Improving murine embryonic stem cell differentiation into cardiomyocytes with neuregulin-1: differential expression of microRNA.

Identification of factors that direct embryonic stem (ES) cell (ESC) differentiation into functional cardiomyocytes is essential for successful use of ESC-based therapy for cardiac repair. Neuregulin-1 (NRG1) and microRNA play important roles in the cardiac differentiation of ESCs. Understanding how NRG1 regulates microRNA will provide new mechanistic insights into the role of NRG1 on ESCs. It may also lead to the discovery of novel microRNAs that are important for ESC cardiac differentiation. The objective of this study was to assess the microRNA expression profile during NRG1-induced ESC cardiac differentiation. Murine ESCs were incubated with a recombinant NRG1ß or an inhibitor of ErbB2 or ErbB4 during hanging drop-induced cardiac differentiation. The expression of cardiac-specific markers and microRNAs was analyzed by RT-PCR and microRNA array, respectively. We found that the expression of NRG1 and the ErbB receptors was increased during hanging drop-induced cardiac differentiation of ESCs. NRG1 stimulation during a specific developmental window enhanced, while inhibition of the ErbB2 or ErbB4 receptor inhibited, cardiac differentiation of ESCs. NRG1 increased the expression of mmu-miR-296-3p and mmu-miR-200c*, and decreased mmu-miR-465b-5p. Inhibition of mmu-miR-296-3p or mmu-miR-200c* decreased, while inhibition of mmu-miR-465-5p increased, the differentiation of ESCs into the cardiac lineage. This is the first report demonstrating that microRNAs are differentially regulated by NRG1-ErbB signaling during cardiac differentiation of ESCs. This study has also identified new microRNAs that are important for ESC cardiac differentiation.

Cytomegalovirus infection causes an increase of arterial blood pressure.

Cytomegalovirus (CMV) infection is a common infection in adults (seropositive 60-99% globally), and is associated with cardiovascular diseases, in line with risk factors such as hypertension and atherosclerosis. Several viral infections are linked to hypertension, including human herpes virus 8 (HHV-8) and HIV-1. The mechanisms of how viral infection contributes to hypertension or increased blood pressure are not defined. In this report, the role of CMV infection as a cause of increased blood pressure and in forming aortic atherosclerotic plaques is examined. Using in vivo mouse model and in vitro molecular biology analyses, we find that CMV infection alone caused a significant increase in arterial blood pressure (ABp) (p<0.01 approximately 0.05), measured by microtip catheter technique. This increase in blood pressure by mouse CMV (MCMV) was independent of atherosclerotic plaque formation in the aorta, defined by histological analyses. MCMV DNA was detected in blood vessel samples of viral infected mice but not in the control mice by nested PCR assay. MCMV significantly increased expression of pro-inflammatory cytokines IL-6, TNF-alpha, and MCP-1 in mouse serum by enzyme-linked immunosorbent assay (ELISA). Using quantitative real time reverse transcriptase PCR (Q-RT-PCR) and Western blot, we find that CMV stimulated expression of renin in mouse and human cells in an infectious dose-dependent manner. Co-staining and immunofluorescent microscopy analyses showed that MCMV infection stimulated renin expression at a single cell level. Further examination of angiotensin-II (Ang II) in mouse serum and arterial tissues with ELISA showed an increased expression of Ang II by MCMV infection. Consistent with the findings of the mouse trial, human CMV (HCMV) infection of blood vessel endothelial cells (EC) induced renin expression in a non-lytic infection manner. Viral replication kinetics and plaque formation assay showed that an active, CMV persistent infection in EC and expression of viral genes might underpin the molecular mechanism. These results show that CMV infection is a risk factor for increased arterial blood pressure, and is a co-factor in aortic atherosclerosis. Viral persistent infection of EC may underlie the mechanism. Control of CMV infection can be developed to restrict hypertension and atherosclerosis in the cardiovascular system.

Genetically modified mouse models used for studying the role of the AT2 receptor in cardiac hypertrophy and heart failure.

The actions of Angiotensin II have been implicated in many cardiovascular conditions. It is widely accepted that the cardiovascular effects of Angiotensin II are mediated by different subtypes of receptors: AT(1) and AT(2). These membrane-bound receptors share a part of their nucleic acid but seem to have different distribution and pathophysiological actions. AT(1) mediates most of the Angiotensin II actions since it is ubiquitously expressed in the cardiovascular system of the normal adult. Moreover AT(2) is highly expressed in the developing fetus but its expression in the cardiovascular system is low and declines after birth. However the expression of AT(2) appears to be modulated by pathological states such as hypertension, myocardial infarction or any pathology associated to tissue remodeling or inflammation. The specific role of this receptor is still unclear and different studies involving in vivo and in vitro experiments have shown conflicting data. It is essential to clarify the role of the AT(2) receptor in the different pathological states as it is a potential site for an effective therapeutic regimen that targets the Angiotensin II system. We will review the different genetically modified mouse models used to study the AT(2) receptor and its association with cardiac hypertrophy and heart failure.

Return to play after treating acute muscle injuries in elite football players with radial extracorporeal shock wave therapy.

BACKGROUND: To compare lay-off times achieved by treating acute muscle injuries in elite football players with a multimodal therapy approach that includes a specific protocol of almost daily radial extracorporeal shock wave therapy (rESWT) with corresponding data reported in the literature. METHODS: We performed a retrospective analysis of treatments and recovery times of muscle injuries suffered by the players of an elite football team competing in the first/second German Bundesliga during one of the previous seasons. RESULTS: A total of 20 acute muscle injuries were diagnosed and treated in the aforementioned season, of which eight (40%) were diagnosed as Type 1a/muscular tightness injuries, five (25%) as Type 2b/muscle strain injuries, four (20%) as Type 3a/partial muscle tear injuries and three (15%) as contusions. All injuries were treated with the previously mentioned multimodal therapy approach. Compared with data reported by Ekstrand et al. (Br J Sports Med 47:769-774, 2013), lay-off times (median/mean) were shortened by 54% and 58%, respectively, in the case of Type 1a injuries, by 50% and 55%, respectively, in the case of Type 2b injuries as well as by 8% and 21%, respectively, in the case of Type 3a injuries. No adverse reactions were observed. CONCLUSIONS: Overall, the multimodal therapy approach investigated in this study is a safe and effective treatment approach for treating Type 1a and 2b acute muscle injuries amongst elite football players and may help to prevent more severe, structural muscle injuries.

Exacerbation of acute viral myocarditis by tobacco smoke is associated with increased viral load and cardiac apoptosis.

Exposure to tobacco smoke is known to have deleterious cardiovascular effects. In this study, we tested whether exposure to tobacco smoke exacerbates the severity of viral myocarditis in mice. Viral myocarditis was generated in 4-week-old male BALB/c mice by injection of Encephalomyocarditis virus (EMCV). Four groups were studied: (1) control (C, no smoke and no virus); (2) smoke only (S, exposure to cigarette smoke for 90 min/day for 15 days); (3) virus only (V); and (4) exposure to smoke for 5 days before plus 10 days following virus injection (S+V). We found that viral inoculation preceded by smoke exposure increased mortality more than twofold compared with virus inoculation alone. In addition, the mRNA level of atrial natriuretic factor was significantly higher in S+V than among any of the other 3 groups. Virus injection significantly decreased cardiac function compared with controls, with further deterioration observed in the S+V group. We also observed a significantly increased rate of apoptosis, with an increased activation of apoptosis-inducing factor in hearts exposed to S+V compared with those exposed to V alone. Our results suggest that preexposure to smoke significantly exacerbates the severity of viral myocarditis, likely through increased viral load and increased cardiomyocyte cell death.

Metabolic modulation and cellular therapy of cardiac dysfunction and failure.

At present the prevalence of heart failure rises along with aging of the population. Current heart failure therapeutic options are directed towards disease prevention via neurohormonal antagonism (beta-blockers, angiotensin converting enzyme inhibitors and/or angiotensin receptor blockers and aldosterone antagonists), symptomatic treatment with diuretics and digitalis and use of biventricular pacing and defibrillators in a special subset of patients. Despite these therapies and device interventions heart failure remains a progressive disease with high mortality and morbidity rates. The number of patients who survive to develop advanced heart failure is increasing. These patients require new therapeutic strategies. In this review two of emerging therapies in the treatment of heart failure are discussed: metabolic modulation and cellular therapy. Metabolic modulation aims to optimize the myocardial energy utilization via shifting the substrate utilization from free fatty acids to glucose. Cellular therapy on the other hand has the goal to achieve true cardiac regeneration. We review the experimental data that support these strategies as well as the available pharmacological agents for metabolic modulation and clinical application of cellular therapy.

Cardiac dysfunction after left permanent cerebral focal ischemia: the brain and heart connection.

BACKGROUND AND PURPOSE: Stroke can lead to cerebrogenic cardiac arrhythmias. We sought to investigate the effect of ischemic stroke on cardiac function in a mouse model of permanent middle cerebral artery occlusion (pMCAO). METHODS: Twenty-four hours after the induction of focal ischemia, cardiac function was measured in mice by endovascular catheterization of the heart. Immediately after hemodynamic measurements, mice were euthanized and brains were excised and sectioned to measure infarct volume and the severity of insular cortex injury. Myocardial damage was evaluated by hematoxylin-eosin staining. Serum and heart levels of norepinephrine (NE) were also determined. RESULTS: Cardiac dysfunction occurred in 9 out of 14 mice that underwent left pMCAO. In these 9 mice, the severity of left insular cortex lesion was greater than the mice with normal heart function. The serum and heart levels of NE were significantly higher in left pMCAO mice with heart dysfunction. Liner regression analysis indicates significant inverse correlation between the severity of left insular cortex damage and heart dysfunction. Mice that underwent right pMCAO did not exhibit cardiac dysfunction. CONCLUSIONS: This study shows that left focal cerebral ischemia can produce cardiac dysfunction, which is associated with the extent of left insular cortex damage. Furthermore, mice exhibiting cardiac dysfunction had elevated levels of NE in the serum and heart.

Neuregulin-1 attenuated doxorubicin-induced decrease in cardiac troponins.

Neuregulin-1 (NRG1) is a potential therapeutic agent for the treatment of doxorubicin (Dox)-induced heart failure. NRG1, however, activates the erbB2 receptor, which is frequently overexpressed in breast cancers. It is, therefore, important to understand how NRG1, via erbB2, protects the heart against Dox cardiotoxicity. Here, we studied NRG1-erbB2 signaling in Dox-treated mice hearts and in isolated neonatal rat ventricular myocytes (NRVM). Male C57BL/6 mice were treated with recombinant NRG1 before and daily after a single dose of Dox. Cardiac function was determined by catheterization. Two-week survival was analyzed by the Kaplan-Meier method. Cardiac troponins [cardiac troponin I (cTnI) and cardiac troponin T (cTnT)] and phosphorylated Akt protein levels were determined in mice hearts and in NRVM by Western blot analysis. Activation of caspases and ubiquitinylation of troponins were determined in NRVM by caspase assay and immunoprecipitation. NRG1 significantly improved survival and cardiac function in Dox-treated mice. NRG1 reduced the decrease in cTnI, cTnT, and cardiac troponin C (cTnC) and maintained Akt phosphorylation in Dox-treated mice hearts. NRG1 reduced the decrease in cTnI and cTnT mRNA and proteins in Dox-treated NRVM. Inhibition of erbB2, phosphoinositide 3-kinase (PI3K), Akt, and mTOR blocked the protective effects of NRG1 on cTnI and cTnT in NRVM. NRG1 significantly reduced Dox-induced caspase activation, which degraded troponins, in NRVM. NRG1 reduced Dox-induced proteasome degradation of cTnI. NRG1 attenuates Dox-induced decrease in cardiac troponins by increasing transcription and translation and by inhibiting caspase activation and proteasome degradation of troponin proteins. NRG1 maintains cardiac troponins by the erbB2-PI3K pathway, which may lessen Dox-induced cardiac dysfunction.

Calcium and cyclic nucleotides affect TNF-alpha-induced stem cell migration.

The purpose of this study was to study the effect of calcium, cyclic AMP (cAMP) and cyclic GMP (cGMP) on embryonic stem cell (ESC) motility during TNF-alpha-induced chemotaxis. ESCs were monitored using a chemotaxis chamber, with different concentrations of calcium or cAMP or cGMP added to the medium. Changes in intracellular calcium ([Ca(2+)](i)) were measured with the fluorescent dye fura-2/AM. We combined migratory parameters in a mathematical model and described it as "mobility". After adding calcium, a dose-dependant increase in cell speed was found. Cyclic AMP increased mobility as well as the [Ca(2+)](i). In contrast, adding dbcGMP resulted in a significant decrease in the mobility of the ESCs. During migration ESCs showed an increase in [Ca(2+)](i). Furthermore, TNF-alpha dramatically increased the movement as well as the directionality of ESCs. These results demonstrate that ESCs are highly motile and respond to different concentrations of calcium in a dose-related manner.

Clozapine-induced myocarditis: role of catecholamines in a murine model.

Clozapine, an atypical antipsychotic, is very effective in the treatment of resistant schizophrenia. However, cardiotoxicity of clozapine, particularly in young patients, has raised concerns about its safety. Increased catecholamines have been postulated to trigger an inflammatory response resulting in myocarditis, dilated cardiomyopathy, and death, although this has not yet been thoroughly studied. Here, we used the mouse to study whether clozapine administration could cause adverse myocarditis associated with an increase in catecholamines. Male Balb/C mice, age ~6 weeks, were administered 5, 10 or 25 mg/kg clozapine daily for 7 and 14 days; one group was administered 25 mg/kg clozapine plus 2 mg/kg propranolol for 14 days. Saline-treated mice served as controls. Heart sections were stained with hematoxylin and eosin for histopathological examination. Plasma catecholamines were measured with HPLC. Myocardial TNF-alpha concentrations were determined by ELISA. Histopathology of clozapine-treated mice showed a significant dose-related increase in myocardial inflammation that correlated with plasma catecholamine levels and release of TNF-alpha. Propranolol significantly attenuated these effects. A hypercatecholaminergic state induced by clozapine could explain the occurrence of myocarditis in some patients. Our data suggest that a beta-adrenergic blocking agent may be effective in reducing the incidence and severity of clozapine-induced myocarditis.

Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia.

Preeclampsia, a syndrome affecting 5% of pregnancies, causes substantial maternal and fetal morbidity and mortality. The pathophysiology of preeclampsia remains largely unknown. It has been hypothesized that placental ischemia is an early event, leading to placental production of a soluble factor or factors that cause maternal endothelial dysfunction, resulting in the clinical findings of hypertension, proteinuria, and edema. Here, we confirm that placental soluble fms-like tyrosine kinase 1 (sFlt1), an antagonist of VEGF and placental growth factor (PlGF), is upregulated in preeclampsia, leading to increased systemic levels of sFlt1 that fall after delivery. We demonstrate that increased circulating sFlt1 in patients with preeclampsia is associated with decreased circulating levels of free VEGF and PlGF, resulting in endothelial dysfunction in vitro that can be rescued by exogenous VEGF and PlGF. Additionally, VEGF and PlGF cause microvascular relaxation of rat renal arterioles in vitro that is blocked by sFlt1. Finally, administration of sFlt1 to pregnant rats induces hypertension, proteinuria, and glomerular endotheliosis, the classic lesion of preeclampsia. These observations suggest that excess circulating sFlt1 contributes to the pathogenesis of preeclampsia.

Antiapoptotic effect of implanted embryonic stem cell-derived early-differentiated cells in aging rats after myocardial infarction.

This study tested whether implanted embryonic stem cell-derived early-differentiated cells (EDCs) lead to improvement in cardiac function by preventing cardiac apoptosis in aging rats after myocardial infarction. Cardiac apoptosis after transplantation of EDCs was assessed in situ by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling reaction (TUNEL) staining as well as by measurements of protein levels of cleaved caspases 3, Bax, and Bcl-2. Our results indicate that cell transplantation improved cardiac function at 6-months observation. The frequency of apoptotic cells in the peri-infarcted myocardium 3 days after cell transplantation was significantly decreased in the cell transplantation group. EDC therapy decreased the protein levels of cleaved caspase 3 and Bax, and increased the level of Bcl-2 in comparison to myocardial infarction control. Additionally, the number of apoptotic cells decreased significantly in cardiomyocytes precocultured with EDCs. This study demonstrates that functional improvement of EDC transplantation may at least in part be related to a reduction in cardiomyocyte apoptosis.

Circadian variation in the onset of myocardial infarction: effect of duration of diabetes.

There are conflicting reports regarding circadian variation in the onset of acute myocardial infarction (MI) among patients with diabetes. We therefore, studied the circadian pattern of the incidence of acute MI in patients (n = 3,882) who were enrolled in the Onset Study stratified by the presence, type, and duration of diabetes. The Onset Study was conducted at 64 U.S. medical centers between August 1989 and September 1996. We used harmonic regression model to evaluate the circadian variation of MI symptom onset in patients with and without diabetes. Subgroup analysis was performed according to the presence, type, and duration of diabetes by the chi(2) test (dividing the day into four 6-h intervals). Patients without diabetes exhibited a prominent morning peak in the incidence of acute MI symptom onset (P < 0.001). In contrast, patients with type 1 diabetes and type 2 diabetes > or =5 years had a marked attenuation of the morning peak. Patients who had type 2 diabetes diagnosed within the previous 5 years had a pattern of onset of acute MI similar to patients without diabetes. Patients with type 1 diabetes and those with type 2 diabetes > or =5 years have an attenuation of the morning peak in acute MI. Inconsistency in observation of such an effect in patients with diabetes in the past may well have been due to difference in the duration of diabetes and thus the variable extent of underlying autonomic dysfunction.

Stem cell therapy in the aging hearts of Fisher 344 rats: synergistic effects on myogenesis and angiogenesis.

OBJECTIVE: Advanced age is a major risk factor for ventricular dysfunction and reduction of cardiac reserve. Finding novel approaches to prevent and attenuate heart dysfunction associated with advanced age is a major therapeutic challenge. The present study was designed to test whether engrafted embryonic stem cells could improve myocardial function in aging hearts. METHODS: Cultured mouse embryonic stem cells used for cell therapy were transfected with green fluorescent protein. Aging rats in the cell-treated group received intramyocardial injection of embryonic stem cells. Hemodynamic measurement, myocyte counting, and evaluation of blood flow were performed 6 weeks after cell transplantation. RESULTS: Embryonic stem cell therapy partially improved cardiac reserve, as reflected by the in vivo response to isoproterenol (INN: isoprenaline) stimulation in aging hearts 6 weeks after cell implantation. The functional benefits from engrafted embryonic stem cells were associated with increased myocyte numbers and enhanced left ventricular blood perfusion in the aging heart. The characteristic phenotype of engrafted embryonic stem cells was identified in the transplanted heart on the basis of green fluorescent protein-positive spots that were further demonstrated to differentiate into cardiac tissue with positive staining for cardiac alpha-myosin heavy chain. CONCLUSIONS: Regenerating cardiomyocytes and increasing regional blood perfusion in the aging heart after embryonic stem cell transplantation synergistically resulted in improvement of cardiac function. Embryonic stem cell transplantation might hold significant clinical potential in attenuating the progressive decrease of cardiac function associated with advanced aging.

Cardiomyocytes overexpressing TNF-alpha attract migration of embryonic stem cells via activation of p38 and c-Jun amino-terminal kinase.

Tumor necrosis factor-alpha (TNF-alpha) plays an important role in the pathogenesis of myocardial infarction. Stem cells are able to regenerate infarcted myocardium. This study investigated whether TNF-alpha was able to induce migration of embryonic stem cells (ESCs) in vitro. We used a Transwell assay in which neonatal rat cardiomyocytes, with or without transfection of TNF-alpha cDNA, were plated in the lower compartments and mouse ESCs tagged with green fluorescent protein were added to the upper compartments. TNF-alpha level was significantly increased in the medium of the lower compartments seeded with TNF-alpha-transfected cardiomyocytes. Compared with the controls, overexpression of TNF-alpha significantly enhanced migration of ESCs to the lower compartments. This enhancement was attenuated by preincubation of ESCs with the antibody against the type II TNF-alpha receptor (TNF-RII), but not by the antibody against the type I TNF-alpha receptor (TNF-RI). Western blot analysis showed that the phosphorylated protein levels of p38 and c-Jun amino-terminal kinase (JNK) were significantly increased in TNF-alpha-treated ESCs. Inhibition of the activity of p38 or JNK significantly attenuated TNF-alpha-induced ESC migration. Our data demonstrate that excessive TNF-alpha stimulates TNF-RII and enhances migration of ESCs in vitro. Activation of p38 and JNK is required for TNF-alpha-enhanced ESC migration.

Gait dynamics in mouse models of Parkinson's disease and Huntington's disease.

BACKGROUND: Gait is impaired in patients with Parkinson's disease (PD) and Huntington's disease (HD), but gait dynamics in mouse models of PD and HD have not been described. Here we quantified temporal and spatial indices of gait dynamics in a mouse model of PD and a mouse model of HD. METHODS: Gait indices were obtained in C57BL/6J mice treated with the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 30 mg/kg/day for 3 days) for PD, the mitochondrial toxin 3-nitropropionic acid (3NP, 75 mg/kg cumulative dose) for HD, or saline. We applied ventral plane videography to generate digital paw prints from which indices of gait and gait variability were determined. Mice walked on a transparent treadmill belt at a speed of 34 cm/s after treatments. RESULTS: Stride length was significantly shorter in MPTP-treated mice (6.6 +/- 0.1 cm vs. 7.1 +/- 0.1 cm, P < 0.05) and stride frequency was significantly increased (5.4 +/- 0.1 Hz vs. 5.0 +/- 0.1 Hz, P < 0.05) after 3 administrations of MPTP, compared to saline-treated mice. The inability of some mice treated with 3NP to exhibit coordinated gait was due to hind limb failure while forelimb gait dynamics remained intact. Stride-to-stride variability was significantly increased in MPTP-treated and 3NP-treated mice compared to saline-treated mice. To determine if gait disturbances due to MPTP and 3NP, drugs affecting the basal ganglia, were comparable to gait disturbances associated with motor neuron diseases, we also studied gait dynamics in a mouse model of amyotrophic lateral sclerosis (ALS). Gait variability was not increased in the SOD1 G93A transgenic model of ALS compared to wild-type control mice. CONCLUSION: The distinct characteristics of gait and gait variability in the MPTP model of Parkinson's disease and the 3NP model of Huntington's disease may reflect impairment of specific neural pathways involved.

Embryonic stem cells cultured in biodegradable scaffold repair infarcted myocardium in mice.

Our previous findings demonstrated that directly injecting embryonic stem cells (ESCs) into ischemic region of the heart improved cardiac function in animals with experimental myocardial infarction (MI). Tissue engineering with stem cells may provide tissue creation and repair. This study was designed to investigate the effectiveness of grafting of ESC-seeded biodegradable patch on infarcted heart. MI in mice was induced by ligation of the left coronary artery. Mouse ESCs were seeded on polyglycolic-acid (PGA) material patches. Three days after culture, an ESC-seeded patch was transplanted on the surface of ischemic and peri-ischemic myocardium. Eight weeks after MI operation and patch transplantation, hemodynamics and cardiac function were evaluated in four (sham-operated, MI, MI + cell-free patch, and MI + ESC-patch) groups of mice. The blood pressure and left ventricular function were significantly reduced in the MI animals. Compared with MI alone and MI + cell-free patch groups, the animals received MI + ESC-seeded patches significantly improved blood pressure and ventricular function. The survival rate of the MI mice grafted with MI + ESC-seeded patches was markedly higher than that in MI alone or MI + cell-free patch animals. GFP-positive tissue was detected in infarcted area with grafting of ESC-seeded patch, which suggests the survivors of ESCs and possible myocardial regeneration. Our data demonstrate that grafting of ESC-seeded bioabsorbable patch can repair infarcted myocardium and improve cardiac function in MI mice. This novel approach of combining stem cells and biodegradable materials may provide a therapeutic modality for repairing injured heart.

The metabolic and cardiovascular effects of hyperthyroidism are largely independent of beta-adrenergic stimulation.

Hyperthyroidism and states of adrenergic hyperactivity have many common clinical features, suggesting similar pathogenic mechanisms of action. The widespread use of beta-adrenergic receptor (betaAR) antagonists (beta-blockers) to treat hyperthyroidism has led to the belief that the physiological consequences of thyroid hormone (TH) excess are mediated in part via catecholamine signaling through betaARs. To test this hypothesis, we compared the response to TH excess in mice lacking the three known betaARs (beta-less) vs. wild-type (WT) mice. Although beta-less mice had a lower heart rate at baseline in comparison to WT mice, the metabolic and cardiovascular responses to hyperthyroidism were equivalent in both WT and beta-less mice. These data indicate that the metabolic and cardiovascular effects of TH excess are largely independent of betaARs. These findings suggest that the efficacy of clinical treatment of hyperthyroidism with beta-blockers is due to antagonism of sympathetic signaling, and that this process functions independently of TH action.

Obesity and the risk of death after acute myocardial infarction.

BACKGROUND: In the general population, obesity is associated with an increased risk of all-cause death. However, the importance of obesity in patients with established coronary heart disease is less well defined. METHODS: As part of the Determinants of Myocardial Infarction Onset Study, we performed a prospective cohort study of 1898 patients hospitalized with confirmed acute myocardial infarction between 1989 and 1994, with a median follow-up of 3.8 years. We assessed all-cause death through December 1995, using the National Death Index. We categorized patients according to WHO criteria for body mass index (BMI). We compared long-term death according to BMI (kg/m2) by using Cox proportional hazards regression. RESULTS: Of the 1898 eligible patients, 607 (32%) were normal weight (18.5 to 24.9 kg/m2), 832 (44%) were overweight (25.0 to 29.9 kg/m2), 331 (17%) were class I obese (30.0 to 34.9 kg/m2), and 128 (7%) were class II or more obese (> or =35.0 kg/m2). A total of 311 patients died during follow-up. After adjustment for potentially confounding risk factors and excluding patients with noncardiac comorbidity, the risk for death appeared to increase linearly, with increasing BMI across all categories (P for trend =.08). The relative risk of death in all obese patients (> or =30 kg/m2) was 1.46, compared with those with normal weight (95% CI, 0.98 to 2.17). CONCLUSIONS: We found that BMI appeared to have a positive, graded relation with post-myocardial infarction death. Whether weight reduction and secondary prevention strategies would reverse this effect in obese population remains to be seen.

Enhancement of nitric oxide production by methylecgonidine in cultured neonatal rat cardiomyocytes.

1. In the present experiments, we investigated the effects of methylecgonidine (MEG) on nitric oxide (NO) production in cultured neonatal rat cardiomyocytes. Incubation of cultured cardiomyocytes with carbachol or MEG for 48 h significantly enhanced NO production. No release was increased from 1.48+/-0.13 microM (mg protein)(-1) for control to 5.73+/-0.19 microM (mg protein)(-1) for 1 microM carbachol treated cells (P<0.001). In addition, incubation with 1 microM MEG enhanced NO production to 5.55+/-0.28 microM (mg protein)(-1). The effects of MEG on NO production were concentration-dependent. The muscarinic antagonist atropine prevented the enhancement of NO production induced by carbachol or MEG. Compared to MEG-induced NO production, cocaine was much less potent. 2. The enhancement of NO production by carbachol or MEG was even greater in cultured cardiomyocytes transfected with the M(2) cDNA. After 48-h incubation with 1 microM carbachol or 1 microM MEG, NO production was increased by 6.5 and 6.7 fold, respectively, in cardiomyocytes overexpressing M(2) receptors. Coincubation with atropine or N(G)-nitro-L-arginine methyl ester abolished the enhancement of NO production. In contrast, NO production enhanced by carbachol or MEG in M(1)- or M(3)-transfected cardiomyocytes was similar to the level in non-transfected cells. 3. Western blot analysis showed that the protein levels of M(1), M(2), and M(3) were significantly increased in cardiomyocytes transfected with the receptor cDNAs, but MEG had no effect on the expressions. It is interesting that both carbachol and MEG caused a significant increase in constitutive endothelial NO synthase (eNOS) only in M(2)-transfected cardiomyocytes, not in non-transfected, M(1)- or M(3)-transfected cells. Again, atropine blocked the MEG-produced induction of eNOS. 4. Our data demonstrate that MEG significantly enhanced NO production in cultured cardiomyocytes and that the enhancement of NO production may result from MEG stimulation of muscarinic M(2) receptors.