Search

The most common ventricular premature contractions (VPCs) originate from the right ventricular outflow tract (RVOT), but the molecular mechanisms of altered cytoskeletons of VPC-induced cardiomyopathy remain unexplored. We created a RVOT bigeminy VPC pig model (n = 6 in each group). Echocardiography was performed. The histopathological alternations in the LV myocardium were analyzed, and next generation sequencing (NGS) and functional enrichment analyses were employed to identify the differentially expressed genes (DEGs) responsible for the histopathological alternations. Finally, a cell silencing model was used to confirm the key regulatory gene and pathway. VPC pigs had increased LV diameters in the 6-month follow-up period. A histological study showed more actin cytoskeleton disorganization and actin accumulation over intercalated disc, Z-line arrangement disarray, increased β-catenin expression, and cardiomyocyte enlargement in the LV myocardium of the VPC pigs compared to the control pigs. The NGS study showed actin cytoskeleton signaling, RhoGDI signaling, and signaling by Rho Family GTPases and ILK Signaling presented z-scores with same activation states. The expressions of Rac family small GTPase 2 (Rac2), the p-cofilin/cofilin ratio, and the F-actin/G-actin ratio were downregulated in the VPC group compared to the control group. Moreover, the intensity and number of actin filaments per cardiomyocyte were significantly decreased by Rac2 siRNA in the cell silencing model. Therefore, the Rac2/cofilin pathway was found to play a crucial role in the sarcomere morphology and Z-line arrangement disarray induced by RVOT bigeminy VPCs.

BACKGROUND: Pacing-induced cardiomyopathy is an undesired outcome in patients with atrioventricular block (AVB), and our animal model showed lipotoxic cardiomyopathy after pacing.OBJECTIVES: The purpose of this study was to explore the mechanisms and clinical outcomes of statins in AVB patients receiving pacing.METHODS: Rat ventricular cardiomyocytes were treated with atorvastatin, liver X receptor (LXR) agonist, and LXR antagonist during pacing. Pigs were divided into 3 groups: right ventricular pacing, pacing with concomitant atorvastatin treatment, and sham control. Clinically, we enrolled 1717 AVB patients who had received a permanent pacemaker from Chang Gung Memorial Hospital Medical database. The primary outcome (cardiovascular death or heart failure [HF] hospitalization) and individual outcome were compared between statin and nonstatin groups after inverse probability of treatment weighting.RESULTS: Lipid accumulation in rat cardiomyocytes by pacing was significantly reduced by treatment with LXR agonist and atorvastatin, whereas LXR antagonist counteracted the atorvastatin effect on lipid expression. Left ventricular ejection fraction (LVEF) was significantly lower in the AVB pig pacing group compared to the group concomitantly treated with atorvastatin. Moreover, lipid accumulation and fibronectin expression were significantly ameliorated by concomitant treatment with atorvastatin. In the clinical study, the statin group had a significantly lower risk of the primary outcome event (hazard ratio [HR] 0.69; 95% confidence interval [CI] 0.56-0.84), less HF hospitalization (HR 0.45; 95% CI 0.30-0.67), and higher LVEF than the nonstatin group.CONCLUSION: In experimental models, atorvastatin ameliorated lipid accumulation in cardiomyocytes and fibrosis in left ventricular myocardium induced by pacing. Clinically, treatment with statins was associated with less HF hospitalization and cardiovascular death in AVB patients receiving pacemaker therapy.

Background MicroRNAs (miRs) regulate gene expression in heart failure. Circulating miRs as biomarkers for heart failure in mitral regurgitation patients (MR) remain unexplored. Methods This case–control study enrolled 32 MR patients with heart failure, 16 asymptomatic MR patients, and 12 control subjects without heart failure. We used next generation sequencing to study the gene expression profiles in the sera, and quantitative RT-PCR to study serum and tissue miRs in the left atria. Results Next generation sequencing analysis and enrichment analysis showed that 25 miRs were differentially expressed in the sera of MR patients with heart failure compared to control subjects. The circulating miR-148b-3p (p=0.002) and miR-409-3p (p=0.010) were significantly down-regulated in the MR patients with heart failure compared to control subjects. However, only circulation miR-148b-3p was significantly down-regulated in the MR patients without heart failure compared to control subjects (p=0.009). The tissue miR-409-3p was significantly down-regulated in the MR patients with heart failure compared to 3 purchased normal controls (p=0.041). Notably, the tissue RASGRP3 mRNA, target gene of miR-409-3p, was significantly up-regulated in the MR patients with heart failure compared to normal controls (p=0.010). The tissue FRY (p=0.010) and GADD45A (p=0.010) mRNAs, target genes of miR-148b-3p, were significantly up-regulated in the MR patients with heart failure compared to normal controls. Conclusions Circulating miR-148b-3p might serve as biomarker for future development of heart failure and miR-409-3p might serve as biomarker for incident heart failure in MR patients.

Illegal, unreported and unregulated (IUU) fishing has become a key issue of increasing concern in the world. Led by the United Nations Food and Agriculture Organization (FAO), recent developments focused on the role of port states as the principal actor against IUU fishing. However, the FAO adopted the Voluntary Guidelines for Flag State Performance (the Guidelines) in 2013, implying that the principal role against IUU fishing still corresponds to flag states. As one of the leading distant-water fishing nations, Taiwan has adopted many domestic regulations in accordance with these international instruments voluntarily, regardless of its statehood debate. Although the adoption of the Guidelines is &ldquo, a real breakthrough&rdquo, against IUU fishing, the instrument is &ldquo, soft law&rdquo, in nature, meaning the implementation of the Guidelines may depend on the political will of states, and the effectiveness and efficiency of the Guidelines are thus highly questionable. Furthermore, the consensus among states to adopt a legally binding agreement on flag state performance in the near future, akin to the development of the Port States Measures Agreement that successfully evolved from a &ldquo, to a &ldquo, hard law&rdquo, basis, is not yet clear.

Background The molecular mechanisms through which high‐demand pacing induce myocardial dysfunction remain unclear. Methods and Results We created atrioventricular block in pigs using dependent right ventricular septal pacing for 6 months. Echocardiography was performed to evaluate dyssynchrony between pacing (n=6) and sham control (n=6) groups. Microarray and enrichment analyses were used to identify differentially expressed genes ( DEG s) in the left ventricular ( LV ) myocardium between pacing and sham control groups. Histopathological and protein changes were also analyzed and an A cell pacing model was also performed. Pacing significantly increased mechanical dyssynchrony. Enrichment analysis using Ingenuity Pathway Analysis and the activation z‐score analysis method demonstrated that there were 5 DEG s ( ABCA 1, APOD , CLU , LY 96, and SERPINF 1) in the LV septum (z‐score=−0.447) and 5 DEG s ( APOD , CLU , LY 96, MSR 1, and SERPINF 1) in the LV free wall (z‐score=−1.000) inhibited the liver X receptor/retinoid X receptor ( LXR / RXR ) pathway, and 4 DEG s ( ACTA 2, MYL 1, PPP 2R3A, and SNAI 2) activated the integrin‐linked kinase ( ILK ) pathway in the LV septum (z‐score=1.000). The pacing group had a larger cell size, higher degree of myolysis and fibrosis, and increased expression of intracellular lipid, inflammatory cytokines, and apoptotic markers than the sham control group. The causal relationships between pacing and DEG s related to LXR / RXR and ILK pathways, apoptosis, fibrosis, and lipid expression after pacing were confirmed in the cell pacing model. Luciferase reporter assay in the cell pacing model also supported inhibition of the LXR pathway by pacing. Conclusions Right ventricular septal‐dependent pacing was associated with persistent LV dyssynchrony–induced cardiomyopathy through inhibition of the LXR / RXR pathway.

Background: Lipid expression is increased in the atrial myocytes of mitral regurgitation (MR) patients. This study aimed to investigate key regulatory genes and mechanisms of atrial lipotoxic myopathy in MR. Methods: The HL-1 atrial myocytes were subjected to uniaxial cyclic stretching for eight hours. Fatty acid metabolism, lipoprotein signaling, and cholesterol metabolism were analyzed by PCR assay (168 genes). Results: The stretched myocytes had significantly larger cell size and higher lipid expression than non-stretched myocytes (all p &lt, 0.001). Fatty acid metabolism, lipoprotein signaling, and cholesterol metabolism in the myocytes were analyzed by PCR assay (168 genes). In comparison with their counterparts in non-stretched myocytes, seven genes in stretched monocytes (Idi1, Olr1, Nr1h4, Fabp2, Prkag3, Slc27a5, Fabp6) revealed differential upregulation with an altered fold change &gt, 1.5. Nine genes in stretched monocytes (Apoa4, Hmgcs2, Apol8, Srebf1, Acsm4, Fabp1, Acox2, Acsl6, Gk) revealed differential downregulation with an altered fold change &lt, 0.67. Canonical pathway analysis, using Ingenuity Pathway Analysis software, revealed that the only genes in the &ldquo, superpathway of cholesterol biosynthesis&rdquo, were Idi1 (upregulated) and Hmgcs2 (downregulated). The fraction of stretched myocytes expressing Nile red was significantly decreased by RNA interference of Idi1 (p &lt, 0.05) and was significantly decreased by plasmid transfection of Hmgcs2 (p = 0.004). Conclusions: The Idi1 and Hmgcs2 genes have regulatory roles in atrial lipotoxic myopathy associated with atrial enlargement.

Apoptosis occurs in atrial cardiomyocytes in mitral and tricuspid valve disease. The purpose of this study was to examine the respective roles of the mitochondrial and tumor necrosis factor-α receptor associated death domain (TRADD)-mediated death receptor pathways for apoptosis in the atrial cardiomyocytes of heart failure patients due to severe mitral and moderate-to-severe tricuspid regurgitation. This study comprised eighteen patients (7 patients with persistent atrial fibrillation and 11 in sinus rhythm). Atrial appendage tissues were obtained during surgery. Three purchased normal human left atrial tissues served as normal controls. Moderately-to-severely myolytic cardiomyocytes comprised 59.7±22.1% of the cardiomyocytes in the right atria and 52.4±12.9% of the cardiomyocytes in the left atria of mitral and tricuspid regurgitation patients with atrial fibrillation group and comprised 58.4±24.8% of the cardiomyocytes in the right atria of mitral and tricuspid regurgitation patients with sinus rhythm. In contrast, no myolysis was observed in the normal human adult left atrial tissue samples. Immunohistochemical analysis showed expression of cleaved caspase-9, an effector of the mitochondrial pathways, in the majority of right atrial cardiomyocytes (87.3±10.0%) of mitral and tricuspid regurgitation patients with sinus rhythm, and right atrial cardiomyocytes (90.6±31.4%) and left atrial cardiomyocytes (70.7±22.0%) of mitral and tricuspid regurgitation patients with atrial fibrillation. In contrast, only 5.7% of cardiomyocytes of the normal left atrial tissues showed strongly positive expression of cleaved caspase-9. Of note, none of the atrial cardiomyocytes in right atrial tissue in sinus rhythm and in the fibrillating right and left atria of mitral and tricuspid regurgitation patients, and in the normal human adult left atrial tissue samples showed cleaved caspase-8 expression, which is a downstream effector of TRADD of the death receptor pathway. Immunoblotting of atrial extracts showed that there was enhanced expression of cytosolic cytochrome c, an effector of the mitochondrial pathways, but no expression of membrane TRADD and cytosolic caspase-8 in the right atrial tissue of mitral and tricuspid regurgitation patients with sinus rhythm, and right atrial and left atrial tissues of mitral and tricuspid regurgitation patients with atrial fibrillation. Taken together, this study showed that mitochondrial pathway for apoptosis was activated in the right atria in sinus rhythm and in the left and right atria in atrial fibrillation of heart failure patients due to mitral and tricuspid regurgitation, and this mitochondrial pathway activation may contribute to atrial contractile dysfunction and enlargement in this clinical setting.

Background. Left atrial enlargement is a mortality and heart failure risk factor in primary mitral regurgitation (MR) patients. Pig models of MR have shown differential expression of genes linked to the renin-angiotensin system. Therefore, the aim of this study was to investigate the key genes of the renin-angiotensin that are expressed differentially in the left atrial myocardium in MR patients. Methods. Quantitative RT-PCR was used to compare gene expression in the renin-angiotensin system in the left atrium in MR patients, aortic valve disease patients, and normal subjects. Results. Plasma angiotensin II concentrations did not significantly differ between MR patients and aortic valve disease patients (P=0.582). Compared to normal controls, however, MR patients had significantly downregulated expressions of angiotensin-converting enzyme, angiotensin I converting enzyme 2, type 1 angiotensin II receptor, glutamyl aminopeptidase, angiotensinogen, cathepsin A (CTSA), thimet oligopeptidase 1, neurolysin, alanyl aminopeptidase, cathepsin G, leucyl/cystinyl aminopeptidase (LNPEP), neprilysin, and carboxypeptidase A3 in the left atrium. The MR patients also had significantly upregulated expressions of MAS1 oncogene (MAS1) and mineralocorticoid receptor compared to normal controls. Additionally, in comparison with aortic valve disease patients, MR patients had significantly downregulated CTSA and LNPEP expression and significantly upregulated MAS1 expression in the left atrium. Conclusions. Expressions of genes in the renin-angiotensin system, especially CTSA, LNPEP, and MAS1, in the left atrium in MR patients significantly differed from expressions of these genes in aortic valve disease patients and normal controls. Notably, differences in expression were independent of circulating angiotensin II levels. The results of this study provide a rationale for pharmacological therapies or posttranslational regulation therapies targeting genes expressed differentially in the renin-angiotensin system to remedy structural remodeling associated with atrial enlargement and heart failure progression in patients with MR.

Background Differentially expressed genes in the left atria of mitral regurgitation (MR) pigs have been linked to peroxisome proliferator-activated receptor (PPAR) signaling pathway in the KEGG pathway. However, specific genes of the PPAR signaling pathway in the left atria of MR patients have never been explored. Methods This study enrolled 15 MR patients with heart failure, 7 patients with aortic valve disease and heart failure, and 6 normal controls. We used PCR assay (84 genes) for PPAR pathway and quantitative RT-PCR to study specific genes of the PPAR pathway in the left atria. Results Gene expression profiling analysis through PCR assay identified 23 genes to be differentially expressed in the left atria of MR patients compared to normal controls. The expressions of APOA1, ACADM, FABP3, ETFDH, ECH1, CPT1B, CPT2, SLC27A6, ACAA2, SMARCD3, SORBS1, EHHADH, SLC27A1, PPARGC1B, PPARA and CPT1A were significantly up-regulated, whereas the expression of PLTP was significantly down-regulated in the MR patients compared to normal controls. The expressions of HMGCS2, ACADM, FABP3, MLYCD, ECH1, ACAA2, EHHADH, CPT1A and PLTP were significantly up-regulated in the MR patients compared to patients with aortic valve disease. Notably, only ACADM, FABP3, ECH1, ACAA2, EHHADH, CPT1A and PLTP of the PPAR pathway were significantly differentially expressed in the MR patients compared to patients with aortic valve disease and normal controls. Conclusions Differentially expressed genes of the PPAR pathway have been identified in the left atria of MR patients compared with patients with aortic valve disease and normal controls.

Eur J Clin Invest 2011; 41 (3): 299–307 Abstract Background Myolysis of atrial cardiomyocytes occurs in patients with severe mitral and tricuspid regurgitation. This morphological remodelling may involve autophagy. Methods This study comprised 20 patients (10 with long-standing persistent atrial fibrillation and 10 with sinus rhythm) with severe mitral and tricuspid regurgitation. Atrial appendageal tissues were obtained during surgery. The appearance of autophagosomes (LC3B) in myocytes can reflect autophagy induction. Complement 9 is used as a reliable marker of oncosis. Results In the fibrillating right atria, 68·4 ± 18·9% of total myocytes showed moderate-to-severe myolysis, while 64·2 ± 15·8% of total myocytes comprised these cells in right atrial myocardium with sinus rhythm. Immunohistochemical study revealed LC3B-positive myocytes in 8·0% of myocytes without myolysis, 11·9% of myocytes with mild myolysis and 49·4% of myocytes with moderate-to-severe myolysis in right atrial myocardium with sinus rhythm (P

Left atrial enlargement associated with mitral regurgitation (MR) predicts a poor prognosis. However, the underlying regulatory mechanisms of atrial remodeling remain unclear. We used high-density oligonucleotide microarrays and enrichment analysis to identify the alteration of RNA expression pattern and biological processes involved in the atrial remodeling of pigs with and without MR. Gene arrays from left atria tissues were compared in 13 pigs (iatrogenic MR pigs [n = 6], iatrogenic MR pigs treated with valsartan [n = 4], and pigs without MR [n = 3]). A total of 22 genes were differentially upregulated by altered fold change2.0 (Log2FC 1), and 49 genes were differentially downregulated by altered fold change0.5 (Log2FC -1) in the left atria of the MR pigs compared with the pigs without MR. Enrichment analysis showed that renin-angiotensin system was identified in the Kyoto Encyclopedia of Genes and Genomes pathway. Notably, 12 of the 22 upregulated genes were identified to be downregulated by valsartan and 10 of the 49 downregulated genes were identified to be upregulated by valsartan. The tissue concentrations of angiotensin II and gene expression of hypertrophic gene, myosin regulatory light chain 2, ventricular isoforms, and fibrosis-related genes were significantly increased in the MR pigs compared with pigs without MR. In conclusion, differentially expressed transcriptome and related biological pathways have been identified in the left atria of the MR pigs compared with pigs without MR. Additionally, some of the differentially expressed genes could be regulated by type I angiotensin II receptor blocker.

Myolysis of atrial cardiomyocytes occurs in patients with severe mitral and tricuspid regurgitation. This morphological remodelling may involve autophagy.This study comprised 20 patients (10 with long-standing persistent atrial fibrillation and 10 with sinus rhythm) with severe mitral and tricuspid regurgitation. Atrial appendageal tissues were obtained during surgery. The appearance of autophagosomes (LC3B) in myocytes can reflect autophagy induction. Complement 9 is used as a reliable marker of oncosis.In the fibrillating right atria, 68·4 ± 18·9% of total myocytes showed moderate-to-severe myolysis, while 64·2 ± 15·8% of total myocytes comprised these cells in right atrial myocardium with sinus rhythm. Immunohistochemical study revealed LC3B-positive myocytes in 8·0% of myocytes without myolysis, 11·9% of myocytes with mild myolysis and 49·4% of myocytes with moderate-to-severe myolysis in right atrial myocardium with sinus rhythm (P0·0001). Similarly, in the fibrillating right atria, LC3B-positive myocytes were observed in 5·9% of myocytes without myolysis, 12·2% of myocytes with mild myolysis and 50·7% of myocytes with moderate-to-severe myolysis (P0·0001). Moreover, in the fibrillating left atria, LC3B-positive myocytes were observed in 4·9% of myocytes without myolysis, 12·6% of myocytes with mild myolysis and 52·0% of myocytes with moderate-to-severe myolysis (P0·0001). None of the atrial myocytes displayed intracellular deposition of complement 9.Induction of autophagy, but not oncosis, occurs in most cases of atrial cardiomyocytes with severe mitral and tricuspid regurgitation, even those without atrial fibrillation, and is closely associated with the development of myolysis in this disease.

Background Most of the atrial cardiomyocytes with positive terminal deoxynucleotidyl transferase (TdT)-mediated dUTP in situ nick end-labelling (TUNEL) reaction are not apoptotic in patients with mitral and tricuspid valve diseases. The TUNEL-positive myocytes with expression of spliceosome assembly factor SC-35, an indicator of increased RNA synthesis, should be living cardiomyocytes. Methods This study analyzed twenty-three patients with significant mitral and tricuspid regurgitation. Fifteen patients had persistent atrial fibrillation, and eight had sinus rhythm. Atrial appendageal tissues were obtained during surgery. Immunohistochemical study was performed. Results Immunohistochemical study of fibrillating right atrial myocardium demonstrated that 44.8±24.6% of myocytes had TUNEL-positive nuclei whereas 39.4±21.4% of myocytes had TUNEL-positive nuclei in sinus right atrial myocardium ( p =0.682). However, most (81.6%) nuclei of TUNEL-positive myocytes in the fibrillating right atria also expressed proliferating cell nuclear antigen (PCNA), an indicator of DNA replication and repair, and most nuclei (91.8%) of TUNEL-positive myocytes also expressed SC-35. In fibrillating left atria, most (88.1%) nuclei of TUNEL-positive myocytes also expressed SC-35. Similarly, in sinus right atrial myocardium, most (78.0%) nuclei of TUNEL-positive myocytes expressed PCNA, and most (91.4%) nuclei of TUNEL-positive myocytes also expressed SC-35, but none expressed Ki-67, a replication-associated antigen. Additionally, the percentage of TUNEL-positive myocytes in the right atria significantly and positively correlated with the percentage of PCNA-positive myocytes (r=0.826, p p Conclusions Most TUNEL-positive atrial cardiomyocytes in patients with mitral and tricuspid regurgitation are living cardiomyocytes.

Background Severe mitral regurgitation (MR) may cause myolysis in the left atrial myocytes. Myolysis may contribute to atrial enlargement. However, the relationship between Rho-associated kinase (ROCK) and myolysis in the left atrial myocytes of MR patients remain unclear. Methods This study comprised 22 patients with severe MR [12 with atrial fibrillation (AF) and ten in sinus rhythm]. Left atrial appendage tissues were obtained during surgery. Normal left atrial tissues were purchased. Immunofluorescence histochemical and immunoblotting studies were performed. Results The expression of ROCK2 in the myolytic left atrial myocytes of MR AF patients (p = 0.009) and MR sinus patients (p = 0.011) were significantly higher than that of the normal subjects. Similarly, the expression of ROCK1 in the myolytic left atrial myocytes of MR AF patients was significantly higher than that of the normal subjects (p = 0.010), and the expression of ROCK1 in the myolytic left atrial myocytes of MR sinus patients was higher than that of the normal subjects (p = 0.091). Immunofluorescence study revealed significant co-localization and juxtaposition of ROCK2 and cleaved caspase-3 in the left atrial myocytes both in the MR AF group (Pearson’s coefficient = 0.74 ± 0.03) and the MR sinus group (Pearson’s coefficient = 0.73 ± 0.02). Similarly, immunofluorescence study revealed significant co-localization and juxtaposition of ROCK1 and cleaved caspase-3 in the left atrial myocytes both in the MR AF group (Pearson’s coefficient = 0.65 ± 0.03) and the MR sinus group (Pearson’s coefficient = 0.65 ± 0.03). Correlation analysis demonstrated that there was a significant direct relationship between the expression of ROCK2 in the myolytic left atrial myocytes and left atrial diameter in the MR patients (p = 0.041; r = 0.440). Moreover, the ratio of phosphorylated myosin-binding subunit of myosin light chain phosphatase (pMBS)/total MBS of left atrial tissues was significantly higher in the MR AF group (p