טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentKirschner Peretz Noa
SubjectDesigning a Culture System for Sustaining Bioelectric,
Biophysical and Bioenergetic Atrial Functions
DepartmentDepartment of Biomedical Engineering
Supervisor Professor Yael Yaniv
Full Thesis textFull thesis text - English Version


Abstract

Atrial myocytes play a significant role in bridging the electrical conductance from the sinoatrial node to the ventricle in the heart. One of the most common cardiac rhythm disturbances, atrial fibrillation (AF), is a condition characterized by irregular activation of the atrium. It is one of the most common cardiac rhythm disturbances and is a main cause of cardiac mortality and morbidity, mainly in adults over 65. The mechanisms that initiate and maintain AF episodes are not completely defined. Therefore, finding a pharmacological cure for this disease continues to motivate researchers. To characterize the changes in bioelectrical, biochemical and bioenergetic signaling that may lead to AF episode, a reliable culture method must be developed. Therefore, we aim to develop a culture method in which atrial cells maintain their shape, their ability to be externally paced in a physiological rate, to be able to sustain their biophysical and bioenergetic properties and to be able to be infected with a GFP adenovirus without changing any cell properties. 

Rabbit atrial cells were maintained in culture for 24h in a medium enriched with a myofilament contraction inhibitor. The functionality of the cells was tested by changes in their volume and their ability to contract in response to electrical stimulation. The biochemical properties were quantified by Ca2 transients and local Ca2 spark release parameters. Also, the cells were immunolabeled in order to examine the sustenance of SERCA and Ryanodine structures in culture.  The bioenergetic function was tested by quantifying Flavoprotein autofluorescence level. Finally, the cells were infected with a GFP adenovirus.  A computational model was applied to examine the ionic basis of the cardiac action potential and energetics in a rabbit atrial cell.   

The morphology and volume of the cells were preserved after 24h in culture. Also, the ability of the cells to contract in response to 1-3 Hz electrical stimulation was maintained in comparison to fresh cells. Preservation of these properties was unique to this method. Moreover, Ca2 transients and local spark parameters (duration, size, amplitude and number per 1s and 1µm) were maintained in cultured cells as opposed to fresh cells. Also, the SERCA and Ryanodine structures were maintained in both fresh and cultured atrial myocytes. The cultured cell Flavoprotein autofluorescence was maintained constant in response to physiological external electrical stimulation, similar to the response of fresh cells. Finally, the cells could also be infected with a GFP adenovirus without modifying any cell properties. The computational model predicts that the ionic channel currents remain constant and that the intracellular and mitochondrial ATP concentrations remain constant, similarly to the NADH and creatine phosphate concentrations.

This new method to culture rabbit atrial cells provides proper sustenance of various atrial myocyte properties. This method has the potential to initiate further comprehension of the energetic and biochemical regulation in the atria, which can lead to novel therapies involving the adaptation of this signaling in the atria, towards eliminating AF events. Our computational model predicts that ionic channels and energetic substrates remain constant in cultured atrial cells.