NIH3T3 fibroblasts and neonatal rat cardiomyocytes were seeded on abraded or non-abraded surfaces. The abraded surfaces were made with silicon carbide polishing film of various grain sizes on polyvinyl coverslips, which were then placed between carbon electrodes connected to an electrical stimulator. Stimulation occurred for 72hr after 24hr of seeding, using square pulses, 1ms duration in 1Hz, with voltages of 2.3 or 4.6V/cm. Pharmacological studies using Cytochalasin D and LY294002 were also performed. Cardiomyocytes' functional properties were examined via excitation threshold (ET) and maximum capture rate (MCR). Elongation and alignment of cells were assessed by image analysis.
Abrasions created with paper of larger grain sizes and greater depth, allowed for better elongation and alignment for fibroblasts and cardiomyocytes. However, electrical field stimulation had different effects on the two cell types. For fibroblast field stimulation, we observed that: i) elongation on non-abraded surfaces was significantly enhanced by electrical field stimulation and ii) such stimulation promoted orientation of fibroblasts in the direction perpendicular to the field lines when the abrasions were also placed perpendicular. For cardiomyocytes, overall we observed that i) elongation on non-abraded surfaces was significantly enhanced by electrical field stimulation and ii) topographical cues were a significantly stronger determinant of cardiomyocyte orientation than the electrical field stimulation. All of this suggests that topographical cues have a stronger effect than electrical stimulation when the factors are combined.
For cardiomyocytes, ET decreased with time due to improved cellular coupling. Higher MCR were seen on non-abraded surfaces, and over 72hr the MCR mostly decreased or stayed the same, possibly due to the rigid substrate. MCR values peaked under different field strength for cells on non-abraded surfaces versus abraded surfaces; thus there is interaction between field stimulation and topographical cues in on MCR.
Pharmacological studies were conducted on cardiomyocytes with abrasions placed parallel to the applied field lines. Cytochalasin D impaired cell elongation and alignment by in response to either field or stimulation by blocking F-actin polymerization. Cells treated with LY294002, a phosphatidyl-inositol 3 kinase (PI3K) pathway blocker, also showed perturbed elongation and alignment, however electrical stimulation could reverse this effect, implying that the effect of stimulation is transduced by a different pathway.
Overall i) elongation of both cell types on non-abraded surfaces was enhanced by electrical stimulation ii) electrical stimulation promoted orientation of fibroblasts perpendicular to the field on the perpendicular abrasions and iii) topographical cues had a stronger effect on cardiomyocyte orientation than the electrical stimulation. Cardiomyocyte orientation and elongation was abolished by inhibition of actin polymerization and only partially affected by inhibition of PI3K pathway.
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Acknowledgements This work was supported by grants from NSERC (DG), CFI (LOF), NIH grant R01HL076485 and University of Toronto Open Fellowships.