Chronic Neuregulin-1ß Treatment Mitigates the Progression of Postmyocardial Infarction Heart Failure in the Setting of Type 1 Diabetes Mellitus by Suppressing Myocardial Apoptosis, Fibrosis, and Key Oxidant-Producing Enzymes.

BACKGROUND: Type 1 diabetes mellitus (DM) patients surviving myocardial infarction (MI) have substantially higher cardiovascular morbidity and mortality compared to their nondiabetic counterparts owing to the more frequent development of subsequent heart failure (HF). Neuregulin (NRG)-1ß is released from cardiac microvascular endothelial cells and acts as a paracrine factor via the ErbB family of tyrosine kinase receptors expressed in cardiac myocytes to regulate cardiac development and stress responses. Because myocardial NRG-1/ErbB signaling has been documented to be impaired during HF associated with type 1 DM, we examined whether enhancement of NRG-1ß signaling via exogenous administration of recombinant NRG-1ß could exert beneficial effects against post-MI HF in the type 1 diabetic heart. METHODS AND RESULTS: Type 1 DM was induced in male Sprague Dawley rats by a single injection of streptozotocin (STZ) (65 mg/kg). Two weeks after induction of type 1 DM, rats underwent left coronary artery ligation to induce MI. STZ-diabetic rats were treated with saline or NRG-1ß (100 µg/kg) twice per week for 7 weeks, starting 2 weeks before experimental MI. Residual left ventricular function was significantly greater in the NRG-1ß-treated STZ-diabetic MI group compared with the vehicle-treated STZ-diabetic MI group 5 weeks after MI as assessed by high-resolution echocardiography. NRG-1ß treatment of STZ-diabetic MI rats was associated with reduced myocardial fibrosis and apoptosis as well as decreased gene expression of key oxidant-producing enzymes. CONCLUSIONS: These results suggest that recombinant NRG-1ß may be a promising therapeutic for HF post-MI in the setting of type 1 DM.

Angiotensin-induced abdominal aortic aneurysms in hypercholesterolemic mice: role of serum cholesterol and temporal effects of exposure.

OBJECTIVE: Understanding variations in size and pattern of development of angiotensin II (Ang II)-induced abdominal aortic aneurysms (AAA) may inform translational research strategies. Thus, we sought insight into the temporal evolution of AAA in apolipoprotein (apo)E(-/-) mice. APPROACH: A cohort of mice underwent a 4-week pump-mediated infusion of saline (n = 23) or 1500 ng/kg/min of Ang II (n = 85) and AAA development was tracked via in vivo ultrasound imaging. We adjusted for hemodynamic covariates in the regression models for AAA occurrence in relation to time. RESULTS: The overall effect of time was statistically significant (p<0.001). Compared to day 7 of AngII infusion, there was no decrease in the log odds of AAA occurrence by day 14 (-0.234, p = 0.65), but compared to day 21 and 28, the log odds decreased by 9.07 (p<0.001) and 2.35 (p = 0.04), respectively. Hemodynamic parameters were not predictive of change in aortic diameter (Δ) (SBP, p = 0.66; DBP, p = 0.66). Mean total cholesterol (TC) was higher among mice with large versus small AAA (601 vs. 422 mg/ml, p<0.0001), and the difference was due to LDL. AngII exposure was associated with 0.43 mm (95% CI, 0.27 to 0.61, p<0.0001) increase in aortic diameter; and a 100 mg/dl increase in mean final cholesterol level was associated with a 12% (95% CI, 5.68 to 18.23, p<0.0001) increase in aortic diameter. Baseline cholesterol was not associated with change in aortic diameter (p = 0.86). CONCLUSIONS: These are the first formal estimates of a consistent pattern of Ang II-induced AAA development. The odds of AAA occurrence diminish after the second week of Ang II infusion, and TC is independently associated with AAA size.

Augmented cardiac formation of oxidatively-induced carbonylated proteins accompanies the increased functional severity of post-myocardial infarction heart failure in the setting of type 1 diabetes mellitus.

SUMMARY: Heart failure (HF) is a dominant cause for the higher mortality of diabetics after myocardial infarction (MI). In the present investigation, we have discovered that higher levels of oxidative stress (OS)-induced carbonylated proteins accompany worsening post-MI HF in the presence of type 1 diabetes. These findings provide a mechanistic link between amplified OS and exacerbation of post-infarction HF in diabetes. BACKGROUND: Type 1 diabetes mellitus (DM) patients surviving myocardial infarction (MI) manifest an increased incidence of subsequent heart failure (HF). We have previously shown that after MI, type 1 DM is associated with accentuated myocardial oxidative stress (OS) and concomitant worsening of left ventricular (LV) function. However, the precise mechanisms whereby type 1 DM-enhanced OS adversely affects HF after MI remain obscure. As carbonylation of proteins is an irreversible post-translational modification induced only by OS that often leads to the loss of function, we analyzed protein-bound carbonyls in the surviving LV myocardium of MI and DM+MI rats in relation to residual LV function. METHODS: Type 1 DM was induced in rats via administration of streptozotocin. Two weeks after induction of type 1 DM, MI was produced in DM and non-DM rats by coronary artery ligation. Residual LV function and remodeling was assessed at 4 weeks post-MI by echocardiography. Myocardial carbonylated proteins were detected through OxyBlot analysis, and identified by mass spectrometry. RESULTS: Compared with MI rats, DM+MI rats exhibited significantly poorer residual LV systolic function and elevated wet to dry weight ratios of the lungs. Protein carbonyl content in cardiac tissue and isolated heart mitochondria of DM+MI rats was 20% and 48% higher, respectively, versus MI rats. Anti-oxidative enzymes and fatty acid utilization proteins were among the carbonylated protein candidates identified. CONCLUSIONS: These findings implicate myocardial protein carbonylation as part of the molecular pathophysiology of aggravated HF in the type 1 diabetic post-infarction heart.

Type 1 diabetes mellitus abrogates compensatory augmentation of myocardial neuregulin-1ß/ErbB in response to myocardial infarction resulting in worsening heart failure.

BACKGROUND: Diabetes mellitus (DM) patients surviving myocardial infarction (MI) exhibit a substantially higher incidence of subsequent heart failure (HF). Neuregulin (NRG)-1 and erythroblastic leukemia viral oncogene homolog (ErbB) receptors have been shown to play a critical role in maintenance of cardiac function. However, whether myocardial NRG-1/ErbB is altered during post-MI HF associated with DM remains unknown. The aim of this study was to determine the impact of type 1 DM on the myocardial NRG-1/ErbB system following MI in relation to residual left ventricular (LV) function. METHODS: Type 1 DM was induced in rats via administration of streptozotocin (65 mg/kg, i.p.). Control rats were injected with citrate buffer (vehicle) only. Two weeks after induction of type 1 DM, MI was produced in DM and non-DM rats by ligation of the left coronary artery. Sham MI rats underwent the same surgical procedure with the exception that the left coronary artery was not ligated. At 4 weeks after surgery, residual in vivo LV function was assessed via echocardiography. Myocardial protein expression of NRG-1ß, ErbB2 and ErbB4 receptors, and MDM2 (a downstream signaling pathway induced by NRG-1 that has been implicated in cell survival) was assessed in the remaining, viable LV myocardium by Western blotting. Changes in ErbB receptor localization in the surviving LV myocardium of diabetic and non-diabetic post-MI rats was determined using immunohistochemistry techniques. RESULTS: At 4 weeks post-MI, echocardiography revealed that LV fractional shortening (FS) and LV ejection fraction (EF) were significantly lower in the DM + MI group compared to the MI group (LVFS: 17.9 ± 0.7 vs. 25.2 ± 2.2; LVEF: 35.5 ± 1.4 vs. 47.5 ± 3.5, respectively; P < 0.05), indicating an increased functional severity of HF among the DM + MI rats. Up-regulation of NRG-1ß and ErbB2 protein expression in the MI group was abrogated in the DM + MI group concurrent with degradation of MDM2, a downstream negative regulator of p53. ErbB2 and ErbB4 receptors re-localized to cardiac myocyte nuclei in failing type 1 diabetic post-MI hearts. CONCLUSIONS: Type 1 DM prevents compensatory up-regulation of myocardial NRG-1/ErbB after MI coincident with an increased severity of HF.

Intravenous glial growth factor 2 (GGF2) isoform of neuregulin-1ß improves left ventricular function, gene and protein expression in rats after myocardial infarction.

AIMS: Recombinant Neuregulin (NRG)-1ß has multiple beneficial effects on cardiac myocytes in culture, and has potential as a clinical therapy for heart failure (HF). A number of factors may influence the effect of NRG-1ß on cardiac function via ErbB receptor coupling and expression. We examined the effect of the NRG-1ß isoform, glial growth factor 2 (GGF2), in rats with myocardial infarction (MI) and determined the impact of high-fat diet as well as chronicity of disease on GGF2 induced improvement in left ventricular systolic function. Potential mechanisms for GGF2 effects on the remote myocardium were explored using microarray and proteomic analysis. METHODS AND RESULTS: Rats with MI were randomized to receive vehicle, 0.625 mg/kg, or 3.25 mg/kg GGF2 in the presence and absence of high-fat feeding beginning at day 7 post-MI and continuing for 4 weeks. Residual left ventricular (LV) function was improved in both of the GGF2 treatment groups compared with the vehicle treated MI group at 4 weeks of treatment as assessed by echocardiography. High-fat diet did not prevent the effects of high dose GGF2. In experiments where treatment was delayed until 8 weeks after MI, high but not low dose GGF2 treatment was associated with improved systolic function. mRNA and protein expression analysis of remote left ventricular tissue revealed a number of changes in myocardial gene and protein expression altered by MI that were normalized by GGF2 treatment, many of which are involved in energy production. CONCLUSIONS: This study demonstrates that in rats with MI induced systolic dysfunction, GGF2 treatment improves cardiac function. There are differences in sensitivity of the myocardium to GGF2 effects when administered early vs. late post-MI that may be important to consider in the development of GGF2 in humans.

Neuregulin in cardiovascular development and disease.

Studies in genetically modified mice have demonstrated that neuregulin-1 (NRG-1), along with the erythroblastic leukemia viral oncogene homolog (ErbB) 2, 3, and 4 receptor tyrosine kinases, is necessary for multiple aspects of cardiovascular development. These observations stimulated in vitro and in vivo animal studies, implicating NRG-1/ErbB signaling in the regulation of cardiac cell biology throughout life. Cardiovascular effects of ErbB2-targeted cancer therapies provide evidence in humans that ErbB signaling plays a role in the maintenance of cardiac function. These and other studies suggest a conceptual model in which a key function of NRG-1/ErbB signaling is to mediate adaptations of the heart to physiological and pathological stimuli through activation of intracellular kinase cascades that regulate tissue plasticity. Recent work implicates NRG-1/ErbB signaling in the regulation of multiple aspects of cardiovascular biology, including angiogenesis, blood pressure, and skeletal muscle responses to exercise. The therapeutic potential of recombinant NRG-1 as a potential treatment for heart failure has been demonstrated in animal models and is now being explored in clinical studies. NRG-1 is found in human serum and plasma, and it correlates with some clinical parameters, suggesting that it may have value as an indicator of prognosis. In this review, we bring together this growing literature on NRG-1 and its significance in cardiovascular development and disease.

The Wnt modulator sFRP2 enhances mesenchymal stem cell engraftment, granulation tissue formation and myocardial repair.

Cell-based therapies, using multipotent mesenchymal stem cells (MSCs) for organ regeneration, are being pursued for cardiac disease, orthopedic injuries and biomaterial fabrication. The molecular pathways that regulate MSC-mediated regeneration or enhance their therapeutic efficacy are, however, poorly understood. We compared MSCs isolated from MRL/MpJ mice, known to demonstrate enhanced regenerative capacity, to those from C57BL/6 (WT) mice. Compared with WT-MSCs, MRL-MSCs demonstrated increased proliferation, in vivo engraftment, experimental granulation tissue reconstitution, and tissue vascularity in a murine model of repair stimulation. The MRL-MSCs also reduced infarct size and improved function in a murine myocardial infarct model compared with WT-MSCs. Genomic and functional analysis indicated a downregulation of the canonical Wnt pathway in MRL-MSCs characterized by significant up-regulation of specific secreted frizzled-related proteins (sFRPs). Specific knockdown of sFRP2 by shRNA in MRL-MSCs decreased their proliferation and their engraftment in and the vascular density of MRL-MSC-generated experimental granulation tissue. These results led us to generate WT-MSCs overexpressing sFRP2 (sFRP2-MSCs) by retroviral transduction. sFRP2-MSCs maintained their ability for multilineage differentiation in vitro and, when implanted in vivo, recapitulated the MRL phenotype. Peri-infarct intramyocardial injection of sFRP2-MSCs resulted in enhanced engraftment, vascular density, reduced infarct size, and increased cardiac function after myocardial injury in mice. These findings implicate sFRP2 as a key molecule for the biogenesis of a superior regenerative phenotype in MSCs.

Emerging role for antioxidant therapy in protection against diabetic cardiac complications: experimental and clinical evidence for utilization of classic and new antioxidants.

Diabetes mellitus (DM) markedly potentiates the risk of cardiovascular morbidity and mortality among individuals with diabetes as compared to the non-diabetic population. After myocardial infarction (MI), DM patients have a higher incidence of death than do non-diabetics. The excess mortality and poor prognosis of these patients results primarily from the development of recurrent MI and heart failure (HF). Although several lines of evidence support a role for increased oxidative stress in a range of cardiovascular diseases, clinical trials examining the therapeutic efficacy of antioxidants have yielded conflicting results. The reasons for these incongruous results is multifactorial. An underlying theme has been lack of patient inclusion based on elevated indices of oxidative stress which could have diluted the population susceptible to benefit in the clinical trials. Laboratory evidence has accumulated indicating that oxidative stress is dramatically accentuated in cardiac abnormalities inherent in DM. In this review, we provide the emergence of experimental and clinical evidence supporting antioxidant supplementation as a cardioprotective intervention in the setting of DM. Specifically, focus will be directed on preclinical animal studies and human clinical trials that have tested the effect of antioxidant supplements on MI and HF events in the presence of DM.

Dietary supplementation with vitamin E ameliorates cardiac failure in type I diabetic cardiomyopathy by suppressing myocardial generation of 8-iso-prostaglandin F2alpha and oxidized glutathione.

BACKGROUND: Diabetic cardiomyopathy has been documented as an underlying cause of heart failure in diabetic patients. Although oxidative stress has been implicated in diabetic cardiomyopathy, much of the current evidence lacks specificity. Furthermore, studies investigating antioxidant protection with vitamin E in this unique cardiac phenomenon have yet to be performed. In the present study, we sought to determine whether vitamin E supplementation can confer cardioprotective effects against diabetic cardiomyopathy in relation to specific and quantitative markers of myocardial oxidative stress. METHODS AND RESULTS: Diabetes was induced in rats by a single injection of streptozotocin. Animals were fed either a basal diet or a diet enriched with 2000 IU of vitamin E per kilogram beginning immediately after induction of diabetes and continued for 8 weeks. Rats were examined for diabetic cardiomyopathy by left ventricular (LV) hemodynamic analysis. Myocardial oxidative stress was assessed by measuring the formation of 8-iso-prostaglandin F2alpha and oxidized glutathione. In the unsupplemented streptozotocin-diabetic rats, LV systolic pressure, rate of pressure increase (+dP/dt), and rate of pressure decay (-dP/dt) were depressed, whereas LV end-diastolic pressure was increased, indicating reduced LV contractility and slowing of LV relaxation. These hemodynamic alterations were accompanied by increased myocardial formation of 8-iso-prostaglandin F2alpha and oxidized glutathione. Vitamin E supplementation improved LV function and significantly attenuated myocardial 8-iso-prostaglandin F2alpha and oxidized glutathione accumulation in streptozotocin-diabetic rats. CONCLUSIONS: These findings demonstrate the usefulness of vitamin E supplementation during the early phases of type I diabetes for the prophylaxis of cardiomyopathy and subsequent heart failure.

Alterations in the diabetic myocardial proteome coupled with increased myocardial oxidative stress underlies diabetic cardiomyopathy.

Diabetic cardiomyopathy has been documented as an underlying etiology of heart failure (HF) among diabetics. Although oxidative stress has been proposed to contribute to diabetic cardiomyopathy, much of the evidence lacks specificity. Furthermore, whether alterations occur at the cardiac proteome level in diabetic cardiac complications with attendant oxidative stress remains unknown. Therefore, we sought to identify cardiac protein changes in relation to myocardial oxidative stress that are specific to diabetic cardiomyopathy. Diabetes was induced in rats by a single injection of streptozotocin (STZ). STZ-treated rats were examined for diabetic cardiomyopathy at 8 weeks post-STZ by left ventricular (LV) hemodynamic analysis. LV systolic pressure (LVSP), rate of pressure rise (+dP/dt), and rate of pressure decay (-dP/dt) were depressed while LV end-diastolic pressure (LVEDP) was increased. Myocardial oxidative stress was increased in STZ-diabetic rats, as indexed by significant increases in myocardial formation of 8-iso PGF(2alpha) and oxidized glutathione (GSSG). In-depth mining of the diabetic myocardial proteome by proteomic analysis utilizing two-dimensional difference gel electrophoresis and mass spectrometry (DIGE/MS) techniques revealed that a high proportion (12 of 24) of the altered proteins that could be identified by mass spectrometry were localized to the mitochondria. Down-regulation of antioxidant and anti-apoptotic proteins was also observed in STZ-diabetic hearts. These results characterize a specific 'type I diabetic' pattern of cardiac proteome changes indicative of diabetic cardiomyopathy presenting with higher oxidative stress, supporting the idea that analysis of isoprostane biosynthesis and protein expression profiles may be useful diagnostically to assess the efficacy of antioxidant therapies as prophylactic treatments against type I diabetes mellitus complications involving the heart.

Greater propensity of diabetic myocardium for oxidative stress after myocardial infarction is associated with the development of heart failure.

Diabetic patients manifest an increased incidence of heart failure (HF) after myocardial infarction (MI), which presages an increase in morbidity and mortality. Although oxidative stress has been implicated in diabetic complications, oxidative stress status associated with comorbid conditions that frequently accompany diabetes remains unknown. Therefore, we examined antioxidants and oxidative stress in the surviving myocardium in relation to ventricular function during diabetic HF following MI. MI was produced in diabetic and nondiabetic rats by ligation of the left coronary artery. At 4 weeks post-MI, LV systolic pressure (LVSP), rate of pressure rise (+dP/dt), and rate of pressure decay (-dP/dt) were depressed to a significantly greater extent in diabetic compared to nondiabetic MI animals. Higher levels of myocardial 8-isoprostane (8-iso PGF(2alpha)), oxidized glutathione (GSSG), as well as greater upregulation of superoxide dismutase (SOD) and catalase (CAT) protein expression paralleled by increases in enzymatic activity was observed in the diabetic MI animals, indicating higher oxidative stress. These data demonstrate a greater derangement of oxidative stress in the surviving tissues of diabetic post-MI rat hearts concomitant with an increased functional severity of HF, and suggest that chronic antioxidant therapy may be useful for the prophylaxis of subsequent HF after MI associated with diabetes.

Reduction in oxidative stress and modulation of heart failure subsequent to myocardial infarction in rats.

BACKGROUND: Patients surviving myocardial infarction (MI) are at a heightened risk for the development of congestive heart failure. This clinical syndrome has been associated with an antioxidant deficit and elevated oxidative stress in the myocardium. Effects of dietary vitamin E, a lipid-soluble antioxidant, on myocardial anti-oxidant enzyme activities, oxidative stress and hemodynamic function, were examined separately in the viable left ventricle (LV) and right ventricle (RV) of rats at 16 weeks post-MI. METHODS AND RESULTS: Animals were fed either a basal diet or a diet enriched with 1500 U of vitamin E/kg beginning two weeks before MI-inducing surgery and continued 16 weeks post-MI. In the MI animals on the basal diet, LV systolic pressure (LVSP) and RVSP were significantly depressed and LV end-diastolic pressure (LVEDP) and RVEDP were significantly elevated. These hemodynamic alterations were accompanied by clinical signs of heart failure including dyspnea, lethargy and cyanotic limbs. Supplementation of MI animals with dietary vitamin E resulted in complete normalization of RVSP and RVEDP. An increase in LVSP and a decrease in LVEDP was observed in the vitamin E-supplemented MI animals, although mild residual LV dysfunction remained. The myocardial enzymatic antioxidants catalase and glutathione peroxidase declined substantially in each of the ventricles of unsupplemented MI animals. Myocardial levels of vitamin E were reduced by 33% in the LV and no change was observed in the RV of the MI animals. Vitamin E-supplemented control animals and MI animals showed a significant increase in vitamin E levels in both ventricles. Myocardial oxidative stress, as assessed by lipid peroxidation and the ratio of reduced to oxidized glutathione, was significantly increased in each of the respective ventricles of untreated MI animals. Supplementation with dietary vitamin E resulted in a substantial increase in the myocardial activities of catalase and glutathione peroxidase in both the LV and RV. Furthermore, an increase in the ratio of reduced to oxidized glutathione concomitant with significantly less lipid peroxidation was also observed in each of the respective ventricles of MI animals supplemented with vitamin E. No overt clinical signs of heart failure were evident in these vitamin E-supplemented animals. CONCLUSIONS: An improved myocardial redox state and endogenous antioxidant reserve with vitamin E therapy, coupled with the modulation of the development of heart failure, lend strong support in favour of a pathophysiological role for increased oxidative stress in the pathogenesis of heart failure, at least in experimental animals. Association between an increase in oxidative stress and cardiac events in patients requires further examination.

Antioxidant and oxidative stress changes in experimental cor pulmonale.

Although right heart failure (RHF) contributes to 20% of all cardiovascular complications, most of the information available on RHF in general is based on the experiences with left heart failure. This study on RHF investigates changes in antioxidants and oxidative stress which are suggested to play a role in the transition from hypertrophy to failure. RHF subsequent to pulmonary hypertension was produced in rats by a single injection of monocrotaline (MCT, 60 mg/kg, i.p.). Based on hemodynamic, clinical and histopathologic observations, the animals were grouped in three functional stages at 1-, 2- and 6-week post-injection periods. In the 1-week group, RV pressure overload and hypertrophy, and a mild increase in antioxidant enzymes was seen. In the 2-week group, compensated HF, a significant increase in antioxidant enzymes, an increase in septal (IVS) wall thickness and leftward displacement of IVS without change in LV free wall were seen. In the 6-week group, lung and liver congestion, RVF and dilation, a decrease in antioxidant enzyme activities, increase in lipid peroxidation and severe bulging of the IVS into the left ventricle were seen. These changes in the hemodynamic, biochemical and histopathologic characteristics suggest that in early stages of MCT-induced pulmonary hypertension at 1 and 2 weeks, RV hypertrophy was accompanied by sustained hemodynamic function and an increase in antioxidant reserve. In the later stage at 6 weeks, clinical RHF was associated with abnormalities of the right heart systolic and diastolic function along with a decrease in antioxidant reserve. These biphasic changes in RV antioxidant enzymes, i.e. an increase during hypertrophy and a decrease in failure may suggest a role of oxidative stress in the pathogenesis of right ventricular dysfunction.