Dysfunction In Diabetic Heart Research Articles

Beyond the matrix: exploring tenascin-c (TNC) as a crucial player in diabetic cardiovascular dysfunction

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Burgermeister Found Wien-Project number : 21001 Introduction Diabetic cardiomyopathy (CMP) is a complex manifestation of diabetes-linked cardiac and vascular dysfunction, but the molecular pathogenesis is still largely unknown. Notably the expression of Tenascin-C (TNC), an extracellular matrix glycoprotein, contributes to the development of cardiovascular diseases and diabetic patients have elevated serum TNC levels. However, the role and mechanism of TNC to diabetic CMP progression remains elusive. Purpose Here, we longitudinally evaluated functional and molecular role of TNC in CMP-induced cardiovascular dysfunction in a murine model. In addition, we used RNA sequencing with cardiac tissues from non-diabetic and diabetic wild type (wt) and TNC KO mice for further investigation. Methods Diabetes was induced in adult male mice, wt (AJ) and TNC-KO strains, by repeated Streptozotocin (STZ) injections (50 mg/kg). Echocardiography, myography, hemodynamics, histology, molecular and cellular analyses were performed to assess the role of TNC in diabetes-associated CV complications. Additionally, RNA-sequencing was analyzed in left ventricular (LV) tissue samples from diabetic and non-diabetic wt and TNC-KO mice providing further molecular insights. Results TNC KO mice showed preserved LV ejection fraction, and endothelium-dependent relaxation (p<0.05 and p<0.001, respectively), reduced cardiac fibrosis and distinctive coronary vessel characteristics, implying enhanced LV perfusion. In addition, the plasma levels of oxidative stress marker malondialdehyde was significantly alleviated in TNC KO diabetic mice in comparison to diabetic wt mice. Cardiomyocytes from diabetic wt mice exhibited stiffness (Fpassive and Factive), which was corrected to control level in cardiomyocytes from TNC KO diabetic hearts. Further, ex vivo isolated heart experiments demonstrated that the administration of recombinant human TNC (80 ng/ml) resulted in a significant reduction in LV hemodynamics (p<0.01) and myocardial energetics (ATP levels, p<0.01). Analysis of RNA sequencing data demonstrated that decreased expression of matrix remodeling genes Serpin1 and Ccn1 links to alleviation of fibrosis in TNC KO diabetic heart. Furthermore, Gene Ontology (GO) enrichment analysis shed light on mitochondrial changes in diabetic hearts. Our pathway analysis also identified that Hsp90aa1 as a crucial gene associated with preprotein import into the mitochondrial membrane, may suggest potential mitochondrial dysfunction in diabetic hearts. Conclusion In conclusion, lack of TNC improved long-term cardiac function in diabetes by reducing cardiac fibrosis, vascular dysfunction, oxidative stress and cardiac energetics. Thus, inhibition of TNC may prevent the diabetic heart from extensive fibrosis and energetics dysfunction that accelerates the development of HF, providing a potential targeted therapy.

Abstract P2051: Fibroblast-specific TGF-b Signaling Promotes Interstitial Fibrosis, Cardiomyocyte Hypertrophy And Cardiac Dysfunction In The Diabetic Heart Through Smad3 Activation

Objectives: Patients with diabetes exhibit a high prevalence of HFpEF associated with interstitial fibrosis and cardiomyocyte hypertrophy. However, the cellular and molecular mechanisms involved in diabetic heart disease remain poorly understood. TGF-bs are potent fibrogenic mediators that are activated in diabetic hearts, and act through a single type 2 receptor, TbRII. We hypothesized that fibroblast-specific actions of TGF-bs may mediate fibrosis and dysfunction in diabetic hearts. Methods and Results: To test the hypothesis, we developed lean and db/db type 2 diabetic TbRII KO mice (FTbRII2KO) using the Col1a2 CreER driver. Fibroblast-specific loss of TGF-b signaling attenuated hypertrophy in db/db mouse hearts. Speckle-tracking echocardiography with strain imaging showed that fibroblast-specific TbRII loss improved systolic and diastolic cardiac function in diabetic animals. In contrast, fibroblast TbRII disruption caused a worsening of both cardiac systolic and diastolic function in lean animals. Attenuated dysfunction in diabetic mice lacking fibroblast TGF-b signaling was associated with reduced collagen fiber thickness and with decreased cardiomyocyte size, suggesting paracrine effects of fibroblasts on cardiomyocytes. To study the downstream cascade responsible for the effects of TGF-b, we generated lean and diabetic mice with fibroblast-specific Smad3 loss. In db/db mice, fibroblast-specific Smad3 loss phenocopied the effects of fibroblast-specific TbRII disruption, suggesting that in diabetic cardiac fibroblasts TGF-b acts through Smad3 signaling. The bioinformatic analyses of transcriptomic profiles of cardiac fibroblasts identified thrombospondin 4 as a candidate mediator of Smad3-dependent diabetes-associated activation. Conclusions: Fibroblast-specific TGF-b actions in diabetes promote cardiac dysfunction through effects on collagen deposition and via paracrine actions on cardiomyocytes. The effects of TGF-b on diabetic cardiac fibroblasts are Smad3-dependent. In contrast to the deleterious effects of fibroblast TGFb/Smad3 signaling in diabetic hearts, TGF-b-activated Smad3-dependent signaling is important to maintain homeostatic function and geometry in normal lean animals.

Cannabinoid Receptor 2-Centric Molecular Feedback Loop Drives Necroptosis in Diabetic Heart Injuries.

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

Abstract 13062: In vivo Knockdown of Proapoptotic Mir-320 Restores Cardiac Function and Angiogenesis in a Mouse Model of Type 2 Diabetes

Background: Non-ischemic diabetic heart disease (NiDHD) is characterised by diastolic dysfunction and decreased or preserved systolic function, eventually resulting in heart failure. The fundamental mechanisms leading to NiDHD are still not known. microRNAs (miRNAs) plays a significant role in the development of NiDHD. Objective: To investigate the pathological role of cardiomyocyte enriched pro-apoptotic miR-320 in the development of NiDHD and to determine if therapeutic knockdown of miR-320 can restore impaired cardiac function and angiogenesis in the diabetic heart. Methods and Results: Cardiac tissue were collected from type-2 diabetic individuals undergoing cardiac surgery showed marked upregulation of miR-320, which was associated with downregulation of its direct target pro-survival insulin growth factor-1 (IGF-1) and anti-apoptotic protein Bcl-2. Analysis of cardiac tissue samples collected from type 2 diabetic mice (BKS.Cg-m+/+Leprdb/J) every 4wks, from 8 to 32wks of age showed activation of miR-320 and downregulation of IGF-1 at the later stages of diabetes. To determine if therapeutic knockdown of miR-320 is beneficial, high glucose cultured adult cardiomyocytes were treated with either locked nucleic acid (LNA) anti-precursor (pre)-miR-320 or scrambled (Scr) sequence. Results showed that LNA-anti-pre-320 significantly restored IGF-1 expression and reduced apoptotic cells death (3.1±05 in Scr vs 1.3±0.6 in LNA-anti-pre-320, P<0.01). Finally, to confirm if the effect can be translated in vivo , 24 weeks old type 2 diabetic mice with established systolic dysfunction were injected with LNA-anti-pre-320 once a week for 4wks. Echocardiography confirmed significant restoration of diastolic function and partial restoration of systolic function. Molecular analysis showed marked reduction in miR-320 expression, which was associated with recovery of IGF-1 and Bcl-2. Further, histological analysis confirmed improved angiogenesis and reduced fibrosis in the LNA-anti-pre-320 treated group. Conclusion: miR-320 is a late responding miRNA that aggravates apoptosis and cardiac dysfunction in diabetic heart, and that therapeutic knockdown of miR-320 is beneficial in partially restoring the deteriorated cardiac function.

Natural remedies medicine derived from flaxseed (secoisolariciresinol diglucoside, lignans, and α-linolenic acid) improve network targeting efficiency of diabetic heart conditions based on computational chemistry techniques and pharmacophore modeling.

Cytokine storms lead to cardiovascular diseases (CVDs). Natural herbal compounds are considered the primary source of active agents with the potential to prevent or treat inflammatory-related pathologies such as CVD and diabetes. Flaxseed contains phytochemicals, including secoisolariciresinol diglucoside (SDG), α-linolenic acid (ALA), and lignans, termed "SAL." Hence, we evaluated the effect of the SAL on the H9c2 cardiac cells in hyperlipidemic and hyperglycemic conditions. Here, candidate hub genes, TNF-α, IL6, SIRT1, NRF1, NPPA, and FGF7, were selected as effective genes in diabetic cardiovascular pathogenesis based on in-silico analysis and chemoinformatic. Myocardial infarction (MI) was induced using H9c2 cardiac cells in hyperlipidemic and hyperglycemic conditions. Real-time qPCR was conducted to assess the expression level of hub genes. This study indicated that SAL compounds bound to the Il-6, SIRT1, and TNF-α active sites as druggable candidate proteins based on the chemoinformatics analysis. This study displayed that the TNF-α, IL6, SIRT1, NRF1, NPPA, and FGF7 network dysfunction in MI models were ameliorated by SAL consumption. Furthermore, SAL compounds improved the function and myogenesis of H9c2 cells in hyperlipidemic and hyperglycemic conditions. Our data suggested that phytochemicals obtained from flaxseed might have proposed potential complementary treatment or preventive strategies for MI. PRACTICAL APPLICATIONS: Phytochemicals obtained from flaxseed (SAL) could reverse diabetic heart dysfunction hallmarks and provide new potential treatment approaches in cardiovascular therapy. SAL could be considered complementary and alternative medicines for treating various disorders/diseases singly or synchronizing with prescription drugs.

Sarco/endoplasmic reticulum calcium ATPase activity is unchanged despite increased myofilament calcium sensitivity in Zucker type 2 diabetic fatty rat heart

Systolic and diastolic dysfunction in diabetes have frequently been associated with abnormal calcium (Ca2+) regulation. However, there is emerging evidence that Ca2+ mishandling alone is insufficient to fully explain diabetic heart dysfunction, with focus shifting to the properties of the myofilament proteins. Our aim was to examine the effects of diabetes on myofilament Ca2+ sensitivity and Ca2+ handling in left ventricular tissues isolated from the same type 2 diabetic rat hearts. We measured the force-pCa relationship in skinned left ventricular cardiomyocytes isolated from 20-week-old type 2 diabetic and non-diabetic rats. Myofilament Ca2+ sensitivity was greater in the diabetic relative to non-diabetic cardiomyocytes, and this corresponded with lower phosphorylation of cardiac troponin I (cTnI) at ser23/24 in the diabetic left ventricular tissues. Protein expression of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), phosphorylation of phospholamban (PLB) at Ser16, and SERCA/PLB ratio were lower in the diabetic left ventricular tissues. However, the maximum SERCA Ca2+ uptake rate was not different between the diabetic and non-diabetic myocardium. Our data suggest that impaired contractility in the diabetic heart is not caused by SERCA Ca2+ mishandling. This study highlights the important role of the cardiac myofilament and provides new insight on the pathophysiology of diabetic heart dysfunction.

SARM1 NAD Hydrolase Deficiency Normalizes Fibrosis and Ameliorates Cardiac Dysfunction in Diabetic Hearts

Decline in cellular NAD levels emerges as a hallmark of diseases such as diabetes and heart failure. Strategies to activate NAD synthesis pathways and replenish NAD levels have shown promises to treat heart disease. However, how NAD consumption mechanisms lead to NAD depletion and disease pathogeneses are less understood. SARM1 is an intracellular NAD hydrolase, and its role in heart disease has never been investigated. We therefore hypothesized that SARM1 deficiency may protect hearts against diabetic cardiomyopathy. Male C57BL/6 wild‐type (WT) and SARM1‐KO mice were challenged with 16‐week diabetic stress initiated by streptozotocin injections (STZ). Longitudinal cardiac function was measured by echocardiography after 8 or 16 weeks of diabetes, and tissue and plasma samples were harvested for biochemical and molecular analyses. Chronic diabetic stress lowered cardiac NAD levels and led to progressive decline in systolic function of WT mice. Diabetic SARM1‐KO mice showed improved systolic function and increased cardiac NAD levels, compared to diabetic WT mice. Progressive decline in diastolic function induced by chronic diabetes was also improved by SARM1 deficiency. Heart weights and hyperglycemia were similar in diabetic WT or diabetic SARM1‐KO mice. Metabolomic analyses of plasma showed significant changes in 58 out of ~300 aqueous metabolites assessed when comparing non‐diabetic WT to diabetic WT plasma, including increased levels of glucose and branched‐chain amino acids. However, the 58 metabolites showed no changes in levels when comparing diabetic WT to diabetic SARM1‐KO plasma. Similarly, we found that 26 out of 85 lipid species showed significant changes when comparing non‐diabetic WT to diabetic WT plasma, and only one of these 26 lipid metabolites showed a significant change between diabetic WT and diabetic SARM1‐KO plasma. The data suggest that cardioprotection in SARM1‐KO mice was likely due to the reversal of cardiac pathogenic mechanisms but not improved systemic metabolic state.To determine how SARM1 deficiency affects cardiac NAD metabolism, expression of genes in NAD metabolic pathways were assessed. Levels of Bst1 and Cd38 did not change in SARM1‐KO hearts, suggesting that SARM1 deficiency did not induce compensatory changes in expression of the other NAD hydrolases. SARM1‐KO hearts showed lower expressions in some of the NAD consuming genes (e.g. Sirtuins) and NAD synthesis genes (e.g. Nmnats). Cardiac dysfunction marker, Nppb expression, was induced in diabetic WT hearts, and was reversed by SARM1 deficiency. Cardiac fibrosis and pro‐fibrotic genes (e.g. ctgf) were induced in diabetic WT hearts and were suppressed by SARM1 deficiency. Using the total SARM1 KO mice, our data support that SARM1 deficiency normalizes decline in NAD levels and pro‐fibrotic pathways to ameliorate diabetic cardiomyopathy. Further experiments are warranted to dissect the cell‐type specific roles of SARM1 in diabetic hearts.

STK35 Gene Therapy Attenuates Endothelial Dysfunction and Improves Cardiac Function in Diabetes.

Diabetic cardiomyopathy (DCM) is characterized by microvascular pathology and interstitial fibrosis that leads to progressive heart failure. The mechanisms underlying DCM pathogenesis remain obscure, and no effective treatments for the disease have been available. In the present study, we observed that STK35, a novel kinase, is decreased in the diabetic human heart. High glucose treatment, mimicking hyperglycemia in diabetes, downregulated STK35 expression in mouse cardiac endothelial cells (MCEC). Knockdown of STK35 attenuated MCEC proliferation, migration, and tube formation, whereas STK35 overexpression restored the high glucose-suppressed MCEC migration and tube formation. Angiogenesis gene PCR array analysis revealed that HG downregulated the expression of several angiogenic genes, and this suppression was fully restored by STK35 overexpression. Intravenous injection of AAV9-STK35 viral particles successfully overexpressed STK35 in diabetic mouse hearts, leading to increased vascular density, suppression of fibrosis in the heart, and amelioration of left ventricular function. Altogether, our results suggest that hyperglycemia downregulates endothelial STK35 expression, leading to microvascular dysfunction in diabetic hearts, representing a novel mechanism underlying DCM pathogenesis. Our study also emerges STK35 is a novel gene therapeutic target for preventing and treating DCM.

Calcium signaling in endocardial and epicardial ventricular myocytes from streptozotocin-induced diabetic rats.

Aims/IntroductionAbnormalities in Ca2+ signaling have a key role in hemodynamic dysfunction in diabetic heart. The purpose of this study was to explore the effects of streptozotocin (STZ)‐induced diabetes on Ca2+ signaling in epicardial (EPI) and endocardial (ENDO) cells of the left ventricle after 5–6 months of STZ injection.Materials and MethodsWhole‐cell patch clamp was used to measure the L‐type Ca2+ channel (LTCC) and Na+/Ca2+ exchanger currents. Fluorescence photometry techniques were used to measure intracellular free Ca2+ concentration.ResultsAlthough the LTCC current was not significantly altered, the amplitude of Ca2+ transients increased significantly in EPI‐STZ and ENDO‐STZ compared with controls. Time to peak LTCC current, time to peak Ca2+ transient, time to half decay of LTCC current and time to half decay of Ca2+ transients were not significantly changed in EPI‐STZ and ENDO‐STZ myocytes compared with controls. The Na+/Ca2+ exchanger current was significantly smaller in EPI‐STZ and in ENDO‐STZ compared with controls.ConclusionsSTZ‐induced diabetes resulted in an increase in amplitude of Ca2+ transients in EPI and ENDO myocytes that was independent of the LTCC current. Such an effect can be attributed, at least in part, to the dysfunction of the Na+/Ca2+ exchanger. Additional studies are warranted to improve our understanding of the regional impact of diabetes on Ca2+ signaling, which will facilitate the discovery of new targeted treatments for diabetic cardiomyopathy.

A novel oral glucagon-like peptide 1 receptor agonist protects against diabetic cardiomyopathy via alleviating cardiac lipotoxicity induced mitochondria dysfunction

Diabetic cardiomyopathy is one of the major cardiovascular complications of diabetes mellitus associated with left ventricular diastolic dysfunction. There are still no specific therapeutic guidelines for the disease. In recent years, glucagon-like peptide 1 receptor agonists were proved to exert cardioprotective effects in comprehensive studies. Therefore, we examined whether a novel oral availably glucagon-like peptide 1 receptor agonist, oral hypoglycemic peptide 2 (OHP2), could protect against diabetic cardiomyopathy in high-fat diets and continuous streptozocin injection induced rat models. After treatment for eight weeks, heart function was evaluated by echocardiography. As expected, OHP2 improved cardiac structure and function beyond glycemic control. Both hyperlipidemia and myocardium lipid accumulation were decreased by OHP2 treatment. In addition, OHP2 reversed oxidative stress and mitochondrial dysfunction in diabetic hearts. In vitro study suggested that OHP2 prevented palmitic acid-induced oxidative stress and mitochondrial dysfunction via suppressing intercellular lipid accumulation. Hence, our present findings pointed out that OHP2 is a promising oral glucagon-like peptide 1 receptor agonist for preventing diabetic cardiomyopathy.

P0974ADIPORON AMELIORATES DIABETIC CARDIOMYOPATHY IN DBDB MICE THROUGH IMPROVING CERAMIDE-DEPENDENT LIPOTOXICITY

Abstract Background and Aims The accumulation of lipid and its metabolites in tissues, including the heart, causes lipotoxicity. Sphingolipids such as ceramides are particularly deleterious. Lowering the accumulation of ceramide can improve metabolic damage of various tissues. Recent reports showed that adiponectin receptors (AdipoRs) have sequence homology with ceramidase enzymes, and an increase of ceramidase activity with overexpression of adiponectin in mice improves ceramide-dependent lipotoxicity. Therefore, we investigated the possible roles of Adiporon, which mimics the effects of adiponectin, in the view of prevention and development of diabetic cardiomyopathy in diabetic mouse model. Method Male db/db mice and db/m controls were fed either a regular diet chow or a diet containing adiporon (30 mg/kg/day p.o. for 4 weeks from 17 to 20 weeks of age) and biochemical and morphological parameters were examined at 20 weeks of age. Results In db/db mice, left ventricular developed pressure (LVDP), heart rate (HR) and coronary flow rate (CFR) were decreased compared to db/m control mice, indicating cardiovascular dysfunction in diabetic heart. The treatment with AdipoRon remarkably improved the diabetes-induced contractile impairment, without any influence on HR. Adioporon also decreased fibrosis, inflammation cell infiltration and accumulations of free fatty acid, triglycerides and ceramide in the heart. In the molecular level, increased expressions of AdipoR1 and AdipoR2 and acid ceramidase activity in the heart were observed in db/db mice with Adiporon administration. Consistent up-regulations of phosphorylated AMPK and PPAR-α level were associated within the same group. Subsequent improvement of enhanced lipid metabolism and decrement of cellular apoptosis with adiporon treatment were also noted. Conclusion Adiporon may control oxidative stress in heart through AMPK and PPAR-α activated pathway and further contribute to prevent deterioration of cardiac function. The protective role of adiporon against the development of diabetic cardiomyopathy seems to occur through a direct action on the heart independently of systemic effects of adiponectin. Our results suggest adiporon as a promising therapeutic agent of diabetic cardiomyopathy through ameliorating ceramide-dependent lipotoxicity.

Mechanisms underlying electro-mechanical dysfunction in the Zucker diabetic fatty rat heart: a model of obesity and type 2 diabetes.

Diabetes mellitus (DM) is a major and worsening global health problem, currently affecting over 450 million people and reducing their quality of life. Type 2 diabetes mellitus (T2DM) accounts for more than 90% of DM and the global epidemic of obesity, which largely explains the dramatic increase in the incidence and prevalence of T2DM in the past 20 years. Obesity is a major risk factor for DM which is a major cause of morbidity and mortality in diabetic patients. The electro-mechanical function of the heart is frequently compromised in diabetic patients. The aim of this review is to discuss the pathophysiology of electro-mechanical dysfunction in the diabetic heart and in particular, the Zucker diabetic fatty (ZDF) rat heart, a well-studied model of T2DM and obesity.

P712Non-neuronal cholinergic system prevents coronary vascular dysfunction in diabetic heart

Abstract Background Diabetic individuals suffer extensive myocardial damage during ischemia due to impaired ATP production and coronary vascular dysfunction. The cardiomyocytes possess a non-neuronal cholinergic system (NNCS) as it has choline acetyltransferase (ChAT) to synthesize acetylcholine (ACh). ACh released from cardiomyocytes activates hypoxia-inducible factor-1 pathway in an auto/paracrine manner under non-hypoxic condition. Activation of this pathway via NNCS promotes angiogenesis and is a promising mechanism to target ischemia in diabetes. Aim To investigate if activation of NNCS could improve the coronary vasculature in diabetic heart. Methods Type-2 diabetic db/db mice with ventricle-specific ChAT transgene (db/db-ChAT-tg) and control db/db mice of 12- and 24-weeks old were used. Catheterization of the jugular vein and carotid artery was performed in combination with synchrotron radiation microangiography to visualize the in-vivo coronary circulation. Changes of the coronary circulation to ACh (10μg/kg/min) and sodium nitroprusside (SNP, 10μg/kg/min) were assessed. Immunofluorescence analysis was performed to measure the density of arterioles and capillaries ex-vivo. Results In comparison to db/db mice, the number of second and third order vessels was higher in the db/db-ChAT-tg mice of 12- and 24-weeks old under baseline condition. In response to ACh and SNP, number of third order vessels were further increased in the db/db-ChAT-tg mice of both ages. However, the magnitude of the diameter changes in db/db-ChAT-tg mice was comparable to that in db/db mice of both ages. Besides, the db/db-ChAT-tg mice had increased density of arterioles and capillaries compared to the db/db mice of both ages. Conclusion NNCS-induced angiogenesis prevents coronary vascular dysfunction in diabetic heart.

Fibroblast growth factor 19 improves cardiac function and mitochondrial energy homoeostasis in the diabetic heart

In diabetic cardiomyopathy, mitochondrial fatty acid oxidation dominates over mitochondrial glucose oxidation, leading to metabolic disturbances. Fibroblast growth factor 19 (FGF19) acts as a metabolic regulator and may have a cardioprotective role on diabetic cardiomyopathy. In this study, we investigated the effects of FGF19 on energy metabolism. FGF19 treatment of diabetic hearts exhibited higher glucose uptake and lower lipid profiles, suggesting changes in energy metabolism. The protective effects of FGF19 prevented ventricular dysfunction in diabetic hearts and improved mitochondrial function by the upregulation of PGC-1α expression. On the other side, knockdown of PGC-1α by siRNA attenuated the effects of FGF19 on the enhancement of mitochondrial function and energy efficiency. Taken together, these results show that FGF19 exhibited improved mitochondrial efficiency, which might be associated with higher cardiac contractility in diabetic hearts. It is also of note that modulation of PGC-1α, which is responsible for the activation by FGF19, may be a therapeutic target for diabetic cardiomyopathy.

Treatment with crocin improves cardiac dysfunction by normalizing autophagy and inhibiting apoptosis in STZ-induced diabetic cardiomyopathy

Background and aimThe association of diabetes mellitus (DM) and poor metabolic control with high incidence of cardiovascular diseases is well established. The aim of this study was to investigate the potential cardioprotective effect of crocin (Crocus sativus L. extract) on diabetic heart dysfunction and to elucidate the mediating molecular mechanisms. Methods and resultsStreptozotocin (STZ)-induced diabetic rats were treated with two different concentrations of crocin (10 or 20 mg/kg), while isolated cardiac myocytes exposed to 25 mM glucose, were treated with 1 or 10 μM of crocin. Treatment of STZ-diabetic rats with crocin resulted in normalization of plasma glucose levels, inhibition of cardiac hypertrophy and fibrosis, and improvement of cardiac contractile function. Heat Shock Response was enhanced. Myocardial AMPK phosphorylation was increased after treatment with crocin, resulting in normalization of autophagy marker proteins (LC3BII/LC3BI ratio, SQSTM1/p62 and Beclin-1), while the diabetes-induced myocardial apoptosis was decreased. Similar results regarding the effect of crocin on autophagy and apoptosis pathways were obtained in isolated cardiac myocytes exposed to high concentration of glucose. ConclusionThe results suggest that crocin improves the deteriorated cardiac function in diabetic animals by enhancing the heat shock response, inhibiting apoptosis and normalizing autophagy in cardiac myocytes. Thus, treatment with crocin may represent a novel approach for treating diabetic cardiomyopathy.

O45-3 - Dpp4 Inhibitor Attenuates Cardiac Systolic Dysfunction in a Murine Dietary Obese Model by Promoting Fibroblast Growth Factor 2-mediated Angiogenesis

Dipeptidyl peptidase-4 (Dpp4) inhibitors are used worldwide to combat diabetes, however, their roles in cardiovascular disorders are yet to be defined. Here we show that a DPP4 inhibitor, linagliptin, contributes for the suppression of capillary rarefaction in diabetic heart and systolic dysfunction. The capillary rarefaction in dietary obese mice associated with high DPP4 level in circulation, and the administration of linagliptin suppressed the development of capillary rarefaction and ameliorated cardiac dysfunction. DNA micro array data showed that early growth response protein 1 (EGR1), known as an angiogenic transcription factor, is significantly reduced in the cardiac tissue upon metabolic stress, and this suppression was inhibited by the administration of linagliptin. Fibroblast growth factor 2 (FGF-2) has putative amino acid sequence cleaved by DPP4, and LC-MS/MS studies showed that DPP4 actually cleaves FGF-2. In vitro studies showed that FGF-2 contributes to up-regulation of Egr1 expression, and this was suppressed with DPP4. Furthermore, our studies suggested that vascular endothelial growth factor a (VEGFa) is the critical angiogenic factor mediating FGF-2-EGR-1 signaling. Our studies suggest that DPP4 inhibits FGF-2/EGR-1/VEGFa signaling in cardiac tissues. Suppression of DPP4 is crucial for the suppression of capillary rarefaction and cardiac dysfunction in diabetic heart.

Inhibition of sarcolemmal FAT/CD36 by sulfo-N-succinimidyl oleate rapidly corrects metabolism and restores function in the diabetic heart following hypoxia/reoxygenation.

AimsThe type 2 diabetic heart oxidizes more fat and less glucose, which can impair metabolic flexibility and function. Increased sarcolemmal fatty acid translocase (FAT/CD36) imports more fatty acid into the diabetic myocardium, feeding increased fatty acid oxidation and elevated lipid deposition. Unlike other metabolic modulators that target mitochondrial fatty acid oxidation, we proposed that pharmacologically inhibiting fatty acid uptake, as the primary step in the pathway, would provide an alternative mechanism to rebalance metabolism and prevent lipid accumulation following hypoxic stress.Methods and resultsHearts from type 2 diabetic and control male Wistar rats were perfused in normoxia, hypoxia and reoxygenation, with the FAT/CD36 inhibitor sulfo-N-succinimidyl oleate (SSO) infused 4 min before hypoxia. SSO infusion into diabetic hearts decreased the fatty acid oxidation rate by 29% and myocardial triglyceride concentration by 48% compared with untreated diabetic hearts, restoring fatty acid metabolism to control levels following hypoxia-reoxygenation. SSO infusion increased the glycolytic rate by 46% in diabetic hearts during hypoxia, increased pyruvate dehydrogenase activity by 53% and decreased lactate efflux rate by 56% compared with untreated diabetic hearts during reoxygenation. In addition, SSO treatment of diabetic hearts increased intermediates within the second span of the Krebs cycle, namely fumarate, oxaloacetate, and the FAD total pool. The cardiac dysfunction in diabetic hearts following decreased oxygen availability was prevented by SSO-infusion prior to the hypoxic stress. Infusing SSO into diabetic hearts increased rate pressure product by 60% during hypoxia and by 32% following reoxygenation, restoring function to control levels.ConclusionsDiabetic hearts have limited metabolic flexibility and cardiac dysfunction when stressed, which can be rapidly rectified by reducing fatty acid uptake with the FAT/CD36 inhibitor, SSO. This novel therapeutic approach not only reduces fat oxidation but also lipotoxicity, by targeting the primary step in the fatty acid metabolism pathway.

Autophagy was involved in the protective effect of metformin on hyperglycemia-induced cardiomyocyte apoptosis and Connexin43 downregulation in H9c2 cells.

Background: Increased cardiomyocyte apoptosis under high glucose condition contributes to diabetic cardiomyopathy. Degradation of cardiac Connexin43 (Cx43) has been associated with cardiac dysfunction in diabetic heart. Clinical and experimental studies suggested that metformin (Met) exhibits cardioprotective properties against diabetes.Aim: The aim of this study was to investigate the effect and underlying signaling mechanisms of metformin on apoptosis and Cx43 expression in H9c2 cells presenting with hyperglycemia conditions.Methods: In the present study, H9c2 cardiac cells were incubated with 5.5 mM glucose, 33.3 mM glucose, 33.3 mM glucose with metformin at two dose (100 μM, 1 mM) for 96 hours, and 1 mM metformin with chloroquine (50 μM) in 33.3 mM glucose medium. Cell viability was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) cell survival assay. Cytotoxicity was determined by the release of lactate dehydrogenase (LDH). The expression of Cx43, autophagic maker protein (LAMP-1, Beclin-1, p62 and LC3) and apoptosis maker protein (Bcl-2 and Bax) were determined by western blot.Results: The results showed that high glucose increased apoptosis and decreased Cx43 expression. Interestingly, metformin attenuated hyperglycemia-increased apoptosis and restored Cx43 expression. Moreover, this treatment caused autophagy as well, which indicated by up-regulation of autophagy-related proteins LAMP-1, Beclin-1, p62 and reduction in the ratio of LC3-II/LC3-I. In addition, administration autophagy inhibitor chloroquine (CQ) did not block the effect of metformin on Cx43 expression while increasing Cx43 content, together with an increased apoptosis.Conclusion: Administration metformin can protect the H9c2 cells against hyperglycemia-induced apoptosis and Cx43 down-regulation, in part, mediated through the induction of autophagy pathway.

Garlic activates SIRT-3 to prevent cardiac oxidative stress and mitochondrial dysfunction in diabetes

BackgroundCardiac complications are major contributor in the mortality of diabetic people. Mitochondrial dysfunctioning is a crucial contributor for the cardiac complications in diabetes, and SIRT-3 remains the major mitochondrial deacetylase. We hypothesized whether garlic has any role on SIRT-3 to prevent mitochondrial dysfunction in diabetic heart. MethodsRats with developed hyperglycemia after STZ injection were divided into two groups; diabetic (Dia) and diabetic+garlic (Dia+Garl). Garlic was administered at a dose of 250mg/kg/day, orally for four weeks. An additional group was maintained to evaluate the effect of raw garlic administration on control rat heart. ResultWe have observed altered functioning of cardiac mitochondrial enzymes involved in metabolic pathways, and increased levels of cardiac ROS with decreased activity of catalase and SOD in diabetic rats. Cardiac mRNA expression of TFAM, PGC-1α, and CO1 was also altered in diabetes. In addition, reduced levels of electron transport chain complexes that observed in Dia group were normalized with garlic administration. This indicates the presence of increased oxidative stress with mitochondrial dysfunctioning in diabetic heart. We have observed reduced activity of SIRT3 and increased acetylation of MnSOD. Silencing SIRT-3 in cells also revealed the same. However, administration of garlic improved the SIRT-3 and MnSOD activity, by deacetylating MnSOD. Increased SOD activity was correlated with reduced levels of ROS in garlic-administered rat hearts. ConclusionCollectively, our results provide an insight into garlic's protection to T1DM heart through activation of SIRT3-MnSOD pathway.

The role of CaMKII in diabetic heart dysfunction.

Diabetes mellitus (DM) is an increasing epidemic that places a significant burden on health services worldwide. The incidence of heart failure (HF) is significantly higher in diabetic patients compared to non-diabetic patients. One underlying mechanism proposed for the link between DM and HF is activation of calmodulin-dependent protein kinase (CaMKIIδ). CaMKIIδ mediates ion channel function and Ca(2+) handling during excitation-contraction and excitation-transcription coupling in the myocardium. CaMKIIδ activity is up-regulated in the myocardium of diabetic patients and mouse models of diabetes, where it promotes pathological signaling that includes hypertrophy, fibrosis and apoptosis. Pharmacological inhibition and knockout models of CaMKIIδ have shown some promise of a potential therapeutic benefit of CaMKIIδ inhibition, with protection against cardiac hypertrophy and apoptosis reported. This review will highlight the pathological role of CaMKIIδ in diabetes and discuss CaMKIIδ as a therapeutic target in DM, and also the effects of exercise on CaMKIIδ.