Abstract 111: Cdk5 is Associated in Cardiac Calcium Channelopathy

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Masayuki Yazawa ◽  
LouJin Song
Keyword(s):  
Dominant Negative ◽  
Calcium Handling ◽  
Human Cardiomyocytes ◽  
Channel Inactivation ◽  
Biochemical Assays ◽  
Direct Binding ◽  
Induced Pluripotent ◽  
Molecular Bases ◽  
Calcium Channelopathy

Cardiac calcium channels play important roles in hearts. Mutations in Ca V 1.2 channels are associated with cardiac arrhythmias including long QT syndrome type 8 (LQTS8). Using human induced pluripotent stem cells (iPSCs) to generate human cardiomyocytes from LQTS8 patients, we reported that the G406R mutation (LQTS8, Timothy syndrome) affected channel inactivation, action potentials and calcium handling in human cardiomyocytes. We found that roscovitine, a cyclin-dependent kinase (CDK) inhibitor, rescued the phenotypes in LQTS8 cardiomyocytes. However, how roscovitine restores the physiological functions in the patient cardiomyocytes remains unclear. Our recent results using roscovitine analogs and CDK inhibitors suggested that CDK5 is involved in the molecular bases of LQTS8. We found that dominant negative forms and shRNA of CDK5 alleviated the phenotypes of the LQTS8 cardiomyocytes and that the expressions of phosphorylated ERK, EGR1 and CDK5R1, a CDK5 activator, significantly increased in the patient cardiomyocytes. The results reveal that calcium overload due to affected Ca V 1.2 inactivation activates ERK/EGR1 pathway, resulting in increased expression of CDK5R1. Biochemical assays supported a direct binding and phosphorylation of Ca V 1.2 channels by CDK5. Overall, the results demonstrated that CDK5 regulates Ca V 1.2 channels and that CDK5 inhibition could be a new therapeutic for LQTS8. This study demonstrates the role of ERK/EGR1/CDK5 in pathogenesis of cardiac arrhythmia. The outcomes of this study provide new insights into Ca V 1.2 channel regulation.

scholarly journals Inhibition of SARS-CoV-2 infection in human cardiomyocytes by targeting the Sigma-1 receptor disrupts cytoskeleton architecture and contractility

2021 ◽  
Author(s):  
José Alexandre Salerno ◽  
Thayana Torquato ◽  
Jairo R. Temerozo ◽  
Livia Goto-Silva ◽  
Mayara Mendes ◽  
...  
Keyword(s):  
Therapeutic Strategy ◽  
Sigma 1 Receptor ◽  
Human Cardiomyocytes ◽  
Sigma 1 ◽  
Induced Pluripotent ◽  
And Function ◽  
Beating Frequency ◽  
Cytoskeleton Integrity

ABSTRACTHeart dysfunction, represented by conditions such as myocarditis and arrhythmia, has been reported in COVID-19 patients. Therapeutic strategies focused on the cardiovascular system, however, remain scarce. The Sigma-1 receptor (S1R) has been recently proposed as a therapeutic target because its inhibition reduces SARS-CoV-2 replication. To investigate the role of S1R in SARS-CoV-2 infection in the heart, we used human cardiomyocytes derived from induced pluripotent stem cells (hiPSC-CM) as an experimental model. Here we show that the S1R antagonist NE-100 decreases SARS-CoV-2 infection and viral replication in hiPSC-CMs. Also, NE-100 reduces cytokine release and cell death associated with infection. Because S1R is involved in cardiac physiology, we investigated the effects of NE-100 in cardiomyocyte morphology and function. We show that NE-100 compromises cytoskeleton integrity and reduces beating frequency, causing contractile impairment. These results show that targeting S1R to challenge SARS-CoV-2 infection may be a useful therapeutic strategy but its detrimental effects in vivo on cardiac function should not be ignored.

scholarly journals Role of Heme-Oxygenase-1 in Biology of Cardiomyocytes Derived from Human Induced Pluripotent Stem Cells

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 522
Author(s):  
Mateusz Jeż ◽  
Alicja Martyniak ◽  
Kalina Andrysiak ◽  
Olga Mucha ◽  
Krzysztof Szade ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Heme Oxygenase ◽  
Pluripotent Stem Cells ◽  
Human Fibroblasts ◽  
Cardiac Differentiation ◽  
Heme Oxygenase 1 ◽  
Human Cardiomyocytes ◽  
Induced Pluripotent

Heme oxygenase-1 (HO-1, encoded by HMOX1) is a cytoprotective enzyme degrading heme into CO, Fe2+, and biliverdin. HO-1 was demonstrated to affect cardiac differentiation of murine pluripotent stem cells (PSCs), regulate the metabolism of murine adult cardiomyocytes, and influence regeneration of infarcted myocardium in mice. However, the enzyme’s effect on human cardiogenesis and human cardiomyocytes’ electromechanical properties has not been described so far. Thus, this study aimed to investigate the role of HO-1 in the differentiation of human induced pluripotent stem cells (hiPSCs) into hiPSC-derived cardiomyocytes (hiPSC-CMs). hiPSCs were generated from human fibroblasts and peripheral blood mononuclear cells using Sendai vectors and subjected to CRISPR/Cas9-mediated HMOX1 knock-out. After confirming lack of HO-1 expression on the protein level, isogenic control and HO-1-deficient hiPSCs were differentiated into hiPSC-CMs. No differences in differentiation efficiency and hiPSC-CMs metabolism were observed in both cell types. The global transcriptomic analysis revealed, on the other hand, alterations in electrophysiological pathways in hiPSC-CMs devoid of HO-1, which also demonstrated increased size. Functional consequences in changes in expression of ion channels genes were then confirmed by patch-clamp analysis. To the best of our knowledge, this is the first report demonstrating the link between HO-1 and electrophysiology in human cardiomyocytes.

Abstract 240: Asb2-dependent Proteolysis of the Cytoskeleton Directs Cardiac Development and Disease

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Abir Yamak ◽  
Dongjian Hu ◽  
Ibrahim J Domian
Keyword(s):  
Heart Development ◽  
Cardiac Development ◽  
Heart Diseases ◽  
Ubiquitin Proteasome System ◽  
Actin Binding ◽  
Human Cardiomyocytes ◽  
Morphological Defects ◽  
Pericardial Edema ◽  
Induced Pluripotent

Congenital heart diseases (CHDs) account for 25% of birth defects and are major risk factors for adult cardiovascular problems. Partial disease penetrance is seen even in autosomal dominant disorders and genotype/phenotype correlations remain a clinical challenge; thus, the need to understand different regulators of cardiac formation. The Ubiquitin-Proteasome System (UPS) is important in controlling protein turnover during organ development but its role in the mammalian heart remains ambiguous. We have identified a specificity subunit of ubiquitin-mediated proteolysis (Asb2) as being specific for the cardiac myogenic lineage. Asb2 was previously shown to regulate hematopoietic and skeletal muscle cell differentiation through targeting filamin proteins (FlnA, B and C), actin-binding proteins important for cytoskeleton stabilization. In our present study, we show that Asb2 is markedly enriched in myocardial progenitor cells and cardiomyocytes. To investigate the role of Asb2 and UPS dependent proteolysis in heart development, we generated two cardiac-specific murine knockouts (KOs): Nkx Cre .Asb2 -/- and Mef2c Cre .Asb2 -/- (deleting Asb2 in early cardiomyocyte progenitors and anterior heart field progenitors, respectively). Both KOs are embryonic lethal with pericardial edema. We used tissue clarifying and confocal microscopy to define the morphological defects of the Asb2 null heart. Moreover, we found that FlnA is overexpressed in the hearts of these mice and its deletion therein partially rescues their lethality. In addition, using transcriptomic analysis on Asb2-null e9.5 hearts, we identified novel potential Asb2 targets in the heart. Finally, to understand the role of Asb2 in the differentiation and function of human cardiomyocytes, we used CRISPR/Cas9 genome editing technique to generate Asb2-null human induced pluripotent stem cells. Collectively, our study provides a novel mechanistic understanding of the role of the UPS proteasome in cardiac development, myocardial function, and disease pathogenesis. Given recent interests in both the UPS and the cytoskeleton as therapeutic targets, our study provides an innovative platform for the development of pharmacotherapy for cardiac disease.

scholarly journals Polyethylene Terephthalate Textiles Enhance the Structural Maturation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Materials ◽  
2019 ◽  
Vol 12 (11) ◽  
pp. 1805 ◽  
Author(s):  
Mari Pekkanen-Mattila ◽  
Martta Häkli ◽  
Risto-Pekka Pölönen ◽  
Tuomas Mansikkala ◽  
Anni Junnila ◽  
...  
Keyword(s):  
Stem Cell ◽  
Polyethylene Terephthalate ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Calcium Handling ◽  
Human Cardiomyocytes ◽  
Induced Pluripotent ◽  
Topographical Cues ◽  
Glass Surfaces

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have the potential to serve as a model for human cardiomyocytes. However, hiPSC-CMs are still considered immature. CMs differentiated from hiPSCs more resemble fetal than adult cardiomyocytes. Putative factors enhancing maturation include in vitro culture duration, culture surface topography, and mechanical, chemical, and electrical stimulation. Stem cell-derived cardiomyocytes are traditionally cultured on glass surfaces coated with extracellular matrix derivatives such as gelatin. hiPSC-CMs are flat and round and their sarcomeres are randomly distributed and unorganized. Morphology can be enhanced by culturing cells on surfaces providing topographical cues to the cells. In this study, a textile based-culturing method used to enhance the maturation status of hiPSC-CMs is presented. Gelatin-coated polyethylene terephthalate (PET)-based textiles were used as the culturing surface for hiPSC-CMs and the effects of the textiles on the maturation status of the hiPSC-CMs were assessed. The hiPSC-CMs were characterized by analyzing their morphology, sarcomere organization, expression of cardiac specific genes, and calcium handling. We show that the topographical cues improve the structure of the hiPSC-CMs in vitro. Human iPSC-CMs grown on PET textiles demonstrated improved structural properties such as rod-shape structure and increased sarcomere orientation.

scholarly journals Potential Dual Role of West Nile Virus NS2B in Orchestrating NS3 Enzymatic Activity in Viral Replication

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 216
Author(s):  
Alanna C. Tseng ◽  
Vivek R. Nerurkar ◽  
Kabi R. Neupane ◽  
Helmut Kae ◽  
Pakieli H. Kaufusi
Keyword(s):  
West Nile Virus ◽  
Enzymatic Activity ◽  
Nonstructural Protein ◽  
Antiviral Drug ◽  
West Nile ◽  
Biochemical Assays ◽  
In Trans ◽  
Viral Polyprotein ◽  
Ns3 Protease

West Nile virus (WNV) nonstructural protein 3 (NS3) harbors the viral triphosphatase and helicase for viral RNA synthesis and, together with NS2B, constitutes the protease responsible for polyprotein processing. NS3 is a soluble protein, but it is localized to specialized compartments at the rough endoplasmic reticulum (RER), where its enzymatic functions are essential for virus replication. However, the mechanistic details behind the recruitment of NS3 from the cytoplasm to the RER have not yet been fully elucidated. In this study, we employed immunofluorescence and biochemical assays to demonstrate that NS3, when expressed individually and when cleaved from the viral polyprotein, is localized exclusively to the cytoplasm. Furthermore, NS3 appeared to be peripherally recruited to the RER and proteolytically active when NS2B was provided in trans. Thus, we provide evidence for a potential additional role for NS2B in not only serving as the cofactor for the NS3 protease, but also in recruiting NS3 from the cytoplasm to the RER for proper enzymatic activity. Results from our study suggest that targeting the interaction between NS2B and NS3 in disrupting the NS3 ER localization may be an attractive avenue for antiviral drug discovery.

Overexpression of miR-431 attenuates hypoxia/reoxygenation-induced myocardial damage via autophagy-related 3

2020 ◽  
Author(s):  
Kang Zhou ◽  
Yan Xu ◽  
Qiong Wang ◽  
Lini Dong
Keyword(s):  
Cell Viability ◽  
Cell Apoptosis ◽  
Myocardial Injury ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Myocardial Damage ◽  
Protective Role ◽  
Human Cardiomyocytes ◽  
Hypoxia Reoxygenation

Abstract Myocardial injury is still a serious condition damaging the public health. Clinically, myocardial injury often leads to cardiac dysfunction and, in severe cases, death. Reperfusion of the ischemic myocardial tissues can minimize acute myocardial infarction (AMI)-induced damage. MicroRNAs are commonly recognized in diverse diseases and are often involved in the development of myocardial ischemia/reperfusion injury. However, the role of miR-431 remains unclear in myocardial injury. In this study, we investigated the underlying mechanisms of miR-431 in the cell apoptosis and autophagy of human cardiomyocytes in hypoxia/reoxygenation (H/R). H/R treatment reduced cell viability, promoted cell apoptotic rate, and down-regulated the expression of miR-431 in human cardiomyocytes. The down-regulation of miR-431 by its inhibitor reduced cell viability and induced cell apoptosis in the human cardiomyocytes. Moreover, miR-431 down-regulated the expression of autophagy-related 3 (ATG3) via targeting the 3ʹ-untranslated region of ATG3. Up-regulated expression of ATG3 by pcDNA3.1-ATG3 reversed the protective role of the overexpression of miR-431 on cell viability and cell apoptosis in H/R-treated human cardiomyocytes. More importantly, H/R treatments promoted autophagy in the human cardiomyocytes, and this effect was greatly alleviated via miR-431-mimic transfection. Our results suggested that miR-431 overexpression attenuated the H/R-induced myocardial damage at least partly through regulating the expression of ATG3.

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