טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentHallas Tova
SubjectInvestigating the molecular, cellular and pathophysiological
features of induced pluripotent stem cells (iPSC)-
derived cardiomyocytes from SCO2
mutated patients
DepartmentDepartment of Medicine
Supervisors Professor Emeritus Ofer Binah
Professor Yeshayahu(Sha Katz
Full Thesis textFull thesis text - English Version


Abstract

Human Cytochrome C Oxidase (COX) is a multimeric protein complex that involves several assembly factors. One of the assembly factors is encoded by the SCO2 gene. Mutations in SCO2 are among the most common causes of isolated COX deficiency, resulting in ATP deficiency. These disorders are characterized by severe encephalomyopathy, severe progressive hypertrophic cardiomyopathy (HCM) and lactic acidosis, collectively leading to death in infancy or early childhood. Our group is the first to generate an induced Pluripotent Stem Cells (iPSC) model of SCO2. We have succeeded in generating cardiomyocytes from two SCO2 patients, both already dead at the time of research. One was a compound heterozygote to the common E140K mutation, and the other was homozygous for the less common G193S mutation. Due to ATP shortage caused by SCO2 mutations, we hypothesized that mutated iPSC-CM will exhibit mitochondrial abnormalities and attenuated responsiveness to positive inotropic interventions which depend on sarcoplasmic reticulum Ca2 stores, regulated by ATP-dependent SERCA activity. The present study had four main aims: (1) to investigate the structural changes of the iPSC derived SCO2 mutated iPSC-CM (2) to investigate the excitation-contraction coupling (ECC) machinery in the mutated iPSC-CM compared to control iPSC-CM. (3) Investigate and compare the Ca2 handling machinery of SCO2G193S and SCO2E140K in response to [Ca2]out, isoproterenol and angiotensin-II (AT-II). (4) Determine SR ca2 release capacity in SCO2-mutated iPSC-CM. (5) Investigate the chronotropic response of SCO2-mutated iPSC-CM to isoproterenol, and analyse their spontaneous beat rate variability compared to control iPSC-CM. Our major findings were: (1) Intracellular structural changes were found in SCO2-mutated iPSC-CM, similar to the intracellular changes previously described in cells obtained from patients’ heart and muscle. These changes include mitochondrial abnormalities, as well as doubled nuclei and large glycogen masses within the cell. (2) While in response to β-adrenergic stimulation healthy iPSC-CM exhibited increased inotropic and lusitropic effects, SCO2G193S and SCO2E140K showed unresponsiveness. The same results were observed when iPSC-CM were subjected to increased concentrations of Ca2. (3) Compared to healthy Control iPSC-CM, caffeine released less Ca2 in SCO2 iPSC-CM. Additionally, while control iPSC-CM demonstrated prompt recovery after caffeine-induced SR Ca2 release, SCO2 iPSC-CM demonstrated ~2 ~ slower recovery. These findings may result from SR low Ca2 storage due to impaired SERCA function in the SCO2 mutated iPSC-CM. (4) The spontaneous electrical activity of SCO2 mutated iPSC-CM exhibited alterations in rate and attenuated BRV properties compared to control, and were associated with adrenergically-mediated arrhythmogenic features. These results indicate that iPSC-CM are useful for investigating the similarities/differences in the pathophysiological consequences underlying SCO2 mutations syndromes compared to healthy control.