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postgraduate thesis: Modelling X-linked dilated cardiomyopathy due to DMD gene mutation by induced pluripotent stem cells-derived cardiomyocytes
Title | Modelling X-linked dilated cardiomyopathy due to DMD gene mutation by induced pluripotent stem cells-derived cardiomyocytes |
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Authors | |
Advisors | |
Issue Date | 2021 |
Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
Citation | Zhu, S. [朱盛]. (2021). Modelling X-linked dilated cardiomyopathy due to DMD gene mutation by induced pluripotent stem cells-derived cardiomyocytes. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. |
Abstract | X-linked dilated cardiomyopathy (XLDCM) is a serious phenotype of dystrophinopathy caused by mutations of the DMD gene, resulting in dilated cardiomyopathy, rapidly deteriorating heart failure, and thus early death. Unlike Duchenne muscular dystrophy, patients with XLDCM do not have major skeletal muscle weakness. Currently, the only curative treatment is heart transplantation. This research, therefore, aimed to establish an effective platform to model XLDCM and screen potential therapeutic drugs. We generated induced pluripotent stem cells (iPSCs) from a patient with XLDCM with a DMD gene c.31+1G>A intron 1 splice site mutation. The iPSCs derived from both the patient and normal control expressed pluripotency markers and showed differentiation capacity into the three germ layers in vivo and were karyotypically normal. The patient-derived iPSCs were genetically identical to the patient specific dystrophin gene mutation. I differentiated them into cardiomyocytes. Cardiac-specific markers were confirmed. The patient-derived iPSC induced cardiomyocytes (iPSC-CMs) did not express full-length dystrophin protein. Deficiencies of dystrophin protein were associated with high osmotic fragility with increased ATP and cTnI release and the patient-derived iPSC-CMs harbored abnormal calcium handling, including prolonged time to the peak of the calcium transient and delayed duration of recovery. I administered membrane sealant Poloxamer 188 and it showed an improvement in calcium handling and could block ATP release from hypotonic stress. Besides, I tested trichostatin A, a histone deacetylases inhibitor, on the patient-derived iPSC-CMs, and found that TSA treatment significantly reduced the time to peak and decay time of the calcium transient in the patient-derived iPSC-CMs. Expression both of calcium related protein, Na+‐Ca2+ exchanger 1 (NCX1) and of mRNA expression was significantly higher in patient-derived iPSC-CMs, compared with the normal control, and were downregulated by TSA treatment. RNA-seq data confirmed the regulation. In enriched KEGG pathway analysis, I also identified some common pathways, including the calcium signal pathway, in normal, patient, and TSA-treated patient group, which was consistent with the pathological function I observed in vitro. We might infer that the rescue of the abnormal calcium handling by TSA may be related to the regulation of NCX1 expression. Additionally, our data showed different responses to different doses of TSA in normal and patient-derived iPSC-CMs. The patient-derived iPSC-CMs seemed to be more vulnerable to high dose of TSA. This study confirmed that our XLDCM patient-derived iPSC-CMs represented an effective platform for disease modeling and drug screening. |
Degree | Doctor of Philosophy |
Subject | Myocardium - Diseases Heart - Dilatation |
Dept/Program | Paediatrics and Adolescent Medicine |
Persistent Identifier | http://hdl.handle.net/10722/322922 |
DC Field | Value | Language |
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dc.contributor.advisor | Chan, HSS | - |
dc.contributor.advisor | Cheung, YF | - |
dc.contributor.advisor | Chan, GCF | - |
dc.contributor.author | Zhu, Sheng | - |
dc.contributor.author | 朱盛 | - |
dc.date.accessioned | 2022-11-18T10:41:47Z | - |
dc.date.available | 2022-11-18T10:41:47Z | - |
dc.date.issued | 2021 | - |
dc.identifier.citation | Zhu, S. [朱盛]. (2021). Modelling X-linked dilated cardiomyopathy due to DMD gene mutation by induced pluripotent stem cells-derived cardiomyocytes. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. | - |
dc.identifier.uri | http://hdl.handle.net/10722/322922 | - |
dc.description.abstract | X-linked dilated cardiomyopathy (XLDCM) is a serious phenotype of dystrophinopathy caused by mutations of the DMD gene, resulting in dilated cardiomyopathy, rapidly deteriorating heart failure, and thus early death. Unlike Duchenne muscular dystrophy, patients with XLDCM do not have major skeletal muscle weakness. Currently, the only curative treatment is heart transplantation. This research, therefore, aimed to establish an effective platform to model XLDCM and screen potential therapeutic drugs. We generated induced pluripotent stem cells (iPSCs) from a patient with XLDCM with a DMD gene c.31+1G>A intron 1 splice site mutation. The iPSCs derived from both the patient and normal control expressed pluripotency markers and showed differentiation capacity into the three germ layers in vivo and were karyotypically normal. The patient-derived iPSCs were genetically identical to the patient specific dystrophin gene mutation. I differentiated them into cardiomyocytes. Cardiac-specific markers were confirmed. The patient-derived iPSC induced cardiomyocytes (iPSC-CMs) did not express full-length dystrophin protein. Deficiencies of dystrophin protein were associated with high osmotic fragility with increased ATP and cTnI release and the patient-derived iPSC-CMs harbored abnormal calcium handling, including prolonged time to the peak of the calcium transient and delayed duration of recovery. I administered membrane sealant Poloxamer 188 and it showed an improvement in calcium handling and could block ATP release from hypotonic stress. Besides, I tested trichostatin A, a histone deacetylases inhibitor, on the patient-derived iPSC-CMs, and found that TSA treatment significantly reduced the time to peak and decay time of the calcium transient in the patient-derived iPSC-CMs. Expression both of calcium related protein, Na+‐Ca2+ exchanger 1 (NCX1) and of mRNA expression was significantly higher in patient-derived iPSC-CMs, compared with the normal control, and were downregulated by TSA treatment. RNA-seq data confirmed the regulation. In enriched KEGG pathway analysis, I also identified some common pathways, including the calcium signal pathway, in normal, patient, and TSA-treated patient group, which was consistent with the pathological function I observed in vitro. We might infer that the rescue of the abnormal calcium handling by TSA may be related to the regulation of NCX1 expression. Additionally, our data showed different responses to different doses of TSA in normal and patient-derived iPSC-CMs. The patient-derived iPSC-CMs seemed to be more vulnerable to high dose of TSA. This study confirmed that our XLDCM patient-derived iPSC-CMs represented an effective platform for disease modeling and drug screening. | - |
dc.language | eng | - |
dc.publisher | The University of Hong Kong (Pokfulam, Hong Kong) | - |
dc.relation.ispartof | HKU Theses Online (HKUTO) | - |
dc.rights | The author retains all proprietary rights, (such as patent rights) and the right to use in future works. | - |
dc.rights | This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. | - |
dc.subject.lcsh | Myocardium - Diseases | - |
dc.subject.lcsh | Heart - Dilatation | - |
dc.title | Modelling X-linked dilated cardiomyopathy due to DMD gene mutation by induced pluripotent stem cells-derived cardiomyocytes | - |
dc.type | PG_Thesis | - |
dc.description.thesisname | Doctor of Philosophy | - |
dc.description.thesislevel | Doctoral | - |
dc.description.thesisdiscipline | Paediatrics and Adolescent Medicine | - |
dc.description.nature | published_or_final_version | - |
dc.date.hkucongregation | 2021 | - |
dc.identifier.mmsid | 991044609101703414 | - |