Vitamin D3 Supplementation Alleviates Left Ventricular Dysfunction in a Mouse Model of Diet-Induced Type 2 Diabetes: Potential Involvement of Cardiac Lipotoxicity Modulation.

PURPOSE: To evaluate the effectiveness of vitamin D3 supplementation, in secondary prevention, on cardiac remodeling and function, as well as lipid profile, in a mouse model of diet-induced type 2 diabetes. METHODS: Mice were fed a high fat and sucrose diet for 10 weeks. Afterward, diet was maintained for 15 more weeks and two groups were formed, with and without cholecalciferol supplementation. A control group was fed with normal chow. Glucose homeostasis and cardiac function were assessed at baseline and at the 10th and 24th weeks. Animals were killed at the 10th and 25th weeks for plasma and cardiac sample analysis. Cardiac lipid profile was characterized by LC-MS/MS. RESULTS: After 10 weeks of diet, mice exhibited pre-diabetes, mild left ventricle hypertrophy, and impaired longitudinal strain, but preserved myocardial circumferential as well as global diastolic and systolic cardiac function. After 15 more weeks of diet, animals presented with well-established type 2 diabetes, pathological cardiac hypertrophy, and impaired regional myocardial function. Cholecalciferol supplementation had no effect on glucose homeostasis but improved cardiac remodeling and regional myocardial function. After 25 weeks, non-supplemented mice exhibited increased myocardial levels of ceramides and diacylglycerol, both of which were normalized by vitamin D3 supplementation. CONCLUSION: This work brought to light the beneficial effects of cholecalciferol supplementation, in secondary prevention, on cardiac remodeling and function in a mouse model of diet-induced type 2 diabetes. Those cardioprotective effects may be, at least in part, attributed to the modulation of myocardial levels of lipotoxic species by vitamin D.

Increased protein S-nitrosylation in mitochondria: a key mechanism of exercise-induced cardioprotection.

Endothelial nitric oxide synthase (eNOS) activation in the heart plays a key role in exercise-induced cardioprotection during ischemia-reperfusion, but the underlying mechanisms remain unknown. We hypothesized that the cardioprotective effect of exercise training could be explained by the re-localization of eNOS-dependent nitric oxide (NO)/S-nitrosylation signaling to mitochondria. By comparing exercised (5 days/week for 5 weeks) and sedentary Wistar rats, we found that exercise training increased eNOS level and activation by phosphorylation (at serine 1177) in mitochondria, but not in the cytosolic subfraction of cardiomyocytes. Using confocal microscopy, we confirmed that NO production in mitochondria was increased in response to H2O2 exposure in cardiomyocytes from exercised but not sedentary rats. Moreover, by S-nitrosoproteomic analysis, we identified several key S-nitrosylated proteins involved in mitochondrial function and cardioprotection. In agreement, we also observed that the increase in Ca2+ retention capacity by mitochondria isolated from the heart of exercised rats was abolished by exposure to the NOS inhibitor L-NAME or to the reducing agent ascorbate, known to denitrosylate proteins. Pre-incubation with ascorbate or L-NAME also increased mitochondrial reactive oxygen species production in cardiomyocytes from exercised but not from sedentary animals. We confirmed these results using isolated hearts perfused with L-NAME before ischemia-reperfusion. Altogether, these results strongly support the hypothesis that exercise training increases eNOS/NO/S-nitrosylation signaling in mitochondria, which might represent a key mechanism of exercise-induced cardioprotection.

Effect of vitamin D supplementation on microvascular reactivity in obese adolescents: A randomized controlled trial.

BACKGROUND AND AIM: Childhood obesity is associated with vitamin D (VD) deficiency and vascular dysfunction. Considering evidence indicates that VD may improve vascular function, this study, for the first time, assessed the effect of VD supplementation on microvascular reactivity in obese adolescents (OA). METHODS AND RESULTS: This randomized controlled trial included 26 OA, receiving fruit juice with (n = 13) or without VD (4000 IU/d; n = 13) over a 3-month lifestyle program, as well as 23 normal-weight adolescents (controls). The primary outcome was the pre-to-post-program change in microvascular reactivity determined by laser speckle contrast imaging with acetylcholine and sodium nitroprusside iontophoresis. Changes in 25 hydroxyvitamin D (25(OH)D), flow-mediated dilation (FMD), nitrate-mediated dilation (NMD), insulin resistance (HOMA-IR) and inflammatory markers (C-reactive protein [CRP]) were monitored. At inclusion, in comparison to controls, OA exhibited lower total and free 25(OH)D, impaired microvascular responses, and impaired FMD, but similar NMD. After the lifestyle program, total and free 25(OH)D increased in all OA, with a greater increase in those receiving VD supplements. HOMA-IR and CRP decreased in all OA. Neither FMD nor NMD were altered in either group. Endothelium-dependent microvascular reactivity only increased in the VD-supplemented group, reaching values comparable to that of controls. Similar results were found when analyzing only OA with a VD deficiency at baseline. CONCLUSION: VD supplementation during a lifestyle program attenuated microvascular dysfunction in OA without altering macrovascular function. REGISTRATION NUMBER FOR CLINICAL TRIAL: NCT02400151.

Vascular endothelial function masks increased sympathetic vasopressor activity in rats with metabolic syndrome.

Sympathetic hyperactivation, a common feature of obesity and metabolic syndrome, is a key trigger of hypertension. However, some obese subjects with autonomic imbalance present a dissociation between sympathetic activity-mediated vasoconstriction and increased blood pressure. Here, we aimed to determine in a rat model of metabolic syndrome whether the endothelium endothelial nitric oxide (NO) synthase (eNOS)-NO pathway contributes to counteract the vasopressor effect of the sympathetic system. Rats were fed a high-fat and high-sucrose (HFS) diet for 15 wk. Sympathovagal balance was evaluated by spectral analysis of heart rate variability and plasmatic catecholamine measurements. Blood pressure was measured in the presence or absence of N-nitro-l-arginine methyl ester (l-NAME) to inhibit the contribution of eNOS. Vascular reactivity was assessed on isolated aortic rings in response to α1-adrenergic agonist. The HFS diet increased sympathetic tone, which is characterized by a higher low on the high-frequency spectral power ratio and a higher plasmatic concentration of epinephrine. Despite this, no change in blood pressure was observed. Interestingly, HFS rats exhibited vascular hyporeactivity (-23.6%) to α1-adrenergic receptor stimulation that was abolished by endothelial removal or eNOS inhibition (l-NAME). In addition, eNOS phosphorylation (Ser1177) was increased in response to phenylephrine in HFS rats only. Accordingly, eNOS inhibition in vivo revealed higher blood pressure in HFS rats compared with control rats (147 vs. 126 mmHg for mean blood pressure, respectively). Restrain of adrenergic vasopressor action by endothelium eNOS is increased in HFS rats and contributes to maintained blood pressure in the physiological range. NEW & NOTEWORTHY Despite the fact that prohypertensive sympathetic nervous system activity is markedly increased in rats with early metabolic syndrome, they present with normal blood pressure. These observations appear to be explained by increased endothelial nitric oxide synthase response to adrenergic stimulation, which results in vascular hyporeactivity to α-adrenergic stimulation, and therefore blood pressure is preserved in the physiological range. Listen to this article's corresponding podcast at http://www.physiology.org/doi/10.1152/ajpheart.00217.2017 .

Exercise does not activate the ß3 adrenergic receptor-eNOS pathway, but reduces inducible NOS expression to protect the heart of obese diabetic mice.

Obesity and diabetes are associated with higher cardiac vulnerability to ischemia-reperfusion (IR). The cardioprotective effect of regular exercise has been attributed to ß3-adrenergic receptor (ß3AR) stimulation and increased endothelial nitric oxide synthase (eNOS) activation. Here, we evaluated the role of the ß3AR-eNOS pathway and NOS isoforms in exercise-induced cardioprotection of C57Bl6 mice fed with high fat and sucrose diet (HFS) for 12 weeks and subjected or not to exercise training during the last 4 weeks (HFS-Ex). HFS animals were more sensitive to in vivo and ex vivo IR injuries than control (normal diet) and HFS-Ex mice. Cardioprotection in HFS-Ex mice was not associated with increased myocardial eNOS activation and NO metabolites storage, possibly due to the ß3AR-eNOS pathway functional loss in their heart. Indeed, a selective ß3AR agonist (BRL37344) increased eNOS activation and had a protective effect against IR in control, but not in HFS hearts. Moreover, iNOS expression, nitro-oxidative stress (protein s-nitrosylation and nitrotyrosination) and ROS production during early reperfusion were increased in HFS, but not in control mice. Exercise normalized iNOS level and reduced protein s-nitrosylation, nitrotyrosination and ROS production in HFS-Ex hearts during early reperfusion. The iNOS inhibitor 1400 W reduced in vivo infarct size in HFS mice to control levels, supporting the potential role of iNOS normalization in the cardioprotective effects of exercise training in HFS-Ex mice. Although the ß3AR-eNOS pathway is defective in the heart of HFS mice, regular exercise can protect their heart against IR by reducing iNOS expression and nitro-oxidative stress.

Hyperglycemia triggers RyR2-dependent alterations of mitochondrial calcium homeostasis in response to cardiac ischemia-reperfusion: Key role of DRP1 activation.

Hyperglycemia increases the heart sensitivity to ischemia-reperfusion (IR), but the underlying cellular mechanisms remain unclear. Mitochondrial dynamics (the processes that govern mitochondrial morphology and their interactions with other organelles, such as the reticulum), has emerged as a key factor in the heart vulnerability to IR. However, it is unknown whether mitochondrial dynamics contributes to hyperglycemia deleterious effect during IR. We hypothesized that (i) the higher heart vulnerability to IR in hyperglycemic conditions could be explained by hyperglycemia effect on the complex interplay between mitochondrial dynamics, Ca2+ homeostasis, and reactive oxygen species (ROS) production; and (ii) the activation of DRP1, a key regulator of mitochondrial dynamics, could play a central role. Using transmission electron microscopy and proteomic analysis, we showed that the interactions between sarcoplasmic reticulum and mitochondria and mitochondrial fission were increased during IR in isolated rat hearts perfused with a hyperglycemic buffer compared with hearts perfused with a normoglycemic buffer. In isolated mitochondria and cardiomyocytes, hyperglycemia increased mitochondrial ROS production and Ca2+ uptake. This was associated with higher RyR2 instability. These results could contribute to explain the early mPTP activation in mitochondria from isolated hearts perfused with a hyperglycemic buffer and in hearts from streptozotocin-treated rats (to increase the blood glucose). DRP1 inhibition by Mdivi-1 during the hyperglycemic phase and before IR induction, normalized Ca2+ homeostasis, ROS production, mPTP activation, and reduced the heart sensitivity to IR in streptozotocin-treated rats. In conclusion, hyperglycemia-dependent DRP1 activation results in higher reticulum-mitochondria calcium exchange that contribute to the higher heart vulnerability to IR.

Protective Role of Chronic Exercise Training in Modulating the Impact of Hyperglycemia on Vascular Sensitivity to Ischemia-Reperfusion.

Hyperglycemia (HG) is associated with increased mortality and morbidity in acute ischemic events. Regardless of the tissue or organs involved, the vascular endothelium is a key target of ischemia-reperfusion (I/R) injury severity. Among endothelium-protective strategies, exercise has been widely described as useful. However, whether this strategy is able to impact the deleterious effect of HG on endothelial function during I/R has never been challenged. For this, 48 male Wistar rats were randomized into 4 groups: sedentary (Sed) or exercised (Ex, 45 min/day, 5 days/week for 5 weeks) rats, treated (hyperglycemic, HG) or not (normoglycemic, NG) with streptozotocin (40 mg/kg, 48 h before procedure). Vascular I/R (120/15 min) was performed by clamping the femoral artery. Arterial and downstream muscular perfusions were assessed using laser speckle contrast imaging. Vascular endothelial function was assessed in vivo 15 min after reperfusion. HG was responsible for impairment of reperfusion blood flow as well as endothelial function. Interestingly exercise was able to prevent those impairments in the HG group. In agreement with the previous results, HG increased reactive oxygen species production and decreased nitric oxide bioavailability whereas exercise training normalized these parameters. It, therefore, appears that exercise may be an effective prevention strategy against the exacerbation of vascular and muscular damage by hyperglycemia during I/R.

Natural Extracts Mitigate the Deleterious Effects of Prolonged Intense Physical Exercise on the Cardiovascular and Muscular Systems.

Muscle fatigue is a common symptom induced by exercise. A reversible loss of muscle force is observed with variable rates of recovery depending on the causes or underlying mechanisms. It can not only affect locomotion muscles, but can also affect the heart, in particular after intense prolonged exercise such as marathons and ultra-triathlons. The goal of our study was to explore the effect of four different natural extracts with recognized antioxidant properties on the contractile function of skeletal (locomotion) and cardiac muscles after a prolonged exhausting exercise. Male Wistar rats performed a bout of exhausting exercise on a treadmill for about 2.5 h and were compared to sedentary animals. Some rats received oral treatment of a natural extract (rosemary, buckwheat, Powergrape®, or rapeseed) or the placebo 24 h and 1 h before exercise. Experiments were performed 30 min after the race and after 7 days of recovery. All natural extracts had protective effects both in cardiac and skeletal muscles. The extent of protection was different depending on muscle type and the duration post-exercise (just after and after one-week recovery), including antiarrhythmic effect and anti-diastolic dysfunction for the heart, and faster recovery of contractility for the skeletal muscles. Moreover, the muscular protective effect varied between natural extracts. Our study shows that an acute antioxidant supplementation can protect against acute abnormal endogenous ROS toxicity, induced here by prolonged exhausting exercise.

Vitamin D Supplementation on Carotid Remodeling and Stiffness in Obese Adolescents.

Obesity is associated with vitamin D (VD) deficiency and arterial stiffness. This randomized control trial assessed the effects of VD supplementation during a weight-loss program on carotid intima-media thickness (IMT) and carotid compliance in obese adolescents. Participants were randomly assigned to receive either a 12-week lifestyle program with VD supplementation (n = 13), a lifestyle program without VD supplementation (n = 13) or a control group composed of normal-weight adolescents (n = 18). Serum total and free 25-hydroxyvitamin D (25(OH)D), IMT and carotid compliance were measured before and after the trial. Insufficiency in 25(OH)D concentration was found in 73% of obese participants compared to 22% among controls. Obese adolescents had lower free 25(OH)D and displayed higher IMT but lower carotid compliance than controls. Free 25(OH)D and IMT were negatively correlated in adolescents displaying VD insufficiency at baseline. After three months, total and free 25(OH)D increased in both groups. The changes of IMT and carotid compliance were similar between groups. The changes in IMT were correlated with the changes in total 25(OH)D in obese adolescents with VD insufficiency at baseline (r = -0.59, p = 0.03). While the lifestyle program with VD supplementation did not affect carotid compliance, IMT reduction was improved in obese adolescents.

Stress-induced protein S-glutathionylation and phosphorylation crosstalk in cardiac sarcomeric proteins - Impact on heart function.

BACKGROUND: The interplay between oxidative stress and other signaling pathways in the contractile machinery regulation during cardiac stress and its consequences on cardiac function remains poorly understood. We evaluated the effect of the crosstalk between ß-adrenergic and redox signaling on post-translational modifications of sarcomeric regulatory proteins, Myosin Binding Protein-C (MyBP-C) and Troponin I (TnI). METHODS AND RESULTS: We mimicked in vitro high level of physiological cardiac stress by forcing rat hearts to produce high levels of oxidized glutathione. This led to MyBP-C S-glutathionylation associated with lower protein kinase A (PKA) dependent phosphorylations of MyBP-C and TnI, increased myofilament Ca2+ sensitivity, and decreased systolic and diastolic properties of the isolated perfused heart. Moderate physiological cardiac stress achieved in vivo with a single 35 min exercise (Low stress induced by exercise, LSE) increased TnI and cMyBP-C phosphorylations and improved cardiac function in vivo (echocardiography) and ex-vivo (isolated perfused heart). High stress induced by exercise (HSE) altered strongly oxidative stress markers and phosphorylations were unchanged despite increased PKA activity. HSE led to in vivo intrinsic cardiac dysfunction associated with myofilament Ca2+ sensitivity defects. To limit protein S-glutathionylation after HSE, we treated rats with N-acetylcysteine (NAC). NAC restored the ability of PKA to modulate myofilament Ca2+ sensitivity and prevented cardiac dysfunction observed in HSE animals. CONCLUSION: Under cardiac stress, adrenergic and oxidative signaling pathways work in concert to alter myofilament properties and are key regulators of cardiac function.

Endothelial function does not improve with high-intensity continuous exercise training in SHR: implications of eNOS uncoupling.

Exercise training is a well-recognized way to improve vascular endothelial function by increasing nitric oxide (NO) bioavailability. However, in hypertensive subjects, unlike low- and moderate-intensity exercise training, the beneficial effects of continuous high-intensity exercise on endothelial function are not clear, and the underlying mechanisms remain unknown. The aim of this study was to investigate the impact of high-intensity exercise on vascular function, especially on the NO pathway, in spontaneous hypertensive rats (SHR). These effects were studied on WKY, sedentary SHR and SHR that exercised at moderate (SHR-MOD) and high intensity (SHR-HI) on a treadmill (1 h per day; 5 days per week for 6 weeks at 55% and 80% of their maximal aerobic velocity, respectively). Endothelial function and specific NO contributions to acetylcholine-mediated relaxation were evaluated by measuring the aortic ring isometric forces. Endothelial nitric oxide synthase (eNOS) expression and phosphorylation (ser1177) were evaluated by western blotting. The total aortic and eNOS-dependent reactive oxygen species (ROS) production was assessed using electron paramagnetic resonance in aortic tissue. Although the aortas of SHR-HI had increased eNOS levels without alteration of eNOS phosphorylation, high-intensity exercise had no beneficial effect on endothelium-dependent vasorelaxation, unlike moderate exercise. This result was associated with increased eNOS-dependent ROS production in the aortas of SHR-HI. Notably, the use of the recoupling agent BH4 or a thiol-reducing agent blunted eNOS-dependent ROS production in the aortas of SHR-HI. In conclusion, the lack of a positive effect of high-intensity exercise on endothelial function in SHR was mainly explained by redox-dependent eNOS uncoupling, resulting in a switch from NO to O2(-) generation.

Effects of a lifestyle program on vascular reactivity in macro- and microcirculation in severely obese adolescents.

CONTEXT AND OBJECTIVE: This study aimed to comprehensively assess the macro- and microcirculation of severely obese adolescents (SOA) and normal-weight counterparts and to determine the longitudinal effects of weight loss on vascular function in SOA. DESIGN, SETTING, PARTICIPANTS, AND OUTCOME MEASURES: Seventeen SOA (body mass index z-score = 4.22 ± 0.73) and 19 puberty-matched normal-weight counterparts (body mass index z-score = -0.02 ± 1.04) were included. The SOA participated in a 4 month weight loss program. Brachial artery flow-mediated dilation and response to sublingual nitrate (nitrate-mediated dilation [NMD]) were assessed by high-resolution ultrasound. Microvascular reactivity was evaluated by laser Doppler flowmetry in response to NMD, iontophoresis of acetylcholine and sodium nitroprusside, and local hyperthermia. Plasma insulin, leptin, resistin, C-reactive protein, myeloperoxidase, and tissue plasminogen activator were measured. RESULTS: At baseline, SOA had similar flow-mediated dilation and impaired NMD in the brachial artery compared to normal-weight adolescents. Similarly, peak responses to acetylcholine and sodium nitroprusside iontophoresis and to local hyperthermia were unaltered, whereas cutaneous blood flow after NMD was lower in the forearm microcirculation of SOA. All plasma measurements were significantly higher in SOA. After the 4-month program, SOA presented a weight reduction of 7.4 ± 3.1%, but neither brachial artery nor microvascular reactivity variables were improved. Significant decreases were detected in plasma leptin, myeloperoxidase, and tissue plasminogen activator. CONCLUSIONS: Macro- and microvascular endothelial function are preserved in adolescents with severe obesity. Conversely, weight loss does not improve their impaired smooth muscle response to exogenous organic nitrate in both vascular beds, despite reducing plasma markers adversely related to vascular homeostasis.

Impact of diet and exercise training-induced weight loss on myocardial mechanics in severely obese adolescents.

OBJECTIVE: Recent findings indicated silent incipient myocardial dysfunction in juvenile obesity despite normal global cardiac function. The present study investigated whether lifestyle intervention is able to favorably impact these obesity-related myocardial abnormalities and whether improvements are related to changes in insulin resistance and cardiac remodeling. DESIGN AND METHODS: Twenty-eight severe obese adolescents (OB) participated in a 9 month lifestyle intervention program (LIP) based on aerobic exercise and diet. Twenty healthy adolescents (CG) served as controls. Conventional echocardiography and myocardial mechanics were obtained at baseline and follow-up along with insulin resistance. RESULTS: Insulin sensitivity improved (P < 0.001) and body weight decreased (P < 0.001) consecutive to LIP. At baseline, OB had depressed longitudinal (L) strain (CG: -18.3 ± 2.6, OB: -14.2 ± 3.6%, P < 0.001) and enhanced twist compared to controls. The LIP in OB restored L strain to normal values (-16.9 ± 3.5%, NS), whereas it did not affect twist mechanics. From stepwise multiple regression analysis, only baseline L strain and changes in BMI Z-score (r(2) -adjusted = 0.49, P < 0.001) emerged as independent predictors of L strain changes. CONCLUSIONS: Juvenile obesity is associated with myocardial mechanic abnormalities that can be partly corrected by lifestyle intervention. Restoration of longitudinal myocardial function occurs in the absence of left ventricular remodeling changes and is not associated with insulin resistance improvements.

Two-dimensional strain and twist by vector velocity imaging in adolescents with severe obesity.

The prevalence of severe obesity is increasing worldwide in adolescents. Whether it is associated with functional myocardial abnormalities remains largely unknown, potentially because of its frequent association with other cardiovascular risk factors and also use of insensitive techniques to detect subclinical changes in myocardial function. We used 2D vector velocity imaging (VVI) to investigate early changes in left ventricular (LV) myocardial function in youths with isolated severe obesity. Thirty-seven asymptomatic severely obese adolescents free of diabetes and hypertension, and 24 lean controls were enrolled. LV longitudinal, basal, and apical circumferential strain, strain rate (SR), rotations, and LV twist were measured. Obese adolescents had greater LV mass and reduced systolic and early diastolic tissue Doppler imaging (TDI) velocities than lean counterparts. L strain (-24%) and systolic and early diastolic SR were also diminished in the obese, whereas no intergroup differences existed for the circumferential deformation indexes. LV twist was more pronounced in the obese (+1.7°, P < 0.01) on account of greater apical rotation only (4.1 ± 0.9 vs. 5.2 ± 1.2°, P < 0.01), potentially compensating for the loss in longitudinal function. Systolic-diastolic coupling, an important component of early filling and diastolic function, was maintained with severe obesity. No intergroup differences were reported regarding time to peak values for all VVI indexes highlighting that dynamics of strain and twist/untwist along the cardiac cycle was preserved with severe obesity. Isolated severe obesity in adolescents, at a preclinical stage, is associated with changes in myocardial deformation and torsional mechanics that could be in part related to alterations in relaxation and contractility properties of subendocardial fibers.

ß-Adrenergic receptors desensitization is not involved in exercise-induced cardiac fatigue: NADPH oxidase-induced oxidative stress as a new trigger.

Prolonged strenuous exercise (PSE) induces transient left ventricular (LV) dysfunction. Previous studies suggest that ß-adrenergic pathway desensitization could be involved in this phenomenon, but it remains to be confirmed. Moreover, other underlying mechanisms involving oxidative stress have been recently proposed. The present study aimed to evaluate the involvement of both the ß-adrenergic pathway and NADPH oxidase (Nox) enzyme-induced oxidative stress in myocardial dysfunction in rats following PSE. Rats were divided into 4 groups: controls (Ctrl), 4-h exercised on treadmill (PSE), and 2 groups in which Nox enzyme was inhibited with apocynin treatment (Ctrl APO and PSE APO, respectively). We evaluated cardiac function in vivo and ex vivo during basal conditions and isoproterenol stress. GSH/GSSG ratio, cardiac troponin I (cTnI) release, and lipid peroxidation (MDA) were evaluated. PSE induced a decrease in LV developed pressure, intrinsic myocardial contractility, and relaxation associated with an increase in plasma cTnI release. Our in vivo and ex vivo results demonstrated no differences in myocardial response to isoproterenol and of effective dose 50 between control and PSE rats. Interestingly, the LV dysfunction was reversed by apocynin treatment. Moreover, apocynin prevented cellular oxidation [GSH/GSSG ratio: PSE APO rats vs. PSE rats in arbitrary units (au): 1.98 ± 0.07 vs. 1.35 ± 0.10; P < 0.001]. However, no differences in MDA were observed between groups. These data suggest that myocardial dysfunction observed after PSE was not due to ß-adrenergic receptor desensitization but could be due to a signaling oxidative stress from the Nox enzyme.