BIN1, Myotubularin, and Dynamin-2 Coordinate T-Tubule Growth in Cardiomyocytes.

BACKGROUND: Transverse tubules (t-tubules) form gradually in the developing heart, critically enabling maturation of cardiomyocyte Ca2+ homeostasis. The membrane bending and scaffolding protein BIN1 (bridging integrator 1) has been implicated in this process. However, it is unclear which of the various reported BIN1 isoforms are involved, and whether BIN1 function is regulated by its putative binding partners MTM1 (myotubularin), a phosphoinositide 3'-phosphatase, and DNM2 (dynamin-2), a GTPase believed to mediate membrane fission. METHODS: We investigated the roles of BIN1, MTM1, and DNM2 in t-tubule formation in developing mouse cardiomyocytes, and in gene-modified HL-1 and human-induced pluripotent stem cell-derived cardiomyocytes. T-tubules and proteins of interest were imaged by confocal and Airyscan microscopy, and expression patterns were examined by RT-qPCR and Western blotting. Ca2+ release was recorded using Fluo-4. RESULTS: We observed that in the postnatal mouse heart, BIN1 localizes along Z-lines from early developmental stages, consistent with roles in initial budding and scaffolding of t-tubules. T-tubule proliferation and organization were linked to a progressive and parallel increase in 4 detected BIN1 isoforms. All isoforms were observed to induce tubulation in cardiomyocytes but produced t-tubules with differing geometries. BIN1-induced tubulations contained the L-type Ca2+ channel, were colocalized with caveolin-3 and the ryanodine receptor, and effectively triggered Ca2+ release. BIN1 upregulation during development was paralleled by increasing expression of MTM1. Despite no direct binding between MTM1 and murine cardiac BIN1 isoforms, which lack exon 11, high MTM1 levels were necessary for BIN1-induced tubulation, indicating a central role of phosphoinositide homeostasis. In contrast, the developing heart exhibited declining levels of DNM2. Indeed, we observed that high levels of DNM2 are inhibitory for t-tubule formation, although this protein colocalizes with BIN1 along Z-lines, and binds all 4 isoforms. CONCLUSIONS: These findings indicate that BIN1, MTM1, and DNM2 have balanced and collaborative roles in controlling t-tubule growth in cardiomyocytes.

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.

AKAP18δ Anchors and Regulates CaMKII Activity at Phospholamban-SERCA2 and RYR.

BACKGROUND: The sarcoplasmic reticulum (SR) Ca2+-ATPase 2 (SERCA2) mediates Ca2+ reuptake into SR and thereby promotes cardiomyocyte relaxation, whereas the ryanodine receptor (RYR) mediates Ca2+ release from SR and triggers contraction. Ca2+/CaMKII (CaM [calmodulin]-dependent protein kinase II) regulates activities of SERCA2 through phosphorylation of PLN (phospholamban) and RYR through direct phosphorylation. However, the mechanisms for CaMKIIδ anchoring to SERCA2-PLN and RYR and its regulation by local Ca2+ signals remain elusive. The objective of this study was to investigate CaMKIIδ anchoring and regulation at SERCA2-PLN and RYR. METHODS: A role for AKAP18δ (A-kinase anchoring protein 18δ) in CaMKIIδ anchoring and regulation was analyzed by bioinformatics, peptide arrays, cell-permeant peptide technology, immunoprecipitations, pull downs, transfections, immunoblotting, proximity ligation, FRET-based CaMKII activity and ELISA-based assays, whole cell and SR vesicle fluorescence imaging, high-resolution microscopy, adenovirus transduction, adenoassociated virus injection, structural modeling, surface plasmon resonance, and alpha screen technology. RESULTS: Our results show that AKAP18δ anchors and directly regulates CaMKIIδ activity at SERCA2-PLN and RYR, via 2 distinct AKAP18δ regions. An N-terminal region (AKAP18δ-N) inhibited CaMKIIδ through binding of a region homologous to the natural CaMKII inhibitor peptide and the Thr17-PLN region. AKAP18δ-N also bound CaM, introducing a second level of control. Conversely, AKAP18δ-C, which shares homology to neuronal CaMKIIα activator peptide (N2B-s), activated CaMKIIδ by lowering the apparent Ca2+ threshold for kinase activation and inducing CaM trapping. While AKAP18δ-C facilitated faster Ca2+ reuptake by SERCA2 and Ca2+ release through RYR, AKAP18δ-N had opposite effects. We propose a model where the 2 unique AKAP18δ regions fine-tune Ca2+-frequency-dependent activation of CaMKIIδ at SERCA2-PLN and RYR. CONCLUSIONS: AKAP18δ anchors and functionally regulates CaMKII activity at PLN-SERCA2 and RYR, indicating a crucial role of AKAP18δ in regulation of the heartbeat. To our knowledge, this is the first protein shown to enhance CaMKII activity in heart and also the first AKAP (A-kinase anchoring protein) reported to anchor a CaMKII isoform, defining AKAP18δ also as a CaM-KAP.

Beneficial effects of exercise initiated before development of hypertrophic cardiomyopathy in genotype-positive mice.

The effect of exercise on disease development in hypertrophic cardiomyopathy (HCM) genotype-positive individuals is unresolved. Our objective was to test the effect of exercise training initiated before phenotype development on cardiac fibrosis, morphology, and function in a mouse model of HCM. Genotype-positive Myh6 R403Q mice exposed to cyclosporine A (CsA) for induction of HCM (HCM mice) were allocated to high-intensity interval treadmill running or sedentary behavior for 6 wk. CsA was initiated from week 4 of the protocol. Cardiac imaging and exercise testing were performed at weeks 0, 3, and 6. After protocol completion, arrhythmia provocation was performed in isolated hearts, and left ventricles (LVs) were harvested for molecular biology and histology. Exercised HCM mice ran farther and faster and exhibited attenuated left atrial (LA) dilatation compared with sedentary mice. Exercised HCM mice had no difference in fibrosis compared with sedentary HCM mice despite lower expression of key extracellular matrix (ECM) genes collagen 1 and 3, fibronectin, and lysyl oxidase, accompanied by increased activation of Akt, GSK3b, and p38. Exercise did not have negative effects on LV function in HCM mice. Our findings indicate mild beneficial effects of exercise initiated before HCM phenotype development, specifically lower ECM gene expression and LA dilatation, and importantly, no detrimental effects.NEW & NOTEWORTHY Genotype-positive hypertrophic cardiomyopathy (HCM) mice had beneficial effects of exercise initiated before phenotype development. Exercised HCM mice had increased exercise capacity, smaller left atria, no increase in hypertrophy, or reduction of function, and a similar degree of fibrosis despite reduction of central extracellular matrix (ECM) genes, including collagens, compared with sedentary HCM mice.

Cardiac Troponin T and NT-proBNP for Prediction of 30-Day Readmission or Death in Patients with Acute Dyspnea: Data from the Akershus Cardiac Examination 2 Study.

INTRODUCTION: N-terminal pro-B-type natriuretic peptide (NT-proBNP) and cardiac troponin T (cTnT) measurements are recommended in patients with acute dyspnea. We aimed to assess the prognostic merit of cTnT compared to NT-proBNP for 30-day readmission or death in patients hospitalized with acute dyspnea. METHODS: We measured cTnT and NT-proBNP within 24 h in 314 patients hospitalized with acute dyspnea and adjudicated the cause of the index admission. Time to first event of readmission or death ≤30 days after hospital discharge was recorded, and cTnT and NT-proBNP measurements were compared head-to-head. RESULTS: Patients who died (12/314) or were readmitted (71/314) within 30 days had higher cTnT concentrations (median: 32.6, Q1-Q3: 18.4-74.2 ng/L vs. median: 19.4, Q1-Q3: 8.4-36.1 ng/L; p for comparison <0.001) and NT-proBNP concentrations (median: 1,753.6, Q1-Q3: 464.2-6,862.0 ng/L vs. median 984, Q1-Q3 201-3,600 ng/L; for comparison p = 0.027) compared to patients who survived and were not readmitted. cTnT concentrations were associated with readmission or death within 30 days after discharge both in the total cohort (adjusted hazard ratio [aHR]: 1.64, 95% confidence interval [CI]: 1.30-2.05) and in patients with heart failure (HF) (aHR: 1.58, 95% CI: 1.14-2.18). In contrast, NT-proBNP concentrations were not associated with short-term events, neither in the total cohort (aHR: 1.10, 95% CI: 0.94-1.30) nor in patients with adjudicated HF (aHR: 1.06, 95% CI: 0.80-1.40). CONCLUSION: cTnT concentrations are associated with 30-day readmission or death in patients hospitalized with acute dyspnea, as well as in patients adjudicated HF.

Integrin α11ß1 and syndecan-4 dual receptor ablation attenuate cardiac hypertrophy in the pressure overloaded heart.

Pathological myocardial hypertrophy in response to an increase in left ventricular (LV) afterload may ultimately lead to heart failure. Cell surface receptors bridge the interface between the cell and the extracellular matrix (ECM) in cardiac myocytes and cardiac fibroblasts and have been suggested to be important mediators of pathological myocardial hypertrophy. We identify for the first time that integrin α11 (α11) is preferentially upregulated among integrin ß1 heterodimer-forming α-subunits in response to increased afterload induced by aortic banding (AB) in wild-type (WT) mice. Mice were anesthetized in a chamber with 4% isoflurane and 95% oxygen before being intubated and ventilated with 2.5% isoflurane and 97% oxygen. For pre- and postoperative analgesia, animals were administered 0.02-mL buprenorphine (0.3 mg/mL) subcutaneously. Surprisingly, mice lacking α11 develop myocardial hypertrophy following AB comparable to WT. In the mice lacking α11, we further show a compensatory increase in the expression of another mechanoreceptor, syndecan-4, following AB compared with WT AB mice, indicating that syndecan-4 compensated for lack of α11. Intriguingly, mice lacking mechanoreceptors α11 and syndecan-4 show ablated myocardial hypertrophy following AB compared with WT mice. Expression of the main cardiac collagen isoforms col1a2 and col3a1 was significantly reduced in AB mice lacking mechanoreceptors α11 and syndecan-4 compared with WT AB.NEW & NOTEWORTHY Despite their putative importance in stress sensing, the specific integrin α-subunit(s) involved in cardiac hypertrophy has not been identified. Here, we show that α11 and syndecan-4 are critical and interdependent mediators of the hypertrophic response to increased LV afterload. We demonstrate in cells lacking both receptors an interdependent reduction in cell attachment to the major cardiac extracellular matrix components, suggesting that their interplay represents an important mechanism for stress sensing in cardiac cells.

Cardiomyocyte-specific overexpression of syndecan-4 in mice results in activation of calcineurin-NFAT signalling and exacerbated cardiac hypertrophy.

BACKGROUND: Cardiomyocyte hypertrophy is a hallmark of cardiac dysfunction in patients with aortic stenosis (AS), and can be triggered by left ventricular (LV) pressure overload in mice by aortic banding (AB). Syndecan-4 is a transmembrane heparan sulphate proteoglycan which is found increased in the myocardium of AS patients and AB mice. The role of syndecan-4 in cardiomyocyte hypertrophy is not well understood. PURPOSE OF THE STUDY: We developed mice with cardiomyocyte-specific overexpression of syndecan-4 (Sdc4-Tg) and subjected these to AB to examine the role of syndecan-4 in hypertrophy and activation of the pro-hypertrophic calcineurin-NFAT signalling pathway. METHODS AND RESULTS: Sdc4-Tg mice showed exacerbated cardiac remodelling upon AB compared to wild type (WT). At 2-6 weeks post-AB, Sdc4-Tg and WT mice showed similar hypertrophic growth, while at 20 weeks post-AB, exacerbated hypertrophy and dysfunction were evident in Sdc4-Tg mice. After cross-breeding of Sdc4-Tg mice with NFAT-luciferase reporter mice, we found increased NFAT activation in Sdc4-Tg hearts after AB. Immunoprecipitation showed that calcineurin bound to syndecan-4 in Sdc4-Tg hearts. Isolated cardiomyocytes from Sdc4-Tg mice showed alterations in Ca2+ fluxes, suggesting that syndecan-4 regulated Ca2+ levels, and thereby, activating the syndecan-4-calcineurin complex resulting in NFAT activation and hypertrophic growth. Similarly, primary cardiomyocyte cultures from neonatal rats showed increased calcineurin-NFAT-dependent hypertrophic growth upon viral Sdc4 overexpression. CONCLUSION: Our study of mice with cardiomyocyte-specific overexpression of Sdc4 have revealed that syndecan-4 is important for activation of the Ca2+-dependent calcineurin-NFAT signalling pathway, hypertrophic remodelling and dysfunction in cardiomyocytes in response to pressure overload.

Circulating MicroRNA-210 Concentrations in Patients with Acute Heart Failure: Data from the Akershus Cardiac Examination 2 Study.

BACKGROUND: MicroRNA (miR)-210 expression is induced by acute and chronic hypoxia and provides prognostic information in patients with aortic stenosis and acute coronary syndrome. We hypothesized that circulating miR-210 concentrations could provide diagnostic and prognostic information in patients with acute heart failure (HF). METHODS: We measured miR-210 concentrations in serum samples on admission from 314 patients hospitalized for acute dyspnea and 9 healthy control subjects. The diagnostic and prognostic properties of miR-210 were tested in patients after adjudication of all diagnoses and with median follow-up of 464 days. RESULTS: All patients and control subjects had miR-210 concentrations within the range of detection, and the analytical variation was low as the coefficient of variation of synthetic spike-in RNA was 4%. Circulating miR-210 concentrations were increased in patients with HF compared to healthy control subjects, but miR-210 concentrations did not separate patients with acute HF (n = 143) from patients with non-HF-related dyspnea (n = 171): the area under the curve was 0.50 (95% CI 0.43-0.57). Circulating miR-210 concentrations were associated with mortality (n = 114) after adjustment for clinical risk factors (hazard ratio 1.65 [95% CI 1.03-2.62] per unit miR-210 increase), but this association was attenuated and not significant after adjustment for established cardiac protein biomarkers. CONCLUSIONS: Circulating miR-210 concentrations are associated with mortality, but do not add to established protein biomarkers for diagnosis or prognosis in patients with acute dyspnea.

Performance of a Novel Research-Use-Only Secretoneurin ELISA in Patients with Suspected Acute Coronary Syndrome: Comparison with an Established Secretoneurin Radioimmunoassay.

BACKGROUND: Circulating secretoneurin (SN) concentrations, as measured by established radioimmunoassay (RIA), risk stratify patients with cardiovascular disease. We now report data for a recently developed research-use-only SN enzyme-linked immunosorbent assay (ELISA) in patients with suspected acute coronary syndrome (ACS). METHODS: SN ELISA was developed according to industry standards and tested in 401 unselected chest pain patients. Blood samples were drawn <24 h from admission, and we adjudicated all hospitalizations as ACS or non-ACS. The mean follow-up was 6.2 years. RESULTS: SN ELISA with 2 monoclonal sheep anti-SN antibodies has a measuring range of 10-250 pmol/L and demonstrates excellent analytical precision and accuracy across the range of SN concentrations. SN measured by ELISA and RIA correlated in the chest pain patients: rho = 0.39, p < 0.001. SN concentrations were higher in ACS patients (n = 161 [40%]) than in non-ACS patients (n = 240) for both assays, with an area under the curve (AUC) of 0.66 (95% CI: 0.61-0.71) for ELISA and 0.59 (0.54-0.65) for RIA. SN concentrations were also higher in nonsurvivors (n = 65 [16%]) than survivors, with an AUC of 0.72 (0.65-0.79) for ELISA versus 0.64 (0.56-0.72) for RIA, p = 0.007, for difference between assays. Adjusting for age, sex, blood pressure, previous myocardial infarction, atrial fibrillation, and heart failure in multivariable analysis, SN concentrations as measured by ELISA, but not RIA, remained associated with mortality, with a hazard ratio of 1.71 (1.03-2.84), p = 0.038. CONCLUSIONS: The novel SN ELISA has excellent performance, higher AUC for diagnosis, and superior prognostic accuracy compared to the established RIA in chest pain patients.

The cardiac syndecan-4 interactome reveals a role for syndecan-4 in nuclear translocation of muscle LIM protein (MLP).

Costameres are signaling hubs at the sarcolemma and important contact points between the extracellular matrix and cell interior, sensing and transducing biomechanical signals into a cellular response. The transmembrane proteoglycan syndecan-4 localizes to these attachment points and has been shown to be important in the initial stages of cardiac remodeling, but its mechanistic function in the heart remains insufficiently understood. Here, we sought to map the cardiac interactome of syndecan-4 to better understand its function and downstream signaling mechanisms. By combining two different affinity purification methods with MS analysis, we found that the cardiac syndecan-4 interactome consists of 21 novel and 29 previously described interaction partners. Nine of the novel partners were further validated to bind syndecan-4 in HEK293 cells (i.e. CAVIN1/PTRF, CCT5, CDK9, EIF2S1, EIF4B, MPP7, PARVB, PFKM, and RASIP). We also found that 19 of the 50 interactome partners bind differently to syndecan-4 in the left ventricle lysate from aortic-banded heart failure (ABHF) rats compared with SHAM-operated animals. One of these partners was the well-known mechanotransducer muscle LIM protein (MLP), which showed direct and increased binding to syndecan-4 in ABHF. Nuclear translocation is important in MLP-mediated signaling, and we found less MLP in the nuclear-enriched fractions from syndecan-4-/- mouse left ventricles but increased nuclear MLP when syndecan-4 was overexpressed in a cardiomyocyte cell line. In the presence of a cell-permeable syndecan-4-MLP disruptor peptide, the nuclear MLP level was reduced. These findings suggest that syndecan-4 mediates nuclear translocation of MLP in the heart.

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.

Caspase-1 induces smooth muscle cell growth in hypoxia-induced pulmonary hypertension.

Lung diseases with hypoxia are complicated by pulmonary hypertension, leading to heart failure and death. No pharmacological treatment exists. Increased proinflammatory cytokines are found in hypoxic patients, suggesting an inflammatory pathogenesis. Caspase-1, the effector of the inflammasome, mediates inflammation through activation of the proinflammatory cytokines interleukin (IL)-18 and IL-1ß. Here, we investigate inflammasome-related mechanisms that can trigger hypoxia-induced pulmonary hypertension. Our aim was to examine whether caspase-1 induces development of hypoxia-related pulmonary hypertension and is a suitable target for therapy. Wild-type (WT) and caspase-1-/- mice were exposed to 10% oxygen for 14 days. Hypoxic caspase-1-/- mice showed lower pressure and reduced muscularization in pulmonary arteries, as well as reduced right ventricular remodeling compared with WT. Smooth muscle cell (SMC) proliferation was reduced in caspase-1-deficient pulmonary arteries and in WT arteries treated with a caspase-1 inhibitor. Impaired inflammation was shown in hypoxic caspase-1-/- mice by abolished pulmonary influx of immune cells and lower levels of IL-18, IL-1ß, and IL-6, which were also reduced in the medium surrounding caspase-1 abrogated pulmonary arteries. By adding IL-18 or IL-1ß to caspase-1-deficient pulmonary arteries, SMC proliferation was retained. Furthermore, inhibition of both IL-6 and phosphorylated STAT3 reduced proliferation of SMC in vitro, indicating IL-18, IL-6, and STAT3 as downstream mediators of caspase-1-induced SMC proliferation in pulmonary arteries. Caspase-1 induces SMC proliferation in pulmonary arteries through the caspase-1/IL-18/IL-6/STAT3 pathway, leading to pulmonary hypertension in mice exposed to hypoxia. We propose that caspase-1 inhibition is a potential target for treatment of pulmonary hypertension.

Circulating Secretoneurin Concentrations After Cardiac Surgery: Data From the FINNish Acute Kidney Injury Heart Study.

OBJECTIVES: Secretoneurin is associated with cardiomyocyte Ca handling and improves risk prediction in patients with acute myocardial dysfunction. Whether secretoneurin improves risk assessment on top of established cardiac biomarkers and European System for Cardiac Operative Risk Evaluation II in patients undergoing cardiac surgery is not known. DESIGN: Prospective, observational, single-center sub-study of a multicenter study. SETTING: Prospective observational study of survival in patients undergoing cardiac surgery. PATIENTS: A total of 619 patients undergoing cardiac surgery. INTERVENTIONS: Patients underwent either isolated coronary artery bypass graft surgery, single noncoronary artery bypass graft surgery, two procedures, or three or more procedures. Procedures other than coronary artery bypass graft were valve surgery, surgery on thoracic aorta, and other cardiac surgery. MEASUREMENTS AND MAIN RESULTS: We measured preoperative and postoperative secretoneurin concentrations and adjusted for European System for Cardiac Operative Risk Evaluation II, N-terminal pro-B-type natriuretic peptide, and cardiac troponin T concentrations in multivariate analyses. During 961 days of follow-up, 59 patients died (9.5%). Secretoneurin concentrations were higher among nonsurvivors compared with survivors, both before (168 pmol/L [quartile 1-3, 147-206 pmol/L] vs 160 pmol/L [131-193 pmol/L]; p = 0.039) and after cardiac surgery (173 pmol/L [129-217 pmol/L] vs 143 pmol/L [111-173 pmol/L]; p < 0.001). Secretoneurin concentrations decreased from preoperative to postoperative measurements in survivors, whereas we observed no significant decrease in secretoneurin concentrations among nonsurvivors. Secretoneurin concentrations were weakly correlated with established risk indices. Patients with the highest postoperative secretoneurin concentrations had worse outcome compared with patients with lower secretoneurin concentrations (p < 0.001 by the log-rank test) and postoperative secretoneurin concentrations were associated with time to death in multivariate Cox regression analysis: hazard ratio lnsecretoneurin 2.96 (95% CI, 1.46-5.99; p = 0.003). Adding postoperative secretoneurin concentrations to European System for Cardiac Operative Risk Evaluation II improved patient risk stratification, as assessed by the integrated discrimination index: 0.023 (95% CI, 0.0043-0.041; p = 0.016). CONCLUSIONS: Circulating postoperative secretoneurin concentrations provide incremental prognostic information to established risk indices in patients undergoing cardiac surgery.

Prognostic Value of Secretoneurin in Patients With Severe Sepsis and Septic Shock: Data From the Albumin Italian Outcome Sepsis Study.

OBJECTIVES: Secretoneurin directly influences cardiomyocyte calcium handling, and circulating secretoneurin levels seem to improve risk prediction in patients with myocardial dysfunction by integrating information on systemic stress, myocardial function, and renal function. Accordingly, in this study, we hypothesized that secretoneurin would improve risk prediction in patients with sepsis and especially in patients with septic shock as these patients are more hemodynamically unstable. DESIGN: Multicenter, interventional randomized clinical trial. SETTING: Multicenter, pragmatic, open-label, randomized, prospective clinical trial testing fluid administration with either 20% human albumin and crystalloids or crystalloid solutions alone in patients with severe sepsis or septic shock (The Albumin Italian Outcome Sepsis). PATIENTS OR SUBJECTS: In total, 540 patients with septic shock and 418 patients with severe sepsis. INTERVENTIONS: Either 20% human albumin and crystalloids or crystalloid solutions alone. MEASUREMENTS AND MAIN RESULTS: We measured secretoneurin on days 1, 2, and 7 after randomization and compared the prognostic value of secretoneurin for ICU and 90-day mortality with established risk indices and cardiac biomarkers in septic shock and severe sepsis. High secretoneurin levels on day 1 were associated with age and serum concentrations of lactate, bilirubin, creatinine, and N-terminal pro-B-type natriuretic peptide. Adjusting for established risk factors and cardiovascular biomarkers, secretoneurin levels on day 1 were associated with ICU (odds ratio, 2.27 [95% CI, 1.05-4.93]; p = 0.04) and 90-day mortality (2.04 [1.02-4.10]; p = 0.04) in patients with septic shock, but not severe sepsis without shock. Secretoneurin levels on day 2 were also associated with ICU (3.11 [1.34-7.20]; p = 0.008) and 90-day mortality (2.69 [1.26-5.78]; p = 0.01) in multivariate regression analyses and improved reclassification in patients with septic shock, as assessed by the net reclassification index. Randomized albumin administration did not influence the associations between secretoneurin and outcomes. CONCLUSIONS: Secretoneurin provides early and potent prognostic information in septic patients with cardiovascular instability.

Prognostic and diagnostic significance of mid-regional pro-atrial natriuretic peptide in acute exacerbation of chronic obstructive pulmonary disease and acute heart failure: data from the ACE 2 Study.

PURPOSE: To compare the diagnostic and prognostic value of mid-regional pro-ANP (MR-proANP) and N-terminal pro-B-type natriuretic peptide (NT-proBNP) in patients with acute dyspnea. METHODS: MR-proANP and NT-proBNP were measured with commercial immunoassays at hospital admission (n = 313), on day 2 (n = 234), and before discharge (n = 91) and compared for diagnosing acute heart failure (HF; n = 143) and to predict mortality among patients with acute HF and acute exacerbation of chronic obstructive pulmonary disease (AECOPD; n = 84) separately. RESULTS: The correlation coefficient between MR-proANP and NT-proBNP was 0.89 (p < 0.001) and the receiver-operating area under the curve (AUC) was 0.85 (95% CI 0.81-0.89) for MR-proANP and 0.86 (0.82-0.90) for NT-proBNP to diagnose acute HF. During a median follow-up of 816 days, mortality rates were 46% in acute HF patients and 42% in AECOPD patients. After adjustment for other risk variables by multivariate Cox regression analysis, MR-proANP and NT-proBNP concentrations were associated with mortality in patients with acute HF, but only MR-proANP were associated with mortality among patients with AECOPD: hazard ratio (lnMR-proANP) 1.98 (95% CI 1.17-3.34). CONCLUSION: MR-proANP and NT-proBNP concentrations provide similar diagnostic and prognostic information in patients with acute HF. In contrast to NT-proBNP, MR-proANP measurements also provided independent prognostic information in AECOPD patients.

Prognostic and diagnostic significance of copeptin in acute exacerbation of chronic obstructive pulmonary disease and acute heart failure: data from the ACE 2 study.

BACKGROUND: Copeptin is a novel biomarker that predicts mortality in lower respiratory tract infections and heart failure (HF), but the diagnostic value of copeptin in acute dyspnea and the prognostic significance of copeptin in acute exacerbation of chronic obstructive pulmonary disease (AECOPD) is not clear. METHOD: We determined copeptin and NT-proBNP concentrations at hospital admission in 314 patients with acute dyspnea who were categorized by diagnosis. Survival was registered after a median follow-up of 816 days, and the prognostic and diagnostic properties of copeptin and NT-proBNP were analyzed in acute HF (n = 143) and AECOPD (n = 84) separately. RESULTS: The median concentration of copeptin at admission was lower in AECOPD compared to acute HF (8.8 [5.2-19.7] vs. 22.2 [10.2-47.9]) pmol/L, p < 0.001), but NT-proBNP discriminated acute HF from non-HF related dyspnea more accurately than copeptin (ROC-AUC 0.85 [0.81-0.89] vs. 0.71 [0.66-0.77], p < 0.0001). Adjusted for basic risk factors, increased copeptin concentrations predicted mortality in AECOPD (HR per log (ln) unit 1.72 [95% CI 1.21-2.45], p = 0.003) and acute HF (1.61 [1.25-2.09], p < 0.001), whereas NT-proBNP concentrations predicted mortality only in acute HF (1.62 [1.27-2.06], p < 0.001). On top of a basic model copeptin reclassified a significant proportion of patients into a more accurate risk strata in AECOPD (NRI 0.60 [0.19-1.02], p = 0.004) and acute HF (0.39 [0.06-0.71], p = 0.020). CONCLUSION: Copeptin is a strong prognostic marker in both AECOPD and acute HF, while NT-proBNP concentrations predict mortality only in patients with acute HF. NT-proBNP levels are superior to copeptin levels to diagnose acute HF in patients with acute dyspnea.

Circulating chromogranin B levels in patients with acute respiratory failure: data from the FINNALI Study.

PURPOSE: Circulating chromogranin B (CgB) levels are increased in situations characterized by systemic and myocardial stress, but whether CgB provides prognostic information in patients with acute respiratory failure (ARF) is unknown. METHODS: We included 584 patients with ARF, defined as ventilatory support >6 h, and with blood samples available on Intensive Care Unit (ICU) admission and day 3 (n = 479). CgB levels were measured by radioimmunoassay and follow-up was 90 days. RESULTS: One-hundred-sixty-nine patients (29%) died during follow-up. Admission CgB levels separated non-survivors from survivors: median 1234 (Q1-3 989-1742) vs. 917 (753-1224) pmol/L, respectively, p < 0.001. CgB levels on ICU admission (logarithmically transformed) were associated with time to death after adjustment for established risk indices available on ICU admission, including N-terminal pro-B-type natriuretic levels: HR 2.62 (95%C.I. 1.82-3.77), p < 0.001. Admission CgB levels also improved prognostication on top of SOFA and SAPS II scores as assessed by Cox regression analyses and the category-free net reclassification index. The area under the curve (AUC) for admission CgB levels to separate survivors and non-survivors was 0.72 (95%CI 0.67-0.76), while the AUC on day 3 was 0.60 (0.54-0.66). CONCLUSIONS: CgB levels measured on ICU admission provided additional prognostic information to established risk indices in ARF patients.

Toll-Like Receptor 9 Promotes Survival in SERCA2a KO Heart Failure Mice.

Aim. Inflammation is important in heart failure (HF). The role of the immune receptor toll-like receptor 9 (TLR9) in HF is not understood and not investigated in diastolic HF. We investigated the role of TLR9 in a murine diastolic HF model caused by cardiomyocyte SERCA2a excision. Methods and Results. We crossed SERCA2a KO and TLR9 KO mice to generate four mouse lines. Tamoxifen-induced cardiomyocyte SERCA2a gene excision was carried out in mice, causing diastolic HF. After 7.6 weeks, cardiac functions and dimensions were analyzed by echocardiography and heart tissues were processed. HF mice depleted of TLR9 demonstrated reduced survival compared to SERC2a KO mice, with a median life expectancy of 58 days compared to 63 days. Both HF groups displayed increased left atrium size, lung weight, fetal gene expressions, monocyte/macrophage infiltration, and fibrosis. However, there were no significant differences between the groups. Conclusion. In mice with SERCA2a KO-induced diastolic HF, the absence of TLR9 reduced median life expectancy. The cause remains elusive, as all investigated HF parameters were unaltered. Still, these findings support a salutary role of TLR9 in some subsets of HF conditions and underline the importance for future studies on the mechanisms of TLR9 in diastolic HF.

Glycosylated Chromogranin A: Potential Role in the Pathogenesis of Heart Failure.

PURPOSE OF REVIEW: Endocrine and paracrine factors influence the cardiovascular system and the heart by a number of different mechanisms. The chromogranin-secretogranin (granin) proteins seem to represent a new family of proteins that exerts both direct and indirect effects on cardiac and vascular functions. The granin proteins are produced in multiple tissues, including cardiac cells, and circulating granin protein concentrations provide incremental prognostic information to established risk indices in patients with myocardial dysfunction. In this review, we provide recent data for the granin proteins in relation with cardiovascular disease, and with a special focus on chromogranin A and heart failure. RECENT FINDINGS: Chromogranin A is the most studied member of the granin protein family, and shorter, functionally active peptide fragments of chromogranin A exert protective effects on myocardial cell death, ischemia-reperfusion injury, and cardiomyocyte Ca2+ handling. Granin peptides have also been found to induce angiogenesis and vasculogenesis. Protein glycosylation is an important post-translational regulatory mechanism, and we recently found chromogranin A molecules to be hyperglycosylated in the failing myocardium. Chromogranin A hyperglycosylation impaired processing of full-length chromogranin A molecules into physiologically active chromogranin A peptides, and patients with acute heart failure and low rate of chromogranin A processing had increased mortality compared to other acute heart failure patients. Other studies have also demonstrated that circulating granin protein concentrations increase in parallel with heart failure disease stage. The granin protein family seems to influence heart failure pathophysiology, and chromogranin A hyperglycosylation could directly be implicated in heart failure disease progression.

Prognostic Value of Secretoneurin in Critically Ill Patients With Infections.

OBJECTIVES: Secretoneurin is produced in neuroendocrine cells, and the myocardium and circulating secretoneurin levels provide incremental prognostic information to established risk indices in cardiovascular disease. As myocardial dysfunction contributes to poor outcome in critically ill patients, we wanted to assess the prognostic value of secretoneurin in two cohorts of critically ill patients with infections. DESIGN: Two prospective, observational studies. SETTING: Twenty-four and twenty-five ICUs in Finland. PATIENTS: A total of 232 patients with severe sepsis (cohort #1) and 94 patients with infections and respiratory failure (cohort #2). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We measured secretoneurin levels by radioimmunoassay in samples obtained early after ICU admission and compared secretoneurin with other risk indices. In patients with severe sepsis, admission secretoneurin levels (logarithmically transformed) were associated with hospital mortality (odds ratio, 3.17 [95% CI, 1.12-9.00]; p = 0.030) and shock during the hospitalization (odds ratio, 2.17 [1.06-4.46]; p = 0.034) in analyses that adjusted for other risk factors available on ICU admission. Adding secretoneurin levels to age, which was also associated with hospital mortality in the multivariate model, improved the risk prediction as assessed by the category-free net reclassification index: 0.35 (95% CI, 0.06-0.64) (p = 0.02). In contrast, N-terminal pro-B-type natriuretic peptide levels were not associated with mortality in the multivariate model that included secretoneurin measurements, and N-terminal pro-B-type natriuretic peptide did not improve patient classification on top of age. Secretoneurin levels were also associated with hospital mortality after adjusting for other risk factors and improved patient classification in cohort #2. In both cohorts, the optimal cutoff for secretoneurin levels at ICU admission to predict hospital mortality was ≈ 175 pmol/L, and higher levels were associated with mortality also when adjusting for Simplified Acute Physiology Score II and Sequential Organ Failure Assessment scores. CONCLUSIONS: Secretoneurin levels provide incremental information to established risk indices for the prediction of mortality and shock in critically ill patients with severe infections.