Interleukin-1 Receptor Antagonist Enhances Islet Engraftment Without Impacting Serum Levels of Nitrite or Osteopontin

Interleukin-1β (IL-1β)-mediated early islet graft dysfunction and loss of islet mass can occur in different phylogenic types of islet transplantation. Large quantities of interleukin-1 receptor antagonist (IL-1RA) have been demonstrated to impede IL-1β-mediated adverse effects on islet grafts in allo- and xenotransplantation. To clarify the influence of IL-1RA on early function and mass change, as well as long-term hypoglycemic effects of islet isografts, we studied streptozotocin-induced diabetic C57BL/6 mice infected with replication-defective adenovirus carrying the mouse IL-1RA cDNA gene. This vector increased the mean serum level of IL-1RA to 8 ng/mL, approximately three times greater than for mice receiving adenovirus carrying the beta-galactosidase (β-Gal) gene. The blood glucose levels declined faster and the insulin content of the graft was significantly higher on day 10 following transplantation among mice receiving mIL-1RA gene than the controls. Nevertheless, the insulin content of the pancreatic remnant did not differ among mice in the IL-1RA, β-Gal, and vehicle control groups. Serum levels of nitrite and osteopontin before and 3 days after islet transplantation did not differ considerably among the IL-1RA, β-Gal, and vehicle groups. Compared with the β-Gal group, temporary posttransplantation hyperglycemia was significantly shortened in the IL-1RA group mice. Removal of graft-bearing kidneys at 13 weeks following transplantation caused recurrence of hyperglycemia in all treated diabetic mice. The insulin content of pancreatic remnants removed at 15 weeks following transplantation was similar in the IL-1RA and β-Gal groups. In conclusion, a mildly elevated serum concentration of IL-1RA protected and enhanced engraftment of islet isografts immediately after transplantation.     

Ex vivo intracoronary gene transfer of adeno‐associated virus 2 leads to superior transduction over serotypes 8 and 9 in rat heart transplants

Heart transplant gene therapy requires vectors with long‐lasting gene expression, high cardiotropism, and minimal pathological effects. Here, we examined transduction properties of ex vivo intracoronary delivery of adeno‐associated virus (AAV) serotype 2, 8, and 9 in rat syngenic and allogenic heart transplants. Adult Dark Agouti (DA) rat hearts were intracoronarily perfused ex vivo with AAV2, AAV8, or AAV9 encoding firefly luciferase and transplanted heterotopically into the abdomen of syngenic DA or allogenic Wistar–Furth (WF) recipients. Serial in vivo bioluminescent imaging of syngraft and allograft recipients was performed for 6 months and 4 weeks, respectively. Grafts were removed for PCR‐, RT‐PCR, and luminometer analysis. In vivo bioluminescent imaging of recipients showed that AAV9 induced a prominent and stable luciferase activity in the abdomen, when compared with AAV2 and AAV8. However, ex vivo analyses revealed that intracoronary perfusion with AAV2 resulted in the highest heart transplant transduction levels in syngrafts and allografts. Ex vivo intracoronary delivery of AAV2 resulted in efficient transgene expression in heart transplants, whereas intracoronary AAV9 escapes into adjacent tissues. In terms of cardiac transduction, these results suggest AAV2 as a potential vector for gene therapy in preclinical heart transplants studies, and highlight the importance of delivery route in gene transfer studies.     

Cardiospecific expression of AAV vectors

Adenoviral vectors have been set for cardiomyocyte gene therapy with considerable transfection rates. However, due to adenoviral immunogenicity, adenoviral transfected cells are frequently subject to specific cellular immune reactions leading to loss of cells and transgene expression. Non‐pathogenic adeno associated vectors lacking immunogenicity have been utilized alternately. Tropism of the most common AAV serotype 2, favoring liver of muscle transfection, has been altered by use of serotypes 1, 6 and 9. We used a model of chronic (4 weeks) regional myocardial transfection of the pig to investigate transfection efficacy of AAV vectors, which were applied to the target region by retroinfusion. Cardiac restriction was ensured by utilization of an MLC‐2v promoter‐CMV‐enhancer construct, fused to a luciferase reporter gene, the activity of which was analyzed. Compared to the wildtype (WT) AAV 2 vector, which barely exceeded background luciferase activity, a vector lacking the heparin binding site of the envelope (AAV2 ΔHep) improved expression only slightly. Substantial increase to 40 000 RLU/mg tissue) was achieved by the addition of VEGF‐A, which enhances vascular permeability. A further 15‐fold increase of luciferase activity was achieved by applying an equal amount of AAV6 virus particles to a pig heart, with a further 5–10 fold increase achieved by utilization of AAV serotype 9. Functionally relevant gene transfer was ensured by applying AAV9 hVEGF/PDGF (0.2/0.4 × 10¹³ virus particles, respectively). 4 weeks after retroinfusion into pig hearts suffering from chronic total LAD‐occlusion (induced by a reduction stent), we found a significant increase of blood flow and regional myocardial function. We conclude that AAV9 is a more efficient AAV vector when compared to WT‐ or ΔHep‐AAV2 or AAV6. Functionally relevant gene expression, as performed by VEGF‐A/PDGF transfection, opens the door for utilization of this vector system for xenotransplant organ priming.     

Cannabinoid 1 receptor promotes cardiac dysfunction, oxidative stress, inflammation, and fibrosis in diabetic cardiomyopathy

Endocannabinoids and cannabinoid 1 (CB(1)) receptors have been implicated in cardiac dysfunction, inflammation, and cell death associated with various forms of shock, heart failure, and atherosclerosis, in addition to their recognized role in the development of various cardiovascular risk factors in obesity/metabolic syndrome and diabetes. In this study, we explored the role of CB(1) receptors in myocardial dysfunction, inflammation, oxidative/nitrative stress, cell death, and interrelated signaling pathways, using a mouse model of type 1 diabetic cardiomyopathy. Diabetic cardiomyopathy was characterized by increased myocardial endocannabinoid anandamide levels, oxidative/nitrative stress, activation of p38/Jun NH(2)-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs), enhanced inflammation (tumor necrosis factor-α, interleukin-1β, cyclooxygenase 2, intracellular adhesion molecule 1, and vascular cell adhesion molecule 1), increased expression of CB(1), advanced glycation end product (AGE) and angiotensin II type 1 receptors (receptor for advanced glycation end product [RAGE], angiotensin II receptor type 1 [AT(1)R]), p47(phox) NADPH oxidase subunit, β-myosin heavy chain isozyme switch, accumulation of AGE, fibrosis, and decreased expression of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA2a). Pharmacological inhibition or genetic deletion of CB(1) receptors attenuated the diabetes-induced cardiac dysfunction and the above-mentioned pathological alterations. Activation of CB(1) receptors by endocannabinoids may play an important role in the pathogenesis of diabetic cardiomyopathy by facilitating MAPK activation, AT(1)R expression/signaling, AGE accumulation, oxidative/nitrative stress, inflammation, and fibrosis. Conversely, CB(1) receptor inhibition may be beneficial in the treatment of diabetic cardiovascular complications.     

Overexpression of metallothionein reduces diabetic cardiomyopathy.

Many diabetic patients suffer from cardiomyopathy, even in the absence of vascular disease. This diabetic cardiomyopathy predisposes patients to heart failure and mortality from myocardial infarction. Evidence from animal models suggests that reactive oxygen species play an important role in the development of diabetic cardiomyopathy. Our laboratory previously developed a transgenic mouse model with targeted overexpression of the antioxidant protein metallothionein (MT) in the heart. In this study we used MT-transgenic mice to test whether an antioxidant protein can reduce cardiomyopathy in the OVE26 transgenic model of diabetes. OVE26 diabetic mice exhibited cardiomyopathy characterized by significantly altered mRNA expression, clear morphological abnormalities, and reduced contractility under ischemic conditions. Diabetic hearts appeared to be under oxidative stress because they had significantly elevated oxidized glutathione (GSSG). Diabetic mice with elevated cardiac MT (called OVE26MT mice) were obtained by crossing OVE26 transgenic mice with MT transgenic mice. Hyperglycemia in OVE26MT mice was indistinguishable from hyperglycemia in OVE26 mice. Despite this, the MT transgene significantly reduced cardiomyopathy in diabetic mice: OVE26MT hearts showed more normal levels of mRNA and GSSG. Typically, OVE26MT hearts were found to be morphologically normal, and elevated MT improved the impaired ischemic contractility seen in diabetic hearts. These results demonstrate that cardiomyocyte-specific expression of an antioxidant protein reduces damage to the diabetic heart.     

AAV-mediated gene transfer to cardiac cells in a heterotopic rat heart transplantation model

INTRODUCTION: Adeno-associated virus (AAV) vectors offer the possibility to transfer genes to a wide range of organ and cell types. To determine the efficiency of AAV-mediated gene transfer to cardiac cells, vectors were administered to the heart under various conditions. METHODS: In Sprague-Dawley rats, AAV vectors based on serotype 2 and coding for beta-galactosidase were injected via coronaries into hypothermic nonbeating and normothermic beating hearts before transplantation. In addition, vectors were injected intravenously or into the thigh muscle. After 28 days all animals were humanely killed and organs explanted for analysis. RESULTS: Transgenic DNA was always detectable in the liver and the heart, irrespective of the application mode. However, transgenic mRNA could not be determined in the transplanted hearts. In contrast, direct injection into the thigh muscle resulted in transgenic mRNA production and marker gene expression. After systemic application, transgenic mRNA was detected in the liver but not in the heart. CONCLUSION: The results of our study indicated that AAV-mediated gene transfer to cardiac cells is possible. However, it was impossible to detect transgenic mRNA or marker gene expression in the transplanted hearts after intracoronary perfusion or systemic injection.     

Heterogeneous cardiac sympathetic denervation and decreased myocardial nerve growth factor in streptozotocin-induced diabetic rats: implications for cardiac sympathetic dysinnervation complicating diabetes

Heterogeneous myocardial sympathetic denervation complicating diabetes has been invoked as a factor contributing to sudden unexplained cardiac death. In subjects with diabetic autonomic neuropathy (DAN), distal left ventricular (LV) denervation contrasts with preservation of islands of proximal innervation, which exhibit impaired vascular responsiveness. The aims of this study were to determine whether this heterogeneous pattern of myocardial sympathetic denervation occurs in a rat model of diabetes and to explore a potential association with regional fluctuations in myocardial nerve growth factor (NGF) protein. Myocardial sympathetic denervation was characterized scintigraphically using the sympathetic neurotransmitter analog C-11 hydroxyephedrine ([11C]HED) and compared with regional changes in myocardial NGF protein abundance and norepinephrine content after 6 and 9 months in nondiabetic (ND) and streptozotocin-induced diabetic (STZ-D) rats. In ND rats, no difference in [11C]HED retention or norepinephrine content was detected in the proximal versus distal myocardium. After 6 months, compared with ND rats, myocardial [11C]HED retention had declined in the proximal segments of STZ-D rats by only 9% (NS) compared with a 33% decrease in the distal myocardium (P < 0.05). Myocardial norepinephrine content was similar in both ND and STZ-D rats. At 6 months, LV myocardial NGF protein content in STZ-D rats decreased by 52% in the proximal myocardial segments (P < 0.01 vs. ND rats) and by 82% distally (P < 0.01 vs. ND rats, P < 0.05 vs. proximal segments). By 9 months, [11C]HED retention had declined in both the proximal and distal myocardial segments of the STZ-D rats by 42% (P < 0.01 vs. ND rats), and LV norepinephrine content and NGF protein were decreased in parallel. Therefore, 6 months of STZ-induced diabetes results in heterogeneous cardiac sympathetic denervation in the rat, with maximal denervation occurring distally, and is associated with a proximal-to-distal gradient of LV NGF protein depletion. It is tempting to speculate that regional fluctuations of NGF protein in the diabetic myocardium contribute to heterogeneous cardiac sympathetic denervation complicating diabetes.     

Increased myocardial oxygen consumption reduces cardiac efficiency in diabetic mice

Altered cardiac metabolism and function (diabetic cardiomyopathy) has been observed in diabetes. We hypothesize that cardiac efficiency, the ratio of cardiac work (pressure-volume area [PVA]) and myocardial oxygen consumption (MVo(2)), is reduced in diabetic hearts. Experiments used ex vivo working hearts from control db/+, db/db (type 2 diabetes), and db/+ mice given streptozotocin (STZ; type 1 diabetes). PVA and ventricular function were assessed with a 1.4-F pressure-volume catheter at low (0.3 mmol/l) and high (1.4 mmol/l) fatty acid concentrations with simultaneous measurements of MVo(2). Substrate oxidation and mitochondrial respiration were measured in separate experiments. Diabetic hearts showed decreased cardiac efficiency, revealed as an 86 and 57% increase in unloaded MVo(2) in db/db and STZ-administered hearts, respectively. The slope of the PVA-MVo(2) regression line was increased for db/db hearts after elevation of fatty acids, suggesting that contractile inefficiency could also contribute to the overall reduction in cardiac efficiency. The end-diastolic and end-systolic pressure-volume relationships in db/db hearts were shifted to the left with elevated end-diastolic pressure, suggesting left ventricular remodeling and/or myocardial stiffness. Thus, by means of pressure-volume technology, we have for the first time documented decreased cardiac efficiency in diabetic hearts caused by oxygen waste for noncontractile purposes.     

AAV based gene delivery to myocardium in rodents and pigs

Objective: To optimize transgene expression levels after AAV‐mediated gene transfer different delivery methods were compared in a rat (A) and porcine (B) heterotopic heart transplantation model. Methods: (A) Heterotopic abdominal heart transplantations were performed in male Lewis rats. After harvesting the donor hearts, the viral vectors were delivered to the graft by the following methods: (1) 0.35 ml saline solution containing AAV2/9‐LacZ (2 × 10¹¹ vector genome, vg) was injected directly into the myocardium (apex) immediately after reperfusion. (2) cardioplegic solution (0.3 ml) containing AAV‐2(HBSD), 2/9‐LacZ vectors was rapidly injected into the aortic root with the pulmonary trunk clamped. Before transplantation the transfected heart was incubated for 20 min in iced cardioplegia. (3) A reperfusion system was applied: For 20 min a cold solution of cardioplegia (5 ml) and AAV‐2(HBSD) or AAV2/9‐LacZ vectors were recirculated through the donor heart. Transplanted grafts were explanted after 3 weeks. To detect and measure marker gene expression X‐gal staining or a luciferase assay was performed. In a second series we compared the effects of the transduction of PD‐L1 to LacZ using the optimum method (intraaortic root injection) in the same heart transplantation model. (B) Heterotopic abdominal HTX was performed in pigs (Landrace, 10–18 kg) following vector application to the donor heart in an in situ‐Langendorff perfusion system (AAV2/GFP and AAV2/Luciferase). Recipients were given tacrolimus (0.3 mg/kg BW), after 21 days the transplanted hearts were explanted for transgene expression analysis. In a second series we compared AAV2/9‐mediated transduction of PD‐L1 and LacZ in the in situ‐Langendorff model and consequent allogeneic heterotopic abdominal heart transplantation. Results: (A) Highest transfection efficiency was observed in the grafts treated with intracoronary infusion of AAV2/9 at the higher dosage, and the expression pattern was global and homogenous in the grafts. hPD‐L1 transduction resulted in no significant difference of survival time and signs of rejection after allogeneic rat heart transplantation. (B) AAV2‐mediated gene delivery was unable to yield sufficient transgene expression after in situ‐Langendorff perfusion of porcine hearts. AAV2/9 based gene transfer of LacZ and hPD‐L1 led to excellent myocardial gene expression lasting up to 2 months. Due to species incompatibility no protective effects were observed in our allogeneic porcine transplantation model. Conclusions: (A) We demonstrated that infusion of AAV vectors into the aortic root with the pulmonary trunk clamped is a simple and efficient method for gene delivery to the donor heart in an allogeneic rat heart transplantation setting. Gene transfer of hPD‐L1 was ineffective to protect against allorejection, on the contrary there was a trend to aggravated rejection. Therefore the exact mechanism of hPD‐L1 in allograft rejection needs further investigation. (B) The in situ‐Langendorff model was developed to allow isolated target organ perfusion with high vector concentrations under physiological conditions. Using this method AAV2/9 mediated gene delivery into porcine hearts proved effective to induce excellent marker gene expression. Expression of hPD‐L1 could also be achieved but was ineffective in pig allotransplantation due to species incompatibility. Double transgenic pig hearts (e.g. Gal‐KO+CD46) can now efficiently be transduced with hPD‐L1 or hCTLA4‐Ig to further optimize long‐term survival after pig‐to‐primate cardiac xenotransplantation.     

MG53 E3 Ligase–Dead Mutant Protects Diabetic Hearts From Acute Ischemic/Reperfusion Injury and Ameliorates Diet-Induced Cardiometabolic Damage

Cardiometabolic diseases, including diabetes and its cardiovascular complications, are the global leading causes of death, highlighting a major unmet medical need. Over the past decade, mitsugumin 53 (MG53), also called TRIM72, has emerged as a powerful agent for myocardial membrane repair and cardioprotection, but its therapeutic value is complicated by its E3 ligase activity, which mediates metabolic disorders. Here, we show that an E3 ligase–dead mutant, MG53-C14A, retains its cardioprotective function without causing metabolic adverse effects. When administered in normal animals, both the recombinant human wild-type MG53 protein (rhMG53-WT) and its E3 ligase–dead mutant (rhMG53-C14A) protected the heart equally from myocardial infarction and ischemia/reperfusion (I/R) injury. However, in diabetic db/db mice, rhMG53-WT treatment markedly aggravated hyperglycemia, cardiac I/R injury, and mortality, whereas acute and chronic treatment with rhMG53-C14A still effectively ameliorated I/R-induced myocardial injury and mortality or diabetic cardiomyopathy, respectively, without metabolic adverse effects. Furthermore, knock-in of MG53-C14A protected the mice from high-fat diet–induced metabolic disorders and cardiac damage. Thus, the E3 ligase–dead mutant MG53-C14A not only protects the heart from acute myocardial injury but also counteracts metabolic stress, providing a potentially important therapy for the treatment of acute myocardial injury in metabolic disorders, including diabetes and obesity.     

Overexpression of the sarcoplasmic reticulum Ca(2+)-ATPase improves myocardial contractility in diabetic cardiomyopathy

Diabetic cardiomyopathy is characterized by reduced cardiac contractility due to direct changes in heart muscle function independent of vascular disease. An important contributor to contractile dysfunction in the diabetic state is an impaired sarcoplasmic reticulum (SR) function, leading to disturbed intracellular calcium handling. We investigated whether overexpression of the SR calcium pump (SERCA2a) in transgenic mice could reduce the impact of diabetes on the development of cardiomyopathy. Diabetes was induced by streptozotocin injection (200 mg/kg), and left ventricular (LV) function was analyzed in isolated hearts 3 weeks later. In diabetic hearts systolic LV pressure was decreased by 15% and maximum speed of relaxation (-dP/dt) by 34%. Functional changes were also assessed in isolated papillary muscles. Active force was reduced by 61% and maximum speed of relaxation by 65% in the diabetic state. The contractile impairment was accompanied by a 30% decrease in SERCA2a protein in diabetic mice. We investigated whether increased SERCA2a expression in transgenic SERCA2a-overexpressing mice could compensate for the diabetes-induced decrease in cardiac function. Under normal conditions, SERCA2a overexpressors show improved contractile performance relative to wild-type (WT) mice (-dP/dt: 3,169 vs. 2,559 mmHg/s, respectively). Measurement of LV function in hearts from diabetic SERCA2a mice revealed systolic and diastolic functions that were similar to WT control mice and markedly improved relative to diabetic WT mice (-dP/dt: 2,534 vs. 1,690 mmHg/s in diabetic SERCA2a vs. diabetic WT mice, respectively). Similarly, the contractile behavior of isolated papillary muscles from diabetic SERCA2a mice was not different from that of control mice. SERCA2a protein expression was higher (60%) in diabetic SERCA2a mice than WT diabetic mice. These results indicate that overexpression of SERCA2a can protect diabetic hearts from severe contractile dysfunction, presumably by improving the calcium sequestration of the SR.     

Expression of connective tissue growth factor is increased in injured myocardium associated with protein kinase C beta2 activation and diabetes

Protein kinase C (PKC) beta isoform activity is increased in myocardium of diabetic rodents and heart failure patients. Transgenic mice overexpressing PKCbeta2 (PKCbeta2Tg) in the myocardium exhibit cardiomyopathy and cardiac fibrosis. In this study, we characterized the expression of connective tissue growth factor (CTGF) and transforming growth factor beta (TGFbeta) with the development of fibrosis in heart from PKCbeta2Tg mice at 4-16 weeks of age. Heart-to-body weight ratios of transgenic mice increased at 8 and 12 weeks, indicating hypertrophy, and ratios did not differ at 16 weeks. Collagen VI and fibronectin mRNA expression increased in PKCbeta2Tg hearts at 4-12 weeks. Histological examination revealed myocyte hypertrophy and fibrosis in 4- to 16-week PKCbeta2Tg hearts. CTGF expression increased in PKCbeta2Tg hearts at all ages, whereas TGFbeta increased only at 8 and 12 weeks. In 8-week diabetic mouse heart, CTGF and TGFbeta expression increased two- and fourfold, respectively. Similarly, CTGF expression increased in rat hearts at 2-8 weeks of diabetes. This is the first report of increased CTGF expression in myocardium of diabetic rodents suggesting that cardiac injury associated with PKCbeta2 activation, diabetes, or heart failure is marked by increased CTGF expression. CTGF could act independently or together with other cytokines to induce cardiac fibrosis and dysfunction.     

The role of poly(ADP-ribose) polymerase activation in the development of myocardial and endothelial dysfunction in diabetes.

Patients with diabetes exhibit a high incidence of diabetic cardiomyopathy and vascular complications, which underlie the development of retinopathy, nephropathy, and neuropathy and increase the risk of hypertension, stroke, and myocardial infarction. There is emerging evidence that the activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) importantly contributes to the development of endothelial dysfunction in a streptozotocin-induced model of diabetes. We investigated the role of PARP activation in the pathogenesis of cardiac dysfunction in streptozotocin-induced and genetic (nonobese diabetic) models of diabetes in rats and mice. Development of diabetes was accompanied by hyperglycemia, cardiac PARP activation, a selective loss of endothelium-dependent vasodilation in the thoracic aorta, and an early diastolic dysfunction of the heart. Treatment with a novel potent phenanthridinone-based PARP inhibitor, PJ34, starting 1 week after the onset of diabetes, restored normal vascular responsiveness and significantly improved cardiac dysfunction, despite the persistence of severe hyperglycemia. The beneficial effect of PARP inhibition persisted even after several weeks of discontinuation of the treatment. Thus, PARP activation plays a central role in the pathogenesis of diabetic cardiovascular (cardiac as well as endothelial) dysfunction. PARP inhibitors may exert beneficial effects against the development of cardiovascular complications in diabetes.     

Optimal elevation of β-cell 11β-hydroxysteroid dehydrogenase type 1 is a compensatory mechanism that prevents high-fat diet-induced β-cell failure

Type 2 diabetes ultimately results from pancreatic β-cell failure. Abnormally elevated intracellular regeneration of glucocorticoids by the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in fat or liver may underlie pathophysiological aspects of the metabolic syndrome. Elevated 11β-HSD1 is also found in pancreatic islets of obese/diabetic rodents and is hypothesized to suppress insulin secretion and promote diabetes. To define the direct impact of elevated pancreatic β-cell 11β-HSD1 on insulin secretion, we generated β-cell-specific, 11β-HSD1-overexpressing (MIP-HSD1) mice on a strain background prone to β-cell failure. Unexpectedly, MIP-HSD1(tg/+) mice exhibited a reversal of high fat-induced β-cell failure through augmentation of the number and intrinsic function of small islets in association with induction of heat shock, protein kinase A, and extracellular signal-related kinase and p21 signaling pathways. 11β-HSD1(-/-) mice showed mild β-cell impairment that was offset by improved glucose tolerance. The benefit of higher β-cell 11β-HSD1 exhibited a threshold because homozygous MIP-HSD1(tg/tg) mice and diabetic Lep(db/db) mice with markedly elevated β-cell 11β-HSD1 levels had impaired basal β-cell function. Optimal elevation of β-cell 11β-HSD1 represents a novel biological mechanism supporting compensatory insulin hypersecretion rather than exacerbating metabolic disease. These findings have immediate significance for current therapeutic strategies for type 2 diabetes.     

Diabetic heart disease: A clinical update

Diabetes mellitus (DM) significantly increases the risk of heart disease, and DM-related healthcare expenditure is predominantly for the management of cardiovascular complications. Diabetic heart disease is a conglomeration of coronary artery disease (CAD), cardiac autonomic neuropathy (CAN), and diabetic cardiomyopathy (DCM). The Framingham study clearly showed a 2 to 4-fold excess risk of CAD in patients with DM. Pathogenic mechanisms, clinical presentation, and management options for DM-associated CAD are somewhat different from CAD among nondiabetics. Higher prevalence at a lower age and more aggressive disease in DM-associated CAD make diabetic individuals more vulnerable to premature death. Although common among diabetic individuals, CAN and DCM are often under-recognised and undiagnosed cardiac complications. Structural and functional alterations in the myocardial innervation related to uncontrolled diabetes result in damage to cardiac autonomic nerves, causing CAN. Similarly, damage to the cardiomyocytes from complex pathophysiological processes of uncontrolled DM results in DCM, a form of cardiomyopathy diagnosed in the absence of other causes for structural heart disease. Though optimal management of DM from early stages of the disease can reduce the risk of diabetic heart disease, it is often impractical in the real world due to many reasons. Therefore, it is imperative for every clinician involved in diabetes care to have a good understanding of the pathophysiology, clinical picture, diagnostic methods, and management of diabetes-related cardiac illness, to reduce morbidity and mortality among patients. This clinical review is to empower the global scientific fraternity with up-to-date knowledge on diabetic heart disease.     

Herpes simplex-mediated gene transfer of nerve growth factor protects against peripheral neuropathy in streptozotocin-induced diabetes in the mouse

Peripheral neuropathy is a common and debilitating complication of diabetes. In animal models, neurotrophic factors can prevent progression of the neuropathy, but adverse effects prevent systemic administration in adequate doses to treat human disease. We examined whether gene transfer with replication-defective genomic herpes simplex virus (HSV) vectors modified to express nerve growth factor (NGF) could be used to prevent progression of neuropathy in mice. Diabetes induced by streptozotocin (STZ) resulted in a sensory neuropathy manifest by a decrease in the foot sensory nerve amplitude (FSA; control = 20 +/- 0.1 microV, treated = 14 +/- 0.1 microV). Transduction of dorsal root ganglia in vivo with an HSV-based vector expressing NGF under the control of the human cytomegalovirus immediate early promoter (vector SHN) or the HSV latency active promoter 2 (vector SLN) by footpad inoculation 2 weeks after STZ administration protected against the decrease in FSA (22 +/- 1.4 microV and 21 +/- 1.7 microV, respectively) measured 4 weeks later. Injection of SHN into inguinal adipose tissue 2 weeks after onset of diabetes also prevented the decrease in FSA (20 +/- 3.3 microV). These results suggest that gene transfer with an NGF-producing herpes-based vector may prove useful in the treatment of diabetic neuropathy.     

Inhibition of superoxide generation and associated nitrosative damage is involved in metallothionein prevention of diabetic cardiomyopathy

The mechanisms of metallothionein prevention of diabetic cardiomyopathy are largely unknown. The present study was performed to test whether inhibition of nitrosative damage is involved in metallothionein prevention of diabetic cardiomyopathy. Cardiac-specific metallothionein-overexpressing transgenic (MT-TG) mice and wild-type littermate controls were treated with streptozotocin (STZ) by a single intraperitoneal injection, and both developed diabetes. However, the development of diabetic cardiomyopathy, revealed by histopathological and ultrastructural examination, serum creatine phosphokinase, and cardiac hemodynamic analysis, was significantly observed only in the wild-type, but not in MT-TG, diabetic mice 2 weeks and 6 months after STZ treatment. Formations of superoxide and 3-nitrotyrosine (3-NT), a marker for peroxynitrite-induced protein damage, were detected only in the heart of wild-type diabetic mice. Furthermore, primary cultures of cardiomyocytes from wild-type and MT-TG mice were exposed to lipopolysaccharide/tumor necrosis factor-alpha for generating intracellular peroxynitrite. Increases in 3-NT formation and cytotoxicity were observed in wild-type, but not in MT-TG, cardiomyocytes. Either urate, a peroxynitrite-specific scavenger, or Mn(111) tetrakis 1-methyl 4-pyridyl porphyrin pentachloride (MnTMPyP), a superoxide dismutase mimic, significantly inhibited the formation of 3-NT along with a significant prevention of cytotoxicity. These results thus suggest that metallothionein prevention of diabetic cardiomyopathy is mediated, at least in part, by suppression of superoxide generation and associated nitrosative damage.     

Kallikrein gene delivery improves serum glucose and lipid profiles and cardiac function in streptozotocin-induced diabetic rats

We investigated the role of the kallikrein-kinin system in cardiac function and glucose utilization in the streptozotocin (STZ)-induced diabetic rat model using a gene transfer approach. Adenovirus harboring the human tissue kallikrein gene was administered to rats by intravenous injection at 1 week after STZ treatment. Human kallikrein transgene expression was detected in the serum and urine of STZ-induced diabetic rats after gene transfer. Kallikrein gene delivery significantly reduced blood glucose levels and cardiac glycogen accumulation in STZ-induced diabetic rats. Kallikrein gene transfer also significantly attenuated elevated plasma triglyceride and cholesterol levels, food and water intake, and loss of body weight gain, epididymal fat pad, and gastrocnemius muscle weight in STZ-induced diabetic rats. However, these effects were blocked by icatibant, a kinin B2 receptor antagonist. Cardiac function was significantly improved after kallikrein gene transfer as evidenced by increased cardiac output and +/-delta P/delta t (maximum speed of contraction/relaxation), along with elevated cardiac sarco(endo)plasmic reticulum (Ca2+ + Mg2+)-ATPase (SERCA)-2a, phosphorylated phospholamban, NOx and cAMP levels, and GLUT4 translocation into plasma membranes of cardiac and skeletal muscle. Kallikrein gene delivery also increased Akt and glycogen synthase kinase (GSK)-3beta phosphorylation, resulting in decreased GSK-3beta activity in the heart. These results indicate that kallikrein through kinin formation protects against diabetic cardiomyopathy by improving cardiac function and promoting glucose utilization and lipid metabolism.     

Beneficial effects of verapamil in diabetic cardiomyopathy

It has been suggested that the occurrence of an intracellular Ca2+ overload may result in the development of diabetic cardiomyopathy, which is associated with depletion of high-energy phosphate stores and a derangement of ultrastructure and cardiac dysfunction. Accordingly, the effects of verapamil, a Ca2+ antagonist, on cardiac function, ultrastructure, and high-energy phosphate stores in the myocardium were evaluated in rats made diabetic by an intravenous injection of streptozocin (65 mg/kg). Four weeks after the induction of diabetes, the animals were treated with three doses (2, 4, or 8 mg.kg-1.day-1) of verapamil for 4 wk until they were used for the measurement of different parameters. Untreated diabetic animals had slower heart rates, depressed rate of contraction and rate of relaxation, lower peak left ventricular systolic pressure, and elevated left ventricular diastolic pressure. All of these changes were significantly improved in diabetic rats receiving verapamil treatment. The beneficial effects of verapamil were more evident with higher doses (8 mg.kg-1.day-1) than with the lower doses (2 mg.kg-1.day-1). The diabetic animals also showed alterations in myocardial high-energy phosphate stores and exhibited evidence of ultrastructural damage; these abnormalities were improved by verapamil treatment without affecting their hyperglycemic status. Our results demonstrate that verapamil is capable of preventing diabetes-induced myocardial changes and support the involvement of Ca2+ in the cardiac pathology during diabetes.     

Improvement of cardiac functions by chronic metformin treatment is associated with enhanced cardiac autophagy in diabetic OVE26 mice

OBJECTIVE

Autophagy is a critical cellular system for removal of aggregated proteins and damaged organelles. Although dysregulated autophagy is implicated in the development of heart failure, the role of autophagy in the development of diabetic cardiomyopathy has not been studied. We investigated whether chronic activation of the AMP-activated protein kinase (AMPK) by metformin restores cardiac function and cardiomyocyte autophagy in OVE26 diabetic mice.

RESEARCH DESIGN AND METHODS

OVE26 mice and cardiac-specific AMPK dominant negative transgenic (DN)-AMPK diabetic mice were treated with metformin or vehicle for 4 months, and cardiac autophagy, cardiac functions, and cardiomyocyte apoptosis were monitored.

RESULTS

Compared with control mice, diabetic OVE26 mice exhibited a significant reduction of AMPK activity in parallel with reduced cardiomyocyte autophagy and cardiac dysfunction in vivo and in isolated hearts. Furthermore, diabetic OVE26 mouse hearts exhibited aggregation of chaotically distributed mitochondria between poorly organized myofibrils and increased polyubiquitinated protein and apoptosis. Inhibition of AMPK by overexpression of a cardiac-specific DN-AMPK gene reduced cardiomyocyte autophagy, exacerbated cardiac dysfunctions, and increased mortality in diabetic mice. Finally, chronic metformin therapy significantly enhanced autophagic activity and preserved cardiac functions in diabetic OVE26 mice but not in DN-AMPK diabetic mice.

CONCLUSIONS

Decreased AMPK activity and subsequent reduction in cardiac autophagy are important events in the development of diabetic cardiomyopathy. Chronic AMPK activation by metformin prevents cardiomyopathy by upregulating autophagy activity in diabetic OVE26 mice. Thus, stimulation of AMPK may represent a novel approach to treat diabetic cardiomyopathy.Autophagy is a physiologic process whereby cytoplasmic components, including long-lived proteins and organelles, are engulfed by a double-membrane structure and targeted for destruction in lysosomes (1). It selectively removes damaged mitochondria as a cytoprotective mechanism for limiting mitochondria-derived oxidative stress and preventing apoptosis (2,3). A low level of constitutive autophagy is important in the heart for maintaining normal cellular function and the quality of proteins and organelles. Defects in this process cause cardiac dysfunction and heart failure, particularly when cellular stress is increased (4). Although autophagy is implicated in various pathologic conditions, including cardiac hypertrophy, cardiomyopathy, and heart failure, there is little information on the pathophysiologic roles of autophagy in the pathogenesis of diabetic cardiomyopathy.Metformin, one of the most commonly prescribed antidiabetic drugs, improves cardiac function and reduces the incidence of myocardial infarction in type 2 diabetic patients (5,6). The UK Prospective Diabetes Study reported that metformin was more effective than sulfonylureas or insulin in reducing all-cause mortality and diabetes-related end points in diabetic patients, even though these agents decreased HbA_1c by comparable magnitudes. These findings suggest that metformin provides cardiovascular protection independent of its hypoglycemic effects (7).Indeed, metformin ameliorates cardiac dysfunctions induced by global ischemia, without affecting blood glucose in nondiabetic animals (8,9), by activating the AMP-activated protein kinase (AMPK) (10,11). AMPK acts as a sensor of cellular energy status and controls several cellular functions in the cardiovascular system, including protein synthesis (12,13), apoptosis (14–16), and autophagy (17,18) in physiologic and pathologic conditions, such as hemodynamic stress (12,13), myocardial ischemia, and reperfusion injury (16,19,20). However, the roles and molecular mechanisms by which AMPK regulates diabetic cardiomyopathy remain to be established.Diabetic cardiomyopathy, which develops in diabetic patients in the absence of coronary artery disease or hypertension (21–24), is a major cause of heart failure in diabetic patients. It is characterized by reduced cardiomyocyte contractility, cardiac apoptosis, mitochondrial pathology, and dysfunction (25,26). Despite the importance of this complication, the underlying mechanisms of diabetic cardiomyopathy are still poorly understood. Thus, this study was designed to test whether decreased autophagy is associated with the development of cardiomyopathy in diabetic OVE26 mice and to evaluate whether metformin improves cardiac function by modulating autophagic activity in diabetes.