The Translational Landscape of the Human Heart.

Gene expression in human tissue has primarily been studied on the transcriptional level, largely neglecting translational regulation. Here, we analyze the translatomes of 80 human hearts to identify new translation events and quantify the effect of translational regulation. We show extensive translational control of cardiac gene expression, which is orchestrated in a process-specific manner. Translation downstream of predicted disease-causing protein-truncating variants appears to be frequent, suggesting inefficient translation termination. We identify hundreds of previously undetected microproteins, expressed from lncRNAs and circRNAs, for which we validate the protein products in vivo. The translation of microproteins is not restricted to the heart and prominent in the translatomes of human kidney and liver. We associate these microproteins with diverse cellular processes and compartments and find that many locate to the mitochondria. Importantly, dozens of microproteins are translated from lncRNAs with well-characterized noncoding functions, indicating previously unrecognized biology.

Dynamics of Cell Generation and Turnover in the Human Heart.

The contribution of cell generation to physiological heart growth and maintenance in humans has been difficult to establish and has remained controversial. We report that the full complement of cardiomyocytes is established perinataly and remains stable over the human lifespan, whereas the numbers of both endothelial and mesenchymal cells increase substantially from birth to early adulthood. Analysis of the integration of nuclear bomb test-derived (14)C revealed a high turnover rate of endothelial cells throughout life (>15% per year) and more limited renewal of mesenchymal cells (<4% per year in adulthood). Cardiomyocyte exchange is highest in early childhood and decreases gradually throughout life to <1% per year in adulthood, with similar turnover rates in the major subdivisions of the myocardium. We provide an integrated model of cell generation and turnover in the human heart.

Calmodulin inhibition of human RyR2 channels requires phosphorylation of RyR2-S2808 or RyR2-S2814.

Calmodulin (CaM) is a Ca-binding protein that binds to, and can directly inhibit cardiac ryanodine receptor calcium release channels (RyR2). Animal studies have shown that RyR2 hyperphosphorylation reduces CaM binding to RyR2 in failing hearts, but data are lacking on how CaM regulates human RyR2 and how this regulation is affected by RyR2 phosphorylation. Physiological concentrations of CaM (100 nM) inhibited the diastolic activity of RyR2 isolated from failing human hearts by ~50% but had no effect on RyR2 from healthy human hearts. Using FRET between donor-FKBP12.6 and acceptor-CaM bound to RyR2, we determined that CaM binds to RyR2 from healthy human heart with a Kd = 121 ±â€¯14 nM. Ex-vivo phosphorylation/dephosphorylation experiments suggested that the divergent CaM regulation of healthy and failing human RyR2 was caused by differences in RyR2 phosphorylation by protein kinase A and Ca-CaM-dependent kinase II. Ca2+-spark measurements in murine cardiomyocytes harbouring RyR2 phosphomimetic or phosphoablated mutants at S2814 and S2808 suggest that phosphorylation of residues corresponding to either human RyR2-S2808 or S2814 is both necessary and sufficient for RyR2 regulation by CaM. Our results challenge the current concept that CaM universally functions as a canonical inhibitor of RyR2 across species. Rather, CaM's biological action on human RyR2 appears to be more nuanced, with inhibitory activity only on phosphorylated RyR2 channels, which occurs during exercise or in patients with heart failure.

Contribution of Impaired Parasympathetic Activity to Right Ventricular Dysfunction and Pulmonary Vascular Remodeling in Pulmonary Arterial Hypertension.

BACKGROUND: The beneficial effects of parasympathetic stimulation have been reported in left heart failure, but whether it would be beneficial for pulmonary arterial hypertension (PAH) remains to be explored. Here, we investigated the relationship between parasympathetic activity and right ventricular (RV) function in patients with PAH, and the potential therapeutic effects of pyridostigmine (PYR), an oral drug stimulating the parasympathetic activity through acetylcholinesterase inhibition, in experimental pulmonary hypertension (PH). METHODS: Heart rate recovery after a maximal cardiopulmonary exercise test was used as a surrogate for parasympathetic activity. RV ejection fraction was assessed in 112 patients with PAH. Expression of nicotinic (α-7 nicotinic acetylcholine receptor) and muscarinic (muscarinic acetylcholine type 2 receptor) receptors, and acetylcholinesterase activity were evaluated in RV (n=11) and lungs (n=7) from patients with PAH undergoing heart/lung transplantation and compared with tissue obtained from controls. In addition, we investigated the effects of PYR (40 mg/kg per day) in experimental PH. PH was induced in male rats by SU5416 (25 mg/kg subcutaneously) injection followed by 4 weeks of hypoxia. In a subgroup, sympathetic/parasympathetic modulation was assessed by power spectral analysis. At week 6, PH status was confirmed by echocardiography, and rats were randomly assigned to vehicle or treatment (both n=12). At the end of the study, echocardiography was repeated, with additional RV pressure-volume measurements, along with lung, RV histological, and protein analyses. RESULTS: Patients with PAH with lower RV ejection fraction (<41%) had a significantly reduced heart rate recovery in comparison with patients with higher RV ejection fraction. In PAH RV samples, α-7 nicotinic acetylcholine receptor was increased and acetylcholinesterase activity was reduced versus controls. No difference in muscarinic acetylcholine type 2 receptor expression was observed. Chronic PYR treatment in PH rats normalized the cardiovascular autonomic function, demonstrated by an increase in parasympathetic activity and baroreflex sensitivity. PYR improved survival, increased RV contractility, and reduced RV stiffness, RV hypertrophy, RV fibrosis, RV inflammation, and RV α-7 nicotinic acetylcholine receptor and muscarinic acetylcholine type 2 receptor expression, as well. Furthermore, PYR reduced pulmonary vascular resistance, RV afterload, and pulmonary vascular remodeling, which was associated with reduced local and systemic inflammation. CONCLUSIONS: RV dysfunction is associated with reduced systemic parasympathetic activity in patients with PAH, with an inadequate adaptive response of the cholinergic system in the RV. Enhancing parasympathetic activity by PYR improved survival, RV function, and pulmonary vascular remodeling in experimental PH.

Pathogenesis and pathophysiology of heart failure with reduced ejection fraction: translation to human studies.

Heart failure represents the end result of different pathophysiologic processes, which culminate in functional impairment. Regardless of its aetiology, the presentation of heart failure usually involves symptoms of pump failure and congestion, which forms the basis for clinical diagnosis. Pathophysiologic descriptions of heart failure with reduced ejection fraction (HFrEF) are being established. Most commonly, HFrEF is centred on a reactive model where a significant initial insult leads to reduced cardiac output, further triggering a cascade of maladaptive processes. Predisposing factors include myocardial injury of any cause, chronically abnormal loading due to hypertension, valvular disease, or tachyarrhythmias. The pathophysiologic processes behind remodelling in heart failure are complex and reflect systemic neurohormonal activation, peripheral vascular effects and localised changes affecting the cardiac substrate. These abnormalities have been the subject of intense research. Much of the translational successes in HFrEF have come from targeting neurohormonal responses to reduced cardiac output, with blockade of the renin-angiotensin-aldosterone system (RAAS) and beta-adrenergic blockade being particularly fruitful. However, mortality and morbidity associated with heart failure remains high. Although systemic neurohormonal blockade slows disease progression, localised ventricular remodelling still adversely affects contractile function. Novel therapy targeted at improving cardiac contractile mechanics in HFrEF hold the promise of alleviating heart failure at its source, yet so far none has found success. Nevertheless, there are increasing calls for a proximal, 'cardiocentric' approach to therapy. In this review, we examine HFrEF therapy aimed at improving cardiac function with a focus on recent trials and emerging targets.

A novel phosphorylation site, Serine 199, in the C-terminus of cardiac troponin I regulates calcium sensitivity and susceptibility to calpain-induced proteolysis.

Phosphorylation of cardiac troponin I (cTnI) by protein kinase C (PKC) is implicated in cardiac dysfunction. Recently, Serine 199 (Ser199) was identified as a target for PKC phosphorylation and increased Ser199 phosphorylation occurs in end-stage failing compared with non-failing human myocardium. The functional consequences of cTnI-Ser199 phosphorylation in the heart are unknown. Therefore, we investigated the impact of phosphorylation of cTnI-Ser199 on myofilament function in human cardiac tissue and the susceptibility of cTnI to proteolysis. cTnI-Ser199 was replaced by aspartic acid (199D) or alanine (199A) to mimic phosphorylation and dephosphorylation, respectively, with recombinant wild-type (Wt) cTn as a negative control. Force development was measured at various [Ca(2+)] and at sarcomere lengths of 1.8 and 2.2 µm in demembranated cardiomyocytes in which endogenous cTn complex was exchanged with the recombinant human cTn complexes. In idiopathic dilated cardiomyopathy samples, myofilament Ca(2+)-sensitivity (pCa50) at 2.2 µm was significantly higher in 199D (pCa50 = 5.79 ± 0.01) compared to 199A (pCa50 = 5.65 ± 0.01) and Wt (pCa50 = 5.66 ± 0.02) at ~63% cTn exchange. Myofilament Ca(2+)-sensitivity was significantly higher even with only 5.9 ± 2.5% 199D exchange compared to 199A, and saturated at 12.3 ± 2.6% 199D exchange. Ser199 pseudo-phosphorylation decreased cTnI binding to both actin and actin-tropomyosin. Moreover, altered susceptibility of cTnI to proteolysis by calpain I was found when Ser199 was pseudo-phosphorylated. Our data demonstrate that low levels of cTnI-Ser199 pseudo-phosphorylation (~6%) increase myofilament Ca(2+)-sensitivity in human cardiomyocytes, most likely by decreasing the binding affinity of cTnI for actin-tropomyosin. In addition, cTnI-Ser199 pseudo-phosphorylation or mutation regulates calpain I mediated proteolysis of cTnI.

Synergistic role of ADP and Ca(2+) in diastolic myocardial stiffness.

Diastolic dysfunction in heart failure patients is evident from stiffening of the passive properties of the ventricular wall. Increased actomyosin interactions may significantly limit diastolic capacity, however, direct evidence is absent. From experiments at the cellular and whole organ level, in humans and rats, we show that actomyosin-related force development contributes significantly to high diastolic stiffness in environments where high ADP and increased diastolic [Ca(2+) ] are present, such as the failing myocardium. Our basal study provides a mechanical mechanism which may partly underlie diastolic dysfunction. Heart failure (HF) with diastolic dysfunction has been attributed to increased myocardial stiffness that limits proper filling of the ventricle. Altered cross-bridge interaction may significantly contribute to high diastolic stiffness, but this has not been shown thus far. Cross-bridge interactions are dependent on cytosolic [Ca(2+) ] and the regeneration of ATP from ADP. Depletion of myocardial energy reserve is a hallmark of HF leading to ADP accumulation and disturbed Ca(2+) handling. Here, we investigated if ADP elevation in concert with increased diastolic [Ca(2+) ] promotes diastolic cross-bridge formation and force generation and thereby increases diastolic stiffness. ADP dose-dependently increased force production in the absence of Ca(2+) in membrane-permeabilized cardiomyocytes from human hearts. Moreover, physiological levels of ADP increased actomyosin force generation in the presence of Ca(2+) both in human and rat membrane-permeabilized cardiomyocytes. Diastolic stress measured at physiological lattice spacing and 37°C in the presence of pathological levels of ADP and diastolic [Ca(2+) ] revealed a 76 ± 1% contribution of cross-bridge interaction to total diastolic stress in rat membrane-permeabilized cardiomyocytes. Inhibition of creatine kinase (CK), which increases cytosolic ADP, in enzyme-isolated intact rat cardiomyocytes impaired diastolic re-lengthening associated with diastolic Ca(2+) overload. In isolated Langendorff-perfused rat hearts, CK inhibition increased ventricular stiffness only in the presence of diastolic [Ca(2+) ]. We propose that elevations of intracellular ADP in specific types of cardiac disease, including those where myocardial energy reserve is limited, contribute to diastolic dysfunction by recruiting cross-bridges, even at low Ca(2+) , and thereby increase myocardial stiffness.

Perturbed length-dependent activation in human hypertrophic cardiomyopathy with missense sarcomeric gene mutations.

RATIONALE: High-myofilament Ca(2+) sensitivity has been proposed as a trigger of disease pathogenesis in familial hypertrophic cardiomyopathy (HCM) on the basis of in vitro and transgenic mice studies. However, myofilament Ca(2+) sensitivity depends on protein phosphorylation and muscle length, and at present, data in humans are scarce. OBJECTIVE: To investigate whether high myofilament Ca(2+) sensitivity and perturbed length-dependent activation are characteristics for human HCM with mutations in thick and thin filament proteins. METHODS AND RESULTS: Cardiac samples from patients with HCM harboring mutations in genes encoding thick (MYH7, MYBPC3) and thin (TNNT2, TNNI3, TPM1) filament proteins were compared with sarcomere mutation-negative HCM and nonfailing donors. Cardiomyocyte force measurements showed higher myofilament Ca(2+) sensitivity in all HCM samples and low phosphorylation of protein kinase A (PKA) targets compared with donors. After exogenous PKA treatment, myofilament Ca(2+) sensitivity was similar (MYBPC3mut, TPM1mut, sarcomere mutation-negative HCM), higher (MYH7mut, TNNT2mut), or even significantly lower (TNNI3mut) compared with donors. Length-dependent activation was significantly smaller in all HCM than in donor samples. PKA treatment increased phosphorylation of PKA-targets in HCM myocardium and normalized length-dependent activation to donor values in sarcomere mutation-negative HCM and HCM with truncating MYBPC3 mutations but not in HCM with missense mutations. Replacement of mutant by wild-type troponin in TNNT2mut and TNNI3mut corrected length-dependent activation to donor values. CONCLUSIONS: High-myofilament Ca(2+) sensitivity is a common characteristic of human HCM and partly reflects hypophosphorylation of PKA targets compared with donors. Length-dependent sarcomere activation is perturbed by missense mutations, possibly via posttranslational modifications other than PKA hypophosphorylation or altered protein-protein interactions, and represents a common pathomechanism in HCM.

Right ventricular diastolic impairment in patients with pulmonary arterial hypertension.

BACKGROUND: The role of right ventricular (RV) diastolic stiffness in pulmonary arterial hypertension (PAH) is not well established. Therefore, we investigated the presence and possible underlying mechanisms of RV diastolic stiffness in PAH patients. METHODS AND RESULTS: Single-beat RV pressure-volume analyses were performed in 21 PAH patients and 7 control subjects to study RV diastolic stiffness. Data are presented as mean ± SEM. RV diastolic stiffness (ß) was significantly increased in PAH patients (PAH, 0.050 ± 0.005 versus control, 0.029 ± 0.003; P<0.05) and was closely associated with disease severity. Subsequently, we searched for possible underlying mechanisms using RV tissue of PAH patients undergoing heart/lung transplantation and nonfailing donors. Histological analyses revealed increased cardiomyocyte cross-sectional areas (PAH, 453 ± 31 µm² versus control, 218 ± 21 µm²; P<0.001), indicating RV hypertrophy. In addition, the amount of RV fibrosis was enhanced in PAH tissue (PAH, 9.6 ± 0.7% versus control, 7.2 ± 0.6%; P<0.01). To investigate the contribution of stiffening of the sarcomere (the contractile apparatus of RV cardiomyocytes) to RV diastolic stiffness, we isolated and membrane-permeabilized single RV cardiomyocytes. Passive tension at different sarcomere lengths was significantly higher in PAH patients compared with control subjects (>200%; Pinteraction <0.001), indicating stiffening of RV sarcomeres. An important regulator of sarcomeric stiffening is the sarcomeric protein titin. Therefore, we investigated titin isoform composition and phosphorylation. No alterations were observed in titin isoform composition (N2BA/N2B ratio: PAH, 0.78 ± 0.07 versus control, 0.91 ± 0.08), but titin phosphorylation in RV tissue of PAH patients was significantly reduced (PAH, 0.16 ± 0.01 arbitrary units versus control, 0.20 ± 0.01 arbitrary units; P<0.05). CONCLUSIONS: RV diastolic stiffness is significantly increased in PAH patients, with important contributions from increased collagen and intrinsic stiffening of the RV cardiomyocyte sarcomeres.

Preserved cross-bridge kinetics in human hypertrophic cardiomyopathy patients with MYBPC3 mutations.

Mutations in the MYBPC3 gene, encoding cardiac myosin binding protein C (cMyBP-C) are frequent causes of hypertrophic cardiomyopathy (HCM). Previously, we have presented evidence for reduced cMyBP-C expression (haploinsufficiency), in patients with a truncation mutation in MYBPC3. In mice, lacking cMyBP-C cross-bridge kinetics was accelerated. In this study, we investigated whether cross-bridge kinetics was altered in myectomy samples from HCM patients harboring heterozygous MYBPC3 mutations (MYBPC3mut). Isometric force and the rate of force redevelopment (k tr) at different activating Ca(2+) concentrations were measured in mechanically isolated Triton-permeabilized cardiomyocytes from MYBPC3mut (n = 18) and donor (n = 7) tissue. Furthermore, the stretch activation response of cardiomyocytes was measured in tissue from eight MYBPC3mut patients and five donors to assess the rate of initial force relaxation (k 1) and the rate and magnitude of the transient increase in force (k 2 and P 3, respectively) after a rapid stretch. Maximal force development of the cardiomyocytes was reduced in MYBPC3mut (24.5 ± 2.3 kN/m(2)) compared to donor (34.9 ± 1.6 kN/m(2)). The rates of force redevelopment in MYBPC3mut and donor over a range of Ca(2+) concentrations were similar (k tr at maximal activation: 0.63 ± 0.03 and 0.75 ± 0.09 s(-1), respectively). Moreover, the stretch activation parameters did not differ significantly between MYBPC3mut and donor (k 1: 8.5±0.5 and 8.8 ± 0.4 s(-1); k 2: 0.77 ± 0.06 and 0.74 ± 0.09 s(-1); P 3: 0.08 ± 0.01 and 0.09 ± 0.01, respectively). Incubation with protein kinase A accelerated k 1 in MYBPC3mut and donor to a similar extent. Our experiments indicate that, at the cMyBP-C expression levels in this patient group (63 ± 6 % relative to donors), cross-bridge kinetics are preserved and that the depressed maximal force development is not explained by perturbation of cross-bridge kinetics.

Affimers targeting proteins in the cardiomyocyte Z-disc: Novel tools that improve imaging of heart tissue.

Dilated Cardiomyopathy is a common form of heart failure. Determining how this disease affects the structure and organization of cardiomyocytes in the human heart is important in understanding how the heart becomes less effective at contraction. Here we isolated and characterised Affimers (small non-antibody binding proteins) to Z-disc proteins ACTN2 (α-actinin-2), ZASP (also known as LIM domain binding protein 3 or LDB3) and the N-terminal region of the giant protein titin (TTN Z1-Z2). These proteins are known to localise in both the sarcomere Z-discs and the transitional junctions, found close to the intercalated discs that connect adjacent cardiomyocytes. We use cryosections of left ventricles from two patients diagnosed with end-stage Dilated Cardiomyopathy who underwent Orthotopic Heart Transplantation and were whole genome sequenced. We describe how Affimers substantially improve the resolution achieved by confocal and STED microscopy compared to conventional antibodies. We quantified the expression of ACTN2, ZASP and TTN proteins in two patients with dilated cardiomyopathy and compared them with a sex- and age-matched healthy donor. The small size of the Affimer reagents, combined with a small linkage error (the distance from the epitope to the dye label covalently bound to the Affimer) revealed new structural details in Z-discs and intercalated discs in the failing samples. Affimers are thus useful for analysis of changes to cardiomyocyte structure and organisation in diseased hearts.

Low expression of the K280N TNNT2 mutation is sufficient to increase basal myofilament activation in human hypertrophy cardiomyopathy.

Background: Hypertrophic cardiomyopathy (HCM) is an autosomal dominant genetic disorder with patients typically showing heterozygous inheritance of a pathogenic variant in a gene encoding a contractile protein. Here, we study the contractile effects of a rare homozygous mutation using explanted tissue and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to gain insight into how the balance between mutant and WT protein expression affects cardiomyocyte function. Methods: Force measurements were performed in cardiomyocytes isolated from a HCM patient carrying a homozygous troponin T mutation (cTnT-K280N) and healthy donors. To discriminate between mutation-mediated and phosphorylation-related effects on Ca2+-sensitivity, cardiomyocytes were treated with alkaline phosphatase (AP) or protein kinase A (PKA). Troponin exchange experiments characterized the relation between mutant levels and myofilament function. To define mutation-mediated effects on Ca2+-dynamics we used CRISPR/Cas9 to generate hiPSC-CMs harbouring heterozygous and homozygous TnT-K280N mutations. Ca2+-transient and cell shortening experiments compared these lines against isogenic controls. Results: Myofilament Ca2+-sensitivity was higher in homozygous cTnT-K280N cardiomyocytes and was not corrected by AP- and PKA-treatment. In cTnT-K280N cells exchanged with cTnT-WT, a low level (14%) of cTnT-K280N mutation elevated Ca2+-sensitivity. Similarly, exchange of donor cells with 45 ± 2% cTnT-K280N increased Ca2+-sensitivity and was not corrected by PKA. cTnT-K280N hiPSC-CMs show elevated diastolic Ca2+ and increases in cell shortening. Impaired cardiomyocyte relaxation was only evident in homozygous cTnT-K280N hiPSC-CMs. Conclusions: The cTnT-K280N mutation increases myofilament Ca2+-sensitivity, elevates diastolic Ca2+, enhances contractility and impairs cellular relaxation. A low level (14%) of the cTnT-K280N sensitizes myofilaments to Ca2+, a universal finding of human HCM.

Models of cardiovascular surgery biobanking to facilitate translational research and precision medicine.

Biobanking in health care has evolved over the last few decades from simple biological sample repositories to complex and dynamic units with multi-organizational infrastructure networks and has become an essential tool for modern medical research. Cardiovascular tissue biobanking provides a unique opportunity to utilize cardiac and vascular samples for translational research into heart failure and other related pathologies. Current techniques for diagnosis, classification, and treatment monitoring of cardiac disease relies primarily on interpretation of clinical signs, imaging, and blood biomarkers. Further research at the disease source (i.e. myocardium and blood vessels) has been limited by a relative lack of access to quality human cardiac tissue and the inherent shortcomings of most animal models of heart disease. In this review, we describe a model for cardiovascular tissue biobanking and databasing, and its potential to facilitate basic and translational research. We share techniques to procure endocardial samples from patients with hypertrophic cardiomyopathy, heart failure with reduced ejection fraction, and heart failure with preserved ejection fraction, in addition to aortic disease samples. We discuss some of the issues with respect to data collection, privacy, biobank consent, and the governance of tissue biobanking. The development of tissue biobanks as described here has significant scope to improve and facilitate translational research in multi-omic fields such as genomics, transcriptomics, proteomics, and metabolomics. This research heralds an era of precision medicine, in which patients with cardiovascular pathology can be provided with optimized and personalized medical care for the treatment of their individual phenotype.

A 2-D gel reference map of the basic human heart proteome.

We have undertaken the identification of basic proteins (pH 6-11) of the human heart using 2-DE. Tissue from the left ventricle of human heart was lysed and proteins were separated in the first dimension on pH 6-11 IPG strips using paper-bridge loading followed by separation on 12% SDS polyacrylamide gels in the second dimension. Proteins were then identified by mass spectrometry and analysed using Proline, a proteomic data analysis platform that was developed in-house. The proteome map contains 176 identified spots with 151 unique proteins and has been made available as part of the UCD-2DPAGE database at http://proteomics-portal.ucd.ie:8082. The associated mass spectrometry data have been submitted to PRIDE (Accession number ♯10098). This reference map, and the other heart reference maps available through the UCD-2DPAGE database, will aid further proteomic studies of heart diseases such as dilated cardiomyopathy and ischaemic heart disease.

Oxidative stress regulates left ventricular PDE5 expression in the failing heart.

BACKGROUND: Phosphodiesterase type 5 (PDE5) inhibition has been shown to exert profound beneficial effects in the failing heart, suggesting a significant role for PDE5 in the development of congestive heart failure (CHF). The purpose of this study is to test the hypothesis that oxidative stress causes increased PDE5 expression in cardiac myocytes and that increased PDE5 contributes to the development of CHF. METHODS AND RESULTS: Myocardial PDE5 expression and cellular distribution were determined in left ventricular samples from patients with end-stage CHF and normal donors and from mice after transverse aortic constriction (TAC)-induced CHF. Compared with donor human hearts, myocardial PDE5 protein was increased approximately equal 4.5-fold in CHF samples, and the increase of myocardial PDE5 expression was significantly correlated with myocardial oxidative stress markers 3'-nitrotyrosine or 4-hydroxynonenal expression (P<0.05). Histological examination demonstrated that PDE5 was mainly expressed in vascular smooth muscle in normal donor hearts, but its expression was increased in both cardiac myocytes and vascular smooth muscle of CHF hearts. Myocardial PDE5 protein content and activity also increased in mice after TAC-induced CHF (P<0.05). When the superoxide dismutase (SOD) mimetic M40401 was administered to attenuate oxidative stress, the increased PDE5 protein and activity caused by TAC was blunted, and the hearts were protected against left ventricular hypertrophy and CHF. Conversely, increased myocardial oxidative stress in superoxide dismutase 3 knockout mice caused a greater increase of PDE5 expression and CHF after TAC. In addition, administration of sildenafil to inhibit PDE5 attenuated TAC-induced myocardial oxidative stress, PDE5 expression, and CHF. CONCLUSIONS: Myocardial oxidative stress increases PDE5 expression in the failing heart. Reducing oxidative stress by treatment with M40401 attenuated cardiomyocyte PDE5 expression. This and selective inhibition of PDE5 protected the heart against pressure overload-induced left ventricular hypertrophy and CHF.

Tryptophan metabolism to kynurenine is a potential novel contributor to hypotension in human sepsis.

OBJECTIVES: To determine whether tryptophan metabolism to kynurenine contributes to the direct regulation of vascular tone in human septic shock. BACKGROUND: Indoleamine 2,3-dioxygenase 1 is an inducible enzyme that converts tryptophan to kynurenine and shares functional similarities with inducible nitric oxide synthase. Recently, kynurenine has been identified as an endothelium-derived relaxing factor produced during inflammation, raising the possibility that this novel pathway may contribute to hypotension in human sepsis. DESIGN: Prospective, matched, single-center, cohort study. SETTINGS: Intensive care unit of a tertiary teaching hospital matched to control subjects from the general medical ward and healthy volunteers. SUBJECTS: Patients (n = 16) with septic shock had indoleamine 2,3-dioxygenase activity assessed as the kynurenine-to-tryptophan ratio, and the severity of hypotension was determined by their inotrope requirements. Healthy and blood pressure-matched nonseptic control subjects were also studied. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Tissues from septic and control patients were stained for the presence of indoleamine 2,3-dioxygenase 1. Indoleamine 2,3-dioxygenase activity increased up to ninefold in patients with septic shock and was significantly higher than in the two control groups (p < .01). Indoleamine 2,3-dioxygenase activity was strongly correlated with inotrope requirements (p < .001). Indoleamine 2,3-dioxygenase protein was expressed in inflamed cardiac tissue as well as in endothelial cells of resistance vessels in hearts and kidneys from subjects who died from sepsis. CONCLUSIONS: : Indoleamine 2,3-dioxygenase 1 is expressed in resistance vessels in human sepsis and Indoleamine 2,3-dioxygenase activity correlates with hypotension in human septic shock. Indoleamine 2,3-dioxygenase 1 is thus a potential novel contributor to hypotension in sepsis.

Protein phosphatase 2A affects myofilament contractility in non-failing but not in failing human myocardium.

Protein phosphatase (PP) type 2A is a multifunctional serine/threonine phosphatase that is involved in cardiac excitation-contraction coupling. The PP2A core enzyme is a dimer, consisting of a catalytic C and a scaffolding A subunit, which is targeted to several cardiac proteins by a regulatory B subunit. At present, it is controversial whether PP2A and its subunits play a critical role in end-stage human heart failure. Here we report that the application of purified PP2AC significantly increased the Ca2+-sensitivity (ΔpCa50=0.05±0.01) of the contractile apparatus in isolated skinned myocytes of non-failing (NF) hearts. A higher phosphorylation of troponin I (cTnI) was found at protein kinase A sites (Ser23/24) in NF compared to failing myocardium. The basal Ca2+-responsiveness of myofilaments was enhanced in myocytes of ischemic (ICM, ΔpCa50=0.10±0.03) and dilated (DCM, ΔpCa50=0.06±0.04) cardiomyopathy compared to NF. However, in contrast to NF myocytes the treatment with PP2AC did not shift force-pCa relationships in failing myocytes. The higher basal Ca2+-sensitivity in failing myocytes coincided with a reduced protein expression of PP2AC in left ventricular tissue from patients suffering from ICM and DCM (by 50 and 56% compared to NF, respectively). However, PP2A activity was unchanged in failing hearts despite an increase of both total PP and PP1 activity. The expression of PP2AB56α was also decreased by 51 and 62% in ICM and DCM compared to NF, respectively. The phosphorylation of cTnI at Ser23/24 was reduced by 66 and 49% in ICM and DCM compared to NF hearts, respectively. Our results demonstrate that PP2A increases myofilament Ca2+-sensitivity in NF human hearts, most likely via cTnI dephosphorylation. This effect is not present in failing hearts, probably due to the lower baseline cTnI phosphorylation in failing compared to non-failing hearts.

Protein kinase G modulates human myocardial passive stiffness by phosphorylation of the titin springs.

The sarcomeric titin springs influence myocardial distensibility and passive stiffness. Titin isoform composition and protein kinase (PK)A-dependent titin phosphorylation are variables contributing to diastolic heart function. However, diastolic tone, relaxation speed, and left ventricular extensibility are also altered by PKG activation. We used back-phosphorylation assays to determine whether PKG can phosphorylate titin and affect titin-based stiffness in skinned myofibers and isolated myofibrils. PKG in the presence of 8-pCPT-cGMP (cGMP) phosphorylated the 2 main cardiac titin isoforms, N2BA and N2B, in human and canine left ventricles. In human myofibers/myofibrils dephosphorylated before mechanical analysis, passive stiffness dropped 10% to 20% on application of cGMP-PKG. Autoradiography and anti-phosphoserine blotting of recombinant human I-band titin domains established that PKG phosphorylates the N2-B and N2-A domains of titin. Using site-directed mutagenesis, serine residue S469 near the COOH terminus of the cardiac N2-B-unique sequence (N2-Bus) was identified as a PKG and PKA phosphorylation site. To address the mechanism of the PKG effect on titin stiffness, single-molecule atomic force microscopy force-extension experiments were performed on engineered N2-Bus-containing constructs. The presence of cGMP-PKG increased the bending rigidity of the N2-Bus to a degree that explained the overall PKG-mediated decrease in cardiomyofibrillar stiffness. Thus, the mechanically relevant site of PKG-induced titin phosphorylation is most likely in the N2-Bus; phosphorylation of other titin sites could affect protein-protein interactions. The results suggest that reducing titin stiffness by PKG-dependent phosphorylation of the N2-Bus can benefit diastolic function. Failing human hearts revealed a deficit for basal titin phosphorylation compared to donor hearts, which may contribute to diastolic dysfunction in heart failure.

Using antibody arrays to detect microparticles from acute coronary syndrome patients based on cluster of differentiation (CD) antigen expression.

Microparticles circulate in plasma and have recently emerged as potential inflammatory markers in cardiovascular disease. They are fragments of cell membranes that express cluster of differentiation (CD) antigens and are present at elevated levels in patients with acute coronary syndrome. We have developed a novel method for the rapid detection of microparticles in plasma using a fluorescence-based antibody array system. Isolated microparticles are captured on anti-CD antibody spots immobilized on a nitrocellulose membrane. These CD antibodies are directed against extracellular epitopes, whereas the intracellular exposed surface of the microparticles is labeled with a fluorescent anti-annexin antibody. The array is then scanned and quantified. A pilot study was undertaken to compare microparticle CD antigen expression in acute coronary syndrome and healthy subjects. Ten CD antigens (44, 45, 54, 62E, 79, 102, 117, 130, 138, and 154) had significantly increased expression in the disease group relative to the healthy controls. These results were then verified using flow cytometry and scanning electron microscopy. Although we have focused our analysis on changes in microparticle CD antigen expression, this technique is amenable to analyzing other surface markers. Microparticles can be derived from a wide variety of cell types, so selection of the primary antibody can be tailored to the cell origin that is to be investigated.