Abstract Body: Abstract
Introduction: Tissue elasticity is essential to proper cell biology and organ function including the heart. Cell culture models on rigid polystyrene dishes are limited in studying the impact of tissue elasticity on specific cardiac cell types such as the cardiac conduction system cells.
Hypothesis: We hypothesized that gelatin hydrogel at varying stiffness can uncover tissue elasticity-dependent changes to cardiac pacemaker cell physiology.
Methods: Neonatal rat ventricular myocytes (NRVMs) were cultured as monolayers on polystyrene culture plates or on 1, 6, and 14 kPa gelatin hydrogels. All culture surface was coated with fibronectin. NRVMs were transduced with Adv-TBX18 or Adv-GFP at day 0 (d0).
Results: The rates of rhythmic contractions were faster in TBX18-NRVMs than in GFP-NRVMs on the gelatin hydrogels from d1 to d7 (n≥4, p<0.05). TBX18-NRVMs cultured on the 14 kPa gelatin hydrogels showed faster contractions than those on 1 kPa or on plastic plates at d3 and d7 (d3: 54±12, 9±5, 0±0 bpm, d7: 31±10, 4±7, 24±10 bpm, n≥4, p<0.05). TBX18-NRVMs exhibited faster Ca²⁺ transients on 14 kPa than on 1 and 6 kPa gelatin hydrogel or on plastic at d8 (33±2 vs. 9±2, 19±8, 11±5 transients/min, respectively, n=3, p<0.05). Hcn4 protein expression was higher in TBX18-NRVMs cultured on 14 kPa than on 1 kPa gelatin hydrogels or plastic plates (n=4/group, p<0.05). Cx43 protein expression was lower in TBX18-NRVMs compared to control at d8 regardless of the basement elasticity (n=4/group, p<0.05). However, Ca²⁺ transient propagation velocity of TBX18-NRVMs was slower on 14 kPa than on 1 kPa hydrogels at d9 (5±1 vs. 8±1 cm/s, n≥3, p<0.05). Vimentin+ fibroblasts were more numerous in TBX18-NRVMs on 14 kPa than on 1 or 6 kPa hydrogels at d6 (40±2 vs. 28±4 or 27±2%, n=4, p<0.05), suggesting that the slower propagation velocity in TBX18-NRVMs on 14 kPa was due to increase in fibroblast content.
Conclusions: The data demonstrate that automaticity of pacemaker cells is augmented by stiffer ECM substrates within the elasticity range of the healthy myocardium. This approach presents an easy-to-deploy in vitro model to study effects of tissue elasticity on cardiac conduction system cell physiology.