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.

[Irisin Alleviates Inflammatory Injury in Diabetic Cardiomyopathy by Regulating NF-κB Pathway].

Objective: To investigate the protective effect of irisin in diabetic cardiomyopathy (DCM) and its mechanism. Methods: A mouse model of DCM was established by high-fat diet combined with the injection of streptozotocin. The mice were assigned to a control group, a DCM group, a DCM+low-dose irisin group, a DCM+high-dose irisin group, and a DCM+pyrrolidine dithiocarbamate (PDTC) (nuclear factor [NF]-κB inhibitor) group. Then, the mice received irisin intervention for 3 weeks after successful modeling. Myocardial morphologic changes were observed by hematoxylin and eosin (HE) staining and Masson staining. The levels of serum creatine kinase (CK) and creatine kinase isoenzyme CK-MB were examined by automatic biochemical analyzer. H9c2 cells were divided into the control group, high glucose and high lipid (HG/HL) group, HG/HL+low-dose irisin group, HG/HL+high-dose irisin group, and HG/HL+PDTC group. CCK-8 assay was conducted to determine cell viability. The expression levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-6 in the myocardial tissue and the cells were determined by ELISA. In addition, nuclear translocation of NF-κB p65 protein and the protein expression level of NF-κB inhibitor protein α (IκBα) in the myocardial tissue and the cells were determined by Western blot. Results: According to the results of animal experiment, low and high doses of irisin could alleviate the pathological injury and fibrosis of myocardial tissue to varying degrees. Irisin inhibited the levels of CK, CK-MB, and inflammatory factors, up-regulated IκB protein expression, and diminished NF-κB nuclear translocation. According to the results of cell experiment, low and high doses of irisin could enhance H9c2 cell viability to varying degrees, increase the level of intracellular IκB proteins, and inhibit NF-κB p65 nuclear translocation and inflammatory factor expression. The changes in these aspects in the DCM+low-dose irisin group and the DCM+high-dose irisin group were similar to those in the DCM+PDTC group. Conclusion: Through inhibiting NF-κB p65 nuclear translocation, irisin may reduce the inflammatory response in the myocardial tissue of DCM mice and H9c2 cells of myocardial injury induced by high glucose and high fat, thereby exerting a protective effect on myocardium.

KLF9 Aggravates Streptozotocin-Induced Diabetic Cardiomyopathy by Inhibiting PPARγ/NRF2 Signalling.

AIMS: Krüppel-like Factor 9 (KLF9) is a transcription factor that regulates multiple disease processes. Studies have focused on the role of KLF9 in the redox system. In this study, we aimed to explore the effect of KLF9 on diabetic cardiomyopathy. METHODS AND RESULTS: Cardiac-specific overexpression or silencing of KLF9 in C57BL/6 J mice was induced with an adeno-associated virus 9 (AAV9) delivery system. Mice were also subjected to streptozotocin injection to establish a diabetic cardiomyopathy model. In addition, neonatal rat cardiomyocytes were used to assess the possible role of KLF9 in vitro by incubation with KLF9 adenovirus or small interfering RNA against KLF9. To clarify the involvement of peroxisome proliferator-activated receptors (PPARγ), mice were subjected to GW9662 injection to inhibit PPARγ. KLF9 was upregulated in the hearts of mice with diabetic cardiomyopathy and in cardiomyocytes. In addition, KLF9 overexpression in the heart deteriorated cardiac function and aggravated hypertrophic fibrosis, the inflammatory response and oxidative stress in mice with diabetic cardiomyopathy. Conversely, cardiac-specific silencing of KLF9 ameliorated cardiac dysfunction and alleviated hypertrophy, fibrosis, the cardiac inflammatory response and oxidative stress. In vitro, KLF9 silencing in cardiomyocytes enhanced inflammatory cytokine release and oxidative stress; KLF9 overexpression increased these detrimental responses. Moreover, KLF9 was found to regulate the transcription of PPARγ, which suppressed the expression and nuclear translocation of nuclear Factor E2-related Factor 2 (NRF2). In mice injected with a PPARγ inhibitor, the protective effects of KLF9 knockdown on diabetic cardiomyopathy were counteracted by GW9662 injection. CONCLUSIONS: KLF9 aggravates cardiac dysfunction, the inflammatory response and oxidative stress in mice with diabetic cardiomyopathy. KLF9 may become a therapeutic target for diabetic cardiomyopathy.

Electrocardiographic and histopathological characterizations of diabetic cardiomyopathy in rats.

Diabetes is a clinical condition that is associated with insulin deficiency and hyperglycemia. Cardiomyopathy, retinopathy, neuropathy, and nephropathy are well known complications of the elevated blood glucose. Diabetic cardiomyopathy is a clinical disorder that is associated with systolic and diastolic dysfunction along with cardiac fibrosis, inflammation, and elevated oxidative stress. In this study, diabetes was induced by intraperitoneal injection of streptozotocin (STZ) 50 mg/kg. We determined the plasma levels of cardiac troponin-T (cTnT) and creatinine kinase MB (CK-MB) by ELISA. Diabetic rats showed abnormal cardiac architecture and increased collagen production. Significant elevation in ST-segment, prolonged QRS, and QT-intervals and increased ventricular rate were detected. Additionally, diabetic rats showed a prolongation in P wave duration and atrial tachyarrhythmia was observed. Plasma levels of cTnT and CK-MB were elevated. In conclusion, these electrocardiographic changes (elevated ST-segment, prolonged QT interval, and QRS complex, and increased heart rate) along with histopathological changes and increased collagen formation could be markers for the development of diabetic cardiomyopathy in rats.

The effects of exercise training on cardiac matrix metalloproteinases activity and cardiac function in mice with diabetic cardiomyopathy.

AIM: To evaluate the effects of exercise training (ET) on cardiac extracellular matrix (ECM) proteins homeostasis and cardiac dysfunction in mice with diabetic cardiomyopathy. METHODS: Thirty-six male C57BL/6 mice were randomized into 3 groups for 8 weeks (12mice/group): Diabetic control-DC: Diabetes was induced by single streptozotocin injection (200 mg/kg i.p.); Diabetic exercise-DE: Diabetic mice underwent ET program on motorized-treadmill (6-times/week, 60min/session); Non-diabetic control-NDC: Vehicle-treated, sedentary, non-diabetic mice served as controls. Before euthanasia, all groups underwent transthoracic echocardiography (TTE). Post-mortem, left-ventricle (LV) samples were histologically analysed for ECM proteins (collagen, elastin), matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). RESULTS: DC group showed significantly higher cardiac contents of collagen and MMP-9 and lower elastic concentration than NDC (p < 0.001). The implementation of ET completely outweighed those diabetes-induced changes (DE vs NDC, p > 0.05). TIMP-1 levels significantly increased across all groups (DC: 18.98 ± 3.47%, DE: 24.24 ± 2.36%, NDC: 46.36 ± 5.91%; p < 0.05), while MMP-9/TIMP-1 ratio followed a reverse pattern. ET tended to increase MMP-2 concentrations versus DC (p = 0.055), but did not achieve non-diabetic levels (p < 0.05). TIMP-2 cardiac concentrations remained unaltered throughout the study (p > 0.05). Importantly, ET ameliorated both LV end-systolic internal diameter (LVESD) (p < 0.001) and the percentage of LV fractional shortening (FS%) (p = 0.006) compared to DC. Despite that favorable effect, the cardiac function level of DE group remained worse than NDC group (%FS: p = 0.002; LVESD: p < 0.001). CONCLUSION: Systemic ET may favorably change ECM proteins, MMP-9 and TIMP-1 cardiac concentrations in mice with diabetic cardiomyopathy. Those results were associated with partial improvement of echocardiography-assessed cardiac function, indicating a therapeutic effect of ET in diabetic cardiomyopathy.

Peroxiredomin-4 ameliorates lipotoxicity-induced oxidative stress and apoptosis in diabetic cardiomyopathy.

Diabetic cardiomyopathy (DCM), one severe complication in the diabetes, leads to high mortality in the diabetic patients. However, the understanding of molecular mechanisms underlying DCM is far from completion. Herein, we investigated the disease-related differences in the proteomes of DCM based on db/db mice and verified the protective roles of peroxiredoxin-4 (Prdx4) in H9c2 cardiomyocytes treated by palmitic acid (PA). Fasting blood glucose (FBG) and cardiac function was detected in the 6-month-old control and diabetic mice. The hearts were then collected and analyzed by a coupled label-free and mass spectrometry approach. In vivo investigation indicated that body weight and FBG of db/db mice markedly increased, and diabetic heart exhibited obvious cardiac hypertrophy and lipid droplet accumulation, and cardiac dysfunction as is indicated by the increases of left ventricle posterior wall thickness in systole (LVPWd) and diastole (LVPWs), and reduction of fractional shortening (FS). We used proteomic analysis and then detected a grand total of 2636 proteins. 175 differentially expressed proteins (DEPs) were markedly detected in the diabetic heart. Thereinto, Prdx4 was markedly down-regulated in the diabetic heart. In vitro experiments revealed that 250 µM PA significantly inhibited viability of H9c2 cell. PA induced much accumulation of lipid droplet in cardiomyocytes and resulted in an increase of mRNA expressions of lipogenic genes (FASN and SCD1) and cardiac hypertrophic genes. Additionally, protein level of Prdx4 evidently reduced in the PA-treated H9c2 cell. It was further found that shRNA-mediated Prdx4 knockdown exacerbated PA-induced oxidative stress and cardiomyocyte apoptosis, whereas overexpressing Prdx4 in the H9c2 cells noteworthily limited PA-induced ROS generation and cardiomyocytes apoptosis. These data collectively reveal the essential role of abnormal Prdx4 in pathological alteration of DCM, and provide potentially therapeutic target for the prevention of DCM.

Engineered cardiac tissues: a novel in vitro model to investigate the pathophysiology of mouse diabetic cardiomyopathy.

Rodent diabetic models, used to understand the pathophysiology of diabetic cardiomyopathy (DCM), remain several limitations. Engineered cardiac tissues (ECTs) have emerged as robust 3D in vitro models to investigate structure-function relationships as well as cardiac injury and repair. Advanced glycation end-products (AGEs), produced through glycation of proteins or lipids in response to hyperglycemia, are important pathogenic factor for the development of DCM. In the current study, we developed a murine-based ECT model to investigate cardiac injury produced by AGEs. We treated ECTs composed of neonatal murine cardiac cells with AGEs and observed AGE-related functional, cellular, and molecular alterations: (1) AGEs (150 µg/mL) did not cause acute cytotoxicity, which displayed as necrosis detected by medium LDH release or apoptosis detected by cleaved caspase 3 and TUNEL staining, but negatively impacted ECT function on treatment day 9; (2) AGEs treatment significantly increased the markers of fibrosis (TGF-ß, α-SMA, Ctgf, Collagen I-α1, Collagen III-α1, and Fn1) and hypertrophy (Nppa and Myh7); (3) AGEs treatment significantly increased ECT oxidative stress markers (3-NT, 4-HNE, HO-1, CAT, and SOD2) and inflammation response markers (PAI-1, TNF-α, NF-κB, and ICAM-1); and (4) AGE-induced pathogenic responses were all attenuated by pre-application of AGE receptor antagonist FPS-ZM1 (20 µM) or the antioxidant glutathione precursor N-acetylcysteine (5 mM). Therefore, AGEs-treated murine ECTs recapitulate the key features of DCM's functional, cellular and molecular pathogenesis, and may serve as a robust in vitro model to investigate cellular structure-function relationships, signaling pathways relevant to DCM and pharmaceutical intervention strategies.

Cardioprotective role of GTS-21 by attenuating the TLR4/NF-κB pathway in streptozotocin-induced diabetic cardiomyopathy in rats.

The cholinergic anti-inflammatory pathway (CAP) was investigated in a variety of inflammatory conditions and constitutes a valuable line in their treatment. In the current study, we investigated the anti-inflammatory effect of GTS-21 (GTS) as a partial selective α7 nicotinic acetylcholine receptor (α7-nAchR) agonist in diabetic cardiomyopathy model in rats. This mechanism was elaborated to study whether it could alleviate the electrocardiographic, histopathological, and molecular levels of Toll-like receptor 4 (TLR4)/nuclear factor κB (NF-κB) pathway proteins. Diabetes was induced by the injection of streptozotocin (STZ) (50 mg/kg). Diabetic rats were treated with GTS (1 or 2 mg/kg/day), methyllycaconitine (MLA), a selective α7-nAchR antagonist (2 mg/kg/day) plus GTS (2 mg/kg/day), or the vehicle. All treatments were given by the intraperitoneal route. Ventricular rate and different electrocardiograph (ECG) anomalies were detected. Plasma levels of cardiac troponin T (cTnT) and creatine kinase MB (CK-MB) were measured by ELISA. Additionally, we elucidated the levels of several proteins involved in the TLR4/NF-κB pathway. Cardiac levels of TLR4 and phosphorylated protein kinase B (p-Akt) were detected by ELISA. The cardiac expression of myeloid differentiation primary response 88 (Myd88), tumor necrosis factor receptor-associated factor 6 (TRAF6), NF-κB, interleukin 1ß (IL-1ß), and active caspase-1 were evaluated by immunohistochemical staining. Finally, the cardiac levels of interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) were determined by ELISA. Diabetic rats showed (i) ECG signs of cardiomyopathy such as significant ST segment elevations, prolonged QRS, QT intervals, and ventricular tachycardia; (ii) increased plasma levels of cTnT and CK-MB; (iii) increased expression of cardiac TLR4; (iv) elevated immunohistochemical expression of cardiac, Myd88, TRAF6, and NF-κB; (v) diminution in the cardiac expression of p-Akt; and (vi) adaptive increases in cardiac expression of TNF-α and IL-6. These effects were ameliorated in diabetic rats treated with both doses of GTS. Pretreatment with MLA did not completely reverse the ameliorative effect of GTS on cTnT, TRAF6, TNF-α, and IL-6, thereby reinforcing the presence of possible α7-nAchR-independent mechanisms. The activation of α7-nAchR with GTS offers a promising prophylactic strategy for diabetic cardiomyopathy by attenuating the TLR4/NF-κB pathway.

[Spermine attenuates high glucose-induced myocardial fibrosis by regulating the cell cycle].

Objective: To investigate the protective effect of spermine (Sp) on diabetic cardiomyopathy (DCM) and high glucose-induced cardiac fibroblasts (CFs), and to explore its mechanism.Methods: ①Animal experiments: 24 male Wistar rats were randomly divided into control group, type 1 diabetes group (TID) and spermine group (TID+Sp, each group n=8). TID rats were induced by streptozocin (STZ, 60 mg/kg), and TID+Sp rat were pretreated with spermine (Sp, 5 mg/(kg·d)) for 2 weeks before STZ injection. After 12 weeks of modeling, blood glucose, insulin levels, ejection fraction (EF) and shortening fraction (FS) were measured, and Masson staining and Sirius red staining were performed in the rat cardiac tissues. ②Cell experiments: primary CFs were extracted from newborn (1-3 d) Wistar rat hearts, and were randomly divided into control group, high-glucose group (HG) and HG+Sp group (n=6 per group). HG group was treated with 40 mmol/L glucose, and the HG+Sp group was pretreated with 5 µmol/L Sp for 30 min before HG treatment. The cell viability of CFs was detected by CCK8, the content of collagen in culture medium was analyzed by ELISA, and protein expressions of cell cycle related proteins (PCNA, CyclinD1 and P27) were detected by Western blot. Results: Compared with control group, the blood glucose and collagen content were increased, and the insulin level and heart function were decreased in the T1D group. Meanwhile, HG induced an increasing of the cell viability, the collagen content in the medium and the expressions of PCNA and CyclinD1, while the expression of P27 was down-regulated. Spermine could reduce the above changes, manifested as improving the cardiac function, regulating the expression of cyclin and reducing the level of myocardial fibrosis. Conclusion: Spermine can alleviate myocardial fibrosis in diabetic cardiomyopathy, which mechanism is related to the regulation of cell cycle.

Early changes in myocyte contractility and cardiac function in streptozotocin-induced type 1 diabetes in rats.

Diabetes can elicit direct deleterious effects on the myocardium, independent of coronary artery disease or hypertension. These cardiac disturbances are termed diabetic cardiomyopathy showing increased risk of heart failure with or without reduced ejection fraction. Presently, there is no specific treatment for this type of cardiomyopathy and in the case of type I diabetes, it may start in early childhood independent of glycemic control. We hypothesized that alterations in isolated myocyte contractility and cardiac function are present in the early stages of experimental diabetes in rats before overt changes in myocardium structure occur. Diabetes was induced by single-dose injection of streptozotocin (STZ) in rats with data collected from control and diabetic animals 3 weeks after injection. Left ventricle myocyte contractility was measured by single-cell length variation under electrical stimulation. Cardiac function and morphology were studied by high-resolution echocardiography with pulsed-wave tissue Doppler imaging (TDI) measurements and three-lead surface electrocardiogram. Triglycerides, cholesterol and liver enzyme levels were measured from plasma samples obtained from both groups. Myocardial collagen content and perivascular fibrosis of atria and ventricle were studied by histological analysis after picrosirius red staining. Diabetes resulted in altered contractility of isolated cardiac myocytes with increased contraction and relaxation time intervals. Echocardiography showed left atrium dilation, increased end-diastolic LV and posterior wall thickness, with reduced longitudinal systolic peak velocity (S') of the septum mitral annulus at the apical four-chamber view obtained by TDI. Triglycerides, aspartate aminotransferase and alkaline phosphatase were elevated in diabetic animals. Intertitial collagen content was higher in atria of both groups and did not differ among control and diabetic animals. Perivascular intramyocardial arterioles collagen did not differ between groups. These results suggest that alterations in cardiac function are present in the early phase in this model of diabetes type 1 and occur before overt changes in myocardium structure appear as evaluated by intersticial collagen deposition and perivascular fibrosis of intramyocardial arterioles.

A study of the effects of SGLT-2 inhibitors on diabetic cardiomyopathy through miR-30d/KLF9/VEGFA pathway.

OBJECTIVE: The aim of this study was to observe the protective effects of SGLT-2 inhibitors on diabetic cardiomyopathy. MATERIALS AND METHODS: After constructing a diabetic rat model, the effects of SGLT-2 (Sodium-Glucose Cotransporter-2) inhibitors and miR-30d on cardiac function in rats were investigated by cardiac echocardiography, hematoxylin-eosin (HE) staining, and immunohistochemical methods. At the same time, changes in autophagy levels in rats were detected by Western blot (WB) experiments. RESULTS: SGLT-2 inhibitors improved the cardiac function of diabetic rats, and alleviated the pathological damage of myocardial tissue. Besides, knocking down miR-30d prevented the decrease of cardiac function in diabetic rats. Moreover, miR-30d could regulate the expression of the KLF9/VEGFA pathway and inhibit autophagy in rats. CONCLUSIONS: SGLT-2 inhibitors can regulate the autophagy level in diabetic rats through the miR-30d/KLF9/VEGFA pathway, thereby improving cardiac function.

Diabetic animal fed with high-fat diet prevents the protective effect of myocardial ischemic preconditioning effect in isolated rat heart perfusion model.

Diabetic heart (diabetes mellitus [DM]) has been shown to attenuate the beneficial effect of ischemic preconditioning (IPC) in rat heart. But the effect of IPC on diabetic rat heart that develops myopathy remains unclear. This study was designed to test the impact of IPC on diabetic cardiomyopathy (DCM) rat heart. Male Wistar rats were grouped as (a) normal, (b) DM (streptozotocin: 65 mg/kg; fed with normal diet), and (c) DCM (streptozotocin: 65 mg/kg; fed with high-fat diet). Isolated rat hearts from each group were randomly subjected to (a) normal perfusion, (b) ischemia-reperfusion (I/R), and (c) IPC procedure. At the end of the perfusion experiments, hearts were analyzed for injury, contractile function, mitochondrial activity, and oxidative stress. The results obtained from hemodynamics, cardiac injury markers, and caspase-3 activity showed that DCM rat displayed prominent I/R-associated cardiac abnormalities than DM rat heart. But the deteriorated physiological performance and cardiac injury were not recovered in both DM and DCM heart by IPC procedure. Unlike normal rat heart, IPC did not reverse mitochondrial dysfunction (determined by electron transport chain enzymes activity, ATP level, and membrane integrity, expression levels of genes like PGC-1ɑ, GSK3ß, complex I, II, and V) in DCM and DM rat heart. The present study demonstrated that IPC failed to protect I/R-challenged DCM rat heart, and the underlying pathology was associated with deteriorated mitochondrial function.

Sodium-glucose cotransporter 2 inhibitor Dapagliflozin attenuates diabetic cardiomyopathy.

BACKGROUND: Diabetes mellitus type 2 (DM2) is a risk factor for developing heart failure but there is no specific therapy for diabetic heart disease. Sodium glucose transporter 2 inhibitors (SGLT2I) are recently developed diabetic drugs that primarily work on the kidney. Clinical data describing the cardiovascular benefits of SGLT2Is highlight the potential therapeutic benefit of these drugs in the prevention of cardiovascular events and heart failure. However, the underlying mechanism of protection remains unclear. We investigated the effect of Dapagliflozin-SGLT2I, on diabetic cardiomyopathy in a mouse model of DM2. METHODS: Cardiomyopathy was induced in diabetic mice (db/db) by subcutaneous infusion of angiotensin II (ATII) for 30 days using an osmotic pump. Dapagliflozin (1.5 mg/kg/day) was administered concomitantly in drinking water. Male homozygous, 12-14 weeks old WT or db/db mice (n = 4-8/group), were used for the experiments. Isolated cardiomyocytes were exposed to glucose (17.5-33 mM) and treated with Dapagliflozin in vitro. Intracellular calcium transients were measured using a fluorescent indicator indo-1. RESULTS: Angiotensin II infusion induced cardiomyopathy in db/db mice, manifested by cardiac hypertrophy, myocardial fibrosis and inflammation (TNFα, TLR4). Dapagliflozin decreased blood glucose (874 ± 111 to 556 ± 57 mg/dl, p < 0.05). In addition it attenuated fibrosis and inflammation and increased the left ventricular fractional shortening in ATII treated db/db mice. In isolated cardiomyocytes Dapagliflozin decreased intracellular calcium transients, inflammation and ROS production. Finally, voltage-dependent L-type calcium channel (CACNA1C), the sodium-calcium exchanger (NCX) and the sodium-hydrogen exchanger 1 (NHE) membrane transporters expression was reduced following Dapagliflozin treatment. CONCLUSION: Dapagliflozin was cardioprotective in ATII-stressed diabetic mice. It reduced oxygen radicals, as well the activity of membrane channels related to calcium transport. The cardioprotective effect manifested by decreased fibrosis, reduced inflammation and improved systolic function. The clinical implication of our results suggest a novel pharmacologic approach for the treatment of diabetic cardiomyopathy through modulation of ion homeostasis.

Knockdown of long non-coding RNA LUCAT1 reverses high glucose-induced cardiomyocyte injury via targeting CYP11B2.

OBJECTIVE: Diabetic cardiomyopathy (DCM) is one of the major complications in patients with diabetes mellitus. Recently, long noncoding RNAs (lncRNAs) have been well concerned for their roles in the progression of multiple diseases, including DCM. In this research, we aimed to explore the role of lncRNA LUCAT1 in cardiomyocyte injury and apoptosis induced by high glucose (HG) in vitro. MATERIALS AND METHODS: High glucose-induced (HG-induced) AC16 cardiomyocytes transfected with LUCAT1 shRNA were constructed. LUCAT1 expression was detected by real-time quantitative polymerase chain reaction (RT-qPCR). Subsequently, cell proliferation and cell apoptosis were detected after LUCAT1 knockdown in HG-induced AC16 cells. Moreover, RT-qPCR and Western blot assay were performed to explore the potential underlying mechanism of LUCAT1 in DCM. RESULTS: The expression of LUCAT1 was significantly upregulated in HG-treated AC16 cardiomyocytes. Moreover, knockdown of LUCAT1 could reverse cardiomyocyte injury and apoptosis through downregulating CYP11B2. CONCLUSIONS: We first demonstrated that knockdown of LUCAT1 could reverse HG-induced cardiomyocyte injury by down-regulating CYP11B2. Our findings might offer a new direction for interpreting the mechanism of DCM development.

Calhex231 Alleviates High Glucose-Induced Myocardial Fibrosis via Inhibiting Itch-Ubiquitin Proteasome Pathway in Vitro.

Diabetic cardiomyopathy (DCM) is a major complication of diabetes, and features myocardial fibrosis as its main pathological feature. Calcium sensing receptor (CaSR) is a G protein-coupled receptor, which involves in myocardial fibrosis by regulation of calcium homeostasis. Calhex231, the CaSR inhibitor, is not clear whether it regulates myocardial fibrosis in DCM. In the present study, type 1 diabetic (T1D) rats and primary neonatal rat cardiac fibroblasts were used to observe the role of Calhex231. In vivo experiments showed that in the T1D group, contractile dysfunction and the deposition of collagen I and III were obvious after 12 weeks. In vitro experiments, we found that high glucose (HG) could increase the expression of CaSR, α-smooth muscle actin (α-SMA), transforming growth factor-ß1 (TGF-ß1) collagen I/III, matrix metalloproteinase-2 (MMP-2), MMP9, along with cardiac fibroblast migration and proliferation. We further demonstrated that CaSR activation increased intracellular Ca2+ concentration and upregulated the expression of Itch (atrophin-1 interacting protein 4), which resulted in increasing the ubiquitination levels of Smad7 and upregulating the expression of p-Smad2, p-Smad3. However, treatment with Calhex231 clearly inhibited the above-mentioned changes. Collectively these results suggest that Calhex231 could inhibit Itch-ubiquitin proteasome and TGF-ß1/Smads pathways, and then depress the proliferation of cardiac fibroblasts, along with the reduction deposition of collagen, alleviate glucose-induced myocardial fibrosis. Our findings indicate an important new mechanism for myocardial fibrosis, and suggest Calhex231 would be a new therapeutic agent for the treatment of DCM.

Tetrahydrocurcumin Ameliorates Diabetic Cardiomyopathy by Attenuating High Glucose-Induced Oxidative Stress and Fibrosis via Activating the SIRT1 Pathway.

Hyperglycemia-induced oxidative stress and fibrosis play a crucial role in the development of diabetic cardiomyopathy (DCM). Tetrahydrocurcumin (THC), a major bioactive metabolite of natural antioxidant curcumin, is reported to exert even more effective antioxidative and superior antifibrotic properties as well as anti-inflammatory and antidiabetic abilities. This study was designed to investigate the potential protective effects of THC on experimental DCM and its underlying mechanisms, pointing to the role of high glucose-induced oxidative stress and interrelated fibrosis. In STZ-induced diabetic mice, oral administration of THC (120 mg/kg/d) for 12 weeks significantly improved the cardiac function and ameliorated myocardial fibrosis and cardiac hypertrophy, accompanied by reduced reactive oxygen species (ROS) generation. Mechanically, THC administration remarkably increased the expression of the SIRT1 signaling pathway both in vitro and in vivo, further evidenced by decreased downstream molecule Ac-SOD2 and enhanced deacetylated production SOD2, which finally strengthened antioxidative stress capacity proven by repaired activities of SOD and GSH-Px and reduced MDA production. Additionally, THC treatment accomplished its antifibrotic effect by depressing the ROS-induced TGFß1/Smad3 signaling pathway followed by reduced expression of cardiac fibrotic markers α-SMA, collagen I, and collagen III. Collectively, these finds demonstrated the therapeutic potential of THC treatment to alleviate DCM mainly by attenuating hyperglycemia-induced oxidative stress and fibrosis via activating the SIRT1 pathway.

FBXL10 regulates cardiac dysfunction in diabetic cardiomyopathy via the PKC ß2 pathway.

Diabetic cardiomyopathy (DCM) is a condition associated with significant structural changes including cardiac tissue necrosis, localized fibrosis, and cardiomyocyte hypertrophy. This study sought to assess whether and how FBXL10 can attenuate DCM using a rat streptozotocin (STZ)-induced DCM model system. In the current study, we found that FBXL10 expression was significantly decreased in diabetic rat hearts. FBXL10 protected cells from high glucose (HG)-induced inflammation, oxidative stress, and apoptosis in vitro. In addition, FBXL10 significantly activated PKC ß2 signaling pathway in H9c2 cells and rat model. The cardiomyocyte-specific overexpression of FBXL10 at 12 weeks after the initial STZ administration attenuated oxidative stress and inflammation, thereby reducing cardiomyocyte death and preserving cardiac function in these animals. Moreover, FBXL10 protected against DCM via activation of the PKC ß2 pathway. In conclusion, FBXL has the therapeutic potential for the treatment of DCM.

Inhibition of JNK and p38 MAPK-mediated inflammation and apoptosis by ivabradine improves cardiac function in streptozotocin-induced diabetic cardiomyopathy.

Inflammation plays a critical role in the development of diabetic cardiomyopathy (DCM), which has been identified as a major predisposing factor for heart failure in diabetic patients. Previous studies indicated that ivabradine (a specific agent for heart rate [HR] reduction) has anti-inflammatory properties, but its role in DCM remains unknown. This study investigated whether ivabradine exerts a therapeutic effect in DCM. C57BL/6J mice were injected intraperitoneally with streptozotocin (STZ) to induce diabetes; then administered with ivabradine or saline (control). After 12 weeks, the surviving mice were analyzed to determine the cardioprotective effect of ivabradine against DCM. Although treatment with ivabradine did not affect blood glucose levels, it attenuated tumor necrosis factor-α, interleukin-1ß, and interleukin-6 messenger RNA (mRNA) expression, inhibited c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) activation, reduced histological abnormalities, myocardial apoptosis and collagen deposition, and improved cardiac function in the diabetic mice. Interestingly, the anti-inflammatory and antiapoptotic properties of ivabradine, but not its inhibitory effect on JNK and p38 MAPK, were observed in high-glucose-cultured neonatal rat ventricular cardiomyocytes. Attenuating inflammation and apoptosis via intramyocardial injection of lentiviruses carrying short hairpin RNA targeting JNK and p38 MAPK validated that the anti-inflammatory and antiapoptotic effects of ivabradine were partly attributed to JNK and p38 MAPK inactivation in diabetic mice. In summary, these data indicate that ivabradine-mediated improvement of cardiac function in STZ-induced diabetic mice may be partly attributed to inhibition of JNK/p38 MAPK-mediated inflammation and apoptosis, which is dependent on the reduction in HR.

Effects of resveratrol on regulation on UCP2 and cardiac function in diabetic rats.

Mitochondrial dysfunction is essential in the development and prognosis of diabetic cardiomyopathy (DCM). Resveratrol (RES) is thought as a mitochondrial protector. In this study, we hypothesized that RES may ameliorate mitochondrial function and consequently improve cardiac function in diabetic rats, and uncoupling protein 2 (UCP2) was involved in the protective effects of RES on DCM. Thirty rats were divided into three groups: normal control, DCM, and DCM+RES groups. DCM was induced by high-fat diet and streptozotocin (STZ) intraperitoneal injection, the rats in DCM+RES group received RES gavage for 16 weeks. RES improved the insulin resistance, and reduced the level of triglyceride, cholesterol, and low density lipoprotein cholesterol (LDLc) in DCM rats (all P < 0.05). Echocardiographic and hemodynamic studies revealed that RES treatment reversed the impaired diastolic and systolic cardiac function in DCM rats. Meanwhile, RES improved myocardial structural disorder and fibrosis, reserved mitochondrial membrane potential level (P < 0.05), and suppressed myocardial apoptosis in DCM rats (P < 0.05). Myocardial mitochondrial respiratory enzyme activities were improved by RES treatment in DCM rats (P < 0.05), accompanied with attenuated reactive oxygen species (ROS) generation (P < 0.05). The expression of UCP2 was further increased by RES treatment both in the myocardium of DCM rats (P < 0.05) and in the H9c2 cardiomyocytes incubated with high-glucose (P < 0.05). The protective effects of RES on high glucose-induced ROS generation, MPTP opening, Cyto c release, and cell apoptosis were all blunted by inhibiting the expression of UCP2 (all P < 0.05). In conclusion, RES treatment improved cardiac function and inhibited cardiomyocyte apoptosis, involving in ameliorating mitochondrial function in diabetic rats. UCP2 mediated the protective effects of RES on diabetic hearts.

Apoptosis of cardiomyocytes in diabetic cardiomyopathy involves overexpression of glycogen synthase kinase-3ß.

To evaluate the role of glycogen synthase kinase-3ß (GSK-3ß) in the apoptosis of cardiomyocytes in diabetic cardiomyopathy (DCM). Diabetes mellitus (DM) in rats was induced by intraperitoneal injection of 1% streptozotocin (STZ), and lithium chloride (LiCl) was used to decrease the expression of GSK-3ß. Hematoxylin/eosin (HE) staining and the terminal deoxyribonucleotide transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay was conducted to evaluate the pathological injury and apoptosis of cardiomyocytes respectively. Western blot was applied to detect the protein expressions of Cleaved-caspase 3, caspase 3, Bax and Bcl-2 in rat cardiomyocytes. Real-time polymerase chain reaction (RT-PCR) was applied to detect the gene expressions of phosphoinositide 3-kinases (PI3K), Akt, and GSK-3ß in rat cardiomyocytes. DM-induced cardiomyocyte injuries, which were presented as capillary basement membrane thickening, interstitial fibrosis, cardiomyocyte hypertrophy and necrosis in HE staining and increased apoptosis detected by TUNEL assay. When comparing with the control group, the mRNA expression of PI3K and Akt in DM group obviously decreased but the mRNA expression of GSK-3ß obviously elevated (P < 0.05). In addition, the ratio of Cleaved-caspase 3/caspase 3 and Bax/Bcl-2 were notably increased in DM group compared with control group (P < 0.05). LiCl, as an inhibitor of GSK-3 apparently reduced the expression of GSK-3ß mRNA (P < 0.05) but not the PI3K and Akt comparing with the DM group. LiCl also attenuated the myocardial injury and apoptosis induced by DM. The myocardial injury induced by DM is associated with the up-regulation of GSK-3ß. LiCl inhibited the expression of GSK-3ß and myocardial apoptosis in diabetic myocardium.