manner. Conclusions: ICIs treatments caused preclinical cardiac injuries by activating myocyte pyroptosis. BACH2 exerted protective effects by promoting GRSF1 transcription and suppressing pyroptosis. Lipoic acid attenuated ICI‐induced cardiotoxicity by upregulating BACH2, which might be a novel therapeutic strategy. Key points: Immune checkpoint inhibitors cause elevated cardiac injuries in humans and mice ICIs cause myocyte pyroptosis and cardiotoxicity not via the adaptive immune system BACH2 ameliorates ICIs‐induced pyroptosis through transcriptionally promoting GRSF1 . Lipoic acid as a transcriptional inducer of BACH2 suppresses ICIs‐induced cardiotoxicity