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171 Amphiphysin ii (bin1) driven transverse tubule formation in cardiac muscle
  1. Jessica Caldwell,
  2. Rebecca Taylor,
  3. David Eisner,
  4. Katharine Dibb,
  5. Andrew Trafford
  1. University of Manchester


Transverse (t)-tubules are vital for maintaining normal contractility of the heart through the tight regulation of excitation coupling. In cardiac disease, such as heart failure, t-tubule loss is closely 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 cardiac disease. Evidence suggests that the protein Amphiphysin II (AmpII) controls t-tubule formation in cardiac muscle and thus, may play a vital role in calcium regulation. Several studies, including our own, have shown that gene silencing of AmpII causes t-tubule loss in both skeletal and cardiac muscle. Furthermore, in non-muscle cells that usually lack t-tubules, expression of some variants of AmpII led to tubule formation. We therefore aimed to extend these observations and determine if AmpII is sufficient to drive t-tubule formation in the heart.

Neonate rat ventricular myocytes (NRVMs) were isolated from 2 day old rats and maintained in culture. Vectors encoding isoforms 5, 8 and 9 of the AmpII 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 negative control. After 48 hours, over-expression of Bin1 was confirmed at both 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. Conversely, tubules were absent in cells only expressing the fluorescent tag (p<0.001). Furthermore, Bin1 isoform 8 expression led to formation of more tubule structures when compared to isoform 5 and 9 (p<0.05).

To determine if Bin1 driven tubules are functional, transfected cells were loaded with the Ca2+ indicator Fluo-8 AM and field stimulated. When compared with untransfected myocytes, expression of Bin1 isoforms 5, 8 and 9 increased the amplitude of the systolic calcium transient (p<0.05). Furthermore, transfection with Bin1 isoforms 5 and 9 led to faster rise and decay of the systolic calcium transient (p<0.05). Transfection with the control vector only had no effect on the calcium handling when compared with untransfected cells.

Over-expression of Bin1 isoforms 5, 8 and 9 led to the formation of tubular structures in NRVMs. Whilst Bin1 isoforms 8 appears to play more of a role in tubule formation in NRVMs, these data suggest that other Bin1 isoforms (5 and 9) may enhance calcium kinetics. These data therefore suggest that Bin1 plays a vital role in tubule formation and development in cardiac myocytes. 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.

  • Transverse Tubules
  • Cardiac Physiology
  • Amphiphysin II

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