Mitochondrial protein interactions and complexes facilitate mitochondrial function. These complexes range from simple dimers to the respirasome supercomplex consisting of oxidative phosphorylation complexes I, III, and IV. To improve understanding of mitochondrial function, we used chemical cross-linking mass spectrometry to identify 2,427 cross-linked peptide pairs from 327 mitochondrial proteins in whole, respiring murine mitochondria. In situ interactions were observed in proteins throughout the electron transport chain membrane complexes, ATP synthase, and the mitochondrial contact site and cristae organizing system (MICOS) complex. Cross-linked sites showed excellent agreement with empirical protein structures and delivered complementary constraints for in silico protein docking. These data established direct physical evidence of the assembly of the complex I-III respirasome and enabled prediction of in situ interfacial regions of the complexes. Finally, we established a database and tools to harness the cross-linked interactions we observed as molecular probes, allowing quantification of conformation-dependent protein interfaces and dynamic protein complex assembly. mitochondria | mass spectrometry | interactome | cross-linking | protein interaction reporter M itochondrial proteins play a diverse role in cellular biology and disease. Mitochondrial dysfunction directly causes multiple inherited diseases (1) and is implicated in common diseases, including neurological developmental disorders (2, 3), neurodegenerative and cardiovascular diseases (4-6), diabetes (7), asthma (8), cancer (9), and age-related disease (10). In mammals, these organelles have evolved to retain more than 1,000 proteins that interact within a complex, i.e., dual membrane architecture (11,12). Within the mitochondrial proteome, the "powerhouse" functions are carried out by the core constituents of the oxidative phosphorylation (OXPHOS) system [complexes I-IV of the electron transport chain (ETC) and ATP synthase (complex V)]. These proteins are necessary for creation of the mitochondrial electrochemical gradient that powers synthesis of ATP. This system includes critical protein-protein interactions within individual OXPHOS complexes as well as "supercomplex" interactions between ETC complexes I, III, and IV in the respirasome (13). Deficient supercomplex formation has been proposed as a critical mitochondrial defect in failing hearts (5,6,14,15), and dynamic rearrangement of supercomplexes has been implicated in noncanonical mitochondrial functions such as antibacterial innate immune responses (16). Assessing these interactions is further complicated by regulatory posttranslational modification and conformational changes of mitochondrial proteins (17)(18)(19)(20). Advances in this area have been impeded, in part, by the lack of large-scale detection of dynamic, sometimes transient, interactions between membrane proteins. Thus, large-scale determination of the protein interactome within mitochondria would provide a valuable tool to adv...

While modern structural biology technologies have greatly expanded the size and type of protein complexes that can now be studied, the ability to derive large-scale structural information on proteins and complexes as they exist within tissues is practically nonexistent. Here, we demonstrate the application of crosslinking mass spectrometry to identify protein structural features and interactions in tissue samples, providing systems structural biology insight into protein complexes as they exist in the mouse heart. This includes insights into multiple conformational states of sarcomere proteins, as well as interactions among OXPHOS complexes indicative of supercomplex assembly. The extension of crosslinking mass spectrometry analysis into the realm of tissues opens the door to increasing our understanding of protein structures and interactions within the context of the greater biological system.

Respirometry is the gold standard measurement of mitochondrial oxidative function, as it reflects the activity of the electron transport chain complexes working together. However, the requirement for freshly isolated mitochondria hinders the feasibility of respirometry in multi‐site clinical studies and retrospective studies. Here, we describe a novel respirometry approach suited for frozen samples by restoring electron transfer components lost during freeze/thaw and correcting for variable permeabilization of mitochondrial membranes. This approach preserves 90–95% of the maximal respiratory capacity in frozen samples and can be applied to isolated mitochondria, permeabilized cells, and tissue homogenates with high sensitivity. We find that primary changes in mitochondrial function, detected in fresh tissue, are preserved in frozen samples years after collection. This approach will enable analysis of the integrated function of mitochondrial Complexes I to IV in one measurement, collected at remote sites or retrospectively in samples residing in tissue biobanks.

Background: Increased fatty acid oxidation (FAO) has long been considered a culprit in the development of obesity/diabetes induced cardiomyopathy. However, enhancing cardiac FAO by removing the inhibitory mechanism of long-chain fatty acids transport into mitochondria via deletion of acetyl-CoA carboxylase 2 (ACC2) does not cause cardiomyopathy in non-obese mice, suggesting that high FAO is distinct from cardiac lipotoxicity. We hypothesize that cardiac pathology associated obesity is attributable to the imbalance of fatty acid supply and oxidation. Thus, we here seek to determine whether further increasing FAO by inducing ACC2 deletion prevents obesity induced cardiomyopathy, and if so, to elucidate the underlying mechanisms. Methods: We induced high FAO in adult mouse hearts by cardiac-specific deletion of ACC2 using a tamoxifen inducible model (ACC2 iKO). Control (Con) and ACC2 iKO mice were subjected to high fat diet (HFD) feeding for 24 weeks to induce obesity. Cardiac function, mitochondria function and mitophagy activity were examined. Results: Despite both Con and ACC2 iKO mice exhibiting similar obese phenotype, increasing FAO oxidation by deletion of ACC2 prevented HFD induced cardiac dysfunction, pathological remodeling as well as mitochondria dysfunction. Similarly, increasing FAO by knock down of ACC2 prevented palmitate induced mitochondria dysfunction and cardiomyocyte death in vitro. Furthermore, HFD suppressed mitophagy activity and caused damaged mitochondria to accumulate in the heart, which was partially attenuated in ACC2 iKO heart. Mechanistically, ACC2 iKO prevented HFD induced downregulation of parkin. During stimulation for mitophagy, mitochondria localized parkin was severely reduced in Con HFD-fed mouse heart, which was partially restored in ACC2 iKO HFD-fed mice. Conclusions: These data show that increasing cardiac FAO alone does not cause cardiac dysfunction but protect against cardiomyopathy in chronically obese mice. The beneficial effect of enhancing cardiac FAO in HFD induced obesity is mediated, in part, by maintenance of mitochondria function through regulating parkin mediated mitophagy. Our findings also suggest that targeting the parkin dependent mitophagy pathway could be an effective strategy against the development of obesity induced cardiomyopathy.

Leigh syndrome is a mitochondrial disease characterized by neurological disorders, metabolic abnormality and premature death. There is no cure for Leigh syndrome; therefore, new therapeutic targets are urgently needed. In Ndufs4-KO mice, a mouse model of Leigh syndrome, we found that Complex I deficiency led to declines in NAD + levels and NAD + redox imbalance. We tested the hypothesis that elevation of NAD + levels would benefit Ndufs4-KO mice. Administration of NAD + precursor, nicotinamide mononucleotide (NMN) extended lifespan of Ndufs4-KO mice and attenuated lactic acidosis. NMN increased lifespan by normalizing NAD + redox imbalance and lowering HIF1a accumulation in Ndufs4-KO skeletal muscle without affecting the brain. NMN up-regulated alpha-ketoglutarate (KG) levels in Ndufs4-KO muscle, a metabolite essential for HIF1a degradation. To test whether supplementation of KG can treat Ndufs4-KO mice, a cell-permeable KG, dimethyl ketoglutarate (DMKG) was administered. DMKG extended lifespan of Ndufs4-KO mice and delayed onset of neurological phenotype. This study identified therapeutic mechanisms that can be targeted pharmacologically to treat Leigh syndrome.

In hypertrophied and failing hearts, fuel metabolism is reprogrammed to increase glucose metabolism, especially glycolysis. This metabolic shift favors biosynthetic function at the expense of ATP production. Mechanisms responsible for the switch are poorly understood. We found that inhibitory factor 1 of the mitochondrial F o F 1 -ATP synthase (ATPIF1), a protein known to inhibit ATP hydrolysis by the reverse function of ATP synthase during ischemia, was significantly upregulated in pathological cardiac hypertrophy induced by pressure overload, myocardial infarction, or α -adrenergic stimulation. Chemical cross-linking mass spectrometry analysis of hearts hypertrophied by pressure overload suggested that increased expression of ATPIF1 promoted the formation of F o F 1 -ATP synthase nonproductive tetramer. Using ATPIF1 gain- and loss-of-function cell models, we demonstrated that stalled electron flow due to impaired ATP synthase activity triggered mitochondrial ROS generation, which stabilized HIF1 α , leading to transcriptional activation of glycolysis. Cardiac-specific deletion of ATPIF1 in mice prevented the metabolic switch and protected against the pathological remodeling during chronic stress. These results uncover a function of ATPIF1 in nonischemic hearts, which gives F o F 1 -ATP synthase a critical role in metabolic rewiring during the pathological remodeling of the heart.

Background: Reduced fatty acid oxidation (FAO) is a hallmark of metabolic remodeling in heart failure. Enhancing mitochondrial long-chain fatty acid uptake by Acetyl-CoA carboxylase 2 (ACC2) deletion increases FAO and prevents cardiac dysfunction during chronic stresses, but therapeutic efficacy of this approach has not been determined. Methods: Male and female ACC2 f/f-MCM (ACC2KO) and their respective littermate controls were subjected to chronic pressure overload by TAC surgery. Tamoxifen injection 3 weeks after TAC induced ACC2 deletion and increased FAO in ACC2KO mice with pathological hypertrophy. Results: ACC2 deletion in mice with pre-existing cardiac pathology promoted FAO in female and male hearts, but improved cardiac function only in female mice. In males, pressure overload caused a downregulation in the mitochondrial oxidative function. Stimulating FAO by ACC2 deletion caused unproductive acyl-carnitine accumulation, which failed to improve cardiac energetics. In contrast, mitochondrial oxidative capacity was sustained in female pressure overloaded hearts and ACC2 deletion improved myocardial energetics. Mechanistically, we revealed a sex-dependent regulation of PPARα signaling pathway in heart failure, which accounted for the differential response to ACC2 deletion. Conclusion: Metabolic remodeling in the failing heart is sex-dependent which could determine the response to metabolic intervention. The findings suggest that both mitochondrial oxidative capacity and substrate preference should be considered for metabolic therapy of heart failure.

Advancements of cross-linking mass spectrometry (XL-MS) for structural analysis of proteins bridges the gap between purified systems and native tissue environments. Here, isobaric quantitative protein interaction reporter technology (iqPIR) was utilized to further extend XL-MS to the first system-wide comparative study of mitochondrial proteins from healthy and diseased murine hearts. The failing heart interactome includes 602 statistically significant cross-linked peptide pairs altered in the disease condition. Structural insight into ketone oxidation metabolons, OXPHOS machinery, and nucleotide transporter hybrid-conformations, support mitochondrial remodeling in failing heart while bringing forth new hypotheses for pathological mechanisms. Application of quantitative cross-linking technology in tissue provides molecularlevel insight to complex biological systems difficult to model in cell culture, thus providing a valuable resource for study of human diseases.