Exposure to air pollution and cardiovascular risk in young children - a pilot project.

OBJECTIVE: To examine relationships between traffic-related air pollution (TRAP) and markers of pre-clinical cardiovascular risk in young children. STUDY DESIGN: We studied a cohort of healthy children ages 2-5 recruited from pediatric primary care sites (n = 122). We obtained child weight, height, blood pressure and hair nicotine levels. A blood sample was obtained for biomarkers of systemic inflammation, oxidation, and prevalence of circulating endothelial progenitor cells. This manuscript represents a secondary analysis. TRAP exposure (particulate levels, nitrogen dioxide, nitrogen oxides, and proximity to major roadways) was assessed using national air pollution data based on child's census tract of residence. RESULTS: TRAP exposure had significant positive associations with prevalence of two of the three EPC subtypes (CD34 + /CD133 + /CD45- and CD133 + /CD45-) in unadjusted correlations. In a linear regression model, adjusting for sex, age, race, ethnicity, body mass index, parental education, child insurance, and secondhand smoke exposure, one EPC subtype (CD133 + /CD45-) had a positive significant correlation to every TRAP measure. No significant relationships between air pollution and measures of inflammation and oxidation was found. CONCLUSION: Our findings of the upregulation of EPCs may signal a response to early vascular damage during early childhood due to air pollution exposure. IMPACT: Traffic-related air pollution (TRAP) - known cardiovascular risk factor during adulthood Current pilot study in very young children shows upregulation of cells which protect the endothelial lining of blood vessels (endothelial progenitor cells, EPCs) Upregulation of EPCs aligns with other cardiovascular risks during childhood (obesity, prematurity, type 1 diabetes) Demonstrated with TRAP exposure lower than EPA threshold Response to air pollution may be protective of cardiovascular damage during early childhood.

Secondhand Smoke Exposure and Preclinical Markers of Cardiovascular Risk in Toddlers.

OBJECTIVE: To investigate relationships between secondhand smoke exposure in young children and several preclinical markers of cardiovascular risk that have been established as relevant to adult populations. STUDY DESIGN: There were 139 children, 2-5 years of age, enrolled in a cross-sectional study. Secondhand smoke exposure was objectively determined by hair nicotine level; a comprehensive panel of clinical markers (morning blood pressure, fasting glucose and insulin, lipid profiles, inflammation) and research markers (markers of oxidation, endothelial stress, and endothelial repair) of cardiovascular risk status were assessed. Univariate and multivariate linear regression were used to evaluate relationships between secondhand smoke exposure and cardiovascular risk markers. RESULTS: Hair nicotine levels were correlated directly with blood pressure and serum C-reactive protein, and inversely correlated with serum high-density lipoprotein cholesterol and endothelial cell progenitor cell prevalence. In multivariate analyses, these relationships remained when controlled for age, sex, body mass index z-score, maternal education, and method of payment. Additionally, in multivariate analyses, hair nicotine level was significantly negatively correlated with total antioxidant capacity. CONCLUSIONS: These results support the view that secondhand smoke exposure in the very young has a detectable relationship with several markers of cardiovascular risk, long before the emergence of clinical disease. Further studies to define mechanisms and strategies to prevent and mitigate these risks early in life are warranted.

Mice deficient in Mkp-1 develop more severe pulmonary hypertension and greater lung protein levels of arginase in response to chronic hypoxia.

The mitogen-activated protein (MAP) kinases are involved in cellular responses to many stimuli, including hypoxia. MAP kinase signaling is regulated by a family of phosphatases that include MAP kinase phosphatase-1 (MKP-1). We hypothesized that mice lacking the Mkp-1 gene would have exaggerated chronic hypoxia-induced pulmonary hypertension. Wild-type (WT) and Mkp-1(-/-) mice were exposed to either 4 wk of normoxia or hypobaric hypoxia. Following chronic hypoxia, both genotypes demonstrated elevated right ventricular pressures, right ventricular hypertrophy as demonstrated by the ratio of the right ventricle to the left ventricle plus septum weights [RV(LV + S)], and greater vascular remodeling. However, the right ventricular systolic pressures, the RV/(LV + S), and the medial wall thickness of 100- to 300-microm vessels was significantly greater in the Mkp-1(-/-) mice than in the WT mice following 4 wk of hypobaric hypoxia. Chronic hypoxic exposure caused no detectable change in eNOS protein levels in the lungs in either genotype; however, Mkp-1(-/-) mice had lower levels of eNOS protein and lower lung NO production than did WT mice. No iNOS protein was detected in the lungs by Western blotting in any condition in either genotype. Both arginase I and arginase II protein levels were greater in the lungs of hypoxic Mkp-1(-/-) mice than those in hypoxic WT mice. Lung levels of proliferating cell nuclear antigen were greater in hypoxic Mkp-1(-/-) than in hypoxic WT mice. These data are consistent with the concept that MKP-1 acts to restrain hypoxia-induced arginase expression and thereby reduces vascular remodeling and the severity of pulmonary hypertension.

Inhaled nitric oxide decreases leukocyte trafficking in the neonatal mouse lung during exposure to >95% oxygen.

Chronic lung injury in the neonate is termed bronchopulmonary dysplasia (BPD). These patients generally require supplemental oxygen therapy, and hyperoxia has been implicated in the pathogenesis of BPD. The concomitant use of oxygen and inhaled NO (iNO) may result in the generation of reactive nitrogen species or may have an anti-inflammatory effect in the neonatal lung. We tested the hypothesis that exposure to >95% O2 in neonatal mice would increase trafficking of leukocytes into the lung and that the addition of iNO to >95% O2 would decrease this leukocyte trafficking. Hyperoxia resulted in fewer alveoli, increased presence of neutrophils and macrophages, and decreased number of mast cells within the lung parenchyma. Adding iNO to hyperoxia prevented the hyperoxia-induced changes and resulted in the numbers of alveoli, neutrophils, macrophages, and mast cells approximating those found in controls (room air exposure). Intercellular adhesion molecule (ICAM) and monocyte chemotactic protein-1 (MCP-1), two factors responsible for leukocyte recruitment, were up-regulated by hyperoxic exposure, but the addition of iNO to the hyperoxic exposure prevented the hyperoxia-induced up-regulation of ICAM and MCP-1. These data demonstrate that iNO alters the hyperoxia-induced recruitment of leukocytes into the lung.

Inhaled nitric oxide prevents 3-nitrotyrosine formation in the lungs of neonatal mice exposed to >95% oxygen.

Inhaled nitric oxide is being evaluated as a preventative therapy for patients at risk for bronchopulmonary dysplasia (BPD). Nitric oxide (NO), in the presence of superoxide, forms peroxynitrite, which reacts with tyrosine residues on proteins to form 3-nitrotyrosine (3-NT). However, NO can also act as an antioxidant and was recently found to improve the oxidative balance in preterm infants. Thus, we tested the hypothesis that the addition of a therapeutically relevant concentration (10 ppm) of NO to a hyperoxic exposure would lead to decreased 3-NT formation in the lung. FVB mouse pups were exposed to either room air (21% O(2)) or >95% O(2) with or without 10 ppm NO within 24 h of birth. In the first set of studies, body weights and survival were monitored for 7 days, and exposure to >95% O(2) resulted in impaired weight gain and near 100% mortality by 7 days. However, the mortality occurred earlier in pups exposed to >95% O(2) + NO than in pups exposed to >95% O(2) alone. In a second set of studies, lungs were harvested at 72 h. Immunohistochemistry of the lungs at 72 h revealed that the addition of NO decreased alveolar, bronchial, and vascular 3-NT staining in pups exposed to both room air and hyperoxia. The lung nitrite levels were higher in animals exposed to >95% oxygen + NO than in animals exposed to >95% oxygen alone. The protein levels of myeloperoxidase, monocyte chemotactic protein-1, and intracellular adhesion molecule-1 were assessed after 72 h of exposure and found to be greatest in the lungs of pups exposed to >95% O(2). This hyperoxia-induced protein expression was significantly attenuated by the addition of 10 ppm NO. We propose that in the presence of >95% O(2), peroxynitrite formation results in protein nitration; however, adding excess NO to the >95% O(2) exposure prevents 3-NT formation by NO reacting with peroxynitrite to produce nitrite and NO(2). We speculate that the decreased protein nitration observed with the addition of NO may be a potential mechanism limiting hyperoxic lung injury.

Decreased cardiac expression of vascular endothelial growth factor and redox imbalance in murine diabetic cardiomyopathy.

Type 1 diabetes is associated with a unique form of cardiomyopathy that is present without atherosclerosis. Redox imbalance and/or changes in vascular endothelial growth factor (VEGF) expression have been associated with diabetes-related cardiomyopathy. However, the mechanisms of these changes and their interrelationships remain unclear. Using a murine type 1 diabetes model, we tested the hypothesis that alterations in cardiac performance are associated with decreased cardiac microvascular prevalence, as well as downregulation of VEGF isoforms. We also investigated oxidative stress as a contributor to regulate individual VEGF isoforms and microvascular rarefaction. Significant and rapid hyperglycemia was observed at 1 wk post-streptozotocin (STZ) and persisted throughout the 5-wk study. Left ventricular (LV) fractional shortening was reduced at week 1 and 5 post-STZ insult relative to age-matched controls. We also observed the early reduction in E/A ratio at 1 wk. Immunostaining for CD31 and digital image analysis demonstrated a 35% reduction in microvessels/myocardial area, indicative of rarefaction, which was highly correlated with fractional shortening. Furthermore, a significant increase in the prevalence of protein 3-nitrotyrosine was observed in the diabetic cardiac tissue, which was inversely associated with microvascular rarefaction. The expressions of three VEGF isoforms were significantly reduced to different extents. The reduction of VEGF(164) was associated with GSSG accumulation. These data demonstrate that the mouse model of STZ-induced diabetes has hallmark features observed in humans with respect to nonischemic systolic and diastolic performance and microvascular rarefaction, which are associated with changes in VEGF isoform expression and redox imbalance in the myocardium.

Vasoprotective endothelial effects of a standardized grape product in humans.

The pathogenesis of coronary lesion development is a multi-factorial process involving a number of different cell types and covariates, and injury and dysfunction of the vascular endothelium is an important marker and likely participant in the initiation and/or progression of most forms of heart disease. In addition to chronic dysfunction of endothelial responses in patients with established heart disease, there is evidence that 'acute insults' can cause measurable dysfunction in vascular response in humans (drug toxicities, hypoxia, high fat meal). Such repeated acute insults may contribute to disease risk in otherwise healthy individuals or promote disease progression in established patients. Consumption of grape products, especially wine, has been linked to lower cardiovascular risk but the vascular endothelial effects of grape products in healthy normal subjects, in the absence of ethanol, have not been evaluated. We therefore tested the hypotheses that 1) a standardized product derived from fresh grapes (GP, acute and chronic consumption) improves endothelial performance in healthy normal young subjects, and 2) that concomitant grape consumption affects the 'acute endothelial insult' caused by a single standardized high fat meal (HF). Acute consumption of GP equivalent to 1.25 cups of fresh grapes caused significant improvement in brachial artery flow mediated dilation (FMD) within 3 h of consumption, when compared to control consumption of sugar solution (p<0.05). No acute changes in heart rate, hemodynamics, or lipid profiles were observed. When this 'dose' was then consumed twice daily for 3 weeks FMD was further improved and total antioxidant capacity in plasma was slightly increased (p<0.05), with no change in heart rate, hemodynamics, or lipid profiles. A single HF meal (900 cal, 49 g total fat) caused a 50% reduction in FMD response when consumed alone, and this effect coincided with increased blood triglyceride levels within 3 h post-consumption. In contrast the concomitant consumption of GP with the HF meal completely prevented this HF-induced vascular endothelial dysfunction (p<0.05), but had no effect on rising triglycerides. These data demonstrate that a modest intake of fresh grapes can have acute favorable effects on vascular endothelial function in normal healthy subjects, that chronic intake can further improve performance and concomitant intake can blunt the 'acute insult' to endothelium caused by a typical western HF meal. This effect is likely to be related to antioxidant effects at the endothelium, rather than changes in blood lipids. These data support epidemiological data of the health benefits of grapes, and demonstrate that 'favorable' food consumption can apparently reduce some toxicities induced by 'unfavorable' food consumption.

Secondhand Smoke Exposure and Sleep-Related Breathing Problems in Toddlers.

BACKGROUND: Adequate sleep during childhood is an important component of overall health and wellbeing for children. Secondhand smoke (SHS) exposure has been linked to a greater risk of sleep-disordered breathing. OBJECTIVE: Our objective was to investigate relationships between SHS exposure and sleep-related breathing problems in healthy toddlers aged 2 to 5 years. We hypothesized that there is an independent relationship between objectively measured SHS exposure and presence of sleep-related breathing problems by parental report. METHODS: A convenience sample of 149 healthy children ages 2 to 5 years was recruited from an academic pediatric primary care center for this cross-sectional study; 138 had complete data that were analyzed. Current SHS exposure was determined by hair nicotine level. Presence of sleep-related breathing problems was assessed by 1 survey item. Inflammation was determined by serum C-reactive protein (CRP) level. Analysis in Stata 15 included a series of multivariate logistic regression models, controlling for individual-level demographics and body mass index z scores according to mediation analysis procedures for dichotomous outcomes. RESULTS: Approximately 24% of parents reported their child snored, gasped, or had difficulty breathing at night sometimes, most of the time, or almost always. Regression models with mediation analysis indicate that SHS exposure significantly increased the odds of reporting the child had sleep-related breathing problems, and 18% of this relationship is explained by log serum CRP levels. CONCLUSIONS: Although the cross-sectional nature of this study limits causality, evidence suggests a relationship exists between SHS exposure, as measured by log hair nicotine and sleep-related breathing problems at night.

Biochemical consequences of the NOS3 Glu298Asp variation in human endothelium: altered caveolar localization and impaired response to shear.

Human endothelial nitric oxide synthase (NOS3) gene polymorphism at Exon 7 (Glu298Asp) has been linked to vascular endothelial dysfunction, but the mechanisms are not defined. Shear is a key modulator of NOS3 function in vivo and association with caveolae is important for the control of NOS3 protein activity. Here we tested the hypothesis that altered enrichment of NOS3 in the caveolar membrane defines Glu298Asp genotype-specific responses and NOS3 activity. Basal caveolar membrane enrichment was carried out to quantitate the NOS3 enrichment in caveolae. Cells were subjected to shear and NOS3 protein levels, phosphorylation, enzyme function were investigated. Variant genotypes had lower NOx production pre- and post-shear, but no genotype-dependent alterations in pNOS3 were observed. Asp variants had significantly lower NOS3 enrichment in the caveolar membrane fraction. Further, immunoprecipitation studies demonstrated that Asp variants had substantially less NOS3/Cav-1 association (approximately 40%) during static conditions. Furthermore, acute shear causes impaired NOS3/Cav-1 dissociation in Asp variants. The results from immunoprecipitation studies were in complete agreement with caveolar membrane preparation findings. Collectively, these data demonstrate functional consequences of the Glu298Asp NOS3 variation and further define disruption of NOS3 caveolar localization and shear-induced mobilization as the primary mechanism responsible for these differences.

Cytoskeletal remodeling of desmin is a more accurate measure of cardiac dysfunction than fibrosis or myocyte hypertrophy.

AIMS: Fibrosis and myocyte hypertrophy are classical remodeling parameters in heart failure (HF); however, an intriguing possibility is that myocytes undergo intracellular remodeling which decrease compliance, contributing to diastolic dysfunction. The most obvious candidates are cytoskeletal proteins. The cytoskeletal protein desmin reinforces the sarcomeres, enabling force generation. As a contributor to sarcomere performance, desmin may represent a better appraisal of dysfunction than fibrosis or myocyte hypertrophy. MAIN METHODS: HF was induced in sheep via coronary microembolization. Echocardiography was performed at baseline, 4-, and 12-months in HF. Desmin, fibrosis, and myocyte hypertrophy from infarcted LV posterior and noninfarcted LV anterior walls were measured using Western blot, immunohistochemistry, and digital image analysis. Multivariate regression analysis was performed, providing structure/function mechanisms. *p<0.05. KEY FINDINGS: EF decreased from 55% to 24%*. LV end-diastolic area (LVEDA) increased 123%* at month-12. Fibrosis increased only in posterior LV whereas myocyte hypertrophy increased in both LV posterior and LV anterior regions but only at month-12. Desmin content progressively increased 121% at month-4 and 182%* at month-12 in both LV posterior and anterior walls. Multivariate linear regression (beta coefficient standardization) demonstrated that desmin was a much better predictor of EF (beta=-0.38*) and LVEDA (beta=0.58*) than fibrosis or myocyte hypertrophy. SIGNIFICANCE: Desmin, fibrosis, and myocyte hypertrophy are temporally and spatially heterogeneous in HF. Desmin content more accurately correlated with remodeling than fibrosis or myocyte hypertrophy, suggesting that intra-myocyte responses, likely related to mechanical stretch, are better predictors of LV function and may represent novel targets for therapeutic intervention.

Preliminary computational modeling of nitric oxide synthase 3 interactions with caveolin-1: influence of exon 7 Glu298Asp polymorphism.

The significance of endothelial nitric oxide synthase 3 (NOS3) activity has been recognized for many years, however it was only recently that the complicated regulation of this constitutively expressed enzyme in endothelial cells was identified. A critical component of the NOS3 regulatory cycle in endothelial cells is its intracellular localization to caveolae. The caveolar coordination of NOS3, more specifically its interaction with caveolin-1 (Cav-1), plays a major role in normal endothelial NOS3 activity and vascular bioavailability of nitric oxide. We have recently shown that the presence of NOS3 exon 7 Glu298Asp polymorphism caused diminished shear-dependent NOS activation, was less extensively associated with caveolae, and had a decreased degree of interaction with Cav-1. Here, we carried out preliminary investigations to identify possible mechanisms of the genotype-dependent endothelial cell responses we observed in our previous investigations. Through this approach we tested the hypothesis that computer simulations could provide insights regarding the contribution of this single nucleotide polymorphism to regulation of the NOS3 isoform. We observed that in the Glu/Asp and Asp/Asp mutant genotypes, the amount of NOS3 associated with Cav-1 was significantly lower. Additionally, we have shown, using a theoretical computational model, that mutation of an amino acid at position 298 might affect the protein-protein interactions and localization of the NOS3 protein. These alterations might also affect the protein function and explain the enhanced disease risk associated with the presence of Glu298Asp polymorphism in the NOS3 protein.

Chronic cardiac resynchronization therapy and reverse ventricular remodeling in a model of nonischemic cardiomyopathy.

While cardiac resynchronization therapy (CRT) has been shown to reduce morbidity and mortality in heart failure (HF) patients, the fundamental mechanisms for the efficacy of CRT are poorly understood. The lack of understanding of these basic mechanisms represents a significant barrier to our understanding of the pathogenesis of HF and potential recovery mechanisms. Our purpose was to determine cellular mechanisms for the observed improvement in chronic HF after CRT. We used a canine model of chronic nonischemic cardiomyopathy. After 15 months, dogs were randomized to continued RV tachypacing (untreated HF) or CRT for an additional 9 months. Six minute walk tests, echocardiograms, and electrocardiograms were done to assess the functional response to therapy. Left ventricular (LV) midmyocardial myocytes were isolated to study electrophysiology and intracellular calcium regulation. Compared to untreated HF, CRT improved HF-induced increases in LV volumes, diameters and mass (p<0.05). CRT reversed HF-induced prolongations in LV myocyte repolarization (p<0.05) and normalized HF-induced depolarization (p<0.03) of the resting membrane potential. CRT improved HF-induced reductions in calcium (p<0.05). CRT did not attenuate the HF-induced increases in LV interstitial fibrosis. Using a translational approach in a chronic HF model, CRT significantly improved LV structure; this was accompanied by improved LV myocyte electrophysiology and calcium regulation. The beneficial effects of CRT may be attributable, in part, to improved LV myocyte function.

Bench-to-bedside review: Developmental influences on the mechanisms, treatment and outcomes of cardiovascular dysfunction in neonatal versus adult sepsis.

Sepsis is a significant cause of morbidity and mortality in neonates and adults, and the mortality rate doubles in patients who develop cardiovascular dysfunction and septic shock. Sepsis is especially devastating in the neonatal population, as it is one of the leading causes of death for hospitalized infants. In the neonate, there are multiple developmental alterations in both the response to pathogens and the response to treatment that distinguish this age group from adults. Differences in innate immunity and cytokine response may predispose neonates to the harmful effects of pro-inflammatory cytokines and oxidative stress, leading to severe organ dysfunction and sequelae during infection and inflammation. Underlying differences in cardiovascular anatomy, function and response to treatment may further alter the neonate's response to pathogen exposure. Unlike adults, little is known about the cardiovascular response to sepsis in the neonate. In addition, recent research has demonstrated that the mechanisms, inflammatory response, response to treatment and outcome of neonatal sepsis vary not only from that of adults, but vary among neonates based on gestational age. The goal of the present article is to review key pathophysiologic aspects of sepsis-related cardiovascular dysfunction, with an emphasis on defining known differences between adult and neonatal populations. Investigations of these relationships may ultimately lead to 'neonate-specific' therapeutic strategies for this devastating and costly medical 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.

Secondhand smoke exposure and endothelial stress in children and adolescents.

OBJECTIVE: Links between secondhand smoke exposure and cardiovascular disease in adults are well established. Little is known about the impact of this exposure on cardiovascular status during childhood. The purpose of this study was to investigate relationships between secondhand smoke exposure in children and adolescents and cardiovascular disease risk--systemic inflammation, endothelial stress, and endothelial repair. METHODS: A total of 145 subjects, aged 9 to 18 years, were studied. Tobacco smoke exposure was determined by hair nicotine level. Cardiovascular risk was assessed by markers of systemic inflammation (C-reactive protein [CRP] and adiponectin); by soluble intercellular adhesion molecule 1 (s-ICAM1), which measures endothelial activation after surface vascular injury; and by endothelial repair. This was measured by prevalence of endothelial progenitor cells (EPCs), which are bone marrow-derived cells that home preferentially to sites of vascular damage. RESULTS: Hair nicotine was directly correlated with s-ICAM1 (r = 0.4090, P < .0001) and negatively correlated with EPC prevalence (r = -0.2002, P = .0195). There was no relationship between hair nicotine and CRP, and a trend toward a weak relationship with adiponectin. Hair nicotine and body mass index were independent variables in a multivariate model predicting s-ICAM1; hair nicotine was the only significant variable in a model predicting EPC prevalence. CONCLUSIONS: Secondhand smoke exposure during childhood and adolescence is detrimental to vascular health because s-ICAM1 is a marker for endothelial activation and stress after vascular surface injury, and EPCs contribute to vascular repair. The fact that body mass index is also a factor in the model predicting s-ICAM1 is concerning, in that 2 risk factors may both contribute to endothelial stress.

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.

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.

Oxidant driven signaling pathways during diabetes: role of Rac1 and modulation of protein kinase activity in mouse urinary bladder.

BACKGROUND: Urinary bladder dysfunction is a complication in diabetes but the mechanisms involved are undefined. Here, we investigated roles of oxidative stress and oxidant driven signaling pathways in a murine model of diabetes, with an emphasis on urothelial vs. smooth muscle regional changes. METHODS: Mice were dosed with streptozotocin (150 mg/kg) or vehicle and studied at 5 weeks. Functional changes were assessed by in vitro cystometry. Immunohistochemical methods and automated digital imaging was used for morphometric and histochemical analysis of bladder tissue regions. RESULTS: We detected significant increases in protein 3-nitrotyrosine in both urothelium and smooth muscle regions during diabetes, demonstrating an increased prevalence of reactive nitrogen species. In light of nitric oxide synthase (NOS) isoforms as potential contributors to increased protein nitration, all three NOS isoforms were studied; region specific increases in NOS1 (urothelium and smooth muscle), NOS2 (urothelium only) but no alterations in NOS3 isoform were detected during diabetes. In contrast, p21-Rac1 (coordinating protein of NADPH oxidase) was significantly increased only in smooth muscle (diabetic vs. controls). We also investigated phosphorylation of ERK, JNK, p38 and Akt using immunohistochemical techniques; each of these was increased during diabetes but with different distributions in the two major regions of bladder tissues viz the smooth muscle and urothelium. CONCLUSIONS: The STZ mouse model of diabetes exhibits bladder dysfunction and structural changes similar to human. Reactive nitrogen species formation occurs in this setting and region specific assessments also revealed that urothelial changes and smooth muscle changes are discrete with respect to mechanisms of reactive nitrogen species (increased production of NO vs. superoxide anion) and activation of oxidant related stress signaling pathways.

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.

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.