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Summary: Background: Circadian (24-h) rhythms are important regulators in physiology and disease, but systemic disease may disrupt circadian rhythmicity. Heart failure (HF) is a systemic disease affecting hormonal regulation. We investigate whether HF affects the rhythmic expression of melatonin and cortisol, main endocrine products of the central clock, and cardiac-specific troponin in patients. We corroborate the functionality of the peripheral clock directly in the organs of translational models, inaccessible in human participants. Methods: We included 46 HF patients (71.7% male, median age of 60 years, NYHA class II (32.6%) or III (67.4%), ischemic cardiomyopathy (43.5%), comorbidities: diabetes 21.7%, atrial fibrillation 30.4%), and 24 matched controls. Blood was collected at seven time-points during a 24-h period (totalling 320 HF and 167 control samples) for melatonin, cortisol, and cardiac troponin T (cTnT) measurements after which circadian rhythms were assessed through cosinor analyses, both on the individual and the group level. Next, we analysed peripheral circadian clock functionality using cosinor analysis in male animal HF models: nocturnal mice and diurnal zebrafish, based on expression of core clock genes in heart, kidneys, and liver, every 4 h during a 24-h period in a light/darkness synchronised environment. Findings: Melatonin and cortisol concentrations followed a physiological 24-h pattern in both patients and controls. For melatonin, acrophase occurred during the night for both groups, with significantly decreased amplitude (median 5.2 vs 8.8, P = 0.0001) and circadian variation ([maximum]/[minimum]) in heart failure patients. For cortisol, mesor showed a significant increase for HF patients (mean 331.9 vs 275.1, P = 0.017) with a difference of 56.8 (95% CI 10.3–103.3) again resulting in a relatively lower variation: median 3.9 vs 6.3 (P = 0.0058). A nocturnal blood pressure dip was absent in 77.8% of HF patients.Clock gene expression profiles (Bmal, Clock, Per, Cry) were similar and with expected phase relations in animal HF models and controls, demonstrating preserved peripheral clock functionality in HF. Furthermore, oscillations in diurnal zebrafish were expectedly in opposite phases to those of nocturnal mice. Concordantly, cTnT concentrations in HF patients revealed significant circadian oscillations. Interpretation: Central clock output is dampened in HF patients while the molecular peripheral clock, as confirmed in animal models, remains intact. This emphasises the importance of taking timing into account in research and therapy for HF, setting the stage for another dimension of diagnostic, prognostic and therapeutic approaches. Funding: Hartstichting.

The Sydney Heart Bank (SHB) is one of the largest human heart tissue banks in existence. Its mission is to provide high-quality human heart tissue for research into the molecular basis of human heart failure by working collaboratively with experts in this field. We argue that, by comparing tissues from failing human hearts with age-matched non-failing healthy donor hearts, the results will be more relevant than research using animal models, particularly if their physiology is very different from humans. Tissue from heart surgery must generally be used soon after collection or it significantly deteriorates. Freezing is an option but it raises concerns that freezing causes substantial damage at the cellular and molecular level. The SHB contains failing samples from heart transplant patients and others who provided informed consent for the use of their tissue for research. All samples are cryopreserved in liquid nitrogen within 40 min of their removal from the patient, and in less than 5-10 min in the case of coronary arteries and left ventricle samples. To date, the SHB has collected tissue from about 450 failing hearts (>15,000 samples) from patients with a wide range of etiologies as well as increasing numbers of cardiomyectomy samples from patients with hypertrophic cardiomyopathy. The Bank also has hearts from over 120 healthy organ donors whose hearts, for a variety of reasons (mainly tissue-type incompatibility with waiting heart transplant recipients), could not be used for transplantation. Donor hearts were collected by the St Vincent's Hospital Heart and Lung transplantation team from local hospitals or within a 4-h jet flight from Sydney. They were flushed with chilled cardioplegic solution and transported to Sydney where they were quickly cryopreserved in small samples. Failing and/or donor samples have been used by more than 60 research teams around the world, and have resulted in more than 100 research papers. The tissues most commonly requested are from donor left ventricles, but right ventricles, atria, interventricular system, and coronary arteries vessels have also been reported. All tissues are stored for long-term use in liquid N or vapor (170-180 °C), and are shipped under nitrogen vapor to avoid degradation of sensitive molecules such as RNAs and giant proteins. We present evidence that the availability of these human heart samples has contributed to a reduction in the use of animal models of human heart failure. [ABSTRACT FROM AUTHOR]

Aims In heart failure transverse-tubule (t-tubule) remodelling disrupts calcium release, and contraction. T-tubules in human failing hearts exhibit increased labelling by wheat germ agglutinin (WGA), a lectin that binds to the dystrophin-associated glycoprotein complex. We hypothesized changes in this complex may explain the increased WGA labelling and contribute to t-tubule remodelling in the failing human heart. In this study we sought to identify the molecules responsible for this increased WGA labelling. Methods and results Confocal and super-resolution fluorescence microscopy and proteomic analyses were used to quantify left ventricle samples from healthy donors and patients with idiopathic dilated cardiomyopathy (IDCM). Confocal microscopy demonstrated both WGA and dystrophin were located at t-tubules. Super-resolution microscopy revealed that WGA labelling of t-tubules is largely located within the lumen while dystrophin was restricted to near the sarcolemma. Western blots probed with WGA reveal a 5.7-fold increase in a 140 kDa band in IDCM. Mass spectrometry identified this band as type VI collagen (Col-VI) comprised of α1(VI), α2(VI), and α3(VI) chains. Pertinently, mutations in Col-VI cause muscular dystrophy. Western blotting identified a 2.4-fold increased expression and 3.2-fold increased WGA binding of Col-VI in IDCM. Confocal images showed that Col-VI is located in the t-tubules and that their diameter increased in the IDCM samples. Super-resolution imaging revealed Col-VI was restricted to the t-tubule lumen where increases were associated with displacement in the sarcolemma as identified from dystrophin labelling. Samples were also labelled for type I, III, and IV collagen. Both confocal and super-resolution imaging identified that these collagens were also present within t-tubule lumen. Conclusion Increased expression and labelling of collagen in IDCM samples indicates fibrosis may contribute to t-tubule remodelling in human heart failure. [ABSTRACT FROM AUTHOR]

Protein phosphatase 1 (PP1) is a key regulator of important cardiac signaling pathways. Dysregulation of PP1 has been heavily implicated in cardiac dysfunctions. Accordingly, pharmacological targeting of PP1 activity is considered for therapeutic intervention in human cardiomyopathies. Recent evidence from animal models implicated previously unrecognized, isoform-specific activities of PP1 in the healthy and diseased heart. Therefore, this study examined the expression of the distinct PP1 isoforms PP1α, β, and γ in human heart failure (HF) and atrial fibrillation (AF) and addressed the consequences of β-adrenoceptor blocker (beta-blocker) therapy for HF patients with reduced ejection fraction on PP1 isoform expression. Using western blot analysis, we found greater abundance of PP1 isoforms α and γ but unaltered PP1β levels in left ventricular myocardial tissues from HF patients as compared to non-failing controls. However, expression of all three PP1 isoforms was higher in atrial appendages from patients with AF compared to patients with sinus rhythm. Moreover, we found that in human failing ventricles, beta-blocker therapy was associated with lower PP1α abundance and activity, as indicated by higher phosphorylation of the PP1α-specific substrate eIF2α. Greater eIF2α phosphorylation is a known repressor of protein translation, and accordingly, we found lower levels of the endoplasmic reticulum (ER) stress marker Grp78 in the very same samples. We propose that isoform-specific targeting of PP1α activity may be a novel and innovative therapeutic strategy for the treatment of human cardiac diseases by reducing ER stress conditions. [ABSTRACT FROM AUTHOR]

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.

Deficiency of energy supply is a major complication contributing to the syndrome of heart failure (HF). Because the concurrent activity profile of mitochondrial bioenergetic enzymes has not been studied collectively in human HF, our aim was to examine the mitochondrial enzyme defects in left ventricular myocardium obtained from explanted end-stage failing hearts. Compared with nonfailing donor hearts, activity rates of complexes I and IV and the Krebs cycle enzymes isocitrate dehydrogenase, malate dehydrogenase, and aconitase were lower in HF, as determined spectrophotometrically. However, activity rates of complexes II and III and citrate synthase did not differ significantly between the two groups. Protein expression, determined by Western blotting, did not differ between the groups, implying posttranslational perturbation. In the face of diminished total glutathione and coenzyme Q10 levels, oxidative modification was explored as an underlying cause of enzyme dysfunction. Of the three oxidative modifications measured, protein carbonylation was increased significantly by 31% in HF (P < 0.01; n = 18), whereas levels of 4-hydroxynonenal and protein nitration, although elevated, did not differ. Isolation of complexes I and IV and F1FoATP synthase by immunocapture revealed that proteins containing iron-sulphur or heme redox centers were targets of oxidative modification. Energy deficiency in end-stage failing human left ventricle involves impaired activity of key electron transport chain and Krebs cycle enzymes without altered expression of protein levels. Augmented oxidative modification of crucial enzyme subunit structures implicates dysfunction due to diminished capacity for management of mitochondrial reactive oxygen species, thus contributing further to reduced bioenergetics in human HF. [ABSTRACT FROM AUTHOR]

Matrix metalloproteinases (MMPs) play an important role in the pathogenesis of heart failure (HF). Our aim was to determine the activities of circulating MMP-2 and MMP-9 in patients with HF in respect of gender, comorbidities and treatment (n=51). We did not reveal any differences in circulating pro-MMP-2 and pro-MMP-9 activities between the patients with HF and without it. However, there was a decrease in activity of pro-MMP-2 in treated hypertensive participants versus healthy ones. In contrast, we observed increased pro-MMP-2 activity in hypertensive participants with coexistent HF versus hypertensive participants without HF. In addition, a decrease in pro-MMP-2 activity was shown in women suffering from HF versus men suffering from HF. In conclusion, potential inhibitory effect of antihypertensive treatment on pro-MMP-2 activity was found. Coexistent HF with hypertension probably reduces the inhibitory effect of antihypertensive treatment on pro-MMP-2 activity. Our data also suggest the role of potential cardioprotective factors influencing the activity of pro-MMP-2 in women. [ABSTRACT FROM AUTHOR]

Aims: Protein kinases are potential therapeutic targets for heart failure, but most studies of cardiac protein kinases derive from other systems, an approach that fails to account for specific kinases expressed in the heart and the contractile cardiomyocytes. We aimed to define the cardiomyocyte kinome (i.e. the protein kinases expressed in cardiomyocytes) and identify kinases with altered expression in human failing hearts. Methods and results: Expression profiling (Affymetrix microarrays) detected > 400 protein kinase mRNAs in rat neonatal ventricular myocytes (NVMs) and/or adult ventricular myocytes (AVMs), 32 and 93 of which were significantly up-regulated or down-regulated (greater than two-fold), respectively, in AVMs. Data for AGC family members were validated by qPCR. Proteomics analysis identified > 180 cardiomyocyte protein kinases, with high relative expression of mitogen- activated protein kinase cascades and other known cardiomyocyte kinases (e.g. CAMKs, cAMP-dependent protein kinase). Other kinases are poorly investigated (e.g. Slk, Stk24, Oxsr1). Expression of Akt1/2/3, BRaf, ERK1/2, Map2k1, Map3k8, Map4k4, MST1/3, p38-MAPK, PKCδ, Pkn2, Ripk1/2, Tnni3k, and Zak was confirmed by immunoblotting. Relative to total protein, Map3k8 and Tnni3k were up-regulated in AVMs vs. NVMs. Microarray data for human hearts demonstrated variation in kinome expression that may influence responses to kinase inhibitor therapies. Furthermore, some kinases were up-regulated (e.g. NRK, JAK2, STK38L) or down-regulated (e.g. MAP2K1, IRAK1, STK40) in human failing hearts. Conclusion: This characterization of the spectrum of kinases expressed in cardiomyocytes and the heart (cardiomyocyte and cardiac kinomes) identified novel kinases, some of which are differentially expressed in failing human hearts and could serve as potential therapeutic targets. [ABSTRACT FROM AUTHOR]

Background Right ventricular (RV) dysfunction contributes to mortality in chronic heart failure (HF). However, the molecular mechanisms of RV failure remain poorly understood, and RV myocardial biomarkers have yet to be developed. Methods and Results We performed RNA sequencing (RNA-seq) on 22 explanted human HF RVs and 5 unused donor human heart RVs (DON RV) and compared results to those recently reported from 16 explanted human LVs We used Bowtie-Tophat for transcript alignment and transcriptome assembly, DESeq for identification of differentially expressed genes (DEGs) and Ingenuity for exploration of gene ontologies. In the HF RV, RNA-seq identified 130,790 total RNA transcripts including 13,272 protein coding genes, 10,831 long non-coding RNA genes and 8,605 pseudogenes. There were 800-1000 DEGs between DON and HF RV comparison groups with differences concentrated in cytoskeletal, basement membrane, extracellular matrix (ECM), inflammatory mediator, hemostasis, membrane transport and transcription factor genes, lncRNAs and pseudogenes. In an unbiased approach, the top 10 DEGs SERPINA3, SERPINA5, LCN6, LCN10, STEAP4, AKR1C1, STAC2, SPARCL1, VSIG4 and F8 exhibited no overlap in read counts between DON and HF RVs, high sensitivities, specificities, predictive values and areas under the receiver operating characteristic curves. STEAP4, SPARCL1 and VSIG4 were differentially expressed between RVs and LVs, supporting their roles as RV-specific myocardial biomarkers. Conclusions Unbiased, comprehensive profiling of the RV transcriptome by RNA-seq suggests structural changes and abnormalities in inflammatory processes and yields specific, novel HF RV vs HF LV myocardial biomarkers not previously identified by more limited transcriptome profiling approaches. [ABSTRACT FROM AUTHOR]

The β-blockers carvedilol and metoprolol provide important therapeutic strategies for heart failure treatment. Therapy with metoprolol facilitates the control by phosphodiesterase PDE3, but not PDE4, of inotropic effects of catecholamines in human failing ventricle. However, it is not known whether carvedilol has the same effect. We investigated whether the PDE3-selective inhibitor cilostamide (0.3 μM) or PDE4-selective inhibitor rolipram (1 μM) modified the positive inotropic and lusitropic effects of catecholamines in ventricular myocardium of heart failure patients treated with carvedilol. Right ventricular trabeculae from explanted hearts of nine carvedilol-treated patients with terminal heart failure were paced to contract at 1 Hz. The effects of (-)-noradrenaline, mediated through β-adrenoceptors (β-adrenoceptors blocked with ICI118551), and (-)-adrenaline, mediated through β-adrenoceptors (β-adrenoceptors blocked with CGP20712A), were assessed in the absence and presence of the PDE inhibitors. The inotropic potency, estimated from -logECs, was unchanged for (-)-noradrenaline but decreased 16-fold for (-)-adrenaline in carvedilol-treated compared to non-β-blocker-treated patients, consistent with the previously reported β-adrenoceptor-selectivity of carvedilol. Cilostamide caused 2- to 3-fold and 10- to 35-fold potentiations of the inotropic and lusitropic effects of (-)-noradrenaline and (-)-adrenaline, respectively, in trabeculae from carvedilol-treated patients. Rolipram did not affect the inotropic and lusitropic potencies of (-)-noradrenaline or (-)-adrenaline. Treatment of heart failure patients with carvedilol induces PDE3 to selectively control the positive inotropic and lusitropic effects mediated through ventricular β-adrenoceptors compared to β-adrenoceptors. The β-adrenoceptor-selectivity of carvedilol may provide protection against β-adrenoceptor-mediated ventricular overstimulation in PDE3 inhibitor-treated patients. PDE4 does not control β- and β-adrenoceptor-mediated inotropic and lusitropic effects in carvedilol-treated patients. [ABSTRACT FROM AUTHOR]

Abstract: In heart failure mitochondrial dysfunction is thought to be responsible for energy depletion and contractile dysfunction. The difficulties in procuring fresh left ventricular (LV) myocardium from humans for assessment of mitochondrial function have resulted in the reliance on surrogate markers of mitochondrial function and limited our understanding of cardiac energetics. We isolated mitochondria from fresh LV wall tissue of patients with heart failure and reduced systolic function undergoing heart transplant or left ventricular assist device placement, and compared their function to mitochondria isolated from the non-failing LV (NFLV) wall tissue with normal systolic function from patients with pulmonary hypertension undergoing heart–lung transplant. We performed detailed mitochondrial functional analyses using 4 substrates: glutamate–malate (GM), pyruvate–malate (PM) palmitoyl carnitine–malate (PC) and succinate. NFLV mitochondria showed preserved respiratory control ratios and electron chain integrity with only few differences for the 4 substrates. In contrast, HF mitochondria had greater respiration with GM, PM and PC substrates and higher electron chain capacity for PM than for PC. Surprisingly, HF mitochondria had greater respiratory control ratios and lower ADP-independent state 4 rates than NFLV mitochondria for GM, PM and PC substrates demonstrating that HF mitochondria are capable of coupled respiration ex vivo. Gene expression studies revealed decreased expression of key genes in pathways for oxidation of both fatty acids and glucose. Our results suggest that mitochondria from the failing LV myocardium are capable of tightly coupled respiration when isolated and supplied with ample substrates. Thus energy starvation in the failing heart may be the result of dysregulation of metabolic pathways, impaired substrate supply or reduced mitochondrial number but not the result of reduced mitochondrial electron transport capacity. [Copyright &y& Elsevier]

Background and Purpose PDE3 and/or PDE4 control ventricular effects of catecholamines in several species but their relative effects in failing human ventricle are unknown. We investigated whether the PDE3-selective inhibitor cilostamide (0.3-1 μM) or PDE4 inhibitor rolipram (1-10 μM) modified the positive inotropic and lusitropic effects of catecholamines in human failing myocardium. Experimental Approach Right and left ventricular trabeculae from freshly explanted hearts of 5 non-β-blocker-treated and 15 metoprolol-treated patients with terminal heart failure were paced to contract at 1 Hz. The effects of (-)-noradrenaline, mediated through β1 adrenoceptors (β2 adrenoceptors blocked with ICI118551), and (-)-adrenaline, mediated through β2 adrenoceptors (β1 adrenoceptors blocked with CGP20712A), were assessed in the absence and presence of PDE inhibitors. Catecholamine potencies were estimated from - logEC50s. Key Results Cilostamide did not significantly potentiate the inotropic effects of the catecholamines in non-β-blocker-treated patients. Cilostamide caused greater potentiation ( P = 0.037) of the positive inotropic effects of (-)-adrenaline (0.78 ± 0.12 log units) than (-)-noradrenaline (0.47 ± 0.12 log units) in metoprolol-treated patients. Lusitropic effects of the catecholamines were also potentiated by cilostamide. Rolipram did not affect the inotropic and lusitropic potencies of (-)-noradrenaline or (-)-adrenaline on right and left ventricular trabeculae from metoprolol-treated patients. Conclusions and Implications Metoprolol induces a control by PDE3 of ventricular effects mediated through both β1 and β2 adrenoceptors, thereby further reducing sympathetic cardiostimulation in patients with terminal heart failure. Concurrent therapy with a PDE3 blocker and metoprolol could conceivably facilitate cardiostimulation evoked by adrenaline through β2 adrenoceptors. PDE4 does not appear to reduce inotropic and lusitropic effects of catecholamines in failing human ventricle. Linked Article This article is commented on by Eschenhagen, pp 524-527 of this issue. To view this commentary visit http://dx.doi.org/10.1111/bph.12168 [ABSTRACT FROM AUTHOR]

Increased dispersion of repolarization has been suggested to underlie increased arrhythmogenesis in human heart failure (HF). However, no detailed repolarization mapping data were available to support the presence of increased dispersion of repolarization in failing human heart. In the present study, we aimed to determine the existence of enhanced repolarization dispersion in the right ventricular (RV) endocardium from failing human heart and examine its association with arrhythmia inducibility. RV free wall preparations were dissected from five failing and five nonfailing human hearts, cannulated and coronary perfused. RV endocardium was optically mapped from an ~6.3 × 6.3 cm2 field of view. Action potential duration (APD), dispersion of APD, and conduction velocity (CV) were quantified for basic cycle lengths (BCL) ranging from 2,000 ms to the functional refractory period. We found that RV APD was significantly prolonged within the failing group compared with the nonfailing group (560 ± 44 vs. 448 ± 39 ms, at BCL = 2,000 ms, P < 0.05). Dispersion of APD was increased in three failing hearts (161 ± 5 vs. 86 ± 19 ms, at BCL < 2,000 ms). APD alternans were induced by rapid pacing in these same three failing hearts. CV was significantly reduced in the failing group compared with the nonfailing group (81 ± 11 vs. 98 ± 8 cm/s, at BCL = 2,000 ms). Arrhythmias could be induced in two failing hearts exhibiting an abnormally steep CV restitution and increased dispersion of repolarization due to APD alternans. Dispersion of repolarization is enhanced across the RV endocardium in the failing human heart. This dispersion, together with APD alternans and abnormal CV restitution, could be responsible for the arrhythmia susceptibility in human HF. [ABSTRACT FROM AUTHOR]

Abstract: The discovery of novel disease biomarkers is a crucial challenge for translational bioinformatics. Demonstration of both their classification power and reproducibility across independent datasets are essential requirements to assess their potential clinical relevance. Small datasets and multiplicity of putative biomarker sets may explain lack of predictive reproducibility. Studies based on pathway-driven discovery approaches have suggested that, despite such discrepancies, the resulting putative biomarkers tend to be implicated in common biological processes. Investigations of this problem have been mainly focused on datasets derived from cancer research. We investigated the predictive andfunctional concordance of five methods for discovering putative biomarkers in four independently-generated datasets from the cardiovascular disease domain. A diversity of biosignatures was identified by the different methods. However, we found strong biological process concordance between them, especially in the case of methods based on gene set analysis. With a few exceptions, we observed lack of classification reproducibility using independent datasets. Partial overlaps between our putative sets of biomarkers and the primary studies exist. Despite the observed limitations, pathway-driven or gene set analysis can predict potentially novel biomarkers and can jointly point to biomedically-relevant underlying molecular mechanisms. [Copyright &y& Elsevier]

The article discusses a study which investigates the role of defective genes in mitochondrial activity leading to heart failure in humans. The experiment involves gathering left ventricular tissue from heart failure patients and those who have healthy hearts, which shows that the defective genes decrease the creation of mitochondria within the heart of patients with the condition. The study proves that defective DNA causes malfunction in the generation of mitochondria and their reduction within patients that have heart failure.

Abstract: If overexpression has been functionally demonstrated in ventricular myocytes from failing human hearts. Altered expression of If-channels as a consequence of electrophysiological remodeling may represent an arrhythmogenic mechanism in heart failure; however, the molecular basis of If overexpression in human cardiac disease is unknown. HCN1, 2 and 4 subtypes, which encode If-channels, have been identified in the heart. The present study was designed to characterize HCN isoform expression in failing and non-failing hearts. Ventricular and atrial samples were obtained from normal or failing hearts explanted from patients with end-stage ischemic cardiomyopathy. If was recorded in patch-clamped left ventricular myocytes. mRNA and protein expression of HCN subunits were measured in both atria and ventricles of control and diseased hearts. HCN2 and HCN4 were detected in human myocardium. Both mRNA and protein levels of HCN2/4 were significantly augmented in failing ventricles (p <0.01 for mRNA, p <0.05 for protein). These results are consistent with the electrophysiological data showing that, in failing ventricular myocytes, If is of larger amplitude and activates at less negative potential. Changes in mRNA and protein expression of both HCN2/4 isoforms in atrial specimens from patients with heart failure mirrored those observed in ventricles (p <0.001 for mRNA, p <0.05 for protein). No disease-dependent alteration was detected for MiRP1, the putative β-subunit of the If-channel. In conclusion, HCN4 is the predominant channel subtype in normal human heart, and its expression is further amplified by disease. HCN upregulation likely contributes to increased If and may play a role in ventricular and atrial arrhythmogenesis in heart failure. [Copyright &y& Elsevier]

Abstract: β-Adrenoceptor blocking agents (β-blockers) that at low concentrations antagonize cardiostimulant effects of catecholamines, but at high concentrations also cause cardiostimulation, have been appearing since the late 1960s. These cardiostimulant β-blockers, coined non-conventional partial agonists, antagonize the effects of catecholamines through a high-affinity site (β1HAR), but cause cardiostimulation mainly through a low-affinity site (β1LAR) of the myocardial β1-adrenoceptor. The experimental non-conventional partial agonist (−)-CGP12177 increases cardiac L-type Ca2+ current density and Ca2+ transients, shortens action potential duration but augments action potential plateau, increases heart rate and force, as well as causes arrhythmic Ca2+ transients and arrhythmic cardiocyte contractions. Other β-blockers, which do not cause cardiostimulation, consistently have lower affinity for β1LAR than β1HAR. These sites were verified and the cardiac pharmacology of non-conventional partial agonists confirmed on recombinant β1-adrenoceptors and on β1-adrenoceptors overexpressed into the heart. A targeted mutation of Asp138 to Glu138 virtually abolished the pharmacology of β1HAR but left intact the pharmacology of β1LAR. Non-conventional partial agonists may be beneficial for the treatment of peripheral autonomic neuropathy but probably due to their arrhythmic propensities, may be harmful for the treatment of chronic heart failure. [Copyright &y& Elsevier]

1. In atrium and ventricle from failing and non-failing human hearts, activation of β1- or β2-adrenoceptors causes increases in contractile force, hastening of relaxation, protein kinase A-catalysed phosphorylation of proteins implicated in the hastening of relaxation, phospholamban, troponin I and C-protein, consistent with coupling of both β1- and β2-adrenoceptors to stimulatory Gsa-protein but not inhibitory Gia-protein. 2. Two ‘affinity states’, namely β1H and β1L, of the β1-adrenoceptor exist. In human heart, noradrenaline elicits powerful increases in contractile force and hastening of relaxation. These effects are blocked with high affinity by β-adenoceptor antagonists, including propranolol, (–)-pindolol, (–)-CGP 12177 and carvedilol. Some beta-blockers, typified by (–)-pindolol and (–)-CGP 12177, not only block the receptor, but also activate it, albeit at much higher concentrations (approximately 2 log units) than those required to antagonize the effects of catecholamines. In human heart, both (–)-CGP 12177 and (–)-pindolol increase contractile force and hasten relaxation. However, the involvement of the β1-adrenoceptor was not immediately obvious because (–)-pindolol- and (–)-CGP 12177-evoked responses were relatively resistant to blockade by (–)-propranolol. Abrogation of cardiostimulant effects of (–)-CGP 12177 in β1-/β2-adrenoceptor double-knockout mice, but not β2-adrenoceptor-knockout mice, revealed an obligatory role of the β1-adrenoceptor. On the basis of these results, two ‘affinity states’ have been designated, the β1H- and β1L-adrenoceptor, where the β1H-adrenoceptor is activated by noradrenaline and blocked with high affinity by beta-blockers and the β1L-adrenoceptor is activated by drugs such as (–)-CGP 12177 and (–)-pindolol and blocked with low affinity by beta-blockers such as (–)-propranolol. The β1H- and β1L-adrenoceptor states are consistent with high- and low-affinity binding sites for (–)-[3H]-CGP 12177 radioligand binding found in cardiac muscle and recombinant β1-adrenoceptors. 3. There are two common polymorphic locations of the β1-adrenoceptor, at amino acids 49 (Ser/Gly) and 389 (Arg/Gly). Their existence has raised several questions, including their role in determining the effectiveness of heart failure treatment with beta-blockers. We have investigated the effect of long-term maximally tolerated carvedilol administration (> 1 year) on left ventricular ejection fraction (LVEF) in patients with non-ischaemic cardiomyopathy (mean left ventricular ejection fraction 23 ± 7%; n = 135 patients). The administration of carvedilol improved LVEF to 37 ± 13% ( P < 0.005); however, the improvement was variable, with 32% of patients showing £ 5% improvement. Upon segregation of patients into Arg389Gly-β1-adrenoceptors, it was found that carvedilol caused a greater increase in left ventricular ejection faction in patients carrying the Arg389 allele with Arg389Arg > Arg389Gly > Gly389Gly. [ABSTRACT FROM AUTHOR]

Abstract: Cardiac myosin-binding protein-C (cMyBP-C) is an important regulator of cardiac contractility, and its phosphorylation by PKA is a mechanism that contributes to increased cardiac output in response to β-adrenergic stimulation. It is presently unknown whether heart failure alters cMyBP-C phosphorylation. The present study determined the level of phosphorylated cMyBP-C in failing human hearts and in a canine model of pacing-induced heart failure. A polyclonal antibody directed against the major phosphorylation site of cMyBP-C (Ser-282) was generated and its specificity was confirmed by PKA phosphorylation with isoprenaline in cardiomyocytes and Langendorff-perfused mouse hearts. Left ventricular myocardial tissue from (i) patients with terminal heart failure (hHF; n =12) and nonfailing donor hearts (hNF; n =6) and (ii) dogs with rapid-pacing-induced end-stage heart failure (dHF; n =10) and sham-operated controls (dNF; n =10) were used for quantification of total cMyBP-C and phospho-cMyBP-C by Western blotting. Total cMyBP-C protein levels were similar in hHF and hNF as well as in dHF and dNF. In contrast, the ratio of phospho-cMyBP-C to total cMyBP-C levels were >50% reduced in hHF and >40% reduced in dHF. In summary, cMyBP-C phosphorylation levels are markedly decreased in human and experimental heart failure. Thus, the compromised contractile function of the failing heart might be in part attributable to reduced cMyBP-C phosphorylation levels. [Copyright &y& Elsevier]

Abstract: Human heart failure is a complex clinical syndrome characterized by extensive abnormalities in the β-adrenergic receptor (βAR) system. Normalization of βAR signalling consistently ameliorates cardiac dysfunction and survival in heart failure, suggesting that βAR dysfunction may be intrinsically linked to the deterioration of cardiac performance. Agonist-dependent phosphorylation of βARs by βAR kinase 1 (βARK1) initiates the processes of desensitization and downregulation, hallmarks of heart failure. Our recent studies have shown that βARK1 forms a cytosolic complex with phosphoinositide 3-kinase (PI3K) and that translocation of βARK1 to the plasma membrane also promotes the βAR-targeting of PI3Ks. At the plasma membrane, the generation of 3′-phosphorylated phosphatidylinositols by PI3K is required in the process of endocytosis, a prodrome to receptor downregulation. A large body of data now indicates that βAR-targeting of PI3Ks is consistently associated with abnormalities of βAR signalling under pathological conditions, including pressure-overload hypertrophy and heart failure from different causes. In this review we will discuss the role of βAR-targeted PI3K activity and novel experimental strategies to disrupt the βARK1/PI3K complex and in turn ameliorate βAR dysfunction and the progression of heart failure. [Copyright &y& Elsevier]

Idiopathic-dilated cardiomyopathy (IDC) is a common primary myocardial disease of unknown etiology associated with apoptosis, cardiac dilatation, progressive heart failure and increased mortality. An elevation of the transcription factor activator protein 2a (AP-2a) is involved in vertebrate embryonic development and oncogenesis. Here, we show that AP-2a protein is expressed in the human heart and increased in human failing myocardium with IDC. Adenovirus-mediated overexpression of human AP-2a triggered apoptosis and increased mRNA levels of Bcl-2 family members Bax and Bcl-x in rat cardiomyocytes. Immunohistological analysis of human myocardium revealed an increased percentage of AP-2a-positive nuclei in IDC and, interestingly, a colocalization of AP-2a-positive but not -negative cells with a caspase-cleaved fragment of poly(ADP-ribose)polymerase. We suggest AP-2a as a novel cardiac regulator implicated in the activation of apoptosis in IDC.Cell Death and Differentiation (2004) 11, 485-493. doi:10.1038/sj.cdd.4401383 Published online 30 January 2004 [ABSTRACT FROM AUTHOR]

Human heart failure carries with it high mortal risks. The deficit in ventricular function results from a depression in power along with a blunting of the force-frequency relationship. Mechanical, myothermal and molecular biological analysis are used to uncover the mechanistic basis for the deficit in performance. At the cross-bridge level the average isometric force-time integral is increased in failing hearts. Despite this increase, the isometric peak force and rate of relaxation are markedly depressed and correlate with a substantial decrease in the amount and rate of calcium cycling per beat. The blunting of the force frequency relationship is shown to be directly related to the frequency dependence of the calcium concentration which in turn can be accounted for by alterations in the SERCA 2 calcium pump and the sarcolemmal Na/Ca exchange protein. There is a positive correlation between sarcoplasmic reticular calcium ATPase and frequency dependent changes in twitch tension and an inverse correlation between Na/Ca exchange protein and diastolic force. This analysis allows the hearts to be divided into two groups based on the Na/Ca exchange and SR calcium ATPase levels that separates the hearts that develop increased diastolic force from those that do not. In both groups of failing hearts the ratio of the Na/Ca exchange protein to the SERCA 2 pump is substantially increased. The phenotypic alterations in Na/Ca exchange protein, SERCA 2 calcium pump and the TnT isoform shift can account for the depression in power output and the associated ventricular dysfunction found in heart failure. [ABSTRACT FROM AUTHOR]

Impaired production of myocardial cyclic adenosine monophosphate (cAMP) is thought to contribute to contractile dysfunction in end stage heart failure, but myocardial cAMP content has not yet been evaluated in heart failure patients in comparison with controls. We therefore measured the myocardial content of cAMP by radioimmunossay in endomyocardial biopsies from patients in different stages of heart failure and in controls and correlated it with biochemical and functional parameters. The myocardial content of norepinephrine was determined by HPLC in the same biopsies in order to assess if the myocardium studied was affected by heart failure. Myocardial cAMP (in fmoLμg−1 non-collagen protein) in 20 patients with heart failure (LVEF: 27 ± 8%, cAMP: 5.8 ± 2.0) was unchanged in comparison with eight controls (LVEF: 64 ± 4.7%, cAMP, 4.9 ± 2.1). In contrast, myocardial norepinephrine (in pg.μg−1 non-collagen protein) in the same biopsies was significantly reduced in heart failure (4.0 ±3.0) in comparison with the same controls (11.5 ±3.0, P <0.0002). Plasma cAMP in 20 heart failure patients (22.0 ± 4.2 pmoLl−1) was not different from controls (22.0± 7.8), whereas plasma norepinephrine was increased (heart failure: 460 ±257pg.ml−1, controls 182 ± 49, P < 0.001).Myocardial cAMP levels are indistinguishable from controls in human heart failure and therefore do not contribute to a further characterization of the cardiac adrenergic system in these patients. This is most likely due to the impossibility of obtaining biopsies with truly unstimulated adenylyl cyclase activity. [ABSTRACT FROM PUBLISHER]

Methods to obtain pressure-volume loops in humans and their application to clinical diagnosis and treatment of cardiac disease are discussed. The method employs a conductance catheter, micromanometer, and inferior vena caval balloon occluder to reversibly and rapidly vary venous return. Multiple cardiac cycles measured during this manoeuvre are used to derive a variety of systolic and diastolic function indices, as well as estimates of vascular loading, and cardiac energetics. Strengths and weaknesses of the method as well as pressure-volume indexes themselves are briefly discussed Lastly, three clinical examples are presented which serve to demonstrate how pressure-volume analysis can provide useful clinical information regarding diagnosis and/or treatment in subjects with heart failure symptoms. [ABSTRACT FROM PUBLISHER]

Little is known about any alterations in sarcoplasmic reticulum (SR) gene expression associated with cardiac diseases of varying degrees of severity. We assessed, using the reverse transcription-polymerase chain reaction (RT-PCR) technique, SR Ca2+ transport protein gene expression in small tissue samples from failing hearts in patients undergoing cardiac surgery. Total RNA was extracted from 30- to 50-mg samples from the hearts of 13 patients with coronary artery disease, congenital heart disease, or valvular heart disease. We used RT-PCR to synthesize and amplify cDNA encoding cardiac SR Ca2+-ATPase, ryanodine receptor (RYR), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The amount of each mRNA in the sample was expressed relative to the amount of GAPDH mRNA. The expression level of each mRNA was correlated with the cardiac functional index. The mRNA levels for Ca2+-ATPase and RYR varied between heart samples, but showed a positive correlation with left ventricular ejection fraction. Ca2+-ATPase mRNA levels showed an inverse relationship with plasma brain natriuretic peptide. In addition, we isolated partial cDNA encoding a human cardiac RYR. The cDNA consisted of 487 nucleotides, and the nucleotide and deduced amino acid sequences showed 93% and 99% homology, respectively, to those of rabbit cardiac RYR. These results suggest that decreased levels of mRNA for SR Ca2+ transport protein could be related to abnormal cardiac function, regardless of the etiology of the heart disease. RT-PCR provides a rapid and economical way of quantifying the expression of multiple genes in small specimens and may, therefore, aid understanding of the pathophysiology and treatment of heart disease. [ABSTRACT FROM AUTHOR]

Background: The present study aimed to characterize the effects of epinine, the active metabolite of ibopamine on tension development in human renal arteries. Methods and results: Experiments were performed on isolated human renal arteries rings obtained during surgery due to kidney tumors ( n = 12). Epinine concentration-dependently relaxed isolated precontracted (PGF) human renal artery rings ( P < 0.05) in the presence of phentolamine, as effectively (epinine − 30 +/− 4 mN, dopamine − 31 +/− 5 mN) and with the same potency as dopamine (epinine EC 0.7 μmol/l (0.4−1.2 μmol/l), dopamine 0.5 μmol/l (0.2−1.7 μmol/l)). This effect was antagonized by the specific D-receptor-antagonist SCH 23390. Effective β-adrenoceptor antagonistic concentrations of propranolol did not affect epinine-induced vasorelaxation. In the absence of α-and β-adrenoceptor-antagonists the potency of epinine to contract renal artery rings was significantly higher compared to dopamine indicating a higher affinity of epinine to α-adrenoceptors. Conclusion: The present study provides evidence for direct vasodilatory effects of epinine via activation of D-receptors on human renal arteries. [ABSTRACT FROM AUTHOR]

In recent years, evidence from various animal experiments has accumulated that emphasizes the role of endothelin-1 in the pathophysiology of several cardiovascular diseases, including congestive heart failure. The recent advent of potent antagonists of this system now allows the assessment of the involvement of endothelin-1 in the maintenance of vascular tone in animals and humans. We report hemodynamic data from two trails in patients with chronic severe congestive heart failure (i.e., reduced left ventricular ejection fraction of <30%, elevated resting pulmonary capillary wedged pressure >15 mmHg, and/or reduced cardiac index of 2.5 L/min/m2 or less) who were treated with the mixed endothelin-type A and type B-receptor antagonist bosentan. In the first study, the acute effect of bosentan (300 mg, intravenous) on hemodynamics and neurohormones was investigated. Bosentan was well tolerated and significantly improved impaired hemodynamics due to systemic and venous vasodilation. In the second trial, bosentan was given orally (0.5 g bid) for 14 days, in addition to conventional triple treatment for congestive heart failure, including digitalis, angiotensin-converting enzyme inhibitors, and diuretics. Cardiac hemodynamics were monitored during the first 24 hours of treatment, and measurements were repeated during the last day of bosentan therapy. Bosentan was well tolerated in these patients as well, and hemodynamic measures were compatible with an additional effect of bosentan after 2 weeks. However, there was a slight increase in heart rate as well. Our result underline the importance of endogenously generated endothelin-1 in congestive heart failure and suggest a potential benefit of endothelin antagonism in such patients. However, long-term studies are needed to establish whether chronic endothelin antagonism has beneficial clinical effects and is capable of improving survival and/or symptoms in severe heart failure patients who remain symptomatic despite standard triple therapy. [ABSTRACT FROM AUTHOR]