Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC161
Effects of detubulation on excitation-contraction coupling in sheep atrial myocytes.
M. A. Richards1, J. D. Clarke1, A. W. Trafford1, K. M. Dibb1
1. Unit of Cardiac Physiology, University of Manchester, Manchester, United Kingdom.
Transverse (t-) tubules are essential for facilitating the synchronous increase in intracellular calcium giving rise to the calcium transient in mammalian ventricular myocytes. It has recently been established that atrial cells of the sheep possess a functional t-tubule network (Dibb et al., 2009) unlike atrial cells of rodents (reviewed by Bootman et al., 2006). Importantly, atrial t-tubules are lost in sheep models of cardiac disease (Dibb et al., 2009, Lenaerts et al., 2009). Here we examine the functional importance of t-tubules for excitation-contraction coupling in sheep atrial cells. Healthy adult sheep were killed in accordance with The Home Office Animal (Scientific Procedures) Act, 1986. Myocytes were enzymatically isolated from the left atrial appendage. Formamide was used to detubulate the myocytes (Brette et al., 2002). T-tubules were visualised using di-4-ANEPPS. Cells were voltage-clamped using the perforated patch technique and stimulated at 0.5Hz. Intracellular calcium was measured using Fura-2 AM. Data are expressed as mean ± SEM and statistical significance was considered to be when p<0.05. Confocal imaging using di-4-ANEPPS confirmed that treatment with formamide could successfully uncouple t-tubules from the sarcolemma in sheep atrial myocytes. Detubulation resulted in a decrease of cell capacitance (104 ± 14 vs. 62 ± 4 pF, p<0.001, n=12-14) without a change in cell length (135 ± 7 vs. 137 ± 4µm, p>0.05, n=6-8) or width (20 ± 3 vs. 23 ± 2µm, p>0.05, n=6-8). Peak ICa-L was reduced in detubulated myocytes (1.5 ± 0.2 vs. 0.4 ± 0.1 pA/pF, p<0.001, n=11-13) and accompanied by a 53% decrease in calcium transient amplitude. In addition, the calcium transient had a slower time to peak (106 ± 15 vs. 275 ± 9 ms, p<0.001, n=10-13), suggesting asynchronous calcium release. These data show that sheep atrial t-tubules are functionally important for E-C coupling. T-tubules constitute 40% of cell capacitance with ~70% of the total ICa-L residing in these structures. Importantly, formamide treatment qualitatively mimics alterations in atrial calcium handling previously observed in a sheep model of heart failure where t-tubules are lost. This suggests t-tubule loss has important functional consequences in cardiac disease.
Where applicable, experiments conform with Society ethical requirements