טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentBiale-Spiegel Idit
SubjectRemodeling of Gap Junctions, Sarcomers and Nuclei by Cyclic
Stretch in NRVM
DepartmentDepartment of Medicine
Supervisor Professor Emeritus Ofer Binah
Full Thesis textFull thesis text - English Version


Abstract

Cardiac hypertrophy and failure are known to interfere with normal electrical function, and the associated increased incidence of sudden death is related to ventricular arrhythmias. Disorganization of gap junctions (GJ) and reduced expression of connexin 43 (Cx43) have been observed in relation to various cardiac pathologies in many studies and may explain increased arrhythmogenesis. Mechanical stretch is thought to play an important role in the remodeling of the cardiac phenotype, and a number of studies have characterized the response of cultured myocytes to mechanical stretch. However, their response to stretch appears to be very complex and warrants further study.

Based  on  the  above  mentioned  considerations,  the  major  goal  of  this  M.Sc. Thesis  was  to  investigate  the structural changes in cardiomyocyte induced by cyclic stretch (CS), emphasizing the influence on the GJ (by the representative Cx43), the sarcomeres (by the sarcomeric protein myomesin) and the nuclei. In addition, to investigate the remodeling of gap junctional intercellular communication (GJIC) by CS, the Lucifer Yellow functional scrape-loading assay was used. The experiments were performed on cultures of neonatal rat ventricular myocytes plated on silicon membranes, which can be stretched cyclically by means of a custom-made device. Our main findings were: (1) Remodeling of GJ by CS, which was first manifested by decrease of Cx43 local density, increase in GJIC and decrease in Cx43 signal entropy parallel to the stretch direction after 12-24 hours of CS. Thereafter the remodeling of GJ by CS was manifested by increase in Cx43 area per cell and GJIC, orientation of Cx43 GJ rows parallel to the stretch direction, and decrease in Cx43 signal entropy perpendicular to the stretch direction after 48-65 hours of CS. (2) Remodeling of the sarcomeres by CS, manifested by orientation of myofibrils parallel to the stretch direction after 12-24 hours and perpendicular to the stretch direction after 48-65 hours of CS. (3) Nuclei remodeling by CS, manifested by increase of nuclei roundness, nuclei orientation parallel to the stretch direction after 12-24 hours of CS and nuclei orientation perpendicular to the stretch direction after 48-65 hours of CS.

In summary, we have shown that CS of 12-65 hours in neonatal rat ventricular myocytes induces a biphasic reaction of Cx43, sarcomeres and nuclei remodeling. The first phase occurs after 12-24 hours of CS. The second occurs after 48-65 hours of CS and corresponds with the self-organization theory of cardiomyocytes in response to mechanical stretch.