Full text
| |
| Author(s): |
Douglas Martins Veronez
Total Authors: 1
|
| Document type: | Doctoral Thesis |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Escola Politécnica (EP/BC) |
| Defense date: | 2018-05-15 |
| Examining board members: |
Idágene Aparecida Cestari;
Maria Claudia Costa Irigoyen;
Cinthia Itiki;
Jose Carlos Teixeira de Barros Moraes;
Mario Ricardo Gongora Rubio
|
| Advisor: | Idágene Aparecida Cestari |
| Abstract | |
This study presents the development and validation of a new approach for the evaluation of intracellular calcium in cultures of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CM), which can be applied to evaluate the effect of drugs on excitation-contraction coupling. The method consists of electrical stimulation and joint measurement of fluorescence in an automated manner and was made possible by the inclusion of an electrical stimulation system in an ELISA (Enzyme-Linked Immunosorbent Assay). A compact electronic stimulator was designed to operate inside a plate reader generating monophasic square pulses with duration of 5 ms and electric field of 8 Vcm-1 applied by platinum-iridium metal microelectrodes to cells in culture. A culture plate used in a plate reader was modified to allow placement of the stimulator and electrodes. Fluorescence intensity of intracellular calcium was measured during electrical stimulation of cell cultures loaded with Ca2+ Fluo-4 AM indicator using a plate reader. The electrical stimulation of the cells generated regularly spaced contractions following the pace of the stimulator at the frequencies of 0.1 Hz, 0.2 Hz, 0.3 Hz and 0.5 Hz. Transient profile parameters were studied after treating cell cultures with Verapamil (0.05, 0.5 and 5.0 µM) the amplitude and the maximum slope of rising phase were progressively reduced with increasing verapamil doses. The data obtained demonstrated that the method presented allows the automated evaluation of calcium transients during the electrical stimulation of hiPSC-CM cultures using the stimulation system in an ELISA reader. These results demonstrated the applicability of the system to the study of changes in the intracellular calcium dynamics induced by drugs in electrically stimulated cells. The system developed is amenable to scaling thus allowing high content automated drug library screening for compounds that target the excitationcontraction coupling in human heart cells in vitro. (AU) | |
| FAPESP's process: | 13/24544-0 - Electrical properties of cardiac muscles following ventricular assist device implantation |
| Grantee: | Douglas Martins Veronez |
| Support Opportunities: | Scholarships in Brazil - Doctorate (Direct) |