Integrated molecular, biochemical, and physiological assessment unravels key extraction method mediated influences on rat neonatal cardiomyocytes

Author(s):Show less -

Jensen, Leonardo ^[1] ; Neri, Elida ^[1] ; Bassaneze, Vinicius ^[1] ; De Almeida Oliveira, Nathalia C. ^[1] ; Dariolli, Rafael ^[1] ; Turaca, Lauro T. ^[1] ; Levy, Debora ^[2] ; Veronez, Douglas ^[3] ; Ferraz, Mariana S. A. ^[4] ; Alencar, Adriano M. ^[4] ; Bydlowski, Sergio P. ^[2] ; Cestari, Idagene A. ^[3] ; Krieger, Jose Eduardo ^[1]

Affiliation

^[1] Univ Sao Paulo, Lab Genet & Mol Cardiol LIM 13, Heart Inst InCor, Sch Med, Sao Paulo - Brazil

^[2] Univ Sao Paulo, Lab Genet & Mol Hematol LIM 31, HC, Sch Med, Sao Paulo - Brazil

^[3] Univ Sao Paulo, Bioengn Div, Heart Inst InCor, Sch Med, Sao Paulo - Brazil

^[4] Univ Sao Paulo, Lab Microrheol & Mol Physiol, Inst Phys, Sao Paulo - Brazil

• Document type: Journal article
• Source: Journal of Cellular Physiology; v. 233, n. 7, p. 5420-5430, JUL 2018.
• Web of Science Citations: 3

Abstract

Neonatal cardiomyocytes are instrumental for disease modeling, but the effects of different cell extraction methods on basic cell biological processes remain poorly understood. We assessed the influence of two popular methods to extract rat neonatal cardiomyocytes, Pre-plating (PP), and Percoll (PC) on cell structure, metabolism, and function. Cardiomyocytes obtained from PP showed higher gene expression for troponins, titin, and potassium and sodium channels compared to PC. Also, PP cells displayed higher levels of troponin I protein. Cells obtained from PC displayed higher lactate dehydrogenase activity and lactate production than PP cells, indicating higher anaerobic metabolism after 8 days of culture. In contrast, reactive oxygen species levels were higher in PP cells as indicated by ethidium and hydroxyethidium production. Consistent with these data, protein nitration was higher in PP cells, as well as nitrite accumulation in cell medium. Moreover, PP cells showed higher global intracellular calcium under basal and 1mM isoprenaline conditions. In a calcium-transient assessment under electrical stimulation (0.5Hz), PP cells displayed higher calcium amplitude than cardiomyocytes obtained from PC and using a traction force microscope technique we observed that PP cardiomyocytes showed the highest relaxation. Collectively, we demonstrated that extraction methods influence parameters related to cell structure, metabolism, and function. Overall, PP derived cells are more active and mature than PC cells, displaying higher contractile function and generating more reactive oxygen species. On the other hand, PC derived cells display higher anaerobic metabolism, despite comparable high yields from both protocols. (AU)

• FAPESP's process: 16/50439-7 - A systems approach to identify and validate novel therapeutic targets to improve cardiac repair and regeneration following myocardial infarction
• Grantee: José Eduardo Krieger
• Support Opportunities: Regular Research Grants
• FAPESP's process: 13/17368-0 - Cardiovascular genomics: mechanisms & novel therapeutics - CVGen mech2ther
• Grantee: José Eduardo Krieger
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 11/19678-1 - Cardiac progenitor cells identification and isolation using aptamers
• Grantee: Vinícius Bassaneze
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 15/50216-5 - Characterization of the optimal hiPSC-derived cardiac cell population for heart regeneration after myocardial infarction
• Grantee: José Eduardo Krieger
• Support Opportunities: Research Grants - Research Partnership for Technological Innovation - PITE