טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentDanon Asaf
SubjectGap Junctional and Electrophysiological Remodeling by
altered activation patterns and Hypoxia in
Neonatal Rat Ventricular Myocytes
DepartmentDepartment of Medicine
Supervisor Professor Emeritus Ofer Binah
Full Thesis textFull thesis text - English Version


Abstract

Introduction: Impulse conduction from cell to cell is accomplished mainly by movement of ions through gap junctions (GJ). Pathologic processes, referred to as GJ remodeling has been postulated to contribute to many of the arrhythmias occurring in the diseased myocardium. The underlying mechanisms responsible for GJ remodeling in diseased conditions are unknown. One hypothesis is that GJ lateralization may be the result of altered activation pathway.

Materials and Methods: NRVM cultures were transferred to a perfusion system, with stable conditions. One hour of acclimation was introduced, during which analysis of the spontaneous activation pathways was performed. At the end of the acclimation period, 7 cultures were stimulated from an alternative site ("stimulation group") while the other 13 cultures were used as control. Electrophysiological properties as well as immunostaining of GJ were compared between the two groups. In the hypoxia experiments, NRVM were exposed to hypoxia for 5 hours.

Results: The baseline electrophysiological characteristics of the control and stimulation groups were similar. CV increased during the recording period in the control group only (6% vs. 19% increase in stimulation and control groups, respectively; P=0.01). dV/dtmax and QRS amplitude did not differ between groups. QRS duration increased in the stimulation group only (P=0.046). GJ distribution was similar in both groups.

Next, we developed an experimental model in living cells. NRVM were transfected with Adenovirus containing GFP gene (Green Fluorescent Protein) attached to Cx43 (Ad-GFP), at the day of culture preparation. Three experiments were performed with the live confocal microscope system, confirming the validity of the model.

The hypoxia experiments: exposure to hypoxia for 5 hrs resulted in increased number of GJ outside the predefined border area between cardiomyocytes.

Discussion: No GJ remodeling was observed with the electrophysiological and microscopy experiments. Further, CV increased after 90 minutes of recording in control experiments only. This observation can also be regarded as resistance of the culture to an external stimulus, and indeed upon cessation of stimulation none of the stimulated cultures continued to propagate in the stimulation direction. The experimental model in living cells was proven to be valid, although did not support the first hypothesis. This model was used to test the second hypothesis as well, demonstrating GJ internalization in response to hypoxia. The later result may be responsible to the slowed conduction seen in the heart during ischemia, contributing to the generation of arrhythmias.