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Summary Muscle atrophy contributes to the poor prognosis of many pathophysiological conditions, but pharmacological therapies are still limited. Muscle activity leads to major swings in mitochondrial [Ca 2+ ], which control aerobic metabolism, cell death, and survival pathways. We investigated in vivo the effects of mitochondrial Ca 2+ homeostasis in skeletal muscle function and trophism by overexpressing or silencing the mitochondrial calcium uniporter (MCU). The results demonstrate that in both developing and adult muscles, MCU-dependent mitochondrial Ca 2+ uptake has a marked trophic effect that does not depend on aerobic control but impinges on two major hypertrophic pathways of skeletal muscle, PGC-1α4 and IGF1-Akt/PKB. In addition, MCU overexpression protects from denervation-induced atrophy. These data reveal a novel Ca 2+ -dependent organelle-to-nucleus signaling route that links mitochondrial function to the control of muscle mass and may represent a possible pharmacological target in conditions of muscle loss.

Publisher Summary This chapter focuses on the development and use of protein chimeras (derived from proteins naturally present in the medusa, aequorea victoria ) that are specifically targeted to the mitochondria, either to the matrix or to the intermembrane space, and can be used to monitor calcium and pH. Aequorin, the pioneer of the targeted recombinant probes, is a Ca 2+ -sensitive photoprotein that emits light on binding of Ca 2+ to its three high affinities Ca 2+ -binding sites. The chapter describes the use of mutants of green fluorescent protein as Ca 2+ and pH probes. Genetically encoded biosensors (chimeric proteins) are intracellular probes of physiological parameters endowed with unique advantages. The advantages include the ability to measure intracellular parameters (such as Ca 2+ and pH) at specific intracellular sites, with little background signal and no probe leakage. Additionally, the use of biosensors is not associated with the toxicity observes with many chemical probes nor with the need of invasive loading procedures. Furthermore, an ideal intracellular probe should be stable, have high parameter sensitivity and specificity, rapid signal response to parameter changes, and good luminescent/optical properties.

It has been known for a long time that cellular Ca2+ overload, occurring in excitotoxicity and various genetic or acquired diseases, leads to energy wasting and cell death, mostly by necrosis. The analysis of the effects of the oncogene Bcl-2 on cellular Ca2+ handling has led to a series of discoveries that suggest another, more specific, role of Ca2+ in cell death, i.e. that of modulating cellular sensitivity to apoptosis. Indeed, mitochondrial Ca2+ loading synergizes with toxic or signalling inputs in favouring the opening of a large-conductance mitochondrial channel, the permeability transition pore (PTP), thus altering mitochondrial structure and causing the release of caspase cofactors, such as cytochrome c, into the cytosol. The antiapoptotic protein Bcl-2 counteracts this effect by partially depleting the endoplasmic reticulum (ER) Ca2+ store (thus reducing mitochondrial Ca2+ loading), whereas the proapoptotic gene products Bax and Bak exert the opposite effect. In this contribution, we review (i) the data on the effect of pro- and antiapoptotic gene products of the Bcl-2 family on cellular Ca2+ homeostasis, (ii) the functional significance of the Ca2+ signalling alteration and (iii) the recent insights into the mechanism of this effect that may account for the apparent discrepancies between the data obtained in different studies.

Publisher Summary This chapter describes the development and use of protein chimeras targeted to either the mitochondrial matrix or intermembrane space. Aequorin, the first of the targeted recombinant probes, is a photoprotein of jellyfish of the genus Aequorea that emits light upon binding of Ca2+ to three high-affinity-binding sites. The second protein probe described in the chapter is luciferase. The chapter investigates how the measurement of luciferase light emission in situ allows monitoring the dynamics of ATP concentration changes in living cells. The use of variants of green fluorescent protein (GFP) as pH probes is also discussed. GFP fluorescence is intense and resistant to photobleaching. Further, spectral variants of GFP have been developed for double or triple label and energy transfer experiments. As a result, GFP has emerged as the most widely employed probe for cell biologists and is used routinely to study protein sorting and trafficking, gene expression, and organelle structure and motility. For some applications, the pH sensitivity of GFP is a disadvantage. The chapter describes how this property can be exploited to probe pH of mitochondria in living cells.

Publisher Summary Aequorin is a 21-kDa protein, isolated from jellyfish of the genus Aequorea that emits blue light in the presence of calcium. The aequorin originally purified from the jellyfish is a mixture of different isoforms called “heterogeneous aequorin.” In its active form, the photo-protein includes an apoprotein and a covalently bound prosthetic group, coelenterazine. Reconstitution of an active aequorin (expressed recombinantly) can also be obtained in living cells by simple addition of coelenterazine to the medium. The chapter describes the recombinant aequorin as tool for monitoring calcium concentration in subcellular compartments. The possibility of using aequorin as a calcium indicator is based on the existence of a well-characterized relationship between the rate of photon emission and the free Ca 2+ concentration. The cloning of the aequorin gene has opened the way to recombinant expression and thus expanded the applications of this tool for investigating Ca 2+ handling in living cells.

Publisher Summary This chapter shows that, through suitable chimeras, green fluorescent protein (GFP) can be targeted to various intracellular locations (the cytosol, the nucleus, the mitochondria, the endoplasmic reticulum, the Golgi, the subplasmalemmal space), thus providing powerful in vivo markers of these compartments. The GFP chimeras described in this chapter are currently utilized in lab for monitoring the dynamic changes of organelle structure that occur in living cells. Two types of instruments are currently used for detecting GFP fluorescence: (a) a wide-field fluorescence imaging system, based on a Zeiss Axiovert inverted microscope and a Princeton Instruments back-illuminated camera, and (b) a Nikon RCM 8000 confocal microscope. In both cases, the microscope is equipped with motorized control of the Z stage, thus allowing the acquisition of three-dimensional images. It is obvious that by confocal imaging, the dense endoplasmic reticulum structure of HeLa cells can be finely resolved, and the continuous rearrangement of organelle structure can be clearly appreciated.