Abstract 10748: Structured Culture Scaffolds Force the Maturation of Calcium Transients in Rat Neonatal Ventricular Myocytes
Neonatal rat ventricular myocytes (NRVM) are widely used to study the physiology and pathology of the myocardium in vitro despite having an immature phenotype which is poorly representative of adult myocardium. We hypothesise that culture scaffolds which force the alignment of NRVM will improve Ca2+ handling properties. NRVM were isolated and seeded on fibronectin coated microgrooved silicone scaffolds (MS) fabricated using photolithography, or on flat silicone scaffolds (FS) (Fig 1). After 3-4 days in culture NRVM were imaged using Rhod-2 and confocal microscopy. The time-to-peak of the Ca2+ transient was shorter in MS at 1Hz field stimulation ((s) Mean+/- SEM: MS: 0.134 +/- 0.01, n=51; FS: 0.19 +/-0.009, n=57; P=0.0003, Mann Whitney Test), 0.5Hz ((s) Mean+/- SEM: MS: 0.18+/- 0.01, n=55; FS: 0.23 +/-0.01, n=52; P=0.0028) and when beating spontaneously ((s) Mean+/- SEM: MS: 0.14+/- 0.02, n=30; FS: 0.21 +/-0.02, n=21; P=0.0177). The time to 50% decline of the Ca2+ transient was reduced at 0.5Hz ((s) Mean+/- SEM: MS: 0.36 +/- 0.02; FS: 0.44 +/-0.02; P=0.0087) and when beating spontaneously ((s) Mean+/- SEM: MS: 0.25+/- 0.02; FS: 0.41 +/-0.07; P= 0.0191), as was the time to 90% decline when beating spontaneously ((s) Mean+/- SEM: MS: 0.68 +/- 0.08; FS: 1.03 +/-0.13; P=0.0171), however the spontaneous beating rate was significantly greater on MS ((Hz) Mean+/- SEM: MS: 0.84 +/- 0.1; FS: 0.52 +/-0.08; P= 0.0199). There was no difference in the amplitude of Ca2+ transients or force-frequency relationship between groups. We conclude that structured culture substrates promote faster Ca2+ transients in RVNM, making them more representative of adult myocardium. Fig 1. (A) NRVM cultured on FS (B) NRVM on FS stained with membrane stain Di-8-ANEPPS (green) (C) Immunohistochemistry: NRVM on FS stained for sarcomeric α-actinin (green) and DAPI (blue) (D) NRVM cultured on MS (E) NRVM on MS stained with Di-8-ANEPPS (F) Immunohistochemistry: NRVM on MS stained for α-actinin and DAPI
- Excitation-contraction coupling (ECC)
- Tissue engineering
- Cardiovascular development
- Myocyte electrophysiology
- © 2011 by American Heart Association, Inc.