96 Finally, KATP channels display metabolic and mechanical co-activation, 99 which may help explain some of the differences between Akt targets experimental and clinical findings on the extent of ATP-reduction needed to activate them. In addition, this insight could shed light on hitherto ill-explored links

between ischaemic and mechanical preconditioning. As will be apparent from the above, the currently available information on the molecular substrates of cardiac SAC poses more questions than it answers. A number of reasons contribute to this. It is notoriously difficult to control and/or quantify the extent and quality of local mechanical stimuli that an individual ion channel is exposed to. 130 Tools to apply strain at whole-cell, tissue, and organ levels exist (including the application of shear stress, axial stretch, or cell volume changes), but there is no commonly implemented ‘gold standard’ for the stimulation of SAC. 27 Furthermore, these techniques have been used with a wide variety of cellular models from different species and developmental stages, making cross comparison of results challenging. In addition, it is difficult to interrelate macroscopic interventions and observations at cell and tissue levels with molecular substrates: in part because there is no ‘zero-strain’ reference even in patch clamp studies. Attempts

to explore causal links from low-level mechanism to integrated response, and back, include changes in gene expression, 87 pharmacological interventions, 131 and computational modelling. 132–135 Further challenges arise from the possibility that ventricular SAC may be localised in T-tubules, caveolae, or intercalated discs. This is thought to explain why patch clamping of single SAC

is so rare in freshly isolated ventricular cardiomyocytes from adult mammals. 130 One possible way around this problem may be to use pre-exposure to α1A receptor stimulation, to aid SAC translocation from T-tubules to the sarcolemma. 59 Another would be pre-stretching of the cardiac tissue prior to cell isolation, as this can cause surface membrane incorporation of caveolae. 43 Batimastat Thirdly, one could isolate the T-tubules using sequential centrifugation of homogenised cardiomyocytes followed by purification of T-tubule membranes by vesicle immuno-isolation and reconstitution into a continuous membrane. 136 It might then be possible to directly patch clamp SAC on the isolated T-tubule membrane. Less invasively, scanning ion conductance microscopy, which generates a three-dimensional topographical map of the cell surface prior to patch clamping, has been suggested as a means to directly target the T-tubule ostium where SAC are more likely to be present. 137 On the other hand, there is evidence to suggest that SAC may activate indirectly via second messenger signalling cascades.