Early Goal-Directed Renal Replacement Therapy in Acute Decompensated Heart Failure Patients with Cardiorenal Syndrome.

INTRODUCTION: The aim of this study was to clarify the efficacy of early goal-directed renal replacement therapy (GDRRT) for treatment of cardiorenal syndrome (CRS) patients after acute decompensated heart failure (ADHF). METHODS: In the retrospective, observational study, we enrolled 54 patients in the early GDRRT group and 63 patients in the late GDRRT group. Baseline characteristics, clinical data at initiation renal replacement therapy time, and the clinical outcome were collected and several parameters were compared and analyzed between 2 groups. RESULTS: The urine volume at GDRRT initiation time in the early group was higher than that in the late GDRRT group (1,060.3 ± 332.1 vs. 300.5 ± 148.3 mL, p < 0.001). Hemodynamic parameters such as mean artery pressure were higher (70.06 ± 32.99 vs. 54.34 ± 40.88 mm Hg, p = 0.012), the heart rate was slower (80.17 ± 15.26 vs. 99.21 ± 25.45 bpm, p = 0.002), and the diameter of inferior vena cava was narrower (22.00 ± 1.91 vs. 25.77 ± 5.5 mm, p = 0.04) in early GDRRT. Primary end point was inhospital all-cause mortality and cardiovascular mortality, which was obviously lower in the early GDRRT group (respectively 24.1 vs. 60.3%, p = 0.002 and 20.3 vs. 50.8%, p = 0.005). The second end point of kidney recovery in the early GDRRT group was much better than that in the latter GDRRT group (p = 0.018). Moreover, urine volume after GDRRT of the early group was more significant than that of the late group (1,432 ± 172 vs. 702 ± 183 mL, p = 0.005). CONCLUSION: This study clarified the effectiveness of the early GDRRT strategy in ADHF patients suffered from CRS, which reduced inhospital mortality and improved the urine output and clinical kidney recovery outcome.

Relationship between Soluble ST2 and Left Ventricular Geometry in Maintenance Hemodialysis Patients.

INTRODUCTION: Left ventricular hypertrophy (LVH) is a highly prevalent presentation of cardiac structural abnormality and a strong predictor of adverse outcomes in maintenance hemodialysis (MHD) patients. Different left ventricular geometry may provide additional clinical information. Soluble ST2 is a novel cardiac prognostic biomarker in MHD patients and is closely related to cardiac remodeling. OBJECTIVE: This study sought to evaluate the association of sST2 and left ventricular structure in a cohort of MHD patients. METHODS: Two hundred eighty-seven patients were enrolled. Left ventricular structure was assessed via transthoracic echocardiography. Left ventricular geometric patterns were defined according to left ventricular mass index and relative wall thickness (RWT). Serum sST2 levels were measured. RESULTS: Prevalence of LVH was 44.9% in the study population. In univariate analysis, sST2 levels were correlated with interventricular septal wall thickness, posterior wall thickness, and RWT. After multivariate adjustment, sST2 was independently correlated with only RWT (p = 0.028). Comparing sST2 concentrations across different LV geometric patterns, we found sST2 levels were significantly increased in patients with concentric cardiac remodeling and concentric LVH. CONCLUSIONS: The present study found that sST2 were significantly increased in patients with concentric remodeling and concentric LVH. ST2/interleukin (IL)-33 signaling might participate in the process of cardiac remodeling via its pro-fibrotic action. Future studies on the mechanism of ST2/IL-33 pathway are needed.

Association of trimethylamine N-Oxide with cardiovascular and all-cause mortality in hemodialysis patients.

BACKGROUND: Trimethylamine-N-Oxide (TMAO) is a proatherogenic and prothrombotic metabolite. Our study examined the association of plasma TMAO level with cardiovascular and all-cause mortality in hemodialysis (HD) patients. METHODS: Patients who were at least 18 years-old and received HD for at least 6 months were enrolled within 6 months. Patients with coronary heart disease, congestive heart failure, arrhythmia, or stroke within 3 months before study onset were excluded. The primary endpoints were cardiovascular and all-cause death, and the secondary endpoint was cerebrovascular death. RESULTS: We recruited 252 patients and divided them into a high-TMAO group (>4.73 µg/mL) and a low-TMAO group (≤4.73 µg/mL). The median follow-up time was 73.4 months (interquartile range: 42.9, 108). A total of 123 patients died, 39 from cardiovascular disease, 19 from cerebrovascular disease, and 65 from other causes. Kaplan-Meier analysis indicated that the high-TMAO group had a greater incidence of cardiovascular death (Log-Rank: p = 0.006) and all-cause death (Log-Rank: p < 0.001). Cox regression analysis showed that high TMAO level was significantly associated with cardiovascular and all-cause mortality. After adjustment for confounding, this association remained significant for cardiovascular mortality (TMAO as a continuous variable: HR: 1.18, 95%CI: 1.07, 1.294, p < 0.001; TMAO as a dichotomous variable: HR: 3.44, 95%CI: 1.68, 7.08, p < 0.001) and all-cause mortality (TMAO as a continuous variable: HR: 1.14, 95%CI: 1.08, 1.21, p < 0.001; TMAO as a dichotomous variable: HR: 2.54, 95%CI: 1.71, 3.76, p < 0.001). CONCLUSIONS: High plasma TMAO level is significantly and independently associated with cardiovascular and all-cause mortality in HD patients.

Sphingosine 1-phosphate alleviates Coxsackievirus B3-induced myocarditis by increasing invariant natural killer T cells.

Sphingosine 1-phosphate (S1P), via binding to its specific receptors of S1PR1, participates in the regulation of both innate and adaptive immunity. Recent reports have identified S1P as a messenger mediating inflammation. However, roles of S1P in Coxsackievirus B3 (CVB3)-induced myocarditis were largely unknown. Here, we investigated the effect of S1P treatment on CVB3-induced myocarditis in vivo. We found that CVB3 infection downregulated S1PR1 expression in spleen and decreased the proportion of invariant natural killer T cells (iNKT) in CD3 positive T cells both in spleen and in blood from left ventricle, which accompanied by severe inflammation lesions and more virus capsid protein (VP1) expression in heart tissue. In comparison, S1P supply upregulated iNKT in the spleen and in blood from left ventricle, which represented the strengthening of anti-inflammatory effects. Indeed, inflammation infiltration, VP1 expression and apoptosis in the myocardium was all downregulated. These results demonstrated that S1P supplement could alleviate CVB3-induced myocarditis.

Roles of AhR/CYP1s signaling pathway mediated ROS production in uremic cardiomyopathy.

PURPOSE: Uremic cardiomyopathy (UCM) is the leading cause of chronic kidney disease (CKD) related mortality. Uremic toxins including indoxyl sulfate (IS) play important role during the progression of UCM. This study was to explore the underlying mechanism of IS related myocardial injury. METHODS: UCM rat model was established through five-sixths nephrectomy to evaluate its effects on blood pressure, cardiac impairment, and histological changes using echocardiography and histological analysis. Additionally, IS was administered to neonatal rat cardiomyocytes (NRCMs) and the human cardiomyocyte cell line AC16. DHE staining and peroxide-sensitive dye 2',7'-dichlorofluorescein diacetate (H2DCFDA) was conducted to assess the reactive oxygen species (ROS) production. Cardiomyocyte hypertrophy was estimated using wheat germ agglutinin (WGA) staining and immunofluorescence. Aryl hydrocarbon receptor (AhR) translocation was observed by immunofluorescence. The activation of AhR was evaluated by immunoblotting of cytochrome P450 1 s (CYP1s) and quantitative real-time PCR (RT-PCR) analysis of AHRR and PTGS2. Additionally, the pro-oxidative and pro-hypertrophic effects were evaluated using the AhR inhibitor CH-223191, the CYP1s inhibitor Alizarin and the ROS scavenger N-Acetylcysteine (NAC). RESULTS: UCM rat model was successfully established, and cardiac hypertrophy, accompanied by increased blood pressure, and myocardial fibrosis. Further research confirmed the activation of the AhR pathway in UCM rats including AhR translocation and downstream protein CYP1s expression, accompanied with increasing ROS production detected by DHE staining. In vitro experiment demonstrated a translocation of AhR triggered by IS, leading to significant increase of downstream gene expression. Subsequently study indicated a close relationship between the production of ROS and the activation of AhR/CYP1s, which was effectively blocked by applying AhR inhibitor, CYP1s inhibitor and siRNA against AhR. Moreover, the inhibition of AhR/CYP1s/ROS pathway collectively blocked the pro-hypertrophic effect of IS-mediated cardiomyopathy. CONCLUSION: This study provides evidence that the AhR/CYP1s pathway is activated in UCM rats, and this activation is correlated with the uremic toxin IS. In vitro studies indicate that IS can stimulate the AhR translocation in cardiomyocyte, triggering to the production of intracellular ROS via CYP1s. This process leads to prolonged oxidative stress stimulation and thus contributes to the progression of uremic toxin-mediated cardiomyopathy.

Identification of resident progenitors labeled with Top2a responsible for proximal tubular regeneration in ischemia reperfusion-induced acute kidney injury.

BACKGROUND: Acute kidney injury is a common fatal disease with complex etiology and limited treatment methods. Proximal tubules (PTs) are the most vulnerable segment. Not only in injured kidneys but also in normal kidneys, shedding of PTs often happens. However, the source cells and mechanism of their regeneration remain unclear. METHODS: ScRNA and snRNA sequencing data of acute injured or normal kidney were downloaded from GEO database to identify the candidate biomarker of progenitor of proximal tubules. SLICE algorithm and CytoTRACE analyses were employed to evaluate the stemness of progenitors. Then the repairing trajectory was constructed through pseudotime analyses. SCENIC algorithm was used to detect cell-type-specific regulon. With spatial transcriptome data, the location of progenitors was simulated. Neonatal/ adult/ aged mice and preconditioning AKI mice model and deconvolution of 2 RNA-seq data were employed for validation. RESULTS: Through cluster identification, PT cluster expressed Top2a specifically was identified to increase significantly during AKI. With relatively strong stemness, the Top2a-labeled PT cluster tended to be the origin of the repairing trajectory. Moreover, the cluster was regulated by Pbx3-based regulon and possessed great segmental heterogeneity. Changes of Top2a between neonatal and aged mice and among AKI models validated the progenitor role of Top2a-labeled cluster. CONCLUSIONS: Our study provided transcriptomic evidence that resident proximal tubular progenitors labeled with Top2a participated in regeneration. Considering the segmental heterogeneity, we find that there is a group of reserve progenitor cells in each tubular segment. When AKI occurs, the reserve progenitors of each tubular segment proliferate and replenish first, and PT-progenitors, a cluster with no obvious PT markers replenish each subpopulation of the reserve cells.

Soluble interleukin-2 receptor predicts acute kidney injury and in-hospital mortality in patients with acute myocardial infarction.

BACKGROUND: Acute kidney injury (AKI) is the most common and critical complication in patients with acute myocardial infarction (AMI). This study aims to evaluate the significance of elevated soluble interleukin 2 receptor (sIL-2R) levels in predicting AKI and mortality. METHODS: A total of 446 patients with AMI were enrolled between January 2020 and July 2022, including 58 patients with AKI and 388 without AKI. The sIL-2R levels were measured using a commercially available chemiluminescence enzyme immunoassay. Logistic regression analysis was used to examine the risk factors for AKI. Discrimination was assessed based on the area under the receiver operating characteristic curve. The model was internally validated using 10-fold cross-validation. RESULTS: During hospitalization, 13% of patients developed AKI following AMI, with higher sIL-2R levels (0.61 ± 0.27 U/L vs. 0.42 ± 0.19 U/L, p = 0.003) and in-hospital all-cause mortality (12.1% vs. 2.6%, P < 0.001). The sIL-2R levels emerged as an independent risk factor for both AKI (OR = 5.08, 95% CI (1.04-24.84, p < 0.045) and in-hospital all-cause mortality (OR = 73.57,95% CI 10.24-528.41, p < 0.001) in AMI patients. The sIL-2R levels were found to be useful biomarkers in prediction of AKI and in-hospital all-cause mortality in patients with AMI (AUC: 0.771 and 0.894, respectively). The respective cutoff values for sIL-2R levels in predicting AKI and in-hospital all-cause mortality were determined to be 0.423 U/L and 0.615 U/L. CONCLUSIONS: The level of sIL-2R was an independent risk factor and predictor for both AKI and in-hospital all-cause mortality in patients with AMI. These findings highlight the potential of sIL-2R as a valuable tool for identifying high-risk patients regarding AKI and in-hospital mortality.

Metagenome-wide analysis uncovers gut microbial signatures and implicates taxon-specific functions in end-stage renal disease.

BACKGROUND: The gut microbiota plays a crucial role in regulating host metabolism and producing uremic toxins in patients with end-stage renal disease (ESRD). Our objective is to advance toward a holistic understanding of the gut ecosystem and its functional capacity in such patients, which is still lacking. RESULTS: Herein, we explore the gut microbiome of 378 hemodialytic ESRD patients and 290 healthy volunteers from two independent cohorts via deep metagenomic sequencing and metagenome-assembled-genome-based characterization of their feces. Our findings reveal fundamental alterations in the ESRD microbiome, characterized by a panel of 348 differentially abundant species, including ESRD-elevated representatives of Blautia spp., Dorea spp., and Eggerthellaceae, and ESRD-depleted Prevotella and Roseburia species. Through functional annotation of the ESRD-associated species, we uncover various taxon-specific functions linked to the disease, such as antimicrobial resistance, aromatic compound degradation, and biosynthesis of small bioactive molecules. Additionally, we show that the gut microbial composition can be utilized to predict serum uremic toxin concentrations, and based on this, we identify the key toxin-contributing species. Furthermore, our investigation extended to 47 additional non-dialyzed chronic kidney disease (CKD) patients, revealing a significant correlation between the abundance of ESRD-associated microbial signatures and CKD progression. CONCLUSION: This study delineates the taxonomic and functional landscapes and biomarkers of the ESRD microbiome. Understanding the role of gut microbiota in ESRD could open new avenues for therapeutic interventions and personalized treatment approaches in patients with this condition.

Aldehyde dehydrogenase-2 deficiency aggravates cardiac dysfunction elicited by endoplasmic reticulum stress induction.

Mitochondrial aldehyde dehydrogenase-2 (ALDH2) has been characterized as an important mediator of endogenous cytoprotection in the heart. This study was designed to examine the role of ALDH2 knockout (KO) in the regulation of cardiac function after endoplasmic reticulum (ER) stress. Wild-type (WT) and ALDH2 KO mice were subjected to a tunicamycin challenge, and the echocardiographic property was examined. Protein levels of six items--78 kDa glucose-regulated protein (GRP78), phosphorylation of eukaryotic initiation factor 2 subunit α (p-eIF2α), CCAAT/enhancer-binding protein homologous protein (CHOP), phosphorylation of Akt, p47(phox) nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and 4-hydroxynonenal--were determined by using Western blot analysis. Cytotoxicity and apoptosis were estimated using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay and caspase-3 activity, respectively. ALDH2 deficiency exacerbated cardiac contractile dysfunction and promoted ER stress after ER stress induction, manifested by the changes of ejection fraction and fractional shortening. In vitro study revealed that tunicamycin significantly upregulated the levels of GRP78, p-eIF2α, CHOP, p47(phox) NADPH oxidase and 4-hydroxynonenal, which was exacerbated by ALDH2 knockdown and abolished by ALDH2 overexpression, respectively. Overexpression of ALDH2 abrogated tunicamycin-induced dephosphorylation Akt. Inhibition of phosphatidylinositol 3-kinase using LY294002 did not affect ALDH2-conferred protection against ER stress, although LY294002 reversed the antiapoptotic action of ALDH2 associated with p47(phox) NADPH oxidase. These results suggest a pivotal role of ALDH2 in the regulation of ER stress and ER stress-induced apoptosis. The protective role of ALDH2 against ER stress-induced cell death was probably mediated by Akt via a p47(phox) NADPH oxidase-dependent manner. These findings indicate the critical role of ALDH2 in the pathogenesis of ER stress in heart disease.

[Role of triggering receptor expressed on myeloid cells-1 on coxsackievirus B3-induced inflammation and cardiomyocyte injury].

OBJECTIVE: To determine the expression of TREM-1 (triggering receptor expressed on myeloid cells-1) in macrophages after coxsackievirus B3 (CVB3) infection and the cardiomyocytes viability after culturing with supernatant of macrophages in the absence and presence of TREM-1 inhibitor LP-17 to explore if TREM-1 is involved in the pathogenesis of CVB3 infection induced inflammation and cardiomyocytes injury. METHODS: TREM-1 mRNA and TREM-1 and DAP-12 protein expression in macrophages were detected by Real-time PCR at 0, 1, 4, 8 and 12 h and by Western blot at 0, 16, 24 and 48 h post CVB3 infection. TNF-α secretion of macrophages was measure by ELISA, vitality and the apoptosis degree of cardiomyocytes was assessed by CCK8 and Annexin V-FITC after the cardiomyocytes were cultured with the supernatant of macrophages in normal control group, CVB3 infection group and LP-17 pretreated CVB3 infection group. RESULTS: TREM-1 mRNA expression was significantly upregulated at 4, 8, and 12 h (peaked at 8 h) and TREM-1 protein expression was significantly upregulated at 16 and 24 h and returned to baseline level at 48 h after CVB3 infection. The protein expression of DAP-12, a direct downstream signaling molecule of TREM-1, also significantly increased at 24 and 48 h post CVB3 infection (P < 0.01). Level of macrophages secreted TNF-α post CVB3 infection was significantly reduced in LP-17 pretreated cells (P < 0.01), LP-17 pretreatment also significantly improved viability and significantly reduced apoptosis of cardiomyocytes cultured with supernatant of CVB3 infected macrophages (P < 0.01). CONCLUSION: TREM-1 might be an important mediator post CVB3 infection and a major player on inducing excess macrophages-related inflammation and resulting in an indirect injury to cardiomyocytes.

Effect Modification of Hyperuricemia, Cardiovascular Risk, and Age on Chronic Kidney Disease in China: A Cross-Sectional Study Based on the China Health and Nutrition Survey Cohort.

Introduction: The question of whether the increased burden of chronic kidney disease (CKD) is caused by the interaction of hyperuricemia and cardiovascular disease (CVD) risk factors or is accelerated by aging remains unresolved. The purpose of this study is to better understand the effect modification of hyperuricemia, cardiovascular risk, and age on CKD among the Chinese population. Methods: This cross-sectional study of 8243 participants was derived from the China Health and Nutrition Survey (CHNS) in 2009. Inclusion criteria included age ≥18 years, non-pregnancy, and no history of high-protein diet prior to blood test. Demographics, comorbidities, health-related behaviors, and serum biomarkers were collected. Interaction association of hyperuricemia, CVD risk and age with CKD were analyzed using Logistic regression. Results: CKD was detected in 359 (27.2%, 95% CI 24.8∼29.7%) of 1321 participants with hyperuricemia and 680 (9.8%, 95% CI 9.1∼10.5%) of 6,922 participants without hyperuricemia, and these patterns remained significant after controlling for age, gender, and Framingham risk score (adjusted odds ratio [aOR] 3.82, 95% CI 3.20∼4.57). We found a negative multiplicative interaction between hyperuricemia and CVD risk on CKD. The aOR in low-CVD risk groups was 5.51 (95% CI 4.03∼7.52), followed by medium-CVD risk groups (aOR: 3.64, 95% CI 2.61∼5.09) and high-CVD risk groups (aOR: 2.89, 95% CI 2.12∼3.96). CVD risk was less associated with CKD in hyperuricemia group (aOR: 0.92, 95% CI 0.68∼1.22) than in non-hyperuricemia group (aOR: 1.43, 95% CI 1.21∼1.70). Furthermore, hyperuricemia and age had a significant additive effect on CKD, with a synergy index of 2.26 (95% CI 1.45∼3.52). Coexisting with older age and hyperuricemia, the likelihood of developing CKD was higher than the sum of the two alone. Conclusion: The link between hyperuricemia and CKD begins at a young age and becomes stronger in the low CVD risk group. For young adults, early detection of hyperuricemia, routine CVD risk assessment, and timely intervention of modifiable factors are warranted.

The ratio of monocytes to lymphocytes multiplying platelet predicts incidence of pulmonary infection-related acute kidney injury.

BACKGROUND: Inflammation is a crucial factor in the pathogenesis and development of acute kidney injury (AKI). Macrophages, as an important innate immune cell, regulate immune response and play a pathophysiological role in AKI. This study aimed to evaluate the predictive capacity of peripheral blood monocytes for the incidence of pulmonary infection-related AKI. METHODS: We recruited 1038 hospitalized patients with pulmonary infections from January 1 to December 31, 2019, in Zhongshan Hospital, Fudan University. Patients were divided into derivation and validation cohorts. Data on demographic characteristics, disease history, and biochemical indexes were retrieved from the electronic medical system. The composite inflammatory indexes were calculated as monocyte/(lymphocyte × platelet ratio) (MLPR). We applied dose-response relationship analyses to delineate the nonlinear odds ratio (OR) in different MLPR levels and integrated it into a logistic model to predict the risk of AKI. RESULTS: The incidence of hospital-acquired AKI was 18.8% in the derivation cohort. Compared to non-AKI, the MLPR levels were significantly higher in AKI patients. Dose-response curve revealed that the increase of AKI risk was faster in the first half of MLPR and then tended to flatten. After classifying the MLPR levels into six groups, the AKI incidence increased from 4.5% to 55.3% with a peaking OR of 24.38. The AUC values of the AKI model only including MLPR were 0.740, and after gradually integrating other covariates, the area under the receiver operating characteristic (AUC) value reached 0.866, which was significantly higher than the AUC of full models without MLPR (0.822). Moreover, the better prediction ability of AKI was observed in the external validation, with an AUC of 0.899. CONCLUSION: MLPR has good predictive efficiency in AKI, which can be used as a simple and easy clinical composite index to effectively predict early pulmonary infection-related AKI.

Improvement of cardiac function after coronary artery bypass grafting surgery reduces the risk of postoperative acute kidney injury.

BACKGROUND: Pre-existing renal dysfunction is an independent risk factor for cardiac surgery-associated acute kidney injury (AKI). We aimed to investigate whether the improvement of postoperative cardiac function after coronary artery bypass grafting (CABG) surgery would affect the risk of AKI in patients with different levels of baseline renal function. METHODS: Data were collected from patients who underwent CABG surgery from January 2018 to April 2019. Patients were divided into normal (GFR ≥ 90 ml/min/1.73 m2 ), non-CKD (60≤GFR < 90 ml/min/1.73 m2 ), and CKD (GFR < 60 ml/min/1.73 m2 ) groups. Improvement in cardiac function was defined as △LVEF (postoperative LVEF-preoperative LVEF) ≥ 10% preoperative LVEF. Patients were further divided into subgroups according to postoperative cardiac function improvement. RESULTS: A total of 1365 patients were enrolled, including 793 (58.1%) in the normal group, 476 (34.9%) in the non-CKD group, and 96 (7.0%) in the CKD group. The AKI incidence in the normal, non-CKD, and CKD groups was 22.2%, 28.4%, and 40.6%, respectively. Patients with improved cardiac function in the non-CKD and CKD groups had significantly lower AKI incidence than those without improved cardiac function (22.8% vs. 36.9%, p = .002% and 32.8% vs. 54.3%, p = .037, respectively). For non-CKD patients with improved cardiac function, the serum creatinine at discharge was significantly lower than its preoperative serum creatinine (0.8 ± 0.5 vs 1.2 ± 0.9 mg/dl, p = .002). Multivariate logistic regression analysis showed that the improvement in cardiac function could reduce the risk for postoperative AKI in non-CKD patients but not in CKD patients. CONCLUSIONS: For patients with renal dysfunction and mildly reduced eGFR (60≤GFR < 90 ml/min/1.73 m2 ), improved cardiac function after CABG surgery can reduce the serum creatinine level and reduce the risk for postoperative AKI.

[Association between expression of chromogranin A and myocardial fibrosis in patients with dilated cardiomyopathy].

OBJECTIVE: To observe the possible correlation between expression of chromogranin A (CGA) and myocardial fibrosis and investigate the potential role of CGA in the development of myocardial fibrosis in patients with dilated cardiomyopathy (DCM). METHODS: Surgical myocardial specimen from 10 DCM patients underwent successful orthotopic cardiac transplantation, and 3 normal myocardial specimen from brain-dead organ donors were obtained. CGA-mRNA, COLI-mRNA, COLIII-mRNA and ADAMTS-1-mRNA were analyzed by real-time PCR. The location and expression of CGA were assessed by immunohistochemistry(INH)with anti-CGA antibody. The collagen specific picrosirius red staining was applied on transversal myocardial slides and the collagen volume fraction (CVF) was calculated. The correlation between CGA and CVF was analyzed. RESULTS: Cytoplasmic expression of CGA assessed by INH showed large amount of strong positive granules densely arranged in the epicardial and endocardial myocardiocytes in DCM specimen while there was only few sparse granules in the normal myocardium (P < 0.05). CVF was significantly higher in DCM myocardial specimen than that in normal specimen (P < 0.001). CGA-mRNA was significantly correlated with COLI-mRNA (r = 0.729), COLIII-mRNA (r = 0.95) and ADAMTS-1-mRNA (r = 0.665, all P < 0.05). Moreover, collagen deposition location was almost identical with the strong positive expression location of CGA. CONCLUSION: We demonstrated for the first time that the deposition of CGA was related with the myocardial fibrosis in DCM heart, therefore, CGA might play an important role by influencing myocardial remodeling and fibrosis in DCM patients.

Endothelial­to­mesenchymal transition in human idiopathic dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is characterized by left ventricular dilation and cardiac fibrosis. Emerging evidence indicated that endothelial­to­mesenchymal transition (Endo­MT) is a crucial event during organ fibrosis. This study was performed to clarify whether Endo­MT contributed to the progression of cardiac fibrosis in DCM. Cardiac samples from patients with DCM and control were obtained. The presence of endothelial markers, cluster of differentiation (CD)31 and vascular endothelial (VE)­cadherin, and mesenchymal markers, α smooth muscle actin (SMA) and fibroblast­specific protein 1 (FSP1) was performed using immunohistochemistry. Co­localization of endothelial markers and mesenchymal markers were identified using confocal immunofluorescence staining. Serum procollagen type I carboxy­terminal propeptide (PICP) and procollagen type III amino­terminal propeptide (PIIINP) were measured by ELISA. Protein levels of Wnt, ß­catenin and Snail were determined using western blot analysis. Immunohistochemistry and double­immunofluorescence staining demonstrated that the expression of CD31 and VE­cadherin were significantly decreased in DCM samples, whereas the FSP­1, and αSMA were significantly increased. CD31 and VE­cadherin labeling indexes were respectively negatively correlated with left ventricular end­diastolic diameter (LVEDD) (CD31 r=­0.82, P<0.01; VE­cadherin r=-0.73, P<0.01), while FSP­1 and αSMA were positively associated with LVEDD (αSMA r=0.65, P<0.01, FSP1 r=0.53, P<0.01) and left ventricular ejection fraction (αSMA r=­0.18, P<0.05; FSP1 r=­0.21, P<0.05). Furthermore, PICP and PIIINP levels were positively associated with the co­expression labeling indexes (CD31/SMA co­labeling index and PICP r=0.727, P<0.01; CD31/SMA co­labeling index and PIIINP r=0.741, P<0.01; VE­Cadherin/FSP­1 co­labeling index and PICP r=0.716, P<0.01; VE­cadherin/FSP­1 co­labeling index and PIIINP r=0.648, P<0.05). Western blot analysis indicated that proteins levels of Wnt signaling and snail were significantly increased in DCM samples. These results suggested that Endo­MT is potentially implicated in the pathogenesis of myocardial fibrosis and remodeling during the development of DCM, indicating a potential therapeutic target for DCM treatment.

The frequency of invariant natural killer T cells correlates with the severity of myocarditis.

Invariant natural killer T cells (iNKT) perform different functions in different diseases. The cells were reported to protect myocarditis. However, the detail relationships between iNKT and Coxsackievirus B3 (CVB3)-induced myocarditis remain unclear. In order to investigate the correlation between the severity of CVB3-induced inflammation infiltration and the proportion of iNKT in the spleen and circulating blood, BALB/c mice were grouped into three groups according to the inflammation infiltration area of heart sections. The proportion of iNKT in CD3-positive cells in the spleen correlated negatively with the inflammation area (linear fit; R(2)=0.93) and virus capsid protein VP1 (linear fit; R(2)=0.84) in the myocardial tissue, while the proportion of iNKT in CD3-positive cells in the PBMC positively correlated with the inflammation area (linear fit; R(2)=0.91) and virus capsid protein VP1 (linear fit; R(2)=0.93) in the myocardial tissue. The results imply that iNKT might be used as a parameter for the diagnosis of myocarditis in clinical practice.

Coxsackievirus B3-induced calpain activation facilitates the progeny virus replication via a likely mechanism related with both autophagy enhancement and apoptosis inhibition in the early phase of infection: an in vitro study in H9c2 cells.

Calpain is a family of neutral cysteine proteinase involved in many physiological and pathological processes including virus replication, autophagy and apoptosis. Previous study has indicated the involvement of calpain in pathogenesis of coxsackievirus B3 (CVB3)-induced myocarditis. Besides, many studies demonstrated that host cell autophagy and apoptosis mechanisms participate in virus life cycle. However, role of calpain in CVB3 replication via autophagy/apoptosis mechanisms has not been reported, which was discussed here in H9c2 cardiomyocytes. The data demonstrated that calpain was activated following CVB3 infection. Calpain inhibition decreased autophagy, indicating role of calpain in enhancing autophagy during CVB3 infection. Both calpain activity and autophagy were involved in facilitating CVB3 replication demonstrated by virus titer and CVB3 capsid protein VP1 expression alterations resulting from calpain inhibitor ALLN and autophagy inhibitor 3MA intervention. We also found that both calpain activity and autophagy suppressed caspase3 activity and host cell apoptosis 5-10h post-infection (p.i.). In summary, the present study shows that CVB3 infection of H9c2 cells hinders caspase3 activity provocation and cell apoptosis at least in the early phase of infection (5-10h p.i.) via calpain-induced autophagy enhancement, which might be a mechanism facilitating CVB3 replication in host cells.

Initial weight and virus dose: two factors affecting the onset of acute coxsackievirus B3 myocarditis in C57BL/6 mouse--a histopathology-based study.

BACKGROUND: C57BL/6 mice have been considered resistant to cardiotropic coxsackievirus B3 (CVB3)-induced myocarditis. However, establishment of viral myocarditis model in C57BL/6 mouse is still a necessity. Here, we discuss the effects of mouse initial body weight and different virus inoculation doses on the onset of acute viral myocarditis in C57BL/6 mouse. METHODS: According to initial body weight, mice were grouped into group A (3-4 weeks, 11-15 g) and group B (5-6 weeks, 18-20 g). Then, each group was divided into three subgroups inoculated with different virus doses: 1000 50% tissue culture infectious dose (TCID(50))/ml, 10,000 TCID(50)/ml, and 100,000 TCID(50)/ml. Virus existence and myocardium inflammatory infiltration conditions were observed and evaluated 7 days postinfection. RESULTS: Immunohistochemistry staining showed that virus capsid protein VP1 appeared in the myocardia of virus-infected mice. Three subgroups in the lower-body-weight group (group A) came out with a higher mortality (40%-100%), while the higher-body-weight group (group B) showed a tendency for body weight decline. Histopathologically, myocardia of the survival cases in the lower-initial-body-weight group got inflammatory infiltration, while almost no inflammation appeared in the dead cases. In the higher-body-weight group, myocardium inflammatory infiltration deteriorated with the increase of virus doses and bared a relatively sound survival rate. CONCLUSIONS: This study indicated that the initial body weight and virus infection dose are two factors affecting the onset of viral myocarditis in C57BL/6 mice. Accordingly, initial body weight of 18-20 g and an inoculation dose of 100,000 TCID(50)/ml × 0.3 ml CVB3 might be an optimal choice to induce acute viral myocarditis in C57BL/6 mice.

Impaired cardiac microvascular endothelial cells function induced by Coxsackievirus B3 infection and its potential role in cardiac fibrosis.

CVB3 virus tropism and tissue access are modulated by cardiac microvascular endothelial cells (CMVECs) in the context of microvasculature. This study was designed to examine biological behaviors of CMVECs following CVB3 infection and its possible effects on cardiac remodeling. Data demonstrated that CVB3 increased caspase-3 activities, Bax/Bcl-2 protein ratio and TGF-ß1 levels in CMVECs, accompanying with elevated microvascular permeability. Double immunofluorescence revealed co-localization of endothelial markers (CD31 and VE-cadherin) and mesenchymal markers (FSP1 and αSMA) in infected CMVECs. Western blot demonstrated that CVB3 significantly decreased the expression of endothelial markers and increased the expression of mesenchymal markers, which were reversed by SB431542 (inhibitor of TGF-ß1), indicating that endothelial-to-mesenchymal transition following CVB3 infection was probably induced by CMVECs-derived TGF-ß1. Excess extracellular matrix was produced by myocardial cells incubated with supernatants of infected CMVECs. Our results displayed that CVB3 induced notable biological changes of CMVECs, which may contribute to cardiac fibrosis.

Astragaloside IV attenuates myocardial fibrosis by inhibiting TGF-ß1 signaling in coxsackievirus B3-induced cardiomyopathy.

Myocardial fibrosis plays an important role in coxsackievirus B3 (CVB3) induced dilated cardiomyopathy. Excessive transforming growth factor (TGF)-ß1 contributes to a pathologic excess of tissue fibrosis. We investigated the effect of astragaloside IV on myocardial fibrosis in CVB3-induced dilated cardiomyopathy. BALB/c mice were inoculated with CVB3 to induce acute viral myocarditis on day 7 (acute VMC group), monthly for 3 months to induce chronic myocarditis (chronic VMC group), and monthly for 9 months to induce dilated cardiomyopathy (DCM group). The same method was used for the DCM+Astra group as that of the DCM group, but former group was given with astragaloside IV-containing drinking water. Compared to DCM group, astragaloside IV treatment significantly increased the survival rate. Histological findings and the collagen volume fraction showed that astragaloside IV decreased fibrosis in heart tissues. Astragaloside IV decreased the level of the serum carboxy-terminal propeptide of procollagen type I (PICP) and the ratio of PICP/ N-terminal type I procollagen propeptide (PINP). Ameliorated myocardial fibrosis was consistent with the downregulated expression of TGF-ß1 and its downstream pSmad2/3 and Smad4 in the myocardium of the DCM+Astra group compared to the DCM group. The level of type I collagen was lower in the DCM+Astra group than the DCM group. The same effect was found in the in vitro study. These findings showed that astragaloside IV had a potent preventive effect on myocardial fibrosis in CVB3-induced dilated cardiomyopathy that might be due to downregulation of TGF-ß1-Smad signaling.