Normal Heart Tissue Research Articles

LncRNA DCRT Protects Against Dilated Cardiomyopathy by Preventing NDUFS2 Alternative Splicing by Binding to PTBP1.

Dilated cardiomyopathy is characterized by left ventricular dilation and continuous systolic dysfunction. Mitochondrial impairment is critical in dilated cardiomyopathy; however, the underlying mechanisms remain unclear. Here, we explored the cardioprotective role of a heart-enriched long noncoding RNA, the dilated cardiomyopathy repressive transcript (DCRT), in maintaining mitochondrial function. The DCRT knockout (DCRT-/-) mice and DCRT knockout cells were developed using CRISPR-Cas9 technology. Cardiac-specific DCRT transgenic mice were generated using α-myosin heavy chain promoter. Chromatin coimmunoprecipitation, RNA immunoprecipitation, Western blot, and isoform sequencing were performed to investigate the underlying mechanisms. We found that the long noncoding RNA DCRT was highly enriched in the normal heart tissues and that its expression was significantly downregulated in the myocardium of patients with dilated cardiomyopathy. DCRT-/- mice spontaneously developed cardiac dysfunction and enlargement with mitochondrial impairment. DCRT transgene or overexpression with the recombinant adeno-associated virus system in mice attenuated cardiac dysfunction induced by transverse aortic constriction treatment. Mechanistically, DCRT inhibited the third exon skipping of NDUFS2 (NADH dehydrogenase ubiquinone iron-sulfur protein 2) by directly binding to PTBP1 (polypyrimidine tract binding protein 1) in the nucleus of cardiomyocytes. Skipping of the third exon of NDUFS2 induced mitochondrial dysfunction by competitively inhibiting mitochondrial complex I activity and binding to PRDX5 (peroxiredoxin 5) and suppressing its antioxidant activity. Furthermore, coenzyme Q10 partially alleviated mitochondrial dysfunction in cardiomyocytes caused by DCRT reduction. Our study revealed that the loss of DCRT contributed to PTBP1-mediated exon skipping of NDUFS2, thereby inducing cardiac mitochondrial dysfunction during dilated cardiomyopathy development, which could be partially treated with coenzyme Q10 supplementation.

The effects of high doses of selenium supplementation on mRNA and protein levels of cMLCK levels and total antioxidant capacity in rat heart tissue

BackgroundHigh doses of selenium are associated with heart disease prevalence in high-risk areas. Cardiac myosin light chain kinase (cMLCK) is an essential enzyme for normal function of heart tissue. Therefore, we studied the effect of high doses of selenium on the expression of cMLCK gene and its protein in normal heart tissue in rats. Materials and methodsTwenty male rats were randomly divided into four groups: control, Se 0.3mg/kg, Se 1.5mg/kg, and Se 3mg/kg. Sodium-selenite was administered orally into drinking water for 20 weeks. Se levels of heart tissue were measured by atomic absorption. Serum creatine phosphokinase (CPK) and total serum antioxidant capacity were measured. Moreover, the concentration of MLCK protein and the gene expression level of cMLCK in normal heart tissue were analyzed. ResultsExcess Se in dietary can significantly increase CPK. Se concentration of heart tissue in the Se 3mg/kg group was significantly higher than the control. cMLCK mRNA levels were decreased by 0.3mg/kg and 3mg/kg sodium selenite intake. There was no significant difference between the three groups for total antioxidant capacity and MLCK protein. ConclusionHigh concentrations of selenium can probably effect on normal function of the heart tissue by changing the expression levels of cMLCK.

Revealing the crucial roles of myxoma cells and CD206+ macrophages in cardiac myxoma progression based on single-cell sequencing.

e23549 Background: Cardiac myxoma is a commonly encountered tumor within the heart that has the potential to be life-threatening. However, the cellular composition and molecular characteristics of this condition are still not well understood. Methods: We performed single-cell sequencing on cardiac myxoma tissues obtained from 5 patients, focusing on the identification and characterization of cellular subtypes, cell trajectory analysis, and intercellular communication analysis. We performed a comparative analysis of the changes in cell types and states between cardiac myxoma and normal heart tissues. Furthermore, we retrospectively collected pathological sections from 49 cardiac myxoma cases and employed the multi-color immunohistochemistry (mIHC) technology to analyze the relationship between cellular infiltration and the severity of disease. Results: In total, 75,641 cells in cardiac myxoma tissues formed 9 distinct cell populations. Myxoma cells exhibited a significant resemblance to fibroblasts, yet they were distinguished by an increased expression of phosphodiesterases and genes associated with cell proliferation, differentiation, adhesion, and migration. Myxoma cells displayed cellular heterogeneity and demonstrated two distinct evolutionary pathways. One involves the transition from stem cell-like cells to stromal cells and the other involves enhancement of blood coagulation function. Immune cells surrounding the myxoma displayed suppressive features, with the presence of CD8+ T cells expressing TOX and GZMK, as well as a decrease in the secretion of cytotoxic molecules, indicating an impaired effector function. Macrophages were found to express growth factors such as EREG and PDGFC which played a role in promoting myxoma growth. Conversely, myxoma cells were also observed to secrete ANGPTL4 and ANXA1 which in turn induced the transformation of CD206+ macrophages. Extensive mIHC experiments indicated that a higher proportion of CD206+macrophages and myxoma cells infiltrating the myxoma tissues, along with their enhanced spatial interaction, were found to significantly contribute to the increased occurrence of embolism in cardiac myxoma patients. Conclusions: In summary, this study presents a comprehensive single-cell atlas of cardiac myxoma, highlighting the heterogeneity of myxoma cells and their collaborative impact on immune cells. These findings shed light on the complex pathobiology of cardiac myxoma and present potential targets for intervention.

Mechanical and electrical uncoupling: the role of cardiac magnetic resonance imaging in arrhythmogenic cardiomyopathy proof of concept

Abstract Funding Acknowledgements None. Introduction Arrhythmogenic Cardiomyopathy (ACM) has a high risk of sudden cardiac death, especially under 60 years old (1:5,000 prevalence). ACM involves the replacement of normal heart tissue with fibro-adipose tissue in specific areas of the right ventricle (RV). Cardiac Magnetic Resonance Imaging (CMR) is vital for ACM stratification and prognosis, focusing on RV volume and ejection fraction. Simulations suggest that ACM's abnormal deformation patterns are primarily due to mechanical changes like reduced contractility and increased stiffness, rather than electrical issues. Ventriculo-arterial coupling (VAC) is a valuable metric for assessing RV efficiency, with the TAPSE/sPAP index reflecting the RV's length-force relationship. CMR assists in ACM stratification and prognosis, with VAC potentially serving as a key predictor of outcomes based on mechanical and structural abnormalities identified through CMR. Purpose Examine and establish a relationship between mechanical uncoupling, ventricular arrhythmias, and findings from CMR in patients with ACM. Methods We examined 25 ACM patients (median age 34, 72% men) using the 2019 Modified Task Force and Padua criteria. We investigated two key factors in ACM: Right ventricular-arterial coupling (TAPSE/SPAP ratio) and clinically relevant ventricular tachycardia (requiring cardioversion). We compared clinical, ECG, and imaging data (transthoracic echocardiogram and CMR as the gold standard). We used Spearman rank tests for correlations, Fisher exact tests for nominal characteristics, and Mann-Whitney-U tests for group comparisons. In predefined regression models involving left ventricular ejection fraction (LVEF) and right ventricular ejection fraction (RVEF) function with VAC, we used flexible nonparametric regressions. A p-value <0.05 was considered statistically significant. The analysis was conducted using SPSSv22 and SAS-University-Edition. Results A positive association of VAC with LVEF (ρ=0.472, p=0.023), RVEF (ρ=0.522, p=0.038) and RV indexed stroke volume (ρ=0.79, p<0.001) was found. Low-rank regression splines showed a mainly positive association of LVEF (df=3 F=20.75 p<0.001), RVEF (df=4 F=24.34 p<0.001) and the RV indexed stroke volume, a positive linear association (df=1 F=31.67 p<0.001). In comparison with patients without crVT, those with crVT had a positive correlation with RV shortening, reduced RVEF (39.6 vs. 32.2%. p=0.025), higher LV mass (38.99 vs. 45.55, p=0.045), and LV end-diastolic volume (LVEDV) (56.99 vs. 68.15 ml/m2, p=0.045) by echocardiogram and CMR respectively; notwithstanding positive associations for VAC were seen with LVEDV (p=0.039), LV mass (p=0.039); and negative with RVEF by CMR (p=0.023), and RV shortening by echo (p=0.026). Conclusions We found that right VAC by echo correlates with RV and LV EF by CMR and RV stroke volume in patients with ACM. RVEF, LV mass, and end-diastolic volume correlate with clinically relevant arrhythmias.

Extracellular vesicles derived from different tissues attenuate cardiac dysfunction in murine MI models

BackgroundExtracellular vesicles (EVs) derived from various cell sources exert cardioprotective effects during cardiac ischemic injury. Our previous study confirmed that EVs derived from ischemic-reperfusion injured heart tissue aggravated cardiac inflammation and dysfunction. However, the role of EVs derived from normal cardiac tissue in myocardial ischemic injury remains elusive.ResultsIn the present study, normal heart-derived EVs (cEVs) and kidney-derived EVs (nEVs) were isolated and intramyocardially injected into mice after myocardial infarction (MI). We demonstrated that administration of both cEVs and nEVs significantly improved cardiac function, reduced the scar size, and alleviated inflammatory infiltration into the heart. In addition, cardiomyocyte apoptosis was inhibited, whereas angiogenesis was enhanced in the hearts receiving cEVs or nEVs treatment. Moreover, intramyocardial injection of cEVs displayed much better cardiac protective efficacy than nEVs in murine MI models. RNA-seq and protein-protein interaction (PPI) network analysis revealed the protective mRNA clusters in both cEVs and nEVs. These mRNAs were involved in multiple signaling pathways, which may synergistically orchestrate to prevent the heart from further damage post MI.ConclusionsCollectively, our results indicated that EVs derived from normal heart tissue may represent a promising strategy for cardiac protection in ischemic heart diseases.

Integrative Proteomic Analysis Reveals the Cytoskeleton Regulation and Mitophagy Difference Between Ischemic Cardiomyopathy and Dilated Cardiomyopathy

Ischemic cardiomyopathy (ICM) and dilated cardiomyopathy (DCM) are the two primary etiologies of end-stage heart failure. However, there remains a dearth of comprehensive understanding the global perspective and the dynamics of the proteome and phosphoproteome in ICM and DCM, which hinders the profound comprehension of pivotal biological characteristics as well as differences in signal transduction activation mechanisms between these two major types of heart failure. We conducted high-throughput quantification proteomics and phosphoproteomics analysis of clinical heart tissues with ICM or DCM, which provided us the system-wide molecular insights into pathogenesis of clinical heart failure in both ICM and DCM. Both protein and phosphorylation expression levels exhibit distinct separation between heart failure and normal control heart tissues, highlighting the prominent characteristics of ICM and DCM. By integrating with omics results, Western blots, phosphosite-specific mutation, chemical intervention, and immunofluorescence validation, we found a significant activation of the PRKACA–GSK3β signaling pathway in ICM. This signaling pathway influenced remolding of the microtubule network and regulated the critical actin filaments in cardiac construction. Additionally, DCM exhibited significantly elevated mitochondria energy supply injury compared to ICM, which induced the ROCK1–vimentin signaling pathway activation and promoted mitophagy. Our study not only delineated the major distinguishing features between ICM and DCM but also revealed the crucial discrepancy in the mechanisms between ICM and DCM. This study facilitates a more profound comprehension of pathophysiologic heterogeneity between ICM and DCM and provides a novel perspective to assist in the discovery of potential therapeutic targets for different types of heart failure.

Discovery of novel benzofuran-based derivatives as acetylcholinesterase inhibitors for the treatment of Alzheimer's disease: Design, synthesis, biological evaluation, molecular docking and 3D-QSAR investigation

A series of novel benzofuran-based compounds 7a-s were designed, synthesized, and investigated in vitro as acetylcholinesterase inhibitors (AChEIs). Compounds 7c and 7e displayed promising inhibitory activity with IC50 values of 0.058 and 0.086 μM in comparison to donepezil with an IC50 value of 0.049 μM.The new molecules' antioxidant evaluation revealed that 7c, 7e, 7j, 7n, and 7q produced the strongest DPPH scavenging activity when compared to vitamin C. As it was the most promising AChEI, compound 7c was selected for further biological evaluation. Acute and chronic toxicity studies exhibited that 7c showed no signs of toxicity or adverse events, no significant differences in the blood profile, and an insignificant difference in hepatic enzymes, glucose, urea, creatinine, and albumin levels in the experimental rat group. Furthermore, 7c did not produce histopathological damage to normal liver, kidney, heart, and brain tissues, ameliorated tissue malonaldehyde (MDA) and glutathione (GSH) levels and reduced the expression levels of the APP and Tau genes in AD rats. Molecular docking results of compounds 7c and 7e showed good binding modes in the active site of the acetylcholinesterase enzyme, which are similar to the native ligand donepezil. 3D-QSAR analysis revealed the importance of the alkyl group in positions 2 and 3 of the phenyl moiety for the activity. Overall, these findings suggested that compound 7c could be deemed a promising candidate for the management of Alzheimer's disease.

Selection of key genes for dilated cardiomyopathy based on machine learning algorithms and assessment of diagnostic accuracy.

The mechanisms of the occurrence and progression of dilated cardiomyopathy are still unclear and further exploration is needed. The upgrading of programming languages and the improvement of biological databases have created conditions for us to explore the structural and functional information of biological molecules at the nucleic acid and protein levels, screen key pathogenic genes, and elucidate pathogenic mechanisms. This study aimed to screen key pathogenic genes using machine learning algorithms and explore the correlation between key genes and immune microenvironment through transcriptome sequencing data sets of myocardial samples from patients with dilated cardiomyopathy, providing new ideas for elucidating the pathogenesis of the disease. The transcriptome sequencing data sets of heart tissue from patients with dilated cardiomyopathy were downloaded from the Gene Expression Omnibus (GEO) database (GSE29819 and GSE21610). Differentially expressed genes (DEGs) were screened between pathological and normal tissues. The key genes were screened using least absolute shrinkage and selection operator (LASSO) regression analysis and random forest tree algorithms. The diagnostic efficiency of the key genes for the disease was evaluated using the receiver operating characteristic (ROC) curve. Compared with the normal heart tissue (control group) samples, there were 213 DEGs in the heart tissue samples of patients with dilated cardiomyopathy (treat group), including 101 upregulated and 102 downregulated genes. CCL5 and CTGF were highly expressed in the treat group compared to the control group. The ROC curve showed that the areas under the curve (AUCs) of CCL5 and CTGF were 0.821 and 0.902, respectively (P<0.05). In the treat group samples, CCL5 was positively correlated with the infiltration content of most immune cell subtypes. CCL5 and CTGF are key disease-causing genes in dilated cardiomyopathy and have good diagnostic efficiency for the disease. CCL5 and CTGF may be related to immune cell enrichment and myocardial fibrosis, respectively.

1542-P: Commercially Available Aspartame-Based Sweetener Damages Morphology of Major Organs Both in Normal and Type 2 Diabetic Rats

Non-nutritive sweeteners (NNS) such as aspartame have become highly popular as sugar substitutes for diabetics. Majority of studies have reported controversial results and used aspartame in its pure form, which is not consumed by people practically. Hence, the present study was aimed at investigating the effects of commercially available aspartame-based sweetener on diabetes related parameters and its associated complications in both normal and type 2 diabetic rats. Seven-week-old male Sprague-Dawley rats were randomly divided into four groups, namely: Normal Control (NC), Toxicological Aspartame (TASP), Diabetic Control (DBC) and Diabetic Aspartame (DASP). Type 2 diabetes was induced experimentally in the DBC and DASP groups only and animals with non-fasting blood glucose levels &amp;gt;300 mg/dL were considered as diabetic. During 13 weeks experimental period, the control groups received normal drinking water, whilst TASP and DASP groups were given aspartame-containing NNS solutions, ad libitum, at concentrations equivalent to the sweetness of 20% and 10% sucrose solution, respectively. Treatment with toxicological aspartame resulted in significantly reduced serum fructosamine and creatinine levels in normal rats, when brain, heart, liver, kidney and pancreatic tissue morphologies were significantly damaged in both normal and diabetic rats. The data of this study suggest that although aspartame-based NNS may not have higher degree of detrimental effects on diabetes associated parameters, the significantly damaged morphology of the major organ tissues as well as deterioration of related serum biomarkers both in normal and diabetic conditions raises a concern of its use as a NNS in various food and food products. Disclosure S.N.Dlamini: None. S.Islam: None. Funding South African Sugar Association (Project 233)

Extracellular vesicle treatment partially reverts epigenetic alterations in chronically ischemic porcine myocardium.

Research has shown epigenetic change via alternation of the methylation profile of human skeletal muscle DNA after Cardio-Pulmonary Bypass (CPB). In this study, we investigated the change in epigenome-wide DNA methylation profiles of porcine myocardium after ischemic insult in the setting of treatment with extracellular vesicle (EV) therapy in normal vs. high-fat diet (HFD) pigs. Four groups of three pigs underwent ameroid constrictor placement to the left circumflex artery (LCx) and were assigned to the following groups: (1) normal diet saline injection; (2) normal diet EV injection; (3) HFD saline injection; and (4) HFD EV injection. DNA methylation was profiled via reduced-representation bisulfite sequencing (RRBS) and compared using a custom bioinformatic pipeline. After initial analysis, 441 loci had a nominal P value < 0.05 when examining the effect of ischemia vs. normal heart tissue on a normal diet in the absence of treatment. 426 loci at P value threshold < 0.05 were identified when comparing the ischemic vs. normal tissue from high-fat diet animals. When examining the effect of EV treatment in ischemic tissue in subjects on a normal diet, there were 574 loci with nominal P value < 0.05 with two loci Fructosamine 3 kinase related protein [(FN3KRP) (P < 0.001)] and SNTG1 (P = 0.03) significant after Bonferroni correction. When examining the effect of EV treatment in ischemic tissue in HFD, there were 511 loci with nominal P values < 0.05. After Bonferroni correction, two loci had P values less than 0.05, betacellulin [(BTC) (P = 0.008)] and [proprotein convertase subtilisin/kexin type 7 (PCSK7) (P = 0.01)]. Alterations in DNA methylation were identified in pig myocardium after ischemic insult, change in diet, and treatment with EVs. Hundreds of differentially methylated loci were detected, but the magnitude of the effects was low. These changes represent significant alterations in DNA methylation and merit further investigation.

Comparison of normal tissue doses in deep inspiration breath-hold and free breathing methods for radiotherapy of left-sided breast cancer using 4D-XCAT digital phantom.

To evaluate normal lung and heart tissue doses for treatment of left-sided breast cancer in deep inspiration breath-hold (DIBH) and free breathing (FB) as a function of breast size and diaphragm displacement using 4D-XCAT digital phantom in a simulation study. 4D-XCAT digital phantom was used to create 36 left-sided breast cancer digital phantom datasets with different breast height (BH) of 40, 50, and 60 mm, breast length (BL) of 16, 17, and 18 mm, and diaphragm excursion of 20, 25, 30, and 35 mm. For each dataset, DIBH and FB treatment plans were prepared using planning computerized radiotherapy-three dimensional (PCRT-3D) treatment planning system (TPS) with superposition computational algorithm. Dose differences in DIBH and FB plans were assessed in terms of mean lung dose (MLD), the lung volume receiving ≤20 Gy (V20), normal tissue complication probability (NTCP) of the lung, mean heart dose (MHD), and the heart volume receiving ≤30 (V30). DIBH reduced mean dose and V20 and NTCP of the lung in all cases, by up to 4.37 Gy, 7.62%, and 18.95%. Mean dose and V30 of the heart were also significantly reduced by 5.02 Gy and 8.23%. The use of DIBH for left-sided breast cancer radiotherapy offers excellent possibilities for sparing critical normal tissue without compromising radiation dose to the target.

Single-cell transcriptomics provides insights into hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a genetic heart disease that is characterized by unexplained segmental hypertrophy that is usually most pronounced in the septum. While sarcomeric gene mutations are often the genetic basis for HCM, the mechanistic origin for the heterogeneous remodeling remains largely unknown. A better understanding of the gene networks driving the cardiomyocyte (CM) hypertrophy is required to improve therapeutic strategies. Patients suffering from HCM often receive a septal myectomy surgery to relieve outflow tract obstruction due to hypertrophy. Using single-cell RNA sequencing (scRNA-seq) on septal myectomy samples from patients with HCM, we identify functional links between genes, transcription factors, and cell size relevant for HCM. The data show the utility of using scRNA-seq on the human hypertrophic heart, highlight CM heterogeneity, and provide a wealth of insights into molecular events involved in HCM that can eventually contribute to the development of enhanced therapies.

Autopsy Histopathologic Cardiac Findings in 2 Adolescents Following the Second COVID-19 Vaccine Dose

Autopsy Histopathologic Cardiac Findings in 2 Adolescents Following the Second COVID-19 Vaccine Dose

CBL knockdown protects cardiomyocytes against hypoxia-reoxygenation injury by downregulating GRB2 expression.

Myocardial ischemia-reperfusion injury (MIRI) is an event that follows a myocardial infarction. As such, close observation and appropriate patient management is paramount in the treatment process of interventional surgery. The pathogenesis of MIRI has not been fully elucidated. Therefore, the aim of the present study was to explore of novel targets for MIRI treatment whilst also determining their possible underlying mechanism of action. The plasma samples used in the present study were collected from 30 patients with ischemic cardiomyopathy and 30 healthy volunteers. H9c2 rat cardiomyoblasts were subjected to hypoxia and reoxygenation (H/R) modeling to establish an in vitro MIRI model. Initially, the expression levels of Cbl proto-oncogene (CBL) in ICM heart tissue, normal heart tissue, H/R-induced H9c2 cells and normal H9c2 cells were detected using quantitative PCR and western blotting. With the application of Cell Counting Kit-8, western blotting and Tunnel assay, the proliferation, oxidative stress and apoptosis of H/R-induced cells were assessed. Moreover, co-IP assay was employed to testify the interaction between CBL and GRB2. The present study revealed that CBL expression was upregulated in patients with ischemic cardiomyopathy and H/R-induced H9c2 cells in comparison with that in normal heart tissue and normal H9c2 cells, respectively. The genetic silence of CBL using small interfering RNA promoted the proliferation and oxidative stress of H/R-induced cells but repressed the apoptosis. The full-length wild-type of growth factor receptor-bound protein 2 (GRB2) was ligated into pcDNA3.1 to achieve GRB2 overexpression, which revealed that GRB2 overexpression reversed the effects of CBL knockdown on cells, suggesting that it may mediate these processes downstream. In conclusion, under hypoxic conditions, CBL knockdown promoted the proliferation and antioxidant capacity of cardiomyocytes whilst inhibiting apoptosis, by downregulating GRB2 expression. These findings revealed the underlying mechanism of action of this pathway, which can be exploited for the prevention or treatment of MIRI.

Nrf2 Promotes Inflammation in Early Myocardial Ischemia-Reperfusion via Recruitment and Activation of Macrophages.

Cardiomyocyte apoptosis in response to inflammation is a primary cause of myocardial ischemia-reperfusion injury (IRI). Nuclear factor erythroid 2 like 2 (Nrf2) reportedly plays an important role in myocardial IRI, but the underlying mechanism remains obscure. Expression data from the normal heart tissues of mice or heart tissues treated with reperfusion for 6 h after ischemia (IR6h) were acquired from the GEO database; changes in biological function and infiltrating immune cells were analyzed. The binding between the molecules was verified by chromatin immunoprecipitation sequencing. Based on confirmation that early myocardial ischemia-reperfusion (myocardial ischemia/reperfusion for 6 hours, IR6h) promoted myocardial apoptosis and inflammatory response, we found that Nrf2, cooperating with Programmed Cell Death 4, promoted transcription initiation of C-C Motif Chemokine Ligand 3 (Ccl3) in myocardial tissues of mice treated with IR6h. Moreover, Ccl3 contributed to the high signature score of C-C motif chemokine receptor 1 (Ccr1)-positive macrophages. The high signature score of Ccr1-positive macrophages leads to the release of pro-inflammatory factors interleukin 1 beta and interleukin 6. This study is the first to elucidate the damaging effect of Nrf2 via remodeling of the immune microenvironment in early myocardial ischemia-reperfusion, which provides us with new perspectives and treatment strategies for myocardial ischemia-reperfusion.

Changes in magnetic resonance imaging relaxation time on postmortem magnetic resonance imaging of formalin-fixed human normal heart tissue

BackgroundPostmortem magnetic resonance imaging (MRI) has been used to investigate the cause of death, but due to time constraints, it is not widely applied to the heart. Therefore, MRI analysis of the heart after formalin fixation was previously performed. However, the changes in MRI signal values based on the fixation time of formalin were not investigated. The objective was to investigate changes over time in the T1- and T2-values of MRI signals in normal areas of hearts removed during autopsy, hearts subsequently fixed in formalin, and heart specimens sliced for the preparation of pathological specimens.MethodsThe study subjects were 21 autopsy cases in our hospital between May 26, 2019 and February 16, 2020 whose hearts were removed and scanned by MRI. The male:female ratio was 14:7, and their ages at death ranged from 9 to 92 years (mean age 65.0 ± 19.7 years). Postmortem (PM)-MRI was conducted with a 0.3-Tesla (0.3-T) scanner containing a permanent magnet. A 4-channel QD head coil was used as the receiver coil. Scans were performed immediately after removal, post-formalin fixation, and after slicing; 7 cases were scanned at all three time points.ResultsThe T1- and T2-values were calculated from the MRI signals of each sample organ at each scanning stage. Specimens were sliced from removed organs after formalin fixation, and the changes in T1- and T2-values over time were graphed to obtain an approximate curve. The median T1-values at each measurement time point tended to decrease from immediately after removal. The T2-values showed the same tendency to decrease, but this tendency was more pronounced for the T1-values.ConclusionMRI signal changes in images of heart specimens were investigated. Formalin fixation shortened both T1- and T2-values over time, and approximation formulae were derived to show these decreases over time. The shortening of T1- and T2-values can be understood as commensurate with the reduction in the water content (water molecules) of the formalin-fixed heart.

The Therapeutic Role of Slit2 in Anti-fibrosis, Anti-inflammation and Anti-oxidative Stress in Rats with Coronary Heart Disease.

To investigate the efficacy of Slit2 in the rats with coronary heart disease (CHD). CHD model were constructed by feeding high-fat food and injecting with pituitrin in rat, followed by recombinant Slit2 treatment, and then the cardiac function was evaluated by echocardiography, and the indicators concerning the cardiomyocyte injury markers and lipoprotein status and oxidative stress were measured. The Slit2 expression in the heart tissues was identified by immunofluorescence. Enzyme-linked immunosorbent assay (ELISA) was carried out to detect inflammatory cytokines, H2DCFDA staining to determine the ROS generation in heart tissues, Masson trichrome staining to observe myocardial fibrosis, and qRT-PCR and Western blotting to detect gene and protein expressions. Slit2 decreased the levels of LDH, CK-MB, cTnI, TG, TC and LDL-C and increased HDL-C level in CHD rats. In the normal heart tissues, Slit2 expression was significantly lower in cardiomyocytes than cardiac fibroblasts. Furthermore, the expressions of VCAM-1, ICAM-1, fibronectin and TGF-β1 were increased in the heart tissues of CHD rats with the obvious myocardial fibrosis, which were dose-dependently reversed by recombinant Slit2. In addition, recombinant Slit2 also dose-dependently increased the activity of NO, SOD, CAT and GSH-Px, and decreased TNF-α, IL-6, MCP-1, MDA and ROS in CHD rats. Slit2 was downregulated in myocardial tissue and plasma of CHD rats. Recombinant Slit2, by regulating the level of blood lipid, can relieve the myocardial fibrosis, inflammation and oxidative stress in 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.

A method for cryopreservation and single nucleus RNA-sequencing of normal adult human interventricular septum heart tissue reveals cellular diversity and function

BackgroundSingle cell sequencing of human heart tissue is technically challenging and methods to cryopreserve heart tissue for obtaining single cell information have not been standardized. Studies published to date have used varying methods to preserve and process human heart tissue, and have generated interesting datasets, but development of a biobanking standard has not yet been achieved. Heart transcription patterns are known to be regionally diverse, and there are few single cell datasets for normal human heart tissue.MethodsUsing pig tissue, we developed a rigorous and reproducible method for tissue mincing and cryopreservation that allowed recovery of high quality single nuclei RNA. We subsequently tested this protocol on normal human heart tissue obtained from organ donors and were able to recover high quality nuclei for generation of single nuclei RNA-seq datasets, using a commercially available platform from 10× Genomics. We analyzed these datasets using standard software packages such as CellRanger and Seurat.ResultsHuman heart tissue preserved with our method consistently yielded nuclear RNA with RNA Integrity Numbers of greater than 8.5. We demonstrate the utility of this method for single nuclei RNA-sequencing of the normal human interventricular septum and delineating its cellular diversity. The human IVS showed unexpected diversity with detection of 23 distinct cell clusters that were subsequently categorized into different cell types. Cardiomyocytes and fibroblasts were the most commonly identified cell types and could be further subdivided into 5 different cardiomyocyte subtypes and 6 different fibroblast subtypes that differed by gene expression patterns. Ingenuity Pathway analysis of these gene expression patterns suggested functional diversity in these cell subtypes.ConclusionsHere we report a simple technical method for cryopreservation and subsequent nuclear isolation of human interventricular septum tissue that can be done with common laboratory equipment. We show how this method can be used to generate single nuclei transcriptomic datasets that rival those already published by larger groups in terms of cell diversity and complexity and suggest that this simple method can provide guidance for biobanking of human myocardial tissue for complex genomic analysis.

MicroRNA-200a represses myocardial infarction-related cell death and inflammation by targeting the Keap1/Nrf2 and β-catenin pathways

BackgroundAcute myocardial infarction (MI) is a main cause of emergency death in the world. MicroRNAs (miRs/miRNAs) are a series of small non-coding RNA molecules, which regulate cardiovascular disorders that involve MI. In this study, we explored the function of miR-200a in MI treatment. MethodsWe observed down-regulation of miR-200a levels and up-regulation of Keap1 and β-catenin levels in H2O2-treated newborn murine ventricular cardiomyocytes (NMVCs) and the infarcted heart tissues of MI mouse models, compared to the non-treated NMVCs and normal heart tissues of healthy mice. ResultsCCK-8 and colony formation assays indicated the reduction in NMVC vitality due to H2O2 treatment and the recovery of cell vitality due to miR-200a overexpression, respectively. Flow cytometry with Annexin and PI staining indicated the inhibition of H2O2-triggered cell apoptosis through ectopically expressed miR-200a. Western blotting and ELISA analyses that detected pro-inflammatory cell factors [interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α] confirmed that miR-200a prevented H2O2-induced NMVC inflammation. Moreover, miR-200a inhibited up-regulation of Keap1 and β-catenin expression in H2O2-treated NMVCs by directly binding with the 3′-UTR regions of both Keap1 and β-catenin. Furthermore, overexpression of Keap1 and β-cateninin in H2O2-treated NMVCs with recovered miR-200a elevated inflammation and apoptosis, respectively. ConclusionThe results showed that miR-200a expression was inhibited in murine cardiomyocytes due to H2O2 stress in MI cardiac tissues and overexpressed miR-200a could protect the cells from death by regulating the Keap1/Nrf2 and β-catenin signal transduction pathways.