Cellular-level analyses of SCN5A mutations in left ventricular noncompaction cardiomyopathy suggest electrophysiological mechanisms for ventricular tachycardia.

Left ventricular noncompaction cardiomyopathy (LVNC) is a cardiovascular disease characterized by arrhythmia and heart failure. In this study, LVNC myocardial samples were collected from patients who underwent heart transplantation and were analyzed using exome sequencing. Approximately half of the LVNC patients carried SCN5A variants, which are associated with clinical symptoms of ventricular tachycardia. To investigate the electrophysiological functions of these SCN5A variants and the underlying mechanism by which they increase arrhythmia susceptibility in LVNC patients, functional evaluations were conducted in CHO-K1 cells and human embryonic stem cell-derived cardiomyocytes (hESC-CMs) using patch-clamp or microelectrode array (MEA) techniques. These findings demonstrated that these SCN5A mutants exhibited gain-of-function properties, leading to increased channel activation and enhanced fast inactivation in CHO-K1 cells. Additionally, these mutants enhanced the excitability and contractility of the cardiomyocyte population in hESC-CMs models. All SCN5A variants induced fibrillation-like arrhythmia and increased the heart rate in cardiomyocytes. However, the administration of Lidocaine, an antiarrhythmic drug that acts on sodium ion channels, was able to rescue or alleviate fibrillation-like arrhythmias and secondary beat phenomenon. Based on these findings, it is speculated that SCN5A variants may contribute to susceptibility to arrhythmia in LVNC patients. Furthermore, the construction of cardiomyocyte models with SCN5A variants and their application in drug screening may facilitate the development of precise therapies for arrhythmia in the future.

Effects of CMYA1 overexpression on cardiac structure and function in mice.

CMYA1 (cardiomyopathy-associated protein 1, also termed Xin) localizes to the intercalated disks (ICDs) of the myocardium and functions to maintain ICD structural integrity and support signal transduction among cardiomyocytes. Our previous study showed that CMYA1 overexpression impairs the function of gap junction intercellular communication processes. Successful model generation was verified based on PCR, western blot analysis, immunohistochemistry, and immunofluorescence analysis. Myocardial CMYA1 expression was confirmed at both the mRNA and the protein levels in the CMYA1-OE transgenic mice. Masson's trichrome staining and electron microscopy revealed myocardial fibrosis and uneven bead width or the interruption of ICDs in the hearts of the CMYA1-OE transgenic mice. Furthermore, the Cx43 protein level was reduced in the CMYA1-OE mice, and co-immunoprecipitation assays of heart tissue protein extracts revealed a physical interaction between CMYA1 and Cx43. Electrocardiogram analysis enabled the detection of an obvious ventricular bigeminy for the CMYA1-OE mice. In summary, analysis of our mouse model indicates that elevated CMYA1 levels may induce myocardial fibrosis, impair ICDs, and downregulate the expression of Cx43. The observed ventricular bigeminy in the CMYA1-OE mice may be mediated by the reduced Cx43 protein level.

Elevated myocardial SORBS2 and the underlying implications in left ventricular noncompaction cardiomyopathy.

BACKGROUND:Left ventricular noncompaction cardiomyopathy (LVNC) is a hereditary heart disease characterized by an excessive trabecular meshwork of deep intertrabecular recesses within the ventricular myocardium. The guidelines for management of LVNC patients aim to improve quality of life by preventing cardiac heart failure. However, the mechanism underlying LVNC-associated heart failure remains poorly understood.METHODS:Using protein mass spectrometry analysis, we established that Sorbin And SH3 Domain Containing 2 (SORBS2) is up-regulated in LVNC hearts without changes to structure proteins. We conducted in vivo experiments wherein the heart tissues of wild-type mice were injected with an AAV9 vector to overexpress SORBS2, followed by analysis using echocardiography, T-tubule analysis and Ca2+ imaging to identify functional and morphological changes. In addition, we analyzed the function and structure of SORBS2 overexpressing human embryonic stem cell (hESC) derived cardiomyocytes (hESC-CM) via immunoblotting, immunohistochemistry, immunofluorescence, and confocal Ca2+ imaging.FINDINGS:LVNC myocardial tissues feature strongly elevated expression of SORBS2, microtubule densification and redistribution of Junctophilin 2 (JP2). SORBS2 interacts with β-tubulin, promoting its polymerization in 293T cells and hESC-derived CMs. In vivo, cardiac dysfunction, β-tubulin densification, JP2 translocation, T-tubule disorganization and Ca2+ handling dysfunction were observed in mice overexpressing SORBS2.INTERPRETATION:We identified a novel mechanism through which SORBS2 interacts with β-tubulin and promotes microtubule densification, eventually effecting JP2 distribution and T-tubule, potentially contributing to heart failure in LVNC disease. FUND: This work was supported by a CAMS Initiative for Innovative Medicine grant (CAMS-I2M, 2016-I2M-1-015 to Y.J.Wei).

Multiple genetic variants in adolescent patients with left ventricular noncompaction cardiomyopathy.

BACKGROUND:Left ventricular noncompaction cardiomyopathy (LVNC) is a primary cardiomyopathy with an unclear aetiology. The clinical symptoms range from asymptomatic to heart failure, arrhythmias and sudden cardiac death. This study aimed to characterize the genetic features and clinical outcomes of LVNC who underwent heart transplantation (HTx) to reveal the potential genetic pathogenesis.METHODS AND RESULTS:We recruited 16 cases who underwent HTx in our hospital. Exome-sequencing was performed to reveal genetic background. Clinical information and histopathology features of patients were investigated. Gene expression profiling of tissue fibrosis were evaluated by quantitative PCR. The median age of patients was 21 years. Of the 16 patients, 14 harboured multiple gene variants involved in LVNC. Ten of the patients harboured biallelic variants and/or truncating variants. Young patients (<18) with biallelic variants and/or truncating variants and lower LVEF (<45%) at initial symptom deteriorated quickly. Except for noncompaction myocardium, myocardial fibrosis was a remarkable pathological feature, and gene profiles related to immune inflammation and extracellular matrix remodelling were upregulated.CONCLUSIONS:This study showed that multiple pathologic variants were underlie genetic mechanism of LVNC who in high risks, suggesting that genetic screening should be applied to the diagnosis of LVNC. LVNC patient with multiple variants should be considered carefully follow-up. Genetics involved in the phenotype and cardiac fibrosis, and is the major causing for LVNC.

Comparative proteomics analysis of myocardium in left ventricular non-compaction cardiomyopathy.

Arrhythmogenic cardiomyopathy: Identification of desmosomal gene variations and desmosomal protein expression in variation carriers.

Arrhythmogenic cardiomyopathy (AC) is an inherited disorder that is predominantly present in the right ventricular myocardium. Mutations in the genes encoding the desmosomal protein are thought to underlie the pathogenesis of AC. Since AC is genetically heterogeneous and phenotypically diverse, modifier genes and environmental factors have an important role in disease expression. The aim of the present study was to identify AC-associated desmosomal gene variations, and examine the expression levels of intercalated disc proteins in AC patients who carry the variations (DSG2 p.Leu797Gln, PKP2 p.Ser249Thr and p.E808fsX30). The results of the present investigation provided information on the search for modifier genes and desmosomal gene mutations, and improved our understanding of the mechanism underlying these AC mutations. Genetic screening of five desmosomal genes (DSG2, DSC2, JUP, PKP2, and DSP) in 23 patients with AC who underwent heart transplantation was performed and the expression levels and localizations of intercalated disc proteins were assessed using western blotting and immunohistochemistry, respectively. The results enabled the identification of three desmosomal gene variations (DSG2 L797Q, PKP2 S249T, and E808fsX30), two of which are reported for the first time. DSG2 L797Q was identified in one patient. The protein expression levels of DSG2 in the L797Q carrier were unchanged compared with the healthy controls, and the expression levels of the other proteins (JUP and Cx43) in the intercalated disc were also similar between the healthy controls, the variation carrier and the case controls. Two variations (S249T and E808fsX30) in PKP2 were identified in one patient, the protein expression levels of PKP2 in this patient were significantly decreased, and the expression levels of the other proteins in the intercalated disc was also decreased. The data suggest that there may be modifier genes and other AC-associated mutations requiring identification, in order to further our understanding of the disease mechanism induced by these mutations.

Lipocalin-2 Promotes Endoplasmic Reticulum Stress and Proliferation by Augmenting Intracellular Iron in Human Pulmonary Arterial Smooth Muscle Cells.

Endoplasmic reticulum (ER) stress, a feature of many conditions associated with pulmonary hypertension (PH), is increasingly recognized as a common response to promote proliferation in the walls of pulmonary arteries. Increased expression of Lipocalin-2 in PH led us to test the hypothesis that Lipocalin-2, a protein known to sequester iron and regulate it intracellularly, might facilitate the ER stress and proliferation in pulmonary arterial smooth muscle cells (PASMCs). In this study, we observed greatly increased Lcn2 expression accompanied with increased ATF6 cleavage in a standard rat model of pulmonary hypertension induced by monocrotaline. In cultured human PASMCs, Lcn2 significantly promoted ER stress (determined by augmented cleavage and nuclear localization of ATF6, up-regulated transcription of GRP78 and NOGO, increased expression of SOD2, and mild augmented mitochondrial membrane potential) and proliferation (assessed by Ki67 staining and BrdU incorporation). Lcn2 promoted ER stress accompanied with augmented intracellular iron levels in human PASMCs. Treatment human PASMCs with FeSO4 induced the similar ER stress and proliferation response and iron chelator (deferoxamine) abrogated the ER stress and proliferation induced by Lcn2 in cultured human PASMCs. In conclusion, Lcn2 significantly promoted human PASMC ER stress and proliferation by augmenting intracellular iron. The up-regulation of Lcn2 probably involved in the pathogenesis and progression of PH.

Profiling of differentially expressed microRNAs in arrhythmogenic right ventricular cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a kind of primary cardiomyopathy characterized by the fibro-fatty replacement of right ventricular myocardium. Currently, myocardial microRNAs have been reported to play critical role in the pathophysiology of cardiovascular pathophysiology. So far, the profiling of microRNAs in ARVC has not been described. In this study, we applied S-Poly (T) Plus method to investigate the expression profile of microRNAs in 24 ARVC patients heart samples. The tissue levels of 1078 human microRNAs were assessed and were compared with levels in a group of 24 healthy controls. Analysis of the area under the receiver operating characteristic curve (ROC) supported the 21 validated microRNAs to be miRNA signatures of ARVC, eleven microRNAs were significantly increased in ARVC heart tissues and ten microRNAs were significantly decreased. After functional enrichment analysis, miR-21-5p and miR-135b were correlated with Wnt and Hippo pathway, which might involve in the molecular pathophysiology of ARVC. Overall, our data suggested that myocardial microRNAs were involved in the pathophysiology of ARVC, miR-21-5p and miR-135b were significantly associated with both the myocardium adipose and fibrosis, which was a potential disease pathway for ARVC and might to be useful as therapeutic targets for ARVC.

RhoA activity increased in myocardium of arrhythmogenic cardiomyopathy patients and affected connexin 43 protein expression in HL-1 cells.

Arrhythmogenic cardiomyopathy (AC) is a familial heart muscle disease with mutations of desmosomal gene and its pathogenesis is related with mutations of desmosomal gene and abnormality of connexin43 (Cx43). One of Rho GTPase, RhoA involves in many pathological processes and is regulated by desmosomal gene PKP2. We aim to identify if PKP2 regulate RhoA activity in myocardium of AC patients, the activity change of RhoA in patients' myocardium and to investigate the effect of active RhoA on the protein expression of Cx43 in myocardial cells. The protein expression level was assessed by western blot and the activity of RhoA was assessed by RhoA protein activation assay. Our results showed that the expression of PKP2 was decreased in myocardium of three patients, one of which carried PKP2 mutations. The activity of RhoA in myocardium was increased in myocardium of AC as compared with healthy control except for the patient with PKP2 mutation, the expression of Cx43 was also increased in those patients with increased activity of RhoA. The results in vitro demonstrated that the increase of active RhoA can cause the change of protein expression of Cx43 in HL-1 cardiomyocytes. In conclusion, regulation of RhoA activity is complex in the myocardium of AC and the activity of RhoA is increased in AC patients without PKP2 mutations. What's more, the active RhoA affects the protein expression of Cx43 in vivo and in vitro, this may be the possible disease mechanism of AC.

Up-regulated lipocalin-2 in pulmonary hypertension involving in pulmonary artery SMC resistance to apoptosis.

A key feature of pulmonary hypertension (PH) is the remodeling of small pulmonary arteries due to abnormal pulmonary artery smooth muscle cell (PASMC) proliferation and resistance to apoptosis. However, the cellular mechanisms underlying how PASMCs in the pathological condition of pulmonary hypertension become resistant to apoptosis remain unknown. It was recently reported that lipocalin 2 (Lcn2) is up-regulated in a wide array of malignant conditions, which facilitates tumorigenesis partly by inhibiting cell apoptosis. In this study, we observed that the expression levels of Lcn2 were significantly elevated in a rat PH model induced with monocrotaline and in patients with congenital heart disease-associated PH (CHD-PH) when compared with respective control. Therefore, we hypothesize that Lcn2 could regulate human PASMC (HPASMC) apoptosis through a mechanism. By the detection of DNA fragmentation using the TUNEL assay, the detection of Annexin V/PI-positive cells using flow cytometry, and the detection of cleaved caspase-3 and caspase-3 activity, we observed that Lcn2 significantly inhibited HPASMC apoptosis induced by serum withdrawal and H2O2 treatment. We also observed that Lcn2 down-regulated the proapoptotic protein Bax, decreased the levels of cellular ROS, and up-regulated the expression of superoxide dismutases (SOD1 and SOD2). In conclusion, Lcn2 significantly inhibits HPASMC apoptosis induced by oxidative stress via decreased intracellular ROS and elevated SODs. Up-regulation of Lcn2 in a rat PH model and CHD-PH patients may be involved in the pathological process of PH.

Cellular-level analyses of SCN5A mutations in left ventricular noncompaction cardiomyopathy suggest electrophysiological mechanisms for ventricular tachycardia.

Left ventricular noncompaction cardiomyopathy (LVNC) is a cardiovascular disease characterized by arrhythmia and heart failure. In this study, LVNC myocardial samples were collected from patients who underwent heart transplantation and were analyzed using exome sequencing. Approximately half of the LVNC patients carried SCN5A variants, which are associated with clinical symptoms of ventricular tachycardia. To investigate the electrophysiological functions of these SCN5A variants and the underlying mechanism by which they increase arrhythmia susceptibility in LVNC patients, functional evaluations were conducted in CHO-K1 cells and human embryonic stem cell-derived cardiomyocytes (hESC-CMs) using patch-clamp or microelectrode array (MEA) techniques. These findings demonstrated that these SCN5A mutants exhibited gain-of-function properties, leading to increased channel activation and enhanced fast inactivation in CHO-K1 cells. Additionally, these mutants enhanced the excitability and contractility of the cardiomyocyte population in hESC-CMs models. All SCN5A variants induced fibrillation-like arrhythmia and increased the heart rate in cardiomyocytes. However, the administration of Lidocaine, an antiarrhythmic drug that acts on sodium ion channels, was able to rescue or alleviate fibrillation-like arrhythmias and secondary beat phenomenon. Based on these findings, it is speculated that SCN5A variants may contribute to susceptibility to arrhythmia in LVNC patients. Furthermore, the construction of cardiomyocyte models with SCN5A variants and their application in drug screening may facilitate the development of precise therapies for arrhythmia in the future.

Effects of CMYA1 overexpression on cardiac structure and function in mice.

CMYA1 (cardiomyopathy-associated protein 1, also termed Xin) localizes to the intercalated disks (ICDs) of the myocardium and functions to maintain ICD structural integrity and support signal transduction among cardiomyocytes. Our previous study showed that CMYA1 overexpression impairs the function of gap junction intercellular communication processes. Successful model generation was verified based on PCR, western blot analysis, immunohistochemistry, and immunofluorescence analysis. Myocardial CMYA1 expression was confirmed at both the mRNA and the protein levels in the CMYA1-OE transgenic mice. Masson's trichrome staining and electron microscopy revealed myocardial fibrosis and uneven bead width or the interruption of ICDs in the hearts of the CMYA1-OE transgenic mice. Furthermore, the Cx43 protein level was reduced in the CMYA1-OE mice, and co-immunoprecipitation assays of heart tissue protein extracts revealed a physical interaction between CMYA1 and Cx43. Electrocardiogram analysis enabled the detection of an obvious ventricular bigeminy for the CMYA1-OE mice. In summary, analysis of our mouse model indicates that elevated CMYA1 levels may induce myocardial fibrosis, impair ICDs, and downregulate the expression of Cx43. The observed ventricular bigeminy in the CMYA1-OE mice may be mediated by the reduced Cx43 protein level.

Elevated myocardial SORBS2 and the underlying implications in left ventricular noncompaction cardiomyopathy.

BACKGROUND:Left ventricular noncompaction cardiomyopathy (LVNC) is a hereditary heart disease characterized by an excessive trabecular meshwork of deep intertrabecular recesses within the ventricular myocardium. The guidelines for management of LVNC patients aim to improve quality of life by preventing cardiac heart failure. However, the mechanism underlying LVNC-associated heart failure remains poorly understood.METHODS:Using protein mass spectrometry analysis, we established that Sorbin And SH3 Domain Containing 2 (SORBS2) is up-regulated in LVNC hearts without changes to structure proteins. We conducted in vivo experiments wherein the heart tissues of wild-type mice were injected with an AAV9 vector to overexpress SORBS2, followed by analysis using echocardiography, T-tubule analysis and Ca2+ imaging to identify functional and morphological changes. In addition, we analyzed the function and structure of SORBS2 overexpressing human embryonic stem cell (hESC) derived cardiomyocytes (hESC-CM) via immunoblotting, immunohistochemistry, immunofluorescence, and confocal Ca2+ imaging.FINDINGS:LVNC myocardial tissues feature strongly elevated expression of SORBS2, microtubule densification and redistribution of Junctophilin 2 (JP2). SORBS2 interacts with β-tubulin, promoting its polymerization in 293T cells and hESC-derived CMs. In vivo, cardiac dysfunction, β-tubulin densification, JP2 translocation, T-tubule disorganization and Ca2+ handling dysfunction were observed in mice overexpressing SORBS2.INTERPRETATION:We identified a novel mechanism through which SORBS2 interacts with β-tubulin and promotes microtubule densification, eventually effecting JP2 distribution and T-tubule, potentially contributing to heart failure in LVNC disease. FUND: This work was supported by a CAMS Initiative for Innovative Medicine grant (CAMS-I2M, 2016-I2M-1-015 to Y.J.Wei).

Multiple genetic variants in adolescent patients with left ventricular noncompaction cardiomyopathy.

BACKGROUND:Left ventricular noncompaction cardiomyopathy (LVNC) is a primary cardiomyopathy with an unclear aetiology. The clinical symptoms range from asymptomatic to heart failure, arrhythmias and sudden cardiac death. This study aimed to characterize the genetic features and clinical outcomes of LVNC who underwent heart transplantation (HTx) to reveal the potential genetic pathogenesis.METHODS AND RESULTS:We recruited 16 cases who underwent HTx in our hospital. Exome-sequencing was performed to reveal genetic background. Clinical information and histopathology features of patients were investigated. Gene expression profiling of tissue fibrosis were evaluated by quantitative PCR. The median age of patients was 21 years. Of the 16 patients, 14 harboured multiple gene variants involved in LVNC. Ten of the patients harboured biallelic variants and/or truncating variants. Young patients (<18) with biallelic variants and/or truncating variants and lower LVEF (<45%) at initial symptom deteriorated quickly. Except for noncompaction myocardium, myocardial fibrosis was a remarkable pathological feature, and gene profiles related to immune inflammation and extracellular matrix remodelling were upregulated.CONCLUSIONS:This study showed that multiple pathologic variants were underlie genetic mechanism of LVNC who in high risks, suggesting that genetic screening should be applied to the diagnosis of LVNC. LVNC patient with multiple variants should be considered carefully follow-up. Genetics involved in the phenotype and cardiac fibrosis, and is the major causing for LVNC.

Comparative proteomics analysis of myocardium in left ventricular non-compaction cardiomyopathy.

Arrhythmogenic cardiomyopathy: Identification of desmosomal gene variations and desmosomal protein expression in variation carriers.

Arrhythmogenic cardiomyopathy (AC) is an inherited disorder that is predominantly present in the right ventricular myocardium. Mutations in the genes encoding the desmosomal protein are thought to underlie the pathogenesis of AC. Since AC is genetically heterogeneous and phenotypically diverse, modifier genes and environmental factors have an important role in disease expression. The aim of the present study was to identify AC-associated desmosomal gene variations, and examine the expression levels of intercalated disc proteins in AC patients who carry the variations (DSG2 p.Leu797Gln, PKP2 p.Ser249Thr and p.E808fsX30). The results of the present investigation provided information on the search for modifier genes and desmosomal gene mutations, and improved our understanding of the mechanism underlying these AC mutations. Genetic screening of five desmosomal genes (DSG2, DSC2, JUP, PKP2, and DSP) in 23 patients with AC who underwent heart transplantation was performed and the expression levels and localizations of intercalated disc proteins were assessed using western blotting and immunohistochemistry, respectively. The results enabled the identification of three desmosomal gene variations (DSG2 L797Q, PKP2 S249T, and E808fsX30), two of which are reported for the first time. DSG2 L797Q was identified in one patient. The protein expression levels of DSG2 in the L797Q carrier were unchanged compared with the healthy controls, and the expression levels of the other proteins (JUP and Cx43) in the intercalated disc were also similar between the healthy controls, the variation carrier and the case controls. Two variations (S249T and E808fsX30) in PKP2 were identified in one patient, the protein expression levels of PKP2 in this patient were significantly decreased, and the expression levels of the other proteins in the intercalated disc was also decreased. The data suggest that there may be modifier genes and other AC-associated mutations requiring identification, in order to further our understanding of the disease mechanism induced by these mutations.

Lipocalin-2 Promotes Endoplasmic Reticulum Stress and Proliferation by Augmenting Intracellular Iron in Human Pulmonary Arterial Smooth Muscle Cells.

Endoplasmic reticulum (ER) stress, a feature of many conditions associated with pulmonary hypertension (PH), is increasingly recognized as a common response to promote proliferation in the walls of pulmonary arteries. Increased expression of Lipocalin-2 in PH led us to test the hypothesis that Lipocalin-2, a protein known to sequester iron and regulate it intracellularly, might facilitate the ER stress and proliferation in pulmonary arterial smooth muscle cells (PASMCs). In this study, we observed greatly increased Lcn2 expression accompanied with increased ATF6 cleavage in a standard rat model of pulmonary hypertension induced by monocrotaline. In cultured human PASMCs, Lcn2 significantly promoted ER stress (determined by augmented cleavage and nuclear localization of ATF6, up-regulated transcription of GRP78 and NOGO, increased expression of SOD2, and mild augmented mitochondrial membrane potential) and proliferation (assessed by Ki67 staining and BrdU incorporation). Lcn2 promoted ER stress accompanied with augmented intracellular iron levels in human PASMCs. Treatment human PASMCs with FeSO4 induced the similar ER stress and proliferation response and iron chelator (deferoxamine) abrogated the ER stress and proliferation induced by Lcn2 in cultured human PASMCs. In conclusion, Lcn2 significantly promoted human PASMC ER stress and proliferation by augmenting intracellular iron. The up-regulation of Lcn2 probably involved in the pathogenesis and progression of PH.

Profiling of differentially expressed microRNAs in arrhythmogenic right ventricular cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a kind of primary cardiomyopathy characterized by the fibro-fatty replacement of right ventricular myocardium. Currently, myocardial microRNAs have been reported to play critical role in the pathophysiology of cardiovascular pathophysiology. So far, the profiling of microRNAs in ARVC has not been described. In this study, we applied S-Poly (T) Plus method to investigate the expression profile of microRNAs in 24 ARVC patients heart samples. The tissue levels of 1078 human microRNAs were assessed and were compared with levels in a group of 24 healthy controls. Analysis of the area under the receiver operating characteristic curve (ROC) supported the 21 validated microRNAs to be miRNA signatures of ARVC, eleven microRNAs were significantly increased in ARVC heart tissues and ten microRNAs were significantly decreased. After functional enrichment analysis, miR-21-5p and miR-135b were correlated with Wnt and Hippo pathway, which might involve in the molecular pathophysiology of ARVC. Overall, our data suggested that myocardial microRNAs were involved in the pathophysiology of ARVC, miR-21-5p and miR-135b were significantly associated with both the myocardium adipose and fibrosis, which was a potential disease pathway for ARVC and might to be useful as therapeutic targets for ARVC.

RhoA activity increased in myocardium of arrhythmogenic cardiomyopathy patients and affected connexin 43 protein expression in HL-1 cells.

Arrhythmogenic cardiomyopathy (AC) is a familial heart muscle disease with mutations of desmosomal gene and its pathogenesis is related with mutations of desmosomal gene and abnormality of connexin43 (Cx43). One of Rho GTPase, RhoA involves in many pathological processes and is regulated by desmosomal gene PKP2. We aim to identify if PKP2 regulate RhoA activity in myocardium of AC patients, the activity change of RhoA in patients' myocardium and to investigate the effect of active RhoA on the protein expression of Cx43 in myocardial cells. The protein expression level was assessed by western blot and the activity of RhoA was assessed by RhoA protein activation assay. Our results showed that the expression of PKP2 was decreased in myocardium of three patients, one of which carried PKP2 mutations. The activity of RhoA in myocardium was increased in myocardium of AC as compared with healthy control except for the patient with PKP2 mutation, the expression of Cx43 was also increased in those patients with increased activity of RhoA. The results in vitro demonstrated that the increase of active RhoA can cause the change of protein expression of Cx43 in HL-1 cardiomyocytes. In conclusion, regulation of RhoA activity is complex in the myocardium of AC and the activity of RhoA is increased in AC patients without PKP2 mutations. What's more, the active RhoA affects the protein expression of Cx43 in vivo and in vitro, this may be the possible disease mechanism of AC.

Up-regulated lipocalin-2 in pulmonary hypertension involving in pulmonary artery SMC resistance to apoptosis.

A key feature of pulmonary hypertension (PH) is the remodeling of small pulmonary arteries due to abnormal pulmonary artery smooth muscle cell (PASMC) proliferation and resistance to apoptosis. However, the cellular mechanisms underlying how PASMCs in the pathological condition of pulmonary hypertension become resistant to apoptosis remain unknown. It was recently reported that lipocalin 2 (Lcn2) is up-regulated in a wide array of malignant conditions, which facilitates tumorigenesis partly by inhibiting cell apoptosis. In this study, we observed that the expression levels of Lcn2 were significantly elevated in a rat PH model induced with monocrotaline and in patients with congenital heart disease-associated PH (CHD-PH) when compared with respective control. Therefore, we hypothesize that Lcn2 could regulate human PASMC (HPASMC) apoptosis through a mechanism. By the detection of DNA fragmentation using the TUNEL assay, the detection of Annexin V/PI-positive cells using flow cytometry, and the detection of cleaved caspase-3 and caspase-3 activity, we observed that Lcn2 significantly inhibited HPASMC apoptosis induced by serum withdrawal and H2O2 treatment. We also observed that Lcn2 down-regulated the proapoptotic protein Bax, decreased the levels of cellular ROS, and up-regulated the expression of superoxide dismutases (SOD1 and SOD2). In conclusion, Lcn2 significantly inhibits HPASMC apoptosis induced by oxidative stress via decreased intracellular ROS and elevated SODs. Up-regulation of Lcn2 in a rat PH model and CHD-PH patients may be involved in the pathological process of PH.