Diabetic Cardiomyopathy In Mice Research Articles

Carvacrol Attenuates Diabetic Cardiomyopathy by Modulating the PI3K/AKT/GLUT4 Pathway in Diabetic Mice.

Background: Diabetic cardiomyopathy (DCM), a common complication of diabetes mellitus, eventually leads to heart failure. Carvacrol is a food additive with diverse bioactivities. We aimed to study the protective effects and mechanisms of carvacrol in DCM. Methods: We used a streptozotocin-induced and db/db mouse model of types 1 and 2 diabetes mellitus (T1DM and T2DM), respectively. Both study groups received daily intraperitoneal injections of carvacrol for 6 weeks. Cardiac remodeling was evaluated by histological analysis. We determined gene expression of cardiac remodeling markers (Nppa and Myh7) by quantitative real-time PCR and cardiac function by echocardiography. Changes of PI3K/AKT signaling were determined with Western blotting. GLUT4 translocation was evaluated by Western blotting and immunofluorescence staining. Results: Compared with control mice, both T1DM and T2DM mice showed cardiac remodeling and left ventricular dysfunction. Carvacrol significantly reduced blood glucose levels and suppressed cardiac remodeling in mice with T1DM and T2DM. At the end of the treatment period, both T1DM and T2DM mice showed lesser cardiac hypertrophy, Nppa and Myh7 mRNA expressions, and cardiac fibrosis, compared to mice administered only the vehicle. Moreover, carvacrol significantly restored PI3K/AKT signaling, which was impaired in mice with T1DM and T2DM. Carvacrol increased levels of phosphorylated PI3K, PDK1, AKT, and AS160 and inhibited PTEN phosphorylation in mice with T1DM and T2DM. Carvacrol treatment promoted GLUT4 membrane translocation in mice with T1DM and T2DM. Metformin was used as the positive drug control in T2DM mice, and carvacrol showed comparable effects to that of metformin on cardiac remodeling and modulation of signaling pathways. Conclusion: Carvacrol protected against DCM in mice with T1DM and T2DM by restoring PI3K/AKT signaling-mediated GLUT4 membrane translocation and is a potential treatment of DCM.

Inhibition of long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 attenuates high glucose-induced cardiomyocyte apoptosis via regulation of miR-181a-5p.

Diabetic cardiomyopathy (DCM) is one of the cardiovascular complications of diabetes mellitus independent of hypertension, coronary disease, and other heart diseases. The development of DCM is multifactorial and hard to detect at an early stage. Long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (Malat1) is emerging as a regulator of DCM, the underlying mechanism of its role in DCM has not been elaborated yet. In this study, we established a mouse DCM model via streptozocin injection as evidenced by cell hypertrophy and cell apoptosis of myocardial tissue, and found that Malat1 expression was upregulated in the myocardium in DCM mice. Meanwhile, elevated expression of pro-apoptotic factors p53, p21, cleaved caspase 3, cleaved caspase 9 and BAX, and down-regulation of anti-apoptotic BCL-2 were observed in DCM myocardium. We further investigated the effect of Malat1 on cardiomyocytes under high glucose condition by silencing Malat1 with its specific short-hairpin RNA. Like in vivo, expression of Malat1 in cardiomyocytes was notably raised, remarkable cell apoptosis and changes in apoptosis-related factors were also observed following high glucose treatment. Besides, we validated that Malat1 acted as a sponge of miR-181a-5p. Inhibition of miR-181a-5p could, at least partially, abolish Malat1 knockdown-induced alteration in cardiomyocytes. In addition, p53, a critical regulator of apoptosis, was validated to be a downstream target of miR-181a-5p. In summary, our findings reveal that Malat1 knockdown attenuates high glucose-induced cardiomyocyte apoptosis via releasing miR-181a-5p, and this mechanism may provide us with new diagnosis target of DCM.

4‐O‐methylhonokiol protects against diabetic cardiomyopathy in type 2 diabetic mice by activation of AMPK‐mediated cardiac lipid metabolism improvement

Diabetic cardiomyopathy (DCM) is characterized by increased left ventricular mass and wall thickness, decreased systolic function, reduced ejection fraction (EF) and ultimately heart failure. The 4‐O‐methylhonokiol (MH) has been isolated mainly from the bark of the root and stem of Magnolia species. In this study, we aimed to elucidate whether MH can effectively prevent DCM in type 2 diabetic (T2D) mice and, if so, whether the protective response of MH is associated with its activation of AMPK‐mediated inhibition of lipid accumulation and inflammation. A total number of 40 mice were divided into four groups: Ctrl, Ctrl + MH, T2D, T2D + MH. Five mice from each group were sacrificed after 3‐month MH treatment. The remaining animals in each group were kept for additional 3 months without further MH treatment. In T2D mice, the typical DCM symptoms were induced as expected, reflected by decreased ejection fraction and lipotoxic effects inducing lipid accumulation, oxidative stress, inflammatory reactions, and final fibrosis. However, these typical DCM changes were significantly prevented by the MH treatment immediately or 3 months after the 3‐month MH treatment, suggesting MH‐induced cardiac protection from T2D had a memory effect. Mechanistically, MH cardiac protection from DCM may be associated with its lipid metabolism improvement by the activation of AMPK/CPT1‐mediated fatty acid oxidation. In addition, the MH treatment of DCM mice significantly improved their insulin resistance levels by activation of GSK‐3β. These results indicate that the treatment of T2D with MH effectively prevents DCM probably via AMPK‐dependent improvement of the lipid metabolism.

The Role of Heme Oxygenase 1 in the Protective Effect of Caloric Restriction against Diabetic Cardiomyopathy.

Type 2 diabetes mellitus (DM2) leads to cardiomyopathy characterized by cardiomyocyte hypertrophy, followed by mitochondrial dysfunction and interstitial fibrosis, all of which are exacerbated by angiotensin II (AT). SIRT1 and its transcriptional coactivator target PGC-1α (peroxisome proliferator-activated receptor-γ coactivator), and heme oxygenase-1 (HO-1) modulates mitochondrial biogenesis and antioxidant protection. We have previously shown the beneficial effect of caloric restriction (CR) on diabetic cardiomyopathy through intracellular signaling pathways involving the SIRT1–PGC-1α axis. In the current study, we examined the role of HO-1 in diabetic cardiomyopathy in mice subjected to CR. Methods: Cardiomyopathy was induced in obese diabetic (db/db) mice by AT infusion. Mice were either fed ad libitum or subjected to CR. In an in vitro study, the reactive oxygen species (ROS) level was determined in cardiomyocytes exposed to different glucose levels (7.5–33 mM). We examined the effects of Sn(tin)-mesoporphyrin (SnMP), which is an inhibitor of HO activity, the HO-1 inducer cobalt protoporphyrin (CoPP), and the SIRT1 inhibitor (EX-527) on diabetic cardiomyopathy. Results: Diabetic mice had low levels of HO-1 and elevated levels of the oxidative marker malondialdehyde (MDA). CR attenuated left ventricular hypertrophy (LVH), increased HO-1 levels, and decreased MDA levels. SnMP abolished the protective effects of CR and caused pronounced LVH and cardiac metabolic dysfunction represented by suppressed levels of adiponectin, SIRT1, PPARγ, PGC-1α, and increased MDA. High glucose (33 mM) increased ROS in cultured cardiomyocytes, while SnMP reduced SIRT1, PGC-1α levels, and HO activity. Similarly, SIRT1 inhibition led to a reduction in PGC-1α and HO-1 levels. CoPP increased HO-1 protein levels and activity, SIRT1, and PGC-1α levels, and decreased ROS production, suggesting a positive feedback between SIRT1 and HO-1. Conclusion: These results establish a link between SIRT1, PGC-1α, and HO-1 signaling that leads to the attenuation of ROS production and diabetic cardiomyopathy. CoPP mimicked the beneficial effect of CR, while SnMP increased oxidative stress, aggravating cardiac hypertrophy. The data suggest that increasing HO-1 levels constitutes a novel therapeutic approach to protect the diabetic heart. Brief Summary: CR attenuates cardiomyopathy, and increases HO-1, SIRT activity, and PGC-1α protein levels in diabetic mice. High glucose reduces adiponectin, SIRT1, PGC1-1α, and HO-1 levels in cardiomyocytes, resulting in oxidative stress. The pharmacological activation of HO-1 activity mimics the effect of CR, while SnMP increased oxidative stress and cardiac hypertrophy. These data suggest the critical role of HO-1 in protecting the diabetic heart.

Catalpol attenuates cardiomyocyte apoptosis in diabetic cardiomyopathy via Neat1/miR-140–5p/HDAC4 axis

The cardioprotection of catalpol and its mechanism in diabetic cardiomyopathy (DCM) remains unclear. Here, mouse cardiomyocytes were treated with high glucose (HG) to establish a model of cellular injury induced by HG. In vitro experiments were carried out and confirmed that Catalpol attenuated HG-induced long noncoding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 (Neat1) expression in mouse cardiomyocytes. Mechanistically, luciferase reporter analysis indicated that Neat1 could decrease the transcription of miR-140–5p to positively regulate histone deacetylase 4 (HDAC4) expression. Notably, overexpression of miR-140–5p or silencing of HDAC4 rescued Neat1-induced cardiomyocyte apoptosis. DCM was induced in male C57BL/6 mice by intraperitoneal injection with streptozotocin (STZ) combined with a high-fat/high-sugar diet. Further in vivo experiments identified that Catalpol alleviated myocardial damage by regulating Neat1/miR-140–5p/HDAC4 axis in DCM mice. Thus, our results demonstrated that Catalpol could exert cardioprotective effect against DCM via Neat1/miR-140–5p/HDAC4 pathway.

Mast cell-deficiency protects mice from streptozotocin-induced diabetic cardiomyopathy

Mast cells (MCs) have been implicated in the pathogenesis of cardiometabolic diseases by releasing pro-inflammatory mediators. Patients and animals with diabetic cardiomyopathy (DCM) also show inflammatory cell accumulation in the heart. Here, we detected MCs in mouse heart after streptozotocin (STZ)-induced DCM. DCM production caused significant systole and diastole interventricular septum and left ventricular (LV) posterior wall thinning, and systolic LV internal dilation in wild-type (WT) mice. DCM production also led to significant reductions of fractional shortening percentage, heart rate, body weight, heart weight, and significant increases of kidney, pancreas, and lung weight to body weight ratios, and blood hemoglobin HbA1c and glucose levels in WT mice. All these changes were improved or disappeared in MC-deficient KitW-sh/W-sh mice. In the myocardium from WT DCM mice, we detected significant decrease of cardiac cell proliferation and increases of cardiac cell death, chemokine expression, macrophage infiltration, inflammatory cytokine expression, and collagen deposition. These changes were also improved or disappeared in KitW-sh/W-sh DCM mice. Adoptive transfer of bone marrow-derived MCs (BMMCs) from WT mice fully or partially reversed these cardiac functional and morphologic changes in KitW-sh/W-sh DCM recipient mice. Yet, adoptive transfer of BMMCs from Il6-/- and Tnf-/- mice failed to make these corrections or at much less extent than the WT BMMCs. Mechanistic studies demonstrated a role of MC and MC-derived IL6 and TNF-α in promoting cardiomyocyte death and cardiac fibroblast TGF-β signaling, and collagen synthesis and deposition. Therefore, MC inhibition may have therapeutic potential in attenuating DCM progression.

Activating Cannabinoid Receptor 2 Protects Against Diabetic Cardiomyopathy Through Autophagy Induction.

Cannabinoid receptor 2 (CB2) has been reported to produce a cardio-protective effect in cardiovascular diseases such as myocardial infarction. Here in this study, we investigated the role of CB2 in diabetic cardiomyopathy (DCM) and its underlying mechanisms. HU308 was used for the selective activation of CB2. Bafilomycin A1 was used for the blockade of autophagy and compound C was used to inhibit AMPK signaling. An streptozotocin (STZ)-induced mice model and high glucose (HG)-challenged cardiomyocytes were applied for study. Cardiac function was detected by echocardiography and Western blot for the detection of autophagy-related and its signaling-related proteins. Transmission electron microscopy was used for the analysis of autophagosome number. Cell viability was detected by Cell Counting Kit-8 (CCK-8) and lactate dehydrogenase (LDH) release assays. We found that activating CB2 by HU308 improved cardiac function in DCM as well as cell viability in cardiomyocytes under HG challenge, while the administration of bafilomycin A1 attenuated the protective effects. HU308 enhanced the level of autophagy in the heart tissues from DCM mice as well as cardiomyocytes under HG challenge. HU308 triggered the AMPK-mTOR-p70S6K signaling pathway, while the administration of compound C attenuated the cardio-protective effect of HU308 in cardiomyocytes under HG challenge. In conclusion, we initially demonstrated that activating CB2 produced a cardio-protective effect in DCM as well as cardiomyocytes under HG challenge through inducing the AMPK-mTOR-p70S6K signaling-mediated autophagy.

Leukocytic toll-like receptor 2 knockout protects against diabetes-induced cardiac dysfunction

Diabetic cardiomyopathy is characterized by the deterioration of the myocardial function. Emerging evidences have indicated that leukocytic toll-like receptor 2 (TLR2) played an important role in the development of diabetic cardiomyopathy. Our study aimed to investigate whether TLR2 knockout (KO) exerted a cardioprotective effect in vivo. The establishment of diabetes model was set up in mice via intraperitoneal injection of streptozotocin (STZ). Results demonstrated that blocking of TLR2 significantly suppressed the enhanced left ventricular end-diastolic dimension (LVEDD), left ventricular end systolic diameter (LVESD) and the reduced the heart rate in diabetic cardiomyopathy mice. The decreased resting cell length, PS, TPS and + dL/dt while increased TR90 and - dL/dt caused by diabetic cardiomyopathy were remarkably inhibited by TLR2 KO. Besides that, the alleviated ΔFFI (360/380), decreased SERCA2a and p-NFATc3 expressions, extended Ca2+ decay time and elevated Calcineurin A induced by diabetic cardiomyopathy were vastly repressed by TLR2 KO in cardiocytes. Moreover, TLR2 gene silence could ameliorate oxidative stress-induced apoptosis, evidences were the up-regulated superoxide generation and Bax/Bcl-2 expression while restrained GSH/GSSG ratio caused by diabetic cardiomyopathy were tremendously repressed in TLR2 KO mice. Furthermore, blocking of TLR2 remarkably attenuated the augmented fibrosis areas of heart tissues in mice with diabetic cardiomyopathy. The result of the enhanced α-SMA and collagenⅠ caused by diabetic cardiomyopathy were suppressed in heart tissues of TLR2 KO mice further validate it. All in all, our study demonstrated that diabetes-induced cardiac dysfunction could be attenuated by TLR2 KO.

CAPE-pNO2 attenuates diabetic cardiomyopathy through the NOX4/NF-κB pathway in STZ-induced diabetic mice.

Diabetic cardiomyopathy (DCM) is one of the most severe cardiovascular complications in diabetes. Caffeic acid para-nitro phenethyl ester (CAPE-pNO2) could ameliorate diabetic nephropathy in the diabetic mice in our previous study. This paper was aimed to investigate the effect of CAPE-pNO2 on DCM and its potential mechanism. The DCM mice were established by intraperitoneal injection with streptozotocin (STZ, 50 mg/kg) for 5 days. When the fasting blood glucose level remains above 11.1 mmol/L, treated the mice with CAPE and CAPE-pNO2 for 8 weeks, then the mice were executed, and the samples of blood and heart tissue were collected for the subsequent experiments. The results showed that CAPE-pNO2 can alleviate CK, LDH, TC and TG levels, as well as depress the activity of ROS by down-regulating the expression of NOX4 and improving SOD activity in the serum of STZ-induced DCM mice. Meanwhile, it can also reduce the content of MDA and inhibit lipid accumulation. Besides, CAPE-pNO2 could repress the expression of TNF-α, IL-1β and IL-6 IL - 6 via the NOX4/NF-κB pathway to improve the development of inflammation. Furthermore, it can suppress the expression of collagen and fibronectin to inhibit myocardial fibrosis through the TGF-β1/Smad pathway, and inhibit ECM deposition by regulating TGF-β1 directly, as shown in cardiac tissue section. Importantly, the above results showed that CAPE-pNO2 had better effects of reversing pathological changes than CAPE in a significant difference of p < 0.05. In brief, CAPE-pNO2 can prevent the heart injury of DCM mice via the NOX4/NF-κB pathway, and shows the improvement effects of anti-fibrosis, anti-oxidative and anti-inflammatory.

Deletion of RasGRF1 Attenuated Interstitial Fibrosis in Streptozotocin-Induced Diabetic Cardiomyopathy in Mice through Affecting Inflammation and Oxidative Stress.

Background: Diabetic cardiomyopathy (DCM) is characterized by cardiac fibrosis and stiffness, which often develops into heart failure. This study investigated the role of Ras protein-specific guanine nucleotide releasing factor 1 (RasGRF1) in the development of DCM. Methods: Forty-eight mice were divided into four groups (n = 12 per group): Group 1: Wild-type (WT) mice, Group 2: RasGRF1 deficiency (RasGRF1−/−) mice. Group 3: Streptozotocin (STZ)-induced diabetic WT mice, Group 4: STZ-induced diabetic RasGRF1−/− mice. Myocardial functions were assessed by cardiac echography. Heart tissues from all of the mice were investigated for cardiac fibrosis, inflammation, and oxidative stress markers. Results: Worse impaired diastolic function with elevation serum interleukin (IL)-6 was found in the diabetic group compared with the non-diabetic groups. Serum IL-6 levels were found to be elevated in the diabetic compared with the non-diabetic groups. However, the diabetic RasGRF1−/− mice exhibited lower serum IL-6 levels and better diastolic function than the diabetic WT mice. The diabetic RasGRF1−/− mice were associated with reduced cardiac inflammation, which was shown by lower invading inflammation cells, lower expression of matrix metalloproteinase 9, and less chemokines compared to the diabetic WT mice. Furthermore, less oxidative stress as well as extracellular matrix deposition leading to a reduction in cardiac fibrosis was also found in the diabetic RasGRF1−/− mice compared with the diabetic WT mice. Conclusion: The deletion of RasGRF1 attenuated myocardial fibrosis and improved cardiac function in diabetic mice through inhibiting inflammation and oxidative stress.

Precision medicine approach: Empagliflozin for diabetic cardiomyopathy in mice with aldehyde dehydrogenase (ALDH) 2 * 2 mutation, a specific genetic mutation in millions of East Asians

A vast majority of type-2 diabetic patients (~65%) die of cardiovascular complications including heart failure (HF). In diabetic hearts, levels of 4-hydroxy-2-nonenal (4HNE), a reactive aldehyde that is produced upon lipid peroxidation, were increased. We also demonstrated that in diabetic hearts, there is a decrease in the activity of aldehyde dehydrogenase (ALDH) 2, a primary detoxifying enzyme present in cardiac mitochondria. A single point mutation at E487K of ALDH2 in East Asians known as ALDH2 * 2 intrinsically lowers ALDH2 activity. We hypothesize that Empagliflozin (EMP), a sodium-glucose cotransporter (SGLT) 2 inhibitor, can ameliorate diabetic cardiomyopathy by decreasing hyperglycemia-mediated 4HNE protein adducts in ALDH2 * 2 mutant mice which serve as a precision medicine tool as they mimic ALDH2 * 2 carriers. We induced type-2 diabetes in 11–14 month-old male and female ALDH2 * 2 mice through a high-fat diet. Chow-fed ALDH2 * 2 mice served as controls. At the end of 4 months, we treated the diabetic ALDH2 * 2 mice with EMP (3 mg/kg/d) or its vehicle (Veh). After 2 months of EMP treatment, cardiac function was assessed by conscious echocardiography after treadmill exercise stress. EMP improved the cardiac function and running distance and duration significantly compared to Veh-treated ALDH2 * 2 diabetic mice. These beneficial effects can be attributed to the EMP-mediated decrease in cardiac mitochondrial 4HNE adducts and increase in the levels of phospho AKT, AKT, phospho Akt substrate of 160 kDa (pAS160), AS160 and GLUT-4 in the skeletal muscle tissue of the ALDH2*2 mutant diabetic mice, respectively. Finally, our data implicate EMP can ameliorate diabetic cardiomyopathy in diabetic ALDH2 * 2 mutant patients.

Effect of RAGE Manipulation on Diabetic Cardiomyopathy in Mice

Advanced glycation end-products (AGE) and their receptors (RAGE) have been implicated in the development of many ailments including diabetes. Soluble RAGE (sRAGE), which acts as a decoy of RAGE, has been studied as a potential therapeutic agent against vascular complications of diabetes. Here we tested the effects of RAGE manipulation in vivo on diabetic cardiomyopathy. We have reported that genetic over-expression of sRAGE produces a consistent high level of plasma sRAGE in mice. In this study, we created a mouse model that has over-expressed sRAGE with either intact or knocked-out RAGE. Diabetes was induced by STZ in following mice with: (1) over-expressed sRAGE and intact RAGE (S group); (2) over-expressed sRAGE and knocked-out RAGE (D group); and (3) wildtypes that have a normal expression of both sRAGE and RAGE (C group). During the 4-month of post-STZ injection, cardiovascular morphology and function were studied using serial Echocardiography, end-point invasive hemodynamic measurements and pathological analysis. A severe hyperglycemia was observed throughout the experiment in all diabetic mice. As expected, a significant reduction of bodyweight, heart weight, heart rate, left ventricular (LV) systolic parameters, LV diastolic parameters, LV Ejection fraction (EF), vascular functional parameters and survival rate (33%) were observed in C group, as indications of diabetic cardiomyopathy, compared to non-diabetic counterparts. Interestingly, in S group, those parameters were identical to that in C group (30% of survival rate in S group). However, in D group, while the effects of hyperglycemia on bodyweight and LV diastolic parameters were similar to that in C and S groups, there was a significantly better survival rate (70%; p<0.05), better LV systolic parameters, better EF and better vascular functional parameters, and lesser reduction in heart weight and heart rate compared to that in C and S groups (Figure). Our results suggested that manipulation of RAGE signaling attenuates the development of diabetic cardiomyopathy. Advanced glycation end-products (AGE) and their receptors (RAGE) have been implicated in the development of many ailments including diabetes. Soluble RAGE (sRAGE), which acts as a decoy of RAGE, has been studied as a potential therapeutic agent against vascular complications of diabetes. Here we tested the effects of RAGE manipulation in vivo on diabetic cardiomyopathy. We have reported that genetic over-expression of sRAGE produces a consistent high level of plasma sRAGE in mice. In this study, we created a mouse model that has over-expressed sRAGE with either intact or knocked-out RAGE. Diabetes was induced by STZ in following mice with: (1) over-expressed sRAGE and intact RAGE (S group); (2) over-expressed sRAGE and knocked-out RAGE (D group); and (3) wildtypes that have a normal expression of both sRAGE and RAGE (C group). During the 4-month of post-STZ injection, cardiovascular morphology and function were studied using serial Echocardiography, end-point invasive hemodynamic measurements and pathological analysis. A severe hyperglycemia was observed throughout the experiment in all diabetic mice. As expected, a significant reduction of bodyweight, heart weight, heart rate, left ventricular (LV) systolic parameters, LV diastolic parameters, LV Ejection fraction (EF), vascular functional parameters and survival rate (33%) were observed in C group, as indications of diabetic cardiomyopathy, compared to non-diabetic counterparts. Interestingly, in S group, those parameters were identical to that in C group (30% of survival rate in S group). However, in D group, while the effects of hyperglycemia on bodyweight and LV diastolic parameters were similar to that in C and S groups, there was a significantly better survival rate (70%; p<0.05), better LV systolic parameters, better EF and better vascular functional parameters, and lesser reduction in heart weight and heart rate compared to that in C and S groups (Figure). Our results suggested that manipulation of RAGE signaling attenuates the development of diabetic cardiomyopathy.

TAX1BP1 overexpression attenuates cardiac dysfunction and remodeling in STZ-induced diabetic cardiomyopathy in mice by regulating autophagy

Diabetic cardiomyopathy is associated with suppressed autophagy and augmented inflammation in the heart. The effects of Tax1 binding protein 1 (TAX1BP1) on both autophagy and inflammation suggest that it may participate in the progression of diabetic cardiomyopathy. Mice were injected with streptozotocin (STZ) to induce experimental diabetes. An adenovirus system was used to induce heart specific TAX1BP1 overexpression 12 weeks after STZ injection.TAX1BP1 expression was significantly decreased in STZ-induced diabetic mouse hearts. TAX1BP1 overexpression in the heart alleviated cardiac hypertrophy and fibrosis, attenuated inflammation, oxidative stress, and apoptosis, and improved cardiac function in STZ-induced diabetic mice. Diabetic mice exhibited decreased autophagy. By contrast, increased autophagy was observed in diabetic mice overexpressing TAX1BP1. TAX1BP1 overexpression promoted autophagic flux, as demonstrated by increased LC3-RFP fluorescence in vitro. Furthermore, the autophagy inhibitor 3-MA abolished the protective effects of TAX1BP1 in vivo. Interestingly, we found that TAX1BP1 increased autophagy via the activation of a non-canonical NF-κB signaling pathway. Conversely, RelB knockdown disrupted the protective effects of TAX1BP1 in cardiomyocytes. TAX1BP1 thus restores the decreased autophagy level, leading to decreased inflammatory responses and oxidative stress and reduced apoptosis in cardiomyocytes.

Resveratrol Combined with Pioglitazone Ameliorates Cardiovascular Complications in db/db Diabetic Mice

Type 2 Diabetes Mellitus patients are predisposed to serious cardiovascular morbidity and mortality. Diabetes causes different structural and functional alterations in the myocardial tissue, which are induced by hyperglycemia, insulin resistance, and hyperlipidemia. The resulting cardiomyopathy is characterized by myocardial fibrosis, dysfunctional remodeling and eventually, clinical heart failure. This study addressed the effects of resveratrol and pioglitazone to ameliorate diabetic cardiovascular complications using a db/db diabetic mice model. Male db/db diabetic mice were randomly assigned to orally receive resveratrol (50 mg/kg, n=5), resveratrol/pioglitazone (50 mg/kg and 20 mg/day, respectively, n=5) for the space of 6 weeks. Non-diabetic lean mice (n=5) were included as a control group. Histopathological analyses were performed in heart and aortic vessel tissue samples using hematoxylin and eosin, as well as Masson Trichromic staining. VCAM-1 immunohistochemistry was also assessed. Blood glucose, hemoglobin A1c, insulin, glycosuria, triglycerides, cholesterol, and LDL cholesterol were all examined. Present data suggest that the combination of pioglitazone plus resveratrol significantly lower circulating levels of insulin, hemoglobin A1c, glucose, triglycerides, cholesterol, and LDL levels. Our results also show that tissue damage to the heart and aortic vessels caused by diabetes can also be improved by the administration of said combination. The present study demonstrates that resveratrol/pioglitazone combination therapy improves carbohydrate and lipid metabolism, as well as improving diabetic cardiomyopathy in diabetic mice by producing a synergistic pharmacological effect. The combination of resveratrol plus pioglitazone has therapeutic potential against diabetic cardiomyopathy.

Andrographolide Ameliorates Diabetic Cardiomyopathy in Mice by Blockage of Oxidative Damage and NF-κB-Mediated Inflammation.

Andrographolide (Andro), a major bioactive component obtained from Andrographis paniculata Nees, has exerted wide antioxidant as well as cytoprotective properties. However, whether Andro treatment could retard the progress of diabetic cardiomyopathy (DCM) remains unknown. In this study, we evaluated the effects of Andro against diabetes-induced myocardial dysfunction and explored the underlying mechanism in STZ-induced diabetic mice. As a result, treatment with Andro dose dependently suppressed cardiac inflammation and oxidative stress, accompanied by decreasing cardiac apoptosis, which subsequently ameliorated cardiac fibrosis and cardiac hypertrophy. Further, Andro blocked hyperglycemia-triggered reactive oxygen species (ROS) generation by suppressing NADPH oxidase (NOX) activation and augmenting nuclear factor erythroid 2-related factor 2 (Nrf2) expression both in vitro and in vivo. Our results suggest that the cardioprotective effects afforded by Andro treatment involve the modulation of NOX/Nrf2-mediated oxidative stress and NF-κB-mediated inflammation. The present study unravels the therapeutic potential of Andro in the treatment of DCM by attenuating oxidative stress, inflammation, and apoptosis.

Changes in the Cardiac GHSR1a-Ghrelin System Correlate With Myocardial Dysfunction in Diabetic Cardiomyopathy in Mice.

Ghrelin and its receptor, the growth hormone secretagogue receptor 1a (GHSR1a), are present in cardiac tissue. Activation of GHSR1a by ghrelin promotes cardiomyocyte contractility and survival, and changes in myocardial GHSR1a and circulating ghrelin track with end-stage heart failure, leading to the hypothesis that GHSR1a is a biomarker for heart failure. We hypothesized that GHSR1a could also be a biomarker for diabetic cardiomyopathy (DCM). We used two models of streptozotocin (STZ)-induced DCM: group 1, adult mice treated with 35 mg/kg STZ for 3 days; and group 2, neonatal mice treated with 70 mg/kg STZ at days 2 and 5 after birth. In group 1, mild fasting hyperglycemia (11 mM) was first detected 8 weeks after the last injection, and in group 2, severe fasting hyperglycemia (20 mM) was first detected 1 to 3 weeks after the last injection. In group 1, left ventricular function was slightly impaired as measured by echocardiography, and Western blot analysis showed a significant decrease in myocardial GHSR1a. In group 2, GHSR1a levels were also decreased as assessed by Cy5-ghrelin(1–19) fluorescence microscopy, and there was a significant negative correlation between GHSR1a levels and glucose tolerance. There were significant positive correlations between GHSR1a and ghrelin and between GHSR1a and sarcoplasmic reticulum Ca2+-ATPase 2a (SERCA2a), a marker for contractility, but not between GHSR1a and B-type natriuretic peptide, a marker for heart failure. We conclude that the subclinical stage of DCM is accompanied by alterations in the myocardial ghrelin-GHSR1a system, suggesting the possibility of a biomarker for DCM.

Fibroblast growth factor 21 inhibition aggravates cardiac dysfunction in diabetic cardiomyopathy by improving lipid accumulation.

Diabetic cardiomyopathy (DCM) is one of the major causes of morbidity and mortality in diabetic patients. Recent studies have demonstrated an increased level of fibroblast growth factor 21 (FGF21) in the plasma of DCM patients, and FGF21 has been proven to be a cardiovascular protector of the heart. The present study aimed to further investigate the pathogenic role of FGF21 in DCM, hypothesizing that a lack of FGF21 may promote the progression of DCM by regulating the lipid metabolism, cardiac hypertrophy and cardiac fibrosis, thus deteriorating the cardiac dysfunction. A total of 44 mice were randomly assigned into the normal (n=6), DCM (n=6), normal + scrambled siRNA (n=6), DCM + scrambled siRNA (n=6), normal + FGF21 siRNA (n=10) and DCM + FGF21 siRNA (n=10) groups. Type 1 diabetes mellitus was induced to mice in the DCM groups by streptozotocin injection, while FGF21 expression was inhibited by FGF21 siRNA. Normal and DCM mice administrated with scrambled siRNA were respectively regarded as the controls for the normal + FGF21 siRNA and DCM + FGF21 siRNA groups. In the DCM group, FGF21 inhibition promoted cardiac hypertrophy and fibrosis, and the expression levels of their indicators, including atrial natriuretic factor, α-skeletal actin, collagen type I and III, and transforming growth factor-β, increased, leading to further decreased cardiac function. In addition, FGF21 inhibition in DCM mice elevated the quantity of lipid droplets and the concentration of heart triglycerides, plasma triglycerides and cholesterol levels, accompanied by downregulation of peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α) and upregulation of cluster of differentiation (CD)36. Thus, the results indicated that FGF21 inhibition exacerbates the cardiac dysfunction by aggravating the lipid accumulation through regulating the expression levels of PGC-1α and CD36. In conclusion, it is suggested that FGF21 may be a potentially useful agent in the treatment of DCM.

SGLT-2 Inhibition with Dapagliflozin Reduces the Activation of the Nlrp3/ASC Inflammasome and Attenuates the Development of Diabetic Cardiomyopathy in Mice with Type 2 Diabetes. Further Augmentation of the Effects with Saxagliptin, a DPP4 Inhibitor.

We assessed whether (1) dapagliflozin (Dapa, an SGLT2-inhibitor) attenuates the deterioration of heart functionNlrp3 and inflammasome activation in diabetic mice. (2) The effects can be augmented with saxagliptin (Saxa), a DDP4-inhibitor. (3) Dapa effect is possibly SGLT2-independent on cardiofibroblasts in vitro. Type 2 diabetic (BTBR ob/ob) and wild-type (WT) mice received vehicle, Dapa, or Dapa+Saxa for 8weeks. Glucose tolerance test and echocardiogram were performed. Cardiofibroblasts from WT and BTBR hearts were incubated with Dapa and exposed to LPS. Left ventricular ejection fraction (LVEF) was 81±1% in the WT and 53±1% in the T2D-cont mice. Dapa and Dapa+Saxa improved LVEF to 68±1 and 74.6±1% in the BTBR mice (p<0.001). The mRNA levels of NALP3, ASC, IL-1β, IL-6, caspase-1, and TNFα were significantly higher in the BTBR compared to the WT hearts; and Dapa and Dapa+Saxa significantly attenuated these levels. Likewise, protein levels of NLRP3, TNFα, and caspase-1 were higher in the BTBR compared to the WT hearts and Dapa, and to a greater extent Dapa+Saxa, attenuated the increase in the BTBR mice. Collagen-1 and collagen-3 mRNA levels significantly increased in the BTBR mice and these increases were attenuated by Dapa and Dapa+Saxa. P-AMPK/total-AMPK ratio was significantly lower in the BTBR mice than in the WT mice. Dapa and Dapa+Saxa equally increased the ratio in the BTBR mice. This in vitro study showed that NALP3, ASC, IL-1β, and caspase-1 mRNA levels were higher in the BTBR cardiofibroblasts and attenuated with Dapa. The effect was AMPK-dependent and SGLT1-independent. Dapa attenuated the activation of the inflammasome, fibrosis, and deterioration of LVEF in BTBR mice. The anti-inflammatory, anti-fibrotic effects are likely SGLT2- and glucose-lowering-independent, as they were replicated in the in vitro model. The effects on remodeling were augmented when Saxa was added to Dapa. Yet, adding Saxa to Dapa did not result in a greater effect on myocardial fibrosis and collagen levels.

Effect of nucleolin on cardiac cell apoptosis in Type 2 diabetic cardiomyopathy mice

To investigate the effect of nucleolin on cardiac cell apoptosis in Type 2 diabetic cardiomyopathy mice. Methods: Mice were fed with high-fat and high-sugar food for 20 weeks (mice were injected intraperitoneally with 60 mg/kg streptozotocin in the 5th and 6th weeks) to establish a mouse model of Type 2 diabetes. The mice were divided into 4 groups: a wild type (WT) control group, a nucleolin transgenic (TG) control group, a WT diabetic group, a TG diabetic group. Diabetes-related indicators were detected at the end of the 8th week. At the end of the 20th week, HE staining was used to observe myocardial morphological changes; TUNEL staining and caspase-3 activity were used to detect the extent of apoptosis of cardiac myocytes. Results: The level of fasting blood glucose was significantly increased in the diabetic group than that in the control group. In WT diabetic group, myocardial disarrangement, fragmentation and dissolution were observed (determined by HE staining); cellular apoptosis (determined by TUNEL staining and caspase-3 activity) also increased markedly in the WT diabetic group. Compared with the wild mice in the diabetic group, myocardial morphological changes and cardiac myocytes apoptosis were alleviated significantly. Conclusion: Nucleolin overexpression affectes the occurrence and development of diabetic cardiomyopathy through inhibition of cardiac myocyte apoptosis.

SIRT1 Activation by Resveratrol Alleviates Cardiac Dysfunction via Mitochondrial Regulation in Diabetic Cardiomyopathy Mice.

Background Diabetic cardiomyopathy (DCM) is a major threat for diabetic patients. Silent information regulator 1 (SIRT1) has a regulatory effect on mitochondrial dynamics, which is associated with DCM pathological changes. Our study aims to investigate whether resveratrol, a SRIT1 activator, could exert a protective effect against DCM. Methods and Results Cardiac-specific SIRT1 knockout (SIRT1KO) mice were generated using Cre-loxP system. SIRT1KO mice displayed symptoms of DCM, including cardiac hypertrophy and dysfunction, insulin resistance, and abnormal glucose metabolism. DCM and SIRT1KO hearts showed impaired mitochondrial biogenesis and function, while SIRT1 activation by resveratrol reversed this in DCM mice. High glucose caused increased apoptosis, impaired mitochondrial biogenesis, and function in cardiomyocytes, which was alleviated by resveratrol. SIRT1 deletion by both SIRT1KO and shRNA abolished the beneficial effects of resveratrol. Furthermore, the function of SIRT1 is mediated via the deacetylation effect on peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), thus inducing increased expression of nuclear respiratory factor 1 (NRF-1), NRF-2, estrogen-related receptor-α (ERR-α), and mitochondrial transcription factor A (TFAM). Conclusions Cardiac deletion of SIRT1 caused phenotypes resembling DCM. Activation of SIRT1 by resveratrol ameliorated cardiac injuries in DCM through PGC-1α-mediated mitochondrial regulation. Collectively, SIRT1 may serve as a potential therapeutic target for DCM.