No evidence of association between subclinical thyroid disorders and common carotid intima medial thickness or atherosclerotic plaque.

BACKGROUND AND AIMS: Increased carotid artery intima-media thickness (IMT) and the presence of plaques have been shown to be predictors of cardiovascular disease. The cardiovascular risk in patients with overt thyroid diseases is related to increased risk of atherosclerosis, but there has been no clear evidence about subclinical disorders. We have assessed whether subclinical thyroid dysfunction is associated with arterial thickening and plaque. METHODS AND RESULTS: The SardiNIA study is a population-based survey on the Italian island of Sardinia. We reviewed data from 5815 subjects (aged 14-102 years), none of whom had overt hyperthyroidism or hypothyroidism or was taking thyroid medication. Serum thyrotropin (TSH), free thyroxine, together with carotid ultrasound IMT and the presence of common carotid plaques were analysed in all subjects. Possible association of IMT and carotid plaques with thyroid parameters was evaluated by univariate and multivariate analyses. IMT was significantly associated with age, sex, smoking, low density lipoprotein cholesterol (LDL), high density lipoprotein cholesterol, pulse pressure (PP), history of arterial hypertension, diabetes, and previous cardiovascular events (p = 0.001 or lower, R(2) = 0.47). Carotid plaques were predicted by age, sex, LDL, PP, history of diabetes, previous cardiovascular events, and the use of statins (p = 0.029 or lower). Thyroid hormone was not predictive of carotid atherosclerosis when adjusted for confounders. CONCLUSION: Thyroid hormone is not associated with increased IMT or with the presence of carotid artery plaque. Our data do not support the idea that treating subclinical disorders might help to prevent arterial remodelling or carotid atherosclerosis.

Relationship between vitamin D status and left ventricular geometry in a healthy population: results from the Baltimore Longitudinal Study of Aging.

OBJECTIVES: The effects of vitamin D on the heart have been studied in patients with cardiac disease, but not in healthy persons. We investigated the relation between vitamin D status and left ventricular (LV) structure and function in community-dwelling subjects without heart disease. DESIGN: The relationship between concentrations of 25-hydroxyvitamin D [25(OH)D], a marker of vitamin D reserve, and LV transthoracic echocardiography measures was analysed in 711 participants in the Baltimore Longitudinal Study of Aging who were without cardiac disease. RESULTS: Mean 25(OH)D in the study population was 32.3 ± 11.4 ng mL(-1) ; only 15.5% of subjects had moderate or severe vitamin D deficiency [25(OH)D < 20 ng mL(-1) ]. Adjusting for age, body mass index, cardiovascular disease risk factors, physical activity, calcium and parathyroid hormone, 25(OH)D was positively correlated with LV thickness (ß 0.095, SE 0.039, P < 0.05) and LV mass index (ß 7.5, SE 2.6, P < 0.01). A significant nonlinear relation between 25(OH)D and LV concentric remodelling was observed. LV remodelling was more likely in participants with 25(OH)D levels <30 ng mL(-1) [odds ratio (OR) 1.24; 95% confidence interval (CI) 0.83-1.85] or ≥38 ng mL(-1) (OR 1.73; 95% CI 1.13-2.65), compared with those with 30-37 ng mL(-1) 25(OH)D. Consistently, LV relative wall thickness was significantly lower (P for trend=0.05), and LV diastolic internal diameter index (P for trend<0.05) and end-diastolic volume index (P for trend<0.05) were significantly higher in subjects with 30-37 ng mL(-1) 25(OH)D compared to the rest of the study population. There was a significant interaction between 25(OH)D and hypertension on the risk of LV hypertrophy (P < 0.05). CONCLUSIONS: In a population-based sample of predominantly vitamin D-sufficient subjects without heart disease, LV geometry was most favourable at intermediate 25(OH)D concentrations.

The relationship between visceral adiposity and left ventricular diastolic function: results from the Baltimore Longitudinal Study of Aging.

BACKGROUND AND AIMS: It is unclear whether subcutaneous and visceral fat are differentially correlated to the decline in left ventricular (LV) diastolic function with aging. This study sought to examine the hypothesis that age-related changes in the regional fat distribution account for changes in LV diastolic function and to explore potential mediators of this association. METHODS AND RESULTS: In this cross-sectional study, we evaluated 843 participants of the Baltimore Longitudinal Study of Aging with echocardiogram, dual-energy X-ray absorptiometry (DEXA), abdominal computed tomography (CT) and blood tests performed at the same visit. LV diastolic function was assessed by parameters of LV relaxation (E/A ratio, Em and Em/Am ratio) and LV filling pressures (E/Em ratio). Total body fat was computed by DEXA, while visceral and subcutaneous fat were determined from abdominal CT. In multivariate models adjusted for demographics, cardiovascular risk factors, antihypertensive medications, physical activity and LV mass, both visceral and subcutaneous fat were associated with LV diastolic dysfunction. When both measures of adiposity were simultaneously included in the same model, only visceral fat was significantly associated with LV diastolic dysfunction. Triglycerides and sex-hormone binding globulin, but not adiponectin and leptin, were found to be significant mediators of the relationship between visceral fat and LV diastolic function, explaining 28-47% of the association. Bootstrapping analyses confirmed the significance of these findings. CONCLUSIONS: Increased visceral adiposity is associated with LV diastolic dysfunction, possibly through a metabolic pathway involving blood lipids and ectopic fat accumulation rather than adipokines.

Quantitative proteomic analysis reveals novel mitochondrial targets of estrogen deficiency in the aged female rat heart.

The incidence of myocardial infarction rises sharply at menopause, implicating a potential role for estrogen (E(2)) loss in age-related increases in ischemic injury. We aimed to identify quantitative changes to the cardiac mitochondrial proteome of aging females, based on the hypothesis that E(2) deficiency exacerbates age-dependent disruptions in mitochondrial proteins. Mitochondria isolated from left ventricles of adult (6 mo) and aged (24 mo) F344 ovary-intact or ovariectomized (OVX) rats were labeled with 8plex isobaric tags for relative and absolute quantification (iTRAQ; n = 5-6/group). Groups studied were adult, adult OVX, aged, and aged OVX. In vivo coronary artery ligation and in vitro mitochondrial respiration studies were also performed in a subset of rats. We identified 965 proteins across groups and significant directional changes in 67 proteins of aged and/or aged OVX; 32 proteins were unique to aged OVX. Notably, only six proteins were similarly altered in adult OVX (voltage-dependent ion channel 1, adenine nucleotide translocator 1, cytochrome c oxidase subunits VIIc and VIc, catalase, and myosin binding protein C). Proteins affected by aging were primarily related to cellular metabolism, oxidative stress, and cell death. The largest change occurred in monoamine oxidase-A (MAO-A), a source of oxidative stress. While acute MAO-A inhibition induced mild uncoupling in aged mitochondria, reductions in infarct size were not observed. Age-dependent alterations in mitochondrial signaling indicate a highly selective myocardial response to E(2) deficiency. The combined proteomic and functional approaches described here offer possibility of new protein targets for experimentation and therapeutic intervention in the aged female population.

Admixture mapping of ankle-arm index: identification of a candidate locus associated with peripheral arterial disease.

BACKGROUND: Peripheral arterial disease (PAD) is associated with significant morbidity and mortality, and has a higher prevalence in African Americans than Caucasians. Ankle-arm index (AAI) is the ratio of systolic blood pressure in the leg to that in the arm, and, when low, is a marker of PAD. METHODS: The authors used an admixture mapping approach to search for genetic loci associated with low AAI. Using data from 1040 African American participants in the observational, population based Health, Aging, and Body Composition Study who were genotyped at 1322 single nucleotide polymorphisms (SNPs) that are informative for African versus European ancestry and span the entire genome, we estimated genetic ancestry in each chromosomal region and then tested the association between AAI and genetic ancestry at each locus. RESULTS: The authors found a region of chromosome 11 that reaches its peak between 80 and 82 Mb associated with low AAI (p<0.001 for rs12289502 and rs9665943, both within this region). 753 African American participants in the observational, population based Cardiovascular Health Study were genotyped at rs9665943 to test the reproducibility of this association, and this association was also statistically significant (odds ratio (OR) for homozygous African genotype 1.59, 95% confidence interval (CI) 1.12 to 2.27). Another candidate SNP (rs1042602) in the same genomic region was tested in both populations, and was also found to be significantly associated with low AAI in both populations (OR for homozygous African genotype 1.89, 95% CI 1.29 to 2.76). CONCLUSION: This study identifies a novel region of chromosome 11 representing an area with a potential candidate gene associated with PAD in African Americans.

Intensity fluctuation spectroscopy monitors contractile activation in "resting" cardiac muscle.

Intensity fluctuations in a laser beam scattered by nonbeating isolated rat cardiac muscle varied directly with the calcium concentrations in the bathing fluid. The steady-state level of these fluctuations varied directly with calcium-dependent force suggesting that the intensity fluctuations reflect an interaction of calcium ions with the myofilaments. The demonstration that both a portion of resting force and the frequency of intensity fluctuations vary directly with calcium even in quiescent conditions indicates that some contractile activation is present in the resting muscle.

Age- and gender-specific awareness, treatment, and control of cardiovascular risk factors and subclinical vascular lesions in a founder population: the SardiNIA Study.

AIM: We investigated the gender-specific control of cardiovascular (CV) risk factors and subclinical vascular lesions in a founder population in Italy. METHODS AND RESULTS: 6148 subjects were enrolled (aged 14-102 years) from four towns. Hypertension (HT), diabetes mellitus (DM) and dyslipidemia (LIP) were defined in accordance with guidelines. A self-reported diagnosis defined awareness of these conditions, and the current use of specific medications as treatment. Prevalence was HT 29.2%, DM 4.8%, LIP 44.1% and was higher in men than in women. Disease prevalence increased with age for every CV risk factor. Men were less likely than women to take anti-HT drugs and to reach BP control (9.9% vs. 16%). Only 17.6% of HT > 65 years had a BP < or =140/90 mmHg, though 48.5% were treated. The use of statins was very low (<1/3 of eligible subjects > 65 years, those with the highest treatment rate). The ratio of control-to-treated HT was lower in subjects with, than in those without, thicker carotid arteries (31.5% vs. 38.8%, p < 0.05) or stiffer aortas (26.0% vs. 40.0%, p < 0.05) or carotid plaques (26.3% vs. 41.1%, p<0.05). CONCLUSION: A large number of subjects at high CV risk are not treated and the management of subclinical vascular lesions is far from optimal.

Education eclipses ethnicity in predicting the development of the metabolic syndrome in different ethnic groups in midlife: the Study of Women's Health Across the Nation (SWAN).

OBJECTIVE: To determine the respective roles of socio-economic status (SES) and ethnicity in the risk of incident metabolic syndrome in middle-aged women. DESIGN AND PARTICIPANTS: A total of 3302 pre- and peri-menopausal women, not receiving hormone therapy at baseline, took part in the Study of Women's Health Across the Nation, a multi-site, community-based, longitudinal study of the menopausal transition. The main outcome measures were to ascertain the prevalence of the metabolic syndrome and the incidence of the metabolic syndrome over 5 years of follow-up. RESULTS: At baseline, the prevalence of the metabolic syndrome was 21% (n = 673). Among 2512 women without metabolic syndrome at baseline, 12.8% (n = 321) developed the metabolic syndrome during 5 years of follow-up. Both ethnicity and SES were significant univariate predictors of incident metabolic syndrome. In multivariate logistic regression models that included age at baseline, menopausal status and site, baseline smoking and alcohol consumption at follow-up visit 1, as well as baseline values of each of the components of the metabolic syndrome, only education was an independent predictor of incident metabolic syndrome. CONCLUSION: Approximately 13% of peri-menopausal women developed the metabolic syndrome during the 5-year follow-up period. Education, but not ethnicity, was an independent predictor of incident metabolic syndrome risk.

Mechanism of myocardial contractile depression by clinical concentrations of ethanol. A study in ferret papillary muscles.

Moderate alcohol intoxication in man, a ubiqitious social event, causes acute but reversible myocardial depression, the mechanism of which is unknown. We investigated whether this depression could be due to a direct effect of ethanol on the process of electromechanical coupling by simultaneously measuring the transmembrane action potential and contraction, or the cytosolic calcium transient (via aequorin photoluminescence) and contraction in isolated ferret right ventricular papillary muscle. Ethanol, in concentrations that are similar to plasma levels in man during intoxication (0.15 vol %), depressed the force of contraction approximately 10%. The step in the electromechanical process that was affected appeared to be the calcium-myofilament interaction, as there was no change in the transmembrane action potential or cytosolic calcium transient. This inhibition was quickly reversed by removal of the ethanol from the perfusate. On the other hand, higher concentrations of ethanol produced changes in contraction, the calcium transient, and the action potential, suggesting multiple levels of inhibition of electromechanical coupling. Increasing the perfusate calcium or use of the calcium channel agonist, BAY-K 8644, increased cytosolic calcium to near maximum but had little effect on contractility, confirming that the relationship between calcium and the myofilaments had been altered. These data suggest that the acute depression in ventricular function seen with alcohol consumption may be due to a direct effect on electromechanical coupling through inhibition of the calcium myofilament interaction.

Calcium oscillations index the extent of calcium loading and predict functional recovery during reperfusion in rat myocardium.

Delayed recovery of contractile function after myocardial ischemia may be due to prolonged recovery of high-energy phosphates, persistent acidosis, increased inorganic phosphate, and/or calcium loading. To examine these potential mechanisms, metabolic parameters measured by 31P nuclear magnetic resonance spectroscopy, and spontaneous diastolic myofilament motion caused by sarcoplasmic reticulum-myofilament calcium cycling indexed by the scattered light intensity fluctuations (SLIF) it produces in laser beam reflected from the heart, were studied in isolated atrioventricularly blocked rat hearts (n = 10) after 65 min of ischemia at 30 degrees C. All metabolic parameters recovered to their full extent 5 min after reperfusion. Developed pressure evidenced a small recovery but then fell abruptly. This was accompanied by an increase in end diastolic pressure to 37 +/- 5 mm Hg and a fourfold increase in SLIF, to 252 +/- 58% of baseline. In another series of hearts initial reperfusion with calcium of 0.08 mM prevented the SLIF rise and resulted in improved developed pressure (74 +/- 3% vs. 39 +/- 13% of control), and lower cell calcium (5.9 +/- 3 vs. 10.3 +/- 1.4 mumol/g dry wt). Thus, during reperfusion, delayed contractile recovery is not associated with delayed recovery of pH, inorganic phosphate, or high-energy phosphates and can be attributed, in part, to an adverse effect of calcium loading which can be indexed by increased SLIF occurring at that time.

Enhanced contractile response and protein kinase activation to threshold levels of beta-adrenergic stimulation in hyperthyroid rat heart.

The contractile response measured as maximum rate of force development to a near threshold concentration of isoproterenol (1 nM) was enhanced in perfused interventricular septa from hyperthyroid (128+/-4% control) compared with euthyroid rats (105+/-2%, P < 0.01). This enhanced contractile response was accompanied by a significant activation of cyclic (c)AMP-dependent protein kinase (protein kinase activity ratio increased from 0.159+/-0.008 to 0.218+/-0.019, P < 0.005, although no significant changes from base line occurred in euthyroid septa, 0.152+/-0.007-0.179+/-0.012). No difference between hyperthyroid and euthyroid hearts was observed in the contractile response to 0.1 mM dibutyryl cAMP (126.5+/-2.5% and 122.0+/-9.2% in hyperthyroid and euthyroid, respectively), and the magnitude of the response to dibutyryl cAMP was comparable with that observed in the hyperthyroid group with 1 nM isoproterenol. These results suggest that the mechanism for enhanced protein kinase activation and contractile response to low concentrations of isoproterenol in the hyperthyroid heart is at or proximal to cAMP generation. The maximum contractile response to isoproterenol (0.5 muM), however, was decreased in hyperthyroid myocardium (192+/-13%) compared with euthyroid (291+/-37%, P < 0.05). Both protein kinase activity ratio (0.356+/-0.017 and 0.344+/-0.013) and the maximum contractile response to Ca(++) (335+/-15 and 340+/-12% control in hyperthyroid and euthyroid, respectively) were similar, suggesting that the mechanism of the diminished maximum response was distal to protein kinase activation but not a function of an altered Ca(++)-troponin interaction. The diminished maximum rate of force development response in the hyperthyroid hearts was accompanied by significantly less shortening of the contraction duration that was 85.6+/-2.1% control in hyperthyroid vs. 66+/-2.8% control in euthyroid, P < 0.001. Although the basal rate of Ca(++) accumulation was greater in microsomes isolated from hyperthyroid than from euthyroid hearts, there was significantly less additional stimulation of Ca(++) accumulation in response to exogenous cAMP and protein kinase in hyperthyroid compared with euthyroid hearts. This reduction may explain the diminished effect of isoproterenol on the shortening of contraction duration in hyperthyroid compared with the euthyroid myocardium, and may explain, at least in part, the diminished maximum contractile response to isoproterenol.

Age-associated changes in beta-adrenergic modulation on rat cardiac excitation-contraction coupling.

Previous studies have demonstrated that the ability of beta-adrenergic receptor (beta AR) stimulation to increase cardiac contractility declines with aging. In the present study, the control mechanisms of excitation-contraction (EC) coupling, including calcium current (ICa), cytosolic Ca2+ (Cai2+) transient and contraction in response to beta AR stimulation were investigated in ventricular myocytes isolated from rat hearts of a broad age range (2, 6-8, and 24 mo). While the baseline contractile performance and the Cai2+ transient did not differ markedly among cells from hearts of all age groups, the responses of the Cai2+ transient and contraction to beta-adrenergic stimulation by norepinephrine (NE) diminished with aging: the threshold concentration and the ED50 increased in rank order with aging; the maximum responses of contraction and Cai2+ transient decreased with aging. Furthermore, the efficacy of beta AR stimulation to increase ICa was significantly reduced with aging, and the diminished responses of the contraction and Cai2+ transient amplitudes to NE were proportional to the reductions in the ICa response. These findings suggest that the observed age-associated reduction in beta AR modulation of the cardiac contraction is, in part at least, due to a deficit in modulation of Cai2+, particularly the activity of L-type calcium channels.

Prolonged contraction duration in aged myocardium.

Isometric performance at 29degreesC was measured in left ventricular trabeculae carneae from young adult (6-mo) and aged (25-mo) rats (n equals 18 in each group). Active tension and maximal rate of tension development did not differ with age, but contraction duration was 255plus or minus6 ms in the young adult and 283plus or minus6 ms in the aged group (P less than0.001). Although catecholamine content per gram heart weight was less in the aged myocardium, additional experiments showed that neither 1 times 10-6 M propranolol nor pretreatment with 6-hydroxydopamine eliminated the age difference in contraction duration. To determine if this age difference resulted from a prolonged active state, electromechanical dissociation and the overshoot of contraction duration during recovery from hypoxia were measured. During paired stimulation greater mechanical refractoriness was found in aged muscles (P less than0.01), but intracellular action potential recordings showed no age difference in the electrical refractory period. On recovery from hypoxia, contraction duration overshoot was 117plus or minus 4percent of control in the young and 138plus or minus 4percent of control in the aged muscles (P less than0.01). The greater electromechanical dissociation and greater overshoot in contraction duration following hypoxia in aged myocardium suggests that prolonged contraction duration in aged myocardium results from a prolonged active state rather than changes in passive properties or myocardial catecholamine content.

p53 and the hypoxia-induced apoptosis of cultured neonatal rat cardiac myocytes.

Myocyte cell loss is a prominent and important pathogenic feature of cardiac ischemia. We have used cultured neonatal rat cardiac myocytes exposed to prolonged hypoxia as an experimental system to identify critical factors involved in cardiomyocyte death. Exposure of myocytes to hypoxia for 48 h resulted in intranucleosomal cleavage of genomic DNA characteristic of apoptosis and was accompanied by increased p53 transactivating activity and protein accumulation. Expression of p21/WAF-1/CIP-1, a well-characterized target of p53 transactivation, also increased in response to hypoxia. Hypoxia did not cause DNA laddering or cell loss in cardiac fibroblasts. To determine whether the increase in p53 expression in myocytes was sufficient to induce apoptosis, normoxic cultures were infected with a replication-defective adenovirus expressing wild-type human p53 (AdCMV.p53). Infected cells expressed high intracellular levels of p53 protein and exhibited the morphological changes and genomic DNA fragmentation characteristic of apoptosis. In contrast, no genomic DNA fragmentation was observed in myocytes infected with the control virus lacking an insert (AdCMV.null) or in cardiac fibroblasts infected with AdCMV.p53. These results suggest that the intracellular signaling pathways activated by p53 might play a critical role in the regulation of hypoxia-induced apoptosis of cardiomyocytes.

Intracellular signaling pathways required for rat vascular smooth muscle cell migration. Interactions between basic fibroblast growth factor and platelet-derived growth factor.

Intracellular signaling pathways activated by both PDGF and basic fibroblast growth factor (bFGF) have been implicated in the migration of vascular smooth muscle cells (VSMC), a key step in the pathogenesis of many vascular diseases. We demonstrate here that, while bFGF is a weak chemoattractant for VSMCs, it is required for the PDGF-directed migration of VSMCs and the activation of calcium/calmodulin-dependent protein kinase II (CamKinase II), an intracellular event that we have previously shown to be important in the regulation of VSMC migration. Neutralizing antibodies to bFGF caused a dramatic reduction in the size of the intracellular calcium transient normally seen after PDGF stimulation and inhibited both PDGF-directed VSMC migration and CamKinase II activation. Partially restoring the calcium transient with ionomycin restored migration and CamKinase II activation as did the forced expression of a mutant CamKinase II that had been "locked" in the active state by site-directed mutagenesis. These results suggest that bFGF links PDGF receptor stimulation to changes in intracellular calcium and CamKinase II activation, reinforcing the central role played by CamKinase II in regulating VSMC migration.

Taxol inhibits neointimal smooth muscle cell accumulation after angioplasty in the rat.

Despite significant improvements in the primary success rate of the medical and surgical treatments for atherosclerotic disease, including angioplasty, bypass grafting, and endarterectomy, secondary failure due to late restenosis continues to occur in 30-50% of individuals. Restenosis and the later stages in atherosclerotic lesions are due to a complex series of fibroproliferative responses to vascular injury involving potent growth-regulatory molecules (such as platelet-derived growth factor and basic fibroblast growth factor) and resulting in vascular smooth muscle cell (VSMC) proliferation, migration, and neointimal accumulation. We show here, based on experiments with both taxol and deuterium oxide, that microtubules are necessary for VSMCs to undergo the multiple transformations contributing to the development of the neointimal fibroproliferative lesion. Taxol was found to interfere both with platelet-derived growth factor-stimulated VSMC migration and with VSMC migration and with VSMC proliferation, at nanomolar levels in vitro. In vivo, taxol prevented medial VSMC proliferation and the neointimal VSMC accumulation in the rat carotid artery after balloon dilatation and endothelial denudation injury. This effect occurred at plasma levels approximately two orders of magnitude lower than that used clinically to treat human malignancy (peak levels achieved in this model were approximately 50-60 nM). Taxol may therefore be of therapeutic value in preventing human restenosis with minimal toxicity.

Age-associated reductions in cardiac beta1- and beta2-adrenergic responses without changes in inhibitory G proteins or receptor kinases.

While an age-associated diminution in myocardial contractile response to beta-adrenergic receptor (beta-AR) stimulation has been widely demonstrated to occur in the context of increased levels of plasma catecholamines, some critical mechanisms that govern beta-AR signaling must still be examined in aged hearts. Specifically, the contribution of beta-AR subtypes (beta1 versus beta2) to the overall reduction in contractile response with aging is unknown. Additionally, whether G protein-coupled receptor kinases (GRKs), which mediate receptor desensitization, or adenylyl cyclase inhibitory G proteins (Gi) are increased with aging has not been examined. Both these inhibitory mechanisms are upregulated in chronic heart failure, a condition also associated with diminished beta-AR responsiveness and increased circulatory catecholamines. In this study, the contractile responses to both beta1-AR and beta2-AR stimulation were examined in rat ventricular myocytes of a broad age range (2, 8, and 24 mo). A marked age-associated depression in contractile response to both beta-AR subtype stimulation was observed. This was associated with a nonselective reduction in the density of both beta-AR subtypes and a reduction in membrane adenylyl cyclase response to both beta-AR subtype agonists, NaF or forskolin. However, the age-associated diminutions in contractile responses to either beta1-AR or beta2-AR stimulation were not rescued by inhibiting Gi with pertussis toxin treatment. Further, the abundance or activity of beta-adrenergic receptor kinase, GRK5, or Gi did not significantly change with aging. Thus, we conclude that the positive inotropic effects of both beta1- and beta2-AR stimulation are markedly decreased with aging in rat ventricular myocytes and this is accompanied by decreases in both beta-AR subtype densities and a reduction in membrane adenylate cyclase activity. Neither GRKs nor Gi proteins appear to contribute to the age-associated reduction in cardiac beta-AR responsiveness.

Enhanced expression of p53 and apoptosis induced by blockade of the vacuolar proton ATPase in cardiomyocytes.

Activation of the vacuolar proton ATPase (VPATPase) has been implicated in the prevention of apoptosis in neutrophils and adult cardiac myocytes. To determine the role of the VPATPase in apoptosis of cardiac myocytes, we used a potent and specific inhibitor of the VPATPase, bafilomycin A1. Bafilomycin A1 alone caused increased DNA laddering of genomic DNA and increased nuclear staining for fragmented DNA in neonatal cardiomyocyte apoptosis in a dose- and time-dependent manner. Intracellular acidification in cardiac myocytes was also observed after 18 h of bafilomycin A1 treatment. Accordingly, bafilomycin A1-treated myocytes also showed increased accumulation of p53 protein and p53-dependent transactivation of gene expression, including a persistent upregulation of p21/wild-type p53 activated fragment 1/cyclin kinase inhibitor protein-1 mRNA. The bafilomycin A1-induced increase in p53 protein levels was accompanied by a marked increase in p53 mRNA accumulation. In contrast, cardiac fibroblasts treated with bafilomycin A1 showed no change in p53 protein expression or pHi and did not undergo apoptosis even after 24 h of treatment. Our data suggest that blockade of the VPATPase induces apoptotic cell death of cardiac myocytes and that this may occur through a p53-mediated apoptotic pathway.

Bcl-2 and Bcl-X(L) block thapsigargin-induced nitric oxide generation, c-Jun NH(2)-terminal kinase activity, and apoptosis.

The proteins Bcl-2 and Bcl-X(L) prevent apoptosis, but their mechanism of action is unclear. We examined the role of Bcl-2 and Bcl-X(L) in the regulation of cytosolic Ca(2+), nitric oxide production (NO), c-Jun NH(2)-terminal kinase (JNK) activation, and apoptosis in Jurkat T cells. Thapsigargin (TG), an inhibitor of the endoplasmic reticulum-associated Ca(2+) ATPase, was used to disrupt Ca(2+) homeostasis. TG acutely elevated intracellular free Ca(2+) and mitochondrial Ca(2+) levels and induced NO production and apoptosis in Jurkat cells transfected with vector (JT/Neo). Buffering of this Ca(2+) response with 1, 2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM) or inhibiting NO synthase activity with N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME) blocked TG-induced NO production and apoptosis in JT/Neo cells. By contrast, while TG produced comparable early changes in the Ca(2+) level (i.e., within 3 h) in Jurkat cells overexpressing Bcl-2 and Bcl-X(L) (JT/Bcl-2 or JT/Bcl-X(L)), NO production, late (36-h) Ca(2+) accumulation, and apoptosis were dramatically reduced compared to those in JT/Neo cells. Exposure of JT/Bcl-2 and JT/Bcl-X(L) cells to the NO donor, S-nitroso-N-acetylpenacillamine (SNAP) resulted in apoptosis comparable to that seen in JT/Neo cells. TG also activated the JNK pathway, which was blocked by L-NAME. Transient expression of a dominant negative mutant SEK1 (Lys-->Arg), an upstream kinase of JNK, prevented both TG-induced JNK activation and apoptosis. A dominant negative c-Jun mutant also reduced TG-induced apoptosis. Overexpression of Bcl-2 or Bcl-X(L) inhibited TG-induced loss in mitochondrial membrane potential, release of cytochrome c, and activation of caspase-3 and JNK. Inhibition of caspase-3 activation blocked TG-induced JNK activation, suggesting that JNK activation occurred downstream of caspase-3. Thus, TG-induced Ca(2+) release leads to NO generation followed by mitochondrial changes including cytochrome c release and caspase-3 activation. Caspase-3 activation leads to activation of the JNK pathway and apoptosis. In summary, Ca(2+)-dependent activation of NO production mediates apoptosis after TG exposure in JT/Neo cells. JT/Bcl-2 and JT/Bcl-X(L) cells are susceptible to NO-mediated apoptosis, but Bcl-2 and Bcl-X(L) protect the cells against TG-induced apoptosis by negatively regulating Ca(2+)-sensitive NO synthase activity or expression.

Sinoatrial nodal cell ryanodine receptor and Na(+)-Ca(2+) exchanger: molecular partners in pacemaker regulation.

The rate of spontaneous diastolic depolarization (DD) of sinoatrial nodal cells (SANCs) that triggers recurrent action potentials (APs) is a fundamental aspect of the heart's pacemaker. Here, in experiments on isolated SANCs, using confocal microscopy combined with a patch clamp technique, we show that ryanodine receptor Ca(2+) release during the DD produces a localized subsarcolemmal Ca(2+) increase that spreads in a wavelike manner by Ca(2+)-induced Ca(2+) release and produces an inward current via the Na(+)-Ca(2+) exchanger (NCX). Ryanodine, a blocker of the sarcoplasmic reticulum Ca(2+) release channel, in a dose-dependent manner reduces the SANC beating rate with an IC(50) of 2.6 micromol/L and abolishes the local Ca(2+) transients that precede the AP upstroke. In voltage-clamped cells in which the DD was simulated by voltage ramp, 3 micromol/L ryanodine decreased an inward current during the voltage ramp by 1.6+/-0.3 pA/pF (SEM, n=4) leaving the peak of L-type Ca(2+) current unchanged. Likewise, acute blockade of the NCX (via rapid substitution of bath Na(+) by Li(+)) abolished SANC beating and reduced the inward current to a similar extent (1.7+/-0.4 pA/pF, n=4), as did ryanodine. Thus, in addition to activation/inactivation of multiple ion channels, Ca(2+) activation of the NCX, because of localized sarcoplasmic reticulum Ca(2+) release, is a critical element in a chain of molecular interactions that permits the heartbeat to occur and determines its beating rate.