טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentNaor Shulamit
SubjectCellular and Molecular Mechanisms of Inherited
Laminopathy in Cardiomyocytes Generated
from Induced Pluripotent Stem Cells
(iPSCs) Derived from Affect...
DepartmentDepartment of Medicine
Supervisor Professor Emeritus Ofer Binah


Abstract

The term laminopathies defines a group of genetic disorders caused by defects in the nuclear envelope, mostly the Lamins. Mutations in the human Lamin genes lead to striated muscle diseases, virtually always involving the heart. Lamins are major structural proteins of the nucleus and are essential for nuclear integrity and the organization of nuclear functions. They form complex protein assemblies with integral proteins of the inner nuclear membrane, transcriptional regulators, histones and chromatin modifiers. Despite the new findings on the role of Lamins in cellular signalling pathways, as well as in ubiquitin-mediated proteasomal degradation, the complex mechanisms of laminopathy are still unclear, mainly due to diverse pathologies and poor genotype-phenotype correlation. In the present study, we investigated in induced Pluripotent Stem Cell (iPSC)-derived cardiomyocytes (iPSC-CMs) the mechanisms of laminopathy caused by E342K missense mutation in Lamin A/C. The aims are: (1) Generate iPSC from two LmnaE342K/WT patients and from healthy subject as control (LmnaWT/WT). (2) Investigate the structural and ultrastructural characteristics of LmnaE342K/WT iPSC-CMs, compared to LmnaWT/WT iPSC-CMs. (3) Investigate the electrophysiological properties of LmnaE342K/WT iPSC-CMs, compared to LmnaWT/WT iPSC-CMs. (4) Investigate LmnaE342K/WT iPSC-CMs response to electrical stimulation, compared to LmnaWT/WT iPSC-CMs. (5) Investigate LmnaE342K/WT iPSC-CMs response to adrenergic stimulation compared to LmnaWT/WT iPSC-CMs. (6) Analyze the heart and beat rate variability in LmnaE342K/WT iPSC-CMs, compared to LmnaWT/WT iPSC-CMs. iPSC were generated from the patients' skin fibroblasts. The major findings were:  (1) LmnaE342K/WT iPSC-CMs exhibited altered Lamin A/C expression and nuclear structural defects. (2) LmnaE342K/WT iPSC-CMs display spontaneous arrhythmias, abnormal electrophysiological features and augmented BRV. (3) LmnaE342K/WT iPSC-CMs presented altered responses to electrical and β-adrenergic stimulation. Notably, although the son is asymptomatic, his mutated cardiomyocytes exhibit the disease phenotype. These findings may result from the nuclear structural defects and disturbances in the transcription of genes, and the activation of important signaling pathways crucial for cardiac activity, specifically for heart conduction. Collectively, these findings show that the mutated cardiomyocytes from LmnaE342K/WT patients recapitulate, in vitro, the electrophysiological abnormalities of inherited laminopathy associated with conduction-system disease in vivo, and are useful for investigating the pathophysiology underlying laminopathy. In addition, LmnaE342K/WT iPSC-CMs can be used for/serve for drug screening and personalized medicine as well as prognostic tool for arrhythmogenecity in laminopathy patients with augmented BRV.