Diabetic Mouse Hearts Research Articles

Role of CCR2 in the Development of Streptozotocin-Treated Diabetic Cardiomyopathy.

CCR2 has been proven to play an important role in diabetes. However, the role of CCR2 in diabetic cardiomyopathy has not been examined. In this study, we investigated the effects of cardiac CCR2 on diabetic cardiomyopathy. We created a model of streptozotocin (STZ)-induced diabetic cardiomyopathy. Expression of CCR2 was upregulated in the hearts of STZ-induced diabetic mice. CCR2 knockout significantly improved STZ-induced cardiac dysfunction and fibrosis. Moreover, deletion of CCR2 inhibited STZ-induced apoptosis and the production of STZ-induced reactive oxygen species in the heart. CCR2 knockout resulted in M2 polarization in hearts of STZ-treated mice. Treatment with a CCR2 inhibitor reversed hyperglycemia-induced cardiac dysfunction in db/db mice. These results suggest that CCR2-induced inflammation and oxidative stress in the heart are involved in the development of diabetic cardiomyopathy and that CCR2 could be a novel target for therapy.

MiR-222 inhibits cardiac fibrosis in diabetic mice heart via regulating Wnt/β-catenin-mediated endothelium to mesenchymal transition.

miR-222 participates in many cardiovascular diseases, but its effect on cardiac remodeling induced by diabetes is unclear. This study evaluated the functional role of miR-222 in cardiac fibrosis in diabetic mice. Streptozotocin (STZ) was used to establish a type 1 diabetic mouse model. After 10 weeks of STZ injection, mice were intravenously injected with Ad-miR-222 to induce the overexpression of miR-222. miR-222 overexpression reduced cardiac fibrosis and improved cardiac function in diabetic mice. Mechanistically, miR-222 inhibited the endothelium to mesenchymal transition (EndMT) in diabetic mouse hearts. Mouse heart fibroblasts and endothelial cells were isolated and cultured with high glucose (HG). An miR-222 mimic did not affect HG-induced fibroblast activation and function but did suppress the HG-induced EndMT process. The antagonism of miR-222 by antagomir inhibited HG-induced EndMT. miR-222 regulated the promoter region of β-catenin, thus negatively regulating the Wnt/β-catenin pathway, which was confirmed by β-catenin siRNA. Taken together, our results indicated that miR-222 inhibited cardiac fibrosis in diabetic mice via negatively regulating Wnt/β-catenin-mediated EndMT.

Abstract 167: Increased Mitophagy and Lysophagy in High Fat Diet and Streptozotocin Induced Diabetic Cardiomyopathy

Heart disease and diabetes are among the leading causes of morbidity and mortality. Diabetes can result in diabetic cardiomyopathy, which further increases the risk of heart failure and mortality in diabetic patients. Diabetic cardiac injury has been associated with altered mitochondrial quality control mechanisms including mitophagy, a selective autophagy in which dysfunctional mitochondria are degraded through the lysosome. Lysophagy is the elimination of injured lysosomes by healthy lysosomes which is extremely important for all forms of autophagy. In the present study, we examined the functional status of mitophagy-lysosome system in the diabetic mouse heart. Cardiac mitophagy and lysophagy were determined by using novel dual fluorescent reporter mice, namely, mt-rosella that express mitochondria-targeted RFP-GFP fusion protein and tfgal3 that express RFP-GFP-galectin3, a galactoside-binding lectin. Mice were fed a high fat diet (60%calories by fat) for 1 month and then injected i.p. with streptozotocin (30 mg/kg/d for 3 days). Mice continued on HFD for additional 3 months. Echocardiographic data showed impaired cardiac function in the diabetic mice. Merged confocal images demonstrated that diabetes increased mitophagy flux in the heart as indicated by the red puncta from mt-Rosella and the mitochondrial LC3-II levels with and without lysosomal protease inhibitors pepstatin A and E64d. In addition, diabetes triggered lysosomal injury in the heart as shown by the increased total number of puncta (yellow plus red) from tfgal3 reporter on the merged confocal images. This was associated with accelerated lysophagy flux as shown by the red puncta, suggesting that the injured lysosomes were being actively degraded in the diabetic heart. The increased lysophagy may have contributed to the increased mitophagy flux since the enhanced elimination of injured lysosomes would be expected to help maintain a pool of healthy lysosomes which is essential for the efficient execution of all forms of autophagy. In summary, our results showed an enhanced mitophagy-lysosome system in the diabetic heart which may be an adaptive response that serves to limit diabetic cardiac injury.

Exenatide Protects Against Cardiac Dysfunction by Attenuating Oxidative Stress in the Diabetic Mouse Heart.

Cardiovascular disease is the major cause of death in patients with diabetes. Current treatment strategies for diabetes rely on lifestyle changes and glucose control to prevent angiopathy and organ failure. Exenatide, a glucagon-like peptide-1 (GLP-1) receptor agonist, is used as an add-on therapy to insulin treatment. Exenatide also has multiple beneficial effects in addition to its hypoglycemic effects, such as preventing hepatic steatosis and protecting against cardiac injury from doxorubicin-induced cardiotoxicity or ischemic reperfusion. However, the mechanisms underlying the cardioprotective effects of exenatide in diabetes have not been fully clarified. To address this issue, we investigated the cardioprotective effects of exenatide in type 1 and type 2 diabetic mice. We found that exenatide simultaneously attenuated reactive oxidative species (ROS) production through increases in the antioxidant enzymes manganese dependent superoxide dismutase (MnSOD) and catalase. Moreover, exenatide decreased tumor protein P53 (p53) expression and prevented cell apoptosis in H9c2 cells. The presence of the catalase inhibitor 3-AT attenuated the effects of exenatide. Overall, the results strongly indicate that exenatide treatment may be protective against the development of diabetic cardiomyopathy.

Atorvastatin Preserves Cardiac Contractility via Reducing Matrix Metalloproteinase-2 Isoforms Expression in a Diabetic Heart Model

Purpose We investigated whether atorvastatin can alleviate diabetic cardiomyopathy (DM CMP) by reducing intracellular matrix metalloproteinase-2 (MMP-2) isoform expression. Methods Rat cardiomyoblasts (H9C2 cells) were tested to determine whether atorvastatin treatment after high glucose could reduce the expression of the two isoforms of MMP-2. For the in vivo study, we used the streptozotocin murine model of Type I DM and age-matched controls. The changes of each MMP-2 isoform in the diabetic mice hearts were determined using quantitative real-time polymerase chain reaction (qRT-PCR). Immunohistochemical stains were conducted to identify MMP-2 isoform expression. Echocardiography was performed to compare and analyze the changes in cardiac function induced by diabetes and atorvastatin. Results Quantitative RT-PCR and immunofluorescence staining showed that the two MMP-2 isoforms strongly induced by high glucose stimulation were attenuated by atorvastatin treatment in H9C2 cells. Although no definite histologic features of diabetic cardiomyopathy were observed in diabetic mice, left ventricular systolic dysfunction observed in diabetic heart was also normalized by atorvastatin treatment by echocardiographic evaluation. These findings accompanied reduced TUNEL staining and mitochondrial morphological change in atorvastatin treated diabetic group. Quantitative RT-PCR and IHC staining showed this abnormal cardiac function was accompanied with the increases in the mRNA levels of the two isoforms of MMP-2 and attenuated with atorvastatin treatment Conclusion Atorvastatin attenuated deterioration of cardiac dysfunction induced by diabetic condition by reducing expression of two isoforms of MMP-2 in vitro and in a Type I diabetes heart model. The roles played by these isoforms and atorvastatin treatment in diabetic cardiomyopathy require further study.

MiR-451 antagonist protects against cardiac fibrosis in streptozotocin-induced diabetic mouse heart

AimsMicroRNAs (miRNAs or miRs) are a large class of small noncoding RNAs. The present study aims to evaluate the effect of miR-451 on cardiac remodeling in diabetic cardiomyopathy. Main methodsMice were injected with streptozotocin (STZ) to induce diabetes. Twelve weeks after final STZ injection, mice were subjected to myocardial injection of adenovirus (Ad)-shmiR-451 to knock down miR-451. Mouse heart endothelial cells (MHECs) were treated with a miR-451 antagomir to inhibit miR451 and were exposed to high glucose. Key findingsSixteen weeks after STZ injection, mice exhibited no significant cardiac hypertrophy but did exhibit serious cardiac fibrosis. MiR-451 knockdown attenuated cardiac fibrosis and improved cardiac function. Moreover, we found that miR-451 knockdown suppressed endothelial-to-mesenchymal transition (EndMT) in diabetic mouse hearts. Hyperglycemia-induced EndMT in MHECs was attenuated by the miR-451 antagomir. Activation of AMPKa1/mTOR was decreased in diabetic mouse heart tissue and hyperglycemia-stimulated MHECs, which was increased following miR-451 knockdown or inhibition. AMPKa1 siRNA abrogated the anti-EndMT effects of miR-451 knockdown in MHECs. SignificancemiR-451 participates in the pathology of diabetic cardiomyopathy via AMPKa1-regulated EndMT in endothelial cells.

Early dysregulation of cardiac-specific microRNA-208a is linked to maladaptive cardiac remodelling in diabetic myocardium

BackgroundThe diabetic heart undergoes remodelling contributing to an increased incidence of heart failure in individuals with diabetes at a later stage. The molecular regulators that drive this process in the diabetic heart are still unknown.MethodsReal-time (RT) PCR analysis was performed to determine the expression of cardiac specific microRNA-208a in right atrial appendage (RAA) and left ventricular (LV) biopsy tissues collected from diabetic and non-diabetic patients undergoing coronary artery bypass graft surgery. To determine the time-dependent changes, cardiac tissue were collected from type 2 diabetic mice at different age groups. A western blotting analysis was conducted to determine the expression of contractile proteins α- and β-myosin heavy chain (MHC) and thyroid hormone receptor-α (TR-α), the negative regulator of β-MHC. To determine the beneficial effects of therapeutic modulation of miR-208a, high glucose treated adult mouse HL-1 cardiomyocytes were transfected with anti-miR-208a.ResultsRT-PCR analysis showed marked upregulation of miR-208a from early stages of diabetes in type 2 diabetic mouse heart, which was associated with a marked increase in the expression of pro-hypertrophic β-MHC and downregulation of TR-α. Interestingly, upregulation of miR-208a preceded the switch of α-/β-MHC isoforms and the development of diastolic and systolic dysfunction. We also observed significant upregulation of miR-208a and modulation of miR-208a associated proteins in the type 2 human diabetic heart. Therapeutic inhibition of miR-208a activity in high glucose treated HL-1 cardiomyocytes prevented the activation of β-MHC and hence the hypertrophic response.ConclusionOur results provide the first evidence that early modulation of miR-208a in the diabetic heart induces alterations in the downstream signaling pathway leading to cardiac remodelling and that therapeutic inhibition of miR-208a may be beneficial in preventing diabetes-induced adverse remodelling of the heart.

Magnesium supplementation improves diabetic mitochondrial and cardiac diastolic function.

In heart failure and type 2 diabetes mellitus (DM), the majority of patients have hypomagnesemia, and magnesium (Mg) supplementation has improved cardiac function and insulin resistance. Recently, we have shown that DM can cause cardiac diastolic dysfunction (DD). Therefore, we hypothesized that Mg supplementation would improve diastolic function in DM. High-fat diet-induced diabetic mouse hearts showed increased cardiac DD and hypertrophy. Mice with DM showed a significantly increased E/e' ratio (the ratio of transmitral Doppler early filling velocity [E] to tissue Doppler early diastolic mitral annular velocity [e']) in the echocardiogram, left ventricular end diastolic volume (LVEDV), incidence of DD, left ventricular posterior wall thickness in diastole (PWTd), and ratio of heart weight to tibia length (HW/TL) when compared with controls. DM mice also had hypomagnesemia. Ventricular cardiomyocytes isolated from DM mice exhibited decreased mitochondrial ATP production, a 1.7- ± 0.2-fold increase of mitochondrial ROS, depolarization of the mitochondrial membrane potential, and mitochondrial Ca2+ overload. Dietary Mg administration (50 mg/ml in the drinking water) for 6 weeks increased plasma Mg concentration and improved cardiac function. At the cellular level, Mg improved mitochondrial function with increased ATP, decreased mitochondrial ROS and Ca2+ overload, and repolarized mitochondrial membrane potential. In conclusion, Mg supplementation improved mitochondrial function, reduced oxidative stress, and prevented DD in DM.

Establishing a Link between Endothelial Cell Metabolism and Vascular Behaviour in a Type 1 Diabetes Mouse Model.

Background/Aims:Vascular complications contribute significantly to the extensive morbidity and mortality rates observed in people with diabetes. Despite well known that the diabetic kidney and heart exhibit imbalanced angiogenesis, the mechanisms implicated in this angiogenic paradox remain unknown. In this study, we examined the angiogenic and metabolic gene expression profile (GEP) of endothelial cells (ECs) isolated from a mouse model with type1 diabetes mellitus (T1DM).Methods:ECs were isolated from kidneys and hearts of healthy and streptozocin (STZ)-treated mice. RNA was then extracted for molecular studies. GEP of 84 angiogenic and 84 AMP-activated Protein Kinase (AMPK)-dependent genes were examined by microarrays. Real time PCR confirmed the changes observed in significantly altered genes. Microvessel density (MVD) was analysed by immunohistochemistry, fibrosis was assessed by the Sirius red histological staining and connective tissue growth factor (CTGF) was quantified by ELISA.Results:The relative percentage of ECs and MVD were increased in the kidneys of T1DM animals whereas the opposite trend was observed in the hearts of diabetic mice. Accordingly, the majority of AMPK-associated genes were upregulated in kidneys and downregulated in hearts of these animals. Angiogenic GEP revealed significant differences in Tgfβ, Notch signaling and Timp2 in both diabetic organs. These findings were in agreement with the angiogenesis histological assays. Fibrosis was augmented in both organs in diabetic as compared to healthy animals.Conclusion:Altogether, our findings indicate, for the first time, that T1DM heart and kidney ECs present opposite metabolic cues, which are accompanied by distinct angiogenic patterns. These findings enable the development of innovative organ-specific therapeutic strategies targeting diabetic-associated vascular disorders.

Abstract 17332: Endothelial-Specific Deletion of Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT) Leads to Microvascular Barrier Dysfunction and Cardiomyopathy

Introduction: Cardiac microvascular hyperpermeability is a key contributor to heart disease in patients with diabetes. Although the link between diabetes and microvascular barrier dysfunction is largely unknown, expression of the transcription factor aryl hydrocarbon receptor nuclear translocator (ARNT) is significantly downregulated in the cardiac microvascular endothelial cells (CMECs) of both diabetic mouse hearts (n=5, p<0.01) and the explanted hearts of patients with diabetes mellitus (n=3, p<0.05). We hypothesize that cardiac microvascular permeability is limited by the expression of ARNT in the endothelium and, consequently that endothelial-cell ARNT (ecARNT) expression is essential for normal cardiac function. Methods and Results: We have recently generated tamoxifen-inducible, endothelial-cell-specific, VE-cadherin-Cre ETR2 ARNT-knockout mice (ecARNT -/- mice). ecARNT deletion is achieved by tamoxifen oral administration for two weeks. Littermates controls are either mice without tamoxifen chow or ARNT flox/flox treated with tamoxifen diet. Induction of the ecARNT -/- mutation led to vascular leakage (as determined via in-vivo endothelial permeability assay) (n=5, p<0.05), which occurred predominantly in the heart, and to increases in matrix metalloproteinase (MMP) expression (microarray analysis), including a 3.8-fold increase in the expression of MMP3 (n=3, p<0.01). Furthermore, MMP3 inhibition attenuated the increase in cell permeability observed in CMECs from ecARNT -/- mouse hearts (as measured via electrical cell-substrate impedance sensing), as well as cardiac vascular leakage in ecARNT -/- mice, while long-term studies indicated that ejection fractions were significantly lower (ecARNT -/- : 22.3±2.6%, Control: 33.2±1.8%;), and Left ventricular end-diastolic diameters were significantly greater (ecARNT -/- : 4.8±0.8 mm, Control: 3.6±0.6 mm; n=8,p<0.01) six months after ecARNT deletion (echocardiography). Conclusion: ARNT-mediated MMP3 downregulation is required for maintaining cardiac microvascular barrier integrity and preserving cardiac function; thus, modulation of the ARNT/MMP3 axis could be a novel approach for the treatment of cardiovascular diseases such as diabetic cardiomyopathy.

Curcumin protects against diabetic cardiomyopathy by promoting autophagy and alleviating apoptosis

The effects of curcumin on regulating cardiac apoptosis and autophagy were analyzed in diabetic models both in vivo and in vitro. In vivo, experimental diabetes was induced in mice by low-dose STZ injection combined with a high-fat diet. In vitro, cultured H9c2 cardiomyoblasts were exposed to high d-glucose concentrations combined with palmitate. Our results showed that apoptosis was increased and autophagy was suppressed in the hearts of diabetic mice, which was ameliorated by curcumin treatment, ultimately improving cardiac function. Moreover, the inhibition of autophagy exacerbated apoptotic death in cardiac cells under diabetic condition. Curcumin activated AMPK and JNK1, which phosphorylated Bcl-2 and Bim and subsequently disrupted their interactions with Beclin1, thereby promoting autophagy and alleviating apoptosis respectively. In addition, AMPK-mediated inhibition of mTORC1 pathway likely played a role in regulating autophagy by curcumin under diabetic condition. Our study suggests that curcumin protects against diabetic cardiomyopathy by modulating the crosstalk between autophagic and apoptotic machinery. Modulation of autophagy may be an effective strategy for the treatment of cardiovascular diseases associated with diabetes.

OR-010 The potential role of MG53 in exercise-mediated modulation of diabetic cardiomyopathy in db/db mice

Objective Diabetic cardiomyopathy is a major complication of Type 2 diabetes. Recent studies have shown that as an E3 ligase targeting insulin receptor (IR) and insulin receptor substrate 1 (IRS1), MG53 could result in insulin resistance and metabolic disorders. However, it is still to be investigated whether MG53 plays a role in the pathogenesis of diabetic cardiomyopathy, as well as whether the effect of exercise intervention on diabetic cardiomyopathy is mediated by MG53.
 Methods 20 db/db mice and 20 m/m mice were randomly assigned to Groups DC (db/db control, N=10), DE (db/db exercise, N=10), MC (m/m control, N=10) and ME (m/m exercise, N=10). The mice of Groups ME and DE were trained to run on the treadmill for 12 weeks in total. For Week 1, the mice ran for 20min at 7-13m/min, four times a week. Since the second week, the mice were trained to run at 13.3m/min, 6d/wk, and the running duration was increased from 30-45min/d for Week 2 to 1h/d since the third week. At the end of the intervention period, IPGTT and IPITT were performed to determine the effect of exercise training. The mice were then euthanized and the heart was removed. Real-time PCR and Western blotting were performed to determine the expression levels of mRNA and proteins.
 Results According to the result of IPGTT and IPITT, glucose concentration was significantly higher in DC compared to MC, and exercise intervention significantly decreased the glucose level of diabetic mice. Heart weight to tibia length ratio was not significantly different between MC and DC, but was higher in DE than in DC. As expected, the mRNA expression of MG53 was significantly higher in the diabetic mice, and was significantly decreased by exercise training. However, the protein level of MG53 was not significantly different between MC and DC, even though exercise intervention caused lower MG53 protein level in DE. The protein level of p-IR-β (Tyr1146) was significantly higher in DC than MC. Exercise intervention significantly decreased the protein level of p-IRS1 (Ser1101) in both the lean and diabetic mice. Diabetes caused a significant decrease in p-AKT (Ser473), while exercise training increased the protein level of p-AKT (Ser473) of the diabetic mice. The mRNA expressions of Ppargc1a, Cpt1b, Acadm, Acadvl, Acacb and Acaa2 were significantly increased in DC compared to MC, suggesting that fat metabolism was enhanced in the hearts of diabetic mice. To the contrary, the enhanced fat metabolism was compromised by exercise intervention, as revealed by the mRNA expression level of Cpt1b, Acadm, Acacb and Acaa2. Besides, the mRNA expression of Pdk1 and Pdk2 were significantly increased in db/db mice, suggesting that glucose metabolism was reduced by diabetes. PPAR-α is an important regulator of fat metabolism. The protein level of PPAR-α was significantly increased in DC compared to MC, and was decreased in DE compared to DC. Besides, the mRNA expression of key components of PPAR-α pathway, including Cd36, Ppargc1b and Fabp3 were all significantly increased in db/db mice, while exercise intervention significantly decreased the mRNA expression of Cd36 and Ppargc1b.
 Conclusions MG53 is involved in the pathogenesis of diabetic cardiomyopathy of db/db mice by inhibiting insulin signaling, downregulating glucose metabolism and promoting fat metabolism. The state of insulin resistance and alteration of substrate utilization brought by diabetes could be improved by 12 week treadmill training, partly through the regulation of PPAR-α pathway.

Isolated perfused working hearts provide valuable additional information during phenotypic assessment of the diabetic mouse heart.

Although murine models for studying the development of cardiac dysfunction in diabetes mellitus are well established, their reported cardiac phenotypes vary. These reported divergences may, in addition to the severity of different models, also be linked to the methods used for cardiac functional assessment. In the present study, we examined the functional changes using conventional transthoracic echocardiography (in vivo) and isolated heart perfusion techniques (ex vivo), in hearts from two mouse models; one with an overt type 2 diabetes (the db/db mouse) and one with a prediabetic state, where obesity was induced by a high-fat diet (HFD). Analysis of left ventricular function in the isolated working hearts from HFD-fed mice, suggested that these hearts develop diastolic dysfunction with preserved systolic function. Accordingly, in vivo examination demonstrated maintained systolic function, but we did not find parameters of diastolic function to be altered. In db/db mice, ex vivo working hearts showed both diastolic and systolic dysfunction. Although in vivo functional assessment revealed signs of diastolic dysfunction, the hearts did not display reduced systolic function. The contrasting results between ex vivo and in vivo function could be due to systemic changes that may sustain in vivo function, or a lack of sensitivity using conventional transthoracic echocardiography. Thus, this study demonstrates that the isolated perfused working heart preparation provides unique additional information related to the development of cardiomyopathy, which might otherwise go unnoticed when only using conventional echocardiographic assessment.

LncRNA HOTAIR functions as a competing endogenous RNA to upregulate SIRT1 by sponging miR-34a in diabetic cardiomyopathy.

TheHOX transcript antisense RNA (HOTAIR) long noncoding RNA (lncRNA), a highly abundant and conserved imprinted gene, has been implicated in many essential biological processes and diseases. However, to date, the significance of HOTAIR in diabetic cardiomyopathy (DCM) has never been investigated. The current study was designed to determine whether DCM can be regulated by HOTAIR and to elucidate the related mechanism. In vivo, streptozotocin (STZ) was injected intraperitoneally to induce type 1 diabetes in mice. Cardiomyocyte specific HOTAIR overexpression was achieved using an adeno-associated virus system 12 weeks after STZ injection. In vitro, H9c2 were used to explore the potential molecular mechanism of HOTAIR in the regulation of high-glucose-induced cardiomyocyte injury. Luciferase reporter assay and RNA immunoprecipitation (RIP) were performed to explore the relationship between HOTAIR, microRNA-34a (miR-34a), and Sirtuin 1 (SIRT1). HOTAIR expression was significantly decreased in diabetic mice hearts. Knockdown of HOTAIR in high glucose-induced H9c2 resulted in increased oxidative injury, inflammation, and apoptosis in vitro. Cardiomyocyte-specific overexpression of HOTAIR improved cardiac function, decreased oxidative stress and inflammation, and attenuated myocyte death in mice treated with STZ. Mechanistically, the expression of SIRT1/forkhead box protein O1 was significantly increased in the HOTAIR-overexpressing hearts compared with the control hearts treated with STZ. Moreover, we found HOTAIR functioned as a molecular sponge of miR-34a in H9c2 and SIRT1 was identified as a target of miR-34a. Furthermore, the protective effects of HOTAIR on DCM was abolished in SIRT1 deficiency mice in vivo. HOTAIR protected against DCM via activation of the SIRT1 expression by sponging miR-34a.

The Dipeptidyl Peptidase 4 Substrate CXCL12 Has Opposing Cardiac Effects in Young Mice and Aged Diabetic Mice Mediated by Ca2+ Flux and Phosphoinositide 3-Kinase γ.

Blood glucose-lowering therapies can positively or negatively affect heart function in type 2 diabetes, or they can have neutral effects. Dipeptidyl peptidase 4 (DPP-4) inhibitors lower blood glucose by preventing the proteolytic inactivation of glucagon-like peptide 1 (GLP-1). However, GLP-1 is not the only peptide substrate of DPP-4. Here, we investigated the GLP-1-independent cardiac effects of DPP-4 substrates. Pointing to GLP-1 receptor (GLP-1R)-independent actions, DPP-4 inhibition prevented systolic dysfunction equally in pressure-overloaded wild-type and GLP-1R knockout mice. Likewise, DPP-4 inhibition or the DPP-4 substrates substance P or C-X-C motif chemokine ligand 12 (CXCL12) improved contractile recovery after no-flow ischemia in the hearts of otherwise healthy young adult mice. Either DPP-4 inhibition or CXCL12 increased phosphorylation of the Ca2+ regulatory protein phospholamban (PLN), and CXCL12 directly enhanced cardiomyocyte Ca2+ flux. In contrast, hearts of aged obese diabetic mice (which may better mimic the comorbid patient population) had diminished levels of PLN phosphorylation. In this setting, CXCL12 paradoxically impaired cardiac contractility in a phosphoinositide 3-kinase γ-dependent manner. These findings indicate that the cardiac effects of DPP-4 inhibition primarily occur through GLP-1R-independent processes and that ostensibly beneficial DPP-4 substrates can paradoxically worsen heart function in the presence of comorbid diabetes.

CELF1 contributes to aberrant alternative splicing patterns in the type 1 diabetic heart

Dysregulated alternative splicing (AS) that contributes to diabetes pathogenesis has been identified, but little is known about the RNA binding proteins (RBPs) involved. We have previously found that the RBP CELF1 is upregulated in the diabetic heart; however, it is unclear if CELF1 contributes to diabetes-induced AS changes. Utilizing genome wide approaches, we identified extensive changes in AS patterns in Type 1 diabetic (T1D) mouse hearts. We discovered that many aberrantly spliced genes in T1D hearts have CELF1 binding sites. CELF1-regulated AS affects key genes within signaling pathways relevant to diabetes pathogenesis. Disruption of CELF1 binding sites impairs AS regulation by CELF1. In sum, our results indicate that CELF1 target RNAs are aberrantly spliced in the T1D heart leading to abnormal gene expression. These discoveries pave the way for targeting RBPs and their RNA networks as novel therapies for cardiac complications of diabetes.

Abstract 274: Regulation of Myocardial Ketone Body Oxidation Capacity by Increased Protein O-GlcNAcylation in Diabetic Heart

Diabetes increases the risk of heart failure independently of underlying coronary artery disease. Perturbations in myocardial substrate utilization have been proposed to contribute to the pathogenesis of cardiac dysfunction. However, the exact mechanisms linking impaired substrate utilization to disease progression remain elusive. Compared to the well-established roles of glucose and fatty acids metabolism in cardiac physiology and pathology, the importance of ketone body (KB) metabolism is less well understood. Several findings suggest the importance of KB in the cardiac health and disease. More recently, two independent studies have simultaneously demonstrated increased reliance on KB utilization by the failing heart in the absence of diabetes. Despite these observations, our understanding of the importance of KB metabolism in a diabetic heart is incomplete. Interestingly, our findings from the hearts of streptozotocin-induced diabetic mice confirmed elevated cardiac β-hydroxybutyrate (a major KB) levels and transcriptional suppression of Oxct1 gene expression that encodes for SCOT (succinyl-CoA:3-oxoacid CoA transferase; a rate limiting enzyme of KB oxidation), consistent with a mismatch between KB availability and utilization. To determine a specific role for glucose delivery in this regulation we used an inducible transgenic mouse model with cardiac-restricted expression of the glucose transporter 4 (GLUT4; mG4H). Interestingly, mG4H mice had reduced cardiac SCOT protein levels and oxidation of KB in isolated working heart suggesting that glucose is sufficient to regulate the ketone metabolic pathway. Hyperglycemia associated with diabetes alter protein function via post-translational O -GlcNAcylation. Interestingly, we found that myocardial SCOT expression was also reduced in a third mouse model with increased cardiac O -GlcNAc levels (overexpression of a dominant negative form of O -GlcNAcase, which normally removes O -GlcNAc; dnOGAh) suggesting that increased protein O -GlcNAcylation is directly involved. Collectively, these studies establish a novel role of glucose and its downstream regulator, O -GlcNAc, in attenuation of KB metabolism in the diabetic heart.

Abstract 540: Magnesium Improves Cardiac Diastolic Function by Modulating Mitochondria

Rationale: A diet rich in saturated fat has been found to induce diabetes mellitus (DM) accompanied by myocardial mitochondrial dysfunction, insulin resistance, and magnesium (Mg) deficiency. The most prominent characteristic of diabetic cardiomyopathy is cardiac diastolic dysfunction (DD). Mg supplementation improves the heart rate variability and arterial elasticity in chronic heart failure. Recently, we have reported that mitochondrial reactive oxygen species (mitoROS) play a critical role in the progress of DD and diastolic heart failure. Therefore, we hypothesized that Mg supplementation would benefit diastolic function by improving mitochondrial function. Methods and Results: High fat diet (HFD)-induced diabetic mouse hearts showed cardiac DD with increased E/e’ (45±2 in DM mice vs. 32±2 in control mice), increased left ventricular diastolic volume (LVDV, 80±1 μL in DM vs. 61±4 μL in control), and increased incidence of DD (9 of 10 mice in DM vs 1 of 10 mice in control; P<0.05 for all). DM mice also showed decreased plasma Mg concentration (0.80±0.04 mmol/L in DM vs. 0.98±0.03 mmol/L in control, P<0.05). Ventricular cardiomyocytes isolated from DM mice exhibited decreased mitochondrial ATP production (75±11% of control), a 1.7±0.2-fold increase in mitoROS, significantly depolarized mitochondrial membrane potential, and mitochondrial Ca 2+ overload (3.7±1.3-fold; P<0.05 vs. control for all). Dietary Mg administration (50 mg/mL, ≈6-8 g/kg/day) for 6 weeks corrected all these parameters with increased plasma Mg concentration (1.5±0.1 mmol/L), improved cardiac diastolic function (E/e’ 37±1; LVDV 54±5 μL; LVPWT 0.77±0.02 mm; the incidence of DD, 2 of 10 mice; P<0.05 vs. DM for all). Mitochondrial function was improved significantly by Mg with increased ATP production, decreased mitoROS, repolarized mitochondrial membrane potential, and decreased mitochondrial Ca 2+ overload in DM mice. Conclusion: These results indicate that Mg supplementation improved mitochondrial function, reduced oxidative stress, and prevented diastolic dysfunction in DM.

LAZ3 protects cardiac remodeling in diabetic cardiomyopathy via regulating miR-21/PPARa signaling

Diabetes contributes to cardiovascular complications and the pathogenesis of cardiac remodeling that can lead to heart failure. We aimed to evaluate the functional role of LAZ3 in diabetic cardiomyopathy (DCM). Streptozotocin (STZ) was used to induce a diabetic mouse model. Three months after induction, the mice were subjected to retro-orbital venous plexus injection of adeno-associated virus 9 (AAV9) that overexpressed LAZ3. Six weeks after the infection, mouse hearts were removed to assess the degree of cardiac remodeling. LAZ3 was down-regulated in the diabetic mouse hearts and high glucose stimulated cardiomyocytes. Knock-down of LAZ3 in cardiomyocytes with LAZ3 siRNA reduced cell viability, increased the inflammatory response and induced oxidative stress and cell apoptosis. Overexpression of LAZ3 by infection with adeno-associated virus (AAV9)-LAZ3 protected against an inflammatory response, oxidative stress and cell apoptosis in both a high glucose stimulated in vitro study and diabetic mouse hearts. We found that LAZ3 increased the activation of PPARa, which increased PGC-1a activation and subsequently augmented NRF2 expression and nuclear translocation. This outcome was confirmed by NRF2 siRNA and a PPARa activator, since NRF2 siRNA abrogated the protective effects of LAZ3 overexpression, while the PPARa activator reversed the deteriorating phenotype of LAZ3 knock-down in both the in vitro and vivo study. Furthermore, LAZ3 decreased miR-21 expression, which resulted in PPARa activation, NRF2 expression and nuclear translocation. In conclusion, LAZ3 protects against cardiac remodeling in DCM by decreasing miR-21, thus regulating PPARa/NRF2 signaling.

Hyperglycaemia-induced epigenetic changes drive persistent cardiac dysfunction via the adaptor p66Shc

AimsHyperglycaemia-induced reactive oxygen species (ROS) are key mediators of cardiac dysfunction. Intensive glycaemic control (IGC) has failed to reduce risk of heart failure in patients with diabetes but the underlying mechanisms remain to be elucidated. The present study investigates whether epigenetic regulation of the pro-oxidant adaptor p66Shc contributes to persistent myocardial dysfunction despite IGC. Methods and resultsp66Shc expression was increased in the heart of diabetic mice, and 3-week IGC by slow-release insulin implants did not revert this phenomenon. Sustained p66Shc upregulation was associated with oxidative stress, myocardial inflammation and left ventricular dysfunction, as assessed by conventional and 2D speckle-tracking echocardiography. In vivo gene silencing of p66Shc, performed during IGC, inhibited ROS production and restored cardiac function. Furthermore, we show that dysregulation of methyltransferase DNMT3b and deacetylase SIRT1 causes CpG demethylation and histone 3 acetylation on p66Shc promoter, leading to persistent transcription of the adaptor. Altered DNMT3b/SIRT1 axis in the diabetic heart was explained by upregulation of miR-218 and miR-34a. Indeed, in human cardiomyocytes exposed to high glucose, inhibition of these miRNAs restored the expression of DNMT3b and SIRT1 and erased the adverse epigenetic signatures on p66Shc promoter. Consistently, reprogramming miR-218 and miR-34a attenuated persistent p66Shc expression and ROS generation. ConclusionsIn diabetic left ventricular dysfunction, a complex epigenetic mechanism linking miRNAs and chromatin modifying enzymes drives persistent p66Shc transcription and ROS generation. Our results set the stage for pharmacological targeting of epigenetic networks to alleviate the clinical burden of diabetic cardiomyopathy.