Douglas B. Sawyer

Opposing Effects of β1- and β2-Adrenergic Receptors on Cardiac Myocyte Apoptosis Role of a Pertussis Toxin–Sensitive G Protein

Background—β-Adrenergic receptor (β-AR) stimulation increases apoptosis in adult rat cardiac (ventricular) myocytes (ARVMs) via activation of adenylyl cyclase. β2-ARs may couple to a Gi-mediated signaling pathway that can oppose the actions of adenylyl cyclase. Methods and Results—In ARVMs, β-AR stimulation for 24 hours increased the number of apoptotic cells as measured by flow cytometry. β-AR–stimulated apoptosis was abolished by the β1-AR–selective antagonist CGP 20712A (P<0.05 versus β-AR stimulation alone) but was potentiated by the β2-AR–selective antagonist ICI 118,551 (P<0.05 versus β-AR stimulation alone). The muscarinic agonist carbachol also prevented β-AR–stimulated apoptosis (P<0.05 versus β-AR stimulation alone), whereas pertussis toxin potentiated the apoptotic action of β-AR stimulation (P<0.05 versus β-AR stimulation alone) and prevented the antiapoptotic action of carbachol. Conclusions—In ARVMs, stimulation of β1-ARs increases apoptosis via a cAMP-dependent mechanism, whereas stimulatio...

Inflammatory Markers and Risk of Heart Failure in Elderly Subjects Without Prior Myocardial Infarction

Background—Experimental studies support a key role for cytokines in left ventricular remodeling. In congestive heart failure (CHF) patients, elevated plasma cytokine levels are associated with worse functional status and adverse prognosis. It is unclear whether cytokine levels can predict the incidence of CHF in asymptomatic individuals. Methods and Results—We investigated the relations of serum interleukin-6 (IL-6), C-reactive protein (CRP), and spontaneous production of tumor necrosis factor-&agr; (TNF&agr;) by peripheral blood mononuclear cell (PBMC) to CHF incidence among 732 elderly Framingham Study subjects (mean age 78 years, 67% women) free of prior myocardial infarction and CHF. On follow-up (mean 5.2 years), 56 subjects (35 women) developed CHF. After adjustment for established risk factors, including the occurrence of myocardial infarction on follow-up, there was a 60 (PBMC TNF&agr;) to 68% (serum IL-6) increase in risk of CHF per tertile increment in cytokine concentration (P =0.04, and 0.03, respectively, for trend). A serum CRP level ≥5 mg/dL was associated with a 2.8-fold increased risk of CHF (P =0.02). Subjects with elevated levels of all 3 biomarkers (serum IL-6 and PBMC TNF&agr; >median values, CRP≥5 mg/dL) had a markedly increased risk of CHF (hazards ratio 4.07 [95% CI 1.34 to 12.37], P =0.01) compared with the other subjects. Conclusions—In our elderly, community-based sample, a single determination of serum inflammatory markers, particularly elevated IL-6, was associated with increased risk of CHF in people without prior myocardial infarction. Additional epidemiological investigations are warranted to confirm the contribution of inflammation to the pathogenesis of CHF in the general population.

Cell Therapy Attenuates Deleterious Ventricular Remodeling and Improves Cardiac Performance After Myocardial Infarction

BackgroundMyocardial infarction (MI) promotes deleterious remodeling of the myocardium, resulting in ventricular dilation and pump dysfunction. We examined whether supplementing infarcted myocardium with skeletal myoblasts would (1) result in viable myoblast implants, (2) attenuate deleterious remodeling, and (3) enhance in vivo and ex vivo contractile performance. Methods and ResultsExperimental MI was induced by 1-hour coronary ligation followed by reperfusion in adult male Lewis rats. One week after MI, 106 myoblasts were injected directly into the infarct region. Three groups of animals were studied at 3 and 6 weeks after cell therapy: noninfarcted control (control), MI plus sham injection (MI), and MI plus cell injection (MI+cell). In vivo cardiac function was assessed by maximum exercise capacity testing and ex vivo function was determined by pressure-volume curves obtained from isolated, red cell-perfused, balloon-in-left ventricle (LV) hearts. MI and MI+cell hearts had indistinguishable infarct sizes of ≈30% of the LV. At 3 and 6 weeks after cell therapy, 92% (13 of 14) of MI+cell hearts showed evidence of myoblast graft survival. MI+cell hearts exhibited attenuation of global ventricular dilation and reduced septum-to-free wall diameter compared with MI hearts not receiving cell therapy. Furthermore, cell therapy improved both post-MI in vivo exercise capacity and ex vivo LV systolic pressures. ConclusionsImplanted skeletal myoblasts form viable grafts in infarcted myocardium, resulting in enhanced post-MI exercise capacity and contractile function and attenuated ventricular dilation. These data illustrate that syngeneic myoblast implantation after MI improves both in vivo and ex vivo indexes of global ventricular dysfunction and deleterious remodeling and suggests that cellular implantation may be beneficial after MI.

Anthracycline Cardiotoxicity: From Bench to Bedside

Anthracyclines remain among the most widely prescribed and effective anticancer agents. Unfortunately, life-threatening cardiotoxicity continues to compromise their usefulness. Despite more than four decades of investigation, the pathogenic mechanisms responsible for anthracycline cardiotoxicity have not been completely elucidated. In addition, new drugs and combination therapies often exacerbate the toxicity. The First International Workshop on Anthracycline Cardiotoxicity, held in fall 2006, in Como, Italy, focused on the state-of-the-art knowledge and discussed the research needed to address the cardiotoxicity of these drugs. Here, we incorporate these discussions into the framework of a broader review of preclinical and clinical issues.

Cardiovascular side effects of cancer therapies: a position statement from the Heart Failure Association of the European Society of Cardiology

The reductions in mortality and morbidity being achieved among cancer patients with current therapies represent a major achievement. However, given their mechanisms of action, many anti‐cancer agents may have significant potential for cardiovascular side effects, including the induction of heart failure. The magnitude of this problem remains unclear and is not readily apparent from current clinical trials of emerging targeted agents, which generally under‐represent older patients and those with significant co‐morbidities. The risk of adverse events may also increase when novel agents, which frequently modulate survival pathways, are used in combination with each other or with other conventional cytotoxic chemotherapeutics. The extent to which survival and growth pathways in the tumour cell (which we seek to inhibit) coincide with those in cardiovascular cells (which we seek to preserve) is an open question but one that will become ever more important with the development of new cancer therapies that target intracellular signalling pathways. It remains unclear whether potential cardiovascular problems can be predicted from analyses of such basic signalling mechanisms and what pre‐clinical evaluation should be undertaken. The screening of patients, optimization of therapeutic schemes, monitoring of cardiovascular function during treatment, and the management of cardiovascular side effects are likely to become increasingly important in cancer patients. This paper summarizes the deliberations of a cross‐disciplinary workshop organized by the Heart Failure Association of the European Society of Cardiology (held in Brussels in May 2009), which brought together clinicians working in cardiology and oncology and those involved in basic, translational, and pharmaceutical science.

Transgenic Mice Overexpressing Mutant PRKAG2 Define the Cause of Wolff-Parkinson-White Syndrome in Glycogen Storage Cardiomyopathy

Background—Mutations in the &ggr;2 subunit (PRKAG2) of AMP-activated protein kinase produce an unusual human cardiomyopathy characterized by ventricular hypertrophy and electrophysiological abnormalities: Wolff-Parkinson-White syndrome (WPW) and progressive degenerative conduction system disease. Pathological examinations of affected human hearts reveal vacuoles containing amylopectin, a glycogen-related substance. Methods and Results—To elucidate the mechanism by which PRKAG2 mutations produce hypertrophy with electrophysiological abnormalities, we constructed transgenic mice overexpressing the PRKAG2 cDNA with or without a missense N488I human mutation. Transgenic mutant mice showed elevated AMP-activated protein kinase activity, accumulated large amounts of cardiac glycogen (30-fold above normal), developed dramatic left ventricular hypertrophy, and exhibited ventricular preexcitation and sinus node dysfunction. Electrophysiological testing demonstrated alternative atrioventricular conduction pathways consistent with WPW. Cardiac histopathology revealed that the annulus fibrosis, which normally insulates the ventricles from inappropriate excitation by the atria, was disrupted by glycogen-filled myocytes. These anomalous microscopic atrioventricular connections, rather than morphologically distinct bypass tracts, appeared to provide the anatomic substrate for ventricular preexcitation. Conclusions—Our data establish PRKAG2 mutations as a glycogen storage cardiomyopathy, provide an anatomic explanation for electrophysiological findings, and implicate disruption of the annulus fibrosis by glycogen-engorged myocytes as the cause of preexcitation in Pompe, Danon, and other glycogen storage diseases.

Inhibition of Copper-Zinc Superoxide Dismutase Induces Cell Growth, Hypertrophic Phenotype, and Apoptosis in Neonatal Rat Cardiac Myocytes In Vitro

Oxidative stress has been implicated in the pathophysiology of myocardial failure. We tested the hypothesis that inhibition of endogenous antioxidant enzymes can regulate the phenotype of cardiac myocytes. Neonatal rat ventricular myocytes in vitro were exposed to diethyldithiocarbamic acid (DDC), an inhibitor of cytosolic (Cu, Zn) and extracellular superoxide dismutase (SOD). DDC inhibited SOD activity and increased intracellular superoxide in a concentration-dependent manner. A low concentration (1 micromol/L) of DDC stimulated myocyte growth, as demonstrated by increases in protein synthesis, cellular protein, prepro-atrial natriuretic peptide, and c-fos mRNAs and decreased sarcoplasmic reticulum Ca(2+)ATPase mRNA. These actions were all inhibited by the superoxide scavenger Tiron (4,5-dihydroxy-1,3-benzene disulfonic acid). Higher concentrations of DDC (100 micromol/L) stimulated myocyte apoptosis, as evidenced by DNA laddering, characteristic nuclear morphology, in situ terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL), and increased bax mRNA expression. DDC-stimulated apoptosis was inhibited by the SOD/catalase mimetic EUK-8. The growth and apoptotic effects of DDC were mimicked by superoxide generation with xanthine plus xanthine oxidase. Thus, increased intracellular superoxide resulting from inhibition of SOD causes activation of a growth program and apoptosis in cardiac myocytes. These findings support a role for oxidative stress in the pathogenesis of myocardial remodeling and failure.

Vascular Endothelial Growth Factor Blockade Promotes the Transition From Compensatory Cardiac Hypertrophy to Failure in Response to Pressure Overload

Cardiac hypertrophy is associated with upregulation of vascular endothelial growth factor (VEGF) in the myocardium. Here, we evaluated the effects of a decoy VEGF receptor on heart morphology and function to a murine model of pressure overload hypertrophy. Mice were administered adenoviral vector encoding a decoy VEGF receptor (Ad-Flk), and their hearts were subjected to pressure overload by transverse aortic constriction (TAC). Treatment with Ad-Flk led to a net reduction in capillary density in hearts subjected to TAC. Ad-Flk also led to a reduction in TAC-induced cardiac hypertrophy and promoted left ventricle dilatation and a loss in contractile function. Treatment with Ad-Flk markedly increased myocardial fibrosis and collagen gene upregulation. In contrast, Ad-Flk had no effect on any of these parameters in sham-treated mice. Administration of a VEGF trap reagent diminished pressure overload cardiac hypertrophy and promoted the progression to heart failure but had no effect on sham-treated animals. These findings suggest that VEGF is required to maintain myocardial capillary density and that reductions in the vascular bed are associated with the transition from compensatory hypertrophy to failure.

β-Adrenergic Receptor–Stimulated Apoptosis in Cardiac Myocytes Is Mediated by Reactive Oxygen Species/c-Jun NH2-Terminal Kinase–Dependent Activation of the Mitochondrial Pathway

Stimulation of &bgr;-adrenergic receptors (&bgr;ARs) causes apoptosis in adult rat ventricular myocytes (ARVMs). The role of reactive oxygen species (ROS) in mediating &bgr;AR-stimulated apoptosis is not known. Stimulation of &bgr;ARs with norepinephrine (10 &mgr;mol/L) in the presence of prazosin (100 nmol/L) for 24 hours increased the number of apoptotic myocytes as determined by TUNEL staining by 3.6- fold. The superoxide dismutase/catalase mimetics Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (MnTMPyP; 10 &mgr;mol/L) and Euk-134 decreased &bgr;AR-stimulated apoptosis by 89±6% and 76±10%, respectively. Infection with an adenovirus expressing catalase decreased &bgr;AR-stimulated apoptosis by 82±15%. The mitochondrial permeability transition pore inhibitor bongkrekic acid (50 &mgr;mol/L) decreased &bgr;AR-stimulated apoptosis by 76±8%, and the caspase inhibitor zVAD-fmk (25 &mgr;mol/L) decreased &bgr;AR-stimulated apoptosis by 62±11%. &bgr;AR-stimulated cytochrome c release was inhibited by MnTMPyP. &bgr;AR stimulation caused c-Jun NH2-terminal kinase (JNK) activation, which was abolished by MnTMPyP. Transfection with an adenovirus expressing dominant-negative JNK inhibited &bgr;AR-stimulated apoptosis by 81±12%, and the JNK inhibitor SP600125 inhibited both &bgr;AR-stimulated apoptosis and cytochrome c release. Thus, &bgr;AR-stimulated apoptosis in ARVMs involves ROS/JNK-dependent activation of the mitochondrial death pathway.

Serum Insulin-like Growth Factor I and Risk for Heart Failure in Elderly Individuals without a Previous Myocardial Infarction: The Framingham Heart Study

Context Biological research and retrospective clinical studies show that insulin-like growth factor I (IGF-I) promotes salutary left ventricular modeling. Contribution This community-based study followed 717 elderly people without known myocardial infarction or heart failure for 5 to 9 years. Participants with higher levels of IGF-I developed heart failure less often than did those with lower levels. Implications High IGF-I levels are associated with decreased risks for heart failure. Cautions High IGF-I levels are also associated with increased risks for cancer. Future research should confirm these associations and explore mechanisms to increase IGF-I levels in people at risk for heart failure without increasing the risk for cancer. The Editors Considerable experimental and clinical evidence supports a key role of insulin-like growth factor I (IGF-I) in the pathogenesis of left ventricular remodeling and heart failure (1). In basic science studies, IGF-I promotes myocardial hypertrophy, increases cardiac contractility, and attenuates myocyte necrosis and apoptosis in models of ischemic, hemodynamic, and toxic cardiac injury (2-7). In clinical reports, patients with congestive heart failure have lower levels of serum IGF-I that correlate with the degree of left ventricular systolic dysfunction, presence of cachexia and skeletal muscle weakness, and indices of neurohormonal and cytokine activation (8, 9). Furthermore, administering IGF-I has been reported to improve left ventricular contractility indices in small clinical case series (10) and experimental models of heart failure (4). Epidemiologic investigations have shown an increasing incidence of congestive heart failure with age (11). Serum IGF-I levels have been reported to decrease with age in community-based investigations (12, 13). These parallel observations suggest that a decrease in serum IGF-I level with age may increase the incidence of heart failure in the elderly (14). However, no previous investigation has prospectively examined the relationship of serum IGF-I level to the incidence of heart failure. We hypothesized, on the basis of the body of scientific evidence noted, that a low serum IGF-I level may be associated with an increased risk for congestive heart failure. Accordingly, we examined the relationship of serum IGF-I levels to the risk for congestive heart failure in an elderly, community-based sample. Methods Study Sample The Framingham Heart Study began in 1948 as a prospective longitudinal epidemiologic investigation of 5209 women and men who are examined every 2 years (15). The 1167 participants who were alive at the time of the 22nd biennial examination cycle (between 1992 and 1994) were eligible for the present investigation. We excluded 450 participants (39%) for the following reasons: prevalent congestive heart failure (84 participants) or myocardial infarction (106 participants) and insufficient blood specimen for IGF-I analyses (260 participants). Participants with a myocardial infarction were excluded because low serum IGF-I level is a risk factor for myocardial infarction (16), which in turn is a powerful risk factor for heart failure (11). After the exclusions, 717 participants (mean age, 78.4 years; 479 women) were eligible. At the baseline examination, all participants underwent a medical history, routine physical examination (including anthropometry and blood pressure measurement), 12-lead electrocardiography, and laboratory assessment of cardiovascular disease risk factors. The institutional review board at Boston Medical Center approved the study, and all participants gave written informed consent. Measurement of Serum IGF-I At the baseline examination, 3 mL of serum from each participant was obtained in the early afternoon (with the participant in a supine position and a nonfasting state). Specimens were immediately transported by courier from Framingham, Massachusetts, to the U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts, and stored at 80 C. Serum on dry ice was then shipped to Endocrine Sciences, Inc. (Calabasas Hills, California). Serum IGF-I was measured by radioimmunoassay after acid ethanol extraction (17) in 3 batches; the intrassay coefficient of variation was less than 4%. Insulin-like growth factorbinding proteins were not measured. Outcome All participants were under continuous surveillance for the development of cardiovascular disease events, including congestive heart failure. Three experienced investigators reviewed all suspected congestive heart failure events by examining hospital records, information from physicians, and pathology reports by using previously described methods (18). Congestive heart failure was diagnosed on the basis of the previously detailed Framingham Heart Study criteria (19); these criteria, which require the simultaneous presence of at least 2 major criteria or 1 major criterion in conjunction with 2 minor criteria to diagnose heart failure, have been validated previously (20). Major criteria included paroxysmal nocturnal dyspnea or orthopnea, jugular venous distention, pulmonary rales, radiographic cardiomegaly, acute pulmonary edema, third heart sound, central venous pressure greater than 16 cm H2O, hepatojugular reflux, and weight loss of at least 4.5 kg in 5 days in response to treatment of heart failure. Minor criteria included bilateral ankle edema, nocturnal cough, dyspnea on ordinary exertion, hepatomegaly, pleural effusion, and heart rate of 120 beats/min or greater. Minor criteria were acceptable only if they were not attributed to another medical condition (such as chronic lung disease, cirrhosis, ascites, or nephrotic syndrome). Statistical Methods We used multivariable Cox proportional-hazards regression models (21) to examine the association of serum IGF-I with the incidence of congestive heart failure. Since there was no effect modification by sex, all analyses were performed for pooled sexes, with sex as a covariate. The distribution of serum IGF-I was slightly skewed positively, and raw values were log-transformed to reduce excessive influence of high values at the upper end of the distribution. Other investigators have used a similar approach (12, 22). Serum IGF-I was treated both as a continuous variable (raw value and with natural logarithmic transformation) and as a categorical variable (values below versus at or above the median value of 140 g/L). Age- and sex-adjusted probabilities of developing congestive heart failure on follow-up were estimated for individuals with serum IGF-I values below versus at or above the median value of 140 g/L from Cox regression models incorporating age and sex as covariates. In multivariable analyses, we adjusted for the following covariates (defined at the baseline examination): age, sex, ratio of serum total cholesterol to high-density lipoprotein (HDL) cholesterol, diabetes mellitus, systolic and diastolic blood pressure, antihypertensive treatment, smoking status, body mass index, clinical valve disease, prevalent atrial fibrillation, electrocardiographic left ventricular hypertrophy, and prevalent cardiovascular disease other than myocardial infarction (individuals with myocardial infarction were excluded, as noted). All 717 participants had complete information on these covariates. Criteria for these covariates have been described previously (18). We examined the following statistical models in hierarchical fashion: multivariable models with all covariates defined at baseline, as detailed, ignoring the occurrence of myocardial infarction on follow-up (model A); models adjusting for covariates at baseline and the occurrence of a myocardial infarction on follow-up (model B) (the latter adjustment is important because low serum IGF-I levels can predispose to the development of congestive heart failure by promoting myocardial infarction [16]); and models in which participants who experienced a myocardial infarction on follow-up were censored at the time of myocardial infarction (model C). Hazard ratios and their 95% CIs were estimated for each SD increase in the log IGF-I and raw IGF-I, and for levels at or above 140 g/L versus below (separately for each statistical model). Additional Analyses In the statistical models adjusting for covariates defined at the baseline examination and occurrence of an interim myocardial infarction (model B), we performed analyses incorporating several interaction terms (serum IGF-I level covariate) to examine for any variation in the effect of serum IGF-I on congestive heart failure hazard (effect modification) according to age, sex, body mass index, presence or absence of diabetes, and total cholesterolHDL cholesterol ratio. High IGF-I levels in conditions such as acromegaly may be associated with cardiac systolic and diastolic dysfunction (23). We therefore evaluated for any nonlinearity (U shape) in the relationship of serum IGF-I level to risk for congestive heart failure. Furthermore, since serum interleukin-6 has been reported to lower IGF-I levels (24) and cytokines have been implicated in the pathogenesis of congestive heart failure (25), we performed supplementary analyses adjusting for serum interleukin-6 in a subgroup of 525 individuals who had both serum interleukin-6 (enzyme-linked immunosorbent assay, R&D Systems, Minneapolis, Minnesota) and IGF-I measurements available. We performed secondary analyses in which death was modeled as a competing outcome to congestive heart failure (26), given the high mortality rate in our elderly cohort. Although left ventricular systolic function was not assessed at the baseline examination, routine echocardiography was done at examination cycle 20, approximately 4 years from the baseline examination. Additional analyses were performed on a subgroup of individuals who attended examination cycle 20 and had normal left ventricular systolic function at that examination. A 2-sided P