Differential expression profiles of plasma exosomal microRNAs in dilated cardiomyopathy with chronic heart failure

As one of the most prevalent heritable cardiovascular diseases, dilated cardiomyopathy (DCM) induces cardiac insufficiency and dysfunction. Although genetic mutation has been identified one of the causes of DCM, the usage of genetic biomarkers such as RNAs for DCM early diagnosis is still being overlooked. In addition, the alternation of RNAs could reflect the progression of the diseases, as an indicator for the prognosis of patients. Therefore, it is beneficial to develop genetic based diagnostic tool for DCM. RNAs are often unstable within circulatory system, leading to the infeasibility for clinical application. Recently discovered exosomal miRNAs have the stability that is then need for diagnostic purpose. Hence, fully understanding of the exosomal miRNA within DCM patients is vital for clinical translation. In this study, we employed the next generation sequencing based on the plasma exosomal miRNAs to comprehensively characterize the miRNAs expression in plasma exosomes from DCM patients exhibiting chronic heart failure (CHF) compared to healthy individuals. A complex landscape of differential miRNAs and target genes in DCM with CHF patients were identified. More importantly, we discovered that 92 differentially expressed miRNAs in DCM patients undergoing CHF were correlated with several enriched pathways, including oxytocin signalling pathway, circadian entrainment, hippo signalling pathway-multiple species, ras signalling pathway and morphine addiction. This study reveals the miRNA expression profiles in plasma exosomes in DCM patients with CHF, and further reveal their potential roles in the pathogenesis of it, presenting a new direction for clinical diagnosis and management of DCM patients with CHF.     

Autophagic vacuoles in cardiomyocytes of dilated cardiomyopathy with initially decompensated heart failure predict improved prognosis

Autophagy is a process of bulk protein degradation and organelle turnover, and is a current therapeutic target in several diseases. The present study aimed to clarify the significance of myocardial autophagy of patients with dilated cardiomyopathy (DCM). Left ventricular endomyocardial biopsy was performed in 250 consecutive patients with DCM (54.9±13.9 years; male, 79%), initially presenting with decompensated heart failure (HF). The association of these findings with HF mortality or recurrence was examined. Myofilament changes, which are apparent in the degenerated cardiomyocytes of DCM, were recognized in 164 patients (66%), and autophagic vacuoles in cardiomyocytes were identified in or near the area of myofilament changes in 86 patients (34%). Morphometrically, fibrosis (odds ratio [OR], 0.96; 95% confidence interval [CI], 0.93 to 0.99) and mitochondrial abnormality (OR, 2.24; 95% CI, 1.23 to 4.08) were independently related with autophagic vacuoles. During the follow-up period of 4.9±3.9 y, 24 patients (10%) died, including 10 (4%) who died of HF, and 67 (27%) were readmitted for HF recurrence. Multivariate analysis identified a family history of DCM (hazard ratio [HR], 2.117; 95% CI, 1.199 to 3.738), hemoglobin level (HR, 0.845; 95% CI, 0.749 to 0.953), myofilament changes (HR, 13.525; 95% CI, 5.340 to 34.255), and autophagic vacuoles (HR, 0.214; 95% CI, 0.114 to 0.400) as independent predictors of death or readmission due to HF recurrence. In conclusion, autophagic vacuoles in cardiomyocytes are associated with a better HF prognosis in patients with DCM, suggesting autophagy may play a role in the prevention of myocardial degeneration.     

Long-term follow-up of patients with myocarditis and idiopathic dilated cardiomyopathy after immunomodulatory therapy

Abstract The randomized clinical trial with interferon-α (IFN) or thymic hormones versus conventional therapy was conducted in patients with myocarditis and idiopathic dilated cardiomyopathy (IDC). We enrolled 180 patients to receive IFN (3–5 million units per day) for 3 months, thymomodulin (10 mg three times per week) for 2 months, or conventional therapy alone. Patients were followed-up for 7 years after the end of treatment. Left ventricular function, exercise tolerance and survival rate were significantly better at long-term follow-up in patients treated with IFN or thymomodulin, than in conventionally treated patients. These results implicate that immune modulating therapy might represent important contribution in the treatment of myocarditis and IDC.     

Beneficial effects of intramyocardial mesenchymal stem cells and VEGF165 plasmid injection in rats with furazolidone induced dilated cardiomyopathy

To explore the impact of myocardial injection of mesenchymal stem cells (MSCs) and specific recombinant human VEGF₁₆₅ (hVEGF₁₆₅) plasmid on collagen remodelling in rats with furazolidone induced dilated cardiomyopathy (DCM). DCM was induced by furazolidone (0.3 mg/bodyweight (g)/day per gavage for 8 weeks). Rats were then divided into four groups: (i) PBS group (n = 18): rats received equal volume myocardial PBS injection; (ii) MSCs group (n = 17): 100 μl culture medium containing 10⁵ MSCs were injected into four sites of left ventricular free wall (25 μl per site); (iii) GENE group (n = 18): pCMVen‐MLC2v‐EGFP‐VEGF₁₆₅ plasmid [5 × 109 pfu (0.2 ml)] were injected into four sites of left ventricular free wall (0.05 ml per site)] and (iv) MSCs+GENE group (n = 17): rats received both myocardial MSCs and pCMVen‐MLC2v‐EGFP‐VEGF₁₆₅ plasmid injections. After 4 weeks, cardiac function was evaluated by echocardiography. Myocardial mRNA expressions of type I, type III collagen and transforming growth factor (TGF)‐β1 were detected by RT‐PCR. The protein expression of hVEGF₁₆₅ was determined by Western blot. Myocardial protein expression of hVEGF₁₆₅ was demonstrated in GENE and MSCs+GENE groups. Cardiac function was improved in MSCs, GENE and MSCs+GENE groups. Collagen volume fraction was significantly reduced and myocardial TGF‐β1 mRNA expression significantly down‐regulated in both GENE and MSCs+GENE groups, collagen type I/III ratio reduction was more significant in MSCs+GENE group than in MSCs or GENE group. Myocardial MSCs and hVEGF₁₆₅ plasmid injection improves cardiac function possibly through down‐regulating myocardial TGF‐β1 expression and reducing the type I/III collagen ratio in this DCM rat model.     

Beneficial effects of edaravone, a novel antioxidant, in rats with dilated cardiomyopathy

Edaravone, a novel antioxidant, acts by trapping hydroxyl radicals, quenching active oxygen and so on. Its cardioprotective activity against experimental autoimmune myocarditis (EAM) was reported. Nevertheless, it remains to be determined whether edaravone protects against cardiac remodelling in dilated cardiomyopathy (DCM). The present study was undertaken to assess whether edaravone attenuates myocardial fibrosis, and examine the effect of edaravone on cardiac function in rats with DCM after EAM. Rat model of EAM was prepared by injection with porcine cardiac myosin 28 days after immunization, we administered edaravone intraperitoneally at 3 and 10 mg/kg/day to rats for 28 days. The results were compared with vehicle-treated rats with DCM. Cardiac function, by haemodynamic and echocardiographic study and histopathology were performed. Left ventricular (LV) expression of NADPH oxidase subunits (p47(phox), p67(phox), gp91(phox) and Nox4), fibrosis markers (TGF-β(1) and OPN), endoplasmic reticulum (ER) stress markers (GRP78 and GADD 153) and apoptosis markers (cytochrome C and caspase-3) were measured by Western blotting. Edaravone-treated DCM rats showed better cardiac function compared with those of the vehicle-treated rats. In addition, LV expressions of NADPH oxidase subunits levels were significantly down-regulated in edaravone-treated rats. Furthermore, the number of collagen-III positive cells in the myocardium of edaravone-treated rats was lower compared with those of the vehicle-treated rats. Our results suggest that edaravone ameliorated the progression of DCM by modulating oxidative and ER stress-mediated myocardial apoptosis and fibrosis.     

The relationship between myocardial fibrosis and myocardial microRNAs in dilated cardiomyopathy: A link between mir‐133a and cardiovascular events

It is unknown whether fibrosis‐associated microRNAs: miR‐21, miR‐26, miR‐29, miR‐30 and miR‐133a are linked to cardiovascular (CV) outcome. The study evaluated the levels of extracellular matrix (ECM) fibrosis and the prevalence of particular microRNAs in patients with dilated cardiomyopathy (DCM) to investigate any correlation with CV events. Methods: Seventy DCM patients (48 ± 12 years, EF 24.4 ± 7.4%) underwent right ventricular biopsy. The control group was comprised of 7 patients with CAD who underwent CABG and intraoperative biopsy. MicroRNAs were measured in blood and myocardial tissue via qPCR. The end‐point was a combination of CV death and urgent HF hospitalization at the end of 12 months. There were differential levels of circulating and myocardial miR‐26 and miR‐29 as well as myocardial miR‐133a when the DCM and CABG groups were compared. Corresponding circulating and myocardial microRNAs did not correlate with one another. There was no correlation between microRNA and ECM fibrosis. By the end of the 12‐month period of the study, CV death had occurred in 6 patients, and a further 19 patients required urgent HF hospitalization. None of the circulating microRNAs was a predictor of the combined end‐point; however, myocardial miR‐133a was an independent predictor in unadjusted models (HR 1.53; 95% CI 1.14‐2.05; P < .004) and adjusted models (HR 1.57; 95% CI 1.14‐2.17; P < .005). The best cut‐off value for the miR‐133a level for the prediction of the combined end‐point was 0.74 ΔCq, with an AUC of 0.67. The absence of a correlation between the corresponding circulating and myocardial microRNAs calls into question their cellular source. This study sheds new light on the role of microRNAs in ECM fibrosis in DCM, which warrants further exploration.     

Single-nucleus RNA sequencing in ischemic cardiomyopathy reveals common transcriptional profile underlying end-stage heart failure

1. Download : Download high-res image (219KB)
2. Download : Download full-size image

     

Plasma microRNA profiling of children with idiopathic dilated cardiomyopathy

Context: Dilated cardiomyopathy (DCM) is the most common cardiomyopathy in children. MicroRNAs (miRNA) are small RNAs which have regulatory functions in many biological processes.

Objective: We aimed to determine miRNA expression levels in plasma of children with DCM.

Materials and methods: Plasma expression levels of 379 miRNAs were compared between 23 DCM and 26 healthy children.

Results: The expression levels of miR-618, miR-875-3p, miR-205, miR-194, miR-302a, miR-147, and miR-544 were found decreased. The expression levels of miR-518f and miR-454 were found increased in DCM patients.

Discussion: miRNA level differences may provide the chance of using these miRNAs as new biomarkers.     

Regional mapping of myocardial hibernation phenotype in idiopathic end‐stage dilated cardiomyopathy

Myocardial hibernation (MH) is a well‐known feature of human ischaemic cardiomyopathy (ICM), whereas its presence in human idiopathic dilated cardiomyopathy (DCM) is still controversial. We investigated the histological and molecular features of MH in left ventricle (LV) regions of failing DCM or ICM hearts. We examined failing hearts from DCM (n = 11; 41.9 ± 5.45 years; left ventricle‐ejection fraction (LV‐EF), 18 ± 3.16%) and ICM patients (n = 12; 58.08 ± 1.7 years; LVEF, 21.5 ± 6.08%) undergoing cardiac transplantation, and normal donor hearts (N, n = 8). LV inter‐ventricular septum (IVS) and antero‐lateral free wall (FW) were transmurally (i.e. sub‐epicardial, mesocardial and sub‐endocardial layers) analysed. LV glycogen content was shown to be increased in both DCM and ICM as compared with N hearts (P < 0.001), with a U‐shaped transmural distribution (lower values in mesocardium). Capillary density was homogenously reduced in both DCM and ICM as compared with N (P < 0.05 versus N), with a lower decrease independent of the extent of fibrosis in sub‐endocardial and sub‐epicardial layers of DCM as compared with ICM. HIF1‐α and nestin, recognized ischaemic molecular hallmarks, were similarly expressed in DCM‐LV and ICM‐LV myocardium. The proteomic profile was overlapping by ~50% in DCM and ICM groups. Morphological and molecular features of MH were detected in end‐stage ICM as well as in end‐stage DCM LV, despite epicardial coronary artery patency and lower fibrosis in DCM hearts. Unravelling the presence of MH in the absence of coronary stenosis may be helpful to design a novel approach in the clinical management of DCM.     

Comparative effects of a vasopeptidase inhibitor vs. an angiotensin convertin enzyme inhibitor on cardiomyocyte apoptosis in rats with heart failure

Apoptosis is involved in ventricular remodeling after myocardial infarction (MI). We investigated the effects of the vasopeptidase inhibitor (VPI) omapatrilat on cardiomyocyte apoptosis and compared it to the angiotensin converting enzyme inhibitor (ACEI) captopril in the rat post-MI model and in cultured neonatal rat cardiomyocytes. Wistar males rats surviving 4 h post-MI were assigned to omapatrilat (40 or 80 mg/kg/day), captopril (160 mg/kg/day) or no treatment. After 56 days, hemodynamic measurements were performed (n = 96) and rats were sacrificed. One group had assessment of cardiac remodeling and detection of DNA fragments by in situ end labelling method (ISEL), while the other had morphologic measurements and DNA laddering assessed. In addition, cultured neonatal rat cardiomyocytes (n = 6) were treated for 72 h with vehicle, captopril or omapatrilat in the presence or absence of the apoptosis inducing agent H₂O₂. Omapatrilat and captopril resulted in similar improvements of hemodynamic measurements, ventricular weight and dilatation, cardiac fibrosis and myocardial cell cross-section in large MI rats. Omapatrilat increased scar thickness more than did captopril. All sham-operated groups had little evidence of apoptosis. In the large MI group, there was a significant increase in ISEL-positive cells in the control (0.095 ± 0.016%) and captopril (0.124 ± 0.024%) groups in comparison with control sham-operated (0.006 ± 0.006%), but this increase was limited to the peri-MI area. Omapatrilat (0.012 ± 0.012% for both doses) prevented the increase in apoptosis in the peri-MI area. Also, omapatrilat but not captopril reduced DNA laddering in large MI. Moreover, in cultured neonatal rat cardiomyocytes, omapatrilat but not captopril reduced apoptosis as assessed by DNA laddering. The VPI omapatrilat, with its combination of NEP and ACE inhibition, suppresses cardiomyocyte apoptosis post-MI and in neonatal cultured rat cardiomyocytes more than the ACEI captopril, but this does not result in significant hemodynamic or morphologic differences between omapatrilat and captopril.     

Cardiac protein changes in ischaemic and dilated cardiomyopathy: a proteomic study of human left ventricular tissue

The development of heart failure (HF) is characterized by progressive alteration of left ventricle structure and function. Previous works on proteomic analysis in cardiac tissue from patients with HF remain scant. The purpose of our study was to use a proteomic approach to investigate variations in protein expression of left ventricle tissue from patients with ischaemic (ICM) and dilated cardiomyopathy (DCM). Twenty-four explanted human hearts, 12 from patients with ICM and 12 with DCM undergoing cardiac transplantation and six non-diseased donor hearts (CNT) were analysed by 2DE. Proteins of interest were identified by mass spectrometry and validated by Western blotting and immunofluorescence. We encountered 35 differentially regulated spots in the comparison CNT versus ICM, 33 in CNT versus DCM, and 34 in ICM versus DCM. We identified glyceraldehyde 3-phophate dehydrogenase up-regulation in both ICM and DCM, and alpha-crystallin B down-regulation in both ICM and DCM. Heat shock 70 protein 1 was up-regulated only in ICM. Ten of the eleven differentially regulated proteins common to both aetiologies are interconnected as a part of a same network. In summary, we have shown by proteomics analysis that HF is associated with changes in proteins involved in the cellular stress response, respiratory chain and cardiac metabolism. Although we found altered expression of eleven proteins common to both ischaemic and dilated aetiology, we also observed different proteins altered in both groups. Furthermore, we obtained that seven of these eleven proteins are involved in cell death and apoptosis processes, and therefore in HF progression.     

Direct inhibition of neutral endopeptidase in vasopeptidase inhibitor-mediated amelioration of cardiac remodeling in rats with chronic heart failure

Vasopeptidase inhibitors possess dual inhibitory actions on neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE) and have beneficial effects on cardiac remodeling. However, the contribution of NEP inhibition to their effects is not yet fully understood. To address the role of cardiac NEP inhibition in the anti-remodeling effects of a vasopeptidase inhibitor, we examined the effects of omapatrilat on the development of cardiac remodeling in rats with left coronary artery ligation (CAL) and those on collagen synthesis in cultured fibroblast cells. In vivo treatment with omapatrilat (30 mg/kg/day for 5 weeks) inhibited cardiac NEP activity in rats with CAL, which was associated with a suppression of both cardiac hypertrophy and collagen deposition. In cultured cardiac fibroblasts, omapatrilat (10^–7~10^–5 M) inhibited NEP activity and augmented the ANP-induced decrease in [³H]-proline incorporation. ONO-BB, an active metabolite of the NEP selective inhibitor ONO-9902, also augmented the ANP-induced response, whereas captopril, an ACE inhibitor, did not. The angiotensin I-induced increase in [³H]-proline incorporation was prevented by omapatrilat and captopril, but not by ONO-BB. The results suggest that vasopeptidase inhibitor suppressed cardiac remodeling in the setting of chronic heart failure, possibly acting through the direct inhibition of cardiac NEP. Vasopeptidase inhibitors may have therapeutic advantages over the classical ACE and NEP inhibitors alone with respect to the regression of cardiac fibrosis.     

Co-expression analysis provides important module and pathways of human dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a heart disease that injured greatly to the people wordwide. Systemic co-expression analysis for this cancer is still limited, although massive clinic experiments and gene profiling analyses had been well performed previously. Here, using the public RNA-Seq data “GSE116250” and gene annotation of Ensembl database, we built the co-expression modules for DCM by Weighted Gene Co-Expression Network Analysis, and investigated the function enrichment and protein-protein interaction (PPI) network of co-expression genes of each module by Database for Annotation, Visualization, and Integrated Discovery and Search Tool for the Retrieval of Interacting Genes/Proteins database, respectively. First, 5,000 genes in the 37 samples were screened and 11 co-expression modules were conducted. The number of genes for each module ranged from 77 to 936, with a mean of 455. Second, interaction relationships of hub-genes between pairwise modules showed great differences, suggesting relatively high-scale independence of the modules. Third, functional enrichments of the co-expression modules exhibited great differences. We found that genes in module 3 were significantly enriched in the pathways of focal adhesion and ubiquitin-mediated proteolysis. This module was inferred as the key module involved in DCM. In addition, PPI analysis revealed that the genes HSP90AA1, CTNNB1, MAPK1, GART, and PPP2CA owned the largest number of adjacency genes, unveiling that they may function importantly during the occurrence of DCM. Focal adhesion and ubiquitin-mediated proteolysis play important roles in human DCM.     

Dilative cardiomyopathy leading to congestive heart failure in a male squirrel monkey (Saimiri sciureus)

A 17-year-old, 1-kg, colony-housed, male squirrel monkey (Samiri sciureus) developed clinical signs of congestive heart failure. The monkey presented with lethargy, increased heart and respiratory rates, and mild abdominal distention. The clinical history, laboratory analysis, and radiographic findings were consistent with heart failure due to dilative cardiomyopathy. Gross and microscopic examination of the heart confirmed a dilative cardiomyopathy. This is the first report describing congestive heart failure caused by dilative cardiomyopathy in a squirrel monkey. Spontaneous dilative cardiomyopathy may be infrequently observed in the squirrel monkeys because they are not routinely housed in the research environment during their advancing years.     

Progression of heart failure: A role for interstitial fibrosis

Progressive deterioration of left ventricular (LV) function is a characteristic feature of the heart failure (HF) state. The mechanism or mechanisms responsible for this hemodynamic deterioration are not known but may be related to progressive intrinsic dysfunction, degeneration and loss of viable cardiocytes. In the present study, we tested the hypothesis that accumulation of collagen in the cardiac interstitium (reactive interstitial fibrosis, RIF), known to occur in HF, results in reduced capillary density (CD=capillary/fiber ratio) and increased oxygen diffusion distance (ODD) which can lead to hypoxia and dysfunction of the collagen encircled myocyte. Studies were performed in LV tissue obtained from 10 dogs with chronic HF (LV ejection fraction 26±1%) produced by multiple sequential intracoronary, microembolizations. In each dog, CD and ODD were evaluated in LV regions that manifested severe RIF (volume fraction 16±2%) and in LV regions of little or no RIF (volume fraction 4±1%). In regions of severe RIF, CD was significantly decreased compared to regions of no RIF (0.92±0.02 vs. 1.05±0.03) (P<0.03). Similarly, ODD was significantly increased in regions of severe RIF compared to regions of no RIF (15.3±0.4 vs. 12.2±0.3 m) (P<0.001). These data suggest that in dogs with chronic HF, constituent myocytes of LV regions which manifest severe RIF may be subjected to chronic hypoxia; a condition that can adversely impact the function and viability of the collagen encircled cardiocyte.     

Identification of micro‐RNA networks in end‐stage heart failure because of dilated cardiomyopathy

Micro‐RNAs regulate gene expression by directly binding to the target mRNAs. The goal of the study was to examine the expression profiling of miRNAs in human failing hearts and identify the key miRNAs that regulate molecular signalling networks and thus contribute to this pathological process. The levels of miRNAs and expressed genes were analysed in myocardial biopsy samples from patients with end‐stage heart failure (n = 14) and those from normal heart samples (n = 8). Four networks were built including the Gene regulatory network, Signal‐Network, miRNA‐GO‐Network and miRNA‐Gene‐Network. According to the fold change in the network and probability values in the microarray cohort, RT‐PCR was performed to measure the expression of five of the 72 differentially regulated miRNAs. miR‐340 achieved statistically significant. miR‐340 was identified for the first time in cardiac pathophysiological condition. We overexpressed miR‐340 in cultured neonatal rat cardiomyocytes to identify whether miR‐340 plays a determining role in the progression of heart failure. ANP, BNP and caspase‐3 were significantly elevated in the miR‐340 transfected cells compared with controls (P < 0.05). The cross‐sectional area of overexpressing miR‐340 cardiomyocytes (1952.22 ± 106.59) was greater (P < 0.0001) than controls (1059.99 ± 45.59) documented by Laser Confocal Microscopy. The changes of cellular structure and the volume were statistical significance. Our study provided a comprehensive miRNA expression profiling in the end‐stage heart failure and identified miR‐340 as a key miRNA contributing to the occurrence and progression of heart failure. Our discoveries provide novel therapeutic targets for patients with heart failure.     

Loss of DNA repair mechanisms in cardiac myocytes induce dilated cardiomyopathy

Cardiomyopathy is a progressive disease of the myocardium leading to impaired contractility. Genotoxic cancer therapies are known to be potent drivers of cardiomyopathy, whereas causes of spontaneous disease remain unclear. To test the hypothesis that endogenous genotoxic stress contributes to cardiomyopathy, we deleted the DNA repair gene Ercc1 specifically in striated muscle using a floxed allele of Ercc1 and mice expressing Cre under control of the muscle-specific creatinine kinase (Ckmm) promoter or depleted systemically (Ercc1^−/D mice). Ckmm-Cre^+/−;Ercc1^−/fl mice expired suddenly of heart disease by 7 months of age. As young adults, the hearts of Ckmm-Cre^+/−;Ercc1^−/fl mice were structurally and functionally normal, but by 6-months-of-age, there was significant ventricular dilation, wall thinning, interstitial fibrosis, and systolic dysfunction indicative of dilated cardiomyopathy. Cardiac tissue from the tissue-specific or systemic model showed increased apoptosis and cardiac myocytes from Ckmm-Cre^+/-;Ercc1^−/fl mice were hypersensitive to genotoxins, resulting in apoptosis. p53 levels and target gene expression, including several antioxidants, were increased in cardiac tissue from Ckmm-Cre^+/−;Ercc1^−/fl and Ercc1^−/D mice. Despite this, cardiac tissue from older mutant mice showed evidence of increased oxidative stress. Genetic or pharmacologic inhibition of p53 attenuated apoptosis and improved disease markers. Similarly, overexpression of mitochondrial-targeted catalase improved disease markers. Together, these data support the conclusion that DNA damage produced endogenously can drive cardiac disease and does so mechanistically via chronic activation of p53 and increased oxidative stress, driving cardiac myocyte apoptosis, dilated cardiomyopathy, and sudden death.