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.

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.

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.

Increased myocardial prevalence of C-reactive protein in human coronary heart disease: direct effects on microvessel density and endothelial cell survival.

BACKGROUND: Elevated plasma C-reactive protein (CRP) is a biomarker of cardiovascular diseases (CVDs), but its potential roles as a participant of the disease process are not well defined. Although early endothelial cell injury and dysfunction are recognized events in CVD, the initiating events are not well established. Here we investigated the local myocardial CRP levels and cardiac microvessel densities in control and CVD tissue samples. Using in vitro methodologies, we investigated the direct effects of CRP on human endothelial cells. METHODS: Cardiac specimens were collected at autopsy within 4 h of death and were classified as normal controls or documented evidence of CVD. The regional prevalence of CRP and the cardiac microvessels (<40 µm) were investigated using immunohistochemistry. For in vitro experiments, human umbilical vein endothelial cells were incubated with CRP. Intracellular oxidant levels were assessed using 2',7'-dichlorofluorescein diacetate fluorescence microscopy, and cell survival was concurrently determined. Effects of chemical antioxidants on endothelial cell survival were also tested. RESULTS: Myocardial CRP levels were elevated in CVD specimens. This was associated with reduced cardiac microvessels, and this rarefaction was inversely correlated to adjacent myocardial CRP prevalence. CRP caused concentration-dependent increases in oxidant production and cell apoptosis. CONCLUSIONS: These findings provide evidence supporting myocardial CRP as a locally produced inflammatory marker and as a potential participant in endothelial toxicity and microvascular rarefaction.

Mitogen-activated protein kinase phosphatase-1 inhibits myocardial TNF-α expression and improves cardiac function during endotoxemia.

AIMS: Myocardial tumour necrosis factor-α (TNF-α) expression induces cardiac dysfunction in endotoxemia. The aim of this study was to investigate the role of mitogen-activated protein kinase phosphatase-1 (MKP1) pathway in myocardial TNF-α expression and cardiac function during endotoxemia. METHODS AND RESULTS: Lipopolysaccharide (LPS) increased MKP1 expression in the myocardium in vivo and in cultured neonatal cardiomyocytes in vitro. LPS-induced extracellular signal-regulated kinase (ERK) 1/2 and p38 phosphorylation in the myocardium was prolonged in MKP1(-/-) mice. Myocardial TNF-α mRNA and protein levels were enhanced in MKP1(-/-) compared with wild-type (WT) mice in endotoxemia, leading to a further decrease in cardiac function. To study if Rac1/p21-activated kinase 1 (PAK1) signalling regulates MKP1 expression, cardiomyocytes were treated with LPS. Inhibition of Rac1 and PAK1 by a dominant negative Rac1 adenovirus (Ad-Rac1N17) and PAK1 siRNA, respectively, blocked LPS-induced MKP1 expression in cardiomyocytes. PAK1 siRNA also decreased p38 and c-Jun N-terminal kinase (JNK) activation, and TNF-α expression induced by LPS. Furthermore, deficiency in either Rac1 or JNK1 decreased myocardial MKP1 expression in endotoxemic mice. CONCLUSION: LPS activates the Rac1/PAK1 pathway, which increases myocardial MKP1 expression via JNK1. MKP1 attenuates ERK1/2 and p38 activation, inhibits myocardial TNF-α expression, and improves cardiac function in endotoxemia. Thus, MKP1 represents an important negative feedback mechanism limiting pro-inflammatory response in the heart during sepsis.

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.

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.

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.

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.

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.

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.

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.

Maturational changes in the regulation of pulmonary vascular tone by nitric oxide in neonatal rats.

Nitric oxide (NO) is an important regulator of vasomotor tone in the pulmonary circulation. We tested the hypothesis that the role NO plays in regulating vascular tone changes during early postnatal development. Isolated, perfused lungs from 7- and 14-day-old Sprague-Dawley rats were studied. Baseline total pulmonary vascular resistance (PVR) was not different between age groups. The addition of KCl to the perfusate caused a concentration-dependent increase in PVR that did not differ between age groups. However, the nitric oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine augmented the K(+)-induced increase in PVR in both groups, and the effect was greater in lungs from 14-day-old rats vs. 7-day-old rats. Lung levels of total endothelial, inducible, and neuronal NOS proteins were not different between groups; however, the production rate of exhaled NO was greater in lungs from 14-day-old rats compared with those of 7-day-old rats. Vasodilation to 0.1 microM of the NO donor spermine NONOate was greater in 14-day lungs than in 7-day lungs, and lung levels of both soluble guanylyl cyclase and cGMP were greater at 14 days than at 7 days. Vasodilation to 100 microM of the cGMP analog 8-(4-chlorophenylthio)guanosine-3',5'-cyclic monophosphate was greater in 7-day lungs than in 14-day lungs. Our results demonstrate that the pulmonary vascular bed depends more on NO production to modulate vascular tone at 14 days than at 7 days of age. The observed differences in NO sensitivity may be due to maturational increases in soluble guanylyl cyclase protein levels.

Atrial glutathione content, calcium current, and contractility.

Atrial fibrillation (AF) is characterized by decreased L-type calcium current (I(Ca,L)) in atrial myocytes and decreased atrial contractility. Oxidant stress and redox modulation of calcium channels are implicated in these pathologic changes. We evaluated the relationship between glutathione content (the primary cellular reducing moiety) and I(Ca,L) in atrial specimens from AF patients undergoing cardiac surgery. Left atrial glutathione content was significantly lower in patients with either paroxysmal or persistent AF relative to control patients with no history of AF. Incubation of atrial myocytes from AF patients (but not controls) with the glutathione precursor N-acetylcysteine caused a marked increase in I(Ca,L). To test the hypothesis that glutathione levels were mechanistically linked with the reduction in I(Ca,L), dogs were treated for 48 h with buthionine sulfoximine, an inhibitor of glutathione synthesis. Buthionine sulfoximine treatment resulted in a 24% reduction in canine atrial glutathione content, a reduction in atrial contractility, and an attenuation of I(Ca,L) in the canine atrial myocytes. Incubation of these myocytes with exogenous glutathione also restored I(Ca,L) to normal or greater than normal levels. To probe the mechanism linking decreased glutathione levels to down-regulation of I(Ca), the biotin switch technique was used to evaluate S-nitrosylation of calcium channels. S-Nitrosylation was apparent in left atrial tissues from AF patients; the extent of S-nitrosylation was inversely related to tissue glutathione content. S-Nitrosylation was also detectable in HEK cells expressing recombinant human cardiac calcium channel subunits following exposure to nitrosoglutathione. S-Nitrosylation may contribute to the glutathione-sensitive attenuation of I(Ca,L) observed in AF.

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.