Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC127
Structured Culture Scaffolds force the Maturation of Calcium Transients in Rat Neonatal Ventricular Myocytes
C. Rao1,2, U. Chaudhry1, S. Abou Al-Saud1, P. Camelliti1, M. H. Yacoub1, T. Athanasiou2, T. Prodromakis3, C. Terracciano1
1. Laboratory of Cellular Electrophysiology, Imperial College, London, United Kingdom. 2. Department of Surgery, Imperial College, London, United Kingdom. 3. Institute of Biomedical Engineering, Imperial College, London, United Kingdom.
Figure 1. A)RVNM on Micropatterning of the PDMS membrane. B)RVNM on Microgrooved PDMS scaffolds. C) Unstructured RVNM. D) Immunohistochemistry of RVNM on Microgrooved PDMS scaffolds.
Figure 2. The time to peak, 50% decay and 90% decay of NRVM field-stimulated at 0.5Hz and 1Hz. The structured constructs are represented by the unshaded bars, the unstructured control NRVM cultures are represented by the black bars. (* p<0.05, ** p<0.005, *** p<0.0005 Mann Whitney Test)
Background: Cardiomyocytes differentiated from pluripotent stem cells and neonatal rat ventricular myocytes (NRVM) are widely advocated as models to study the physiology, pathology and pharmacology of the myocardium in vitro. The application of these cell lines is however limited by their immature phenotype which is poorly representative of adult myocardium. For this reason we aim to investigate the potential of structured culture scaffolds which force the alignment of myocytes on calcium transients, an important aspect of myocyte physiology. Methods: Two structured constructs were fabricated as previously described in the literature: microgrooved polydimethylsiloxane (PDMS) scaffolds [1-3] and micropatterned fibronectin on flat PDMS membranes [1,2] (Figure 1). NRVMs were isolated using a method described previously and with complete compliance to Home Office guidance . NRVMs were then seeded at a density of 2 million cells per 60mm culture dish containing micropatterned PDMS or 2/3 million cells per well in a 12 well plate containing PDMS scaffolds. After 24 hours preparations were washed with prewarmed DMEM and complete medium to remove dead and non-adherent cells . To visualise Ca2+ transients NRVMs were loaded with Rhod-2 AM (60μM, Invitrogen) in DMEM at 37C° for 30 minutes, then washed and incubated in prewarmed DMEM with 2% FBS (Invitogen) for 30 minutes to de-esterify. Rhod-2 AM was excited at 552-nm, and the emitted fluorescence was collected through a 581-nm long-pass filter. Confocal line scanning was performed (LSM 510, Carl Zeiss Micro Imaging) whilst NRVMs were beating spontaneously and whilst field stimulated at 0.5Hz, 1Hz and 2Hz. Time-to-peak was taken as the time taken for the ratio signal to go from baseline fluorescence to peak fluorescence. 50% and 90% decay were the time taken for the Rhod-2 AM Ca2+ transient to decline by 50% or 90% of the transient amplitude from peak fluorescence . Results The mean time-to-peak was significantly shorter in the Structured Constructs compared with control cells (P<0.0005, Mann Whitney Test) as was the time to 50% (P<0.0005, Mann Whitney Test) and 90% decay (P<0.005, Mann Whitney Test) when the RNVM were paced at 1Hz (Figure 2). Similar findings were seen when the RNVM were paced at 0.5Hz and when the cells beat spontaneously (Figure 2). This trend was not seen at 2Hz pacing in any group as there was not sufficient time between stimulation for the Ca2+ transients to return to baseline. Conclusion: Structured culture scaffolds affect Ca cycling of RVNM calcium transients. The mechanisms underlying these observations need to be elucidated and further characterisation of the effect of structured culture scaffolds and other aspects of cardiomyocyte electrophysiology is required.
Where applicable, experiments conform with Society ethical requirements