Left Ventricle Samples Research Articles

Optimisation of Reference Genes for Gene-Expression Analysis in a Rabbit Model of Left Ventricular Diastolic Dysfunction

Left ventricular diastolic dysfunction (LVDD) is characterized by the disturbance of ventricle’s performance due to its abnormal relaxation or to its increased stiffness during the diastolic phase. The molecular mechanisms underlying LVDD remain unknown. We aimed to identify normalization genes for accurate gene-expression analysis of LVDD using quantitative real-time PCR (RT-PCR) in a new rabbit model of LVDD. Eighteen rabbits were fed with a normal diet (n = 7) or a 0.5% cholesterol-enriched diet supplemented with vitamin D2 (n = 11) for an average of 14.5 weeks. We validated the presence of LVDD in this model using echocardiography for diastolic function assessment. RT-PCR was performed using cDNA derived from left ventricle samples to measure the stability of 10 genes as candidate reference genes (Gapdh, Hprt1, Ppia, Sdha, Rpl5, Actb, Eef1e1, Ywhaz, Pgk1, and G6pd). Using geNorm analysis, we report that Sdha, Gapdh and Hprt1 genes had the highest stability (M <0.2). By contrast, Hprt1 and Rpl5 genes were found to represent the best combination for normalization when using the Normfinder algorithm (stability value of 0.042). Comparison of both normalization strategies highlighted an increase of natriuretic peptides (Bnp and Anp), monocytes chemotactic protein-1 (Mcp-1) and NADPH oxidase subunit (Nox-2) mRNA expressions in ventricle samples of the hypercholesterolemic rabbits compared to controls (P<0.05). This increase correlates with LVDD echocardiographic parameters and most importantly it molecularly validates the presence of the disease in our model. This is the first study emphasizing the selection of stable reference genes for RT-PCR normalization in a rabbit model of LVDD.

Mitochondrial proteome remodeling in ischemic heart failure

AimsMitochondrial dysfunction is an important part of the decline in cardiac function in heart failure. We hypothesized for hypothesized that there would be specific abnormalities in mitochondrial function and proteome with the progression of ischemic heart failure (HF). Main methodsWe used a high left anterior descending artery (LAD) ligation in 3–4month old male rats to generate HF. Rats were studied 9weeks post-ligation. Key findingsElectron microscopy of left ventricle samples showed mitochondrial changes including decreased size, increased number, abnormal distribution, and cristae loss. Mitochondria in ischemic HF exhibited decreased total ATP, impaired mitochondrial respiration, as well as reduced complex I activity. Analysis of LV mitochondrial proteins by mass spectrometry was performed, and 31 differentially expressed proteins (p<0.05) of more than 500 total proteins were identified. Of these proteins, 15 were up-regulated and 16 were down-regulated in the failing heart. A set of complex I proteins was significantly decreased, consistent with the impairment of complex I activity. There were distinct changes in mitochondrial function and proteome in ischemic HF. Although there were similarities, the distinction between the reported proteomic changed with TAC pressure overload induced HF and ischemic HF in the current study suggested different pathological mechanisms. SignificanceSpecific changes in mitochondrial protein expression, which correlate with changes in mitochondrial function, have been identified in ischemic HF for the first time.

A Novel Method for Isolating and Culturing Human Cardiomyocytes from Cryopreserved Tissues

Current animal models fail to accurately mimic human heart biology. We have developed a novel method to isolate viable human cardiomyocytes (HCMs) from cryopreserved heart transplant donors. The Sydney Heart Bank is an effective and unique resource for isolating HCMs from donors as young as 2 months to 65 years. Preliminary data were collected from 2 donors aged 4 and 59 years. Left ventricle (LV) samples were frozen (−196°C) after being excised by transplant surgeons. LV tissues stored for 48 and 30 months, respectively, were then warmed gradually over 24 hours to 37°C. 300 μm thick cryosections were digested with collagenase type B and D [Mollova et al.. 2013 PNAS. 110:1446], gently agitated and then plated. Cell viability of isolated HCMs was evaluated using CellTracker CMFDA and calcein-AM. Our findings showed that isolated HCMs are metabolically active and viable. HCMs that were metabolically challenged using FCCP (trifluorocarbonylcyanide phenylhydrazone) increased their oxygen consumption. Immunohistochemical analyses using antibodies against cardiac markers showed that isolated HCMs express sarcomeric alpha-actinin, connexin43 and cardiac troponin T. Addition of BDM (2,3-butanedione monoxime, an inhibitor of myosin ATPase activity) allowed a much higher yield of viable HCMs (20-25% compared to 5% in the absence of BDM). However, the use of BDM in these culture seems to inhibit contractility of freshly isolated HCMs. Current studies are investigating optimal culture conditions to control contractility and viability of HCMs that will be used to replace current animal models. Taken together, our studies show that viable HCMs can be isolated and cultured from LV samples and may be used to investigate cardiomyocyte biology and pathology.

Pregnancy differentially regulates the collagens types I and III in left ventricle from rat heart.

The pathologic cardiac remodeling has been widely documented; however, the physiological cardiac remodeling induced by pregnancy and its reversion in postpartum are poorly understood. In the present study we investigated the changes in collagen I (Col I) and collagen III (Col III) mRNA and protein levels in left ventricle from rat heart during pregnancy and postpartum. Col I and Col III mRNA expression in left ventricle samples during pregnancy and postpartum were analyzed by using quantitative PCR. Data obtained from gene expression show that Col I and Col III in left ventricle are upregulated during pregnancy with reversion in postpartum. In contrast to gene expression, the protein expression evaluated by western blot showed that Col I is downregulated and Col III is upregulated in left ventricle during pregnancy. In conclusion, the pregnancy differentially regulates collagens types I and III in heart; this finding could be an important molecular mechanism that regulates the ventricular stiffness in response to blood volume overload present during pregnancy which is reversed in postpartum.

Expression Quantitative Trait Locus (eQTL) analysis in human heart for elucidation of causal genes at loci identified in genome-wide association studies

In recent years genome-wide association studies (GWAS) have uncovered a plethora of chromosomal loci associated with a variety of cardiac traits. As in most GWAS, a considerable portion of identified signals occur at inter-genic regions of the genome. Trait-associated single nucleotide polymorphisms (SNPs) located in these non-coding regions likely exert their effect through modulation of gene expression. Thus interrogating these variants for association with differential transcript abundance by expression quantitative trait locus (eQTL) analysis is a highly relevant means to unravel the causal genes at loci identified by GWAS. This will in turn further our understanding of the molecular mechanisms underlying cardiac traits. In this study, for the first time, we carried out an eQTL analysis in human heart. A total of 129 human left ventricle samples were collected at four collaborating centers. Samples were acquired from non-diseased hearts that were considered suitable for transplantation, yet not used for logistical reasons. All individuals were of Western European descent. Genome-wide transcript abundance and genotypes were assessed using the Illumina HumanHT-12 and HumanOmniExpress microarrays, respectively. After pre-processing and stringent quality control of transcript and genotypic data, each transcript was tested for association with genotypes genome-wide using linear modelling in R, correcting for age, gender and center effects. We identified 770 independent cis-eQTLs (SNPs within 1 Mb of transcript) mapping to 457 unique transcripts (FDR < 1%). Overlaying these eQTLs with cardiac GWAS loci identified strong candidate genes for functional studies. One of these involves an eQTL effect of rs9912468, a robust modifier of QRS, on PRKCA (p = 1.52E-11). Our findings provide evidence for the role of PRKCA in mediating the effect of the rs9912468-tagged haplotype on QRS duration. Additionally, we identified several trans-eQTL hotspots in the vicinity of transcription factors, regulating targets predominantly involved in immune response, signalling and cardiac muscle structure related processes, all known to impact on the heart function. An on-going effort entails overlaying the eQTLs with known cardiac regulatory regions, such as binding regions of cardiac specific transcription factors (TBX3, TBX5, NKX2-5, GATA4, SRF) and enhancers, identified through ChIP-sequencing in the ENCODE project and other studies. Our study illustrates the power of integrating gene expression, phenotype and genotype data in a systems genetics approach, in identifying novel causal genes for human cardiac phenotypes.

Detection of differentially methylated gene promoters in failing and nonfailing human left ventricle myocardium using computation analysis

Human dilated cardiomyopathy (DCM) is characterized by congestive heart failure and altered myocardial gene expression. Epigenetic changes, including DNA methylation, are implicated in the development of DCM but have not been studied extensively. Clinical human DCM and nonfailing control left ventricle samples were individually analyzed for DNA methylation and expressional changes. Expression microarrays were used to identify 393 overexpressed and 349 underexpressed genes in DCM (GEO accession number: GSE43435). Gene promoter microarrays were utilized for DNA methylation analysis, and the resulting data were analyzed by two different computational methods. In the first method, we utilized subtractive analysis of DNA methylation peak data to identify 158 gene promoters exhibiting DNA methylation changes that correlated with expression changes. In the second method, a two-stage approach combined a particle swarm optimization feature selection algorithm and a discriminant analysis via mixed integer programming classifier to identify differentially methylated gene promoters. This analysis identified 51 hypermethylated promoters and six hypomethylated promoters in DCM with 100% cross-validation accuracy in the group assignment. Generation of a composite list of genes identified by subtractive analysis and two-stage computation analysis revealed four genes that exhibited differential DNA methylation by both methods in addition to altered gene expression. Computationally identified genes (AURKB, BTNL9, CLDN5, and TK1) define a central set of differentially methylated gene promoters that are important in classifying DCM. These genes have no previously reported role in DCM. This study documents that rigorous computational analysis applied to microarray analysis of healthy and diseased human heart samples helps to define clinically relevant DNA methylation and expressional changes in DCM.

Thymidine kinase and mtDNA depletion in human cardiomyopathy: epigenetic and translational evidence for energy starvation

This study addresses how depletion of human cardiac left ventricle (LV) mitochondrial DNA (mtDNA) and epigenetic nuclear DNA methylation promote cardiac dysfunction in human dilated cardiomyopathy (DCM) through regulation of pyrimidine nucleotide kinases. Samples of DCM LV and right ventricle (n = 18) were obtained fresh at heart transplant surgery. Parallel samples from nonfailing (NF) controls (n = 12) were from donor hearts found unsuitable for clinical use. We analyzed abundance of mtDNA and nuclear DNA (nDNA) using qPCR. LV mtDNA was depleted in DCM (50%, P < 0.05 each) compared with NF. No detectable change in RV mtDNA abundance occurred. DNA methylation and gene expression were determined using microarray analysis (GEO accession number: GSE43435). Fifty-seven gene promoters exhibited DNA hypermethylation or hypomethylation in DCM LVs. Among those, cytosolic thymidine kinase 1 (TK1) was hypermethylated. Expression arrays revealed decreased abundance of the TK1 mRNA transcript with no change in transcripts for other relevant thymidine metabolism enzymes. Quantitative immunoblots confirmed decreased TK1 polypeptide steady state abundance. TK1 activity remained unchanged in DCM samples while mitochondrial thymidine kinase (TK2) activity was significantly reduced. Compensatory TK activity was found in cardiac myocytes in the DCM LV. Diminished TK2 activity is mechanistically important to reduced mtDNA abundance and identified in DCM LV samples here. Epigenetic and genetic changes result in changes in mtDNA and in nucleotide substrates for mtDNA replication and underpin energy starvation in DCM.

Molecular Changes of the Angiotensin Converting Enzyme 2 Pathway in Myocardial Tissue from Heart Failure Patients Undergoing Heart Transplantation

Background: The angiotensin-converting enzyme homologue ACE2 and the peptide angiotensin 1-7 (Ang 1-7) have antifibrotic effects, but it is not understood whether they have a protective role in cardiac remodeling. We aimed to analyze the Ang 1-7 Mas receptor and ACE2 expresion levels and their relationship with fibrotic and remodeling factors in human heart failure (HF) and compared them with non-pathological human hearts.Methods: This was a human case-control study in which two groups of samples were analyzed. Controls were organ donors without cardiovascular pathology, but whose heart could not be used for transplantation. Cases were patients with end-stage HF and left ventricle samples were harvested right after the heart transplantation surgery. They were either kept with liquid nitrogen until their analysis or were processed for paraffin embedding. We quantified mRNA expression by real time PCR and ACE2 protein levels by western blot. The collagen deposition was stained by sirius red and quantified with morphometric analysis.Results: Expression of the MMP3 stromelysin mRNA was only detectable in 10 out of 33 pathological samples and in 1 out of 13 control samples. Expression of components of the ACE2 pathway and fibrotic factors were higher in patients with detectable MMP3 expression than in patients with non-detectable MMP3. No differences were found between patients with MMP3 expression or no MMP3 expression regarding etiology, functional class, ventricle dilatation or medication.Conclusions: Only a subset of myocardiums from HF patients is active in the remodeling process at the moment of heart transplantation, as indicated by the MMP3 expression and the higher levels of fibrotic factors. Angiotensin 1-7 Mas receptor expression and ACE2 protein levels are increased in this myocardium subset, suggesting a role for the ACE2 in this process. Overall, we encountered molecular differences in transplanted hearts from patients with similar clinical characteristics.

A New Functional Measure of Contractility in Human Cardiomyopathies

Hypertrophic and familial dilated cardiomyopathies are arguably the most common forms of inherited myocardial dysfunction. Both disease states result in deterioration of cardiac function and quality of life as a consequence of extensive remodelling of the chamber walls.Here we use a novel technique SPontaneous Oscillatory Contractions - or SPOCs to assess changes in contraction and relaxation phases using a range of explanted human heart samples. We examine left ventricle (LV) samples from: (1) patients with hypertrophic cardiomyopathy (HCM); (2) patients with familial dilated cardiomyopathy (FDCM); and (3) and aged-matched non-failing donors. The SPOC parameters of interest are: (i) SPOC amplitudes; (ii) the rates of lengthening (relaxation); (iii) the rates of shortening (contraction); and (iv) their respective SPOC periods.On average, samples from HCM patients exhibited significantly slower rates of lengthening and shorter SPOC periods, while FDCM patients displayed significantly longer lengthening and shortening SPOC periods, compared to donors. Impaired shortening is indicative of diastolic dysfunction while impaired shortening indicates a systolic dysfunction. We observed extensive changes in SPOC parameters that were mutation-specific. The MYBPC3 mutation exhibited shorter SPOC period and faster shortening rates while the samples with the TNNI3 mutation had a higher amplitude and slower shortening rates. Their SPOC data are consistent with the relatively mild phenotype associated with their respective mutations. Furthermore, SPOC is also sensitive to the progressive deterioration in LV ejection fraction.SPOC analysis is a promising tool that provides a quantitative insights into cardiac contractility. It may allow us to unravel other significant differences between familial cardiomyopathies and donor hearts. The SPOC data agree well with patient clinical phenotypes.

Analysis of the arrhythmogenic substrate in human heart failure

The mechanism of sudden cardiac death in patients with heart failure (HF) is uncertain. Both electrical instability and structural remodelling could be factors that lead to fatal arrhythmias. We sought to analyse the expression of the sodium (SCN5A) and potassium (KCND3) channels as well as the fibrosis content in the ventricles of human HF and of non-diseased hearts under different post-mortem intervals. We analysed normal human hearts as controls [n=20 for the right ventricle (RV) and n=13 for the left ventricle (LV)] and human hearts from HF patients, which were obtained at the time of cardiac transplantation, as cases (n=48 for RV and n=34 for LV). Transcription of the SCN5A (probes SCN5A E4-5, E11-12, and E28) and KCND3 channels and of COLLAGEN I and III were assayed by real-time polymerase chain reaction. In addition, paraffin sections were used to analyse the percentage of collagen deposition in both cases and controls. KCND3 mRNA expression in the LV was lower in the cases than in controls (P<.001). Higher levels of SCN5A mRNA were found in the HF samples when analysed with probe SCN5A E4-5 (P<.05). SCN5A expression was lower in the controls with longer post-mortem interval (n=4) than in the controls with a shorter post- mortem interval (n=16, P<.01). KCND3 mRNA levels were also different between the two control groups (P<.05). Collagen deposition was higher in the LV tissues of the cases when compared to controls (P<.001), and it was higher in the LV from HF patients than in the RV (P<.05). Furthermore, collagen deposition was higher in the LV samples from patients with implanted cardiac defibrillator (ICD) therapy than in the LV of patients with no ICD therapy (P<.05). These data indicate that ionic and structural remodelling could be pathophysiological mechanisms of cardiac arrhythmias in HF patients.

253 IMPACT OF LOW BIRTH WEIGHT ON THE EXPRESSION OF THE RENIN-ANGIOTENSIN SYSTEM, FACTORS WHICH REGULATE AUTOPHAGY, FIBROSIS AND CAPILLARY DENSITY IN THE HEART DURING EARLY POSTNATAL LIFE

Background: Low birth weight (LBW) is associated with increased hypertension in offspring. RAS is important in hypertension but is also implicated in cardiac angiogenesis, remodelling and autophagy. We aimed to investigate the effects of LBW on the cardiac RAS and these cardiac processes. Methods: Plasma renin activity and ANGII in average birth weight (ABW) and LBW lambs at d20 were determined using radioimmunoassay. Left ventricle samples were collected from the lambs at d21 for the analysis of gene and protein expression. Results: There was increased plasma renin activity and ANGII concentrations in LBW compared to ABW lambs. There was increased ACE2 mRNA and decreased abundance of ATR1 but no changes in abundance of ACE or ATR2 in LBW compared to ABW lambs. There was increased phospho-Akt protein and decreased capillary density in the heart of LBW lambs compared to ABW lambs. There was decreased collagen I and II mRNA expression but no evidence of cardiac remodelling or fibrosis in the LBW lambs. There was decreased FoxO1 and FoxO3a protein in the LBW lambs compared to ABW lambs, but no changes in autophagy proteins: phospho-FoxO1, phospho-FoxO3a, Beclin1, LC3B and LAMP1 was not altered in LBW lambs. Conclusion: Although circulating and local cardiac RAS components were altered in LBW lambs there was no increase in fibrosis or autophagy in the heart of the LBW lamb. LBW resulted in decreased capillary density during early postnatal life and this may result in an increased risk of impaired heart function in later life.

Abstract 5675: Potential use of proteosome pathway inhibitor (bortezomib) in combination with anthracyclines to treat multiple myeloma and pancreatic cancer

Abstract Anthracyclines are widely used to treat cancers, but may also cause a cumulative dose-related cardiotoxicity. One possible mechanism of cardiotoxicity relates to effects on the cardiomyocyte proteasomal/ubiquitin system (UPS), the major protein recycling system in the cell. The goal of the research is to determine the impact of anthracyclines on the UPS and modulate cardiotoxicity by using other drugs in combination. Rats were treated chronically with a clinically relevant dose of daunorubicin (DNR) or the vehicle (controls) over 9 weeks. Another group received DNR pretreated with ICRF (dexrazoxane; a known protectant against anthracycline cardiotoxicity). The hearts were harvested one week later and analyzed. Isometric atrial preparations from rats treated with DNR showed decreased contractility compared to those pretreated with ICRF or with DNR vehicle only. Ubiquitin ligase atrogin-1 has been associated with muscle atrophy and cancer cachexia. In western blot analysis of common E3 ligases in left ventricle samples from rats treated with DNR, Atrogin-1 showed a significant reduction (P&amp;lt;0.05) compared to animals pretreated with ICRF or DNR vehicle. This suggests that chronic anthracycline cardiotoxicity is accompanied by ubiquitin function. Bortezomib, a proteasomal inhibitor, is approved for treatment of multiple myeloma (MM) and is being investigated for treatment of pancreatic cancer with other drugs. To understand the antineoplastic interaction of a proteosome inhibitor (bortezomib) with an anthracycline (doxorubicin), we performed dose-response assays using MM and pancreatic cancer cell lines. Our studies indicate that, compared to bortezomib alone, 0.05 µM of doxorubicin in combination with bortezomib, enhanced cytotoxicity of bortezomib in the two cell lines. Thus, bortezomib and anthracycline combination therapy may provide enhanced tumoricidal activity for MM and pancreatic cancer compared to bortezomib alone. It is important to understand the mechanisms of the effect of bortezomib and anthracyclines on ubiquitin-proteasome pathway. (Supported by a St. Luke's Health System Mountain States Tumor and Medical Research Institute grant) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5675. doi:1538-7445.AM2012-5675

Comparison of human cardiac gene expression profiles in paired samples of right atrium and left ventricle collected in vivo

Studies of expressed genes in human heart provide insight into both physiological and pathophysiological mechanisms. This is of importance for extended understanding of cardiac function as well as development of new therapeutic drugs. Heart tissue for gene expression studies is generally hard to obtain, particularly from the ventricles. Since different parts of the heart have different functions, expression profiles should likely differ between these parts. The aim of the study was therefore to compare the global gene expression in cardiac tissue from the more accessible auricula of the right atrium to expression in tissue from the left ventricle. Tissue samples were collected from five men undergoing aortic valve replacement or coronary artery bypass grafting. Global gene expression analysis identified 542 genes as differentially expressed between the samples extracted from these two locations, corresponding to ~2% of the genes covered by the microarray; 416 genes were identified as abundantly expressed in right atrium, and 126 genes were abundantly expressed in left ventricle. Further analysis of the differentially expressed genes according to available annotations, information from curated pathways and known protein interactions, showed that genes with higher expression in the ventricle were mainly associated with contractile work of the heart. Transcription in biopsies from the auricula of the right atrium on the other hand indicated a wider area of functions, including immunity and defense. In conclusion, our results suggest that biopsies from the auricula of the right atrium may be suitable for various genetic studies, but not studies directly related to muscle work.

Characteristics of interstitial fibrosis and inflammatory cell infiltration in right ventricles of systemic sclerosis-associated pulmonary arterial hypertension.

Objective. Systemic sclerosis-associated pulmonary arterial hypertension (SScPAH) has a disturbed function of the right ventricle (RV) when compared to idiopathic PAH (IPAH). Systemic sclerosis may also affect the heart. We hypothesize that RV differences may occur at the level of interstitial inflammation and—fibrosis and compared inflammatory cell infiltrate and fibrosis between the RV of SScPAH, IPAH, and healthy controls. Methods. Paraffin-embedded tissue samples of RV and left ventricle (LV) from SScPAH (n = 5) and IPAH (n = 9) patients and controls (n = 4) were picrosirius red stained for detection of interstitial fibrosis, which was quantified semiautomatically. Neutrophilic granulocytes (MPO), macrophages (CD68), and lymphocytes (CD45) were immunohistochemically stained and only interstitial leukocytes were counted. Presence of epi- or endocardial inflammation, and of perivascular or intimal fibrosis of coronary arteries was assessed semiquantitatively (0–3: absent to extensive). Results. RV's of SScPAH showed significantly more inflammatory cells than of IPAH (cells/mm2, mean ± sd MPO 11 ± 3 versus 6 ± 1; CD68 11 ± 3 versus 6 ± 1; CD45 11 ± 1 versus 5 ± 1 , P < .05) and than of controls. RV interstitial fibrosis was similar in SScPAH and IPAH (4 ± 1 versus 5 ± 1%, P = .9), and did not differ from controls (5 ± 1%, P = .8). In 4 SScPAH and 5 IPAH RV's foci of replacement fibrosis were found. No differences were found on epi- or endocardial inflammation or on perivascular or intimal fibrosis of coronary arteries. Conclusion. SScPAH RVs display denser inflammatory infiltrates than IPAH, while they do not differ with respect to interstitial fibrosis. Whether increased inflammatory status is a contributor to altered RV function in SScPAH warrants further research.

Characterization of a Mutant Rat Model with Altered Titin Isoform Expression

Titin isoform expression is related to human cardiac disease. A mutant rat model with dramatically altered titin isoform expression has been described (Greaser et al. J Mol. Cell. Cardiol. 44:483, 2009), and the ultrastructural and physiological properties of mutant and wild type rats were compared in this study. Electron micrographs of homozygous mutant ventricles showed normal structure in most areas, but occasional regions of Z line streaming, myofibrillar disarray, lipofuchin granules, and myofibril degeneration were observed as found previously in human heart failure. Dobutamine administration caused an increased heart rate in wild type (Wt), heterozygotes (Ht) and homozygote mutants (Hm), but the dobutamine effects on preload recruitable stroke work and maximal systolic elastance were significantly blunted in the mutant groups. Maximal exercise capacity of Wt rats was significantly longer than that of Hm. Electrophoretic myosin heavy chain analysis of left ventricle (LV) samples showed no differences between Wt, Ht, or Hm in the beta myosin heavy chain proportions. Gene expression patterns in LV were conducted with Affymetrix GeneChip Rat Genome 230 2.0 microarrays using WT and Hm LV at three developmental stages (day 1, day 20 and day 49). A Student t-test with a p value cut-off of 0.05 and a minimum 1.5-fold change reveals changes in 372 mutation-specific transcripts (188 known and 96 un-annotated genes). A number of titin associated genes were up-regulated (Myot, T-cap, DARP, FHL1), and this up-regulation was verified by QPCR. Hierarchical clustering revealed gene expression patterns of Wt and Hm LV were related to their titin protein gel pattern. Predefined pathways and functional categories annotated by KEGG, Biocarta, and GO using the DAVID bioinformatic resource indicated involvement of TGF Beta 2, CTGF-regulated fibrosis, Trdn-Casq interaction-regulated RyR channel, and cAMP-dependent pathways. Supported by NIH HL77196.

Induction of Mitogen-Activated Protein Kinases Is Proportional to the Amount of Pressure Overload

Pressure overload has been shown to induce mitogen activated protein kinases (MAPKs) and reactivate the atrial natriuretic factor in the heart. To test the sensitivity of these signals to pressure overload, we assayed the activity of MAPKs extracellular signal-regulated kinase, c-Jun N-terminal kinase 1, and p38 in protein lysates from the left ventricle (LV) or white blood cells (WBC) isolated from aortic banded mice with varying levels of pressure overload. In separated mice we measured atrial natriuretic factor mRNA levels by Northern blotting. As expected, a significant induction of atrial natriuretic factor mRNA levels was observed after aortic banding, and it significantly correlated with the trans-stenotic systolic pressure gradient but not with the LV weight:body weight ratio. In contrast, a significant correlation with systolic pressure gradient or LV weight:body weight ratio was observed for all of the MAPK activity detected in LV samples or WBCs. Importantly, LV activation of MAPKs significantly correlated with their activation in WBCs from the same animal. To test whether MAPK activation in WBCs might reflect uncontrolled blood pressure levels in humans, we assayed extracellular signal-regulated kinase, c-Jun N-terminal kinase 1, and p38 activation in WBCs isolated from normotensive volunteers, hypertensive patients with controlled blood pressure values, or hypertensive patients with uncontrolled blood pressure values. Interestingly, in hypertensive patients with controlled blood pressure values, LV mass and extracellular signal-regulated kinase phosphorylation were significantly reduced compared with those in hypertensive patients with uncontrolled blood pressure values. These results suggest that MAPKs are sensors of pressure overload and that extracellular signal-regulated kinase activation in WBCs might be used as a novel surrogate biomarker of uncontrolled human hypertension.

Atrial fibrillation, amyloidosis, myocarditis and viral infection

The semi-quantitative histologic and immunohistochemical analyses were carried out on the biopsy and autopsy samples of right atrium, interatrial septum and left ventricle of 110 patients. The antibodies against calcineurin, natriuretic peptide, prealbumin (transthyretin), λ- and κ-light chains, АА amyloid, Ki-67-antigen, VP1-antigen of enterovirus, herpes simplex virus, cytomegalovirus, LMP- antigen of Epstein-Barr virus, adenovirus, parvovirus B19 were used. All cases with persistent atrial fibrillation are characterized by myocarditis, amyloidosis signs and by the presence of enterovirus and parvovirus B19 antigens, and more rarely by adenovirus.

Re‐expression of alpha skeletal actin as a marker for dedifferentiation in cardiac pathologies

Differentiation of foetal cardiomyocytes is accompanied by sequential actin isoform expression, i.e. down-regulation of the ‘embryonic’ alpha smooth muscle actin, followed by an up-regulation of alpha skeletal actin (αSKA) and a final predominant expression of alpha cardiac actin (αCA). Our objective was to detect whether re-expression of αSKA occurred during cardiomyocyte dedifferentiation, a phenomenon that has been observed in different pathologies characterized by myocardial dysfunction. Immunohistochemistry of αCA, αSKA and cardiotin was performed on left ventricle biopsies from human patients after coronary bypass surgery. Furthermore, actin isoform expression was investigated in left ventricle samples of rabbit hearts suffering from pressure- and volume-overload and in adult rabbit ventricular cardiomyocytes during dedifferentiation in vitro. Atrial goat samples up to 16 weeks of sustained atrial fibrillation (AF) were studied ultrastructurally and were immunostained for αCA and αSKA. Up-regulation of αSKA was observed in human ventricular cardiomyocytes showing down-regulation of αCA and cardiotin. A patchy re-expression pattern of αSKA was observed in rabbit left ventricular tissue subjected to pressure- and volume-overload. Dedifferentiating cardiomyocytes in vitro revealed a degradation of the contractile apparatus and local re-expression of αSKA. Comparable αSKA staining patterns were found in several areas of atrial goat tissue during 16 weeks of AF together with a progressive glycogen accumulation at the same time intervals. The expression of αSKA in adult dedifferentiating cardiomyocytes, in combination with PAS-positive glycogen and decreased cardiotin expression, offers an additional tool in the evaluation of myocardial dysfunction and indicates major changes in the contractile properties of these cells.

The role of cardiomyocyte apoptosis in mechanisms of ischemic myocardial remodeling

Here the role of cardiomyocyte apoptosis in mechanisms of ischemic myocardial remodeling was investigated. The samples of left ventricle were obtained on 50 patients with the diagnosed atherosclerotic disease of coronary arteries and heart failure. Obtained data allow to approve about the maximal importance of cardiomyocyte apoptosis at an early stages of ischemic myocardial remodeling. The role of cardiomyocyte apoptosis was decreased at a high volume of left ventricle. The received results suggest about an importance of cardiomyocyte apoptosis as an marker of ischemic myocardial remodeling and heart failure.

Comparison of Blood Activation in the Wound, Active Vent, and Cardiopulmonary Bypass Circuit

During cardiopulmonary bypass, aspirated blood exhibits strong activation features, but the triggering event remains unclear. Contact of blood with the pericardial cavity and surgical wound has been advocated as the main trigger, but suction forces are also considered as a possible contributor. We thus designed a study to identify the possible causes involved in this activation. In 10 patients, we analyzed hemostasis activation markers and inflammatory mediators in blood collected in the pericardial cavity and in blood actively aspirated from the left ventricle without any contact with the pericardial cavity. In addition, the same variables were determined in blood sampled in the cardiopulmonary bypass circuit. Markers of tissue factor pathway activation and of thrombin generation, microparticles, free hemoglobin, interleukin 6, and tumor necrosis factor-alpha were significantly increased in pericardial samples as compared with the left ventricle and cardiopulmonary bypass circuit samples. All measured variables were similar between left ventricle and cardiopulmonary bypass samples, except free hemoglobin, interleukin 6, and microparticle levels, which were significantly higher in the left ventricle. Blood contact with the pericardial cavity induces strong hemolysis, inflammatory mediator release, and coagulation activation, driven by tissue factor pathway activation. By contrast, suction forces applied to left ventricular blood poorly contribute to blood trauma and activation. Comparison of pericardial and left ventricular blood shows that contact with the pericardial cavity, and not suction forces, is the leading cause of blood activation. The specific trigger for blood trauma and activation present in the pericardial cavity remains to be identified.