Calcium-Dependent Turnover of the Cardiac Sarcolemma Analyzed by Non-Invasive Capacitance Recording and Optical Methods in Intact Cardiac Tissue

Abstract

We have described two types of massive Ca-activated endocytosis (MEND) in fibroblasts, HEK293 cells and cardiac myocytes (JGP, 137, Numbers 1 and 2, 2011). On the one hand, large Ca transients can promote subsequent internalization of >50% of the cell surface by mechanisms that require PIP2 synthesis. On the other hand, large Ca transients can promote internalization of >50% of the cell surface without PIP2, in the presence of high cytoplasmic Ca, when the cytoplasmic polyamine content is increased. Both MENDforms appear to internalize selectively membrane with a high content of liquid-ordered (Lo) domains, and neither MEND form uses classical endocytic proteins. To probe the physiological function of these and other endocytic mechanisms, we developed a non-invasive method to monitor capacitance in intact cardiac tissues, and we have combined this method with optical methods to monitor the disposition of transporters with respect to the sarolemma (e.g. Na/Ca exchangers via an extracellular pHluorin fusion). Using right ventricular strips from young adult mice, large-diameter (0.5 mm) pipettes are placed on the tissue surface, and transcellular voltage gradients are generated in tissue below the tip by sinusoidal voltage oscillations at frequencies of 10 to 100 kHz. Tentatively, we have identified a large membrane pool that can be inserted into the sarcolemma by cAMP- and Ca-dependent processes. These insertion processes are countered over time by large-scale retrieval processes that depend on ornithine decarboxylase activity (i.e. require synthesis of polyamines under β-adrenergic control). Further consistent with an important physiological role of MEND-related endocytic mechanisms, lysophosphatidylcholine's and other amphipaths can promote large-scale membrane retrieval in intact cardiac tissue. Advantages and potential pitfalls of the new electrophysiological methods will be discussed.