טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentDolnikov Katya
SubjectDifferentiation and Maturation of Functional Properties of
Human Embryonic Stem Cells-Derived Cardiomyocytes
DepartmentDepartment of Medicine
Supervisors Professor Emeritus Ofer Binah
Professor Emeritus Joseph Itskovitz
Full Thesis textFull thesis text - English Version


Abstract

Heart failure is one of the leading causes of morbidity and mortality in the industrialized world. The current treatment for heart failure is limited. Recent years have witnessed numerous publications concerning the therapeutic efficacy of myocardial cell therapy utilizing cardiomyocytes from different sources, including human embryonic stem cell-derived cardiomyocytes (hESC-CMs). In view of the therapeutic potential of hESC-CMs we investigated their functional properties, focusing on the key aspects of excitation-contraction coupling and mechanical function. The functional properties of hESC-CMs were investigated by recording simultaneously [Ca2]i transients and contractions. Additionally, we performed Western blot analysis of the key Ca2 -handling proteins SERCA2, calsequestrin, phospholamban, and Na/Ca2 exchanger (NCX).

Our major findings are: (1) hESC-CMs display temporally related [Ca2]i transients and contractions. (2) hESC-CMs feature negative force-frequency relations and lack of post-rest potentiation. (3) Ryanodine, thapsigargin, and caffeine did not affect the [Ca2]i transient and contraction parameters, indicating that in the hESC-CMs clones studied contraction depends on transsarcolemmal Ca2 influx rather than on sarcoplasmic reticulum Ca2 release. (4) Whereas hESC-CMs expressed SERCA2 and NCX at levels comparable to those of the adult human myocardium, calsequestrin and phospholamban were absent in these cells. Collectively, these findings demonstrate that in contrast to the mature myocardium, in hESC-CMs Ca2-induced Ca2 release does not contribute to contraction and Ca2 utilized by the contractile machinery is provided by transsarcolemmal influx.

During the initial processes of embryonic heart development, 1,4,5-IP3 regulated Ca2 signaling, rather than SR Ca2 release is considered important in establishing the cytoplasmic Ca2 oscillations and contraction. Therefore, we hypothesized that hESC-CMs contain functional 1,4,5-IP3 operated intracellular Ca2 stores. To test this hypothesis we investigated the effect of angiotensin II (AT-II) and endothelin 1 (ET-1) on hESC-CMs [Ca2]i transients and contractions.

Our major findings are:  (1) AT-II and ET-1 increased the contraction amplitude, as well as the maximal rate of contraction and relaxation (2) The effects of AT-II were blocked by 2-APB (1,4,5-IP3 receptor blocker) and U73122 (a phospholipase C blocker). (3) hESC-CMs express IP3 type I and II receptors, as well as calreticulin, the major Ca2 buffering protein in 1,4,5-IP3-operated Ca2 stores.

Collectively, we conclude that Ca2 handling machinery of hESC-CMs differs from the mature myocardium. While SR is either absent or immature and CICR release does not contribute significantly to E-C coupling in these cells, 1,4,5-IP3-operated Ca2 stores are functional and can modulate [Ca2]i transient and contraction parameters upon appropriate physiological stimuli.