Ph.D Thesis

Ph.D StudentSchick-Tuval Revital
SubjectCellular and Molecular Mechanisms of Dilated
Cardiomyopathy in Cardiomyocytes Generated from
Induced Pluripotent Stem Cells(iPSC)
Derived from Affected...
DepartmentDepartment of Medicine
Supervisors PROFESSOR EMERITUS Ofer Binah
Full Thesis textFull thesis text - English Version


Dilated cardiomyopathy (DCM), the most common cardiomyopathy, is a myocardial disorder defined by ventricular chamber enlargement and dilatation, and systolic dysfunction that can result in progressive heart failure, supraventricular and ventricular arrhythmias. Despite extensive research in recent years, the complex pathological mechanisms of DCM are still unclear, mainly because numerous mutations in different gene families result in a similar outcome: depressed ventricular function. Titin (TTN) is a giant (up to 4000kD) sarcomere protein that is the largest known polypeptide, and is expressed in cardiac and skeletal muscles. The titin protein is a fundamental part of the sarcomere and is located within the sarcomere as a third filament around 1μm in length connecting between Z-line and M-line. In addition, titin functions as a molecular bi-directional spring and therefore is responsible for the passive elasticity of muscle and sarcomere length. In the present study we investigated in induced Pluripotent Stem Cell (iPSC)-derived cardiomyocytes (iPSC-CM) the mechanisms of DCM caused by mutations in two different proteins: 1) adenine and two base pairs insertion mutations in the sarcomere structural protein titin; 2) E342K missense mutation in the nuclear envelope protein lamin. The aims are: (I) Generate and characterize iPSC clones from titin-mutated DCM patient. (II) Investigate the main features of the cardiac excitation contraction coupling (ECC) machinery and the adrenergic responsiveness in the titin-mutated cardiomyocytes. (III) Investigate the electrophysiological properties and the adrenergic responsiveness of the titin- and lamin-mutated cardiomyocytes. (IV) Study the heart/beat rate variability in cardiomyocytes derived from iPSC generated from the titin- and lamin-mutated DCM patients. iPSC were generated from the patients' skin fibroblasts. The major findings were:  (1) titin-mutated iPSC-CM exhibited diminished (compared to healthy) inotropic and lusitropic responses to several positive inotropic agents such as isoproterenol, increased [Ca2]out and angiotensin-II. In addition, healthy and mutated iPSC-CM responded differently to caffeine and mutated cells displayed slower recovery period. These findings may result from reduction in expression levels of several calcium handling proteins such as phospholamban and SERCA2 as well as other proteins interacting with titin through its kinase domain which is absent in the mutated cells. (2) Spontaneous delayed afterdepolarizations (DADs) and abnormal automaticity were observed in lamin-mutated iPSC-CM generated from father and son carrying the E342K missense mutation. Notably, although the son is asymptomatic, his mutated cardiomyocytes exhibit the disease phenotype. Collectively, these findings show that the mutated cardiomyocytes from DCM patients recapitulate abnormalities of the inherited cardiomyopathies. Thus, novel insights attained from research on cardiomyopathies may also provide better treatment for the patients affected with the mutated genes.