Diabetic Mouse Hearts Research Articles

Acetylation of α-tubulin on Lys40, a new way to modulate cardiac glucose transport

Cardiovascular disease remains the leading cause of death in the diabetic population. Diabetic patients develop a specific heart condition called diabetic cardiomyopathy that can evolve to heart failure and that is characterized by altered metabolism in the early stages of the disease. Our group previously demonstrated that overfueling the heart with branched-chain amino acids, ketone bodies or fatty acids, substrates that are upregulated in diabetes, inhibits glucose entry in cardiomyocytes through a global rise in protein acetylation that interferes with glucose transporter 4 (GLUT4) translocation to the plasma membrane. However, the acetylated proteins involved in this process were not yet identified. We investigated the role of α-tubulin, an important cytoskeleton element responsible for GLUT4 translocation, that is known to be acetylable on Lys40 (K40). Acetylation level of α-tubulin on K40 was evaluated in a mouse model of diabetes (high-fat diet, 4 months) and in primary cultured adult rat cardiomyocytes. Its acetylation level was modulated by using a pharmacological inhibitor of its deacetylase (tubacin 10 μM, 5 hrs) and by overexpressing a non-acetylable form of α-tubulin (mCherry α-tubulin K40A). GLUT4 translocation was assessed by fluorescent immunostaining in cells expressing the fusion protein HA-GLUT4-GFP. Glucose transport was measured by following the detritiation rate of 2-3H-glucose in insulin-sensitive and insulin-resistant cardiomyocytes. Acetylation of α-tubulin on K40 was significantly enhanced in the heart of diet-induced diabetic mice. Experimentally increasing its acetylation level in cardiomyocytes with tubacin impaired GLUT4 translocation to the plasma membrane and reduced both basal and insulin-dependent glucose uptake. On the other hand, decreasing α-tubulin K40 acetylation via the overexpression of the K40A form of α-tubulin stimulated glucose transport. Interestingly, reducing α-tubulin K40 acetylation similarly promoted glucose entry in insulin-resistant conditions. Cardiac glucose transport can be regulated by modulation of α-tubulin K40 acetylation, which may lead to new therapeutic strategies preventing the metabolic changes occurring during the development of diabetic cardiomyopathy.

Mitochondrial damage and activation of the cytosolic DNA sensor cGAS–STING pathway lead to cardiac pyroptosis and hypertrophy in diabetic cardiomyopathy mice

Diabetic cardiomyopathy (DCM) is a serious cardiac complication of diabetes that currently lacks specific treatment. The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway has been suggested to contribute to the pathogenesis of cardiovascular diseases. However, whether cGAS-STING is involved in the development of DCM has not been established. Our study aimed to determine the role of cGAS-STING in the initiation of nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3) inflammasome-induced cardiac pyroptosis and chronic inflammation during the pathogenesis of DCM. C57BL/6J mice were preinjected with adeno-associated virus 9 (AAV9) intravenously via the tail vein to specifically knock down myocardial STING. After four weeks, mice with myocardium-specific knockdown of STING received injections of streptozotocin (STZ; 50 mg/kg) and a high-fat diet to induce diabetes. Measurements included echocardiography, immunohistochemical analyses, wheat germ agglutinin (WGA) staining, and western blotting. Here, we showed that the cGAS-STING signaling pathway was activated in diabetic hearts, which was indicated by the increased phosphorylation of TANK-binding kinase 1 (TBK1) and interferon (IFN) regulatory factor 3 (IRF3), leading to the activation of the NLRP3 inflammasome in the hearts of diabetic mice and proinflammatory cytokine release into serum. Moreover, STING knockdown via adeno-associated virus-9 (AAV9) in diabetic mouse heart alleviated cardiac pyroptosis and the inflammatory response, prevented diabetes-induced hypertrophy, and restored cardiac function. Mechanistically, we showed that palmitic acid (PA)-induced lipotoxicity impairs mitochondrial homeostasis, producing excessive mitochondrial reactive oxygen species (mtROS), which results in oxidative damage to mitochondrial DNA (mtDNA) and its release into the cytoplasm while switching on cGAS-STING-mediated pyroptosis in cardiomyocytes, thereby worsening the progression of diabetic cardiomyopathy. Our study demonstrated that activation of the cGAS-STING pathway caused by mitochondrial oxidative damage and mtDNA escape induced by free fatty acids promoted pyroptosis and proinflammatory responses in cardiomyocytes in a NLRP3 inflammasome-dependent manner, thus promoting myocardial hypertrophy during the progression of DCM.

Inhibition of Cardiac Glucose Transport Corrects Diabetic Cardiomyopathy Even Without Alleviation of Hyperglycemia

Healthy hearts rely more on fatty acid (FA) rather than glucose utilization. It remains unclear whether diabetic cardiomyopathy (DbCM) is accounted for by glucotoxicity or lipotoxicity. Previously, we discovered that either insulin deficiency or insulin resistance causes FOXO1‐KLF5 activation in human and murine hearts, which drives cardiac lipotoxicity and oxidative stress. Now, we investigate how glucose activates cardiac KLF5 and causes DbCM. We hypothesized that higher cardiac glucose content in diabetes potentiates FOXO1‐KLF5 activation and causes glucolipotoxicity.We mimicked Type‐1 diabetes (T1D) in C57BL/6 mice via 5 daily intraperitoneal injections of streptozotocin (STZ). In contrast to late T1D (12 Wks post‐STZ), cardiac KLF5 expression levels are not increased in the early T1D (4 Wks post‐STZ). However, mice developed cardiac dysfunction in early T1D. Seahorse analysis in adult cardiomyocytes isolated from mice with early T1D showed suppression of FA dependence in the expense of higher glucose dependence compared to non‐diabetic mice. To confirm whether hyperglycemia or higher cardiac glucose content accounts for cardiac dysfunction in early T1D, we applied anti‐hyperglycemia treatment (Dapagliflozin, DAPA, SGLT2 inhibitor) or GLUT1 inhibition (STF‐31). Either of the two treatments restored cardiac FA dependence and prevented both glucose preference and cardiac dysfunction in early T1D. GC‐MS analysis in hearts of mice with late T1D, which have shifted back to increased FA dependence, showed increased cardiac glucose content ‐presumably unused glucose‐ and subsequent increase of cardiac KLF5, as previously shown, which exacerbates cardiac dysfunction. New data revealed that GLUT1 levels were increased in late T1D compared to early T1D, as well as that this is driven by KLF5 activation. The lack of activation of KLF5 expression in early T1D, which reverses in late T1D, is mirrored by FOXO1 transcriptional activity as shown by expression of FOXO1 targets and lower FOXO1 acetylation in late T1D, which is controlled by Sirtuin‐1. Analysis of hearts from mice with late T1D and a human cardiomyocyte cell line (AC16) that was treated with high glucose indicated higher Sirtuin‐1 expression and Sirtuin‐1 binding on FOXO1. Accordingly, prevention of glucose transport to hearts of diabetic mice via treatment with either DAPA or STF‐31 for 12 Wks lowered cardiac expression of KLF5 and its targets and improved cardiac function.Conclusively, DbCM begins in early T1D with lower mitochondrial FA utilization that is compensated by higher glucose utilization and is exacerbated in late T1D with activation of SIRT1‐FOXO1‐KLF5 axis that causes combined lipotoxicity and glucotoxicity. Inhibition of GLUT1 alleviates DbCM via prevention of both early glucose dependence and late KLF5 activation.

ERK/RSK-mediated phosphorylation of Y-box binding protein-1 aggravates diabetic cardiomyopathy by suppressing its interaction with deubiquitinase OTUB1

Diabetic cardiomyopathy (DCM) is a major complication of diabetes, but its underlying mechanisms still remain unclear. The multifunctional protein Y-box binding protein-1 (YB-1) plays an important role in cardiac pathogenesis by regulating cardiac apoptosis, cardiac fibrosis, and pathological remodeling, whereas its role in chronic DCM requires further investigation. Here, we report that the phosphorylation of YB-1 at serine102 (S102) was markedly elevated in streptozotocin-induced diabetic mouse hearts and in high glucose-treated cardiomyocytes, whereas total YB-1 protein levels were significantly reduced. Coimmunoprecipitation experiments showed that YB-1 interacts with the deubiquitinase otubain-1, but hyperglycemia-induced phosphorylation of YB-1 at S102 diminished this homeostatic interaction, resulting in ubiquitination and degradation of YB-1. Mechanistically, the high glucose-induced phosphorylation of YB-1 at S102 is dependent on the upstream extracellular signal-regulated kinase (ERK)/Ras/mitogen-activated protein kinase (p90 ribosomal S6 kinase [RSK]) signaling pathway. Accordingly, pharmacological inhibition of the ERK pathway using the upstream kinase inhibitor U0126 ameliorated features of DCM compared with vehicle-treated diabetic mice. We demonstrate that ERK inhibition with U0126 also suppressed the phosphorylation of the downstream RSK and YB-1 (S102), which stabilized the interaction between YB-1 and otubain-1 and thereby preserved YB-1 protein expression in diabetic hearts. Taken together, we propose that targeting the ERK/RSK/YB-1 pathway could be a potential therapeutic approach for treating DCM.

The Chinese herbal medicine Fufang Zhenzhu Tiaozhi protects against diabetic cardiomyopathy by alleviating cardiac lipotoxicity-induced oxidative stress and NLRP3-dependent inflammasome activation

BackgroundFufang Zhenzhu Tiao Zhi (FTZ) formula is a Chinese herbal preparation used in the clinical treatment of disorders of glucolipid metabolism. Given its effective actions on the regulation of lipid dysfunction and its anti-inflammatory and antioxidative effects, we designed this study to investigate the cardioprotective effect and possible mechanism of FTZ in diabetic cardiomyopathy (DCM) mice. MethodsFTZ was administered to diabetic mice by oral gavage daily at a dose of 1.2 g/kg or 2.4 g/kg bodyweight for 8 weeks. Doppler echocardiography, H&E, and WGA staining were used to evaluate cardiac function and structure in the mice. The levels of proinflammatory cytokines and lipids in serum were detected with corresponding commercial kits. Immunofluorescence staining and flow cytometry were used to detect oxidation damage and pyroptosis in myocardial cells. RT–PCR and western blotting were used to analyze the protein and mRNA expression levels of NLRP3 inflammasome-related genes. ResultsOur study indicated that FTZ improved cardiac function, attenuated heart hypertrophy, improved serum lipid and proinflammatory cytokine levels, and restrained oxidative stress and NLRP3 inflammasome-induced inflammatory activities in diabetic mouse hearts. The in vitro data suggested that FTZ suppressed intercellular lipid accumulation as well as palmitic acid (PA)-induced oxidative stress and NLRP3 inflammasome-dependent pyroptosis in cardiomyocytes. ConclusionOur present findings indicate that FTZ inhibits DCM by inhibiting both oxidative stress and NLRP3 inflammasome activation induced by cardiac lipotoxicity.

Myeloid differential protein-2 inhibition improves diabetic cardiomyopathy via p38MAPK inhibition and AMPK pathway activation

Myeloid differential protein-2 (MD2) has been shown to play a critical role in the progression of diabetic cardiomyopathy (DCM). This study aims to explore the non-inflammatory mechanisms mediated by MD2 in DCM and to test the therapeutic effects of MD2 inhibitor C30 on DCM. Streptozotocin (STZ) was used to construct DCM model in wild-type and MD2 knockout mice. The collected heart samples were subjected to RNA-sequencing assay. Gene set enrichment analysis of the RNA-seq data indicated that MD2 knockout was associated with energy metabolism pathways in diabetic mouse heart. Further data showed that AMPK pathway was impaired under high glucose condition, which was mediated by p38MAPK activation. MD2 knockout or pharmacological inhibitor C30 completely rescued AMPK signaling through p38MAPK inhibition. Importantly, C30 treatment significantly prevented myocardial damage and dysfunction in T1DM mice evidenced by improved cardiac function and reduced cardiomyocyte apoptosis and cardiac fibrosis. Furthermore, the therapeutic effect of C30 on DCM was correlated to p38MAPK inhibition and AMPK pathway activation in vivo and in vitro. In conclusion, MD2 inhibition exhibits therapeutic effects on DCM through p38MAPK inhibition and AMPK activation, which enables MD2 a promising target for DCM treatment by suppressing metaflammation and improving cardiac metabolism.

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.

Metabolomics study reveals the alteration of fatty acid oxidation in the hearts of diabetic mice by empagliflozin.

Empagliflozin (Empa, SGLT2 inhibitor) is widely used in clinical situations for the management of diabetes. It has beneficial effects in reducing cardiac dysfunction and heart failure. However, rare studies have reported the potential mechanisms of Empa reaction. Here, we treated db/db diabetic mice with Empa and collected the heart tissue for metabolomics study. We found that db/db mice showed obvious differences in metabolomics profile compared with db/m mice. Many amino acid metabolism pathways and glycerophospholipids and fatty acyl carnitines were significantly enriched in db/db mice. Detailed analysis revealed the alteration of fatty acid oxidation in db/db mice. Interestingly, many metabolites in the fatty acid oxidation pathway, such as myristoleic acid, 12-hydroxydodecanoic acid, (15-carboxypentadecanoyl)carnitine, decanoylcarnitine, and propionylcarnitine, were significantly rescued by Empa treatment. These results suggest that fatty acid oxidation is one of the targets for Empa treatment in the heart of db/db mice. This study provided new possibilities for the development of therapeutic interventions for diabetic cardiomyopathy.

Abstract 12315: Circulating Gut Microbial Peptides Aggravate Cardiac Inflammation and Promote DNA Hypomethylation at the TLR-2 Promoter Region in Type 2 Diabetic Mice

Introduction: Epigenetic modifications, especially DNA methylation, influence key biological processes, including inflammation, and are considered as a “switch” between gene silencing and gene activation. Promoter DNA hypomethylation activates TLR-2, a modulator of inflammation, and may impact cardiac function in T2D. Hypothesis: The gut microbial peptide peptidoglycan (PGN) enters the circulation via a leaky gut and activates TLR-2 in cardiac tissue by DNA hypomethylation contributing to cardiac dysfunction in T2D. Methods: T2D ( db/db ) and wild-type mice were euthanized after four months of diabetes. Assessment of gut permeability, gut and systemic inflammation, and hypomethylation (5hmC) of cardiac tissue was performed using IHC, qPCR, ELISA, and western blot analysis. In-vitro studies were conducted in PGN-treated cardiomyocytes with and without 2-hydroxyglutarate (2HG). Results: db/db mice showed cardiac dysfunction as measured by reduced percent of LVEF (46.2±2.7 vs. 65.8±1.8; p<0.001), LVFS(22.7±1.6 vs. 35.4±1.3; p<0.001), and increased LV mass (192.4±17.0 vs. 105.8±12.4; p<0.006) compared with WT controls. db/db mice also exhibited higher gut permeability as observed by reduction of tight junction proteins (ZO-1, p120-catenin, and β-catenin) and increased levels of plasma FABP-2. The levels of PGN were higher in plasma (84.19 ± 13.56 vs. 27.21 ± 8.4; p<0.003) and cardiac tissue (1361 ± 236.1 vs. 113 ± 25.58; p<0.001) of db/db mice. The expression of TLR-2 (2.26 folds; p<0.003), pro-inflammatory cytokines IL-1β (p<0.008), and TNF-α (p<0.0004) were significantly up-regulated compared to WT mice. Expression of 5hmC was significantly higher (18.48±1.32 vs. 5.91±0.28; p<0.001) in the hearts of diabetic mice. Increased expression of 5hmC, ten-eleven translocation 1-3 (TET-1, 2, 3), IL-1β, IL-6, and TNF-α were also observed in PGN treated cardiomyocytes. 2HG treatment blocked PGN- mediated DNA hypomethylation and inflammation in cardiomyocytes. Conclusions: Circulating gut peptides have deleterious effects on cardiac health in diabetic mice by promoting DNA hypomethylation of TLR2 and increased inflammation. Strategies aiming to restore gut barrier integrity may beneficially impact diabetic cardiovascular complications.

Abstract 14079: Targeted Deletion of Cardiac miR-17-92 Cluster Exacerbates Ischemic Myocardial Injury in Diabetic Mice Through Induction of Sphingolipid Biosynthesis and Cardiomyocyte Inflammation

Background: The miR-17-92 cluster is indispensable for normal cardiac development and its deletion leads to cardiovascular disease. Aberrancies in bioactive sphingolipid metabolism, specifically ceramide (CER), contribute to diabetic cardiomyopathy, where induction of ceramide synthases (CerS) predicts elevated risk of cardiovascular complications. We investigated the role of miR-17-92 in suppression of ceramide synthesis to attenuate cardiac inflammation following myocardial ischemia/reperfusion (I/R) injury in diabetic mice. Methods and Results: Hearts from adult high-fat-diet fed diabetic tamoxifen-inducible cardiac-specific miR-17-92-deficient (miR-17-92-def) and wild type (WT) mice were subjected to 30 min of myocardial ischemia (I) and 60 min of reperfusion (R) in Langendorff mode. Myocardial infarct size was exacerbated with severe ventricular dysfunction in diabetic miR-17-92-def mice as compared to diabetic WT mice ( Fig. A ). Cardiomyocytes isolated from diabetic miR-17-92-def and WT mice were subjected to 40 min simulated ischemia and 2-hr reoxygenation (SI/RO). Immunofluorescence staining demonstrated an enhanced inflammasome formation with ASC (apoptosis speck-like protein with a caspase recruitment domain) aggregation ( Fig. B ) and caspase-1 activation ( Fig. C ) following SI/RO in cardiomyocytes isolated from diabetic miR-17-92-def mice. Lipidomics analysis revealed that C16:0 ceramide, a product of Ceramide Synthase 6 (CerS6), was induced after I/R injury and was more prominent in the hearts of diabetic miR-17-92-def mice (Fig. D). Western blot analysis revealed that CerS6 expression was nearly undetectable in the hearts of diabetic WT mice, but was dramatically induced by I/R in diabetic miR-17-92-def mice ( Fig. E ). Conclusion: The deletion of cardiac miR-17-92 exacerbates myocardial I/R injury under diabetic condition through accumulation of CER by inducing CerS6 and stimulating inflammation in cardiomyocytes.

Phosphoproteomics analysis of diabetic cardiomyopathy in aging-accelerated mice and effects of D-pinitol.

The molecular mechanisms of diabetic cardiomyopathy (DCM) development and D-pinitol (DP) in its treatment remain unclear. The present study is to explore the underlying mechanism of DCM in an elderly diabetic mouse model and to seek the protective targets of DP by phosphoproteomics. We used streptozotocin to induce diabetes in SAMP8 and DP (150mg/kg/day) intragastrically administrated to diabetic mice for 8weeks. The heart tissues were harvested for label-free phosphoproteomic analysis from diabetic mice. Some differentially regulated phosphorylation sites were confirmed by parallel reaction monitoring. Our results showed that 612 phosphorylation sites on 454 proteins had their phosphorylation levels significantly changed in the heart of untreated diabetic mice (DM). Of these phosphorylation sites, 216 phosphorylation sites on 182 proteins were normalized after DP treatment. We analyzed the functional signaling pathways in the heart of DP treated diabetic mice (DMT), including glucagon signaling pathway, insulin signaling pathway, mitophagy, apoptosis, and longevity regulating pathway. Two consensus motifs identified were targeted by Src and epidermal growth factor receptor between DMT and DM groups. Our study might help to better understand the mechanism of DCM, provide novel targets for estimating the protective effects of DP.

Weighted Gene Co-Expression Network Analysis Identifies ANGPTL4 as a Key Regulator in Diabetic Cardiomyopathy via FAK/SIRT3/ROS Pathway in Cardiomyocyte.

BackgroundDiabetic cardiomyopathy (DbCM) is characterized by initial impairment of left ventricular relaxation followed by contractile dysfunction. Despite intensive research, the exact mechanism remains so far unsolved.MethodsWe constructed weighted gene co-expression network analysis (WGCNA) to screen gene modules that were closely related with DbCM based on the GSE5606 dataset, which contained expression data of the cardiac left ventricle in a rodent model of streptozotocin (STZ)-induced DbCM. Then, the most related hub gene, angiopoietin-like 4 (ANGPTL4), was selected for functional ex vivo and in vitro assays. In our experiments, STZ-induced diabetic mice (C57BL/6J) and human cardiomyocytes (AC16) were used to study the functional roles and potential mechanisms of ANGPTL4 in DbCM.ResultsWGCNA analysis revealed the yellow and green modules were most correlated with DbCM, and identified ANGPTL4 as one of the most significantly upregulated hub genes (ANGPTL4, ACOT1, DECR1, HMGCS2, and PDK4). Consistent with the bioinformatic analysis, the amount of ANGPTL4 was significantly upregulated in diabetic mouse heart. DbCM group, compared with the control group, had increased phosphorylation of focal adhesion kinase (FAK), reduced SIRT3 expression, increased SOD2 acetylation, upregulated NADPH oxidase activation, elevated reactive oxygen species (ROS) produciton, and enhanced apoptosis in the diabetic mouse heart. Moreover, ANGPTL4 induced apoptosis via FAK/SIRT3/ROS pathway in human cardiomyocytes (AC16) under high glucose condition in vitro.These effects were abrogated by treatment of two independent siRNA for ANGPTL4, whereas exogenous recombinant ANGPLT4 protein treatment exacerbated those effects in AC16.ConclusionWe found ANGPTL4, ACOT1, DECR1, HMGCS2, and PDK4 were significantly increased in diabetic heart. ANGPTL4 could promote cardiac apoptosis via a FAK/SIRT3/ROS dependent signaling pathway in DbCM.

Diabetic Aldehyde Dehydrogenase 2 Mutant (ALDH2*2) Mice Are More Susceptible to Cardiac Ischemic-Reperfusion Injury Due to 4-Hydroxy-2-Nonenal Induced Coronary Endothelial Cell Damage.

BackgroundAldehyde dehydrogenase‐2 (ALDH2), a mitochondrial enzyme, detoxifies reactive aldehydes such as 4‐hydroxy‐2‐nonenal (4HNE). A highly prevalent E487K mutation in ALDH2 (ALDH2*2) in East Asian people with intrinsic low ALDH2 activity is implicated in diabetic complications. 4HNE‐induced cardiomyocyte dysfunction was studied in diabetic cardiac damage; however, coronary endothelial cell (CEC) injury in myocardial ischemia‐reperfusion injury (IRI) in diabetic mice has not been studied. Therefore, we hypothesize that the lack of ALDH2 activity exacerbates 4HNE‐induced CEC dysfunction which leads to cardiac damage in ALDH2*2 mutant diabetic mice subjected to myocardial IRI.Methods and ResultsThree weeks after diabetes mellitus (DM) induction, hearts were subjected to IRI either in vivo via left anterior descending artery occlusion and release or ex vivo IRI by using the Langendorff system. The cardiac performance was assessed by conscious echocardiography in mice or by inserting a balloon catheter in the left ventricle in the ex vivo model. Just 3 weeks of DM led to an increase in cardiac 4HNE protein adducts and, cardiac dysfunction, and a decrease in the number of CECs along with reduced myocardial ALDH2 activity in ALDH2*2 mutant diabetic mice compared with their wild‐type counterparts. Systemic pretreatment with Alda‐1 (10 mg/kg per day), an activator of both ALDH2 and ALDH2*2, led to a reduction in myocardial infarct size and dysfunction, and coronary perfusion pressure upon cardiac IRI by increasing CEC population and coronary arteriole opening.ConclusionsLow ALDH2 activity exacerbates 4HNE‐mediated CEC injury and thereby cardiac dysfunction in diabetic mouse hearts subjected to IRI, which can be reversed by ALDH2 activation.

MO626EFFECTS OF RAMIPRIL IN LUNG AND KIDNEY ANGIOTENSIN CONVERTING ENZYME 2 (ACE2) EXPRESSION IN A TYPE 2 DIABETIC MURINE MODEL: LESSONS FOR COVID-19 INFECTION

Background and AimsAngiotensin converting enzyme 2 (ACE2) is one of the components of the renin-angiotensin system (RAS) that mainly degrades angiotensin II to angiotensin-(1-7). ACE2 is predominantly expressed in the kidney and the heart, but it has been evidenced in type 2 alveolar lung cells, where it acts as a receptor for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this context, a controversy arose as to whether the use of RAS blockers could increase ACE2 lung expression and the risk infection by COVID-19. This study aimed to investigate the effect of an ACE inhibitor (Ramipril) on ACE2 expression in experimental diabetes.Method12 weeks old diabetic db/db mice (n=7) were given ramipril (8 mg/Kg/day) during 8 weeks or the respective vehicle. db/m (n=7) vehicle-treated non-diabetic mice were included as controls. ACE2 mRNA expression and enzymatic activity were studied in kidney, heart and lung samples of these animals to identify if the diabetic condition or treatment with ramipril modulated ACE2 expression.ResultsIn vehicle-treated diabetic db/db animals, ACE2 mRNA expression was significantly increased in the kidney (p<0.001) and ramipril treatment reversed this effect (p=0.026). In the heart, ACE2 expression decreased in db/db when compared to db/m littermates (p=0.035) and ramipril had no effect. We found no differences in ACE2 gene expression in the lung. Besides, ACE2 enzymatic activity was increased in the kidney (29%) and also in the lung (16%) of db/db mice when compared to controls. Ramipril treatment decreased ACE2 activity a 19% in the lung and had no effect in the kidney when compared to untreated db/db (see figure). In the heart, ACE2 activity tended to decrease in db/db mice (29%) when compared to db/m and ramipril increased ACE2 activity (18%) but did not exceed the cardiac ACE2 activity of the db/m.MO626 Figure: ACE2 mRNA expression and activity. ACE2 mRNA expression in the kidney (A), lung (B) and heart (C). ACE2 enzymatic activity in the kidney (A), lung (E) and heart (F).ConclusionACE2 is increased in the kidney and the lung, and decreased in the heart of diabetic mice. Ramipril treatment restores ACE2 levels. The results suggest that ACE inhibitors do not increase ACE2 expression and the activity decrease exerted in the lung may be protective against COVID-19 infection.

Canagliflozin attenuates lipotoxicity in cardiomyocytes and protects diabetic mouse hearts by inhibiting the mTOR/HIF-1α pathway

SummaryLipotoxicity plays an important role in the development of diabetic heart failure (HF). Canagliflozin (CAN), a marketed sodium-glucose co-transporter 2 inhibitor, has significantly beneficial effects on HF. In this study, we evaluated the protective effects and mechanism of CAN in the hearts of C57BL/6J mice induced by high-fat diet/streptozotocin (HFD/STZ) for 12 weeks in vivo and in HL-1 cells (a type of mouse cardiomyocyte line) induced by palmitic acid (PA) in vitro. The results showed that CAN significantly ameliorated heart functions and inflammatory responses in the hearts of the HFD/STZ-induced diabetic mice. CAN significantly attenuated the inflammatory injury induced by PA in the HL-1 cells. Furthermore, CAN seemed to bind to the mammalian target of rapamycin (mTOR) and then inhibit mTOR phosphorylation and hypoxia-inducible factor-1α (HIF-1α) expression. These results indicated that CAN might attenuate lipotoxicity in cardiomyocytes by inhibiting the mTOR/HIF-1α pathway and then show protective effects on diabetic hearts.

Knockout of interleukin-17A diminishes ventricular arrhythmia susceptibility in diabetic mice via inhibiting NF-κB-mediated electrical remodeling.

Interleukin-17A (IL-17), a potent proinflammatory cytokine, has been shown to participate in cardiac electrical disorders. Diabetes mellitus is an independent risk factor for ventricular arrhythmia. In this study, we investigated the role of IL-17 in ventricular arrhythmia of diabetic mice. Diabetes was induced in both wild-type and IL-17 knockout mice by intraperitoneal injection of streptozotocin (STZ). High-frequency electrical stimuli were delivered into the right ventricle to induce ventricular arrhythmias. We showed that the occurrence rate of ventricular tachycardia was significantly increased in diabetic mice, which was attenuated by IL-17 knockout. We conducted optical mapping on perfused mouse hearts and found that cardiac conduction velocity (CV) was significantly decreased, and action potential duration (APD) wasprolonged in diabetic mice, which were mitigated by IL-17 knockout. We performed whole-cell patch clamp recordings from isolated ventricular myocytes, and found that the densities of Ito, INa and ICa,L were reduced, the APDs at 50% and 90% repolarization were increased, and early afterdepolarization (EAD) was markedly increased in diabetic mice. These alterations were alleviated by the knockout of IL-17. Moreover, knockout of IL-17 alleviated the downregulation of Nav1.5 (the pore forming subunit of INa), Cav1.2 (the main component subunit of ICa,L) and KChIP2 (potassium voltage-gated channel interacting protein 2, the regulatory subunit of Ito) in the hearts of diabetic mice. The expression of NF-κB was significantly upregulated in the hearts of diabetic mice, which was suppressed by IL-17 knockout. In neonatal mouse ventricular myocytes, knockdown of NF-κB significantly increased the expression of Nav1.5, Cav1.2 and KChIP2. These results imply that IL-17 may represent a potential target for the development of agents against diabetes-related ventricular arrhythmias.

Treatment with XMU-MP-1 erases hyperglycaemic memory in hearts of diabetic mice

Hyperglycaemic memory refers to the damages occurred under early hyperglycaemic environment in organs of diabetic patients persisting after intensive glycaemic control. Mammalian sterile 20-like kinase 1 (Mst1) contributes to the development of diabetic cardiomyopathy. Here, we investigated the role of Mst1 in hyperglycaemic memory and test the effect of XMU-MP-1, a Mst1 inhibitor, on hyperglycaemic memory in hearts. Eight weeks after induction of type 1 diabetes by injection with streptozotocin (STZ) in mice, glycaemic control was obtained by means of insulin treatment and maintained for 4 additional weeks. In the diabetic mice, insulin treatment alone did not reduce phosphorylation of Mst1 or improve cardiac function. Treatment with XMU-MP-1 alone immediately after induction of diabetes for 12 weeks did not improve myocardial function in mice. But treatment with XMU-MP-1 for the later 4 weeks relieved myocardial dysfunction when glycaemic control was obtained by insulin treatment simultaneously. Mst1 deficiency and glycaemic control synergistically improved myocardial function and reduced apoptosis in myocardium of diabetic mice. Mechanistically, when Mst1 was deficient or inhibited by XMU-MP-1, AMPK was activated and mitochondrial dysfunction was attenuated. In vitro, treatment with AMPK activator reversed the detrimental effects of Mst1 overexpression in cultured cardiomyocytes. XMU-MP-1 might thus be envisaged as a complement for insulin treatment against diabetic cardiomyopathy.

MiR-20a-5p overexpression prevented diabetic cardiomyopathy via inhibition of cardiomyocyte apoptosis, hypertrophy, fibrosis and JNK/NF-κB signalling pathway.

Diabetic cardiomyopathy (DCM) is a common cardiovascular disease. A declined miR-20a-5p was observed in hearts of diabetic mice, while its effect on DCM remains unknown. Herein, we established streptozotocin-induced DCM rat model and high glucose-stimulated H9C2 model of DCM. Then they were treated with adenovirus expressing miR-20a-5p to explore the function of miR-20a-5p. Insulin tolerance test and intraperitoneal glucose tolerance test assay revealed that miR-20a-5p reduced blood glucose level. Besides, miR-20a-5p improved cardiac dysfunction reflected by reduced heart weight/body weight and left ventricular diastolic pressure, and increased left ventricular systolic pressure and ±LV dp/dt max. MiR-20a-5p prevented cardiomyocyte apoptosis, along with the upregulated c-caspase-3, bax and downregulated bcl-2. Moreover, miR-20a-5p alleviated cardiac hypertrophy as the parameters of atrial natriuretic peptide, B-type natriuretic peptide and MyHC-β decreased. Also, miR-20a-5p attenuated the cardiac fibrosis demonstrated by decreased transforming growth factor-β1, collagen I levels and the inflammatory response manifested by reduced interleukin-6, tumour necrosis factor-α and IL-1β production. Furthermore, miR-20a-5p prevented Jun NH2-terminal kinase (JNK) phosphorylation and nuclear factor-κB (NF-κB) p65nuclear translocation. Similarly, the effects of miR-20a-5p on DCM were confirmed in our in vitro experiments. Additionally, ROCK2 is a possible target gene of miR-20a-5p. ROCK2 overexpression reversed the protective effect of miR-20a-5p on DCM. Overall, miR-20a-5p may effectively ameliorate DCM through improving cardiac metabolism, and subsequently inhibiting inflammation, apoptosis, hypertrophy, fibrosis and JNK/NF-κB pathway via modulating ROCK2.

Rebuttal from Manoja K. Brahma, Adam R. Wende and Kyle S. McCommis.

Drs Karwi and Lopaschuk have provided compelling evidence of the potential for ketone bodies to provide an extra source of ATP for the heart. The foundation of our opposing views most likely stems from comparing analyses of the acute effects of ketone bodies in predominantly ex vivo studies to that which occurs in vivo with more chronic ketosis. Likewise, we must all be more prudent when comparing cardiac metabolic adaptations to physiological ketosis to those observed with ‘pure’ ketosis as occurs with consumption of ketone esters or salts, or ketone infusion. In physiological states of ketosis such as fasting or consumption of a low-carbohydrate, high-fat ketogenic diet, hearts preferentially downregulate ketolytic enzymes, decreasing ketone oxidation and enhancing fatty acid oxidation (Wentz et al. 2010; McCommis et al. 2020). This downregulation of ketolytic enzymes also occurs in the hearts of diabetic mice, or in hearts overexpressing glucose transporter 4 with enhanced glucose uptake and utilization (Brahma et al. 2020). However, cardiac ketone metabolism may be directly enhanced if delivery of lipids or glucose is not concurrently elevated. Recent studies suggest that ketone ester supplementation enhanced cardiac ketone uptake in humans (Monzo et al. 2020), and ketone ester-enriched diets increased the expression of ketolytic enzymes in the rat heart (Yurista et al. 2020). These studies imply that elevated ketone extraction is associated with increased oxidation, but neither of these studies directly measured oxidation. We agree that ketone utilization appears to be more important in failing hearts. Failing hearts from humans and rodent models display enhanced expression of the ketolytic enzymes β-hydroxybutyrate dehydrogenase 1 (BDH1) and succinyl-CoA:3-oxoacid CoA transferase (SCOT) and have been shown to extract more ketones than non-failing hearts (Aubert et al. 2016; Bedi et al. 2016; Horton et al. 2019; Monzo et al. 2020; Murashige et al. 2020). Also, cardiac-specific deletion of BDH1 or SCOT in mice exacerbates cardiac dysfunction and remodelling when challenged by pressure overload (Schugar et al. 2014; Horton et al. 2019). But these knockout mouse models provide perhaps the most significant evidence that ketone body metabolism is not critical for normal hearts, as deletion of BDH1 or SCOT does not directly induce cardiac dysfunction, and these hearts can even normally adapt to physiological ketosis (Schugar et al. 2014; Horton et al. 2019). To conclude, further investigation is needed to better understand the importance of ketone body metabolism in heart failure, as well as in response to ketosis as a potential therapy for heart failure. Readers are invited to give their views on this and the accompanying CrossTalk articles in this issue by submitting a brief (250 word) comment. Comments may be submitted up to 6 weeks after publication of the article, at which point the discussion will close and the CrossTalk authors will be invited to submit a ‘Last Word’. Please email your comment, including a title and a declaration of interest, to [email protected]. Comments will be moderated and accepted comments will be published online only as ‘supporting information’ to the original debate articles once discussion has closed. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. None. All authors conceived, wrote, and edited this manuscript. All authors have read and approved the final version of this manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. A.R.W. is supported by NIH R01 HL133011. K.S.M. is supported by NIH R00 HL136658.

Ophiopogonin D alleviates diabetic myocardial injuries by regulating mitochondrial dynamics

Ethnopharmacological relevanceOphiopogonin D (OP-D) is a steroidal saponin extracted from Ophiopogon japonicus (Thunb.) Ker Gawl. (Liliaceae), that has been traditionally used to treat cough, sputum, and thirst in some Asian countries. Recently, various pharmacological roles of OP-D have been identified, including anti-inflammatory, cardioprotective, and anti-cancer effects. However, whether OP-D can prevent diabetic myocardial injury remains unknown. Aim of the studyIn this study, we aimed to observe the effects of OP-D on the diabetic myocardium. Materials and methodsLeptin receptor-deficient db/db mice were used as an animal model for type 2 diabetes. The effects of OP-D on blood glucose, blood lipids, myocardial ultrastructure, and mitochondrial function in mice were observed after four weeks of intragastric administration. Palmitic acid was used to stimulate cardiomyocytes to establish a myocardial lipotoxicity model. Cell apoptosis, mitochondrial morphology, and function were observed. ResultsBlood glucose and blood lipid levels were significantly increased in db/db mice, accompanied by myocardial mitochondrial injury and dysfunction. OP-D treatment reduced blood lipid levels in db/db mice and relieved mitochondrial injury and dysfunction. OP-D inhibited palmitic acid induced-mitochondrial fission and dysfunction, reduced endogenous apoptosis, and improved cell survival rate in H9C2 cardiomyocytes. Both in vivo and in vitro models showed increased phosphorylation of DRP1 at Ser-616, reduced phosphorylation of DRP1 at Ser-637, and reduced expression of fusion proteins MFN1/2 and OPA1. Meanwhile, immunofluorescence co-localization analysis revealed that palmitic acid stimulated the translocation of DRP1 protein from the cytoplasm to the mitochondria in H9C2 cardiomyocytes. The imbalance of mitochondrial dynamics, protein expression, and translocation of DRP1 were effectively reversed by OP-D treatment. In isolated mice ventricular myocytes, palmitic acid enhanced cytoplasmic Ca2+ levels and suppressed contractility in ventricular myocytes, accompanied by activation of calcineurin, a key regulator of DRP1 dephosphorylation at Ser-637. OP-D reversed the changes caused by palmitic acid. ConclusionsOur findings indicate that OP-D intervention could alleviate lipid accumulation and mitochondrial injury in diabetic mouse hearts and palmitic acid-stimulated cardiomyocytes. The cardioprotective effect of OP-D may be mediated by the regulation of mitochondrial dynamics.