Human Heart Failure Patients Research Articles

Abstract 85: Cardiac Pathology Is Worsened by Loss of the Cell Junction Protein Claudin-5 and Improved by Claudin-5 Gene Therapy

Claudin-5 is a cell-cell adhesion protein that plays established roles in the blood-brain barrier and cancer. We were the first to demonstrate that it plays a role in the heart with implications for heart failure and cardiomyopathy. Several animal models with cardiac pathology show reduced cardiac claudin-5 levels. Importantly, 60% of human heart failure patients show reduced levels of cardiac claudin-5, independent of changes in other cell junction proteins in the heart, supporting that the loss of cardiac claudin-5 has clinical relevance. To test our hypothesis that claudin-5 plays an important role in protecting the heart, we used mouse models to demonstrate whether circumventing reductions in cardiac claudin-5 is able to improve cardiac histology and physiology, and whether ablation of claudin-5 is able to induce cardiac pathology. We now show that maintaining cardiac claudin-5 levels using gene therapy (cldn5 transduction using an adeno-associated virus vector) significantly reduced cardiac pathology in mice which otherwise demonstrate heart failure and cardiomyopathy indicators. We also present preliminary evidence of the effects of knocking out the cldn5 gene specifically in cardiomyocytes using the inducible cre-loxP system.

Abstract 10: Antifibrotic Effect of CCN5 in a Murine Model of Heart Failure

CCN family members are matricellular proteins with diverse roles in cell function. Recently, we showed that the differential expression of CCN2 and CCN5 during cardiac remodeling suggests that these two members of the CCN family play opposing roles during the development of cardiac hypertrophy and fibrosis. Since it is reported that an underlying morphological correlate of diastolic dysfunction is cardiac fibrosis, which leads to increased stiffness of the heart, we aimed to evaluate the role of CCN5 on cardiac fibrosis and function by the gene delivery using the cardiotropic AAV9 vector. We generated pressure-overload heart failure models in mouse by TAC operation. After 8-10 weeks of TAC on mice, HF was confirmed by Echocardiography. In those HF mice, AAV9-GFP (control) and AAV9-CCN5 were addressed by IV. Two more months later, cardiac function was evaluated by echocardiography and invasive hemodynamics. Protein and RNA expression levels of CCN5, several types of collagen and conventional TGF-beta signaling related genes were evaluated by western blot and quantitative real time PCR analysis. First, we were able to achieve about 4-5 fold increase of CCN5 expression by AAV9-CCN5 injection without any change in heart function. Second, CCN5 expression level in blood was not significantly altered after AAV9-CCN5 gene transfer because it may be the result of the cardiac tropism of the vector used. The HF model by TAC surgery was confirmed with echocardiography (FS (%)). Overall average FS (%) in HF was 41.87+/− 5.27 (n=16) and in non-surgery control mice was 58.39 +/− 2.06(n=4). After AAV9 injection, cardiac function of CCN5 injected mice was sustained but AAV9-GFP injected mice showed severe cardiac dysfunction and dilation (AAV-GFP (24.29+/− 9.11) vs AAV-CCN5 (42.66 +/− 4.73)). Third, western blot analysis showed that the downstream effectors, namely TGF-beta signaling pathways were significantly down-regulated in CCN5 injected mice. In addition, fibrotic area of the heart was tremendously reduced. Finally, CCN5 expression is significantly decreased in human heart failure patients compared to those in nonfailing donors. Taken together our data would indicate that CCN5 may be a promising therapeutic target to reduce cardiac fibrosis.

DHRS7c, a novel cardiomyocyte‐expressed gene that is down‐regulated by adrenergic stimulation and in heart failure

Although cardiac diseases account for the highest mortality and morbidity rates in Western society, there is still a considerable gap in our knowledge of genes that contribute to cardiac (dys)function. Here we screened for gene expression profiles correlated to heart failure. By expression profiling we identified a novel gene, termed DHRS7c, which was significantly down-regulated by adrenergic stimulation and in heart failure models. Dhrs7c is a short chain dehydrogenase/reductase (SDR) and is localized to the endo/sarcoplasmic reticulum. Dhrs7c is strongly conserved in vertebrates, and mRNA and protein expression levels were highest in heart and skeletal muscle followed by skin, but were not detectable in other organs. In vitro, both α- and β-adrenergic stimulation repressed Dhrs7c expression in neonatal cardiomyocytes and this could be mimicked by the direct activation of protein kinase C and adenylate cyclase, the respective intracellular targets of these hormones. In contrast, endothelin-1, which also provoked strong hypertrophy development in vitro, did not repress Dhrs7c expression. The latter suggests adrenergic specificity and indicates that down-regulation is not a prerequisite for hypertrophy development. In vivo adrenergic stimulation could also down-regulate Dhrs7c expression. Finally, we confirmed that expression was also down-regulated in two different models of failure and, importantly, also in biopsies from human heart failure patients. Our results show that the expression of Dhrs7c, a novel endo/sarcoplasmic reticulum-localized SDR, is inversely correlated with adrenergic stimulation and heart failure development.

1H nuclear magnetic resonance based metabolic urinary profiling of patients with ischemic heart failure

Objectives We sought to identify metabolic pathways characterizing human heart failure (HF) using 1NMR based urinary metabolomic analysis in conjunction with multivariate statistics. Design and methods Patients with systolic HF of ischemic origin (n = 15) and healthy controls (n = 20) participated in this study. Patients with type 2 diabetes mellitus were excluded. Results The results showed that the urine of the HF patients had higher levels of metabolites for acetate (p < 0.05) and acetone (p < 0.01) compared to the healthy controls. In addition, there was a perturbation in methylmalonate metabolism as shown by increased urinary levels of methylmalonic acid (p < 0.001) in the HF patients. HF patients also had increased urinary levels of cytosine (p < 0.01) and phenylacetylglycine (p < 0.01) and decreased 1-methylnicotinamide (p < 0.05) compared to healthy controls. Conclusions TCA cycle metabolites and fatty acid metabolism were modified in the HF patients, indicating altered energy metabolism. Moreover, perturbations of metabolism in nucleotide and methylmalonate were observed.

Hold Me Tight

Hold Me Tight

Rate-dependent action potential alternans in human heart failure implicates abnormal intracellular calcium handling

Alternans in action potential voltage (APV-ALT) at heart rates <110 bpm is a novel index to predict ventricular arrhythmias. However, the rate dependency of APV-ALT and its mechanisms in failing versus nonfailing human myocardium are poorly understood. It is hypothesized that APV-ALT in human heart failure (HF) reflects abnormal calcium handling. Using a modeling and clinical approach, our objectives were to (1) determine how APV-ALT varies with pacing rate and (2) ascertain whether abnormalities in calcium handling explain the rate dependence of APV-ALT in HF. APV-ALT was analyzed at several cycle lengths (CLs) using a dynamic pacing protocol applied to a human left ventricle wedge model with various alterations in calcium handling. Modeled APV-ALT was used to predict APV-ALT in left ventricle monophasic action potentials recorded from HF (n = 3) and control (n = 2) patients with the same pacing protocol. Reducing the sarcoplasmic reticulum calcium uptake current < or =25%, the release current < or =11%, or the sarcolemmal L-type calcium channel current < or =43% of control predicted APV-ALT to arise at CL > or =600 ms and then increase in magnitude by >400% for CL <400 ms. In HF patients, APV-ALT arose at CL = 600 ms and then increased in magnitude by >500% at CL <350 ms. For all other model alterations and for control patients, APV-ALT occurred only at CL <500 ms. APV-ALT shows differing rate dependence in HF versus control patients, arising at slower rates in HF and predicted by models with abnormal calcium handling. Future studies should investigate whether APV-ALT at slow rates identifies patients with deranged calcium handing, including HF patients before decompensation or at risk for arrhythmias.

Cardiolipin biosynthesis and remodeling enzymes are altered during development of heart failure

Cardiolipin (CL) is responsible for modulation of activities of various enzymes involved in oxidative phosphorylation. Although energy production decreases in heart failure (HF), regulation of cardiolipin during HF development is unknown. Enzymes involved in cardiac cardiolipin synthesis and remodeling were studied in spontaneously hypertensive HF (SHHF) rats, explanted hearts from human HF patients, and nonfailing Sprague Dawley (SD) rats. The biosynthetic enzymes cytidinediphosphatediacylglycerol synthetase (CDS), phosphatidylglycerolphosphate synthase (PGPS) and cardiolipin synthase (CLS) were investigated. Mitochondrial CDS activity and CDS-1 mRNA increased in HF whereas CDS-2 mRNA in SHHF and humans, not in SD rats, decreased. PGPS activity, but not mRNA, increased in SHHF. CLS activity and mRNA decreased in SHHF, but mRNA was not significantly altered in humans. Cardiolipin remodeling enzymes, monolysocardiolipin acyltransferase (MLCL AT) and tafazzin, showed variable changes during HF. MLCL AT activity increased in SHHF. Tafazzin mRNA decreased in SHHF and human HF, but not in SD rats. The gene expression of acyl-CoA: lysocardiolipin acyltransferase-1, an endoplasmic reticulum MLCL AT, remained unaltered in SHHF rats. The results provide mechanisms whereby both cardiolipin biosynthesis and remodeling are altered during HF. Increases in CDS-1, PGPS, and MLCL AT suggest compensatory mechanisms during the development of HF. Human and SD data imply that similar trends may occur in human HF, but not during nonpathological aging, consistent with previous cardiolipin studies.

Association of Body Weight and Body Condition with Survival in Dogs with Heart Failure

Obesity is a risk factor for cardiovascular disease in people, but overweight and obese human heart failure patients have improved survival compared with normal--or underweight controls--the obesity paradox. The purpose of this study was to determine if there is an association of body weight and body condition with survival in dogs with heart failure. That body condition and changes in body weight are predictors of survival in dogs with heart failure. One hundred and eight dogs with heart failure (International Small Animal Cardiac Health Council stages 2, 3a, or 3b) secondary to dilated cardiomyopathy or chronic valvular disease. Medical records were reviewed, and data regarding initial body weight and body condition score (BCS), subsequent changes in body weight, and treatment were collected. Survival times were determined for dogs that were discharged from the hospital and lived >24 hours. Survival was significantly different between dogs that gained, lost, or maintained body weight over the course of their disease (P= .04), with dogs that gained weight surviving the longest. BCS and medications were not significantly associated with survival time; however, n-3 fatty acid intake was associated with longer survival time (P= .009). These results suggest that changes in body weight might be an important consideration in the survival of dogs with heart failure.

Carvedilol Exhibits No Tolerance to α 1-Receptor Blockade in Class C Heart Failure Subjects

Introduction: Myocardial α1-receptor (α1R) stimulation produces myocyte hypertrophy in animal models. Increased α1R signal transduction occurs in human heart failure (HF) patients due to increased sympathetic tone and α1R up-regulation. These findings suggest a possible role for α1R antagonists to reverse pathologic remodeling in HF. Nonetheless, α1R blockade remains a controversial target in HF due to reports of tolerance in other HF investigations. Hypothesis: No tolerance occurs to carvedilol α1R blockade in HF patients.

Proteomics analysis of leukocyte membrane proteins from human heart failure patients using an antibody microarray platform

Proteomics analysis of leukocyte membrane proteins from human heart failure patients using an antibody microarray platform

Reversal of amyloid-induced heart disease in desmin-related cardiomyopathy

Amyloid oligomers, similar to the toxic entities found in Alzheimer's disease patients and in other amyloid-based diseases, are present in cardiomyocytes derived from human heart-failure patients and in animal models of desmin-related cardiomyopathy (DRM). The R120G mutation in alpha-B-crystallin (CryAB) causes DRM and is characterized by aggresomes containing CryAB(R120G) and amyloid oligomer. In this study, we show that aggresome levels do not correlate with disease. Blocking aggresome formation results in increased levels of toxic amyloid oligomer and decreased cardiomyocyte viability. We confirmed the primary toxicity of intrasarcoplasmic amyloid accumulation in the cardiomyocytes by ectopic expression of the amyloidogenic peptide PQ81, which consists of multiple repeats of a polyglutamine tract. We then addressed the issue of disease reversibility by placing CryAB(R120G) under inducible cardiomyocyte-specific expression in transgenic mice. The mice developed aggresomes and contained high concentrations of amyloid oligomer in the heart, resulting in cardiac disease. Cessation of CryAB(R120G) expression in symptomatic mice improved cardiac function and rescued all of the animals from premature death. Rescue was accompanied by significant decreases in amyloid oligomer without a significant reduction in aggresomes. Blocking cardiac amyloid oligomer formation, even after cardiac dysfunction presents, may be a therapeutic strategy in DRM as well as in other types of cardiac disease in which significant amyloid accumulation occurs.

Elevated myocardial and lymphocyte GRK2 expression and activity in human heart failure

The G protein-coupled receptor kinase-2 (GRK2 or beta-ARK1) regulates beta-adrenergic receptors (beta-ARs) in the heart, and its cardiac expression is elevated in human heart failure (HF). We sought to determine whether myocardial levels and activity of GRK2 could be monitored using white blood cells, which have been used to study cardiac beta-ARs. Moreover, we were interested in determining whether GRK2 levels in myocardium and lymphocytes may be associated with beta-AR dysfunction and HF severity. In myocardial biopsies from explanted failing human hearts, GRK activity was inversely correlated with beta-AR-mediated cAMP production (R(2)=-0.215, P<0.05, n=24). Multiple regression analysis confirmed that GRK activity participates with beta-AR density to regulate catecholamine-sensitive cAMP responses. Importantly, there was a direct correlation between myocardial and lymphocytes GRK2 activity (R(2)=0.5686, P<0.05, n=10). Lymphocyte GRK activity was assessed in HF patients with various ejection fractions (EFs) (n=33), and kinase activity was significantly higher in patients with lower EFs and was higher with increasing NYHA class (P<0.001). Myocardial GRK2 expression and activity are mirrored by lymphocyte levels of this kinase, and its elevation in HF is associated with the loss of beta-AR responsiveness and appears to increase with disease severity. Therefore, lymphocytes may provide a surrogate for monitoring cardiac GRK2 in human HF.

Augmented sympathetic neural response to simulated obstructive apnoea in human heart failure.

Sleep apnoea in heart failure increases mortality risk, possibly as a result of greater activation of the sympathetic nervous system. In healthy subjects, simulated central apnoeas (holding breath) and obstructive apnoeas (Mueller manoeuvres) increase muscle sympathetic activity equally, primarily through chemoreceptor stimulation. In heart failure, however, Mueller manoeuvres cause greater reductions in blood pressure than breath holds. We hypothesized that in heart failure, the summation of arterial baroreceptor unloading and chemoreceptor stimulation would increase sympathetic activity more during obstructive than central apnoeas. Healthy human subjects and heart failure patients (seven of each) performed 15-s breath holds and 15-s Mueller manoeuvres. Breath holds evoked a progressive increase in muscle sympathetic nerve activity in both groups, but had no effect on blood pressure. In healthy subjects, breath holds and Mueller manoeuvres caused equal peaks in sympathetic activity. In contrast, in heart failure patients, Mueller manoeuvres caused a progressive decrease in blood pressure (P < 0.05) and greater increases in sympathetic activity than breath holds (P < 0.01). In heart failure, simulated obstructive apnoea elicits greater increases in sympathetic activity than simulated central apnoea, due to its additional hypotensive effect. These present findings offer novel insight into the potential role of sleep apnoea in augmenting sympathetic activity and accelerating disease progression in heart failure.

Augmented sympathetic neural response to simulated obstructive apnoea in human heart failure

Sleep apnoea in heart failure increases mortality risk, possibly as a result of greater activation of the sympathetic nervous system. In healthy subjects, simulated central apnoeas (holding breath) and obstructive apnoeas (Mueller manoeuvres) increase muscle sympathetic activity equally, primarily through chemoreceptor stimulation. In heart failure, however, Mueller manoeuvres cause greater reductions in blood pressure than breath holds. We hypothesized that in heart failure, the summation of arterial baroreceptor unloading and chemoreceptor stimulation would increase sympathetic activity more during obstructive than central apnoeas. Healthy human subjects and heart failure patients (seven of each) performed 15-s breath holds and 15-s Mueller manoeuvres. Breath holds evoked a progressive increase in muscle sympathetic nerve activity in both groups, but had no effect on blood pressure. In healthy subjects, breath holds and Mueller manoeuvres caused equal peaks in sympathetic activity. In contrast, in heart failure patients, Mueller manoeuvres caused a progressive decrease in blood pressure (P&lt;0.05) and greater increases in sympathetic activity than breath holds (P&lt;0.01). In heart failure, simulated obstructive apnoea elicits greater increases in sympathetic activity than simulated central apnoea, due to its additional hypotensive effect. These present findings offer novel insight into the potential role of sleep apnoea in augmenting sympathetic activity and accelerating disease progression in heart failure.

Sarcoplasmic reticulum Ca2+-ATPase modulates cardiac contraction and relaxation.

The cardiac SR Ca(2+)-ATPase (SERCA2a) regulates intracellular Ca(2+)-handling and thus, plays a crucial role in initiating cardiac contraction and relaxation. SERCA2a may be modulated through its accessory phosphoprotein phospholamban or by direct phosphorylation through Ca(2+)/calmodulin dependent protein kinase II (CaMK II). As an inhibitory component phospholamban, in its dephosphorylated form, inhibits the Ca(2+)-dependent SERCA2a function, while protein kinase A dependent phosphorylation of the phospho-residues serine-16 or Ca(2+)/calmodulin-dependent phosphorylation of threonine-17 relieves this inhibition. Recent evidence suggests that direct phosphorylation at residue serine-38 in SERCA2a activates enzyme function and enhances Ca(2+)-reuptake into the sarcoplasmic reticulum (SR). These effects that are mediated through phosphorylation result in an overall increased SR Ca(2+)-load and enhanced contractility. In human heart failure patients, as well as animal models with induced heart failure, these modulations are altered and may result in an attenuated SR Ca(2+)-storage and modulated contractility. It is also believed that abnormalities in Ca(2+)-cycling are responsible for blunting the frequency potentiation of contractile force in the failing human heart. Advanced gene expression and modulatory approaches have focused on enhancing SERCA2a function via overexpressing SERCA2a under physiological and pathophysiological conditions to restore cardiac function, cardiac energetics and survival rate.

Clinical overview of the novel inotropic agent toborinone

Toborinone (OPC-18790, Otsuka Pharmaceutical Co. Ltd, 2(1H) -quinolone,6-[3-[ [3,4-dimethoxyphenyl)methyl] amino]-2-hydroxy prop oxyl]-,(.+-.)-) is a novel iv. inotropic agent. Positive inotropic effects are produced by PDE inhibition with the resulting increase in cAMP and intracellular calcium levels. Unlike other inotropic agents that increase cAMP, there is an absence of positive chronotropic effects, which are attributed to prolongation of the action potential due to blockade of delayed rectifier currents. There is also marked venous and arterial vasodilating properties. The absence of heart rate increases results in decreased myocardial oxygen consumption compared with conventional inotropes. Studies in human heart failure patients have been consistent with previous work in animal studies, confirming the effects of toborinone as being positive inotropy (relatively weak), marked arterial and venous vasodilatation and absence of increase in myocardial oxygen consumption. Data regarding safety in larger clinical trials, particularly regarding arrhythmias, is at present unavailable. This information will determine whether this agent becomes an accepted iv. therapeutic option for congestive heart failure.

Rationale for the use of drugs in the treatment of cardiovascular disease 3. Positive inotropes

THE quest for the ideal positive inotrope has led to the development of many agents, to the extent that even a limited review of the literature can be a bewildering...

Studies on the mechanism of action of the bipyridine milrinone on the heart

Milrinone is a positive inotropic and vasodilator agent when tested in experimental animals and in human heart-failure patients. It is generally believed that milrinone acts by inhibiting phosphodiesterase IV, thus increasing cyclic AMP, [Ca++]i and cardiac contractile force and relaxation. Maximal force produced by milrinone is greater when single-dose response curves are compared to cumulative dose-response curves. In vitro, milrinone produces a tachyphylaxis, the extent of which is both dose- and time-dependent. Recovery of tachyphylaxis is both dose- and time-dependent and is not influenced by inhibitors of protein or RNA synthesis. There is a specific cross-tachyphylaxis between milrinone and amrinone, theophylline, papaverine, and Bay K8644. This tachyphylaxis may explain the low maximal contractile response of the cumulative dose-response observed in isolated tissues. Milrinone increased cyclic AMP in dog and guinea pig cardiac muscle. As previously shown by Endoh et al., milrinone in low doses produced a biphasic effect on cyclic AMP. The early increase (first 60-70 s) in cyclic AMP shows a good correlation with contractile force changes. If cyclic AMP is determined at maximal contractile force this correlation was poor. Here we also present instances where the increase in cyclic AMP after milrinone (determined at maximal effect) does not correlate with the contractile response. The cross-tachyphylaxis of milrinone with Bay K8644 suggests that milrinone has an action on the sarcolemmal Ca++ channels. Bay K8644 suppresses the positive inotropic effect of catecholamines by 50%, but not the cyclic AMP response. The inotropic effect of milrinone, in contrast to norepinephrine is highly sensitive to [Ca++]0, stimulation rate, and [K+]0. In this respect milrinone behaves more like Bay K8644. We postulate that the main inotropic action of milrinone is due to a sarcolemmal effect. The early cyclic AMP production described could be in the sarcolemmal compartment and this may explain some of the similarities of milrinone's actions with those of Bay K8644. The tachyphylaxis observed with the inotropic effect of milrinone does not extend to the decreases in relaxation time. This and other findings to be discussed suggest that the positive inotropic and reduction in relaxation time by milrinone depend on different mechanisms, possibly through differential compartmentalization of cyclic AMP.