טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentSofia Segal
SubjectThe Effect of Eliminating Contraction on Preserving
Pacemaker Cell Morphology and Function
in Culture
DepartmentDepartment of Biomedical Engineering
Supervisor Professor Yaniv Yael
Full Thesis textFull thesis text - English Version


Abstract

Pacemaker cells residing in the sinoatrial node generate the regular heartbeat. Thus, dysfunction of these cells can lead to a disturbance in the heart function. In order to understand the mechanisms that lead to heart dysfunction, first the dynamics of these mechanisms must be measured at different physiological and pathophysiological conditions. For that target, cultured cells that can be genetically manipulated and/or virally infected to introduce probes to measure this signaling are necessary. However, culturing rabbit pacemaker cells leads to a loss of their physiological spontaneous action potential (AP) firing rate and deterioration of signaling that determine the pacemaker cell function. We aim to develop a culture method that sustains the pacemaker cell AP firing rate along with biochemical signaling that maintains its function. We hypothesize that inhibiting the contraction during the culture period will preserve pacemaker function.

Rabbit pacemaker cells were maintained in culture for 48h in a medium enriched with a myofilament contraction inhibitor, either BDM (2,3-Butanedione 2-monoxime) or blebbistatin.   The area of the cells was maintained at the same level as fresh cells with either BDM or blebbistatin. Importantly, the cells could be successfully infected with a GFP adenovirus using either culture conditions. Finally, phosphorylation of phospholamban and ryanodine reminded intact during culture when either blebbistatin or BDM were presence However, blebbistatin maintains the AP firing rate and Ca2 transient characteristics closer to fresh level than BDM. Action potential parameters, Ca2 transients, and local Ca2 spark parameters were similar in the cultured cells with blebbistatin and in fresh cells. Our computational model predicts that (i) preservation of energy by eliminating contraction and maintaining pH helps to conserve the pacemaker function in culture, (ii) the side effects of BDM reduce its effectiveness compared to blebbistatin. Thus, eliminating contraction by blebbistatin during the culture period preserves rabbit pacemaker cell functions.