טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentZeevi-Levin Naama
SubjectGap Junctional Remodeling by Hypoxia in Cultured Neonatal
Rat Ventricular Myocytes
DepartmentDepartment of Medicine
Supervisors Professor Emeritus Ofer Binah
Professor Andrew Peter Levy


Abstract

The coordinated mechanical activity of the heart is dependent upon orderly electrical activation of the individual myocytes. This process occurs at gap junctions, specialized regions of the membranes of adjacent cells containing arrays of densely packed intercellular channels that directly connect the cytoplasmic compartments of neighboring cells and permit intercellular passage of ions and small molecules. The gap junction channels are composed of members of a multigene family of proteins referred to as connexins of which Connexin43 (Cx43) is the predominant connexin expressed by cardiomyocytes. Cx43 is found in abundance in ventricular and atrial cardiomyocytes of all mammalian species, including man.

Cardiovascular disease is the leading cause of morbidity and mortality in the developed world. Arrhythmias are a common, serious and often fatal complication of many forms of heart diseases. Altered gap junctional coupling of ventricular myocytes plays an important role in arrhythmogenesis in ischemic heart disease. Since hypoxia is a major component of ischemia, we tested the hypothesis that hypoxia causes gap junctional remodeling accompanied by conduction disturbances. Cultured neonatal rat ventricular myocytes (NRVM) were exposed to hypoxia (1% O2) in the time range of 15 min to 30 hrs. Cx43 expression was analyzed and conduction velocity measured using the Micro-Electrode-Array (MEA) data acquisition system, which for the electrophysiological experiments, enabled us to use cultures of NRVM as their own control and conduct measurements before and after the hypoxic insult. Whereas at 15 minutes of hypoxia, total-Cx43 as well as the relative abundance of the phosphorylated and nonphosphorylated isoforms was increased, conduction velocity was unaffected. At 5 hrs of hypoxia, total-Cx43 protein was decreased by 50%, while the nonphosphorylated-Cx43 isoform was unchanged. Longer exposures to hypoxia led to a decrease in nonphosphorylated-Cx43. Confocal analyses demonstrated a 55% decrease in gap junctional Cx43 fluorescence signal, a 55% decrease in gap junctional number and a 26% decrease in size. The changes in Cx43 were not accompanied by changes in mRNA levels. The reduction in Cx43 protein levels was associated with a ~20% decrease in conduction velocity compared to normoxic cultures. Our study shows that short-term hypoxia (5 hrs) decreased gap junctional Cx43 protein expression and conduction velocity, which in the compromised myocardium, is likely to constitute an arrhythmogenic substrate.