Getting heart cells on the same wavelength: infrared triggering of Ca2+ transients in cardiac myocytes

Calcium (Ca2+) is a ubiquitous second messenger that is essential for numerous biological processes, including neurotransmitter release, muscle contraction and activation of signalling pathways that regulate gene expression. Because a single ion often has to simultaneously play different roles, information is encoded in the amplitude, kinetics, and spatial localization of Ca2+ signals. Determining the source of a cellular Ca2+ signal is not trivial, however. In a cell such as a cardiac myocyte, for instance, the instantaneous change in [Ca2+] may depend on: (1) Ca2+ fluxes across the cell membrane through channels and transporters; (2) Ca2+ binding to and release from intracellular buffering proteins; (3) Ca2+ release from, and reuptake into, the sarcoplasmic reticulum; (4) Ca2+ transport into and out of the nucleus; and (5) Ca2+ fluxes across mitochondrial membranes. Establishing the functional role of any particular Ca2+ flux is greatly aided when specific tools to activate or inhibit that flux exist. Whole cell patch clamp, for instance, has been instrumental for controlling, and thereby determining the function of, currents through voltage-dependent Ca2+ channels in the cell membrane. Definitive experiments regarding other pathways, such as SR Ca2+ release, have frequently employed relatively specific pharmacological tools. Mitochondrial Ca2+ fluxes, however, have proved somewhat of an exception. The tools that exist to manipulate these fluxes are generally less specific than those used to probe other pathways, and, partly as a result, controversies have persisted regarding the kinetics and amplitude of beat-to-beat changes in mitochondrial [Ca2+] (O’Rourke & Blatter, 2009). Optical methods for measuring and perturbing intracellular signalling have enormous strengths, including precise timing and localization. Optical techniques have been used extensively to measure [Ca2+] in heart cells and to elevate [Ca2+] through flash photolysis of caged [Ca2+]. More recently electrical activity has been directly perturbed in heart cells expressing light sensitive ion channels (Arrenberg et al. 2010). All of these techniques, however, require altering the native cells by either adding an exogenous compound or inducing expression of a non-native protein. Optical methods that can be used in native, unperturbed tissue therefore offer an additional potential advantage. In this issue of The Journal of Physiology, Dittami et al. (2011) present novel and surprising results that offer the promise of a new, highly welcome tool for investigating mitochondrial Ca2+ fluxes in cardiac myocytes and perhaps in other cell types. In cultured neonatal rat ventricular myocytes, these authors examined the effects of brief (4 ms) pulses of infrared (IR) radiation (1862 nm wavelength). When delivered to quiescent myocytes, these pulses triggered transient increases in intracellular [Ca2+]. Interesting features of these Ca2+ transients included the following: (1) IR-triggered transients were smaller in amplitude, more spatially homogeneous, and faster to decay compared with spontaneous Ca2+ transients observed in the same cells; (2) IR pulses delivered during spontaneous Ca2+ transients produced initial decreases in [Ca2+], which were followed by subsequent increases; and (3) pharmacological experiments suggest that mitochondria serve as the primary source of Ca2+ during IR-triggered transients. Recent work has demonstrated that IR pulses can cause myocyte contraction (Smith et al. 2008) – the present study provides a significant advance by helping to clarify the biological mechanism and by proposing that mitochondria are the most important IR-sensitive source of [Ca2+]. As with any unusual and novel observation, the work raises additional questions and suggests future experiments to provide greater understanding. In this case important questions include the following: (1) Given that neonatal and adult myocytes exhibit qualitative differences in Ca2+ handling (Wang & Sobie, 2008), can similar increases in [Ca2+] be triggered by IR pulses in adult cells? It is important to note that either a positive or negative result would be important: the former would validate the method for probing adult Ca2+ fluxes whereas the latter would suggest a developmental difference in mitochondrial Ca2+ handling. (2) Can the IR-induced Ca2+ transients be spatially localized? Dittami et al. delivered IR radiation to the cells through an optical fibre with diameter 400 μm, much larger than individual cells. Microscopic IR illumination may reveal interesting subcellular non-uniformity. (3) What is the wavelength dependence of the response? Given that 1862 nm IR radiation was used in this study whereas shorter-wavelength pulses (780 nm) were applied previously (Smith et al. 2008), it is possible that adjusting the wavelength may provide a means to tune the cellular response. If so, such studies may provide insights into the structural elements that cause particular proteins to respond to IR pulses. In the longer term, variations of this technique may provide a valuable tool for activating specific mitochondrial Ca2+ transport pathways. This could help to address important questions that have remained controversial despite years of investigation. For instance, while it is generally acknowledged that cardiac mitochondria accumulate Ca2+ during prolonged rapid pacing, the speed and extent of mitochondrial [Ca2+] changes during single contractions remain unclear (O’Rourke & Blatter, 2009). Additional important questions include: (1) how the close spatial relationship between mitochondria and ryanodine receptors influences cross-signalling between mitochondria and the SR; and (2) how disease states perturb particular mitochondrial Ca2+ fluxes. The technique described in this issue by Dittami et al. may prove valuable for addressing such questions, and, more broadly, for helping us to understand the coupling between cardiac Ca2+ handling and metabolism.