Regular invaginations in the surface membrane of ventricular myocytes, known as transverse tubules (t-tubules), enable synchronous intracellular Ca release, facilitating contraction. Evidence shows t-tubules are remodelled in several models of heart failure (HF), leading to delayed asynchronous Ca release. It remains unclear how t-tubule structure and function change during cardiac disease. Previous data indicates a role for Amphiphysin (AMPII) where KO models of AMPII lead to t-tubule disruption, similar to observed in HF (1). We aim to investigate if a) t-tubules and AMPII are reduced in a sheep model of HF; b) t-tubule biogenesis depends on the presence of AMPII; c) loss of AMPII leads to asynchronous Ca release. Under isoflurane anaesthesia (2-4% v/v oxygen) sheep were instrumented with a pacemaker and pacing lead. Post-operative analgesia (meloxicam 0.5mg/kg) and antibiosis (enrofloxacin 2.5mg/kg) were provided for 24hrs. HF was induced by right ventricular tachypacing at 210bpm for 4-5wks (2). Upon sacrifice, left ventricular (LV) and atrial (LA) myocytes were isolated and tissue samples were snap frozen. T-tubule density was assessed using di4ANEPPS and confocal microscopy (3). Data presented as mean±SEM statistical significance p<0.05 (t-test). The distance at which 50% of pixels in sheep HF cells are from a membrane compared with control increases (18±3.3% p<0.05 n=7). Western blotting on samples obtained from LV and LA showed a reduction in AMPII protein in HF (LV 24±7.5%, LA 35±12.3% n=7 p<0.05). Western blotting shows a reduction in AMPII in atria compared to ventricle in; rat, 58±6.7%, ferret, 55±9.8%, sheep, 27±9.6%, n=7. Transfecting rat ventricular myocytes with siRNA against AMPII causes t-tubule loss. The distance at which 50% of pixels in target cells are from a membrane compared with scrambled cells increases 50±6.6% p<0.05 n=20. Western blotting on transfected cells shows AMPII reduces 14±3.2% p<0.05 n=18 in target cells, compared with scrambled. Pearson’s coefficient performed on paired data shows the distance at which 50% pixels in target cells are from a membrane increases as AMPII decreases p<0.05 n=18. Immunocytochemistry was used to confirm loss of AMPII in transfected cells. Transfected cells, loaded with Fluo-3 AM and field stimulated, showed asynchronous Ca release. The STDEV of time to reach 50% peak fluorescence for each pixel of the line scan increases 159±43.8% p<0.05 n=9 in cells transfected with AMPII siRNA compared with cells transfected with scrambled siRNA. AMPII protein expression is reduced in a sheep model of tachypacing induced HF in both atria and ventricle and in the atria of species that lack t-tubules. There is also a loss of t-tubules in cells transfected with siRNA against AMPII, suggesting AMPII plays a role in the maintenance of t-tubules. This reduction results in asynchrous Ca release, likely to cause reduced contractility and HF.