Regulation of cardiomyocyte t-tubule structure by preload and afterload: Roles in cardiac compensation and decompensation.

Mechanical load is a potent regulator of cardiac structure and function. Although high workload during heart failure is associated with disruption of cardiomyocyte t-tubules and Ca2+ homeostasis, it remains unclear whether changes in preload and afterload may promote adaptive t-tubule remodelling. We examined this issue by first investigating isolated effects of stepwise increases in load in cultured rat papillary muscles. Both preload and afterload increases produced a biphasic response, with the highest t-tubule densities observed at moderate loads, whereas excessively low and high loads resulted in low t-tubule levels. To determine the baseline position of the heart on this bell-shaped curve, mice were subjected to mildly elevated preload or afterload (1 week of aortic shunt or banding). Both interventions resulted in compensated cardiac function linked to increased t-tubule density, consistent with ascension up the rising limb of the curve. Similar t-tubule proliferation was observed in human patients with moderately increased preload or afterload (mitral valve regurgitation, aortic stenosis). T-tubule growth was associated with larger Ca2+ transients, linked to upregulation of L-type Ca2+ channels, Na+-Ca2+ exchanger, mechanosensors and regulators of t-tubule structure. By contrast, marked elevation of cardiac load in rodents and patients advanced the heart down the declining limb of the t-tubule-load relationship. This bell-shaped relationship was lost in the absence of electrical stimulation, indicating a key role of systolic stress in controlling t-tubule plasticity. In conclusion, modest augmentation of workload promotes compensatory increases in t-tubule density and Ca2+ cycling, whereas this adaptation is reversed in overloaded hearts during heart failure progression. KEY POINTS: Excised papillary muscle experiments demonstrated a bell-shaped relationship between cardiomyocyte t-tubule density and workload (preload or afterload), which was only present when muscles were electrically stimulated. The in vivo heart at baseline is positioned on the rising phase of this curve because moderate increases in preload (mice with brief aortic shunt surgery, patients with mitral valve regurgitation) resulted in t-tubule growth. Moderate increases in afterload (mice and patients with mild aortic banding/stenosis) similarly increased t-tubule density. T-tubule proliferation was associated with larger Ca2+ transients, with upregulation of the L-type Ca2+ channel, Na+-Ca2+ exchanger, mechanosensors and regulators of t-tubule structure. By contrast, marked elevation of cardiac load in rodents and patients placed the heart on the declining phase of the t-tubule-load relationship, promoting heart failure progression. The dependence of t-tubule structure on preload and afterload thus enables both compensatory and maladaptive remodelling, in rodents and humans.

Dual or single antiplatelet therapy after coronary surgery for acute coronary syndrome (TACSI trial): Rationale and design of an investigator-initiated, prospective, multinational, registry-based randomized clinical trial.

The TACSI trial (ClinicalTrials.gov Identifier: NCT03560310) tests the hypothesis that 1-year treatment with dual antiplatelet therapy with acetylsalicylic acid (ASA) and ticagrelor is superior to only ASA after isolated coronary artery bypass grafting (CABG) in patients with acute coronary syndrome. The TACSI trial is an investigator-initiated pragmatic, prospective, multinational, multicenter, open-label, registry-based randomized trial with 1:1 randomization to dual antiplatelet therapy with ASA and ticagrelor or ASA only, in patients undergoing first isolated CABG, with a planned enrollment of 2200 patients at Nordic cardiac surgery centers. The primary efficacy end point is a composite of time to all-cause death, myocardial infarction, stroke, or new coronary revascularization within 12 months after randomization. The primary safety end point is time to hospitalization due to major bleeding. Secondary efficacy end points include time to the individual components of the primary end point, cardiovascular death, and rehospitalization due to cardiovascular causes. High-quality health care registries are used to assess primary and secondary end points. The patients will be followed for 10 years. The TACSI trial will give important information useful for guiding the antiplatelet strategy in acute coronary syndrome patients treated with CABG.

Sirtuin1, not NAMPT, possesses anti-inflammatory effects in epicardial, pericardial and subcutaneous adipose tissue in patients with CHD.

BACKGROUND: Inflammation in cardiac adipose tissue (AT) is associated with atherosclerosis. We investigated whether the epicardial-, pericardial and pre-sternal subcutaneous AT (EAT, PAT and SAT) expression of Sirtuin1 (SIRT1) and nicotinamide phosphoribosyl transferase (NAMPT) are involved in the inflammatory process in coronary heart disease (CHD), and potentially associated to nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome-related markers, macrophage polarization markers, cell markers and the cardiometabolic profile. METHODS: In this cohort study performed between 2016 and 2018, EAT, PAT and SAT biopsies were retrieved from 52 CHD patients (77% men, median age 67) undergoing open-chest coronary artery bypass grafting (CABG), and 22 patients (50% men, median age 69) undergoing aortic valve replacement serving as controls. AT samples were snap-frozen at - 80 °C until RNA extraction and AT expression of actual markers, relatively quantified by PCR. Circulating SIRT1 and NAMPT were measured with Enzyme-linked immunosorbent assays (ELISAs). Non-parametric statistical tests were mainly used, including Friedman's test coupled to Wilcoxon signed-rank test and Spearman Correlation. RESULTS: SIRT1 and NAMPT levels were similar in CHD and controls. In CHD, SIRT1 and NAMPT were inter-correlated in all AT compartments (r = 0.37-0.56, p < 0.01, all), and differently expressed between compartments, with the highest expression in SAT, significantly different from EAT (p < 0.01, both). Circulating SIRT1 and NAMPT levels were inversely associated (r = - 0.32, p = 0.024). In EAT and SAT, SIRT1 expression was inversely associated with IL-18 (r = - 0.43 and r = - 0.38, p < 0.01, both), whereas NAMPT expression was positively associated with the NLRP3 inflammasome-related markers in all compartments (r = 0.37-0.55, p < 0.01, all). While SIRT1 and NAMPT correlated to nitric oxide synthase 2 (NOS2), especially in SAT (r = 0.50-0.52, p ≤ 0.01, both), SIRT1 expression was related to endothelial cells, and NAMPT to macrophages. SIRT1 levels were correlated to weight and waist (r = 0.32 and r = 0.38, p < 0.03, both) and inversely to triglycerides and glycated haemoglobin (HbA1c) (r = - 0.33-- 0.37, p < 0.03, all), the latter positively correlated to NAMPT concentration (r = 0.39, p = 0.010). CONCLUSION: The study indicates that targeting SIRT1, with its anti-inflammatory properties, may be a novel anti-inflammatory strategy in preventing atherosclerosis and CHD progression. NAMPT may be an early player in AT inflammation, mediating/reflecting a pro-inflammatory state. TRIAL REGISTRATION: Registration: Clinicaltrials.gov ID: NCT02760914, registered the 5th of February 2016, http://clinicaltrials.gov/NCT02760914.

Moderate Loss of the Extracellular Matrix Proteoglycan Lumican Attenuates Cardiac Fibrosis in Mice Subjected to Pressure Overload.

INTRODUCTION: The heart undergoes myocardial remodeling during progression to heart failure following pressure overload. Myocardial remodeling is associated with structural and functional changes in cardiac myocytes, fibroblasts, and the extracellular matrix (ECM) and is accompanied by inflammation. Cardiac fibrosis, the accumulation of ECM molecules including collagens and collagen cross-linking, contributes both to impaired systolic and diastolic function. Insufficient mechanistic insight into what regulates cardiac fibrosis during pathological conditions has hampered therapeutic so-lutions. Lumican (LUM) is an ECM-secreted proteoglycan known to regulate collagen fibrillogenesis. Its expression in the heart is increased in clinical and experimental heart failure. Furthermore, LUM is important for survival and cardiac remodeling following pressure overload. We have recently reported that total lack of LUM increased mortality and left ventricular dilatation, and reduced collagen expression and cross-linking in LUM knockout mice after aortic banding (AB). Here, we examined the effect of LUM on myocardial remodeling and function following pressure overload in a less extreme mouse model, where cardiac LUM level was reduced to 50% (i.e., moderate loss of LUM). METHODS AND RESULTS: mRNA and protein levels of LUM were reduced to 50% in heterozygous LUM (LUM+/-) hearts compared to wild-type (WT) controls. LUM+/- mice were subjected to AB. There was no difference in survival between LUM+/- and WT mice post-AB. Echocardiography revealed no striking differences in cardiac geometry between LUM+/- and WT mice 2, 4, and 6 weeks post-AB, although markers of diastolic dysfunction indicated better function in LUM+/- mice. LUM+/- hearts revealed reduced cardiac fibrosis assessed by histology. In accordance, the expression of collagen I and III, the main fibrillar collagens in the heart, and other ECM molecules central to fibrosis, i.e. including periostin and fibronectin, was reduced in the hearts of LUM+/- compared to WT 6 weeks post-AB. We found no differences in collagen cross-linking between LUM+/- and WT mice post-AB, as assessed by histology and qPCR. CONCLUSIONS: Moderate lack of LUM attenuated cardiac fibrosis and improved diastolic dysfunction following pressure overload in mice, adding to the growing body of evidence suggesting that LUM is a central profibrotic molecule in the heart that could serve as a potential therapeutic target.

Complement Activation in Association with Markers of Neutrophil Extracellular Traps and Acute Myocardial Infarction in Stable Coronary Artery Disease.

Complement activation and neutrophil extracellular traps (NETs) have both been suggested to drive atherosclerotic plaque progression. Although experimental studies suggest interplay between these two innate immunity components, the relevance in patients with coronary artery disease (CAD) is unclear. The aim of this study was to assess associations between complement activation and NETs in patients with stable CAD and examine the role of complement activation on clinical outcome. Blood samples from a cohort of patients with angiographically verified stable CAD (n = 1001) were analyzed by ELISA for the terminal complement complex (TCC) and by relative quantification for gene expression of the C5a receptor 1 (C5aR1) as markers of complement activation. As markers of NETs, dsDNA was analyzed by fluorescent nucleic acid stain and myeloperoxidase-DNA (MPO-DNA) by ELISA. Clinical outcome was defined as unstable angina, nonhemorrhagic stroke, acute myocardial infarction (MI), or death (n = 106, whereof 36 MI). Levels of TCC and C5aR1 were not significantly correlated to dsDNA (TCC: r = -0.045, p = 0.153; C5aR1: r = -0.060, p = 0.434) or MPO-DNA (TCC: r = 0.026, p = 0.414; C5aR1: r = 0.123, p = 0.107). When dividing TCC and C5aR1 levels into quartiles (Q), levels of MPO-DNA differed significantly across quartiles (TCC: p = 0.008, C5aR1: 0.049), while dsDNA did not (TCC: p = 0.181, C5aR1: p = 0.771). Patients with TCC levels in Q4 had significantly higher levels of MPO-DNA than Q1-3 (p = 0.019), and C5aR1 levels in Q3-4 had significantly higher levels of MPO-DNA than Q1-2 (p = 0.046). TCC levels did not differ between patients experiencing a clinical endpoint or not, but high levels were associated with increased risk of acute MI (OR. 1.97, 95% CI: 0.99-3.90, p = 0.053) during two-year follow up, also when adjusted for relevant covariates. In conclusion, TCC and C5aR1 were moderately associated with the NET marker MPO-DNA, and TCC levels were related to the risk of future MI in this cohort of patients with stable CAD.

Facilitators of and barriers to reducing thirty-day readmissions and improving patient-reported outcomes after surgical aortic valve replacement: a process evaluation of the AVRre trial.

BACKGROUND: The Aortic Valve Replacement Readmission (AVRre) randomized control trial tested whether a telephone intervention would reduce hospital readmissions following surgical aortic valve replacement (SAVR). The telephone support provided 30 days of continuous phone-support (hotline) and two scheduled phone-calls from the hospital after discharge. The intervention had no effect on reducing 30-day all-cause readmission rate (30-DACR) but did reduce participants' anxiety compared to a control group receiving usual care. Depression and participant-reported health state were unaffected by the intervention. To better understand these outcomes, we conducted a process evaluation of the AVRre trial to gain insight into the (1) the dose and fidelity of the intervention, (2) mechanism of impacts, and (3) contextual factors that may have influenced the outcomes. METHODS: The process evaluation was informed by the Medical Research Council framework, a widely used set of guidelines for evaluating complex interventions. A mix of quantitative (questionnaire and journal records) and qualitative data (field notes, memos, registration forms, questionnaire) was prospectively collected, and retrospective interviews were conducted. We performed descriptive analyses of the quantitative data. Content analyses, assisted by NVivo, were performed to evaluate qualitative data. RESULTS: The nurses who were serving the 24/7 hotline intervention desired to receive more preparation before intervention implementation. SAVR patient participants were highly satisfied with the telephone intervention (58%), felt safe (86%), and trusted having the option of calling in for support (91%). The support for the telephone hotline staff was perceived as a facilitator of the intervention implementation. Content analyses revealed themes: "gap in the care continuum," "need for individualized care," and "need for easy access to health information" after SAVR. Differences in local hospital discharge management practices influenced the 30-DACR incidence. CONCLUSIONS: The prospective follow-up of the hotline service during the trial facilitated implementation of the intervention, contributing to high participant satisfaction and likely reduced their anxiety after SAVR. Perceived less-than-optimal preparations for the hotline could be a barrier to AVRre trial implementation. Integrating user experiences into a mixed-methods evaluation of clinical trials is important for broadening understanding of trial outcomes, the mechanism of impact, and contextual factors that influence clinical trials. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02522663. Registered on 11 August 2015.

Thyroid and Glucocorticoid Hormones Promote Functional T-Tubule Development in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

RATIONALE: Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are increasingly being used for modeling heart disease and are under development for regeneration of the injured heart. However, incomplete structural and functional maturation of hiPSC-CM, including lack of T-tubules, immature excitation-contraction coupling, and inefficient Ca-induced Ca release remain major limitations. OBJECTIVE: Thyroid and glucocorticoid hormones are critical for heart maturation. We hypothesized that their addition to standard protocols would promote T-tubule development and mature excitation-contraction coupling of hiPSC-CM when cultured on extracellular matrix with physiological stiffness (Matrigel mattress). METHODS AND RESULTS: hiPSC-CM were generated using a standard chemical differentiation method supplemented with T3 (triiodothyronine) and/or Dex (dexamethasone) during days 16 to 30 followed by single-cell culture for 5 days on Matrigel mattress. hiPSC-CM treated with T3+Dex, but not with either T3 or Dex alone, developed an extensive T-tubule network. Notably, Matrigel mattress was necessary for T-tubule formation. Compared with adult human ventricular cardiomyocytes, T-tubules in T3+Dex-treated hiPSC-CM were less organized and had more longitudinal elements. Confocal line scans demonstrated spatially and temporally uniform Ca release that is characteristic of excitation-contraction coupling in the heart ventricle. T3+Dex enhanced elementary Ca release measured by Ca sparks and promoted RyR2 (ryanodine receptor) structural organization. Simultaneous measurements of L-type Ca current and intracellular Ca release confirmed enhanced functional coupling between L-type Ca channels and RyR2 in T3+Dex-treated cells. CONCLUSIONS: Our results suggest a permissive role of combined thyroid and glucocorticoid hormones during the cardiac differentiation process, which when coupled with further maturation on Matrigel mattress, is sufficient for T-tubule development, enhanced Ca-induced Ca release, and more ventricular-like excitation-contraction coupling. This new hormone maturation method could advance the use of hiPSC-CM for disease modeling and cell-based therapy.

Shedding of syndecan-4 promotes immune cell recruitment and mitigates cardiac dysfunction after lipopolysaccharide challenge in mice.

Inflammation is central to heart failure progression. Innate immune signaling increases expression of the transmembrane proteoglycan syndecan-4 in cardiac myocytes and fibroblasts, followed by shedding of its ectodomain. Circulating shed syndecan-4 is increased in heart failure patients, however the pathophysiological and molecular consequences associated with syndecan-4 shedding remain poorly understood. Here we used lipopolysaccharide (LPS) challenge to investigate the effects of syndecan-4 shedding in the heart. Wild-type mice (10mg/kg, 9h) and cultured neonatal rat cardiomyocytes and fibroblasts were subjected to LPS challenge. LPS increased cardiac syndecan-4 mRNA without altering full-length protein. Elevated levels of shedding fragments in the myocardium and blood from the heart confirmed syndecan-4 shedding in vivo. A parallel upregulation of ADAMTS1, ADAMTS4 and MMP9 mRNA suggested these shedding enzymes to be involved. Echocardiography revealed reduced ejection fraction, diastolic tissue velocity and prolonged QRS duration in mice unable to shed syndecan-4 (syndecan-4 KO) after LPS challenge. In line with syndecan-4 shedding promoting immune cell recruitment, expression of immune cell markers (CD8, CD11a, F4/80) and adhesion receptors (Icam1, Vcam1) were attenuated in syndecan-4 KO hearts after LPS. Cardiomyocytes and fibroblasts exposed to shed heparan sulfate-substituted syndecan-4 ectodomains showed increased Icam1, Vcam1, TNFα and IL-1ß expression and NF-κB-activation, suggesting direct regulation of immune cell recruitment pathways. In cardiac fibroblasts, shed ectodomains regulated expression of extracellular matrix constituents associated with collagen synthesis, cross-linking and turnover. Higher syndecan-4 levels in the coronary sinus vs. the radial artery of open heart surgery patients suggested that syndecan-4 is shed from the human heart. Our data demonstrate that shedding of syndecan-4 ectodomains is part of the cardiac innate immune response, promoting immune cell recruitment, extracellular matrix remodeling and mitigating cardiac dysfunction in response to LPS.

Molecular basis of calpain cleavage and inactivation of the sodium-calcium exchanger 1 in heart failure.

Cardiac sodium (Na(+))-calcium (Ca(2+)) exchanger 1 (NCX1) is central to the maintenance of normal Ca(2+) homeostasis and contraction. Studies indicate that the Ca(2+)-activated protease calpain cleaves NCX1. We hypothesized that calpain is an important regulator of NCX1 in response to pressure overload and aimed to identify molecular mechanisms and functional consequences of calpain binding and cleavage of NCX1 in the heart. NCX1 full-length protein and a 75-kDa NCX1 fragment along with calpain were up-regulated in aortic stenosis patients and rats with heart failure. Patients with coronary artery disease and sham-operated rats were used as controls. Calpain co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes and left ventricle lysate. Immunoprecipitations, pull-down experiments, and extensive use of peptide arrays indicated that calpain domain III anchored to the first Ca(2+) binding domain in NCX1, whereas the calpain catalytic region bound to the catenin-like domain in NCX1. The use of bioinformatics, mutational analyses, a substrate competitor peptide, and a specific NCX1-Met(369) antibody identified a novel calpain cleavage site at Met(369). Engineering NCX1-Met(369) into a tobacco etch virus protease cleavage site revealed that specific cleavage at Met(369) inhibited NCX1 activity (both forward and reverse mode). Finally, a short peptide fragment containing the NCX1-Met(369) cleavage site was modeled into the narrow active cleft of human calpain. Inhibition of NCX1 activity, such as we have observed here following calpain-induced NCX1 cleavage, might be beneficial in pathophysiological conditions where increased NCX1 activity contributes to cardiac dysfunction.

Attenuated development of cardiac fibrosis in left ventricular pressure overload by SM16, an orally active inhibitor of ALK5.

Pressure overload-induced TGF-ß signaling activates cardiac fibroblasts (CFB) and leads to increased extracellular matrix (ECM) protein synthesis including fibrosis. Excessive ECM accumulation may in turn affect cardiac function contributing to development of heart failure. The aim of this study was to examine the effects of SM16, an orally active small molecular inhibitor of ALK5, on pressure overload-induced cardiac fibrosis. One week after aortic banding (AB), C57Bl/6J mice were randomized to standard chow or chow with SM16. Sham operated animals served as controls. Following 4 weeks AB, mice were characterized by echocardiography and cardiovascular magnetic resonance before sacrifice. SM16 abolished phosphorylation of SMAD2 induced by AB in vivo and by TGF-ß in CFB in vitro. Interestingly, Masson Trichrome and Picrosirius Red stained myocardial left ventricular tissue revealed reduced development of fibrosis and collagen cross-linking following AB in the SM16 treated group, which was confirmed by reduced hydroxyproline incorporation. Furthermore, treatment with SM16 attenuated mRNA expression following induction of AB in vivo and stimulation with TGF-ß in CFB in vitro of Col1a2, the cross-linking enzyme LOX, and the pro-fibrotic glycoproteins SPARC and osteopontin. Reduced ECM synthesis by CFB and a reduction in myocardial stiffness due to attenuated development of fibrosis and collagen cross-linking might have contributed to the improved diastolic function and cardiac output seen in vivo, in combination with reduced lung weight and ANP expression by treatment with SM16. Despite these beneficial effects on cardiac function and development of heart failure, mice treated with SM16 exhibited increased mortality, increased LV dilatation and inflammatory heart valve lesions that may limit the use of SM16 and possibly also other small molecular inhibitors of ALK5, as future therapeutic drugs.

Identifying pathogenic processes by integrating microarray data with prior knowledge.

BACKGROUND: It is of great importance to identify molecular processes and pathways that are involved in disease etiology. Although there has been an extensive use of various high-throughput methods for this task, pathogenic pathways are still not completely understood. Often the set of genes or proteins identified as altered in genome-wide screens show a poor overlap with canonical disease pathways. These findings are difficult to interpret, yet crucial in order to improve the understanding of the molecular processes underlying the disease progression. We present a novel method for identifying groups of connected molecules from a set of differentially expressed genes. These groups represent functional modules sharing common cellular function and involve signaling and regulatory events. Specifically, our method makes use of Bayesian statistics to identify groups of co-regulated genes based on the microarray data, where external information about molecular interactions and connections are used as priors in the group assignments. Markov chain Monte Carlo sampling is used to search for the most reliable grouping. RESULTS: Simulation results showed that the method improved the ability of identifying correct groups compared to traditional clustering, especially for small sample sizes. Applied to a microarray heart failure dataset the method found one large cluster with several genes important for the structure of the extracellular matrix and a smaller group with many genes involved in carbohydrate metabolism. The method was also applied to a microarray dataset on melanoma cancer patients with or without metastasis, where the main cluster was dominated by genes related to keratinocyte differentiation. CONCLUSION: Our method found clusters overlapping with known pathogenic processes, but also pointed to new connections extending beyond the classical pathways.

Slow Ca²âº sparks de-synchronize Ca²âº release in failing cardiomyocytes: evidence for altered configuration of Ca²âº release units?

In heart failure, cardiomyocytes exhibit slowing of the rising phase of the Ca(2+) transient which contributes to the impaired contractility observed in this condition. We investigated whether alterations in ryanodine receptor function promote slowing of Ca(2+) release in a murine model of congestive heart failure (CHF). Myocardial infarction was induced by left coronary artery ligation. When chronic CHF had developed (10 weeks post-infarction), cardiomyocytes were isolated from viable regions of the septum. Septal myocytes from SHAM-operated mice served as controls. Ca(2+) transients rose markedly slower in CHF than SHAM myocytes with longer time to peak (CHF=152 ± 12% of SHAM, P<0.05). The rise time of Ca(2+) sparks was also increased in CHF (SHAM=9.6 ± 0.6 ms, CHF=13.2 ± 0.7 ms, P<0.05), due to a sub-population of sparks (≈20%) with markedly slowed kinetics. Regions of the cell associated with these slow spontaneous sparks also exhibited slowed Ca(2+) release during the action potential. Thus, greater variability in spark kinetics in CHF promoted less uniform Ca(2+) release across the cell. Dyssynchronous Ca(2+) transients in CHF additionally resulted from T-tubule disorganization, as indicated by fast Fourier transforms, but slow sparks were not associated with orphaned ryanodine receptors. Rather, mathematical modeling suggested that slow sparks could result from an altered composition of Ca(2+) release units, including a reduction in ryanodine receptor density and/or distribution of ryanodine receptors into sub-clusters. In conclusion, our findings indicate that slowed, dyssynchronous Ca(2+) transients in CHF result from alterations in Ca(2+) sparks, consistent with rearrangement of ryanodine receptors within Ca(2+) release units.

Macrophage polarization markers in subcutaneous, pericardial, and epicardial adipose tissue are altered in patients with coronary heart disease.

Background: Epicardial and pericardial adipose tissue (EAT and PAT) surround and protect the heart, with EAT directly sharing the microcirculation with the myocardium, possibly presenting a distinct macrophage phenotype that might affect the inflammatory environment in coronary heart disease (CHD). This study aims to investigate the expression of genes in different AT compartments driving the polarization of AT macrophages toward an anti-inflammatory (L-Galectin 9; CD206) or pro-inflammatory (NOS2) phenotype. Methods: EAT, PAT, and subcutaneous (SAT) biopsies were collected from 52 CHD patients undergoing coronary artery bypass grafting, and from 22 CTRLs undergoing aortic valve replacement. L-Galectin9 (L-Gal9), CD206, and NOS2 AT gene expression and circulating levels were analyzed through RT-PCR and ELISA, respectively. Results: L-Gal9, CD206, and NOS2 gene expression was similar in all AT compartments in CHD and CTRLs, as were also L-Gal9 and CD206 circulating levels, while NOS2 serum levels were higher in CHD (p = 0.012 vs. CTRLs). In CTRLs, NOS2 expression was lower in EAT vs. SAT (p = 0.007), while in CHD patients CD206 expression was lower in both SAT and EAT as compared to PAT (p = 0.003, p = 0.006, respectively), suggestive of a possible macrophage reprogramming toward a pro-inflammatory phenotype in EAT. In CHD patients, NOS2 expression in SAT correlated to that in PAT and EAT (p = 0.007, both), CD206 expression correlated positively to L-Gal9 (p < 0.001) only in EAT, and CD206 expression associated with that of macrophage identifying markers in all AT compartments (p < 0.001, all). In CHD patients, subjects with LDL-C above 1.8 mmol/L showed significantly higher NOS2 expression in PAT and EAT as compared to subjects with LDL-C levels below (p < 0.05), possibly reflecting increased cardiac AT pro-inflammatory activation. In SAT and PAT, CD206 expression associated with BMI in both CHD and CTRLs (p < 0.05, all), and with L-Gal9 in EAT, however only in CTRLs (p = 0.002). Conclusion: CHD seems to be accompanied by an altered cardiac, and especially epicardial AT macrophage polarization. This may represent an important pathophysiological mechanism and a promising field of therapy targeting the excessive AT inflammation, in need of further investigation.

Lumican accumulates with fibrillar collagen in fibrosis in hypertrophic cardiomyopathy.

AIMS: Familial hypertrophic cardiomyopathy (HCM) is the most common form of inherited cardiac disease. It is characterized by myocardial hypertrophy and diastolic dysfunction, and can lead to severe heart failure, arrhythmias, and sudden cardiac death. Cardiac fibrosis, defined by excessive accumulation of extracellular matrix (ECM) components, is central to the pathophysiology of HCM. The ECM proteoglycan lumican is increased during heart failure and cardiac fibrosis, including HCM, yet its role in HCM remains unknown. We provide an in-depth assessment of lumican in clinical and experimental HCM. METHODS: Left ventricular (LV) myectomy specimens were collected from patients with hypertrophic obstructive cardiomyopathy (n = 15), and controls from hearts deemed unsuitable for transplantation (n = 8). Hearts were harvested from a mouse model of HCM; Myh6 R403Q mice administered cyclosporine A and wild-type littermates (n = 8-10). LV tissues were analysed for mRNA and protein expression. Patient myectomy or mouse mid-ventricular sections were imaged using confocal microscopy, direct stochastic optical reconstruction microscopy (dSTORM), or electron microscopy. Human foetal cardiac fibroblasts (hfCFBs) were treated with recombinant human lumican (n = 3) and examined using confocal microscopy. RESULTS: Lumican mRNA was increased threefold in HCM patients (P < 0.05) and correlated strongly with expression of collagen I (R2  = 0.60, P < 0.01) and III (R2  = 0.58, P < 0.01). Lumican protein was increased by 40% in patients with HCM (P < 0.01) and correlated with total (R2  = 0.28, P = 0.05) and interstitial (R2  = 0.30, P < 0.05) fibrosis. In mice with HCM, lumican mRNA increased fourfold (P < 0.001), and lumican protein increased 20-fold (P < 0.001) in insoluble ECM lysates. Lumican and fibrillar collagen were located together throughout fibrotic areas in HCM patient tissue, with increased co-localization measured in patients and mice with HCM (patients: +19%, P < 0.01; mice: +13%, P < 0.01). dSTORM super-resolution microscopy was utilized to image interstitial ECM which had yet to undergo overt fibrotic remodelling. In these interstitial areas, collagen I deposits located closer to (-15 nm, P < 0.05), overlapped more frequently with (+7.3%, P < 0.05) and to a larger degree with (+5.6%, P < 0.05) lumican in HCM. Collagen fibrils in such deposits were visualized using electron microscopy. The effect of lumican on collagen fibre formation was demonstrated by adding lumican to hfCFB cultures, resulting in thicker (+53.8 nm, P < 0.001), longer (+345.9 nm, P < 0.001), and fewer (-8.9%, P < 0.001) collagen fibres. CONCLUSIONS: The ECM proteoglycan lumican is increased in HCM and co-localizes with fibrillar collagen throughout areas of fibrosis in HCM. Our data suggest that lumican may promote formation of thicker collagen fibres in HCM.

Inflammation and Syndecan-4 Shedding from Cardiac Cells in Ischemic and Non-Ischemic Heart Disease.

Circulating biomarkers reflecting cardiac inflammation are needed to improve the diagnostics and guide the treatment of heart failure patients. The cardiac production and shedding of the transmembrane proteoglycan syndecan-4 is upregulated by innate immunity signaling pathways. Here, we investigated the potential of syndecan-4 as a blood biomarker of cardiac inflammation. Serum syndecan-4 was measured in patients with (i) non-ischemic, non-valvular dilated cardiomyopathy (DCM), with (n = 71) or without (n = 318) chronic inflammation; (ii) acute myocarditis (n = 15), acute pericarditis (n = 3) or acute perimyocarditis (23) and (iii) acute myocardial infarction (MI) at day 0, 3 and 30 (n = 119). Syndecan-4 was investigated in cultured cardiac myocytes and fibroblasts (n = 6-12) treated with the pro-inflammatory cytokines interleukin (IL)-1ß and its inhibitor IL-1 receptor antagonist (IL-1Ra), or tumor necrosis factor (TNF)α and its specific inhibitor infliximab, an antibody used in treatment of autoimmune diseases. The levels of serum syndecan-4 were comparable in all subgroups of patients with chronic or acute cardiomyopathy, independent of inflammation. Post-MI, syndecan-4 levels were increased at day 3 and 30 vs. day 0. IL-1Ra attenuated IL-1ß-induced syndecan-4 production and shedding in vitro, while infliximab had no effect. In conclusion, syndecan-4 shedding from cardiac myocytes and fibroblasts was attenuated by immunomodulatory therapy. Although its circulating levels were increased post-MI, syndecan-4 did not reflect cardiac inflammatory status in patients with heart disease.

Inhibition of the extracellular enzyme A disintegrin and metalloprotease with thrombospondin motif 4 prevents cardiac fibrosis and dysfunction.

AIMS: Heart failure is a condition with high mortality rates, and there is a lack of therapies that directly target maladaptive changes in the extracellular matrix (ECM), such as fibrosis. We investigated whether the ECM enzyme known as A disintegrin and metalloprotease with thrombospondin motif (ADAMTS) 4 might serve as a therapeutic target in treatment of heart failure and cardiac fibrosis. METHODS AND RESULTS: The effects of pharmacological ADAMTS4 inhibition on cardiac function and fibrosis were examined in rats exposed to cardiac pressure overload. Disease mechanisms affected by the treatment were identified based on changes in the myocardial transcriptome. Following aortic banding, rats receiving an ADAMTS inhibitor, with high inhibitory capacity for ADAMTS4, showed substantially better cardiac function than vehicle-treated rats, including ∼30% reduction in E/e' and left atrial diameter, indicating an improvement in diastolic function. ADAMTS inhibition also resulted in a marked reduction in myocardial collagen content and a down-regulation of transforming growth factor (TGF)-ß target genes. The mechanism for the beneficial effects of ADAMTS inhibition was further studied in cultured human cardiac fibroblasts producing mature ECM. ADAMTS4 caused a 50% increase in the TGF-ß levels in the medium. Simultaneously, ADAMTS4 elicited a not previously known cleavage of TGF-ß-binding proteins, i.e. latent-binding protein of TGF-ß and extra domain A-fibronectin. These effects were abolished by the ADAMTS inhibitor. In failing human hearts, we observed a marked increase in ADAMTS4 expression and cleavage activity. CONCLUSION: Inhibition of ADAMTS4 improves cardiac function and reduces collagen accumulation in rats with cardiac pressure overload, possibly through a not previously known cleavage of molecules that control TGF-ß availability. Targeting ADAMTS4 may serve as a novel strategy in heart failure treatment, in particular, in heart failure with fibrosis and diastolic dysfunction.

Cardiac O-GlcNAc signaling is increased in hypertrophy and heart failure.

Reversible protein O-GlcNAc modification has emerged as an essential intracellular signaling system in several tissues, including cardiovascular pathophysiology related to diabetes and acute ischemic stress. We tested the hypothesis that cardiac O-GlcNAc signaling is altered in chronic cardiac hypertrophy and failure of different etiologies. Global protein O-GlcNAcylation and the main enzymes regulating O-GlcNAc, O-GlcNAc transferase (OGT), O-GlcNAcase (OGA), and glutamine-fructose-6-phosphate amidotransferase (GFAT) were measured by immunoblot and/or real-time RT-PCR analyses of left ventricular tissue from aortic stenosis (AS) patients and rat models of hypertension, myocardial infarction (MI), and aortic banding (AB), with and without failure. We show here that global O-GlcNAcylation was increased by 65% in AS patients, by 47% in hypertensive rats, by 81 and 58% post-AB, and 37 and 60% post-MI in hypertrophic and failing hearts, respectively (P < 0.05). Noticeably, protein O-GlcNAcylation patterns varied in hypertrophic vs. failing hearts, and the most extensive O-GlcNAcylation was observed on proteins of 20-100 kDa in size. OGT, OGA, and GFAT2 protein and/or mRNA levels were increased by pressure overload, while neither was regulated by myocardial infarction. Pharmacological inhibition of OGA decreased cardiac contractility in post-MI failing hearts, demonstrating a possible role of O-GlcNAcylation in development of chronic cardiac dysfunction. Our data support the novel concept that O-GlcNAc signaling is altered in various etiologies of cardiac hypertrophy and failure, including human aortic stenosis. This not only provides an exciting basis for discovery of new mechanisms underlying pathological cardiac remodeling but also implies protein O-GlcNAcylation as a possible new therapeutic target in heart failure.

Troponin T is a better predictor than creatine kinase-MB of long-term mortality after coronary artery bypass graft surgery.

BACKGROUND: Elevations of creatine kinase-MB (CK-MB) and cardiac troponin T (cTnT) have an uncertain long-term prognostic value after coronary artery bypass graft (CABG) surgery. We aimed to test the hypothesis that CK-MB and cTnT are predictors of long-term survival after CABG and to assess which of these 2 biomarkers is the better predictor. METHODS: A total of 1,350 consecutive patients undergoing isolated on-pump CABG had CK-MB and cTnT measured at 7, 20, and 44 hours, postoperatively. The end point was all-cause mortality, and during the median follow-up time of 6.1 years, 207 patients (15.3%) died. RESULTS: Both peak CK-MB and peak cTnT independently predicted long-term mortality (hazard ratio [HR] 1.003, 95% confidence interval [CI] 1.001-1.005, P = .007, and HR 1.31, 95% CI 1.17-1.46, P <.001, respectively) when analyzed in separate multivariate Cox models, adjusting for baseline demographic characteristics and perioperative risk factors. However, when analyzed simultaneously in the same Cox model, cTnT was a significant predictor (HR 1.31, 95% CI 1.13-1.51, P <.001), whereas CK-MB was not (P = .99). Similar results were found when the biomarkers were analyzed together in a Cox model adjusting for European System for Cardiac Operative Risk Evaluation. The differences in mortality between the biomarker groups were consistent also when analyzing strict quartiles of peak values of CK-MB and cTnT (P = .81 and P = .001, respectively). CONCLUSIONS: Both CK-MB and cTnT are predictors of mortality after CABG surgery; however, our data suggest that cTnT is a better predictor of long-term mortality after CABG surgery than CK-MB.

Endothelin-1 in the human myocardium and circulating plasma: evaluation before, during and after correction of aortic stenosis with aortic valve replacement.

OBJECTIVES: Due to the pathological effects of endothelin-1 (ET-1) on cardiomyocytes and the extracellular matrix, ET-1 levels may impact on the prognosis of aortic stenosis (AS) patients operated with aortic valve replacement (AVR). We examined ET-1 levels in AS patients throughout the whole AVR process, thus exposing potential therapeutic windows of opportunity. METHODS: Plasma ET-1 levels were measured before and 2 days, 6 and 12 months after AVR in 22 patients with AS. Myocardial ET-1 was measured in biopsies from 7 patients undergoing AVR. Peroperatively, plasma ET-1 was analyzed in the coronary sinus and radial artery before aortic cross-clamp and at 5 and 20 min of reperfusion, in a second group of 30 patients. RESULTS: Circulating ET-1 levels were transiently increased 2.6-fold 2 days following AVR. Myocardial ET-1 protein was 2.1-fold higher in patients with AS compared to controls. Plasma levels of ET-1 correlated to echocardiographic markers of diastolic dysfunction postoperatively. There was no increase in plasma ET-1 during early reperfusion, but veno-arterial differences indicated potential cardiac ET-1 extraction. CONCLUSIONS: Plasma ET-1 increases 2 days following AVR and myocardial ET-1 protein levels are increased in patients with AS before AVR. Peroperatively, no plasma ET-1 augmentation or release from the heart was observed in AS patients.