Tectorigenin protects against cardiac fibrosis in diabetic mice heart via activating the adiponectin receptor 1-mediated AMPK pathway.

Diabetic cardiomyopathy (DCM) is a common severe complication of diabetes that occurs independently of hypertension, coronary artery disease, and valvular cardiomyopathy, eventually leading to heart failure. Previous studies have reported that Tectorigenin (TEC) possesses extensive anti-inflammatory and anti-oxidative stress properties. In this present study, the impact of TEC on diabetic cardiomyopathy was examined. The model of DCM in mice was established with the combination of a high-fat diet and STZ treatment. Remarkably, TEC treatment significantly attenuated cardiac fibrosis and improved cardiac dysfunction. Concurrently, TEC was also found to mitigate hyperglycemia and hyperlipidemia in the DCM mouse. At the molecular level, TEC is involved in the activation of AMPK, both in vitro and in vivo, by enhancing its phosphorylation. This is achieved through the regulation of endothelial-mesenchymal transition via the AMPK/TGFß/Smad3 pathway. Furthermore, it was demonstrated that the level of ubiquitination of the adiponectin receptor 1 (AdipoR1) protein is associated with TEC-mediated improvement of cardiac dysfunction in DCM mice. Notably the substantial reduction of myocardial fibrosis. In conclusion, TEC improves cardiac fibrosis in DCM mice by modulating the AdipoR1/AMPK signaling pathway. These findings suggest that TEC could be an effective therapeutic agent for the treatment of diabetic cardiomyopathy.

Identify Tcea3 as a novel anti-cardiomyocyte hypertrophy gene involved in fatty acid oxidation and oxidative stress.

Background: Chronic pressure overload triggers pathological cardiac hypertrophy that eventually leads to heart failure. Effective biomarkers and therapeutic targets for heart failure remain to be defined. The aim of this study is to identify key genes associated with pathological cardiac hypertrophy by combining bioinformatics analyses with molecular biology experiments. Methods: Comprehensive bioinformatics tools were used to screen genes related to pressure overload-induced cardiac hypertrophy. We identified differentially expressed genes (DEGs) by overlapping three Gene Expression Omnibus (GEO) datasets (GSE5500, GSE1621, and GSE36074). Correlation analysis and BioGPS online tool were used to detect the genes of interest. A mouse model of cardiac remodeling induced by transverse aortic constriction (TAC) was established to verify the expression of the interest gene during cardiac remodeling by RT-PCR and western blot. By using RNA interference technology, the effect of transcription elongation factor A3 (Tcea3) silencing on PE-induced hypertrophy of neonatal rat ventricular myocytes (NRVMs) was detected. Next, gene set enrichment analysis (GSEA) and the online tool ARCHS4 were used to predict the possible signaling pathways, and the fatty acid oxidation relevant pathways were enriched and then verified in NRVMs. Furthermore, the changes of long-chain fatty acid respiration in NRVMs were detected using the Seahorse XFe24 Analyzer. Finally, MitoSOX staining was used to detect the effect of Tcea3 on mitochondrial oxidative stress, and the contents of NADP(H) and GSH/GSSG were detected by relevant kits. Results: A total of 95 DEGs were identified and Tcea3 was negatively correlated with Nppa, Nppb and Myh7. The expression level of Tcea3 was downregulated during cardiac remodeling both in vivo and in vitro. Knockdown of Tcea3 aggravated cardiomyocyte hypertrophy induced by PE in NRVMs. GSEA and online tool ARCHS4 predict Tcea3 involved in fatty acid oxidation (FAO). Subsequently, RT-PCR results showed that knockdown of Tcea3 up-regulated Ces1d and Pla2g5 mRNA expression levels. In PE induced cardiomyocyte hypertrophy, Tcea3 silencing results in decreased fatty acid utilization, decreased ATP synthesis and increased mitochondrial oxidative stress. Conclusion: Our study identifies Tcea3 as a novel anti-cardiac remodeling target by regulating FAO and governing mitochondrial oxidative stress.

Limonin stabilises sirtuin 6 (SIRT6) by activating ubiquitin specific peptidase 10 (USP10) in cardiac hypertrophy.

BACKGROUND AND PURPOSE: Limonin, a naturally occurring tetracyclic triterpenoid, has extensive pharmacological effects. Its role in cardiac hypertrophy remains to be elucidated. We investigated its effects on cardiac hypertrophy along with the potential mechanisms involved. EXPERIMENTAL APPROACH: The effects of limonin on cardiac hypertrophy in C57/BL6 mice caused by aortic banding, plus neonatal rat cardiac myocytes (NRCMs) stimulated with phenylephrine to induce cardiomyocyte hypertrophy in vitro were investigated. KEY RESULTS: Limonin markedly improved the cardiac function and heart weight in aortic banded mice. Limonin-treated mice and NRCMs also produced fewer cardiac hypertrophy markers than those treated with the vehicle in the hypertrophic groups. Sustained aortic banding- or phenylephrine-stimulation impaired cardiac sirtuin 6 (SIRT6) protein levels, which were partially reversed by limonin associated with enhanced activity of PPARα. Sirt6 siRNA inhibited the anti-hypertrophic effects of limonin in vitro. Interestingly, limonin did not influence Sirt6 mRNA levels, but regulated ubiquitin levels. Thus, the protein biosynthesis inhibitor, cycloheximide and proteasome inhibitor, MG-132, were used to determine SIRT6 protein expression levels. Under phenylephrine stimulation, limonin increased SIRT6 protein levels in the presence of cycloheximide, but it did not influence SIRT6 expression in the presence of MG-132, suggesting that limonin promotes SIRT6 levels by inhibiting its ubiquitination degradation. Furthermore, limonin inhibited the degradation of SIRT6 by activating ubiquitin-specific peptidase 10 (USP10), while Usp10 siRNA prevented the beneficial effects of limonin. CONCLUSION AND IMPLICATIONS: Limonin mediates the ubiquitination and degradation of SIRT6 by activating USP10, providing an attractive therapeutic target for cardiac hypertrophy.

Screening of Lipid Metabolism-Related Gene Diagnostic Signature for Patients With Dilated Cardiomyopathy.

Background: Dilated cardiomyopathy (DCM) is characterized by enlarged ventricular dimensions and systolic dysfunction and poor prognosis. Myocardial lipid metabolism appears abnormal in DCM. However, the mechanism of lipid metabolism disorders in DCM remains unclear. Methods: A gene set variation analysis (GSVA) were performed to estimate pathway activity related to DCM progression. Three datasets and clinical data downloaded from the Gene Expression Omnibus (GEO), including dilated cardiomyopathy and donor hearts, were integrated to obtain gene expression profiles and identify differentially expressed genes related to lipid metabolism. GO enrichment analyses of differentially expressed lipid metabolism-related genes (DELs) were performed. The clinical information used in this study were obtained from GSE21610 dataset. Data from the EGAS00001003263 were used for external validation and our hospital samples were also tested the expression levels of these genes through RT-PCR. Subsequently, logistic regression model with the LASSO method for DCM prediction was established basing on the 7 DELs. Results: GSVA analysis showed that the fatty acid metabolism was closely related to DCM progression. The integrated dataset identified 19 DELs, including 8 up-regulated and 11 down-regulated genes. A total of 7 DELs were identified by further external validation of the data from the EGAS00001003263 and verified by RT-PCR. By using the LASSO model, 6 genes, including CYP2J2, FGF1, ETNPPL, PLIN2, LPCAT3, and DGKG, were identified to construct a logistic regression model. The area under curve (AUC) values over 0.8 suggested the good performance of the model. Conclusion: Integrated bioinformatic analysis of gene expression in DCM and the effective logistic regression model construct in our study may contribute to the early diagnosis and prevention of DCM in people with high risk of the disease.

Diosmetin Protects against Cardiac Hypertrophy via p62/Keap1/Nrf2 Signaling Pathway.

An important pathophysiological consequence of pressure overload-induced cardiac hypertrophy is adverse cardiac remodeling, including structural changes in cardiomyocytes and extracellular matrix. Diosmetin (DIO), a monomethoxyflavone isolated from citrus fruits, had antioxidative stress effects in multiple organs. The purpose of this study was to examine the biological effect of diosmetin on pathological cardiac hypertrophy. In mice, diosmetin treatment reduced cardiac hypertrophy and dysfunction in an aortic banding- (AB-) induced pressure overload model and reducing myocardial oxidative stress by increasing antioxidant gene expression. In vitro, diosmetin (10 or 50 µm, 12 h or 24 h) protected PE-induced cardiomyocyte hypertrophy in neonatal rat cardiomyocytes. Mechanistically, diosmetin inhibited autophagy by activating the PI3K/Akt pathway. In particular, diosmetin induced the accumulation of p62 and its interaction with Keap1, promoted the nuclear translocation of Nrf2, and increased the expression of antioxidant stress genes in the process of cardiac hypertrophy. Furthermore, knockdown of p62 in rat primary cardiomyocytes abrogate the protective effect of diosmetin on cardiomyocyte hypertrophy. Similarly, the Nrf2 inhibitor ML385 obviously abolished the above effects by diosmetin treatment. In conclusion, our results suggest that diosmetin protects cardiac hypertrophy under pressure overload through the p62/Keap1/Nrf2 signaling pathway, suggesting the potential of diosmetin as a novel therapy for pathological cardiac hypertrophy.

Lupeol protects against cardiac hypertrophy via TLR4-PI3K-Akt-NF-κB pathways.

Inflammation and apoptosis are main pathological processes that lead to the development of cardiac hypertrophy. Lupeol, a natural triterpenoid, has shown anti-inflammatory and anti-apoptotic activities as well as potential protective effects on cardiovascular diseases. In this study we investigated whether lupeol attenuated cardiac hypertrophy and fibrosis induced by pressure overload in vivo and in vitro, and explored the underlying mechanisms. Cardiac hypertrophy was induced in mice by transverse aortic constriction (TAC) surgery, and in neonatal rat cardiomyocytes (NRCMs) by stimulation with phenylephrine (PE) in vitro. We showed that administration of lupeol (50 mg ·kg-1· d-1, i.g., for 4 weeks) prevented the morphological changes and cardiac dysfunction and remodeling in TAC mice, and treatment with lupeol (50 µg/mL) significantly attenuated the hypertrophy of PE-stimulated NRCMs, and blunted the upregulated hypertrophic markers ANP, BNP, and ß-MHC. Furthermore, lupeol treatment attenuated the apoptotic and inflammatory responses in the heart tissue. We revealed that lupeol attenuated the inflammatory responses including the reduction of inflammatory cytokines and inhibition of NF-κB p65 nuclear translocation, which was mediated by the TLR4-PI3K-Akt signaling. Administration of a PI3K/Akt agonist 740 Y-P reversed the protective effects of lupeol in TAC mice as well as in PE-stimulated NRCMs. Moreover, pre-treatment with a TLR4 agonist RS 09 abolished the protective effects of lupeol and restored the inhibition of PI3K-Akt-NF-κB signaling by lupeol in PE-stimulated NRCMs. Collectively, our results demonstrate that the lupeol protects against cardiac hypertrophy via anti-inflammatory mechanisms, which results from inhibiting the TLR4-PI3K-Akt-NF-κB signaling.