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Background: Identification of novel biomarkers could provide prognostic information and improve risk stratification in patients with aortic stenosis (AS). YKL-40 (chitinase-3-like protein 1), a protein involved in atherogenesis, is upregulated in human calcific aortic valves. We hypothesized that circulating YKL-40 would be elevated and associated with the degree of AS severity and outcome in patients with symptomatic AS. Methods: Plasma YKL-40 was analyzed in 2 AS populations, one severe AS (n=572) with outcome measures and one with mixed severity (n=67). YKL-40 expression in calcified valves and in an experimental pressure overload model was assessed. Results: We found (1) patients with AS had upregulated circulating YKL-40 compared with healthy controls (median 109 versus 34 ng/mL, P P Conclusions: YKL-40 levels were elevated in AS and associated with mortality but not with other metrics of disease severity including the degree of AS severity. Despite scientific rationale for its role in AS, the clinical utility of circulating YKL-40 as a biomarker is limited. Registration: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT01794832.

Myocardial infarction (MI) triggers a reparative response involving fibroblast proliferation and differentiation driving extracellular matrix modulation necessary to form a stabilizing scar. Recently, it was shown that a genetic variant of the base excision repair enzyme NEIL3 was associated with increased risk of MI in humans. Here, we report elevated myocardial NEIL3 expression in heart failure patients and marked myocardial upregulation of Neil3 after MI in mice, especially in a fibroblast-enriched cell fraction. Neil3−/− mice show increased mortality after MI caused by myocardial rupture. Genome-wide analysis of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) reveals changes in the cardiac epigenome, including in genes related to the post-MI transcriptional response. Differentially methylated genes are enriched in pathways related to proliferation and myofibroblast differentiation. Accordingly, Neil3−/− ruptured hearts show increased proliferation of fibroblasts and myofibroblasts. We propose that NEIL3-dependent modulation of DNA methylation regulates cardiac fibroblast proliferation and thereby affects extracellular matrix modulation after MI. © 2017 The Authors. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

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.

Palmitate triggers inflammatory responses in several cell types, but its effects on cardiac fibroblasts are at present unknown. The aims of the study were to (1) assess the potential of palmitate to promote inflammatory signaling in cardiac fibroblasts through TLR4 and the NLRP3 inflammasome and (2) characterize the cellular phenotype of cardiac fibroblasts exposed to palmitate. We examined whether palmitate induces inflammatory responses in cardiac fibroblasts from WT, NLRP3-/- and ASC-/-mice (C57BL/6 background). Exposure to palmitate caused production of TNF, IL-6 and CXCL2 via TLR4 activation. NLRP3 inflammasomes are activated in a two-step manner. Whereas palmitate did not prime the NLRP3 inflammasome, it induced activation in LPS-primed cardiac fibroblasts as indicated by IL-1β, IL-18 production and NLRP3-ASC co-localization. Palmitate-induced NLRP3 inflammasome activation in LPS-primed cardiac fibroblasts was associated with reduced AMPK activity, mitochondrial reactive oxygen species production and mitochondrial dysfunction. The cardiac fibroblast phenotype caused by palmitate, in an LPS and NLRP3 independent manner, was characterized by decreased cellular proliferation, contractility, collagen and MMP-2 expression, as well as increased senescence-associated β-galactosidase activity, and consistent with a state of cellular senescence. This study establishes that in vitro palmitate exposure of cardiac fibroblasts provides inflammatory responses via TLR4 and NLRP3 inflammasome activation. Palmitate also modulates cardiac fibroblast functionality, in a NLRP3 independent manner, resulting in a phenotype related to cellular senescence. These effects of palmitate could be of importance for myocardial dysfunction in obese and diabetic patients.

Background— Chromogranin B (CgB) is a member of the granin protein family. Because CgB is often colocalized with chromogranin A (CgA), a recently discovered cardiac biomarker, we hypothesized that CgB is regulated during heart failure (HF) development. Methods and Results— CgB regulation was investigated in patients with chronic HF and in a post–myocardial infarction HF mouse model. Animals were phenotypically characterized by echocardiography and euthanized 1 week after myocardial infarction. CgB mRNA levels were 5.2-fold increased in the noninfarcted part of the left ventricle of HF animals compared with sham-operated animals ( P r =0.74, P =0.04) but not with CgA mRNA levels ( r =0.20, P =0.61). CgB protein levels were markedly increased in both the noninfarcted (110%) and the infarcted part of the left ventricle (70%) but unaltered in other tissues investigated. Myocardial CgB immunoreactivity was confined to cardiomyocytes. Norepinephrine, angiotensin II, and transforming growth factor-β increased CgB gene expression in cardiomyocytes. Circulating CgB levels were increased in HF animals (median levels in HF animals versus sham, 1.23 [interquartile range, 1.03 to 1.93] versus 0.98 [0.90 to 1.04] nmol/L; P =0.003) and in HF patients (HF patients versus control, 1.66 [1.48 to 1.85] versus 1.47 [1.39 to 1.58] nmol/L; P =0.007), with levels increasing in proportion to New York Heart Association functional class ( P =0.03 for trend). Circulating CgB levels were only modestly correlated with CgA ( r =0.31, P =0.009) and B-type natriuretic peptide levels ( r =0.27, P =0.014). Conclusions— CgB production is increased and regulated in proportion to disease severity in the left ventricle and circulation during HF development.

Background— Inflammation has been implicated in the pathogenesis of heart failure (HF), but knowledge about the production and role of inflammatory actors remains incomplete. On the basis of its role in vascular inflammation, vascular proliferation, and matrix degradation, we hypothesized a role for the chemokine CXCL16 in the pathogenesis of myocardial remodeling and development of HF. Methods and Results— Our main findings were (1) patients with chronic HF (n=188) had increased plasma levels of CXCL16, which correlated with disease severity. (2) Left ventricular tissue from patients with end-stage HF (n=8) showed enhanced CXCL16 levels compared with nonfailing left ventricular (n=6) as assessed by Western blotting. (3) In mice with postmyocardial infarction HF, expression of CXCL16, as assessed by real-time RT-PCR, was increased in the infarcted and the noninfarcted areas of left ventricular 3 and 7 days after coronary ligation, indicating early onset of CXCL16 production. Furthermore, mice exposed to aortic banding had enhanced CXCL16 expression in left ventricular, indicating that CXCL16 expression is not related to ischemia alone. (4) In vitro, CXCL16 promoted proliferation and impaired collagen synthesis in myocardial fibroblasts, and in cardiomyocytes and myocardial fibroblasts, CXCL16 increased matrix metalloproteinase activity, primarily reflecting increased matrix metalloproteinase-2 levels. (5) By using specific inhibitors, we showed that the effect of CXCL16 on fibroblasts involved activation of Jun N-terminal kinase. Conclusion— We show enhanced myocardial CXCL16 expression in experimental and clinical HF. The effect of CXCL16 on cardiomyocytes and fibroblasts suggests a role for CXCL16 in matrix remodeling and ultimately in the development of HF.

Several lines of evidence suggest that inflammatory processes mediated by cytokines are involved in the pathogenesis of heart failure (HF). However, the regulation of cytokine expression and the role of cytokines during HF development are not well understood. To address this issue, we have examined alterations in gene expression during HF progression by microarray technology in non-infarcted left ventricular (LV) murine tissue at various time points after myocardial infarction (MI). The highest number of regulated genes was found five days after MI. In total, we identified 14 regulated genes encoding cytokines with no previous association to HF. The strongest up-regulation was found for the chemokine fractalkine (CX3CL1). In human failing hearts we detected a 3-fold increase in CX3CL1 protein production, and both cardiomyocytes and fibrous tissue revealed immunoreactivity for CX3CL1 and its specific receptor CX3CR1. We also found that the circulating level of CX3CL1 was increased in patients with chronic HF in accordance with disease severity (1.6-fold in NYHA II, 2.2-fold in NYHA III and 2.9-fold in NYHA IV). In vitro experiments demonstrated that CX3CL1 production could be induced by inflammatory cytokines known to be highly expressed in HF. CX3CL1 itself induced the expression of markers of cardiac hypertrophy and protein phosphatases in neonatal cardiomyocytes. Given the increased CX3CL1 production in both an experimental HF model and in patients with chronic HF as well as its direct effects on cardiomyocytes, we suggest a role for CX3CL1 and its receptor CX3CR1 in the pathogenesis of HF.

Background Mitochondrial DNA (mtDNA) and possibly nuclear DNA (nDNA) are released as danger-associated molecular patterns during cardiac stress, and may activate several innate immune receptors. The purpose of this study was to investigate the regulation of these danger-associated molecular patterns during human heart failure (HF). Methods and Results Plasma levels of mtDNA and nDNA from HF patients (n = 84) were analyzed by reverse transcriptase-polymerase chain reaction and compared with controls (n = 72). Increased levels of mtDNA were found in New York Heart Association (NYHA) I-II and NYHA III-IV. There was evidence of increased nDNA in NYHA III-IV compared with controls and NYHA I-II. Kaplan-Meier analysis revealed higher mortality in patients with high nDNA levels, whereas high levels of mtDNA were associated with survival. Conclusions Plasma levels of mtDNA and nDNA are elevated in human HF associated with increased and decreased mortality, respectively. This study may suggest a rationale for exploring interventions within inflammatory signaling pathways activated by nucleic acids as novel targets in treatment of HF.

Finsen, Alexandra Vanessa, Per Reidar Woldbaek, Jian Li, Jiaping Wu, Torstein Lyberg, Theis Tonnessen, and Geir Christensen. Increased syndecan expression following myocardial infarction indicates a role in cardiac remodeling. Physiol Genomics 16: 301-308, 2004. First published November 18, 2003; 10.1152/physi-olgenomics. 00144.2002.—The purpose of this study was to identify essential genes involved in myocardial growth and remodeling following myocardial infarction (MI). Left ventricular noninfarcted tissues from six mice subjected to MI under general anesthesia and from six sham-operated mice were obtained 1 wk after primary surgery and analyzed by means of cDNA filter arrays. Out of a total of 1,176 genes, 641 were consistently expressed, twenty-three were upregulated and thirteen downregulated. Five genes were only expressed following MI. Syndecan-3, a transmembranous heparan sulfate proteoglycan, was found to be upregulated together with a transcriptional activator of syndecans, Wilms tumor protein 1 (WT-1). Northern blotting demonstrated a significant upregulation of syndecan-1, -2, -3, and -4, WT-1, fibronectin, and basic fibroblast growth factor (FGF) receptor 1. Furthermore, Western blot analysis showed statistically significant increases in protein levels for syndecan-3 and -4. In conclusion, we have identified a subset of genes with increased expression in noninfarcted left ventricular tissue following MI, including syndecans 1–4, WT-1, fibronectin, collagen 6A, and FGF receptor 1. Since the syndecans link the cytoskeleton to the extracellular matrix and function as required coreceptors for FGF, we suggest a role for the syndecans in cardiac remodeling following MI.

Objectives Experimental studies have shown involvement of Wnt signalling in heart failure (HF). We hypothesized that secreted frizzled-related protein 3 (sFRP3), a modulator of Wnt signalling, is related to the progression of HF. Design Circulating sFRP3 was measured in 153 HF patients and compared with 25 healthy controls. The association of sFRP3 with mortality was evaluated in 1202 patients (GISSI-HF trial). sFRP3 mRNA expression was assessed in failing human and murine left ventricles (LV), and cellular localization was determined after fractioning of myocardial tissue. In vitro studies were carried out in cardiac fibroblasts subjected to cyclic mechanical stretch. Results (i) Heart failure patients had significantly raised serum sFRP3 levels compared with controls, (ii) during a median follow-up of 47 months, 315 patients died in the GISSI-HF substudy. In univariable Cox regression, tertiles of baseline sFRP3 concentration were significantly associated with all-cause and cardiovascular mortality. After adjustment for demographic and clinical variables, but not for CRP and NT-proBNP, the associations with mortality remained significant for the third tertile (all-cause, HR 1.45, P = 0.011; cardiovascular, HR 1.66, P = 0.003), (iii) sFRP3 mRNA expression was increased in failing human LV, with a decline following LV assist device therapy. LV from post-MI mice showed an increased sFRP3 mRNA level, particularly in cardiac fibroblasts, and (iv) mechanical stretch enhanced sFRP3 expression and release in myocardial fibroblasts. Conclusion There is an association between increased sFRP3 expression and adverse outcome in HF, suggesting that the failing myocardium itself contributes to an increase in circulating sFRP3.

Aims N ucleotide-binding oligomerization domain- L ike R eceptor with a P yrin domain 3 (NLRP3) is considered necessary for initiating a profound sterile inflammatory response. NLRP3 forms multi-protein complexes with A poptosis-associated S peck-like protein containing a C aspase recruitment domain (ASC) and Caspase-1, which activate pro-interleukin-1β (IL-1β) and pro-IL-18. The role of NLRP3 in cardiac cells is not known. Thus, we investigated the expression and function of NLRP3 during myocardial ischaemia. Methods and results Myocardial infarction (MI) was induced in adult C57BL/6 mice and Wistar rats by ligation of the coronary artery. A marked increase in NLRP3, IL-1β, and IL-18 mRNA expression was found in the left ventricle after MI, primarily located to myocardial fibroblasts. In vitro studies in cells from adult mice showed that myocardial fibroblasts released IL-1β and IL-18 when primed with lipopolysaccharide and subsequently exposed to the danger signal adenosine triphosphate, a molecule released after tissue damage during MI. When hearts were isolated from NLRP3-deficient mice, perfused and subjected to global ischaemia and reperfusion, a marked improvement of cardiac function and reduction of hypoxic damage was found compared with wild-type hearts. This was not observed in ASC-deficient hearts, potentially reflecting a protective role of other ASC-dependent inflammasomes or inflammasome-independent effects of NLRP3. Conclusion This study shows that the NLRP3 inflammasome is up-regulated in myocardial fibroblasts post-MI, and may be a significant contributor to infarct size development during ischaemia–reperfusion.

Evidence from both experimental and clinical trials indicates that inflammatory mediators are of importance in the pathogenesis of chronic heart failure (HF) contributing to cardiac remodeling and peripheral vascular disturbances. Several studies have shown raised levels of inflammatory cytokines such as tumor necrosis factor (TNF)α, interleukin (IL)-1β and IL-6 in HF patients in plasma and circulating leukocytes, as well as in the failing myocardium itself. There is strong evidence that these mediators are involved in processes leading to cardiac remodeling such as hypertrophy, fibrosis and apoptosis. Some of these cytokines can also give useful prognostic information as reliable biomarkers in this disorder. In general, immunomodulating treatments have, with a few exceptions, been neutral or even harmful. However, the negative results of anti-TNF studies, for instance, do not necessarily argue against the ‘cytokine hypothesis’. These studies just underscore the challenges in developing treatment modalities that can modulate the cytokine network in HF patients and result in beneficial net effects. Future studies should identify the crucial actors and their mechanisms of action in the immunopathogenesis of chronic HF and, in particular, clarify the balance between adaptive and maladaptive effects of these molecules. Such studies are a prerequisite for the development of new treatment strategies that target inflammatory and immunopathogenic mechanisms in HF. In this review article, these issues are thoroughly discussed, and we also argue for the possibility of future therapeutic targets such as mediators in innate immunity, chemokines and mediators in matrix remodeling.

Background: CCL19 and CCL21, acting through CCR7, are termed homeostatic chemokines. Based on their role in concerting immunological responses and their proposed involvement in tissue remodeling, we hypothesized that these chemokines could play a pathogenic role in heart failure (HF). Methodology/Principal Findings: Our main findings were: (i) Serum levels of CCL19 and particularly CCL21 were markedly raised in patients with chronic HF (n = 150) as compared with healthy controls (n = 20). A CCL21 level above median was independently associated with all-cause mortality. (ii) In patients with HF following acute myocardial infarction (MI; n = 232), high versus low CCL21 levels 1 month post-MI were associated with cardiovascular mortality, even after adjustment for established risk factors. (iii). Explanted failing human LV tissue (n = 29) had markedly increased expression of CCL21 as compared with non-failing myocardium (n = 5). (iv) Our studies in CCR7−/− mice showed improved survival and attenuated increase in markers of myocardial dysfunction and wall stress in post-MI HF after 1 week, accompanied by increased myocardial expression of markers of regulatory T cells. (v) Six weeks post-MI, there was an increase in markers of myocardial dysfunction and wall stress in CCR7 deficient mice. Conclusions/Significance: High serum levels of CCL21 are independently associated with mortality in chronic and acute post-MI HF. Our findings in CCR7 deficient mice may suggest that CCL21 is not only a marker, but also a mediator of myocardial failure. However, while short term inhibition of CCR7 may be beneficial following MI, a total lack of CCR7 during long-term follow-up could be harmful. publishedVersion

Sustained pressure overload leads to compensatory myocardial hypertrophy and subsequent heart failure, a leading cause of morbidity and mortality. Further unraveling of the cellular processes involved is essential for development of new treatment strategies. We have investigated the hypothesis that the transmembrane Z-disc proteoglycan syndecan-4, a coreceptor for integrins, connecting extracellular matrix proteins to the cytoskeleton, is an important signal transducer in cardiomyocytes during development of concentric myocardial hypertrophy following pressure overload. Echocardiographic, histochemical and cardiomyocyte size measurements showed that syndecan-4 -/- mice did not develop concentric myocardial hypertrophy as found in wild-type mice, but rather left ventricular dilatation and dysfunction following pressure overload. Protein and gene expression analyses revealed diminished activation of the central, pro-hypertrophic calcineurinnuclear factor of activated T-cell (NFAT) signaling pathway. Cardiomyocytes from syndecan-4-/- -NFAT-luciferase reporter mice subjected to cyclic mechanical stretch, a hypertrophic stimulus, showed minimal activation of NFAT (1.6-fold) compared to 5.8-fold increase in NFAT-luciferase control cardiomyocytes. Accordingly, overexpression of syndecan-4 or introducing a cell-permeable membrane-targeted syndecan-4 polypeptide (gain of function) activated NFATc4 in vitro. Pulldown experiments demonstrated a direct intracellular syndecan-4-calcineurin interaction. This interaction and activation of NFAT were increased by dephosphorylation of serine 179 (pS179) in syndecan-4. During pressure overload, phosphorylation of syndecan-4 was decreased, and association between syndecan-4, calcineurin and its co-activator calmodulin increased. Moreover, calcineurin dephosphorylated pS179, indicating that calcineurin regulates its own binding and activation. Finally, patients with hypertrophic myocardium due to aortic stenosis had increased syndecan-4 levels with decreased pS179 which was associated with increased NFAT activation. In conclusion, our data show that syndecan-4 is essential for compensatory hypertrophy in the pressure overloaded heart. Specifically, syndecan-4 regulates stretch-induced activation of the calcineurin-NFAT pathway in cardiomyocytes. Thus, our data suggest that manipulation of syndecan-4 may provide an option for therapeutic modulation of calcineurin-NFAT signaling. © 2011 Finsen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Rationale Inflammatory mechanisms have been suggested to play a role in the development of heart failure (HF), but a role for chemokines is largely unknown. Based on their role in inflammation and matrix remodeling in other tissues, we hypothesized that CXCL13 and CXCR5 could be involved in cardiac remodeling during HF. Objective We sought to analyze the role of the chemokine CXCL13 and its receptor CXCR5 in cardiac pathophysiology leading to HF. Methods and Results Mice harboring a systemic knockout of the CXCR5 (CXCR5−/−) displayed increased mortality during a follow-up of 80 days after aortic banding (AB). Following three weeks of AB, CXCR5−/− developed significant left ventricular (LV) dilatation compared to wild type (WT) mice. Microarray analysis revealed altered expression of several small leucine-rich proteoglycans (SLRPs) that bind to collagen and modulate fibril assembly. Protein levels of fibromodulin, decorin and lumican (all SLRPs) were significantly reduced in AB CXCR5−/− compared to AB WT mice. Electron microscopy revealed loosely packed extracellular matrix with individual collagen fibers and small networks of proteoglycans in AB CXCR5−/− mice. Addition of CXCL13 to cultured cardiac fibroblasts enhanced the expression of SLRPs. In patients with HF, we observed increased myocardial levels of CXCR5 and SLRPs, which was reversed following LV assist device treatment. Conclusions Lack of CXCR5 leads to LV dilatation and increased mortality during pressure overload, possibly via lack of an increase in SLRPs. This study demonstrates a critical role of the chemokine CXCL13 and CXCR5 in survival and maintaining of cardiac structure upon pressure overload, by regulating proteoglycans essential for correct collagen assembly.

In ischemic congestive heart failure (CHF), anemia is associated with poor prognosis. Whether anemia develops in nonischemic CHF is uncertain. The hematopoietic inhibitors TNF-α and nitric oxide (NO) are activated in ischemic CHF. We examined whether mice with ischemic or nonischemic CHF develop anemia and whether TNF-α and NO are involved. We studied mice ( n = 7–9 per group) with CHF either due to myocardial infarction (MI) or to overexpression of the Ca2+-binding protein calsequestrin (CSQ) or to induced cardiac disruption of the sarcoplasmic reticulum Ca2+-ATPase 2 gene (SERCA2 KO). Hematopoiesis was analyzed by colony formation of CD34+bone marrow cells. Hemoglobin concentration was 14.0 ± 0.4 g/dl (mean ± SD) in controls, while it was decreased to 10.1 ± 0.4, 9.7 ± 0.4, and 9.6 ± 0.3 g/dl in MI, CSQ, and SERCA2 KO, respectively ( P < 0.05). Colony numbers per 100,000 CD34+cells in the three CHF groups were reduced to 33 ± 3 (MI), 34 ± 3 (CSQ), and 39 ± 3 (SERCA2 KO) compared with 68 ± 4 in controls ( P < 0.05). Plasma TNF-α nearly doubled in MI, and addition of anti-TNF-α antibody normalized colony formation. Inhibition of colony formation was completely abolished with blockade of endothelial NO synthase in CSQ and SERCA2 KO, but not in MI. In conclusion, the mechanism of anemia in CHF depends on the etiology of cardiac disease; whereas TNF-α impairs hematopoiesis in CHF following MI, NO inhibits blood cell formation in nonischemic murine CHF.

Sarco(endo)plasmic reticulum calcium ATPases (SERCA) are cellular pumps that transport Ca(2+) into the sarcoplasmic reticulum (SR). Serca2 is the most widely expressed gene family member. The very early embryonic lethality of Serca2(null) mouse embryos has precluded further evaluation of loss of Serca2 function in the context of organ physiology. We have generated mice carrying a conditional Serca2(flox) allele which allows disruption of the Serca2 gene in an organ-specific and/or inducible manner. The model was tested by mating Serca2(flox) mice with MLC-2v(wt/Cre) mice and with alphaMHC-Cre transgenic mice. In heterozygous Serca2(wt/flox)MLC-2v(wt/Cre) mice, the expression of SERCA2a and SERCA2b proteins were reduced in the heart and slow skeletal muscle, in accordance with the expression pattern of the MLC-2v gene. In Serca2(flox/flox) Tg(alphaMHC-Cre) embryos with early homozygous cardiac Serca2 disruption, normal embryonic development and yolk sac circulation was maintained up to at least embryonic stage E10.5. The Serca2(flox) mouse is the first murine conditional gene disruption model for the SERCA family of Ca(2+) ATPases, and should be a powerful tool for investigating specific physiological roles of SERCA2 function in a range of tissues and organs in vivo both in adult and embryonic stages.

The sarco(endo)plasmic reticulum calcium ATPase 2 (SERCA2) transports Ca(2+) from cytosol into the sarcoplasmic reticulum (SR) of cardiomyocytes, thereby maintaining the store of releasable Ca(2+) necessary for contraction. Reduced SERCA function has been linked to heart failure, and loss of SERCA2 in the adult mammalian heart would be expected to cause immediate severe myocardial contractile dysfunction and death. We investigated heart function in adult mice with an inducible cardiomyocyte-specific excision of the Atp2a2 (Serca2) gene (SERCA2 KO). Seven weeks after induction of Serca2 gene excision, the mice displayed a substantial reduction in diastolic function with a 5-fold increase in the time constant of isovolumetric pressure decay (tau). However, already at 4 weeks following gene excision less than 5% SERCA2 protein was found in myocardial tissue. Surprisingly, heart function was only moderately impaired at this time point. Tissue Doppler imaging showed slightly reduced peak systolic tissue velocity and a less than 2-fold increase in tau was observed. The SR Ca(2+) content was dramatically reduced in cardiomyocytes from 4-week SERCA2 KO mice, and Ca(2+) transients were predominantly generated by enhanced Ca(2+) flux through L-type Ca(2+) channels and the Na(+)-Ca(2+) exchanger. Moreover, equivalent increases in cytosolic [Ca(2+)] in control and SERCA2 KO myocytes induced greater cell shortening in SERCA2 KO, suggesting enhanced myofilament responsiveness. Our data demonstrate that SR-independent Ca(2+) transport mechanisms temporarily can prevent major cardiac dysfunction despite a major reduction of SERCA2 in cardiomyocytes.

Our aim was to establish parameters describing systolic and diastolic function in mice after myocardial infarction (MI) that distinguish MI with pulmonary congestion from MI without congestion. Echocardiography, left ventricular (LV) catheterization, and infarct size measurements were performed on days 3, 5, 7, and 14 after ligation of the left coronary artery in C57BL/6 mice. Sham-operated mice were used as controls (Sham). MI mice with lung weight normalized to tibial length >125% of the average in the corresponding Sham group were considered to have pulmonary congestion (MIchf). MI mice with a smaller increase were called MI nonfailing (MInf). An infarct >40% of total LV circumference measured in two-dimensional long axis distinguished MIchf from MInf on both an average and an individual basis. Mean maximum rate of rise of LV pressure, LV fractional shortening, and posterior wall shortening velocity were significantly lower in MIchf compared with Sham at all time points and to MInf at 7 days. The diastolic parameters mitral flow deceleration velocity, LV end-diastolic pressure, and maximum rate of decline in LV pressure (LVdP/d tmin) discriminated the MIchf groups from Sham at all time points. Mitral flow deceleration velocity and LVdP/d tmin separated MIchf from MInf at 7 days. In addition to distinguishing all the groups on an average basis, left atrial diameter distinguished all MIchf animals from Sham and MInf. In conclusion, significantly increased left atrial diameter and infarct size >40% of total LV circumference may serve as major criteria for heart failure with pulmonary congestion after MI in mice.

Purpose: Immune activation plays a nodal role in the pathogenesis of heart failure (HF) and Toll-like receptor 9 (TLR9) signaling significantly impacts the development of systolic HF. However, the consequence of altered TLR9 activity in diastolic HF is unknown. Methods: We engaged in a 3-generation breeding strategy using αMHC-MerCreMer Serca2a flox/flox mice crossed with TLR9-/- mice to generate several comparable mouse lines. Employing these various genetic mouse lines we addressed the importance of TLR9 on progression of diastolic HF, the latter by temporally restricted depletion of cardiac myocyte SERCA2a. Mice were allocated to two substudies; 1) a 12 week survival study registering spontaneous death or signs of severe morbidity according to pre-specified criteria (leading to euthanasia) and 2) a study examining mice at baseline, 3 weeks and 6 weeks by MRI and echocardiography. After imaging at 6 weeks mice were euthanized and tissue and blood were harvested. Results: All mice depleted of cardiomyocyte SERCA2a, but none with the SERCA2a gene intact, reached our pre-specified end-parameter within 73 days. The presence of TLR9 in this model of diastolic HF led to significant improvement in survival with a median life expectancy of 62.5 days as compared to 58 days when the TLR9 gene was deleted (p=0.007). In accordance with these findings serial imaging demonstrated an earlier onset of left ventricular restrictive filling abnormalities in the HF group depleted of TLR9. Significantly larger left atria, lower end-systolic and end-diastolic left ventricular volumes and lower stroke volumes could be detected. Conclusions: The presence of TLR9 augments the development of diastolic HF induced by SERCA2a depletion. This observation may pave the road for novel diastolic HF treatment strategies. ![Figure][1] Survival in days after HF induction [1]: pending:yes

Accumulation of DNA damage due to impaired base excision repair (BER) may play a role in the pathogenesis of myocardial failure. The DNA glycosylase Neil3 is known to carry out BER during oxidative stress induced DNA damage, but is also thought to have a role in stem cell biology. Based on this, we hypothesized that Neil3 plays a role in myocardial remodeling during heart failure (HF). qPCR showed an increased expression of Neil3 in myocardial biopsies from patients with severe HF, and that the Neil3 expression significantly decreased following improvement of myocardial function following treatment with left ventricular assist device. In a murine model of myocardial infarction (MI) we found a strong upregulation of Neil3 on day 3, 7 and 21 after MI. At day 7, myocardial cell isolation revealed a low Neil3 expression in cardiomyocytes, while a 2.5- and a 4.5-fold increased expression was found in endothelial cells and fibroblasts, respectively, following MI compared to sham. In leukocytes an equally high expression of Neil3 was observed in both sham and MI. Using Neil3 knock-out (KO) mice we found a significantly higher mortality in KO compared to wild type (WT) mice after MI ( 73% vs. 40%), despite smaller infarct sizes in the Neil3 KO, as determined by MRI. The first week post MI, all animals died from cardiac rupture. Zymography on protein extracts from the infarcted tissue revealed significantly increased levels of MMP2 in the Neil3 KO, 3 days after MI. Immunohistochemistry (IHC), at the same time point, showed similar numbers of inflammatory cells measured by the presence of Ly6G+ neutrophils, Mac2+ monocytes/macrophages and CD45+ leukocytes. At day 6 a significantly higher number of Ki67+ proliferating cells were observed in the Neil3 KO infarcted areas. In accordance with this, primary cardiac fibroblasts lacking Neil3 showed a higher proliferation rate, measured both by BrdU and scrape wound assay. Furthermore, we found a significantly higher mRNA expression of both the myofibroblast marker α-SMA, and collagen type III in cardiac fibroblasts isolated from Neil3 KO mice 7 days post MI, indicating increased levels of activated fibroblasts and altered fibroblast function. IHC 6 days post MI showed the same tendency towards more α-SMA-positive fibroblasts and more collagen by Sirius red in the Neil3 KO infarcts, however these differences were not statistically significant. In conclusion, our data suggest that after MI, Neil3 plays a role in regulating fibroblast proliferation and function, affecting wound healing and extracellular matrix composition and quality, altogether reducing mortality.