Heart Tissue Of Patients Research Articles

Cardiac secreted-HSP90alpha in cardiomyocyte hypertrophy by N-Cadherin mediated signaling under pressure overload

Abstract Background Left ventricular hypertrophy (LVH) in response to pressure overload is strongly associated with adverse cardiovascular outcomes. Heat shock protein 90 (HSP90) has been reported to be involved in cardiac remodeling under stress. Aims Here, we evaluate whether serum HSP90α (an isoform of HSP90) increases and associates with cardiac hypertrophy in patients with hypertension or severe aortic stenosis (AS), and explore the potential mechanisms in experimental pressure overload mouse model. Methods Serum HSP90α levels in patients with hypertension or aortic stenosis (AS) were determined by ELISA. Pressure overload mouse model was built by a transverse aortic constriction (TAC). Cardiac function was assessed by echocardiography. Cardiac hypertrophy was examined by histology, western blot and RNA analysis. Results Serum levels of HSP90α were higher in patients with hypertension or AS than in the control group, respectively, and the levels positively correlated with LVH. HSP90α levels also increased in heart tissues of patients with obstructive hypertrophic cardiomyopathy (HCM), and mice after TAC, in parallel with the hypertrophic markers. TAC induced the enhanced expression and secretion of HSP90α from cardiomyocytes and cardiac fibroblasts. Knockdown of HSP90α or blockade of extracellular HSP90α (eHSP90α) prevent the development of LVH under pressure overload in vivo and in vitro. By Nano-HPLC-MS/MS analysis, we identified eHSP90α interacted with N-cadherin in cardiomyocyte surface, which induced the activation of β-catenin and promoted β-catenin to bind TCF7 (a member of TCF/Lef family) in nucleus, enhanced the transcription of hypertrophic genes, contributing to cardiac hypertrophy and dysfunction under pressure overload. Conclusions Our data demonstrated that cardiac secreted-HSP90α promoted myocardial hypertrophy by N-Cadherin mediated β-catenin/TCF7 signaling under pressure overload. It indicates the therapeutic potential of targeting HSP90α-initiated signaling against cardiac hypertrophy and dysfunction under pressure overload.

Role of inflammation in the development of atrial fibrillation

This study analyzed and demonstrated the role of inflammatory mechanisms and inflammation markers in the development of atrial fibrillation, their significance in the structural and electrical remodeling of the atria, and pharmacological agents that can be effective in reducing inflammation. Data were obtained from the analysis of retrospective and prospective studies and systematic reviews. The available domestic and foreign scientific studies indexed in PubMed, Google Scholar, and eLibrary.ru were analyzed. Atrial fibrillation is one of the most common arrhythmias in adults and is associated with many complications and mortality. The pathophysiological mechanisms of this arrhythmia remain completely unclear, and their search continues at the molecular level. Atrial fibrillation causes electrical and structural changes in the myocardium, which lead to further pathological transformations of the heart, and some of them are associated with inflammation, which has been demonstrated in studies on an experimental model and heart tissues of patients with this rhythm disorder. Whether inflammation is the cause of the development of this arrhythmia or its consequence is not clearly understood. Statins, corticosteroids, colchicine, genetically engineered biological drugs, which have a specific application point in the inflammatory cascade, and some other drugs, such as anticoagulants, polyunsaturated fatty acids, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, and mineralocorticoid receptor antagonists, also take place in the treatment of atrial fibrillation. However, their applications are not clearly specified. Currently, research continuously aims at finding effective ways to prevent and treat this nosology. Thus, promising ways to reduce the role of inflammation in the occurrence, recurrence, diagnosis, and treatment of atrial fibrillation are relevant in the development of precision medicine.

Long non-coding RNA TERRA regulates DNA damage and survival of endothelial cells and cardiomyocytes: implications for aging

Abstract Introduction Ageing is the major risk factor for cardiovascular disease. Long non-coding RNAs are emerging as novel regulators of cellular functions and contributors to cardiovascular ageing. One of the hallmarks of aging is telomere attrition. Non-coding transcripts called Telomeric repeat-containing RNA (TERRA) are molecules of 0.2–10kb in length which are transcribed from the subtelomeres and telomeres of chromosomes and might play a role in cardiovascular ageing. Purpose This study aims to characterize the role of TERRA in aging of the cardiovascular system. Methods and results TERRA molecules from different chromosomes were upregulated in the hearts of old mice compared to young mice (p=0.002). Increased TERRA expression was also shown in heart tissue of patients with ischemic heart disease compared to donors (p=0.001). In vitro an upregulation of the TERRA molecule transcribed from chromosome 20 (h20q-TERRA) was found with increasing passage in human umbilical vein endothelial cells (HUVECs) (p=0.014) and IPSC-derived cardiomyocytes (p=0.011). After h20q-TERRA knockdown with LNA GapmeRs, HUVECs show less sprout formation in a spheroid assay compared to negative control transfected HUVECs (p=0.002), without showing a change in migration (p=0.205) or proliferation (p=0.114). H20q-TERRA knockdown revealed an increase in apoptosis (p=0.015) and telomeric DNA damage (p=0.011) and a decrease in telomere length (p<0.001), while lentiviral TERRA-repeat overexpression had the opposite effect (p=0.016, p=0.031, p<0.001, resp.). Apoptosis (p=0.012) and telomeric DNA damage (p=0.007) were also increased after the knockdown of h20q-TERRA in human cardiomyocytes. An apoptosis pathway profiler array in HUVECs showed that the expression of the antioxidant PON2 was decreased after knockdown of h20q-TERRA (p=0.040). PON2 expression was increased after TERRA overexpression (p=0.003). RNA immunoprecipitation revealed that TERRA can bind to PON2. Silencing PON2 in TERRA overexpressing cells diminished the TERRA-mediated decrease in caspase activation, suggesting a detrimental role for PON2 in caspase activation and endothelial cell survival. Conclusion Our data demonstrates that TERRA is upregulated with ageing and plays a role in endothelial and cardiomyocyte function and survival. Funding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): Horizon 2020

Identification of a long noncoding RNA Gm17501 as a novel negative regulator of cardiac hypertrophy

Pathological cardiac hypertrophy is an independent risk factor for the development of heart failure. Long noncoding RNAs (lncRNAs), an emerging class of non-protein-coding transcripts, are involved in regulation of multiple cardiac diseases through diverse molecular mechanism, whereas the role of cytoplasmic lncRNAs in regulating cardiac hypertrophy remains unclear. In this study, we identified a novel and functional long noncoding RNA Gm17501, which was predominantly expressed in the cytoplasm of cardiomyocytes. The expression level of lncRNA Gm17501 was altered in cardiac hypertrophy induced by pressure overload and phenylephrine treatment. Moreover, lncRNA Gm17501 expression was decreased in the heart tissue of patients with heart failure. Silencing lncRNA Gm17501 aggravated cardiac hypertrophy under pathological stress. Inhibition of lncRNA Gm17501 did not alter the expression of nearby genes but decreased mRNA level of calcium handling proteins which were involved in cardiac contraction. Therefore, the cytoplasmic lncRNA Gm17501 might protect cardiomyocytes against hypertrophy, possibly by maintaining calcium signaling pathway.

Integrated insights into the mechanisms underlying sepsis-induced myocardial depression using a quantitative global proteomic analysis

Sepsis-induced myocardial depression is common among patients in the intensive care unit; however, the exact mechanisms underlying this condition remain unclear. We investigated differences in the expression of specific proteins and determined the potential functions of the proteins in a rat model of lipopolysaccharide-induced septic shock. Left ventricular tissue was excised from 16 rats (sepsis group, 8; control group, 8) and analysed. Quantitative analysis of the global proteome was performed using 4D label-free technique. Bioinformatic analyses were conducted based on differentially expressed (DE) proteins. Parallel reaction monitoring (PRM) validation for selected proteins and western blotting for selected global protein modifications in heart tissues were also performed. As a result, out of 3653 proteins identified, 108 were expressed differentially between the two groups. The bioinformatic analyses revealed that DE proteins play important roles in metabolism- and immune-related pathways. PRM results supported the plausibility and reliability of the proteomics data. Modification of heart tissue acetyllysine, succinyllysine, 2-hydroxyisobutyryllysine, and lactyllysine revealed clear differences between the two groups, indicating the effects of protein modification. Our study suggested that expression patterns of global proteins in heart tissue were different between the two groups. These results provide new valuable information on the possible mechanisms underlying sepsis-induced myocardial depression. SignificanceThe expression patterns of global proteins in the heart tissues of patients with sepsis and control groups remain unknown. In this study, we used the 4D label-free proteomics technique to compare differentially expressed (DE) proteins between the sepsis and control groups. We identified 3653 proteins, 108 of which were expressed differentially between the sepsis and control groups. Further bioinformatic analyses revealed that DE proteins play critical roles in metabolism- and immune-related processes and pathways. Interestingly, modification of heart tissue acetyllysine, succinyllysine, 2-hydroxyisobutyryllysine, and lactyllysine revealed clear differences between the sepsis and control groups. The findings of this study improve our understanding of the basic molecular mechanisms underlying sepsis-induced myocardial depression.

Identification and Validation of Ferroptosis-Related Biomarkers in Septic Cardiomyopathy via Bioinformatics Analysis.

Septic cardiomyopathy (SCM) is a cardiac dysfunction caused by severe sepsis and septic shock that increases the risk of heart failure and death and its molecular mechanism remains unclear. Ferroptosis, a novel form of programmed cell death, has been reported to be present in the heart tissue of patients with sepsis, which demonstrated that ferroptosis may be a potential mechanism of myocardial injury in SCM. Therefore, we explored the role of ferroptosis-related genes (FRGs) in SCM and aimed to identify pivotal ferroptosis-related targets in SCM and potential therapeutic targets involved in the pathological process of SCM. To explore the regulatory mechanisms of ferroptosis in SCM, we identified differentially expressed genes (DEGs) in SCM and FRGs by bioinformatics analysis, and further identified hub genes. And the crucial microRNAs (miRNAs)-FRGs regulatory network was subsequently constructed. Finally, several candidate drugs associated with the hub genes were predicted, and Real-time quantitative reverse Transcription PCR (qRT-PCR) and western blotting analysis were performed to confirm the abnormal expression of hub genes. In this study, we identified several FRGs that may be involved in the pathogenesis of SCM, which helps us further clarify the role of ferroptosis in SCM and deeply understand the molecular mechanisms and potential therapeutic targets of SCM.

MiR-29b-3p Inhibitor Alleviates Hypomethylation-Related Aberrations Through a Feedback Loop Between miR-29b-3p and DNA Methylation in Cardiomyocytes.

As a member of the miR-29 family, miR-29b regulates global DNA methylation through target DNA methyltransferases (DNMTs) and acts as both a target and a key effector in DNA methylation. In this study, we found that miR-29b-3p expression was inversely correlated with DNMT expression in the heart tissues of patients with congenital heart disease (CHD), but whether it interacts with DNMTs in cardiomyocytes remains unknown. Further results revealed a feedback loop between miR-29b-3p and DNMTs in cardiomyocytes. Moreover, miR-29b-3p inhibitor relieved the deformity of hypomethylated zebrafish and restored the DNA methylation patterns in cardiomyocytes, resulting in increased proliferation and renormalization of gene expression. These results suggest mutual regulation between miR-29b-3p and DNMTs in cardiomyocytes and support the epigenetic normalization of miRNA-based therapy in cardiomyocytes.

RADIATION-INDUCED DAMAGE TO THE CARDIOVASCULAR SYSTEM AFTER RADIATION THERAPY IN WOMEN WITH BREAST CANCER.

Despite current research in the development of drugbased cytostatic and targeted therapies in the treatment of breast cancer, radiation therapy (RT) is an important component in the treatment of this pathology in the postoperative period. At the same time, an important problem is the development of pathological changes in the heart in the longterm period, which worsen the quality of life and increase the risk of cardiac death 10 or more years after RT The problem of heart damage due to RT is gaining new significance due to the existing increase in the number of cancer patients requiring radiation exposure in the modern world. RT of malignant tumors of the breast and organs of the chest cavity causes damage to all structures of the heart, including the coronary arteries, valves, conducting system, pericardium. The frequency of heart damage increases in proportion to the time that has passed after RT. According to some authors, the prevalence of clinically significant radiationinduced heart diseases 5-10 years after RT is 10-30%, and the frequency of asymptomatic lesions is 88%. The duration of the latent period can reach 15-20 years. The work has an analysis of longterm studies of the presence of pathological changes that are found in the heart tissues of patients with breast cancer, as a result of radiation therapy.

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.

Circular RNA hsa_circ_105039 promotes cardiomyocyte differentiation by sponging miR‑17 to regulate cyclinD2 expression.

Previously it was found that hsa_circ_105039 was underexpressed in the heart tissue of patients with congenital heart disease (CHD). However, the function and mechanism of hsa_circ_105039 in CHD are unclear. In the present study, induced pluripotent stem (iPS) cells were differentiated into cardiomyocytes using 1% dimethyl sulfoxide (DMSO). Cell differentiation, viability, migration and apoptosis were measured before and following hsa_circ_105039 knockdown or overexpression. The results indicated that hsa_circ_105039 overexpression promoted cell differentiation, viability and migration; whereas apoptosis was simultaneously repressed. A luciferase reporter assay verified that hsa_circ_105039 acted as a sponge for microRNA (miR)-17 and that cyclinD2 was a direct target of miR-17. Furthermore, differentiation-related genes and proteins were analyzed by reverse transcription-quantitative PCR and western blotting, respectively. The results showed that hsa_circ_105039 could also upregulate the expression of differentiation-related genes and proteins, including natriuretic peptide A, cardiac troponin I, GATA-binding protein 4 and homobox transcription factor, in iPS cells. The results suggested that hsa_circ_105039 exerted a protective effect by promoting miR-17/cyclinD2 in DMSO-induced iPS cardiomyocytes, which indicated that hsa_circ_105039 is a potential key molecule for the diagnosis of CHD.

Enrichment analysis of differentially expressed genes in chronic heart failure.

The study explores the differentially expressed genes in the heart tissue of patients with chronic heart failure (CHF) and normal heart tissue, thus providing information for further research on the pathogenesis of CHF. The Gene Expression Omnibus (GEO) database was used to download the whole transcriptome sequencing results of CHF patients (GSE2656, n=49). Transcriptome sequencing results of 44 normal left ventricular tissues were randomly screened and downloaded using the Genotype-Tissue Expression (GTEX) database (n=44). We explored the differentially expressed genes between CHF tissue and normal heart tissue. Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis were performed for differentially expressed genes. Growth hormone-releasing hormone (GHRH) was used as a representative differential gene for serological sample verification by the enzyme linked immunosorbent assay (ELISA). A total of 902 differentially expressed genes between CHF and normal heart tissues were screened, including 354 up-regulated genes and 548 down-regulated genes. GO enrichment analysis showed that the differentially expressed genes were significantly enriched in the extracellular and sequence-specific DNA binding domains. KEGG enrichment demonstrated that the differential genes were enriched in neuroactive ligand-receptor interaction, the calcium signaling pathway, vascular smooth muscle contraction, and other signaling pathways. ELISA results showed that the expression level of GHRH in patients with heart failure was significantly higher than that in healthy subjects (P<0.05). A total of 902 differentially expressed genes were found in CHF tissues compared with normal heart tissues. Signaling pathways such as neuroactive ligand-receptor interaction, the calcium ion signaling pathway, and vascular smooth muscle contraction may be related to the pathogenesis of CHF.

TAX1BP1 protects against myocardial infarction-associated cardiac anomalies through inhibition of inflammasomes in a RNF34/MAVS/NLRP3-dependent manner

Acute myocardial infarction (MI), one of the most common cardiovascular emergencies, is a leading cause of morbidity and mortality. Ample evidence has revealed an essential role for inflammasome activation and autophagy in the pathogenesis of acute MI. Tax1-binding protein 1 (TAX1BP1), an adaptor molecule involved in termination of proinflammatory signaling, serves as an important selective autophagy adaptor, but its role in cardiac ischemia remains elusive. This study examined the role of TAX1BP1 in myocardial ischemic stress and the underlying mechanisms involved. Levels of TAX1BP1 were significantly downregulated in heart tissues of patients with ischemic heart disease and in a left anterior descending (LAD) ligation-induced model of acute MI. Adenovirus carrying TAX1BP1 was delivered into the myocardium. The acute MI induced procedure elicited an infarct and cardiac dysfunction, the effect of which was mitigated by TAX1BP1 overexpression with little effect from viral vector alone. TAX1BP1 nullified acute MI-induced activation of the NLRP3 inflammasome and associated mitochondrial dysfunction. TAX1BP1 overexpression suppressed NLRP3 mitochondrial localization by inhibiting the interaction of NLRP3 with mitochondrial antiviral signaling protein (MAVS). Further investigation revealed that ring finger protein 34 (RNF34) was recruited to interact with TAX1BP1 thereby facilitating autophagic degradation of MAVS through K27-linked polyubiquitination of MAVS. Knockdown of RNF34 using siRNA nullified TAX1BP1 yielded protection against hypoxia-induced MAVS mitochondrial accumulation, NLRP3 inflammasome activation and associated loss of mitochondrial membrane potential. Taken together, our results favor a cardioprotective role for TAX1BP1 in acute MI through repression of inflammasome activation in a RNF34/MAVS-dependent manner.

A single injection of periostin decreases cardiac voltage-gated Na+ channel in rat ventricles.

Changes in electrophysiological properties, such as ion channel expression and activity, are closely related to arrhythmogenesis during heart failure (HF). However, a causative factor for the electrical remodeling in HF has not been determined. Periostin (POSTN), a matricellular protein, is increased in heart tissues of patients with HF. In the present study, we investigated whether a single injection of POSTN affects the electrophysiological properties in rat ventricles. After male Wistar rats were intravenously injected with recombinant rat POSTN (64 µg/kg, 24 hr), electrocardiogram (ECG) was recorded. Whole-cell patch clamp was performed to measure action potential (AP) and Na+ current (INa) in isolated ventricular myocytes. Protein expression of cardiac voltage-gated Na+ channel (NaV1.5) in isolated ventricles was examined by Western blotting. In ECG, POSTN-injection significantly increased RS height. POSTN-injection significantly delayed time to peak in AP and decreased INa in the isolated ventricular myocytes. POSTN-injection decreased NaV1.5 expression in the isolated ventricles. It was confirmed that POSTN (1 µg/ml, 24 hr) decreased INa and NaV1.5 protein expression in neonatal rat ventricular myocytes. This study for the first time demonstrated that a single injection of POSTN in rats decreased INa by suppressing NaV1.5 expression in the ventricular myocytes, which was accompanied by a prolongation of time to peak in AP and an increase of RS height in ECG.

Long non-coding RNA TERRA influences DNA damage and survival of endothelial cells and cardiomyocytes

Abstract Ageing is the major risk factor for cardiovascular disease. Current therapies are mainly based on proteins, while targeting long non-coding RNAs (lncRNAs) is largely unexplored. Long non-coding RNAs are emerging as novel regulators of cellular functions and contributors to cardiovascular ageing. Although telomeres are heterochromatic regions, non-coding transcripts called Telomeric repeat-containing RNA (TERRA) are transcribed from the telomeres of most chromosomes. The transcription of TERRA starts at the subtelomere and ends in the telomere, leading to molecules of 0.2–10kb. This study aims to characterize the role of TERRA in the cardiovascular system. TERRA molecules from different chromosomes were upregulated in the hearts of old mice compared to young mice (p=0.002). An increased TERRA expression was also shown in heart tissue of patients with ischemic heart disease compared to donor heart tissue (p=0.001). In vitro an upregulation of the TERRA molecule transcribed from chromosome 20 (h20q-TERRA) was found in old passage human umbilical vein endothelial cells (HUVECs) (P15–17) compared to young HUVECs (P3) (p=0.014). IPSC-derived cardiomyocytes also increased the expression of h20q-TERRA with increasing passage (p=0.011). After knockdown of h20q-TERRA with LNA GapmeRs HUVECs show less sprout formation in a spheroid assay compared to negative control transfected HUVECs (p=0.002), without showing a change in migration (p=0.205) or proliferation (p=0.114). H20q-TERRA knockdown revealed that there was more apoptosis (p=0.015), more DNA damage as measured with the comet assay (p&amp;lt;0.001), increased γH2AX levels (p=0.029) and an increase in γH2AX colocalization with the telomere (p=0.011). The amount of phosphorylated P53 was also increased after knockdown (p=0.038), while it was decreased after TERRA overexpression (p=0.001). Inhibition of the ATM-yH2AX-P53 pathway did however not reduce the increased apoptosis after knockdown (KU60019 p=0.399; siP53 p=0.303). Silencing the m18-TERRA molecule in mouse endothelial H5V cells led to an increase in caspase activity (p=0.004) similar to what was shown in HUVECs. In addition, increased caspase activity (p=0.012), increased γH2AX levels (p&amp;lt;0.001) and increased γH2AX colocalization with the telomere (p=0.007) was also shown after silencing of h20q-TERRA in human cardiomyocytes. In summary, our data demonstrates that TERRA is upregulated with ageing and plays a role in endothelial and cardiomyocyte function and survival. These data show that TERRA transcripts are induced in cardiovascular ageing and are essential for endothelial cell function. Funding Acknowledgement Type of funding source: Public grant(s) – EU funding. Main funding source(s): Horizon 2020

Production and characterization of elastomeric cardiac tissue-like patches for Myocardial Tissue Engineering

Cardiovascular disease remains the leading cause of death. Damaged heart muscle is the etiology of heart failure. Heart failure is the most frequent cause of hospital and emergency room admissions. As a differentiated organ, current therapeutics and techniques can not repair or replace the damaged myocardial tissue. Myocardial tissue engineering is one of the promising treatment modalities for repairing damaged heart tissue in patients with heart failure. In this work, random Polylactic acid (PLA), Polylactic acid/Polyethylene glycol (PLA/PEG) and random and aligned Polylactic acid/Polyethylene glycol/Collagen (PLA/PEG/COL) nanofiber patches were successfully produced by the electrospinning technique. In vitro cytotoxic test (MTT), morphological (SEM), molecular interactions between the components (FT-IR), thermal analysis (DSC), tensile strength and physical analysis were carried out after production. The resulting nanofiber patches exhibited beadless and smooth structures. When the fiber diameters were examined, it was observed that the collagen doped random nanofiber patches had the lowest fiber diameter value (755 nm). Mechanical characterization results showed that aligned nanofiber patches had maximum tensile strength (5.90 MPa) values compared to PLA, PLA/PEG, and PLA/PEG/COL (random). In vitro degradation test reported that aligned patch had the highest degradation ratio. The produced patches displayed good alignment with tissue on cardiomyocyte cell morphology studies. In conclusion, newly produced patches have noticeable potential as a tissue-like cardiac patch for regeneration efforts after myocardial infarction.

Variability in Cardiac miRNA-122 Level Determines Therapeutic Potential of miRNA-Regulated AAV Vectors

Systemically delivered adeno-associated viral vector serotype 9 (AAV9) effectively transduces murine heart, but provides transgene expression also in liver and skeletal muscles. Improvement of the selectivity of transgene expression can be achieved through incorporation of target sites (TSs) for miRNA-122 and miRNA-206 into the 3′ untranslated region (3′ UTR) of the expression cassette. Here, we aimed to generate such miRNA-122- and miRNA-206-regulated AAV9 vector for a therapeutic, heart-specific overexpression of heme oxygenase-1 (HO-1). We successfully validated the vector functionality in murine cell lines corresponding to tissues targeted by AAV9. Next, we evaluated biodistribution of transgene expression following systemic vector delivery to HO-1-deficient mice of mixed C57BL/6J × FVB genetic background. Although AAV genomes were present in the hearts of these animals, HO-1 protein expression was either absent or significantly impaired. We found that miRNA-122, earlier described as liver specific, was present also in the hearts of C57BL/6J × FVB mice. Various levels of miRNA-122 expression were observed in the hearts of other mouse strains, in heart tissues of patients with cardiomyopathy, and in human induced pluripotent stem cell-derived cardiomyocytes in which we also confirmed such posttranscriptional regulation of transgene expression. Our data clearly indicate that therapeutic utilization of miRNA-based regulation strategy needs to consider inter-individual variability.

Indexes of Angiogenic Activation in Myocardial Samples of Patients with Advanced Chronic Heart Failure.

Background and objectives: Ischemic and idiopathic heart failure are characterized by reactive cardiac fibrosis and impaired vasculogenesis involving pro-angiogenic factors such as angiogenin, angiopoietin-1 (Ang-1), and angiopoietin-2 (Ang-2), as demonstrated in experimental models of heart failure. However, differences in the molecular pathways between these cardiomyopathies are still unclear. In this short communication, we evaluate and compare the expression of pro-angiogenic molecules in the heart tissue of patients with advanced chronic heart failure (CHF) of ischemic vs. nonischemic etiology. Materials and Methods: We obtained heart tissue at transplantation from left ventricular walls of 16 explanted native hearts affected by either ischemic (ICM) or nonischemic dilated cardiomyopathy (NIDCM). Tissue samples were examined using immunohistochemistry for angiogenic molecules. Results: We found immunopositivity (I-pos) for angiopoietin-1 mainly in the cardiomyocytes, while we observed I-pos for Ang-2 and Tie-2 receptor mainly in endothelial cells. Expression of Procollagen-I (PICP), angiogenin, Ang-1, and Tie-2 receptor was similar in ICM and NIDCM. In contrast, endothelial immunopositivity for Ang-2 was higher in ICM samples than NIDCM (p = 0.03). Conclusions: In our series of CHF heart samples, distribution of Ang-1 and angiogenin was higher in cardiomyocytes while that of Ang-2 was higher in endothelial cells; moreover, Ang-2 expression was higher in ICS than NIDCM. Despite the small series examined, these findings suggest different patterns of angiogenic stimulation in ICM and NIDCM, or at least a more altered endothelial integrity in ICD. Our data may contribute to a better understanding of the angiogenesis signaling pathways in CHF. Further studies should investigate differences in the biochemical processes leading to heart failure.

Toll-like receptor-mediated inflammation markers are strongly induced in heart tissue in patients with cardiac disease under both ischemic and non-ischemic conditions

BackgroundA sustained low grade inflammatory state is a recognized feature of various diseases, including cardiovascular disease. This state of chronic inflammation involves activation of Toll-like receptor (TLR) signaling. However, little is known regarding the genetic profile of TLR components in cardiac tissue from patients with cardiac disease. MethodsIn this study we investigated the genetic profile of 84 TLR markers in a unique set of cardiac tissue from patients that had undergone either coronary artery bypass grafting (CABG) or aortic valve replacement (AVR). In addition, we compared the gene data from the cardiac tissue with the same gene profile in blood as well as circulating cytokines to elucidate possible targets in blood that could be used to estimate the inflammatory state of the heart in cardiac disease. ResultsWe found a marked upregulation of TLR-induced inflammation in cardiac tissue from both patient groups compared to healthy controls. The inflammation appeared to be primarily mediated through TLR1, 3, 7, 8 and 10, resulting in a marked induction of mediators of the innate immune response. Furthermore, the gene expression data in combination with unbiased multivariate analysis suggested a difference in inflammatory response in ischemic cardiac tissue compared to non-ischemic cardiac tissue. Serum levels of IL-13 were significantly elevated in both CABG and AVR patients compared to controls, whereas other cytokines did not appear to coincide with cardiac TLR-induced inflammation. ConclusionsWe propose that cardiac disease in humans may be mediated by local cardiac TLR signaling under both ischemic and non-ischemic conditions.

HDAC11 regulates interleukin-13 expression in CD4+ T cells in the heart

Background and aimsImmune deregulation is a causative factor in pathogenesis of myocarditis. Histone deacetylases (HDAC) involve multiple biochemical activities in the cell. This study aims to elucidate the role of HDAC11 in the regulation of interleukin (IL)-13-expression in CD4+ T cells of heart tissue in patients with myocarditis (MCD). MethodsAfter heart transplantation, surgically removed hearts were collected from patients with advanced heart failure and MCD or dilated cardiomyopathy (DCM). CD4+ T cells were isolated from the heart samples and analyzed by immune assay. The association between IL-13 over production by CD4+ T cells in heart tissue and the pathogenesis of MCD was analyzed. ResultsT helper (Th) 2-biased inflammation was observed in hearts tissue of MCD patients with advanced heart failure. CD4+ T cells isolated from MCD heart tissue showed lower levels of HDAC11 expression than that isolated from DCM heart tissue. HDAC11 was negatively correlated with IL-13 expression in the CD4+ T cells. A complex of HDAC11 and E4 binding protein-4 (E4BP4; the transcription factor of IL13) was detected in the CD4+ T cells, which restricted the binding between E4BP4 and the Il13 promoter to repress the Il13 gene transcription. Reconstitution of HDAC11 in MCD CD4+ T cells reduced the expression of IL-13, while inhibition of HDAC11 in DCM CD4+ T cells increased the IL-13 expression. ConclusionsHDAC11 is a regulatory molecule in Th2 response and plays a critical role in the restriction of the biased IL-13 expression in CD4+ T cells of the heart.

Altered protein quality control in heart tissue of patients with hypertrophic cardiomyopathy

Background: Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease with a prevalence of 1:200 and is caused by mutations in genes encoding sarcomere proteins. Age of onset and disease-severity vary greatly between patients. The mechanisms that underlie this variation are unknown. Recent findings suggest an important role for secondary disease-modifiers to underlie HCM susceptibility. Because keeping mutant protein levels at a low level is an important function of the protein quality control (PQC) system, we hypothesize that impairment of the PQC system may contribute to HCM development.