Mitochondrial MIsCUes in liver regeneration
Authors: Mateus T. Guerra
Journal: Hepatology (2016)

Beyond their well-known role as the energy factories of eukaryotic cells, mitochondria are also essential regulators of cell death. Interestingly, both processes are dependent on the concentration of Ca2+ within the mitochondrial matrix. It is thus predicted that manipulation of the expression or function of mitochondrial Ca2+ signaling proteins would have major consequences to cellular physiology including hepatocyte function. Indeed, Antony et al. now show that the lack of a mitochondrial Ca2+ (Ca2+mit) gatekeeper protein (mitochondrial calcium uptake 1 [MICU1]), an essential component of the Ca2+ import mechanism of the mitochondrial membrane, prevents complete liver regeneration after partial hepatectomy in mice. This is shown to result from a sustained increase in Ca2+mit and the consequent necrotic cell death.1 Ca2+ uptake into energized mitochondria was first documented in the mid-1960s, and those findings were followed by studies demonstrating that Ca2+ positively regulates the activity of three enzymes of the citric acid cycle and thus adenosine triphosphate (ATP) synthesis (reviewed in De Stefani et al.2). Later on, it was shown that prolonged elevations of Ca2+mit could lead to the formation of the permeability transition pore (PTP) on the inner mitochondrial membrane, allowing for the diffusion of cytochrome C to the cytosol and the activation of caspase-dependent apoptotic cascades (reviewed in De Stefani et al.2). Despite detailed biophysical data that clearly supported the existence of a regulated Ca2+ entry machinery on the inner mitochondrial membrane, the identity of the so-called mitochondrial Ca2+ uniporter was not discovered until 2011, when two groups independently cloned and characterized the mitochondrial calcium uniporter (MCU) as the bona fide Ca2+ transporter of the mitochondrial membrane.3, 4 These exciting findings were followed by some disappointment when the first MCU knockout mouse study was published because the animals only displayed mild defects in skeletal muscle performance in extreme conditions of strenuous physical exercise.5 The missing piece of the puzzle turned out to be the protein MICU1. First described as a component of the yet unidentified Ca2+mit uniporter,6 MICU1 was later shown to directly bind to and act as a regulator of MCU (reviewed in De Stefani et al.2). Under basal cytoplasmic Ca2+ concentrations, MICU1 prevents MCU opening; and in conditions of higher (cytoplasmic Ca2+), it acts to facilitate MCU transporter activity.7 Therefore, it was shown in in vitro studies in which MICU1 expression was altered that Ca2+ can more freely cross the inner mitochondrial membrane and accumulate in the mitochondrial matrix in the absence of MICU1. Also, mutations in MICU1 (both large deletions and splice-site variants) that result in protein loss have been described in humans. Accordingly, these mutations are associated with a range of muscular and neurological manifestations that are linked to elevated basal and stimulated (Ca2+mit) levels. The study by Antony and collaborators provides the first in vivo evidence of MICU1's role in liver function. The authors demonstrated that mice lacking MICU1 undergo normal embryonic development but die soon after birth, presumably from respiratory malfunction. They then engineered MICU1 hepatocyte-specific knockdown (MICU1 KD) mice in which global gene expression and liver histology are similar to wild-type mice. Moreover, the authors show that KD of MICU1 in hepatocytes increases the rate of Ca2+ uptake in the mitochondria in lower micromolar conditions when compared to control hepatocytes. The lack of a gross phenotype in the MICU1 KD mice under basal conditions prompted the authors to perform 70% partial hepatectomy to examine the stress response in MICU1 KD livers. Interestingly, DNA synthesis is halted and cyclin D expression and bromodeoxyuridine incorporation are reduced in MICU1 KD livers. These animals failed to progress from the initial inflammatory phase (within the first 30 hours postsurgery) to the proliferative phase of liver regeneration, despite normal levels of key proliferative mediators such as tumor necrosis factor alpha, interleukin-6, c-Fos, and c-Jun in MICU1 KD livers. Concurrently, liver histology was indicative of hepatocellular damage, and indicators of hepatocyte injury (alanine aminotransferase, total and direct bilirubin) were significantly elevated in KD animals, suggesting that low expression of MICU1 also induced hepatocyte cell death during liver regeneration. Interestingly, hepatocyte death does not occur through classic apoptotic caspase activation but rather through necrosis as no cleaved caspase-3, Bax activation, or cytochrome C release was detected in MICU1 KD livers. At first glance, these findings are intriguing given the well-described connection between elevated Ca2+mit levels and formation of the PTP and subsequent caspase activation in several cell systems. In addition, buffering of Ca2+mit in the liver was shown to accelerate regeneration after partial hepatectomy by reducing apoptosis.8 However, mitochondrial Ca2+ overload has also been shown to induce necrotic cell death.9 Accordingly, pharmacological inhibition of the PTP both prevented necrosis and rescued the proliferation defect in MICU1 KD livers. One question that thus remains to be explored is the mechanism by which PTP formation induces necrotic, rather than apoptotic, cell death in hepatocytes during liver regeneration in conditions of elevated mitochondrial calcium. One possibility is that the opening of the PTP causes a collapse of the mitochondrial membrane potential and thus severely compromises ATP generation through oxidative phosphorylation. Alternatively, one could speculate that the initial elevation in Ca2+mit caused by the KD of MICU1 could drive the tricarboxylic acid cycle to produce higher levels of ATP and in the process promote the generation of increased levels of reactive oxygen species that in turn could mediate the necrotic cell death. Data from a skeletal muscle-specific knockout mouse for MCU, the mitochondrial Ca2+ uniporter, would favor the first hypothesis. In such conditions, MCU acts to supply Ca2+ on demand according to the ATP requirements of the myocyte. Given that MICU1 is a master regulator of MCU, one can envision an essential role for MICU1 in this process. Nonetheless, experimental data are not yet available for either of the hypotheses. The work by Antony and collaborators also indirectly presents the broader question of whether MICU1 and Ca2+mit are involved in the pathogenesis of additional liver diseases. The most appealing possibility would be nonalcoholic fatty liver disease. Deregulation of energy metabolism is one of the hallmarks of this spectrum of diseases, placing mitochondria as central organelles in this process. Indeed, recent evidence from human liver biopsies from nonalcoholic fatty liver disease patients, which was the subject of a recent Hepatology Elsewhere, demonstrated that mitochondrial metabolic function in the liver undergoes beneficial adaptive changes during the progression from normal to steatosis and is lost in steatohepatitis.10 Moreover, studies in animal models of fatty liver disease have shown an increase in physical association between the endoplasmic reticulum and mitochondria in the so-called mitochondria-associated membranes.11 These physical contact points between the two organelles are thought to work as preferential conduits of Ca2+ between the endoplasmic reticulum and mitochondria. In fact, an increased transfer of Ca2+ from the endoplasmic reticulum stocks to the mitochondrial matrix was reported in these animal models, which leads to mitochondrial dysfunction and worsening of steatosis. Also, pharmacological interventions that uncouple mitochondrial metabolism, i.e., promote consumption of substrates such as fatty acids and generate heat as opposed to ATP, are effective ways to improve indicators of nonalcoholic fatty liver disease and insulin resistance in diet-induced obesity in rats. It remains to be seen whether changes in Ca2+mit are drivers of lipid accumulation in the liver or simply an adaptation to the increased metabolic demand generated by the elevated energetic input and which are the potential mechanisms involved. In addition, the challenge persists in translating findings of in vitro and animal studies to human therapy. Nonetheless, the work by Antony and colleagues places MICU1 and more importantly Ca2+mit as attractive targets for therapeutic intervention in a range of liver diseases. Mateus T. Guerra, Ph.D. Section of Digestive Diseases and Yale Liver Center Department of Internal Medicine Yale University School of Medicine New Haven, CT Author names in bold designate shared co-first authorship.