Alterations in connexin 43 during diabetic cardiomyopathy: competition of tyrosine nitration versus phosphorylation.

BACKGROUND: Cardiac conduction abnormalities are observed early in the progression of type 1 diabetes (T1D), but the mechanism(s) involved are undefined. Connexin 43, a critical component of ventricular gap junctions, depends on tyrosine phosphorylation status to modulate channel conductance; changes in connexin 43 content, distribution, and/or phosphorylation status may be involved in cardiac rhythm disturbances. We tested the hypothesis that cardiac content and/or distribution of connexin 43 is altered in a rat model of T1D cardiomyopathy, investigating a mechanistic role for tyrosine. METHODS: Electrocardiographic analyses were conducted during the progression of diabetic cardiomyopathy in rats dosed with streptozotocin (STZ; 65 mg/kg) 3, 7, and 35 days after the induction of diabetes. Following functional analyses, we conducted immunohistochemical and immunoprecipitation studies to assess alterations in connexin 43. RESULTS: There was significant evidence of ventricular conduction abnormalities (QRS complex, Q-T interval) as early as 7 days after STZ, persisting throughout the study. Connexin 43 levels were increased 7 days after STZ and remained elevated throughout the study. Connexin 40 content was unchanged relative to controls throughout the study. Changes in connexin 43 distribution were also observed: connexin 43 staining was dispersed from myocyte short axis junctions. Connexin 43 tyrosine phosphorylation declined during the progression of diabetes, with concurrent increases in tyrosine nitration. CONCLUSIONS: The data suggest that changes in connexin 43 content and distribution occur during experimental diabetes and likely contribute to alterations in cardiac function, and that oxidative modification of tyrosine-mediated signaling may play a mechanistic role.

Bioenergetic and oxidative effects of free 3-nitrotyrosine in culture: selective vulnerability of dopaminergic neurons and increased sensitivity of non-dopaminergic neurons to dopamine oxidation.

Protein bound and free 3-nitrotyrosine (3NT) levels are elevated in neurodegenerative diseases and have been used as evidence for peroxynitrite generation. Intrastriatal injection of free 3NT causes dopaminergic neuron injury and represents a new mouse model of Parkinson's disease (PD). We are investigating the nature of free 3NT neurotoxicity. In primary ventral midbrain cultures, free 3NT damaged dopaminergic neurons, while adjacent non-dopaminergic neurons were unaffected. Combined treatment with free 3NT and subtoxic amounts of dopamine caused extensive death of non-dopaminergic forebrain neurons in culture. Free 3NT alone directly inhibited mitochondrial complex I, decreased ATP, sensitized neurons to mitochondrial depolarization, and increased superoxide production. Subtoxic concentrations of rotenone (instead of free 3NT) caused similar results. Additionally, free 3NT and dopamine combined increased extraneuronal hydrogen peroxide and decreased intraneuronal glutathione levels more than dopamine alone. Oxidative and bioenergetic processes have been proposed to contribute to neurodegeneration in PD. As free 3NT is a compound that is increased in PD, damages dopamine neurons in vivo and in vitro and has detrimental effects on neuronal bioenergetics, it is possible that free 3NT is an endogenous contributing factor to neuronal loss, in addition to being a marker of oxidative and nitrative processes.

Cardiac dysfunction in the R6/2 mouse model of Huntington's disease.

Recent evidence suggests that mutant huntingtin protein-induced energetic perturbations contribute to neuronal dysfunction in Huntington's disease (HD). Given the ubiquitous expression of huntingtin, other cell types with high energetic burden may be at risk for HD-related dysfunction. Early-onset cardiovascular disease is the second leading cause of death in HD patients; a direct role for mutant huntingtin in this phenomenon remains unevaluated. Here we tested the hypothesis that expression of mutant huntingtin is sufficient to induce cardiac dysfunction, using a well-described transgenic model of HD (line R6/2). R6/2 mice developed cardiac dysfunction by 8 weeks of age, progressing to severe failure at 12 weeks, assessed by echocardiography. Limited evidence of cardiac remodeling (e.g. hypertrophy, fibrosis, apoptosis, beta(1) adrenergic receptor downregulation) was observed. Immunogold electron microscopy demonstrated significant elevations in nuclear and mitochondrial polyglutamine presence in the R6/2 myocyte. Significant alterations in mitochondrial ultrastructure were seen, consistent with metabolic stress. Increased cardiac lysine acetylation and protein nitration were observed and were each significantly associated with impairments in cardiac performance. These data demonstrate that mutant huntingtin expression has potent cardiotoxic effects; cardiac failure may be a significant complication of this important experimental model of HD. Investigation of the potential cardiotropic effects of mutant huntingtin in humans may be warranted.

Bacterial lipopolysaccharide enhances cardiac dysfunction but not retroviral replication in murine AIDS: roles of macrophage infiltration and toll-like receptor 4 expression.

Cardiovascular disease is an important complication of human immunodeficiency virus/acquired immune deficiency syndrome (AIDS), but the mechanism(s) involved are poorly understood. Although co-infecting pathogens have been implicated as an important factor in AIDS progression, no studies have investigated these interactions in cardiac tissue. We recently demonstrated that the murine AIDS model (LPBM5 retroviral infection) mimics human immunodeficiency virus-related cardiac dysfunction and pathology. We tested the hypothesis that subseptic lipopolysaccharide exposure (LPS) would enhance LPBM5 progression and exacerbate cardiovascular dysfunction during murine AIDS development. LPS (5 mg/kg, Escherichia coli 0111:B4) was administered at 1, 6, and 8 weeks during LPBM5 infection, and cardiac performance was evaluated at 10 weeks using noninvasive echocardiography. LPS alone had no significant effects, whereas it amplified abnormalities in cardiac structure and function observed in murine AIDS. Cardiac dysfunction was associated with selective increases in nonfocal infiltration of CD68(+) cells and correlated with the extent of cardiac dysfunction. Retroviral progression and cardiac retroviral content remained unaltered, but cardiac toll-like receptor 4 was increased in retrovirus + LPS. We provide first-time evidence of multipathogen enhancements to retrovirus-related cardiac complications and implicate innate immune responses, not co-pathogen-induced retroviral replication, as the primary mechanism in this setting.

Exenatide versus insulin glargine in patients with suboptimally controlled type 2 diabetes: a randomized trial.

BACKGROUND: Physicians may use either insulin or exenatide injections for patients with type 2 diabetes mellitus who have poor glycemic control despite taking oral blood glucose-lowering drugs. OBJECTIVE: To compare effects of exenatide and insulin glargine on glycemic control in patients with type 2 diabetes mellitus that is suboptimally controlled with metformin and a sulfonylurea. DESIGN: 26-week multicenter, open-label, randomized, controlled trial. SETTING: 82 outpatient study centers in 13 countries. PATIENTS: 551 patients with type 2 diabetes and inadequate glycemic control (defined as hemoglobin A1c level ranging from 7.0% to 10.0%) despite combination metformin and sulfonylurea therapy. INTERVENTION: Exenatide, 10 microg twice daily, or insulin glargine, 1 daily dose titrated to maintain fasting blood glucose levels of less than 5.6 mmol/L (<100 mg/dL). MEASUREMENTS: Hemoglobin A1c level, fasting plasma glucose level, body weight, 7-point self-monitored blood glucose, standardized test-meal challenge, safety, and tolerability. RESULTS: Baseline mean hemoglobin A1c level was 8.2% for patients receiving exenatide and 8.3% for those receiving insulin glargine. At week 26, both exenatide and insulin glargine reduced hemoglobin A1c levels by 1.11% (difference, 0.017 percentage point [95% CI, -0.123 to 0.157 percentage point]). Exenatide reduced postprandial glucose excursions more than insulin glargine, while insulin glargine reduced fasting glucose concentrations more than exenatide. Body weight decreased 2.3 kg with exenatide and increased 1.8 kg with insulin glargine (difference, -4.1 kg [CI, -4.6 to -3.5 kg]). Rates of symptomatic hypoglycemia were similar, but nocturnal hypoglycemia occurred less frequently with exenatide (0.9 event/patient-year versus 2.4 events/patient-year; difference, -1.6 events/patient-year [CI, -2.3 to -0.9 event/patient year]). Gastrointestinal symptoms were more common in the exenatide group than in the insulin glargine group, including nausea (57.1% vs. 8.6%), vomiting (17.4% vs. 3.7%) and diarrhea (8.5% vs. 3.0%). LIMITATIONS: The trial was open-label and did not assess clinical complications related to diabetes. Of the 551 participants, 19.4% of those receiving exenatide and 9.7% of those receiving insulin glargine withdrew from the study. Only 21.6% of the insulin glargine group and 8.6% of the exenatide group achieved the target level for fasting plasma glucose of less than 5.6 mmol/L (<100 mg/dL). CONCLUSIONS: Exenatide and insulin glargine achieved similar improvements in overall glycemic control in patients with type 2 diabetes that was suboptimally controlled with oral combination therapy. Exenatide was associated with weight reduction and had a higher incidence of gastrointestinal adverse effects than insulin glargine.

Activated Akt-1 in specific cell populations during multi-stage skin carcinogenesis.

The goal of the present study was to identify specific populations of cells that contain activated Akt-1, as determined by the presence ofphosphorylated Akt at serine 473 (p Akt), during development of skin tumors using a murine multi-stage carcinogenesis model. Nucleated papillomas cells as well as both epidermal and follicular keratinocytes in hyperplastic skin contained increased pAkt compared to skin treated only with acetone or 7, 12 dimethylbenz[a]anthracene (DMBA). Although the numbers of both mast cells and neutrophils were significantly increased in the stroma of papillomas (p<0.0005; p<0.0001, respectively), only mast cells contained pAkt. The amount of total Akt protein was similar regardless of time or treatment group examined. The present results suggest that activation of Akt-1 may provide specific populations of epidermal keratinocytes that develop into skin tumors with the ability to resist terminal differentiation and have enhanced proliferation during multi-stage skin carcinogenesis. In addition, mast cells which contain activated Akt-1 may persist within the stroma of papillomas during skin tumor development and progression through this signaling pathway, thereby contributing to a pro-oxidant and proangiogenic microenvironment.

HIV/AIDS-related cardiovascular disease.

Recent advances in antiretroviral therapies have enhanced survival of HIV/AIDS patients, but cardiovascular complications have emerged as important issues in this growing patient population. Although the antiviral drug therapies apparently yield cardiac and/or vascular toxicities themselves, several other factors associated with HIV pathogenesis have also been implicated. This brief review provides an overview of the significance and complexities of HIV/ AIDS-related cardiovascular complications and addresses some important mechanistic aspects that may contribute to this important clinical problem.

Cardiomyopathy in a murine model of AIDS: evidence of reactive nitrogen species and corroboration in human HIV/AIDS cardiac tissues.

OBJECTIVE: Cardiomyopathy and other vascular complications are now recognized as significant components of HIV/AIDS pathogenesis. Although the mechanisms involved in cardiomyopathy are poorly defined, a role for direct retroviral action and/or focal infiltration of activated immune cells have been postulated. Here we investigated mechanisms in retrovirus associated cardiomyopathy using a well-defined mouse model of acquired immunodeficiency. METHODS: Mice were dosed with LPBM5 retrovirus; cardiac performance was assessed by echocardiography followed by tissue collection at 5 and 10 weeks post-infection. RESULTS: Contractile deficits were observed at 5 and 10 weeks post-retrovirus infection and preceded the development of overt immunodeficiency. Selective and widespread cardiac infiltration of CD68+ cells, but not neutrophils, mast cells, or eosinophils was also observed at both 5 and 10 weeks. LPBM5 retrovirus was readily detectable in cardiac samples by RT-PCR. Time dependent increases in cardiac protein nitration (biomarker of reactive nitrogen species) were observed and were correlated to the extent of cardiac dysfunction whereas no changes in NOSII occurred at 5 and 10 weeks. We corroborated the mouse findings using cardiac tissues and clinical findings from human HIV/AIDS autopsies. CONCLUSIONS: These studies demonstrated that cardiac myocyte protein nitration in AIDS related cardiomyopathies, rather than focal immune cell lesions characterize retrovirus associated cardiomyopathies and differentiate them from non-retroviral cardiomyopathies.

Effects of peroxynitrite on isolated cardiac trabeculae: selective impact on myofibrillar energetic controllers.

Formation of peroxynitrite and cardiac protein nitration have been implicated in multiple cardiac disease states, but their contributions to disease initiation remain undefined. We have previously observed nitration of myofibrillar regions of cardiac myocytes in several experimental and clinical settings of cardiac myocyte dysfunction and postulated that oxidative insult to key components of the contractile apparatus may be initiating events. Here we tested the hypothesis that peroxynitrite alters myofibrillar contractile function, and investigated a mechanistic role for nitration in this process. Isolated rat ventricular trabeculae were exposed to physiologically relevant concentrations of peroxynitrite and ATP-dependent contractile responses were measured. Maximal trabecular force generation was significantly impaired following 300 nM peroxynitrite exposures. Several myofibrillar proteins demonstrated increased tyrosine nitration, the most significant increases occurred in the myosin heavy chain and the myofibrillar isoform of creatine kinase. Additional functional experiments were conducted using phosphocreatine (high energy phosphate substrate for myofibrillar creatine kinase) as the primary energy substrate. Myofibrillar creatine kinase-dependent force generation was impaired at peroxynitrite concentrations as low as 50 nM, suggesting potent inactivation of the enzyme. Extent of tyrosine nitration of myofibrillar creatine kinase was negatively correlated to myofibrillar creatine kinase-dependent force generation. These data demonstrate that the cardiac contractile apparatus is highly sensitive to peroxynitrite, and that MM-CK may be a uniquely vulnerable target.

Effects of angiotensin II on vascular endothelial cells: formation of receptor-mediated reactive nitrogen species.

Angiotensin II (ANG II) participates in many cardiovascular disease states, but the mechanisms involved are not completely defined. Doses of ANG II that do not affect blood pressure significantly can still cause early changes in vascular endothelial performance and cell-specific protein 3-nitrotyrosine formation (protein-3NT, marker of peroxynitrite formation) in vivo. Here, we have tested the hypothesis that ANG II induces endothelial cell peroxynitrite (ONOO-) formation in vitro, and investigated the mechanisms involved. Endothelial cells were incubated with ANG II (1nM-250 microM), and protein nitration was assessed by immunoblotting. ANG II caused concentration-dependent increases in protein-3NT above detectable basal control levels, at concentrations greater than 100nM. This response was inhibited significantly by co-incubation with losartan or diphenyleneiodonium chloride. Endothelial cell lysates incubated with nitrated protein standards demonstrated significant protein-3NT modification activity only in the presence of serum. However, endothelial cell lysates did not modify the free amino acid form of 3NT (free-3NT) in identical experimental conditions, assessed by capillary electrophoresis. Finally, free-3NT was cytotoxic to cultured endothelial cells (fitted LC(50)=98 microM). These data demonstrate that stimulation of angiotensin receptor subtype 1 by ANG II can cause increased endothelial cell protein nitration in vitro in the absence of other cell types or stimuli, at concentrations that are pathophysiologically relevant. Furthermore, endothelial cells selectively modified nitrated protein tyrosine residues only in the presence of a cofactor(s), and did not modify the free modified amino acid. Protein nitration may be a regulated endothelial signaling process, while free-3NT may be toxic to endothelial cells.

A simplified method for identification of human cardiac myosin heavy-chain isoforms.

Cardiac myosin is a central participant in the cross-bridge cycling that mediates myocyte contraction and consists of multiple subunits that mediate both hydrolysis of ATP and mechanical production of contractile force Two isoforms of myosin heavy chain (MHC- alpha and MHC- beta ) are known to exist in mammalian cardiac tissue, and it is within this myosin subunit that ATPase activity resides. These isoforms differ by less than 0.2% in total molecular mass and amino acid sequence, but, strikingly, influence the rate and efficiency of energy utilization for generation of contractile force. Changes in the MHC- alpha /MHC- beta ratio has been classically viewed as an adaptation of a failing myocyte in both animal models and humans; however, their measurement has traditionally required specialized preparations and materials for sufficient resolution. Here we describe a greatly simplified method for routine assessments of myosin isoform composition in human cardiac tissues. The primary advantages of our approach include higher throughput and reduced supply costs with no apparent loss of statistical power, reproducibility or achieved results. Use of this more convenient method may provide enhanced access to an otherwise specialized technique and could provide additional opportunity for investigation of cardiac myocyte adaptive changes.

Peroxynitrite-induced inhibition and nitration of cardiac myofibrillar creatine kinase.

Although cardiac peroxynitrite formation and attendant protein nitration is an established event in both acute and chronic settings of cardiac failure, the putative intracellular targets involved remain incompletely defined. We have recently shown that the myofibrillar isoform of creatine kinase (a critical energetic controller of cardiomyocyte contractility) may be a particularly sensitive target of peroxynitrite-induced nitration and inactivation in vivo. However, the kinetic and mechanistic aspects of this interaction remain undefined. Here we tested the hypothesis that myofibrillar creatine kinase is sensitive to inhibition by peroxynitrite, and investigated the mechanistic role for tyrosine nitration in this process. Peroxynitrite potently and irreversibly inhibited myofibrillar creatine kinase capacity (Vmax), at concentrations as low as 100 nM, while substrate affinity (Km) was unaffected. Concentration-dependent nitration of myofibrillar creatine kinase was observed. The extent of nitration was linearly related to peroxynitrite concentration and highly correlated to the extent of myofibrillar creatine kinase inhibition. This inhibition was not reversible by treatment with free cysteine (250 microM), but pre-incubation with substrate (phosphocreatine and/or ATP) provided significant protection of MM-CK from both nitration and inhibition. These results suggest that myofibrillar creatine kinase is a highly sensitive target of peroxynitrite-mediated inhibition, and that nitration may mediate this inhibition.

Intracellular distribution of peroxynitrite during doxorubicin cardiomyopathy: evidence for selective impairment of myofibrillar creatine kinase.

Cardiac peroxynitrite and protein nitration are increased during doxorubicin cardiotoxicity, but the intracellular targets and functional consequences have not been defined. We investigated the intracellular distribution of protein nitration during doxorubicin cardiotoxicity in mice. Following in vivo cardiac function assessments by echocardiography, cardiac tissues were prepared for immunohistochemistry and electron microscopy 5 days after doxorubicin (20 mg kg(-1)) or vehicle control. Increased cardiac 3-nitrotyrosine was observed using light microscopy in doxorubicin treated animals. Immunogold electron microscopy (55,000x) revealed increased myofibrillar and mitochondrial 3-nitrotyrosine levels following doxorubicin, but cellular 3-nitrotyrosine density was 2 fold higher in myofibrils. We therefore investigated the actions of peroxynitrite on intact cardiac contractile apparatus. Skinned ventricular trabeculae were exposed to physiologically relevant peroxynitrite concentrations (50 or 300 nM) for 1 h, then Ca(2+) induced contractile responses were measured in the presence of ATP (4 mM) or phosphocreatine (12 mM) as high energy phosphate supplier. ATP maximal force generation was unaltered after 50 nM peroxynitrite, but phosphocreatine/ATP response was reduced (0.99+/-0.63 vs 1.59+/-0.11), suggesting selective inactivation of myofibrillar creatine kinase (MM-CK). Reduction of ATP maximal force was observed at 300 nM peroxynitrite and phosphocreatine/ATP response was further reduced (0.64+/-0.30). Western blotting showed concentration dependent nitration of MM-CK in treated trabeculae. Similarly, cardiac tissues from doxorubicin treated mice demonstrated increased nitration and inactivation of MM-CK compared to controls. These results demonstrate that peroxynitrite-related protein nitration are mechanistic events in doxorubicin cardiomyopathy and that the cardiac myofibril is an important oxidative target in this setting. Furthermore, MM-CK may be a uniquely vulnerable target to peroxynitrite in vivo.