A study of proteomics and transcriptional analysis of X-linked dilated cardiomyopathy patient specific induced pluripotent stem cells derived 3-D cardiac tissue strips

Dr Chan, Hoi Shan Sophelia   (Principal Investigator (PI))

Dr Sharma Rakesh   (Co-Investigator)

Professor Chan Godfrey Chi Fung   (Co-Investigator)

Dr Javed Asif   (Co-Investigator)

Professor Li Ronald Adolphus   (Co-Investigator)

18

2020-02-02

2021-08-01

55460

A study of proteomics and transcriptional analysis of X-linked dilated cardiomyopathy patient specific induced pluripotent stem cells derived 3-D cardiac tissue strips

Cardiac tissue engineering, induced pluripotent stem cells, Proteomics, Transcriptomes, X-linked dilated cardiomyopathy

Disease Model Development,Stem Cell Biology

201910159292

Seed Fund for PI Research – Basic Research

2019

Completed

Key issues of the problem being addressed X-linked dilated cardiomyopathy (XLDCM) is a serious condition with high morbidity and mortality. Patients with mutation in dystrophin gene, developed XLDCM, a cardio-specific phenotype with progressive dilated cardiomyopathy but minimal skeletal muscles involvement. Most patients presented with early signs of heart failure and deteriorated quickly despite medical treatment so resulted in early death. Currently there is no curative treatment and heart transplantation is often required. Absent of dystrophin in the cardiac muscles of these patients caused the cardiomyopathy. The dystrophin protein is a major component of the dystrophin-glycoprotein complex on the skeletal and cardiac muscle cell membrane maintaining the healthy function of the muscle cells. Reduced or absence of dystrophin increases the susceptibility of the muscle cells to the mechanical stress and therefore early muscle cell damage, inflammation, necrosis and eventually fibrosis and fatty replacement in both skeletal and cardiac muscles leading to skeletal muscle weakness and heart failure due to cardiomyopathy. Previous studies on the dystrophin isoform expression on patients with XLDCM having splice site mutation in intron 1 confirmed persistent pattern of dystrophin isoforms expression. There is absence of all muscle(M)-, brain(B) and Purkinje(P) dystrophin isoforms expression in the cardiac muscles, but an upregulation of the brain(B) and Purkinji(P) dystrophin isoforms in the skeletal muscles [1,2,3,4] that may explain the skeletal muscle sparing effect. What specific regulatory mechanisms and transcription factors are involved in this skeletal-muscle-specific P and B dystrophin isoform activation, and whether these two isoforms can also be upregulated in the cardiac muscles, is important question to be answered. Work done by us Earlier we have reported a patient initially presented with isolated dilated cardiomyopathy with significant ventricular dysfunction and heart failure eventually required heart transplantation. He did not have any muscle weakness upon presentation and his creatine kinase levels were normal. Two years after heart transplantation, he developed two episodes of transient significant weakness followed by vigorous exercise and viral infection respectively with raised creatine kinase level. His creatine kinase level remained elevated afterwards up to few thousands U/L. His muscle biopsy over quadriceps showed minimal structural disruption with normal dystrophin immunohistochemical staining, but utrophin was markedly upregulation. Dystrophinopathy was suspected and DMD gene mutation study subsequently confirmed a splice site mutation at intron 1 of the DMD gene c.31+1G>A. We reviewed the literatures and found that previous patients with mutation in the exon 1 and intron 1 boundary have similar cardiac specific phenotype, so a high genotypic-phenotypic correlation [5]. In order to study the possible underlying genetic regulatory mechanisms of the upregulation of the B and P dystrophin isoforms in the skeletal muscles we decide to generate an integration-free induced pluripotent stem cells (iPSCs) derived from the peripheral blood mononuclear cells (PBMCs) of our XLDCM patient. We have developed three xenobiotic-free patient-derived iPSC lines derived from our XLDCM patient peripheral blood mononuclear cells (PBMCs) using Sendai virus to deliver Yamanaka factors (Klf4, Oct3/4, Sox2, and cMyc) necessary for efficient generation of iPSCs, and similar for the healthy control. These iPSCs exhibited the morphology and growth properties of embryonic stem (ES) cells and expressed ES cell marker genes. The XLDCM patient-derived iPSC line confirmed to carry the unique 5’ splice site mutation in intron 1. We have completed the comprehensive characteristics of iPSCs by immunofluorescent staining (Figure 3), qPCR of stem cell markers expression, in vitro and in vivo differentiation, and the chromosomal karyotyping. The whole exome sequencing is under evaluation. We have further differentiated the patient-derived iPSCs to cardiomyocytes and similarly for the normal control. Pathological study comparing dystrophin isoforms expression using qPCR and western blot of patient-differentiated cardiomyocytes and that from normal control confirmed the absence of full-length dystrophin isoforms in the cardiomyocytes of the patients but not in the control. The functional study with osmotic stress test and the calcium handling and CK expression again confirmed the significant difference in the cardiomyocytes from the patients when compare to that from normal control subject. References: 1. Muntoni, F., et al., Transcription of the dystrophin gene in normal tissues and in skeletal muscle of a family with X-linked dilated cardiomyopathy. Am J Hum Genet, 1995. 56(1): p. 151-7. 2. Bastianutto, C., et al., Dystrophin muscle enhancer 1 is implicated in the activation of non-muscle isoforms in the skeletal muscle of patients with X-linked dilated cardiomyopathy. Hum Mol Genet, 2001. 10(23): p. 2627-35. 3. Kimura, S., et al., Novel mutation in splicing donor of dystrophin gene first exon in a patient with dilated cardiomyopathy but no clinical signs of skeletal myopathy. J Child Neurol, 2007. 22(7): p. 901-6. 4. Kimura, S., et al., Novel mutation in splicing donor of dystrophin gene first exon in a patient with dilated cardiomyopathy but no clinical signs of skeletal myopathy. J Child Neurol, 2007. 22(7): p. 901-6. 5. Sophelia HS Chan, et al. X-linked dilated cardiomyopathy with mutaiton in the 5’splice site intron 1 of dystrophin gene with utrophin upregulation. J Pediatr Neurol 2018; 16(01): 029-034 DOI: 10.1055/s-0037-1603997 6. Burridge, PW, et al. Chemically Defined Culture and Cardiomyocyte Differentiation of Human Pluripotent Stem Cells. Curr Protoc Hum Genet, 2015. 87: 21.3.1–21.3.15. 7. Turnbull, IC, et al. Advancing functional engineered cardiac tissues toward a preclinical model of human myocardium. FASEB J, 2014. 28(2):644-54. Objectives of this study We hope through this XLDCM patient-derived iPSCs platform as disease model, to further study the underlying molecular signalling pathway and regulatory mechanisms driving the current pathological expression causing the disease phenotype, through proteomics and transcriptome study. We also hope through this XLDCM patient-derived iPSCs platform as disease model, to further study the post translational modification causing the disease phenotype, through proteomics and transcriptome study. We also hope to study whether the changes in proteomics and transcriptome analysis can provide effective markers for drug screening using this XLDCM patient-derived iPSCs platform as disease model.