Edited by Jeffrey E. Pessin Acyl-CoA thioesterases (Acots) hydrolyze fatty acyl-CoA esters. Acots in the mitochondrial matrix are poised to mitigate ␤-oxidation overload and maintain CoA availability. Several Acots associate with mitochondria, but whether they all localize to the matrix, are redundant, or have different roles is unresolved. Here, we compared the suborganellar localization, activity, expression, and regulation among mitochondrial Acots (Acot2,-7,-9, and-13) in mitochondria from multiple mouse tissues and from a model of Acot2 depletion. Acot7,-9, and-13 localized to the matrix, joining Acot2 that was previously shown to localize there. Mitochondria from heart, skeletal muscle, brown adipose tissue, and kidney robustly expressed Acot2,-9, and-13; Acot9 levels were substantially higher in brown adipose tissue and kidney mitochondria, as was activity for C4:0-CoA, a unique Acot9 substrate. In all tissues, Acot2 accounted for about half of the thioesterase activity for C14:0-CoA and C16:0-CoA. In contrast, liver mitochondria from fed and fasted mice expressed little Acot activity, which was confined to long-chain CoAs and due mainly to Acot7 and Acot13 activities. Matrix Acots occupied different functional niches, based on substrate specificity (Acot9 versus Acot2 and-13) and strong CoA inhibition (Acot7,-9, and-13, but not Acot2). Interpreted in the context of ␤-oxidation, CoA inhibition would prevent Acotmediated suppression of ␤-oxidation, while providing a release valve when CoA is limiting. In contrast, CoA-insensitive Acot2 could provide a constitutive siphon for long-chain fatty acyl-CoAs. These results reveal how the family of matrix Acots can mitigate ␤-oxidation overload and prevent CoA limitation. Acyl-CoA thioesterases (Acots) 2 hydrolyze acyl-CoA into CoA and an acyl chain and are classified into two families based on functional domain. Type I Acots are members of the superfamily of ␣/␤-hydrolases, are found only in mammals, and have a high degree of similarity (1). Humans and rodents possess Type I Acots residing within the cytoplasm (Acot1), mitochondria (Acot2), and peroxisomes (Acot3-6 in rodents, Acot3-4 in humans) (1). In contrast, Type II Acots (Acot7-15) share little similarity beyond a hotdog fold domain (2). Some possess StAR-related lipid transfer domains (2) or can interact with a Start domain protein (3) and are found in the cytoplasm (Acot7-14), mitochondria (Acot7-13 and Acot15), and peroxisomes (Acot8) (2). Dual localization is also possible (Acot7,-11, and-13). Type I and II Acots have a signature substrate specificity that includes saturated and unsaturated fatty acyl-CoAs of different chain lengths and, in fewer cases, other CoA esters (3-5). Acots are predicted to have high biological relevance because their substrates, acyl-CoA esters, are also substrates for other enzymes that serve major metabolic pathways, such as mitochondrial ␤-oxidation, and can serve as allosteric or covalent regulators. In fact, genetic manipulation in mice of Type I or II Acots is associated ...

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Friedreich’s ataxia (FRDA) is an inherited disorder caused by depletion of frataxin (FXN), a mitochondrial protein required for iron–sulfur cluster (ISC) biogenesis. Cardiac dysfunction is the main cause of death. Yet pathogenesis, and, more generally, how the heart adapts to FXN loss, remain poorly understood, though are expected to be linked to an energy deficit. We modified a transgenic (TG) mouse model of inducible FXN depletion that permits phenotypic evaluation of the heart at different FXN levels, and focused on substrate-specific bioenergetics and stress signaling. When FXN protein in the TG heart was 17% of normal, bioenergetics and signaling were not different from control. When, 8 weeks later, FXN was ~ 97% depleted in the heart, TG heart mass and cardiomyocyte cross-sectional area were less, without evidence of fibrosis or apoptosis. mTORC1 signaling was activated, as was the integrated stress response, evidenced by greater phosphorylation of eIF2α relative to total eIF2α, and decreased protein translation. We interpret these results to suggest that, in TG hearts, an anabolic stimulus was constrained by eIF2α phosphorylation. Cardiac contractility was maintained in the 97%-FXN-depleted hearts, possibly contributed by an unexpected preservation of β-oxidation, though pyruvate oxidation was lower. Bioenergetics alterations were matched by changes in the mitochondrial proteome, including a non-uniform decrease in abundance of ISC-containing proteins. Altogether, these findings suggest that the FXN depleted heart can suppress a major ATP demanding process such as protein translation, which, together with some preservation of β-oxidation, could be adaptive, at least in the short term.

Friedreich's ataxia is an inherited disorder caused by depletion of frataxin (Fxn), a mitochondrial protein involved in iron-sulfur cluster biogenesis. Cardiac dysfunction is the main cause of death; pathogenesis remains poorly understood but is expected to be linked to an energy deficit. In mice with adult-onset Fxn loss, bioenergetics analysis of heart mitochondria revealed a time-and substrate-dependent decrease in oxidative phosphorylation (oxphos). Oxphos was lower with substrates that depend on Complex I and II, but preserved for lipid substrates, especially through electron entry into Complex III via the electron transfer flavoprotein dehydrogenase. This differential substrate vulnerability is consistent with the half-lives for mitochondrial proteins.Cardiac contractility was preserved, likely due to sustained β-oxidation. Yet, a stress response was stimulated, characterized by activated mTORC1 and the p-eIF2α/ATF4 axis. This study exposes an unrecognized mechanism that maintains oxphos in the Fxn-depleted heart. The stress response that nonetheless occurs suggests energy deficit-independent pathogenesis. KEYWORDSFrataxin; bioenergetics; β-oxidation; cardiac metabolism; mitochondrial disease; integrated stress response cardiac hypertrophy (Huang et al., 2013, Seznec, Simon et al., 2004, Stram, Wagner et al., 2017, Wagner, Pride et al., 2012, which is not easily explained by elevated peIF2α and an ensuing decrease in global translation. Taken together, the mitochondrial disease literature suggests that multiple signaling pathways can be altered to potentially drive phenotypes. Thus, in any given model, it would be useful to broadly understand disrupted signaling. A broader understanding could expand the possible therapeutic targets and also reveal if disease heterogeneity needs to be considered in the context of FRDA treatments. The CKM mouse model of Fxn loss has been useful because it exhibits severe cardiac dysfunction (Huang et al., 2013, Martin, Abraham et al., 2017, Seznec et al., 2004); indeed it has been used to demonstrate the potential of gene replacement therapy in the heart (Belbellaa, Reutenauer et al., 2019, Perdomini, Belbellaa et al., 2014). Yet, this model features complete depletion of Fxn from birth and thus might reflect a developmental response. Moreover, the model has a rapid time course, making it more challenging to disentangle causes from consequences of severe pathology. A recently developed model of adult-onset Fxn depletion (Chandran, Gao et al., 2017) has several advantages for the study of the pathogenesis of cardiomyopathy in FRDA: the model avoids a developmental context, and has a wider window of time without overt major cardiac pathology. We have used this model to investigate the progression of cardiac mitochondrial metabolism and nutrient and stress signaling changes with the goal of obtaining insight into the pathogenesis of Fxn loss in the heart, specifically with regard to the impact on energy metabolism and how changes in energy metabolism might drive pathology. RESUL...

Edited by F. Anne StephensonThe relevance of mitochondrial phosphate carrier (PiC), encoded by SLC25A3, in bioenergetics is well accepted. However, little is known about the mechanisms mediating the cellular impairments induced by pathological SLC25A3 variants. To this end, we investigated the pathogenicity of a novel compound heterozygous mutation in SLC25A3. First, each variant was modeled in yeast, revealing that substituting GSSAS for QIP within the fifth matrix loop is incompatible with survival on non-fermentable substrate, whereas the L200W variant is functionally neutral. Next, using skin fibroblasts from an individual expressing these variants and HeLa cells with varying degrees of PiC depletion, PiC loss of ϳ60% was still compatible with uncompromised maximal oxidative phosphorylation (oxphos), whereas lower maximal oxphos was evident at ϳ85% PiC depletion. Furthermore, intact mutant fibroblasts displayed suppressed mitochondrial bioenergetics consistent with a lower substrate availability rather than phosphate limitation. This was accompanied by slowed proliferation in glucose-replete medium; however, proliferation ceased when only mitochondrial substrate was provided. Both mutant fibroblasts and HeLa cells with 60% PiC loss showed a less interconnected mitochondrial network and a mitochondrial fusion defect that is not explained by altered abundance of OPA1 or MFN1/2 or relative amount of different OPA1 forms. Altogether these results indicate that PiC depletion may need to be profound (>85%) to substantially affect maximal oxphos and that pathogenesis associated with PiC depletion or loss of function may be independent of phosphate limitation when ATP requirements are not high.