scholarly journals LAMP-2B regulates human cardiomyocyte function by mediating autophagosome–lysosome fusion

2018 ◽  
Vol 116 (2) ◽  
pp. 556-565 ◽  
Author(s):  
Congwu Chi ◽  
Andrea Leonard ◽  
Walter E. Knight ◽  
Kevin M. Beussman ◽  
Yuanbiao Zhao ◽  
...  
Keyword(s):  
Heart Failure ◽  
Membrane Protein ◽  
Pluripotent Stem Cells ◽  
Coiled Coil ◽  
Gene Correction ◽  
Danon Disease ◽  
Human Cardiomyocytes ◽  
Coiled Coil Domain ◽  
Induced Pluripotent ◽  
Isoform B

Mutations in lysosomal-associated membrane protein 2 (LAMP-2) gene are associated with Danon disease, which often leads to cardiomyopathy/heart failure through poorly defined mechanisms. Here, we identify the LAMP-2 isoform B (LAMP-2B) as required for autophagosome–lysosome fusion in human cardiomyocytes (CMs). Remarkably, LAMP-2B functions independently of syntaxin 17 (STX17), a protein that is essential for autophagosome–lysosome fusion in non-CMs. Instead, LAMP-2B interacts with autophagy related 14 (ATG14) and vesicle-associated membrane protein 8 (VAMP8) through its C-terminal coiled coil domain (CCD) to promote autophagic fusion. CMs derived from induced pluripotent stem cells (hiPSC-CMs) from Danon patients exhibit decreased colocalization between ATG14 and VAMP8, profound defects in autophagic fusion, as well as mitochondrial and contractile abnormalities. This phenotype was recapitulated by LAMP-2B knockout in non-Danon hiPSC-CMs. Finally, gene correction of LAMP-2 mutation rescues the Danon phenotype. These findings reveal a STX17-independent autophagic fusion mechanism in human CMs, providing an explanation for cardiomyopathy in Danon patients and a foundation for targeting defective LAMP-2B–mediated autophagy to treat this patient population.

Abstract 182: Mimetic peptide overcomes dysregulated L-Type Calcium Channel density and recovers myocardial function

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Francesca Rusconi ◽  
Michele Miragoli ◽  
Elisa Di Pasquale ◽  
Marcella Rocchetti ◽  
Paola Ceriotti ◽  
...  
Keyword(s):  
Heart Failure ◽  
Pluripotent Stem Cells ◽  
Cardiac Contraction ◽  
Proof Of Concept ◽  
Mimetic Peptide ◽  
Human Cardiomyocytes ◽  
Voltage Dependent ◽  
Effective Delivery ◽  
A Cell ◽  
Induced Pluripotent

Voltage dependent L-Type calcium-channels (LTCCs) are located on the cardiomyocyte membrane and regulate cardiac contraction and rhythmicity. In human pathologies, such as heart failure (HF), decreased inward calcium current (I Ca ) is frequently observed. Here, we generated a mimetic peptide (MP) that targets LTCCs and restores impaired intracellular calcium homeostasis through a novel mechanism. Effective delivery of MP, fused with a cell penetrating peptide, was found to correct Ca2+ alterations in a mouse model of HF, in human cardiomyocytes derived from induced pluripotent stem-cells. These data provide a proof-of-concept supporting a therapeutic role for MP to treat human diseases related to LTCC abnormalities. Category: heart failure biology

TMCC3 localizes at the three-way junctions for the proper tubular network of the endoplasmic reticulum

2019 ◽  
Vol 476 (21) ◽  
pp. 3241-3260
Author(s):  
Sindhu Wisesa ◽  
Yasunori Yamamoto ◽  
Toshiaki Sakisaka
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Mammalian Cells ◽  
Coiled Coil ◽  
Transmembrane Domains ◽  
Domain Family ◽  
Coiled Coil Domain ◽  
U2os Cells ◽  
Er Membrane

The tubular network of the endoplasmic reticulum (ER) is formed by connecting ER tubules through three-way junctions. Two classes of the conserved ER membrane proteins, atlastins and lunapark, have been shown to reside at the three-way junctions so far and be involved in the generation and stabilization of the three-way junctions. In this study, we report TMCC3 (transmembrane and coiled-coil domain family 3), a member of the TEX28 family, as another ER membrane protein that resides at the three-way junctions in mammalian cells. When the TEX28 family members were transfected into U2OS cells, TMCC3 specifically localized at the three-way junctions in the peripheral ER. TMCC3 bound to atlastins through the C-terminal transmembrane domains. A TMCC3 mutant lacking the N-terminal coiled-coil domain abolished localization to the three-way junctions, suggesting that TMCC3 localized independently of binding to atlastins. TMCC3 knockdown caused a decrease in the number of three-way junctions and expansion of ER sheets, leading to a reduction of the tubular ER network in U2OS cells. The TMCC3 knockdown phenotype was partially rescued by the overexpression of atlastin-2, suggesting that TMCC3 knockdown would decrease the activity of atlastins. These results indicate that TMCC3 localizes at the three-way junctions for the proper tubular ER network.

scholarly journals Pharmacological response of human cardiomyocytes derived from virus-free induced pluripotent stem cells

2011 ◽  
Vol 91 (4) ◽  
pp. 577-586 ◽  
Author(s):  
Ashish Mehta ◽  
Ying Ying Chung ◽  
Alvin Ng ◽  
Fahamy Iskandar ◽  
Shirhan Atan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Human Cardiomyocytes ◽  
Pharmacological Response ◽  
Induced Pluripotent

Abstract 498: Krueppel-like Factor 15 Modulation: A Crispr-based Re-establishment of Homeostatic Transcription in Diseased Mouse and Human Cardiomyocytes

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Eric Schoger ◽  
Lavanya M Iyer ◽  
Claudia Noack ◽  
Cheila Rocha ◽  
Mareike Jassyk ◽  
...  
Keyword(s):  
Heart Failure ◽  
Gene Activation ◽  
Specific Inhibitor ◽  
Hek293 Cells ◽  
Beta Catenin ◽  
Human Cardiomyocytes ◽  
Guide Rnas ◽  
Heart Failure Progression ◽  
Induced Pluripotent ◽  
Crispr Activation

An altered transcriptomic profile of cardiomyocytes is a hallmark of cardiac remodelling and heart failure. Re-activation of gene programs necessary for myocardial homeostasis and inhibition of deleterious signalling pathways, such as Wnt/beta-catenin signalling, is therefore therapeutically desirable, however challenging to achieve tissue-specifically. We identified Krueppel-like factor 15 (KLF15) as a cardiac specific inhibitor of Wnt/beta-catenin signalling, lost during heart failure progression in the mouse and human heart. To re-establish endogenous Klf15 expression, we designed a cardiomyocyte specific CRISPR activation mouse model expressing enzymatically inactive Cas9 (dCas9) fused to a transcriptional activator (VPR) under Myh6 promoter control. The dCas9VPR protein is directed by guide RNAs (gRNA), delivered by AAV9, to the promoter region of Klf15 and induces gene expression. We tested activation of Klf15 in the adult myocardium by injecting 15 weeks old Myh6-dCas9VPR mice systemically with AAV9 carrying Klf15 gRNAs and increasing Klf15 transcript levels 8 weeks later up to 1.6 fold compared to controls. Since we found a 0.5 fold change of Klf15 expression upon transverse aortic constriction compared to sham operated mice, we expect the CRISPR activation induced Klf15 activation to be sufficient to re-establish Klf15 expression. As a translational approach and since we demonstrated KLF15-WNT regulation in human cardiomyocytes, we integrated dCas9VPR into human induced pluripotent stem cells (hIPSC) by targeted genome editing at the AAVS1 safe harbour locus and confirmed dCas9VPR expression in hIPSC and in hIPSC-derived cardiomyocytes (hIPSC-CM). We tested KLF15 gRNAs in HEK293 cells and selected the most efficient candidate gRNAs to test Klf15 activation in dCas9VPR-IPSC resulting in an up to 1.7 fold activation compared to controls. For gene activation in dCas9VPR hIPSC-CM, we are currently testing lentivirus constructs to deliver up to 3 gRNAs simultaneously to tailor KLF15 expression. With these prerequisites, we aim to re-activate master regulators of normal cardiomyocyte function in failing cardiomyocytes at physiologically relevant levels, such as KLF15, to test potential therapeutic targets for the prevention of heart failure progression.

Modeling Congenital Amegakaryocytic Thrombocytopenia Using Patient-Specific Induced Pluripotent Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 703-703
Author(s):  
Naoya Takayama ◽  
Shinji Hirata ◽  
Ryoko Jono-Ohnishi ◽  
Sou Nakamura ◽  
Sho-ichi Hirose ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Conflicts Of Interest ◽  
Receptor Gene ◽  
Patient Specific ◽  
Gene Correction ◽  
Donor Skin ◽  
Induced Pluripotent

Abstract Abstract 703 Patient-specific, induced pluripotent stem cells (iPSCs) enable us to study disease mechanisms and drug screening. To clarify the phenotypic alterations caused by the loss of c-MPL, the thrombopoietin (TPO) receptor, we established iPSCs derived from skin fibroblasts of a patient who received curative bone marrow transplantation for congenital amegakarycytic thrombocytopenia (CAMT) caused by the loss of the TPO receptor gene, MPL. The resultant CAMT-iPSCs exhibited mutations corresponding to the original donor skin. Then using an in vitro culture system yielding hematopoietic progenitor cells (HPCs), we evaluated the role of MPL on the early and late phases of human hematopoiesis. Although CAMT-iPSCs generated CD34+ HPCs, per se, their colony formation capability was impaired, as compared to control CD34+ HPCs. Intriguingly, both Glycophorin A (GPA)+ erythrocyte development and CD41+ megakaryocyte yields from CAMT-iPSCs were also impaired, suggesting that MPL is indispensable for MEP (megakaryocyte erythrocyte progenitors) development. Prospective analysis along with the hematopoietic hierarchy revealed that, in CAMT-iPSCs but not control iPSCs expressing MPL, mRNA expression and phosphorylation of putative signaling molecules downstream of MPL are severely impaired, as is the transition from CD34+CD43+CD41-GPA- MPP (multipotent progenitors) to CD41+GPA+ MEP. Additional analysis also indicated that c-MPL is required for maintenance of a consistent supply of megakaryocytes and erythrocytes from MEPs. Conversely, complimentary transduction of MPL into CAMT-iPSCs using a retroviral vector restored the defective erythropoiesis and megakaryopoiesis; however, excessive MPL signaling appears to promote aberrant megakaryopoiesis with CD42b (GPIba)-null platelet generation and impaired erythrocyte production. Taken together, our findings demonstrate the usefulness of CAMT-iPSCs for validation of functionality in the human hematopoiesis system. For example, it appears that MPL is not indispensable for the emergence of HPCs, but is indispensible for their maintenance, and for subsequent MEP development. Our results also strongly indicate that an appropriate expression level of an administered gene is necessary to achieve curative gene correction / therapy using patient-derived iPSCs. Disclosures: No relevant conflicts of interest to declare.

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