Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA028

Poster Communications

Amphiphysin II (Bin 1) driven transverse tubule formation in cardiac muscle.

J. Caldwell1, R. Taylor1, D. Eisner1, K. Dibb1, A. Trafford1

1. Cardiac Physiology, University of Manchester, Manchester, United Kingdom.


Regular invaginations in the surface membrane of cardiac myocytes, known as transverse tubules (t-tubules), are vital for maintaining normal contractility of the heart through the regulation of excitation contraction coupling. In cardiac diseases, such as heart failure, t-tubule loss is associated with decreased synchrony of calcium release from the sarcoplasmic reticulum, resulting in impaired contractility. Thus, determining the mechanisms that control t-tubule formation is essential for understanding this disease. Evidence suggests that the protein Amphiphysin II controls cardiac t-tubule formation and may play a vital role in calcium regulation (Lee et al, 2002; Caldwell et al, 2014). We therefore aimed to extend these observations and determine if Amphiphysin II is sufficient to drive t-tubule formation in the heart. Neonatal rat ventricular myocytes (NRVMs) were isolated from 2 day old Wistar rats (n=14 litters) and maintained in culture. Vectors encoding isoforms 5, 8 and 9 of the Amphiphysin II gene (Bin1) with a C-terminal mKate2 fluorescent protein tag were transiently expressed in NRVMs using FuGENE 6 lipofection. A vector containing the mKate2 fluorescent tag only was used as control. After 48hrs, over-expression of Bin1 was confirmed at the mRNA and protein level. Tubule formation was assessed using the membrane dye FM-464 and confocal microscopy. Of cells successfully transfected with Bin1, 95% had developed tubule structures (n=6 litters). Conversely, tubules were absent in cells only expressing the fluorescent tag control. To determine if Bin1 driven tubules are functional, transfected cells were loaded with the calcium indicator Fluo-8 AM and field stimulated. The systolic calcium transient amplitude was larger in cells transfected with Bin1 isoforms 5 (18.5±11.5%, n=5 litters), 8 (29.8±11.8%, n=7 litters) and 9 (29.73±11.9%, n=4 litters), when compared with untransfected myocytes (p<0.05, SPSS Statistics). Furthermore, transfection with Bin1 isoforms 5 and 9 led to faster rise (63±42.6%, 81±49.2% respectively) and decay (83±45.7%, 122±94.5% respectively) of the systolic calcium transient (p<0.05, SPSS Statistics, n= 4-5 litters). Transfection with the control vector only had no effect on calcium handling when compared with untransfected cells. Additionally, the calcium handling proteins NCX & RyR co-localised with Bin1 induced t-tubules when assessed using immunocytochemistry (Manders = 0.58 (NCX); 0.54 (RyR), n=2 litters). These data suggest that Bin1 plays a vital role in tubule formation in cardiac myocytes and specific Bin1 isoforms may enhance calcium kinetics. Given the importance of t-tubules to normal excitation contraction coupling and their perturbation in heart failure we therefore suggest that Bin1 might be a novel therapeutic target to restore t-tubules in disease.

Where applicable, experiments conform with Society ethical requirements