Ph.D Thesis

Ph.D StudentReisner Yotam
SubjectRemodeling of Impules Conduction in Cultured Ventricular
Myocytes by Biomechanical Forces and Hypertrophic
DepartmentDepartment of Medicine
Supervisors PROFESSOR EMERITUS Ofer Binah
PROF. Noam Ziv
Full Thesis textFull thesis text - English Version


Background: Myocardial hypertrophy is an early stage in the course of heart failure. Heart failure and hypertrophy are associated with increased risk for cardiac death and arrythmogenesis. Endothelin-1 (ET-1) and mechanical load play an important role in remodeling of the myocardium. The thesis hypothesis was that long term exposure to load and ET-1 is accompanied by remodeling of impulse conduction and the electrical properties of cardiac myocytes, due to remodeling of gap junction expression. The thesis hypothesis was tested in the in vitro setting on cultures of neonatal rat ventricular myocytes grown on micro-electrode array (MEA) culture plates. An integral part of testing the hypothesis was the characterization of the model system including the effects of culture age, tissue architecture, beat rate, gap junction uncoupling and the relationship between the slope of the extracellular signal and conduction velocity.

Methods: Neonatal rat ventricular myocytes were cultured on special culture plates containing an 8x8 matrix of microscopic electrodes - the MEA system. Designated algorithms were constructed to generate activation maps, calculate conduction velocity and analyze the waveform of the extracellular signal. On day 5 of the culture, the myocytes were exposed either to ET-1 or to afterload induced by collagen coated glass microspheres. Expression of the gap junction protein connexin43 (Cx43) was measured using blotting and quantitative confocal microscopy.

Results: While ET-1 caused hypertrophy, as measured by the increase in cell surface area, a-actinin staining intensity, nuclear size and ANP mRNA expression, exposure to microspheres did not affect any of the above parameters of hypertrophy. ET-1 induced hypertrophy was accompanied by a decrease in conduction velocity and an increase in the spike peak negative slope (APNslope) that were apparent already 3 hrs following exposure. The decrease in conduction velocity was partially blocked by bosantan. Exposure to microspheres did not affect conduction velocity, but decreased APNslope. An increase in Cx43 protein level was observed concomitant with a decrease in spot number and staining intensity following exposure to ET-1. Microspheres did not change Cx43 expression.

Conclusions: Long term exposure to ET-1 induces hypertrophy accompanied by remodeling of impulse conduction and the waveform of the extracellular signal. Total Cx43 protein level increased, but a decrease in the gap junction number and size was observed and may be the cause for the observed changes in conduction. 24 hrs exposure to microsphere-induced load did not cause remodeling of conduction and Cx43 expression.