Diabetic Cardiomyopathy Model Research Articles

Identification of fibrosis-associated lncRNAs in diabetic cardiomyopathy patients.

Our study endeavours to ascertain the plasma-derived long noncoding ribonucleic acids (lncRNA) and messenger RNA (mRNA) expression profiles through gene microarray analysis, aiming to elucidate their potential biological roles in the development and progression of diabetic cardiomyopathy (DCM), particularly with respect to myocardial fibrosis. We conducted gene chip experiments to discern differences in lncRNA and mRNA expression profiles between diabetic cardiomyopathy and type 2 diabetes mellitus (T2DM). Differentially expressed mRNAs were subjected to functional enrichment analysis, thereby enabling the identification of key genes. Subsequently, we established an interaction network connecting lncRNAs with mRNAs. To validate myocardial fibrosis-related mRNAs, we further developed a rat model of diabetic cardiomyopathy. We identified 688 differentially expressed lncRNAs and 341 differentially expressed mRNAs, which were primarily enriched in creatine metabolism, small guanosine triphosphate hydrolase (GTPase)-mediated signal transduction, and fatty acid degradation processes. Our analyses revealed 8 core genes (SMD11, DRG1, RPS26, EIF2S1, UBE3A, CEBPZ, NUP153, and EMD) associated with diabetic cardiomyopathy. An investigation into the lncRNA-mRNA coexpression network underscored 4 lncRNAs (lnc-NEK10-3, lnc-KDM4A-2, lnc-PCYOX1-3, and lnc-CDCP2-1) as significantly linked to differentially expressed fibrosis-associated mRNAs. The expression levels of transmembrane protein 173 (TMEM173) and toll-like receptor 7 (TLR7) were found to be significantly higher in DCM compared to normal controls, whereas cathepsin L1 (CTSL) and forkhead box O3 (FOXO3) displayed significantly lower expression levels relative to those of normal controls. Our study disclosed a subset of lncRNAs and mRNAs that are implicated in diabetic cardiomyopathy and myocardial fibrosis, thereby presenting themselves as promising biomarkers and therapeutic targets for the management of both diabetic cardiomyopathy and myocardial fibrosis.

Novel mitochondria-targeted therapy AP39 limits mitochondrial dysregulation and diastolic dysfunction in 2D & 3D human models of diabetic cardiomyopathy

Abstract Introduction Mitochondrial dysregulation has been implicated in many complications of diabetes. Mitochondrial-targeted therapies AP39 and SS31 are renoprotective, but their cardioprotective potential in diabetes has not been fully resolved. Purpose We investigated AP39 and SS31 in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-hCM) and endothelial cells (iPSC-hEC) independently in 2D culture, as well as collectively in a 3D cardiac organoid (iPSC-hCO) model also comprising fibroblasts (iPSC-hFB), under type 2 diabetes (T2D)-like conditions. Methods Cells (iPSC-hCM, iPSC-hEC, iPSC-hFB; foreskin-2 cell line) were exposed to either 5.55mM glucose (control) or T2D conditions (20mM glucose plus fatty acids, endothelin-1 and cortisol) ± AP39 (50nM) or SS31 (1µM) for 48h. Cardiac organoids were subjected to 5.55mM glucose (control) or T2D for 4 days, with AP39 (50nM) or SS31 (1µM) added on day 2. Data are expressed as mean±SEM (fold) with n=4-6 from 2-3 independent experiments. One-way ANOVA with Dunnett`s post hoc analysis was performed with P<0.05 considered significant. Results 2D human cells: After 48h, T2D increased mitochondrial superoxide in iPSC-hCM (2.4±0.1 fold vs control) and iPSC-hEC (3.2±0.29 fold vs control), both P<0.05 (measured by MitoSOX). This was blunted by both AP39 (to 1.1±0.17) and SS31 (to 1.5±0.17) in T2D iPSC-hCM (P<0.05), with similar impact in iPSC-hEC, to 2.9±0.26 and 1.3±0.16 fold, both P<0.05. T2D increased cell death in both iPSC-hCM (1.6±0.09 fold vs control) and iPSC-hEC (1.6±0.12 fold, both P<0.05 measured by propidium iodide staining. Interestingly, AP39 selectively blunted cell death in T2D iPSC-hCM (to 1.1±0.06, P<0.05), mimicked by SS31 in T2D iPSC-hEC only (to 1.0±0.10, P<0.05). Further, T2D-induced mitochondrial hyperpolarisation in iPSC-hCM (1.5±0.09 fold vs control, assessed using TMRM), was only protected by AP39 (1.2±0.06 fold, P<0.05), but not SS31. 3D human cardiac organoids: After 4 days, T2D increased total contraction duration (1.6±0.10 fold control), time to peak (1.7±0.11) and relaxation duration (1.5±0.13, indicative of diastolic dysfunction), assessed by Olympus IX71 with a DP72 camera. All 3 contractile parameters were attenuated with AP39 (to 1.5±0.06, 1.3±0.09 and 1.0±0.04 fold, respectively) or SS31 (to 1.1±0.05, 1.2±0.15 and 1.2±0.12 fold, respectively), all P<0.05. T2D increased mitochondrial superoxide (2.7±0.16 fold control), blunted by AP39 (to 1.8±0.16) and SS31 (to 1.9±0.17), both P<0.05. Cellular injury measured by LDH was evident in T2D iPSC-hCO (2.1±0.23 fold control) was limited by AP39 (to 1.2±0.09) and SS31 (to 1.1±0.07), both P<0.05. Conclusion Our results highlight the cardioprotective potential of AP39 and SS31 to limit mitochondrial dysregulation, cell death and contractile dysfunction in human in vitro models of T2D. These findings may facilitate their clinical progression as therapeutic options for diabetic cardiomyopathy.

Sacubitril/valsartan improves diastolic left ventricular stiffness with increased titin phosphorylation via cGMP-PKG activation in diabetic mice

Titin, a giant sarcomeric protein, regulates diastolic left ventricular (LV) passive stiffness as a molecular spring and could be a therapeutic target for diastolic dysfunction. Sacubitril/valsartan (Sac/Val), an angiotensin receptor neprilysin inhibitor, has been shown to benefit patients with heart failure with preserved ejection fraction. The effect of Sac/Val is thought to be due to the enhancement of the cGMP/PKG pathway via natriuretic peptide. In this study, the effects of Sac/Val on LV diastolic dysfunction are demonstrated in a mouse diabetic cardiomyopathy model focusing on titin phosphorylation. Sac/Val-treated diabetic mice showed a greater increase in myocardial levels of cGMP-PKG than Val-treated and control mice. Conductance catheter analysis showed a significant reduction in LV stiffness in diabetic mice, but not in non-diabetic mice. Notably, diastolic LV stiffness was significantly reduced in Sac/Val-treated diabetic hearts compared with Val-treated or vehicle-treated diabetic mice. The phosphorylation level of titin (N2B), which determines passive stiffness and modulates active contraction, was higher in Sac/Val-treated hearts compared with Val-treated hearts in diabetic mice. Given that alteration of titin phosphorylation through PKG contributes to myocardial stiffness, the beneficial effects of Sac/Val in heart failure might be partly attributed to the induction of titin phosphorylation.

Identification of immune feature genes and intercellular profiles in diabetic cardiomyopathy

BACKGROUND Diabetic cardiomyopathy (DCM) is a multifaceted cardiovascular disorder in which immune dysregulation plays a pivotal role. The immunological molecular mechanisms underlying DCM are poorly understood. AIM To examine the immunological molecular mechanisms of DCM and construct diagnostic and prognostic models of DCM based on immune feature genes (IFGs). METHODS Weighted gene co-expression network analysis along with machine learning methods were employed to pinpoint IFGs within bulk RNA sequencing (RNA-seq) datasets. Single-sample gene set enrichment analysis (ssGSEA) facilitated the analysis of immune cell infiltration. Diagnostic and prognostic models for these IFGs were developed and assessed in a validation cohort. Gene expression in the DCM cell model was confirmed through real time-quantitative polymerase chain reaction and western blotting techniques. Additionally, single-cell RNA-seq data provided deeper insights into cellular profiles and interactions. RESULTS The overlap between 69 differentially expressed genes in the DCM-associated module and 2483 immune genes yielded 7 differentially expressed immune-related genes. Four IFGs showed good diagnostic and prognostic values in the validation cohort: Proenkephalin (Penk) and retinol binding protein 7 (Rbp7), which were highly expressed, and glucagon receptor and inhibin subunit alpha, which were expressed at low levels in DCM patients (all area under the curves > 0.9). SsGSEA revealed that IFG-related immune cell infiltration primarily involved type 2 T helper cells. High expression of Penk (P < 0.0001) and Rbp7 (P = 0.001) was detected in cardiomyocytes and interstitial cells and further confirmed in a DCM cell model in vitro . Intercellular events and communication analysis revealed abnormal cellular phenotype transformation and signaling communication in DCM, especially between mesenchymal cells and macrophages. CONCLUSION The present study identified Penk and Rbp7 as potential DCM biomarkers, and aberrant mesenchymal-immune cell phenotype communication may be an important aspect of DCM pathogenesis.

Intermedin Alleviates Diabetic Cardiomyopathy by Up-Regulating CPT-1β through Activation of the Phosphatidyl Inositol 3 Kinase/Protein Kinase B Signaling Pathway.

Diabetic cardiomyopathy (DCM), one of the most serious long-term consequences of diabetes, is closely associated with myocardial fatty acid metabolism. Carnitine palmitoyltransferase-1β (CPT-1β) is the rate-limiting enzyme responsible for β-oxidation of long-chain fatty acids. Intermedin (IMD) is a pivotal bioactive small molecule peptide, participating in the protection of various cardiovascular diseases. However, the role and underlying mechanisms of IMD in DCM are still unclear. In this study, we investigated whether IMD alleviates DCM via regulating CPT-1β. A rat DCM model was established by having rats to drink fructose water for 12 weeks. A mouse DCM model was induced by feeding mice a high-fat diet for 16 weeks. We showed that IMD and its receptor complexes levels were significantly down-regulated in the cardiac tissues of DCM rats and mice. Reduced expression of IMD was also observed in neonatal rat cardiomyocytes treated with palmitic acid (PA, 300 μM) in vitro. Exogenous and endogenous IMD mitigated cardiac hypertrophy, fibrosis, dysfunction, and lipid accumulation in DCM rats and IMD-transgenic DCM mice, whereas knockout of IMD worsened these pathological processes in IMD-knockout DCM mice. In vitro, IMD alleviated PA-induced cardiomyocyte hypertrophy and cardiac fibroblast activation. We found that CPT-1β enzyme activity, mRNA and protein levels, and acetyl-CoA content were increased in T2DM patients, rats and mice. IMD up-regulated the CPT-1β levels and acetyl-CoA content in T2DM rats and mice. Knockdown of CPT-1β blocked the effects of IMD on increasing acetyl-CoA content and on inhibiting cardiomyocyte hypertrophy and cardiac fibroblast activation. IMD receptor antagonist IMD17-47 and the phosphatidyl inositol 3 kinase (PI3K)/protein kinase B (Akt) inhibitor LY294002 reversed the effects of IMD on up-regulating CPT-1β and acetyl-CoA expression and on inhibiting cardiomyocyte hypertrophy and cardiac fibroblast activation. We revealed that IMD alleviates DCM by up-regulating CPT-1β via calcitonin receptor-like receptor/receptor activity-modifying protein (CRLR/RAMP) receptor complexes and PI3K/Akt signaling. IMD may serve as a potent therapeutic target for the treatment of DCM.

Abstract We095: Using Human Cardiac Organoids to Elucidate the Cardioprotective Potential of Mitochondrial-Targeted Therapies in a Preclinical Model of Diabetic Cardiomyopathy

Introduction: Mitochondrial dysregulation is implicated in many complications of type 2 diabetes (T2D). Mitochondrial-targeted therapies SkQ1, AP39 and SS31 limit renal T2D complications. We sought to obtain the first evidence of the cardioprotective potential of SkQ1, AP39 and SS31 in T2D, using a human induced pluripotent stem cell (iPSC)-derived 3D multicellular cardiac organoid model of diabetic cardiomyopathy. Methods: Human cardiac organoids (foreskin-2 cell-line) generated from human iPSC-derived cardiomyocytes, cardiac fibroblasts and endothelial cells were subjected to 5.55mM glucose (control) or T2D-like conditions (20mM glucose, fatty acids, endothelin-1 and cortisol) for 4 days with supplementation of either SkQ1 (50nM), AP39 (50nM) or SS31 (1µM) on day 2. On day 4, endpoint parameters were evaluated in cardiac organoids, including contractile function (captured via Olympus IX71), mitochondrial superoxide levels (MitoSOX assay), mitochondrial membrane potential (TMRM assay), and cellular injury (lactate dehydrogenase; LDH). Data are expressed as mean±SEM (fold control) with n =4-6 from 2-3 independent experiments. One-way ANOVA with Dunnett`s post hoc analysis was performed with P<0.05 considered significant. Results: After 4 days, T2D-conditions increased total contraction duration (1.55±0.10-fold control), time-to-peak (1.71±0.11-fold control, indicative of systolic dysfunction) and relaxation duration (1.46±0.13-fold control, indicative of diastolic dysfunction) in cardiac organoids. All 3 functional parameters were attenuated by AP39 (to 1.15±0.06, 1.32±0.09 and 1.04±0.04-fold control, respectively), or by SS31 (to 1.10±0.05, 1.12±0.15 and 1.09±0.12-fold control, respectively; all P<0.05), but not SkQ1. T2D-conditions increased mitochondrial superoxide (2.68±0.16-fold control), which was blunted by all 3 interventions, SkQ1 to 2.00±0.29, AP39 to 1.82±0.16 and SS31 to 1.89±0.17 (all P<0.05). Cellular injury was evident in T2D cardiac organoids (2.07±0.23-fold control), which was again limited by SkQ1 (to 1.06±0.11), AP39 (to 1.18±0.09) or SS31 (to 1.08±0.07), all P<0.05. Conclusion: These findings in human cardiac organoids highlight the cardioprotective potential of mitochondrial-targeted therapies to limit T2D-induced cardiac dysfunction, mitochondrial dysregulation and cellular injury, potentially facilitating clinical progression of candidates such as AP39 and SS31 as new therapies for patients with diabetic cardiomyopathy.

Abstract Mo048: Cereblon as a potential biomarker for early detection of diabetic cardiomyopathy

Type 2 diabetes mellitus (T2DM) patients have a higher risk of heart failure and cause cardiomyopathy without other cardiac disease. There is no specific biomarker for discriminating patients with diabetic cardiomyopathy (DCM). In this study, we investigated the cereblon (CRBN) level in mice and human samples to determine its potential as a biomarker for early detection of diabetic cardiomyopathy (DCM). C57BL/db/db male mice at 6 and 8 weeks of age, as the early DCM model with obesity and T2DM were investigated. We confirmed that mitochondrial function was decreased in the 8 weeks old db/db mice without alterations of cardiac function and structures. Compared to the control mice, CRBN protein level was significantly increased and phosphorylation of AMPK, a negative regulator by CRBN, was significantly decreased in the heart tissue of 8 weeks old db/db mice. Blood CRBN level was also significantly increased in both 6 and 8 weeks old db/db mice compared with control mice. In blood of diabetic patients, CRBN level was significantly correlated with IL-6, IL-1b, TNF-a, IGFBP-7, and adiponectin, which are previously reported biomarkers of DCM. However, CRBN level was not correlated with cardiac troponin I, NT-proBNP, and ST2, which are biomarkers of myocardial injury and heart failure. Furthermore, the CRBN level showed a higher tendency in patients with diastolic dysfunction without systolic dysfunction compared to patients with normal function and systolic dysfunction, although the difference was not statistically significant. In conclusion, cardiac mitochondrial dysfunction occured and CRBN levels of heart and blood increased prior to cardiac functional and structural changes in the early DCM mouse model, and CRBN might serve as a potential biomarker for the early detection of DCM in T2DM patients.

Compound Yuye Decoction protects diabetic rats against cardiomyopathy by inhibiting myocardial apoptosis and inflammation via regulating the PI3K/Akt signaling pathway

To explore the therapeutic mechanism of compound Yuye Decoction against diabetic cardiomyopathy (DCM). Drugbank, Gene Cards, OMIM and PharmGKb databases were used to obtain DCM-related targets, and the core targets were identified and functionally annotated by protein-protein interaction network analysis followed by GO and KEGG enrichment analysis. The "Traditional Chinese Medicine-Key Component-Key Target-Key Pathway" network was constructed using Cytoscape 3.9.1, and molecular docking was carried out for the key components and the core targets. In the animal experiment, Wistar rat models of DCM were treated with normal saline or Yuye Decoction by gavage at low (0.29 g/kg) and high (1.15 g/kg) doses for 8 weeks, and the changes in cardiac electrophysiology and histopathology were evaluated. The changes in serum levels of LDH, CK, and CK-MB were examined, and myocardial expressions of PI3K, P-PI3K, Akt, P-AKT, BAX, IL-6, and TNF-α were detected using Western blotting. We identified 61 active compounds in Yuye Decoction with 1057 targets, 3682 DCM-related disease targets, and 551 common targets between them. Enrichment of the core targets suggested that apoptosis, inflammation and the PI3K/Akt pathways were the key signaling pathways for DCM treatment. Molecular docking studies showed that the active components in Yuye Decoction including gold amidohydroxyethyl ester and kaempferol had strong binding activities with AKT1 and PIK3R1. In DCM rat models, treatment with Yuye Decoction significantly alleviated myocardial pathologies, reduced serum levels of LDH, CK and CK-MB, lowered myocardial expressions of BAX, IL-6 and TNF-α, and increased the expressions of P-PI3K and P-AKT. The therapeutic effect of compound Yuye Decoction against DCM is mediated by its multiple active components that act on multiple targets and pathways to inhibit cardiomyocyte apoptosis and inflammatory response by regulating the PI3K/Akt signaling pathway.

Nimbolide protects against diabetic cardiomyopathy by regulating endoplasmic reticulum stress and mitochondrial function via the Akt/mTOR pathway

Nimbolide has been demonstrated to possess protective properties against gestational diabetes mellitus and diabetic retinopathy. However, the role and molecular mechanism of nimbolide in diabetic cardiomyopathy (DCM) remain unknown. Diabetes was induced in rats via a single injection of streptozotocin (STZ) and then the diabetic rats were administered nimbolide (5 mg/kg and 20 mg/kg) or dimethyl sulfoxide daily for 12 weeks. H9c2 cardiomyocytes were exposed to high glucose (25 mM glucose) to mimic DCM in vitro. The protective effects of nimbolide against DCM were evaluated in vivo and in vitro. The potential molecular mechanism of nimbolide in DCM was further explored. We found that nimbolide dose-dependently decreased blood glucose and improved body weight of diabetic rats. Additionally, nimbolide dose-dependently improved cardiac function, alleviated myocardial injury/fibrosis, and inhibited endoplasmic reticulum (ER) stress and apoptosis in diabetic rats. Moreover, nimbolide dose-dependently improved mitochondrial function and activated the Akt/mTOR signaling. We consistently demonstrated the cardioprotective effects of nimbolide in an in vitro model of DCM. The involvement of ER stress and mitochondrial pathways were further confirmed by using inhibitors of ER stress and mitochondrial division. By applying a specific Akt inhibitor SC66, the cardioprotective effects of nimbolide were partially blocked. Our study indicated that nimbolide alleviated DCM by activating Akt/mTOR pathway. Nimbolide may be a novel therapeutic agent for DCM treatment.

Effect of NLRP3 gene knockdown on pyroptosis and ferroptosis in diabetic cardiomyopathy injury

Diabetic cardiomyopathy (DCM) is a chronic disease caused by diabetes mellitus, which is recognized as a worldwide challenging disease. This study aimed to investigate the role and the potential mechanism of knocking down the NACHT-, LRR- and PYD domains-containing protein 3 (NLRP3), an inflammasome associated with onset and progression of various diseases, on high glucose or diabetes -induced cardiac cells pyroptosis and ferroptosis, two regulated non-necrosis cell death modalities discovered recent years. In the present study, both in vivo and in vitro studies were conducted simultaneously. Diabetic rats were induced by 55 mg/kg intraperitoneal injection of streptozotocin (STZ). Following the intraperitoneal injection of MCC950 (10 mg/kg), On the other hand, the DCM model in H9C2 cardiac cells was simulated with 35 mmol/L glucose and a short hairpin RNA vector of NLRP3 were transfected to cells. The results showed that in vivo study, myocardial fibers were loosely arranged and showed inflammatory cell infiltration, mitochondrial cristae were broken and the GSDMD-NT expression was found notably increased in the DM group, while the protein expressions of xCT and GPX4 was significantly decreased, both of which were reversed by MCC950. High glucose reduced the cell viability and ATP level in vitro, accompanied by an increase in LDH release. All of the above indicators were reversed after NLRP3 knockdown compared with the HG treated alone. Moreover, the protein expressions of pyroptosis- and ferroptosis-related fators were significantly decreased or increased, consistent with the results shown by immunofluorescence. Furthermore, the protective effects of NLRP3 knockdown against HG were reversed following the mtROS agonist rotenone (ROT) treatment. In conclusion, inhibition of NLRP3 suppressed DM-induced myocardial injury. Promotion of mitochondrial ROS abolished the protective effect of knockdown NLRP3, and induced the happening of pyroptosis and ferroptosis. These findings may present a novel therapeutic underlying mechanism for clinical diabetes-induced myocardial injury treatment.

Cannabinoid 2 Receptor Activation Protects against Diabetic Cardiomyopathy through Inhibition of AGE/RAGE-Induced Oxidative Stress, Fibrosis, and Inflammasome Activation.

Oxidative stress, fibrosis, and inflammasome activation from advanced glycation end product (AGE)-receptor of advanced glycation end product (RAGE) interaction contribute to diabetic cardiomyopathy (DCM) formation and progression. Our study revealed the impact of β-caryophyllene (BCP) on activating cannabinoid type 2 receptors (CB2Rs) against diabetic complication, mainly cardiomyopathy and investigated the underlying cell signaling pathways in mice. The murine model of DCM was developed by feeding a high-fat diet with streptozotocin injections. After the development of diabetes, the animals received a 12-week oral BCP treatment at a dose of 50 mg/kg/body weight. BCP treatment showed significant improvement in glucose tolerance and insulin resistance and enhanced serum insulin levels in diabetic animals. BCP treatment effectively reversed the heart remodeling and restored the phosphorylated troponin I and sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a expression. Ultrastructural examination showed reduced myocardial cell injury in DCM mice treated with BCP. The preserved myocytes were found to be associated with reduced expression of AGE/RAGE in DCM mice hearts. BCP treatment mitigated oxidative stress by inhibiting expression of NADPH oxidase 4 and activating phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/nuclear factor erythroid 2-related factor 2 (Nrf2) signaling. Also, BCP suppressed cardiac fibrosis and endothelial-to-mesenchymal transition in DCM mice by inhibiting transforming growth factor β (TGF-β)/suppressor of mothers against decapentaplegic (Smad) signaling. Further, BCP treatment suppressed nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome activation in DCM mice and alleviated cellular injury to the pancreatic tissues evidenced by significant elevation of the number of insulin-positive cells. To demonstrate a CB2R-dependent mechanism of BCP, another group of DCM mice were pretreated with AM630, a CB2R antagonist. AM630 was observed to abrogate the beneficial effects of BCP in DCM mice. Taken together, BCP demonstrated the potential to protect the myocardium and pancreas of DCM mice mediating CB2R-dependent mechanisms. SIGNIFICANCE STATEMENT: BCP, a CB2R agonist, shows protection against DCM. BCP attenuates oxidative stress, inflammation, and fibrosis in DCM via activating CB2Rs. BCP mediating CB2R activation favorably modulates AGE/RAGE, PI3K/AKT/Nrf2β and TGF-β/Smad and (NLRP3) inflammasome in diabetic cardiomyopathy.

Letter to the Editor: “Effect of grape seed extract on β-catenin gene expression and hyperglycemia in rats induced by streptozotocin”

This letter is related to the article titled "Effect of grape seed extract on β-catenin gene expression and hyperglycemia in rats induced by streptozotocin" published in Volume 39, Is sue 2 of the Eurasian Journal of Veterinary Sciences in 2023. First of all, we would like to thank you for the study whose aim was to determine the effect of grape seed extract (GSE) on body weight, blood glucose, and β-catenin gene expres sion in experimental Type 2 diabetic rats. We read this ar ticle with interest and have one suggestion for the authors. The authors reported that the effect of grape seed extract on blood glucose, body weight, and B-catenin gene expression levels determined in the Wnt/β-catenin signaling pathway in Type 2 Diabetes Mellitus. Instead of examining the expression levels of the tissue ma terials obtained due to the experimental design at a single gene level, next-generation sequencing, and total transcrip tome level will allow the expression levels of many genes expressed in the relevant pathway to be determined. In this regard, a literature search on the Web of Science with the keywords "streptozotocin", "diabetes", and "RNA-seq" reve aled 29 studies in the last four years. Among these studies; there is a study on RNA-seq analysis in diabetic male mouse liver (Ge et al 2020). Again, different functional, cellular, and molecular models of diabetic cardiomyopathy were exami ned in Type I and Type II mouse models administered with Streptozotocin (Marino et al 2023). Elzinga et al. (2023) reported research results in which diabetic kidney disease and neuropathy were examined at the transcriptome level in type I and type II diabetic mice. In the currently planned and realized experimental design, it is recommended that next-generation sequencing analyses in liver and pancre atic tissues be performed in a short time in terms of cost, time, and more information content. It is envisaged that further investigation of Type 2 Diabetes Mellitus disease at the transcriptome level in animal experimental models with grape seed extract and publication of the analysis results in international journals will have a serious interest potential for researchers working on this subject

MARK4 aggravates cardiac dysfunction in mice with STZ-induced diabetic cardiomyopathy by regulating ACSL4-mediated myocardial lipid metabolism

Diabetic cardiomyopathy is a specific type of cardiomyopathy. In DCM, glucose uptake and utilization are impaired due to insulin deficiency or resistance, and the heart relies more heavily on fatty acid oxidation for energy, resulting in myocardial lipid toxicity-related injury. MARK4 is a member of the AMPK-related kinase family, and improves ischaemic heart failure through microtubule detyrosination. However, the role of MARK4 in cardiac regulation of metabolism is unclear. In this study, after successful establishment of a diabetic cardiomyopathy model induced by streptozotocin and a high-fat diet, MARK4 expression was found to be significantly increased in STZ-induced DCM mice. After AAV9-shMARK4 was administered through the tail vein, decreased expression of MARK4 alleviated diabetic myocardial damage, reduced oxidative stress and apoptosis, and facilitated cardiomyocyte mitochondrial fusion, and promoted myocardial lipid oxidation metabolism. In addition, through the RNA-seq analysis of differentially expressed genes, we found that MARK4 deficiency promoted lipid decomposition and oxidative metabolism by downregulating the expression of ACSL4, thus reducing myocardial lipid accumulation in the STZ-induced DCM model.

Diabetic cardiomyopathy: a focus on sodium-glucose cotransporter type 2 inhibitors

Diabetic cardiomyopathy (DCM) is a complication of type 2 diabetes mellitus (T2DM) capable of progressing to the development of symptomatic heart failure (HF), independently of traditional risk factors for it, such as coronary artery disease and hypertension. There is no specific treatment for DCM; however, sodium-glucose cotransporter 2 inhibitors (SGLT2i) are hypoglycemic drugs that act on SGLT2 channels, inhibiting glucose reabsorption in the kidney. In addition, they have cardioprotective effects, which is why their mechanisms at the cardiac level have been studied. According to the results of preclinical studies, SGLT2i act by interfering in the pathophysiology of DCM. Their main effects are: improvement in diastolic function and left ventricular ejection fraction (LVEF), attenuation in the progress of cardiac fibrosis, reduction of oxidative stress and proinflammatory markers. The clinical trials in humans specifically with DCM that have been carried out are limited; however, randomized clinical trials in patients with HF have shown benefits with SGLT2i, regardless of glycemic control in the reduction of hospitalization for HF and mortality from cardiovascular causes. In summary, SGLT2i suggest a treatment in DCM due to their cardiovascular benefits, in preclinical and clinical models of DCM and in the glycemic control of T2DM.

Adropin Treatment Restores Cardiac Function and Metabolic Flexibility in Diabetic Cardiomyopathy

Background: Diabetic cardiomyopathy (DCM) is a major complication of diabetes, and has been recognized as a cause of heart failure independent of other common risk factors. Metabolic inflexibility is a hallmark feature, where increased free fatty acid availability and decreased myocardial glucose uptake lead to an over-reliance on fatty acid oxidation, reducing cardiac work effciency. Energetic ineffciency in diabetic hearts may have profound implications for cardiac function under conditions of increased workload, and therefore therapeutic approaches are warranted. Adropin is a liver- and brain-secreted peptide hormone shown to regulate fuel metabolism in the heart. Adropin levels are significantly reduced in obese and diabetic human subjects, and this decrease is linked to increased adiposity, insulin resistance, and impaired glucose tolerance. Our lab recently showed that acute adropin treatment restores glucose oxidation activity in the hearts of prediabetic obese mice. However, the effects of chronic adropin exposure in the heart remains unknown. Hypothesis: We hypothesize that adropin treatment will exhibit beneficial effects on cardiac fuel metabolism that may lead to long-term improvements in functional output. Methods: We used a preclinical model of DCM to investigate the effects of long-term adropin treatment on cardiac structural and metabolic remodeling. Mice were treated with adropin daily for four weeks, and subjected to echocardiography, glucose tolerance tests, histopathology, RNA-sequencing, and metabolomics. Results: We report that long-term adropin treatment restores normal cardiac structure and metabolic function in the diabetic heart, by inhibiting flux of non-oxidative glycolytic intermediates into the hexosamine biosynthetic pathway. Conclusions: Our findings highlight the therapeutic potential of adropin signaling to attenuate cardiac remodeling in DCM. The project described was supported by NIDDK 1F31DK134089 and NHLBI R01HL147861. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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/TGFβ/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.

Mechanism of Shexiang Tongxin Dropping Pills in treating diabetic cardiomyopathy based on network pharmacology and animal experiments

This study aimed to explore the mechanism of Shexiang Tongxin Dropping Pills(STDP) in treating diabetic cardiomyopathy(DCM) based on network pharmacology, molecular docking, and animal experiments. BATMAN, TCMSP, and GeneCards were searched for the active ingredients and targets of STDP against DCM. STRING and Cytoscape were used to build the protein-protein interaction(PPI) network and "drug-active ingredient-target" network. Gene Ontology(GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analysis of the targets were carried out based on DAVID. The molecular docking of key receptor proteins with corresponding active ingredients was performed using AutoDock Vina. The rat model of DCM was established by a high-fat diet combined with intraperitoneal injection of streptozotocin. Rats were assigned into control, model, low-(20 mg·kg~(-1)) and high-dose(40 mg·kg~(-1)) STDP, and metformin(200 mg·kg~(-1)) groups. After 8 weeks of continuous administration, the cardiac function, myocardial pathological changes, and myocardial collagen fiber deposition of rats in each group were detected by echocardiography, hematoxylin-eosin(HE) staining, and Sirius red staining, respectively. The myocardial hypertrophy was detected by WGA staining. The expression levels of p38 mitogen-activated protein kinase(p38), phosphorylation-p38(p-p38), c-Jun N-terminal kinase(JNK), phosphorylation-JNK(p-JNK), caspase-3, and C-caspase-3 in the myocardial tissue of rats in each group were measured by Western blot. The network pharmacology predicted 199 active ingredients and 1 655 targets of STDP and 463 targets of DCM. One hundred and thirty-four potential targets of STDP for treating DCM were obtained, and the AGE-RAGE signaling pathway in diabetic complications was screened out. Molecular docking results showed that miltirone, dehydromiltirone, and tryptanthrin had strong binding affinity with RAGE. The results of animal experiments confirmed that STDP effectively protected the cardiac function of DCM rats. Compared with the DCM model group, the STDP groups showed significantly down-regulated protein levels of p-p38, p-JNK, and C-caspase-3. To sum up, STDP may protect the cardiac function of DCM rats by regulating the AGE-RAGE signaling pathway.

Identification of key genes of diabetic cardiomyopathy in hiPSCs-CMs based on bioinformatics analysis.

Diabetic cardiomyopathy (DbCM) is one of the most common vascular complications of diabetes, and can cause heart failure and threaten the life of patients. The pathogenesis is complex, and key genes have not fully identified. In this study, bioinformatics analysis was used to predict DbCM-related gene targets. Published datasets from the NCBI Gene Expression Omnibus with accession numbers GSE62203 and GSE197850 were selected for analysis. Differentially expressed genes (DEGs) were identified by the online tool GEO2R. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the DAVID online database. Protein-protein interaction network construction and hub gene identification were performed using STRING and Cytoscape. We used 30mM and 1μM hydrocortisone-stimulated AC16 cells as an in vitro model of diabetic cardiomyopathy. Quantitative real-time PCR (qRT-PCR) was performed to validate the expression levels of hub genes. A total of 73 common DEGs were identified in both datasets, including 47 upregulated and 26 downregulated genes. GO and KEGG pathway enrichment analyses revealed that the DEGs were significantly enriched in metabolism, hypoxia response, apoptosis, cell proliferation regulation, and cytoplasmic and HIF signalling pathways. The top 10 hub genes were LDHA, PGK1, SLC2A1, ENO1, PFKFB3, EGLN1, MYC, PDK1, EGLN3 and BNIP3. In our in vitro study, we found that PGK1, SLC2A1, PFKFB3, EGLN1, MYC, EGLN3 and BNIP3 were upregulated, ENO1 was downregulated, and LDHA was unchanged. Except for PGK1 and ENO1, these hub genes have been previously reported to be involved in DbCM. In summary, we identified DEGs and hub genes and first reported PGK1 and ENO1 in DbCM, which may serve as potential candidate genes for DbCM targeted therapy.

LncRNA HCG18 affects diabetic cardiomyopathy and its association with miR-9-5p/IGF2R axis

This paper aimed to investigate the role of lncRNA HCG18 (HCG18) in the progression of diabetic cardiomyopathy (DCM) and potential mechanisms. Streptozocin (STZ) was used to induce DCM model in rats, which was confirmed by blood glucose concentration, body weight, and HE staining. Myocardial apoptosis was detected by TUNEL. H9c2 cardiomyocytes were used to construct cell models of DCM through treatment of high glucose. The results showed that HCG18 was overexpressed in STZ induced DCM rat model and high glucose induced H9c2 cardiomyocytes. Si-HCG18 significantly increased cell viability, reduced cell apoptosis, attenuated activities of myocardial enzymes and enhanced activities of antioxidant enzymes in STZ induced DM model and high glucose induced H9c2 cardiomyocytes, while the results of upregulation of HCG18, in high glucose induced H9c2 cardiomyocytes, were opposite with that of si-HCG18. MiR-9-5p was a target of HCG18, and which was down-regulated in cardiomyocytes of DCM. The overexpression of miR-9-5p could neutralize the high glucose induced cardiomyocyte injury, and the silence of miR-9-5p could reverse the effect of si-HCG18 on high glucose induced cardiomyocytes. MiR-9-5p could directly target to IGF2R, and IGF2R was overexpressed in cardiomyocytes of DCM. Up-regulation of IGF2R can reverse the protective effect of si-HCG18 on cardiomyocytes. Taken together, HCG18 is significantly increased in cardiomyocytes of DCM. Down-regulation of HCG18 can improve cardiomyocyte injury through miR-9-5p/IGF2R axis in DCM.

GDF11 mitigates high glucose-induced cardiomyocytes apoptosis by inhibiting the ALKBH5-FOXO3-CDR1as/Hippo signaling pathway

Diabetic cardiomyopathy remains a formidable health challenge with a high mortality rate and no targeted treatments. Growth differentiation factor 11 (GDF11) has shown promising effects on cardiovascular diseases; however, its role and the underlying mechanism in regulating diabetic cardiomyopathy remain unclear. In this study, we developed mouse models of diabetic cardiomyopathy using leptin receptor-deficient (db/db) mice and streptozocin-induced C57BL/6 mice. The diabetic cardiomyopathy model mice exhibited apparent structural damage in cardiac tissues and a significant increase in the expression of apoptosis-related proteins. Notably, we observed a significant decreased expression of GDF11 in the myocardium of mice with diabetic cardiomyopathy. Moreover, GDF11 cardiac-specific knock-in mice (transgenic mice) exhibited improved cardiac function and reduced apoptosis. Moreover, exogenous administration of GDF11 mitigated high glucose-induced cardiomyocyte apoptosis. Mechanistically, we demonstrated that GDF11 alleviated high glucose-induced cardiomyocytes apoptosis by inhibiting the activation of the alkylation repair homolog 5 (ALKBH5)-forkhead box group O3a (FOXO3)-cerebellar degeneration-related protein 1 transcript (CDR1as)/Hippo signaling pathway. Consequently, this novel mechanism effectively counteracted myocardial cell apoptosis, providing valuable insights into potential therapeutic strategies for clinical diabetic cardiomyopathy.