Clustering of cardiovascular risk factors and carotid intima-media thickness: The USE-IMT study.

BACKGROUND: The relation of a single risk factor with atherosclerosis is established. Clinically we know of risk factor clustering within individuals. Yet, studies into the magnitude of the relation of risk factor clusters with atherosclerosis are limited. Here, we assessed that relation. METHODS: Individual participant data from 14 cohorts, involving 59,025 individuals were used in this cross-sectional analysis. We made 15 clusters of four risk factors (current smoking, overweight, elevated blood pressure, elevated total cholesterol). Multilevel age and sex adjusted linear regression models were applied to estimate mean differences in common carotid intima-media thickness (CIMT) between clusters using those without any of the four risk factors as reference group. RESULTS: Compared to the reference, those with 1, 2, 3 or 4 risk factors had a significantly higher common CIMT: mean difference of 0.026 mm, 0.052 mm, 0.074 mm and 0.114 mm, respectively. These findings were the same in men and in women, and across ethnic groups. Within each risk factor cluster (1, 2, 3 risk factors), groups with elevated blood pressure had the largest CIMT and those with elevated cholesterol the lowest CIMT, a pattern similar for men and women. CONCLUSION: Clusters of risk factors relate to increased common CIMT in a graded manner, similar in men, women and across race-ethnic groups. Some clusters seemed more atherogenic than others. Our findings support the notion that cardiovascular prevention should focus on sets of risk factors rather than individual levels alone, but may prioritize within clusters.

Race/Ethnic Differences in the Associations of the Framingham Risk Factors with Carotid IMT and Cardiovascular Events.

BACKGROUND: Clinical manifestations and outcomes of atherosclerotic disease differ between ethnic groups. In addition, the prevalence of risk factors is substantially different. Primary prevention programs are based on data derived from almost exclusively White people. We investigated how race/ethnic differences modify the associations of established risk factors with atherosclerosis and cardiovascular events. METHODS: We used data from an ongoing individual participant meta-analysis involving 17 population-based cohorts worldwide. We selected 60,211 participants without cardiovascular disease at baseline with available data on ethnicity (White, Black, Asian or Hispanic). We generated a multivariable linear regression model containing risk factors and ethnicity predicting mean common carotid intima-media thickness (CIMT) and a multivariable Cox regression model predicting myocardial infarction or stroke. For each risk factor we assessed how the association with the preclinical and clinical measures of cardiovascular atherosclerotic disease was affected by ethnicity. RESULTS: Ethnicity appeared to significantly modify the associations between risk factors and CIMT and cardiovascular events. The association between age and CIMT was weaker in Blacks and Hispanics. Systolic blood pressure associated more strongly with CIMT in Asians. HDL cholesterol and smoking associated less with CIMT in Blacks. Furthermore, the association of age and total cholesterol levels with the occurrence of cardiovascular events differed between Blacks and Whites. CONCLUSION: The magnitude of associations between risk factors and the presence of atherosclerotic disease differs between race/ethnic groups. These subtle, yet significant differences provide insight in the etiology of cardiovascular disease among race/ethnic groups. These insights aid the race/ethnic-specific implementation of primary prevention.

Extracellular superoxide dismutase ameliorates skeletal muscle abnormalities, cachexia, and exercise intolerance in mice with congestive heart failure.

BACKGROUND: Congestive heart failure (CHF) is a leading cause of morbidity and mortality, and oxidative stress has been implicated in the pathogenesis of cachexia (muscle wasting) and the hallmark symptom, exercise intolerance. We have previously shown that a nitric oxide-dependent antioxidant defense renders oxidative skeletal muscle resistant to catabolic wasting. Here, we aimed to identify and determine the functional role of nitric oxide-inducible antioxidant enzyme(s) in protection against cardiac cachexia and exercise intolerance in CHF. METHODS AND RESULTS: We demonstrated that systemic administration of endogenous nitric oxide donor S-nitrosoglutathione in mice blocked the reduction of extracellular superoxide dismutase (EcSOD) protein expression, as well as the induction of MAFbx/Atrogin-1 mRNA expression and muscle atrophy induced by glucocorticoid. We further showed that endogenous EcSOD, expressed primarily by type IId/x and IIa myofibers and enriched at endothelial cells, is induced by exercise training. Muscle-specific overexpression of EcSOD by somatic gene transfer or transgenesis (muscle creatine kinase [MCK]-EcSOD) in mice significantly attenuated muscle atrophy. Importantly, when crossbred into a mouse genetic model of CHF (α-myosin heavy chain-calsequestrin), MCK-EcSOD transgenic mice had significant attenuation of cachexia with preserved whole body muscle strength and endurance capacity in the absence of reduced HF. Enhanced EcSOD expression significantly ameliorated CHF-induced oxidative stress, MAFbx/Atrogin-1 mRNA expression, loss of mitochondria, and vascular rarefaction in skeletal muscle. CONCLUSIONS: EcSOD plays an important antioxidant defense function in skeletal muscle against cardiac cachexia and exercise intolerance in CHF.

Mechanisms involved in increased sensitivity to adenosine A(2A) receptor activation and hypoxia-induced vasodilatation in porcine coronary arteries.

Hypoxia-induced coronary vasorelaxation is a compensatory mechanism increasing blood flow. We hypothesized that hypoxia shares pathways with adenosine and causes vasorelaxation through the adenosine A(2A) receptor and force suppression by increasing cAMP and phosphorylated heat shock protein (HSP)20. Adenosine receptors in porcine left anterior descending coronary arteries (LAD) were examined by RT-PCR and isometric tension recording in myographs. Vasorelaxation was induced by adenosine, 1% oxygen, or both in the absence or presence of ZM241385, an adenosine A(2A) receptor antagonist. cAMP was determined by ELISA and p-HSP20/HSP20 and p-MLC/MLC were determined by immunoblotting and densitometric analyses. In coronary arteries exposed to 1% oxygen, there was increased sensitivity to adenosine, the adenosine A2 selective agonist NECA, and the adenosine A(2A) selective receptor agonist CGS21680. ZM241385 shifted concentration-response curves for CGS21680 to the right, whereas the adenosine A1 antagonist DPCPX, the adenosine A2B receptor antagonist MRS1754 and the adenosine A3 receptor antagonist MRS1523 failed to reduce vasodilatation induced by CGS21680. 1% oxygen or adenosine increased cAMP accumulation and HSP20 phosphorylation without changing T850-MYPT1 and MLC phosphorylation. ZM241385 failed to change 1% oxygen-induced vasodilation, cAMP accumulation, HSP20 phosphorylation and MLC phosphorylation. The PKA inhibitor Rp-8-CPT-cAMPS significantly reduced vasorelaxation induced by 1% oxygen or CGS21680. Our findings suggest that the increased sensitivity to adenosine, NECA, and CGS21680 at 1% oxygen involves adenosine A(2A) receptors. Adenosine and 1% oxygen induce vasorelaxation in PGF2α-contracted porcine coronary arteries partly by force suppression caused by increased cAMP and phosphorylation of HSP20.

Increased expression of heat shock protein 20 and decreased contractile stress in obstructed rat bladder.

PURPOSE: Bladder outlet obstruction induces detrusor hypertrophy and it can eventually lead to decreased bladder smooth muscle contractility. Heat shock protein 20 is the proposed mediator of force suppression in vascular smooth muscle. We investigated whether heat shock protein 20 could also mediate the decreased contractility observed in partially obstructed rat bladders. MATERIALS AND METHODS: Female Wistar rats (Harlan Laboratories, Indianapolis, Indiana) were randomized to partial urethral ligation or sham ligation. After 3 weeks the rats were sacrificed, and the bladders were harvested, frozen, homogenized and analyzed for heat shock protein 20 content by Western blot immunoreactivity. The content of myosin regulatory light chain, a constitutively expressed protein, was determined as a control. Bladder smooth muscle strips were dissected from some rats and mounted for force generation measurement. RESULTS: At cystectomy obstructed bladders were significantly heavier and had more residual urine compared to sham operated bladders. Heat shock protein 20 immunoreactivity was significantly increased a mean +/- 1 SEM of 1.9 +/- 0.3-fold in obstructed vs sham operated bladders. Control protein myosin regulatory light chain immunoreactivity did not significantly differ in obstructed and sham operated bladders. Maximal stress, that is force per cross-sectional area, was significantly decreased in obstructed vs sham operated bladders. Human bladder was found to express immunoreactive heat shock protein 20. CONCLUSIONS: We noted that partially obstructed rat bladders 1) express higher levels of heat shock protein 20 and 2) generate less stress than sham operated bladders. These data suggest the possibility that heat shock protein 20 over expression could at least partially mediate the decreased contractile activity observed with partial bladder outlet obstruction. The mechanism for increased heat shock protein 20 expression is unknown but it may involve increased mechanical stress or hypoxia from urethral obstruction. Human bladder expressed immunoreactive heat shock protein 20, suggesting that a similar mechanism could potentially occur in humans. If confirmed in humans, patients with clinical conditions that result in detrusor hypocontractility could potentially benefit from pharmacological interventions aimed at inhibiting heat shock protein 20.

The latch-bridge hypothesis of smooth muscle contraction.

In contrast to striated muscle, both normalized force and shortening velocities are regulated functions of cross-bridge phosphorylation in smooth muscle. Physiologically this is manifested as relatively fast rates of contraction associated with transiently high levels of cross-bridge phosphorylation. In sustained contractions, Ca2+, cross-bridge phosphorylation, and ATP consumption rates fall, a phenomenon termed "latch". This review focuses on the Hai and Murphy (1988a) model that predicted the highly non-linear dependence of force on phosphorylation and a directly proportional dependence of shortening velocity on phosphorylation. This model hypothesized that (i) cross-bridge phosphorylation was obligatory for cross-bridge attachment, but also that (ii) dephosphorylation of an attached cross-bridge reduced its detachment rate. The resulting variety of cross-bridge cycles as predicted by the model could explain the observed dependencies of force and velocity on cross-bridge phosphorylation. New evidence supports modifications for more general applicability. First, myosin light chain phosphatase activity is regulated. Activation of myosin phosphatase is best demonstrated with inhibitory regulatory mechanisms acting via nitric oxide. The second modification of the model incorporates cooperativity in cross-bridge attachment to predict improved data on the dependence of force on phosphorylation. The molecular basis for cooperativity is unknown, but may involve thin filament proteins absent in striated muscle.

Serine 68 phospholemman phosphorylation during forskolin-induced swine carotid artery relaxation.

BACKGROUND: Phospholemman (PLM) is an abundant phosphoprotein in the plasma membrane of cardiac, skeletal and smooth muscle. It is a member of the FXYD family of proteins that bind to and regulate the Na,K-ATPase. Protein kinase A (PKA) is known to phosphorylate PLM on serine 68 (S68), although the functional effect of S68 PLM phosphorylation is unclear. We therefore evaluated S68 PLM phosphorylation in swine carotid arteries. METHODS: Two anti-PLM antibodies, one to S68 phosphorylated PLM and one to unphosphorylated PLM, were made to PLM peptides in rabbits and tested with purified PLM and PKA-treated PLM. Swine carotid arteries were mounted isometrically, contracted, relaxed with forskolin and then homogenized. Proteins were separated on SDS gels and the intensity of immunoreactivity to the two PLM antibodies determined on immunoblots. RESULTS: The antipeptide antibody 'C2' primarily reacted with unphosphorylated PLM, and the antipeptide antibody 'CP68' detected S68 PLM phosphorylation. Histamine stimulation of intact swine carotid artery induced a contraction, increased the CP68 PLM antibody signal and reduced the C2 PLM antibody signal. High extracellular [K(+)] depolarization induced a contraction without altering the C2 or CP68 PLM signal. Forskolin-induced relaxation of histamine or extracellular [K(+)] contracted arteries correlated with an increased CP68 signal. Nitroglycerin-induced relaxation was not associated with changes in the C2 or CP68 PLM signal. CONCLUSIONS: These data suggest that a contractile agonist increased S68 PLM phosphorylation. Agents that increase [cAMP], but not agents that increase [cGMP], increased S68 PLM phosphorylation. S68 PLM phosphorylation may be involved in cAMP-dependent regulation of smooth muscle force.

An upper airway resonator model of high-frequency inspiratory sounds in children with sleep-disordered breathing.

The goal of this study was to determine how high-frequency inspiratory sounds (HFIS) are generated by sleeping children with obstructive sleep-disordered breathing (OSDB). We hypothesized that HFIS are generated when a high-velocity jet of air, generated by a narrowed upper airway, induces the upper airway to act as a resonating chamber. We tested two predictions of this hypothesis: 1) the upper airway is narrowed in children who make HFIS and 2) the length of the upper airway, calculated from HFIS harmonic intervals, is similar to that calculated from magnetic resonance imaging (MRI) scans. The study was conducted in the setting of a sleep laboratory. Participants included 29 children between 6 and 12 yr of age with adenotonsillar hypertrophy suspected of having OSDB. Minimum cross-sectional airway area and airway long dimensions (lips to larynx or soft palate) were measured in awake children with MRIs. Later that night, sound was recorded with a microphone suspended above their bed while the children underwent polysomnography. Sounds were later analyzed with fast Fourier transforms. We found that sleeping children who generated HFIS had significantly narrower upper airways compared with children who did not make HFIS [minimum airway area 20.5 +/- 4.4 vs. 70.9 +/- 22.5 mm(2) (mean +/- SE), respectively; P = 0.02]. There was a significant inverse correlation between the log(10) of the narrowest airway area and the number of HFIS recorded per hour (r(2) = 0.55, P < 0.00001). The harmonics characteristics of HFIS predicted that they were generated by sound resonating in chamber whose length was 12.0 +/- 0.9 cm, which is similar to the MRI measured distance from the lips to the larynx of 12.8 +/- 0.4 cm. In conclusion, these data suggest that children generate HFIS when 1) they have a narrowed upper airway and 2) their upper airway acts as a resonating chamber.

Children with obstructive sleep-disordered breathing generate high-frequency inspiratory sounds during sleep.

STUDY OBJECTIVES: We observed that some children with adenotonsillar hypertrophy and obstructive sleep-disordered breathing (SDB) make high-frequency inspiratory sounds (HFIS) during sleep. Our objective was to determine whether HFIS occur in most children with obstructive SDB and adenotonsillar hypertrophy and whether adenotonsillectomy reduces HFIS. DESIGN: Prospective consecutive-entry trial. SETTING: Sleep laboratory. PARTICIPANTS: Twenty-six children between 6 and 12 years of age with adenotonsillar hypertrophy suspected of having obstructive SDB. MEASUREMENTS AND RESULTS: We performed polysomnography and measured sounds during sleep with a microphone suspended above the bed. Sounds were recorded on a computer at 44 kHz, analyzed with fast Fourier transformation for frequency content. HFIS were sounds occurring during an inspiration with frequencies greater than 2 kHz. HFIS were different from the low-frequency (< 2 kHz) sounds described in snoring adults. HFIS usually occurred in consecutive breaths, occasionally exceeding 100. We counted the number of HFIS that occurred per hour of sleep. Children who made more HFIS had more obstructive SDB than did those who did not make the HFIS, and there was a significant positive correlation between the number of HFIS and the obstructive apnea-hypopnea index. Children with more than 3 apneas and hypopneas per hour of sleep all made at least 10 HFIS per hour, and all children who had more than 10 HFIS per hour had obstructive apnea-hypopnea index values greater than 1. Children with adenotonsillar hypertrophy made more HFIS than did those children whose tonsils and adenoids had been removed. CONCLUSIONS: HFIS may be a marker of disturbed breathing during sleep in children with adenotonsillar hypertrophy.

Heat induced HSP20 phosphorylation without increased cyclic nucleotide levels in swine carotid media.

BACKGROUND: Heat pretreatment of swine carotid artery has been shown to increase ser16-heat shock protein 20 (HSP20) phosphorylation and suppress force, i.e., reduce force with only minimal reduction in ser19-myosin regulatory light chain (MRLC) phosphorylation. RESULTS: We further investigated this response in intact histamine stimulated swine carotid artery rings. There was a heat threshold such that increased ser16-HSP20 phosphorylation and force suppression were observed between 43 degrees C and 46 degrees C. The increased ser16-HSP20 phosphorylation persisted up to 16 hours after 44.5 degrees C heat treatment. Pretreatment of swine carotid media at 44.5 degrees C increased ser16-HSP20 phosphorylation without increases in [cAMP] or [cGMP], suggesting an alternate mechanism, perhaps phosphatase inhibition, for the increase in ser16-HSP20 phosphorylation. Heat pretreatment at 47.5 degrees C reduced force by decreasing MRLC phosphorylation rather than by large increases in ser16-HSP20 phosphorylation. HSP20 phosphorylation at the putative PKC site did not change with any treatment. CONCLUSION: These results demonstrate that multiple mechanisms can induce force suppression that is correlated with ser16-HSP20 phosphorylation: 1) nitrovasodilators via cGMP, 2) forskolin via cAMP, and 2) thermal stress in a cyclic nucleotide independent manner.