Adaptation of the heart to Frataxin depletion: Evidence that integrated stress response can predominate over mTORC1 activation.

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 ß-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 ß-oxidation, could be adaptive, at least in the short term.

Multiple mitochondrial thioesterases have distinct tissue and substrate specificity and CoA regulation, suggesting unique functional roles.

Acyl-CoA thioesterases (Acots) hydrolyze fatty acyl-CoA esters. Acots in the mitochondrial matrix are poised to mitigate ß-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 ß-oxidation, CoA inhibition would prevent Acot-mediated suppression of ß-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 ß-oxidation overload and prevent CoA limitation.

Natural and Induced Mitochondrial Phosphate Carrier Loss: DIFFERENTIAL DEPENDENCE OF MITOCHONDRIAL METABOLISM AND DYNAMICS AND CELL SURVIVAL ON THE EXTENT OF DEPLETION.

The 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 ∼60% was still compatible with uncompromised maximal oxidative phosphorylation (oxphos), whereas lower maximal oxphos was evident at ∼85% 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.

Cytokine Profiling in Low- and High-Density Small Extracellular Vesicles from Epidermoid Carcinoma Cells.

Exosomes or small extracellular vesicles (sEVs) are membrane-bound nanoparticles that carry various macromolecules and act as autocrine and paracrine signaling messengers. In this study, sEVs from epidermoid carcinoma cells influenced by membrane presentation of the glycoprotein desmoglein 2 and its palmitoylation state were investigated. In this study, sEVs were isolated by sequential ultracentrifugation followed by iodixanol density gradient separation. They were then subjected to multiplex profiling of cytokines associated with the surface of intact sEVs. The results revealed a previously undescribed active sorting of cytokines onto the surface of low-density and high-density sEV subpopulations. Specifically, an altered surface presentation of desmoglein 2 decreased FGF-2 and VEGF in low-density sEVs. In addition, in response to desmoglein 2, IL-8 and RANTES were increased in low-density sEVs but only slightly decreased in high-density sEVs. Finally, IL-6 and G-CSF were increased dramatically in high-density sEVs. This comprehensive analysis of the cytokine production profile by squamous cell carcinoma‒derived sEVs highlights their contribution to immune evasion, pro-oncogenic and proangiogenic activity, and the potential to identify diagnostic disease biomarkers.