Ventricular Tissue Samples Research Articles

Myocardial inflammation is associated with impaired mitochondrial oxidative capacity in ischaemic cardiomyopathy.

Myocardial inflammation and impaired mitochondrial oxidative capacity are hallmarks of heart failure (HF) pathophysiology. The extent of myocardial inflammation in patients suffering from ischaemic cardiomyopathy (ICM) or dilated cardiomyopathy (DCM) and its association with mitochondrial energy metabolism are unknown. We aimed at establishing a relevant role of cardiac inflammation in the impairment of mitochondrial energy production in advanced ischaemic and non-ischaemic HF. We included 81 patients with stage D HF (ICM, n=44; DCM, n=37) undergoing left ventricular assist device implantation (n=59) or heart transplantation (n=22) and obtained left ventricular tissue samples during open heart surgery. We quantified mitochondrial oxidative capacity, citrate synthase activity (CSA) and fibrosis and lymphocytic infiltration. We considered infiltration of >14 CD3+ cells/mm2 relevant inflammation. Patients with ICM or DCM did not differ regarding age (61.5±5.7 vs. 56.5±12.7years, P=0.164), sex (86% vs. 84% male, P=0.725), type 2 diabetes mellitus (34% vs. 18%, P=0.126) or chronic kidney disease (8% vs. 11%, P=0.994). ICM exhibited oxidative capacity reduced by 23% compared to DCM (108.6±41.4 vs. 141.9±59.9pmol/(s*mg), P=0.006). Maximum production of reactive oxygen species was not significantly different between ICM and DCM (0.59±0.28 vs. 0.69±0.36pmol/(s*ml), P=0.196). Mitochondrial content, detected by CSA, was lower in ICM (359.6±164.1 vs. 503.0±198.5nmol/min/mg protein, P=0.002). Notably, relevant inflammation was more common in ICM (27% vs. 6%, P=0.024), and the absolute number of infiltrating leucocytes correlated with lower oxidative capacity (r=-0.296, P=0.019). Fibrosis was more prevalent in ICM (20.9±21.2 vs. 7.2±5.6% of area, P=0.002), but not associated with oxidative capacity (r=-0.13, P=0.327). More than every fourth ICM patient with advanced HF displays myocardial inflammation in the range of inflammatory cardiomyopathy associated with reduced mitochondrial oxidative capacity. Future studies may evaluate inflammation in ICM at earlier stages in standardised fashion to explore the therapeutic potential of immunosuppression to influence trajectories of HF in ICM.

The myocardium of human dilated nonischemic cardiomyopathy harbors unique tissue-resident lymphocyte subsets

Abstract Purpose The etiology of dilated nonischemic cardiomyopathy (NICM) is often unclear, but thought to be multifactorial, involving an interplay between genetic factors and direct myocardial damage caused by infection, autoimmunity, or toxins. Tissue-resident lymphocytes are non-circulating immune cells which are retained within various tissues and play integral roles in maintaining homeostasis, responding to infection, regulating inflammation, and mediating tissue repair. Despite their likely involvement in various cardiac pathologies, the presence of tissue-resident lymphocytes in the human heart has not yet been well-characterized, neither in health nor in disease. Methods Human left ventricular tissue and paired peripheral blood samples were obtained from six adult organ donors with normal heart function who died of noncardiac causes and from the explants of six adult patients with end-stage nonischemic cardiomyopathy. Heart tissue was processed using mechanical and enzymatic digestion. Mononuclear cells were isolated from both heart and paired blood by density centrifugation. Isolated mononuclear cells were surface stained with an antibody cocktail and interrogated using multidimensional flow cytometry. Results The adult human heart contains a diverse immune cell population (Fig 1A). Both healthy and NICM cardiac lymphocytes express distinct phenotypic profiles which resemble those of resident lymphocytes found across various tissues, distinct from those isolated from paired blood. Frequencies of effector memory T (Tem) cells (CD45RA- CCR7-) were higher in heart than in blood among both CD4+ and CD8+ T-cells. In heart compared to blood, significantly more Tem expressed CD69, a canonical marker of tissue-resident memory T (Trm) cells (Fig 1B). Cardiac Trm, especially CD8+ Trm, expressed classical Trm signature markers including integrins CD103 (epithelial binding) and CD49a (collagen binding). NICM Trm expressed more CD49a than healthy heart Trm (Fig 1C). NK-cells also demonstrated higher CD69 expression (trNK) in heart than in blood, with associated expression of CD103, CD49a, and CXCR6 (Fig 1D). Compared to healthy hearts, NICM trNK were predominantly CD56bright/CD16- (Fig 1E and 1F) and expressed less CD57 (Fig 1D), overall indicating a less mature state with lower cytotoxic potential. Confocal imaging of cardiac tissue revealed CD69-expressing tissue-resident lymphocytes within the cardiac interstitium. Conclusions The adult human heart contains diverse populations of lymphocytes which express canonical markers of tissue residency, similar to lymphocytes resident in other tissues. Compared to healthy hearts, NICM hearts have more CD49a+ Trm, perhaps related to greater fibrosis, and also harbor a distinct subset of immature tissue-resident NK cells which may shed light on pathophysiology. Future work is focused on better exploring the functional phenotypes of these resident cells and better localizing them within the myocardium.FA - Figure 1

Beyond the matrix: exploring tenascin-c (TNC) as a crucial player in diabetic cardiovascular dysfunction

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Burgermeister Found Wien-Project number : 21001 Introduction Diabetic cardiomyopathy (CMP) is a complex manifestation of diabetes-linked cardiac and vascular dysfunction, but the molecular pathogenesis is still largely unknown. Notably the expression of Tenascin-C (TNC), an extracellular matrix glycoprotein, contributes to the development of cardiovascular diseases and diabetic patients have elevated serum TNC levels. However, the role and mechanism of TNC to diabetic CMP progression remains elusive. Purpose Here, we longitudinally evaluated functional and molecular role of TNC in CMP-induced cardiovascular dysfunction in a murine model. In addition, we used RNA sequencing with cardiac tissues from non-diabetic and diabetic wild type (wt) and TNC KO mice for further investigation. Methods Diabetes was induced in adult male mice, wt (AJ) and TNC-KO strains, by repeated Streptozotocin (STZ) injections (50 mg/kg). Echocardiography, myography, hemodynamics, histology, molecular and cellular analyses were performed to assess the role of TNC in diabetes-associated CV complications. Additionally, RNA-sequencing was analyzed in left ventricular (LV) tissue samples from diabetic and non-diabetic wt and TNC-KO mice providing further molecular insights. Results TNC KO mice showed preserved LV ejection fraction, and endothelium-dependent relaxation (p<0.05 and p<0.001, respectively), reduced cardiac fibrosis and distinctive coronary vessel characteristics, implying enhanced LV perfusion. In addition, the plasma levels of oxidative stress marker malondialdehyde was significantly alleviated in TNC KO diabetic mice in comparison to diabetic wt mice. Cardiomyocytes from diabetic wt mice exhibited stiffness (Fpassive and Factive), which was corrected to control level in cardiomyocytes from TNC KO diabetic hearts. Further, ex vivo isolated heart experiments demonstrated that the administration of recombinant human TNC (80 ng/ml) resulted in a significant reduction in LV hemodynamics (p<0.01) and myocardial energetics (ATP levels, p<0.01). Analysis of RNA sequencing data demonstrated that decreased expression of matrix remodeling genes Serpin1 and Ccn1 links to alleviation of fibrosis in TNC KO diabetic heart. Furthermore, Gene Ontology (GO) enrichment analysis shed light on mitochondrial changes in diabetic hearts. Our pathway analysis also identified that Hsp90aa1 as a crucial gene associated with preprotein import into the mitochondrial membrane, may suggest potential mitochondrial dysfunction in diabetic hearts. Conclusion In conclusion, lack of TNC improved long-term cardiac function in diabetes by reducing cardiac fibrosis, vascular dysfunction, oxidative stress and cardiac energetics. Thus, inhibition of TNC may prevent the diabetic heart from extensive fibrosis and energetics dysfunction that accelerates the development of HF, providing a potential targeted therapy.

Targeting NLRP3 signaling reduces myocarditis-induced arrhythmogenesis and cardiac remodeling

BackgroundMyocarditis substantially increases the risk of ventricular arrhythmia. Approximately 30% of all ventricular arrhythmia cases in patients with myocarditis originate from the right ventricular outflow tract (RVOT). However, the role of NLRP3 signaling in RVOT arrhythmogenesis remains unclear.MethodsRats with myosin peptide–induced myocarditis (experimental group) were treated with an NLRP3 inhibitor (MCC950; 10 mg/kg, daily for 14 days) or left untreated. Then, they were subjected to electrocardiography and echocardiography. Ventricular tissue samples were collected from each rat’s RVOT, right ventricular apex (RVA), and left ventricle (LV) and examined through conventional microelectrode and histopathologic analyses. In addition, whole-cell patch-clamp recording, confocal fluorescence microscopy, and Western blotting were performed to evaluate ionic currents, intracellular Ca2+ transients, and Ca2+-modulated protein expression in individual myocytes isolated from the RVOTs.ResultsThe LV ejection fraction was lower and premature ventricular contraction frequency was higher in the experimental group than in the control group (rats not exposed to myosin peptide). Myocarditis increased the infiltration of inflammatory cells into cardiac tissue and upregulated the expression of NLRP3; these observations were more prominent in the RVOT and RVA than in the LV. Furthermore, experimental rats treated with MCC950 (treatment group) improved their LV ejection fraction and reduced the frequency of premature ventricular contraction. Histopathological analysis revealed higher incidence of abnormal automaticity and pacing-induced ventricular tachycardia in the RVOTs of the experimental group than in those of the control and treatment groups. However, the incidences of these conditions in the RVA and LV were similar across the groups. The RVOT myocytes of the experimental group exhibited lower Ca2+ levels in the sarcoplasmic reticulum, smaller intracellular Ca2+ transients, lower L-type Ca2+ currents, larger late Na+ currents, larger Na+–Ca2+ exchanger currents, higher reactive oxygen species levels, and higher Ca2+/calmodulin-dependent protein kinase II levels than did those of the control and treatment groups.ConclusionMyocarditis may increase the rate of RVOT arrhythmogenesis, possibly through electrical and structural remodeling. These changes may be mitigated by inhibiting NLRP3 signaling.

A transcriptomic analysis of correlation between mitochondrial function and energy metabolism remodeling in mice with myocardial fibrosis following myocardial infarction

To investigate the changes of mitochondrial respiratory function during myocardial fibrosis in mice with myocardial infarction (MI) and its correlation with the increase of glycolytic flux. Forty C57BL/6N mice were randomized into two equal groups to receive sham operation or ligation of the left anterior descending coronary artery to induce acute MI. At 28 days after the operation, 5 mice from each group were euthanized and left ventricular tissue samples were collected for transcriptomic sequencing. FPKM method was used to calculate gene expression levels to identify the differentially expressed genes (DEGs) in MI mice, which were analyzed using GO and KEGG databases to determine the pathways affecting the disease process. Heat maps were drawn to show the differential expressions of the pathways and the related genes in the enrichment analysis. In primary cultures of neonatal mouse cardiac fibroblasts (CFs), the changes in mitochondrial respiration and glycolysis levels in response to treatment with the pro-fibrotic agonist TGF-β1 were analyzed using Seahorse experiment. The mouse models of MI showed significantly increased diastolic and systolic left ventricular diameter (P < 0.05) and decreased left ventricular ejection fraction (P < 0.0001). A total of 124 up-regulated and 106 down-regulated DEGs were identified in the myocardial tissues of MI mice, and GO and KEGG enrichment analysis showed that these DEGs were significantly enriched in fatty acid metabolism, organelles and other metabolic pathways and in the mitochondria. Heat maps revealed fatty acid beta oxidation, mitochondrial dysfunction and increased glycolysis levels in MI mice. In the primary culture of CFs, treatment with TGF-β1 significantly reduced the basal and maximum respiratory levels and increased the basal and maximum glycolysis levels (P < 0.0001). During myocardial fibrosis, energy metabolism remodeling occurs in the CFs, manifested by lowered mitochondrial function and increased energy generation through glycolysis.

Abstract P3123: Sex-related Differences In Cardiac Biomarkers And Ventricular Fibrosis In A Minipig Model Of Hypertrophic Cardiomyopathy

Hypertrophic Cardiomyopathy (HCM) is characterized by ventricular hypertrophy and stiffness resulting in part from interstitial fibrosis. While associated with autosomal dominant mutations in sarcomere proteins, it has been reported that males and females may have different clinical presentations and disease courses. We have previously shown that minipigs carrying the R403Q mutation in MYH7 demonstrate a phenotype that recapitulates human non-obstructive HCM with increased hypertrophy, markers of cardiac stress, and diastolic dysfunction. Under the hypothesis that sex-dependent differences in cardiac physiology can independently modulate disease phenotype, we evaluated ventricular fibrosis and cardiac biomarkers in both male and female mutant pigs. Purpose-bred Yucatan R403Q MYH7 heterozygous mutant minipigs of either sex (M: n=8, F: n=6) and wild-type littermates (M: n=4, F: n=14) were used; for the purposes of these comparisons, cloned male pigs were also included (R403Q: 8). Males were castrated, and all pigs received soybean-based feed. Circulating levels of both cardiac troponin T (cTnT) and brain natriuretic peptide (BNP), markers of cardiac injury and stress, were assessed. Interstitial fibrosis was assessed in left ventricular free wall (LV-FW) and apical tissue samples utilizing picrosirius red staining and semi-automated quantification (HALO). Male HCM pigs died earlier (&lt;9M of age, P&lt;0.05) and had higher serum biomarkers than females (cTnT levels (M: 143±17 vs F: 44±7 ng/L, P&lt;0.05; BNP 1536±335 vs 4881±414 ng/L, P&lt;0.05) Consistent with the higher cTnT levels, male HCM pigs had greater fibrosis areas than females in both LV-FW (11±3% vs 7±2%, P&lt;0.05) and apical regions (13±1% vs. 8±2%, respectively, P&lt;0.05). All HCM pigs had significantly increased fibrotic area vs WT controls (R403Q: 10±3% vs WT: 3±2%, P&lt;0.05). Our findings demonstrate that despite a common genotype, female and male carriers of the HCM pathogenic R403Q mutation present different disease phenotypes, with castrated males showing greater fibrotic burden. Future studies to understand the mechanistic drivers of these differences should not only help elucidate the role of sex in HCM disease progression, but also the development of sex-informed therapies.

Meta-Analysis of Mechano-Sensitive Ion Channels in Human Hearts: Chamber- and Disease-Preferential mRNA Expression.

The cardiac cell mechanical environment changes on a beat-by-beat basis as well as in the course of various cardiac diseases. Cells sense and respond to mechanical cues via specialized mechano-sensors initiating adaptive signaling cascades. With the aim of revealing new candidates underlying mechano-transduction relevant to cardiac diseases, we investigated mechano-sensitive ion channels (MSC) in human hearts for their chamber- and disease-preferential mRNA expression. Based on a meta-analysis of RNA sequencing studies, we compared the mRNA expression levels of MSC in human atrial and ventricular tissue samples from transplant donor hearts (no cardiac disease), and from patients in sinus rhythm (underlying diseases: heart failure, coronary artery disease, heart valve disease) or with atrial fibrillation. Our results suggest that a number of MSC genes are expressed chamber preferentially, e.g., CHRNE in the atria (compared to the ventricles), TRPV4 in the right atrium (compared to the left atrium), CACNA1B and KCNMB1 in the left atrium (compared to the right atrium), as well as KCNK2 and KCNJ2 in ventricles (compared to the atria). Furthermore, 15 MSC genes are differentially expressed in cardiac disease, out of which SCN9A (lower expressed in heart failure compared to donor tissue) and KCNQ5 (lower expressed in atrial fibrillation compared to sinus rhythm) show a more than twofold difference, indicative of possible functional relevance. Thus, we provide an overview of cardiac MSC mRNA expression in the four cardiac chambers from patients with different cardiac diseases. We suggest that the observed differences in MSC mRNA expression may identify candidates involved in altered mechano-transduction in the respective diseases.

The effect of cardioplegic supplementation with sildenafil on cardiac energetics in a piglet model of cardiopulmonary bypass and cardioplegic arrest with warm or cold cardioplegia.

Cardioplegic cardioprotection strategies used during paediatric open-heart surgery remain suboptimal. Sildenafil, a phosphodiesterase 5 (PDE-5) inhibitor, has been shown to be cardioprotective against ischemia/reperfusion injury in a variety of experimental models and this study therefore tested the efficacy of supplementation of cardioplegia with sildenafil in a piglet model of cardiopulmonary bypass and arrest, using both cold and warm cardioplegia protocols. Piglets were anaesthetized and placed on coronary pulmonary bypass (CPB), the aorta cross-clamped and the hearts arrested for 60 min with cardioplegia with or without sildenafil (10 nM). Twenty minutes after removal of cross clamp (reperfusion), attempts were made to wean the pigs from CPB. Termination was carried out after 60 min reperfusion. Throughout the protocol blood and left ventricular tissue samples were taken for analysis of selected metabolites (using HPLC) and troponin I. In both the cold and warm cardioplegia protocols there was evidence that sildenafil supplementation resulted in faster recovery of ATP levels, improved energy charge (a measure of metabolic flux) and altered release of hypoxanthine and inosine, two purine catabolites. There was no effect on troponin release within the studied short timeframe. In conclusion, sildenafil supplementation of cardioplegia resulted in improved cardiac energetics in a translational animal model of paediatric CPB surgery.

ANTIFIBROTIC POTENTIAL OF ANGIOTENSIN (1-7) IN HEMODYNAMICALLY OVERLOADED RAT HEART

Objective: Heart failure is a clinical syndrome characterized by cardiac dysfunction due to structural abnormalities of the myocardium that results in the inability of the heart to eject sufficient volume of the blood to the circulation. HF is a major cause of morbidity and mortality in the world. The ECM is a highly dynamic structure controlling the proper functioning of heart muscle. ECM remodelling with enhanced deposition of collagen due to hemodynamic overload impairs cardiomyocyte adhesion and electrical coupling that contributes to cardiac mechanical dysfunction and arrhythmias. Design and method: We aimed to explore ECM and connexin-43 (Cx43) signalling pathways in hemodynamically overloaded rat heart as well as the possible implication of Ang(1-7) to prevent/attenuate adverse myocardial remodelling. Males, 8 weeks-old, normotensive Hannover Spraque-Dawley rats (HSD), hypertensive Ren-2 transgenic rats (TGR) and Ang(1-7)-transgenic rats (TGR(A1-7)) underwent aortocaval fistula (ACF) to produce volume overload. 5 weeks later, were right and left ventricular tissue samples used for biometric, biochemical, and proteomic analyzes. Results: Cardiac hypertrophy in response to volume overload was significantly less pronounced in TGR(A1-7) comparing to HSD rats. Moreover, a marker of fibrosis, hydroxyproline, was increased in both ventricles of volume overloaded TGR while reduced in Ang(1-7) right heart ventricle. Protein level and activity of MMP-2 were reduced in both ventricles of volume overloaded TGR/TGR(A1-7) compared to HSD. SMAD2/3 protein levels were decreased in the right ventricle of TGR(A1-7) compared to HSD/TGR in response to volume overload. In parallel, Cx43 and pCx43 implicated in electrical coupling were increased in TGR(A1-7) versus HSD/TGR. PKC↑ and PKC↗ showed a decrease in the TGR group compared to HSD and a decrease in the TGR(A1-7) ACF group compared to HSD and TGR after ACF in both chambers. Conclusions: It can be concluded that Ang(1-7) exhibits cardioprotective and anti-fibrotic potential in condition of cardiac volume overload. If we look at the results comprehensively, it can be concluded that Ang(1-7) exhibits cardioprotective and antifibrotic potential in condition of cardiac volume overload. Supported by grants VEGA 2/0006/23, APVV 21-0410.

Genome-wide profiling of miRNA-gene regulatory networks in mouse postnatal heart development-implications for cardiac regeneration.

After birth, mammalian cardiomyocytes substantially lose proliferative capacity with a concomitant switch from glycolytic to oxidative mitochondrial energy metabolism. Micro-RNAs (miRNAs) regulate gene expression and thus control various cellular processes. Their roles in the postnatal loss of cardiac regeneration are however still largely unclear. Here, we aimed to identify miRNA-gene regulatory networks in the neonatal heart to uncover role of miRNAs in regulation of cell cycle and metabolism. We performed global miRNA expression profiling using total RNA extracted from mouse ventricular tissue samples collected on postnatal day 1 (P01), P04, P09, and P23. We used the miRWalk database to predict the potential target genes of differentially expressed miRNAs and our previously published mRNA transcriptomics data to identify verified target genes that showed a concomitant differential expression in the neonatal heart. We then analyzed the biological functions of the identified miRNA-gene regulatory networks using enriched Gene Ontology (GO) and KEGG pathway analyses. Altogether 46 miRNAs were differentially expressed in the distinct stages of neonatal heart development. For twenty miRNAs, up- or downregulation took place within the first 9 postnatal days thus correlating temporally with the loss of cardiac regeneration. Importantly, for several miRNAs, including miR-150-5p, miR-484, and miR-210-3p there are no previous reports about their role in cardiac development or disease. The miRNA-gene regulatory networks of upregulated miRNAs negatively regulated biological processes and KEGG pathways related to cell proliferation, while downregulated miRNAs positively regulated biological processes and KEGG pathways associated with activation of mitochondrial metabolism and developmental hypertrophic growth. This study reports miRNAs and miRNA-gene regulatory networks with no previously described role in cardiac development or disease. These findings may help in elucidating regulatory mechanism of cardiac regeneration and in the development of regenerative therapies.

Subtyping based on immune cell fractions reveal heterogeneity of cardiac fibrosis in end-stage heart failure.

A central issue hindering the development of effective anti-fibrosis drugs for heart failure is the unclear interrelationship between fibrosis and the immune cells. This study aims at providing precise subtyping of heart failure based on immune cell fractions, elaborating their differences in fibrotic mechanisms, and proposing a biomarker panel for evaluating intrinsic features of patients' physiological statuses through subtype classification, thereby promoting the precision medicine for cardiac fibrosis. We inferred immune cell type abundance of the ventricular samples by a computational method(CIBERSORTx) based on ventricular tissue samples from 103 patients with heart failure, and applied K-means clustering to divide patients into two subtypes based on their immune cell type abundance. We also designed a novel analytic strategy: Large-Scale Functional Score and Association Analysis (LAFSAA), to study fibrotic mechanisms in the two subtypes. Two subtypes of immune cell fractions: pro-inflammatory and pro-remodeling subtypes, were identified. LAFSAA identified 11 subtype-specific pro-fibrotic functional gene sets as the basis for personalised targeted treatments. Based on feature selection, a 30-gene biomarker panel (ImmunCard30) established for diagnosing patient subtypes achieved high classification performance, with the area under the receiver operator characteristic curve corresponding to 0.954 and 0.803 for the discovery and validation sets, respectively. Patients with the two subtypes of cardiac immune cell fractions were likely having different fibrotic mechanisms. Patients' subtypes can be predicted based on the ImmunCard30 biomarker panel. We envision that our unique stratification strategy revealed in this study will unravel advance diagnostic techniques for personalised anti-fibrotic therapy.

Ventricular stabilization with a customized decellularized cardiac ECM-based scaffold after myocardial infarction alters gene expression in a rodent LAD-ligation model.

Objectives: Decellularized extracellular matrix (dECM) is increasingly used in a wide range of regenerative medicine applications and may also offer the potential to support injured myocardium. Here, we evaluated the myocardial gene expression pattern after myocardial infarction (MI) in a standardized rodent LAD-ligation model with and without ventricular stabilization with a customized, cardiac dECM-based scaffold (cdECM). Methods: MI was induced in male Wistar rats by standard LAD-ligation and confirmed 14 days post-intervention by echocardiographic parameters (FAS<40%). Cardiac ECM from donor rats was used to generate individual cdECM-scaffolds (tissue engineered myocardial sleeve, TEMS), which were epicardially implanted after confirmed MI for ventricular stabilization. After 4 and 8 weeks heart function was assessed by echocardiography, rats were sacrificed and explanted hearts were analyzed. In addition to histological analysis, standardized anterior left ventricular wall myocardial tissue samples were assessed by quantitative real-time PCR evaluating the specific gene expression pattern for immunomodulatory (IL-10, TGFBR2, TNFα), pro-angiogenic (VEGFA, FGF2, PGF, PDGFB), pro-survival (HGF, SDF1, IGF1, AKT1), remodeling-associated (TIMP1, MMP2, MMP9) and infarction-specific (NPPA, NPPB) markers. Results: Ventricular stabilization led to integration of the TEMS-scaffold into the myocardial scar with varying degrees of cellular infiltration, as well as significantly improved echocardiographic parameters demonstrating attenuation of maladaptive cardiac remodeling. Further, TEMS implantation after MI altered the myocardial gene expression pattern. Differences in gene expression were most striking after 4 weeks with significantly reduced expression of NPPA (0.36 ± 0.26 vs 0.75 ± 0.40; p < 0.05), NPPB (0.47 ± 0.25 vs 0.91 ± 0.429; p < 0.01), TGFBR2 (0.68 ± 0.16 vs 0.90 ± 0.14; p < 0.01) and PDGFB (0.81 ± 0.13 vs 1.06 ± 0.14; p < 0.01) as well as increased expression of IL-10 (5.93 ± 5.67 vs 1.38 ± 0.60; p < 0.05), PGF (1.48 ± 0.38 vs 1.09 ± 0.25; p < 0.05) and IGF1 (1.67 ± 0.70 vs 1.03 ± 0.42; p < 0.05). However, after 8 weeks differences in the gene expression patterns of remodeling-associated, and pro-angiogenic markers could still be observed between groups. Conclusion: Ventricular stabilization via TEMS implantation after MI did not only led to biological integration of the cdECM-scaffolds into the host tissue and improved functional cardiac parameters, but also altered 4 and 8 week gene expression of infarcted myocardium, possibly contributing to reducing chronic deteriorating effects while increasing the potential for myocardial regeneration.

Statins Protect Against Early Stages of Doxorubicin-induced Cardiotoxicity Through the Regulation of Akt Signaling and SERCA2

BackgroundDoxorubicin-induced cardiomyopathy (DICM) is one of the complications that can limit treatment for a significant number of cancer patients. In animal models, the administration of statins can prevent the development of DICM. Therefore, the use of statins with anthracyclines potentially could enable cancer patients to complete their chemotherapy without added cardiotoxicity. The precise mechanism mediating the cardioprotection is not well understood. The purpose of this study is to determine the molecular mechanism by which rosuvastatin confers cardioprotection in a mouse model of DICM. MethodsRosuvastatin was intraperitoneally administered into adult male mice at 100 μg/kg daily for 7 days, followed by a single intraperitoneal doxorubicin injection at 10 mg/kg. Animals continued to receive rosuvastatin daily for an additional 14 days. Cardiac function was assessed by echocardiography. Optical calcium mapping was performed on retrograde Langendorff perfused isolated hearts. Ventricular tissue samples were analyzed by immunofluorescence microscopy, Western blotting, and quantitative polymerase chain reaction. ResultsExposure to doxorubicin resulted in significantly reduced fractional shortening (27.4% ± 1.11% vs 40% ± 5.8% in controls; P < 0.001) and re-expression of the fetal gene program. However, we found no evidence of maladaptive cardiac hypertrophy or adverse ventricular remodeling in mice exposed to this dose of doxorubicin. In contrast, rosuvastatin-doxorubicin-treated mice maintained their cardiac function (39% ± 1.26%; P < 0.001). Mechanistically, the effect of rosuvastatin was associated with activation of Akt and phosphorylation of phospholamban with preserved sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2 (SERCA2)-mediated Ca2+ reuptake. These effects occurred independently of perturbations in ryanodine receptor 2 function. ConclusionsRosuvastatin counteracts the cardiotoxic effects of doxorubicin by directly targeting sarcoplasmic calcium cycling.

Abstract GS112: An Epigenome-wide Association Study Paired With Cell-type-specific Data Identifies Key Regulators Of Heart Failure

Background: Heart Failure (HF) is driven by the interactions of multiple genetic, epigenetic, and environmental factors both within and between the cell types of the heart. Our lab uses the Hybrid Mouse Diversity Panel (HMDP), a large cohort of inbred mouse strains, to perform systems genetics analyses of isoproterenol-induced cardiac hypertrophy and failure by leveraging the benefits of a curated model organism population to identify key drivers of phenotype. We are now expanding this study into the DNA methylome, which has been convincingly linked to cardiac-associated phenotypic changes in prior studies. Methods and Results: Using Reduced Representational Bisulfite Sequencing, we profiled left ventricular tissue samples from 88 HMDP strains subjected to isoproterenol challenge (30 mg/kg/day for 21 days) and matched control animals. We identified nearly 170,000 CpGs whose methylation status varies across the HMDP and 179 significant associations (FDR of 5%) between CpG methylation and phenotypic variation using the epigenome-wide association study algorithm MACAU, including 37 associations linking CpG methylation in unchallenged hearts to 19 post-challenge phenotypes. To identify high-confidence candidate genes, we combined our loci with data from the Wellcome Trust Mouse Genomes Resource, transcriptomic and metabolomic data from the HMDP, Hi-C data, and cell-type-specific gene expression and methylation data from healthy and failing mouse hearts. We are systematically querying the resulting 78 candidates using in silico and in vitro approaches. These genes include Mospd3 , which is associated with right ventricular hypertrophy and whose knockdown in vitro results in reduced cardiomyocyte hypertrophy and changes to hypertrophy-related gene expression. Also observed were Atp9a , whose expression levels are associated with significant global DNA methylation changes between control and ISO-treated animals and whose knockdown likewise causes a reduction in observed cellular hypertrophy, and Mdga1 , whose promoter methylation status is linked to changes in 5% of expressed genes of the heart during heart failure. Further analysis and in vivo study of these loci will further our understanding of the role of DNA methylation in heart failure.

Gene Expression Profiles at Different Time Points after Acute Myocardial Infarction in Mice.

To explore the mRNA differential expressions and the sequential change pattern in acute myocardial infarction (AMI) mice. The AMI mice relevant dataset GSE4648 was downloaded from Gene Expression Omnibus (GEO). In the dataset, 6 left ventricular myocardial tissue samples were selected at 0.25, 1, 4, 12, 24 and 48 h after operation in AMI group and sham control group, and 6 left ventricular myocardial tissue samples were selected in blank control group, a total of 78 samples were analyzed. Differentially expressed genes (DEGs) were analyzed by R/Bioconductor package limma, functional pathway enrichment analysis was performed by clusterProfiler, protein-protein interaction (PPI) network was constructed by STRING database and Cytoscape software, the key genes were identified by Degree topological algorithm, cluster sequential changes on DEGs were analyzed by Mfuzz. A total of 1 320 DEGs were associated with the development of AMI. Functional enrichment results included cellular catabolic process, regulation of inflammatory response, development of muscle system and vasculature system, cell adhesion and signaling pathways mainly enriched in mitogen-activated protein kinase (MAPK) signaling pathway. The key genes of AMI included MYL7, TSC22D2, HSPA1A, BTG2, NR4A1, RYR2 were up-regulated or down-regulated at 0.25-48 h after the occurrence of AMI. The functional signaling pathway of DEGs and the sequential expression of key genes in AMI may provide a reference for the forensic identification of AMI.

SPONTANEOUSLY HYPERTENSIVE RATS EXHIBIT ENHANCED CARDIAC CONNEXIN-43 AND REDUCED PRO-FIBROTIC MARKERS IN RESPONSE TO COLD ACCLIMATION

Objective: We have previously shown that down-regulation of myocardial gap junction channel protein, connexin-43 (Cx43), and up-regulation of extracellular matrix (ECM) proteins in hearts of spontaneously hypertensive rats (SHR) promote occurrence of lethal arrhythmia. Cold acclimation has a potential for reducing cardiovascular risk, especially in setting of various pathological conditions, but there is a gap of knowledge concern the heart response to adaptive thermogenesis. The purpose of the study is to explore myocardial Cx43 and ECM proteins in genetically hairless strain of SHR (SHRM) that represents rewarding model of increased adaptive thermogenesis. Design and method: Adult hairless males and females SHRM strain that harbors mutated desmoglein-4 gene and their wild type SHR were housed at standard 22 C (that is below thermo-neutrality for these animals) and 12:12 hour light: dark cycle. WKY control rats were used as reference normotensive strain. Heart samples of the left ventricular tissue from euthanatized rats were used for the proteomics of Cx43, protein kinases, which phosphorylates Cx43, and for ECM markers TGF-, SMAD2/3, MMP-2, collagen-1. Immunofluorescence detection of Cx43 was performed to examine its myocardial topology. Results: Cx43 protein and its phosphorylated status was reduced in males and females SHR vs WKY rat hearts. In contrast, adaptive thermogenesis resulted in increased both parameters in SHRM regardless the sex. Moreover, pathological topology of Cx43 attributed to its enhanced localization at lateral sides of the cardiomyocytes in SHR was attenuated in SHRM. In addition, there were gentle differences in protein levels of PKA, PKG, PKC, MAPK, Akt between SHR and SHRM. Protein level of TGF- and SMAD 2/3 were increased in SHR compare to WKY rat hearts but decreased in SHRM unlike collagen-1 that was not altered in SHRM compare to SHR. Conclusions: Findings revealed beneficial up-regulation of myocardial Cx43 along with down-regulation of extracellular matrix proteins in response of hairless SHR to increased thermogenesis.

Prior Freezing Has Minimal Impact on the Contractile Properties of Permeabilized Human Myocardium.

BackgroundExperiments measuring the contractile properties of human myocardium are important for translational research but complicated by the logistical difficulties of acquiring specimens. Accordingly, many groups perform contractile assays using samples that are acquired from patients at one institution and shipped to another institution for experiments. This necessitates freezing the samples and performing subsequent assays using chemically permeabilized preparations. It is unknown how prior freezing affects the contractile function of these preparations.Methods and ResultsTo examine the effects of freezing we measured the contractile function of never‐frozen and previously frozen myocardial samples. Samples of left ventricular tissue were obtained from 7 patients who were having a ventricular assist device implanted. Half of each sample was chemically permeabilized and used immediately for contractile assays. The other half of the sample was snap frozen in liquid nitrogen and maintained at –180 °C for at least 6 months before being thawed and tested in a second series of experiments. Maximum isometric force measured in pCa 4.5 solution, passive force measured in pCa 9.0 solution, and Hill coefficients were not influenced by prior freezing (P=0.07, P=0.14, and P=0.27 respectively). pCa50 in never‐frozen samples (6.11±0.04) was statistically greater (P<0.001) than that measured after prior freezing (5.99±0.04) but the magnitude of the effect was only ≈0.1 pCa units.ConclusionsWe conclude that prior freezing has minimal impact on the contractile properties that can be measured using chemically permeabilized human myocardium.

Insurance lesions: Does a second lesion make a difference?

In radiofrequency ablation procedures for cardiac arrhythmia, the efficacy of creating repeated lesions at the same location ("insurance lesions") remains poorly studied. We assessed the effect of type of tissue, power, and time on the resulting lesion geometry during such multiple ablation procedures. A custom ex vivo ablation model was used to assess lesion formation. An ablation catheter was oriented perpendicular to the tissue and used to create lesions that varied by type of tissue (atrial or ventricular free wall), power (30 or 50 W), and time (30, 40, or 50 s for standard ablations and 5, 10, or 15 s for high-power, short-duration [HPSD] ablations). Lesion dimensions were recorded and then analyzed. Radiofrequency ablations were performed on 57 atrial tissue samples (28 HPSD, 29 standard) and 28 ventricular tissue samples (all standard). With ablation parameters held constant, performing multiple ablations significantly increased lesion depth in ventricular tissue when ablations were performed at 30 W for 50 s. No other set of ablation parameters was shown to affect the width or depth of the resulting lesions in either tissue type. Multiple ablations created with the same power and time, delivered within 30 s of each other at the same exact location, offer no meaningful benefit in lesion depth or width over single ablations, with the exception of ventricular ablation at 30 W for 50 s. Given the risks associated with excessive ablation, our results suggest that this practice should be re-evaluated by clinical electrophysiologists.

Protective Effect of Cardiomyocyte-Specific Prolyl-4-Hydroxylase 2 Inhibition on Ischemic Injury in a Mouse MI Model.

Our earlier studies showed that inhibiting prolyl-4-hydroxylase enzymes (PHD-1 and PHD-3) improves angiogenesis, heart function, and limb perfusion in mouse models via stabilizing hypoxia-inducible transcription factor-alpha (HIF-1α). The present study explored the effects of the prolyl-4-hydroxylase enzyme, PHD-2, on ischemic heart failure using cardiac-specific PHD-2 gene knockout (KO) mice (PHD2 -/- ). Adult wild-type (WT) and PHD2 -/- mice, 8-12 weeks old, were subjected to myocardial infarction (MI) by irreversibly ligating the left anterior descending (LAD) coronary artery. All sham group mice underwent surgery without LAD ligation. Animals were divided into 4 groups: (1) wild-type sham (WTS); (2) wild-type myocardial infarction (WTMI); (3) PHD2KO sham (PHD2 -/- S); (4) PHD2KO myocardial infarction (PHD2 -/- MI). Left ventricular tissue samples collected at various time points after surgery were used for microRNA expression profiling, Western blotting, and immunohistochemical analysis. Volcano plot analysis revealed 19 differentially-expressed miRNAs in the PHD2 -/- MI group compared with the WTMI group. Target analysis using Ingenuity Pathway Analysis showed several differentially regulated miRNAs targeting key signaling pathways such as Akt, VEGF, Ang-1, PTEN, apoptosis, and hypoxia pathways. Western blot analysis showed increased HIF-1α, VEGF, phospho-AKT, β-catenin expression and reduced Bax expression for the PHD2 -/- MI group compared with the WTMI group. Echocardiographic analysis showed preserved heart functions, and picrosirius red staining revealed decreased fibrosis in PHD2 -/- MI compared with the WTMI group. PHD2 inhibition showed preserved heart function, enhanced angiogenic factor expression, and decreased apoptotic markers after MI. Overall, cardiac PHD2 gene inhibition is a promising candidate for managing cardiovascular diseases.

Proteomic mapping of atrial and ventricular heart tissue in patients with aortic valve stenosis

Aortic valve stenosis (AVS) is one of the most common valve diseases in the world. However, detailed biological understanding of the myocardial changes in AVS hearts on the proteome level is still lacking. Proteomic studies using high-resolution mass spectrometry of formalin-fixed and paraffin-embedded (FFPE) human myocardial tissue of AVS-patients are very rare due to methodical issues. To overcome these issues this study used high resolution mass spectrometry in combination with a stem cell-derived cardiac specific protein quantification-standard to profile the proteomes of 17 atrial and 29 left ventricular myocardial FFPE human myocardial tissue samples from AVS-patients. In our proteomic analysis we quantified a median of 1980 (range 1495–2281) proteins in every single sample and identified significant upregulation of 239 proteins in atrial and 54 proteins in ventricular myocardium. We compared the proteins with published data. Well studied proteins reflect disease-related changes in AVS, such as cardiac hypertrophy, development of fibrosis, impairment of mitochondria and downregulated blood supply. In summary, we provide both a workflow for quantitative proteomics of human FFPE heart tissue and a comprehensive proteomic resource for AVS induced changes in the human myocardium.