Pro-Inflammatory Biomarkers in Stable Versus Acutely Decompensated Heart Failure With Preserved Ejection Fraction.

BACKGROUND: Underlying inflammation has been increasingly recognized in heart failure with a preserved ejection fraction (HFpEF). In this study we tested the hypothesis that pro-inflammatory biomarkers are elevated in patients with acutely decompensated HFpEF (AD-HFpEF) compared with patients with stable HFpEF (S-HFpEF). METHODS AND RESULTS: Using a post hoc analysis the serum biomarkers tumor necrosis factor-alpha, high-sensitivity C-reactive protein interleukin 6 and pentraxin 3 (PTX3) and clinical, demographic, echocardiographic-Doppler and clinical outcomes data were analyzed in HFpEF patients enrolled in NHLBI Heart Failure Research Network clinical trials which enrolled patients with either AD-HFpEF or S-HFpEF. Compared to S-HFpEF, AD-HFpEF patients had higher levels of PTX3 (3.08 ng/mL versus 1.27 ng/mL, P<0.0001), interleukin-6 (4.14 pg/mL versus 1.71 pg/mL, P<0.0001), tumor necrosis factor-alpha (11.54 pg/mL versus 8.62 pg/mL, P=0.0015), and high-sensitivity C-reactive protein (11.90 mg/dL versus 3.42 mg/dL, P<0.0001). Moreover, high-sensitivity C-reactive protein, interleukin-6 and PTX3 levels were significantly higher in AD-HFpEF compared with S-HFpEF patients admitted for decompensated HF within the previous year. PTX3 was positively correlated with left atrial volume index (r=0.41, P=0.0017) and left ventricular mass (r=0.26, P=0.0415), while tumor necrosis factor-alpha was inversely correlated with E/A ratio (r=-0.31, P=0.0395). CONCLUSIONS: Levels of pro-inflammatory biomarkers are strikingly higher in AD-HFpEF compared with S-HFpEF patients. PTX3 and tumor necrosis factor-alpha are correlated with echocardiographic-Doppler evidence of diastolic dysfunction. Taken together these data support the concept that a heightened pro-inflammatory state has a pathophysiologic role in the development of AD-HFpEF.

Abundance, localization, and functional correlates of the advanced glycation end-product carboxymethyl lysine in human myocardium.

Advanced glycation end-products (AGEs) play a role in the pathophysiology of diabetes mellitus (DM) and possibly hypertension (HTN). In experimental DM, AGEs accumulate in myocardium. Little is known about AGEs in human myocardium. We quantified abundance, localization, and functional correlates of the AGE carboxymethyl lysine (CML) in left ventricular (LV) myocardium from patients undergoing coronary bypass grafting (CBG). Immunoelectron microscopy was used to quantify CML in epicardial biopsies from 98 patients (71 M, 27 F) with HTN, HTN + DM or neither (controls), all with normal LV ejection fraction. Myofilament contraction-relaxation function was measured in demembranated myocardial strips. Echocardiography was used to quantify LV structure and function. We found that CML was abundant within cardiomyocytes, but minimally associated with extracellular collagen. CML counts/µm2 were 14.7% higher in mitochondria than the rest of the cytoplasm (P < 0.001). There were no significant sex or diagnostic group differences in CML counts [controls 45.6 ± 3.6/µm2 (±SEM), HTN 45.8 ± 3.6/µm2, HTN + DM 49.3 ± 6.2/µm2; P = 0.85] and no significant correlations between CML counts and age, HgbA1c or myofilament function indexes. However, left atrial volume was significantly correlated with CML counts (r = 0.41, P = 0.004). We conclude that in CBG patients CML is abundant within cardiomyocytes but minimally associated with collagen, suggesting that AGEs do not directly modify the stiffness of myocardial collagen. Coexistent HTN or HTN + DM do not significantly influence CML abundance. The correlation of CML counts with LAV suggests an influence on diastolic function independent of HTN, DM or sex whose mechanism remains to be determined.

Human epicardial cell-conditioned medium contains HGF/IgG complexes that phosphorylate RYK and protect against vascular injury.

AIMS: The aim of this study was to evaluate the paracrine activity of human epicardial-derived cells (hEPDCs) to screen for secreted vasoprotective factors and develop therapeutics to treat vascular reperfusion injury. METHODS AND RESULTS: Epicardial cells support cardiac development, repair, and remodelling after injury in part, through paracrine activity. We hypothesized that secreted ligands from hEPDCs would protect vascular integrity after myocardial infarction (MI) with reperfusion. During simulated ischaemia in culture (24-48 h), concentrated hEPDC-conditioned medium (EPI CdM) increased survival of primary cardiac endothelial cells. In a rat MI model, EPI CdM treatment reduced vascular injury in vivo after reperfusion. By phospho-receptor tyrosine kinase (RTK) arrays, ELISA, and neutralizing antibody screens, we identified hepatocyte growth factor (HGF) as a key vasoprotective factor in EPI CdM. Unexpectedly, we observed that some of the HGF in EPI CdM formed complexes with polyclonal IgG. Following reperfusion, preparations of HGF/IgG complexes provided greater vascular protection than free HGF with IgG. HGF/IgG complexes localized to blood vessels in vivo and increased HGF retention time after administration. In subsequent screens, we found that 'related to tyrosine kinase' (RYK) receptor was phosphorylated after exposure of cardiac endothelial cells to HGF/IgG complexes, but not to free HGF with IgG. The enhanced protection conferred by HGF/IgG complexes was lost after antibody blockade of RYK. Notably, the HGF/IgG complex is the first 'ligand' shown to promote phosphorylation of RYK. CONCLUSION: Early treatment with HGF/IgG complexes after myocardial ischaemia with reperfusion may rescue tissue through vasoprotection conferred by c-Met and RYK signalling.

Cardiac myosin isoforms exhibit differential rates of MgADP release and MgATP binding detected by myocardial viscoelasticity.

We measured myosin crossbridge detachment rate and the rates of MgADP release and MgATP binding in mouse and rat myocardial strips bearing one of the two cardiac myosin heavy chain (MyHC) isoforms. Mice and rats were fed an iodine-deficient, propylthiouracil diet resulting in ~100% expression of ß-MyHC in the ventricles. Ventricles of control animals expressed ~100% α-MyHC. Chemically-skinned myocardial strips prepared from papillary muscle were subjected to sinusoidal length perturbation analysis at maximum calcium activation pCa 4.8 and 17°C. Frequency characteristics of myocardial viscoelasticity were used to calculate crossbridge detachment rate over 0.01 to 5mM [MgATP]. The rate of MgADP release, equivalent to the asymptotic value of crossbridge detachment rate at high MgATP, was highest in mouse α-MyHC (111.4±6.2s(-1)) followed by rat α-MyHC (65.0±7.3s(-1)), mouse ß-MyHC (24.3±1.8s(-1)) and rat ß-MyHC (15.5±0.8s(-1)). The rate of MgATP binding was highest in mouse α-MyHC (325±32 mM(-1) s(-1)) then mouse ß-MyHC (152±23 mM(-1) s(-1)), rat α-MyHC (108±10 mM(-1) s(-1)) and rat ß-MyHC (55±6 mM(-1) s(-1)). Because the events of MgADP release and MgATP binding occur in a post power-stroke state of the myosin crossbridge, we infer that MgATP release and MgATP binding must be regulated by isoform- and species-specific structural differences located outside the nucleotide binding pocket, which is identical in sequence for these four myosins. We postulate that differences in the stiffness profile of the entire myosin molecule, including the thick filament and the myosin-actin interface, are primarily responsible for determining the strain on the nucleotide binding pocket and the subsequent differences in the rates of nucleotide release and binding observed among the four myosins examined here.

Zinc-induced cardiomyocyte relaxation in a rat model of hyperglycemia is independent of myosin isoform.

It has been reported previously that diabetic cardiomyopathy can be inhibited or reverted with chronic zinc supplementation. In the current study, we hypothesized that total cardiac calcium and zinc content is altered in early onset diabetes mellitus characterized in part as hyperglycemia (HG) and that exposure of zinc ion (Zn2+) to isolated cardiomyocytes would enhance contraction-relaxation function in HG more so than in nonHG controls. To better control for differential cardiac myosin isoform expression as occurs in rodents after ß-islet cell necrosis, hypothyroidism was induced in 16 rats resulting in 100% ß-myosin heavy chain expression in the heart. ß-Islet cell necrosis was induced in half of the rats by streptozocin administration. After 6 wks of HG, both HG and nonHG controls rats demonstrated similar myofilament performance measured as thin filament calcium sensitivity, native thin filament velocity in the myosin motility assay and contractile velocity and power. Extracellular Zn2+ reduced cardiomyocyte contractile function in both groups, but enhanced relaxation function significantly in the HG group compared to controls. Most notably, a reduction in diastolic sarcomere length with increasing pacing frequencies, i.e., incomplete relaxation, was more pronounced in the HG compared to controls, but was normalized with extracellular Zn2+ application. This is a novel finding implicating that the detrimental effect of HG on cardiomyocyte Ca2+ regulation can be amelioration by Zn2+. Among the many post-translational modifications examined, only phosphorylation of ryanodine receptor (RyR) at S-2808 was significantly higher in HG compared to nonHG. We did not find in our hypothyroid rats any differentiating effects of HG on myofibrillar protein phosphorylation, lysine acetylation, O-linked N-acetylglucosamine and advanced glycated end-products, which are often implicated as complicating factors in cardiac performance due to HG. Our results suggest that the relaxing effects of Zn2+ on cardiomyocyte function are more pronounced in the HG state due an insulin-dependent effect of enhancing removal of cytosolic Ca2+ via SERCA2a or NCX or by reducing Ca2+ influx via L-type channel or Ca2+ leak through the RyR. Investigations into the effects of Zn2+ on these mechanisms are now underway.

Myosin cross-bridge dynamics in patients with hypertension and concentric left ventricular remodeling.

BACKGROUND: Hypertension (HTN) causes concentric left ventricular remodeling, defined as an increased relative wall thickness or overt left ventricular hypertrophy, and associated diastolic dysfunction. HTN and concentric remodeling are also common precursors to heart failure with a preserved ejection fraction. It is not known whether the myofilament contributes to diastolic dysfunction in patients with concentric remodeling. METHODS AND RESULTS: Intraoperative myocardial biopsies were obtained in 15 male patients undergoing coronary bypass grafting, all with normal left ventricular ejection fraction and wall motion. Eight patients had a history of HTN and concentric remodeling. Seven without HTN or remodeling served as controls. Myocardial strips were dissected and demembranated with detergent. Isometric tension was measured and sinusoidal length perturbation analysis performed at sarcomere length 2.2 µm and pCa 8 to 4.5. Sinusoidal analysis provides estimates of cross-bridge dynamics, including rate constants of attachment and detachment and cross-bridge attachment time. The normalized isometric tension-pCa relation was similar in HTN and controls. However, cross-bridge attachment time was significantly prolonged at submaximal [Ca(2+)] (pCa ≥6.5) in HTN patients. Analysis of protein phosphorylation revealed ≈25% reduction in phosphorylation of troponin I in HTN patients (P<0.05). CONCLUSIONS: Compared with controls, patients with HTN and concentric remodeling display prolonged cross-bridge attachment time at submaximal [Ca(2+)] without a change in the tension-pCa relation. Prolonged cross-bridge attachment time implicates altered cross-bridge dynamics as a cause of slowed relaxation in these patients. This finding was associated with reduced phosphorylation of troponin I, suggesting decreased phosphorylation of protein kinase A/G sites as a mechanism.

Unique single molecule binding of cardiac myosin binding protein-C to actin and phosphorylation-dependent inhibition of actomyosin motility requires 17 amino acids of the motif domain.

Cardiac myosin binding protein-C (cMyBP-C) has 11 immunoglobulin or fibronectin-like domains, C0 through C10, which bind sarcomeric proteins, including titin, myosin and actin. Using bacterial expressed mouse N-terminal fragments (C0 through C3) in an in vitro motility assay of myosin-generated actin movement and the laser trap assay to assess single molecule actin-binding capacity, we determined that the first N-terminal 17 amino acids of the cMyBP-C motif (the linker between C1 and C2) contain a strong, stereospecific actin-binding site that depends on positive charge due to a cluster of arginines. Phosphorylation of 4 serines within the motif decreases the fragments' actin-binding capacity and actomyosin inhibition. Using the laser trap assay, we observed individual cMyBP-C fragments transiently binding to a single actin filament with both short (~20 ms) and long (~300 ms) attached lifetimes, similar to that of a known actin-binding protein, α-actinin. These experiments suggest that cMyBP-C N-terminal domains containing the cMyBP-C motif tether actin filaments and provide one mechanism by which cMyBP-C modulates actomyosin motion generation, i.e. by imposing an effective viscous load within the sarcomere.

Erythropoietin induces positive inotropic and lusitropic effects in murine and human myocardium.

Initial clinical studies indicate a potential beneficial effect of erythropoietin (EPO) in patients with anemia and heart failure. Here, we investigate the direct contractile effects of erythropoietin on myocardial tissue. Treatment with EPO (50U/mL) using excitable murine and human left ventricular muscle preparations resulted in a 37% and 62% increase in twitch tension, respectively (P<0.05). Isolated murine cardiomyocytes exposed to EPO demonstrated a 41% increase in peak sarcomere shortening (P=0.012). Using compounds that specifically stimulate a non-erythropoietic EPO receptor yielded similar increases in contractile dynamics. Cardiomyocyte Ca(2+)dynamics showed an 18% increase in peak calcium in EPO treated cardiomyocytes over controls (P=0.03). Studies in muscle strips skinned after EPO treatment demonstrated a phosphorylation dependant increase in the viscous modulus as well as an increase in oscillatory work. The EPO mediated increase in peak sarcomere shortening was abrogated by PI3-K blockade via wortmannin and by non-isozyme specific PKC blockade by chelerythrine. Finally, EPO treatment resulted in an increase in PKCε in the particulate cellular fraction, indicating activation of this isoform. EPO exhibits direct positive inotropic and lusitropic effects in cardiomyocytes and ventricular muscle preparation. These effects are mediated through PI3-K and PKCε isoform signaling to directly affect both calcium release dynamics and myofilament function.

Resistance training alters skeletal muscle structure and function in human heart failure: effects at the tissue, cellular and molecular levels.

Reduced skeletal muscle function in heart failure (HF) patients may be partially explained by altered myofilament protein content and function. Resistance training increases muscle function, although whether these improvements are achieved by correction of myofilament deficits is not known. To address this question, we examined 10 HF patients and 14 controls prior to and following an 18 week high-intensity resistance training programme. Evaluations of whole muscle size and strength, single muscle fibre size, ultrastructure and tension and myosin-actin cross-bridge mechanics and kinetics were performed. Training improved whole muscle isometric torque in both groups, although there were no alterations in whole muscle size or single fibre cross-sectional area or isometric tension.Unexpectedly, training reduced the myofibril fractional area of muscle fibres in both groups. This structural change manifested functionally as a reduction in the number of strongly bound myosin-actin cross-bridges during Ca²âº activation. When post-training single fibre tension data were corrected for the loss of myofibril fractional area, we observed an increase in tension with resistance training. Additionally, training corrected alterations in cross-bridge kinetics (e.g. myosin attachment time) in HF patients back to levels observed in untrained controls. Collectively, our results indicate that improvements in myofilament function in sedentary elderly with and without HF may contribute to increased whole muscle function with resistance training. More broadly, these data highlight novel cellular and molecular adaptations in muscle structure and function that contribute to the resistance-trained phenotype.

Biomarker profiles as descriptors of left ventricular remodeling after acute myocardial infarction.

BACKGROUND: Biomarker expression can predict subsequent cardiovascular events. The goal of this study was to determine the pattern of expression in blood of a broad array of cytokines and growth factors taken 24-72 h after an ST elevation myocardial infarction (STEMI) involving the left anterior descending (LAD) coronary artery. METHODS: Blood was taken from 16 patients with LAD STEMI. Cytokine and growth factor expressions were quantified with the use of a Milliplex cytokine/chemokine array analysis that tested 42 analytes. Results from patients were compared with those in blood from 20 healthy volunteers. RESULTS: Most (15/16) participants had positive remodeling without reduction in left ventricular function during follow-up. Analytes were grouped based on their function into those that activate class 1 T-helper cells (Th1 activates cell-mediated immunity), those that activated a Th2 response (activates humoral immunity and attenuates cell-mediated immunity), chemokines (attract leukocytes), and growth factors (promote a healing response). Elevation of cytokines involved in the Th2 response predominated over the Th1 response demonstrating a balance favoring tolerance and limiting activation of cell-mediated immunity. The concentration of selected chemokines favoring cell-mediated immunity was not elevated. The concentration of selected growth factors was increased. CONCLUSION: The cytokine expression, 24-72 h after an LAD STEMI, suggests that positive ventricular remodeling is associated with growth factor expression and limitation of cell-mediated immunity. Subsequent studies are warranted to determine whether deviation from this pattern identifies patients at an increased risk of adverse remodeling after myocardial infarction.

Blood gene expression signatures associate with heart failure outcomes.

Gene expression signatures in blood correlate with specific diseases. Such signatures may serve as valuable diagnostic and prognostic tools in disease management. Blood gene expression signatures associated with heart failure may be applied to predict prognosis, monitor disease progression, and optimize treatment. Blood gene expression profiles were generated for 71 subjects with heart failure and 15 controls without heart failure, using the Affymetrix GeneChip U133Plus2.0. Survival analysis identified 197 "mortality genes" that were significantly associated with patient outcome. Functional categorization showed that genes associated with T cell receptor signaling were most significantly overpresented. Cluster analysis of these T cell receptor signaling genes significantly categorized heart failure patients into three risk groups (P = 0.031) that were distinct from the three risk groups categorized by New York Heart Association (NYHA) Classification (P = 0.0002). By combining the analysis of clinical assessment (NYHA class) with T cell receptor signaling gene expression, we proposed a model that demonstrated an even greater differentiation of patients at risk (P = 0.0001). In this discovery study, we identified blood expression signatures associated with heart failure patient outcomes. Characterization of these mortality genes helped identify a set of T cell receptor signaling genes that may be of utility in predicting survival of heart failure patients. These data raise the possibility of prospectively risk stratifying patients with heart failure by integrating blood gene expression signatures with current clinical assessment.

Effect of resistance training on physical disability in chronic heart failure.

PURPOSE: Patients with chronic heart failure (CHF) report difficulty performing activities of daily living. To our knowledge, however, no study has directly measured performance in activities of daily living in these patients to systematically assess their level of physical disability. Moreover, the contribution of skeletal muscle weakness to physical disability in CHF remains unclear. Thus, we measured performance in activities of daily living in CHF patients and controls, its relationship to aerobic capacity and muscle strength, and the effect of resistance exercise training to improve muscle strength and physical disability. METHODS: Patients and controls were assessed for performance in activities of daily living, self-reported physical function, peak aerobic capacity, body composition, and muscle strength before and after an 18-wk resistance training program. To remove the confounding effects of several disease-related factors (muscle disuse, hospitalization, acute illness), we recruited controls with similar activity levels as CHF patients and tested patients >6 months after any disease exacerbation/hospitalization. RESULTS: Performance in activities of daily living was 30% lower (P < 0.05) in CHF patients versus controls and was related to both reduced aerobic capacity (P < 0.001) and muscle strength (P < 0.01). Moreover, resistance training improved (P < 0.05 to P < 0.001) physical function and muscle strength in patients and controls similarly, without altering aerobic capacity. CONCLUSIONS: CHF patients are characterized by marked physical disability compared with age- and physical activity-matched controls, which is related to reduced aerobic capacity and muscle strength. CHF patients respond to resistance training with normal strength/functional adaptations. Our results support muscle weakness as a determinant of physical disability in CHF and show that interventions that increase muscle strength (resistance training) reduce physical disability.

Chronic heart failure reduces Akt phosphorylation in human skeletal muscle: relationship to muscle size and function.

Patients with chronic heart failure (HF) frequently lose muscle mass and function during the course of the disease. A reduction in anabolic stimuli to the muscle has been put forth as a potential mechanism underlying these alterations. The present study examined the hypothesis that skeletal muscle tissue from HF patients would show reduced IGF-1 expression and phosphorylation of signaling molecules downstream of receptor activation. To isolate the unique effect of HF on these variables, we limited the confounding effects of muscle disuse and/or acute disease exacerbation by recruiting controls (n = 11) with similar physical activity levels as HF patients (n = 11) and by testing patients at least 6 mo following any bouts of disease exacerbation/hospitalization. IGF-1 expression in skeletal muscle was similar between patients and controls. Despite this, HF patients were characterized by reduced levels of phospho-Akt/Akt (S473; -43%; P < 0.05), whereas no differences were found in total Akt protein content or phospho- or total protein content of mammalian target of rapamycin (mTOR; S2448), glycogen synthase kinase-3ß (GSK-3ß; S9), eukaryotic translation initiation factor 4E binding protein-1 (eIF4E-BP; T37/46), p70 ribosomal S6 kinase (p70 S6K; T389), or eIF2Bε (S540). Reduced phospho-Akt/Akt levels and phospho-mTOR/mTOR were related to decreased skeletal muscle myosin protein content (r = 0.602; P < 0.02) and knee extensor isometric torque (r = 0.550; P < 0.05), respectively. Because patients and controls were similar for age, muscle mass, and physical activity, we ascribe the observed alterations in Akt phosphorylation and its relationship to myosin protein content to the unique effects of the HF syndrome.

Ventricular resynchronization by implementation of direct his bundle pacing in a patient with congenital complete AV block and newly diagnosed cardiomyopathy.

Congenital complete atrioventricular block (CCAVB) is usually due to failure of AV nodal conduction with preservation of the His Purkinje system, typically present at birth. While most patients with CCAVB ultimately require pacemaker therapy to restore physiologic heart rates, recent studies have suggested that chronic right ventricular (RV) pacing in patients with CCAVB can have detrimental effects on cardiac structure and function, and may account for a 7-10% incidence of congestive heart failure in these patients. Since the His Purkinje system is preserved in CCAVB, this patient population could be uniquely well served by direct His bundle pacing (DHBP) which would be expected to restore physiologic activation of both ventricles. We present a case of a young woman who presented with RV pacing-induced cardiomyopathy who responded dramatically to DHBP.

Chronic heart failure decreases cross-bridge kinetics in single skeletal muscle fibres from humans.

Skeletal muscle function is impaired in heart failure patients due, in part, to loss of myofibrillar protein content, in particular myosin. In the present study, we utilized small-amplitude sinusoidal analysis for the first time in single human skeletal muscle fibres to measure muscle mechanics, including cross-bridge kinetics, to determine if heart failure further impairs contractile performance by altering myofibrillar protein function. Patients with chronic heart failure (n = 9) and controls (n = 6) were recruited of similar age and physical activity to diminish the potentially confounding effects of ageing and muscle disuse. Patients showed decreased cross-bridge kinetics in myosin heavy chain (MHC) I and IIA fibres, partially due to increased myosin attachment time (t(on)). The increased t(on) compensated for myosin protein loss previously found in heart failure patients by increasing the fraction of the total cycle time myosin is bound to actin, resulting in a similar number of strongly bound cross-bridges in patients and controls. Accordingly, isometric tension did not differ between patients and controls in MHC I or IIA fibres. Patients also had decreased calcium sensitivity in MHC IIA fibres and alterations in the viscoelastic properties of the lattice structure of MHC I and IIA fibres. Collectively, these results show that heart failure alters skeletal muscle contraction at the level of the myosin-actin cross-bridge, leading to changes in muscle mechanics which could contribute to impaired muscle function. Additionally, we uncovered a unique kinetic property of MHC I fibres, a potential indication of two distinct populations of cross-bridges, which may have important physiological consequences.

Atrial contractile protein content and function are preserved in patients with coronary artery disease and atrial fibrillation.

OBJECTIVES: Atrial fibrillation (AF) causes atrial contractile dysfunction. The focus of this study was to determine whether the contractile deficit of human AF is the result of altered contractile protein abundance and/or function. METHODS: Atrial tissue from patients with chronic AF undergoing open-heart surgery was compared with the tissue from patients in normal sinus rhythm (NSR). Myosin isoform composition and content were determined. Intact native thin filament and cardiac myosin contractile protein performance were independently assessed in an in-vitro motility assay. RESULTS: Myosin isoform expression and total myosin content were not different between AF and NSR. Calcium-activated native thin filament function was similar between AF and NSR as measured by calcium sensitivity and maximal activation. Myosin isolated from the atria of AF and NSR groups showed similar unloaded shortening speeds and isometric force generation. CONCLUSION: Unlike human ventricular dysfunction where contractile protein function is directly affected, the contractile deficit of AF is not the result of alterations in myosin content or contractile protein function.