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The outer membrane of gram-negative bacteria is a barrier to chemical and physical stress. Phospholipid transport between the inner and outer membranes has been an area of intense investigation and, in E. coli K-12, it has recently been shown to be mediated by YhdP, TamB, and YdbH, which are suggested to provide hydrophobic channels for phospholipid diffusion, with YhdP and TamB playing the major roles. However, YhdP and TamB have different phenotypes suggesting distinct functions. It remains unclear whether these functions are related to phospholipid metabolism. We investigated a synthetic cold sensitivity caused by deletion of fadR, a transcriptional regulator controlling fatty acid degradation and unsaturated fatty acid production, and yhdP, but not by ΔtamB ΔfadR or ΔydbH ΔfadR. Deletion of tamB recuses the ΔyhdP ΔfadR cold sensitivity further demonstrating the phenotype is related to functional diversification between these genes. The ΔyhdP ΔfadR strain shows a greater increase in cardiolipin upon transfer to the non-permissive temperature and genetically lowering cardiolipin levels can suppress cold sensitivity. These data also reveal a qualitative difference between cardiolipin synthases in E. coli, as deletion of clsA and clsC suppresses cold sensitivity but deletion of clsB does not. Moreover, increased fatty acid saturation is necessary for cold sensitivity and lowering this level genetically or through supplementation of oleic acid suppresses the cold sensitivity of the ΔyhdP ΔfadR strain. Together, our data clearly demonstrate that the diversification of function between YhdP and TamB is related to phospholipid metabolism. Although indirect regulatory effects are possible, we favor the parsimonious hypothesis that YhdP and TamB have differential phospholipid-substrate transport preferences. Thus, our data provide a potential mechanism for independent control of the phospholipid composition of the inner and outer membranes in response to changing conditions based on regulation of abundance or activity of YhdP and TamB., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Rai et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Acute kidney injury (AKI) is a systemic disease that affects energy metabolism in various remote organs in murine models of ischemic AKI. However, AKI-mediated effects in the liver have not been comprehensively assessed. After inducing ischemic AKI in 8-10-week-old, male C57BL/6 mice, mass spectrometry metabolomics revealed that the liver had the most distinct phenotype 24 h after AKI versus 4 h and 7 days. Follow up studies with in vivo [ 13 C 6 ]-glucose tracing on liver and kidney 24 h after AKI revealed 4 major findings: (1) increased flux through glycolysis and the tricarboxylic (TCA) cycle in both kidney and liver; (2) depleted hepatic glutathione levels and its intermediates despite unchanged level of reactive oxygen species, suggesting glutathione consumption exceeds production due to systemic oxidative stress after AKI; (3) hepatic ATP depletion despite unchanged rate of mitochondrial respiration, suggesting increased ATP consumption relative to production; (4) increased hepatic and renal urea cycle intermediates suggesting hypercatabolism and upregulation of the urea cycle independent of impaired renal clearance of nitrogenous waste. Taken together, this is the first study to describe the hepatic metabolome after ischemic AKI in a murine model and demonstrates that there is significant liver-kidney crosstalk after AKI., (© 2024. The Author(s).)

Background: Hepatic mitochondrial dysfunction is a major cause of fat accumulation in the liver. Individuals with fatty liver conditions have hepatic mitochondrial structural abnormalities and a switch in the side chain composition of the mitochondrial phospholipid, cardiolipin, from poly- to monounsaturated fatty acids. Linoleic acid (LA), an essential dietary fatty acid, is required to remodel nascent cardiolipin (CL) to its tetralinoleoyl cardiolipin (L 4 CL, CL with 4 LA side chains) form, which is integral for mitochondrial membrane structure and function to promote fatty acid oxidation. It is unknown, however, whether increasing LA in the diet can increase hepatic L 4 CL concentrations and improve mitochondrial respiration in the liver compared with a diet rich in monounsaturated and saturated fatty acids., Objectives: The main aim of this study was to test the ability of a diet fortified with LA-rich safflower oil (SO), compared with the one fortified with lard (LD), to increase concentrations of L 4 CL and improve mitochondrial respiration in the livers of mice., Methods: Twenty-four (9-wk-old) C57 BL/J6 male mice were fed either the SO or LD diets for ∼100 d, whereas food intake and body weight, fasting glucose, and glucose tolerance tests were performed to determine any changes in glycemic control., Results: Livers from mice fed SO diet had higher relative concentrations of hepatic L 4 CL species compared with LD diet-fed mice (P value = 0.004). Uncoupled mitochondria of mice fed the SO diet, compared with LD diet, had an increased baseline oxygen consumption rate (OCR) and succinate-driven respiration (P values = 0.03 and 0.01). SO diet-fed mice had increased LA content in all phospholipid classes compared with LD-fed mice (P < 0.05)., Conclusions: Our findings reveal that maintaining or increasing hepatic L 4 CL may result in increased OCR in uncoupled hepatic mitochondria in healthy mice whereas higher oleate content of CL reduced mitochondrial function shown by lower OCR in uncoupled mitochondria., (Copyright © 2023. Published by Elsevier Inc.)

Aim: Barth syndrome (BTHS) is a rare X-linked genetic disease in which mitochondrial oxidative phosphorylation is impaired due to a mutation in the TAFAZZIN gene. The protein kinase C delta (PKCδ) signalosome exists as a high molecular weight complex in mitochondria and controls mitochondrial oxidative phosphorylation., Method: Here, we examined PKCδ levels in mitochondria of aged-matched control and BTHS patient B lymphoblasts and its association with a higher molecular weight complex in mitochondria., Result: Immunoblot analysis of blue-native polyacrylamide gel electrophoresis mitochondrial fractions revealed an increase in total PKCδ protein expression in BTHS lymphoblasts compared to controls. In contrast, PKCδ associated with a higher molecular weight complex was markedly reduced in BTHS patient B lymphoblasts compared to controls. Given the decrease in PKCδ associated with a higher molecular weight complex in mitochondria, we examined the uptake of creatine, a compound whose utilization is enhanced upon high energy demand. Creatine uptake was markedly elevated in BTHS lymphoblasts compared to controls., Conclusion: We hypothesize that reduced PKCδ within this higher molecular weight complex in mitochondria may contribute to the bioenergetic defects observed in BTHS lymphoblasts and that enhanced creatine uptake may serve as one of several compensatory mechanisms for the defective mitochondrial oxidative phosphorylation observed in these cells., Competing Interests: Conflicts of interest All authors declared that there are no conflicts of interest.

Cardiolipin is a hallmark phospholipid of mitochondrial membranes. Despite established significance of cardiolipin in supporting respiratory supercomplex organization, a mechanistic understanding of this lipid-protein interaction is still lacking. To address the essential role of cardiolipin in supercomplex organization, we report cryo-EM structures of a wild type supercomplex (IV 1 III 2 IV 1 ) and a supercomplex (III 2 IV 1 ) isolated from a cardiolipin-lacking Saccharomyces cerevisiae mutant at 3.2-Å and 3.3-Å resolution, respectively, and demonstrate that phosphatidylglycerol in III 2 IV 1 occupies similar positions as cardiolipin in IV 1 III 2 IV 1 . Lipid-protein interactions within these complexes differ, which conceivably underlies the reduced level of IV 1 III 2 IV 1 and high levels of III 2 IV 1 and free III 2 and IV in mutant mitochondria. Here we show that anionic phospholipids interact with positive amino acids and appear to nucleate a phospholipid domain at the interface between the individual complexes, which dampen charge repulsion and further stabilize interaction, respectively, between individual complexes., (© 2023. The Author(s).)

The mechanisms responsible for heart failure in single-ventricle congenital heart disease are unknown. Using explanted heart tissue, we showed that failing single-ventricle hearts have dysregulated metabolic pathways, impaired mitochondrial function, decreased activity of carnitine palmitoyltransferase activity, and altered functioning of the tricarboxylic acid cycle. Interestingly, nonfailing single-ventricle hearts demonstrated an intermediate metabolic phenotype suggesting that they are vulnerable to development of heart failure in the future. Mitochondrial targeted therapies and treatments aimed at normalizing energy generation could represent a novel approach to the treatment or prevention of heart failure in this vulnerable group of patients., Competing Interests: This work was supported by the National Institutes of Health National Heart Lung and Blood Institute (R01 HL126928[SDM, CCS, BLS], R01 HL126928-03S1[AMG]), the National Center for Advancing Translational Sciences Colorado Clinical and Translational Sciences Institute awards (UL1-TR002535, KL2-TR002534, and TL1-TR002533), the Addison Scott Memorial Fund, the Boedecker Foundation, the Nair Family, the Rose Community Foundation, and the Jack Cooper Millisor Chair in Pediatric Heart Disease. Dr. Sucharov has served as a scientific founder at miRagen, Inc. All other authors have reported that they have no relationships relevant to the content of this paper to disclose., (© 2023 The Authors.)

Cardiolipin (CL), the major mitochondrial phospholipid, regulates the activity of many mitochondrial membrane proteins. CL composition is shifted in heart failure with decreases in linoleate and increases in oleate side chains, but whether cardiolipin composition directly regulates metabolism is unknown. This study defines cardiolipin composition in rat heart and liver at three distinct ages to determine the influence of CL composition on beta-oxidation (ß-OX). CL species, expression of ß-OX and glycolytic genes, and carnitine palmitoyltransferase (CPT) activity were characterized in heart and liver from neonatal, juvenile, and adult rats. Ventricular myocytes were cultured from neonatal, juvenile, and adult rats and cardiolipin composition and CPT activity were measured. Cardiolipin composition in neonatal rat ventricular cardiomyocytes (NRVMs) was experimentally altered and mitochondrial respiration was assessed. Linoleate-enrichment of CL was observed in rat heart, but not liver, with increasing age. ß-OX genes and CPT activity were generally higher in adult heart and glycolytic genes lower, as a function of age, in contrast to liver. Palmitate oxidation increased in NRVMs when CL was enriched with linoleate. Our results indicate (1) CL is developmentally regulated, (2) linoleate-enrichment is associated with increased ß-OX and a more oxidative mitochondrial phenotype, and (3) experimentally induced linoleate-enriched CL in ventricular myocytes promotes a shift from pyruvate metabolism to fatty acid ß-OX.

Cardiolipin (CL) is a phospholipid unique to the inner mitochondrial membrane that supports respiratory chain structure and function and is demonstrated to be influenced by types of dietary fats. However, the influence of dietary fat on CL species and how this best supports mitochondrial function in brown adipose tissue (BAT), which exhibits an alternative method of energy utilization through the uncoupling of the mitochondrial proton gradient to generate heat, is not well understood. Therefore, the aim of our study was to evaluate metabolic parameters, interscapular BAT CL quantity, species, and mitochondrial function in mice consuming isocaloric moderate-fat diets with either lard (LD; similar fatty acid profile to western dietary patterns) or safflower oil high in linoleic acid (SO), shown to be metabolically favorable in large clinical meta-analyses. Mice fed the SO diet exhibited decreased adiposity, improved insulin sensitivity, and enrichment of LA-containing CL species in BAT CL. Furthermore, mice fed the SO diet exhibit higher levels of OXPHOS complex proteins and increased oxygen consumption in BAT. Our findings demonstrate that dietary consumption of LA-rich oil improves metabolic parameters, increases LA-containing CL species, and improves BAT function when compared to the consumption of lard in mice during diet-induced weight gain.

Barth syndrome (BTHS) is a rare X-linked genetic disease caused by mutations in TAFAZZIN. The tafazzin (Taz) protein is a cardiolipin remodeling enzyme required for maintaining mitochondrial function. Patients with BTHS exhibit impaired mitochondrial respiratory chain and metabolic function and are susceptible to serious infections. B lymphocytes (B cells) play a vital role in humoral immunity required to eradicate circulating antigens from pathogens. Intact mitochondrial respiration is required for proper B-cell function. We investigated whether Taz deficiency in mouse B cells altered their response to activation by anti-cluster of differentiation 40 (anti-CD40) + interleukin-4 (IL-4). B cells were isolated from 3-4-month-old wild type (WT) or tafazzin knockdown (TazKD) mice and were stimulated with anti-CD40 + IL-4 for 24 h and cellular bioenergetics, surface marker expression, proliferation, antibody production, and proteasome and immunoproteasome activities determined. TazKD B cells exhibited reduced mRNA expression of Taz, lowered levels of cardiolipin, and impairment in both oxidative phosphorylation and glycolysis compared to WT B cells. In addition, anti-CD40 + IL-4 stimulated TazKD B cells expressed lower levels of the immunogenic surface markers, cluster of differentiation 86 (CD86) and cluster of differentiation 69 (CD69), exhibited a lower proliferation rate, reduced production of immunoglobulin M and immunoglobulin G, and reduced proteasome and immunoproteasome proteolytic activities compared to WT B cells stimulated with anti-CD40 + IL-4. The results indicate that Taz is required to support T-cell-dependent signaling activation of mouse B cells., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Barth Syndrome (BTHS) is a rare X-linked genetic disorder caused by mutation in the TAFAZZIN gene. Tafazzin (Taz) deficiency in BTHS patients results in an increased risk of infections. Mesenchymal stem cells (MSCs) are well known for their immune-inhibitory function. We examined how Taz-deficiency in murine MSCs impact their ability to modulate the function of lipopolysaccharide (LPS)-activated wild type (WT) B lymphocytes. MSCs from tafazzin knockdown (TazKD) mice exhibited a reduction in mitochondrial cardiolipin compared to wild type (WT) MSCs. However, mitochondrial bioenergetics and membrane potential were unaltered. In contrast, TazKD MSCs exhibited increased reactive oxygen species generation and increased glycolysis. The increased glycolysis was associated with an elevated proliferation, phosphatidylinositol-3-kinase expression and expression of the immunosuppressive markers indoleamine-2,3-dioxygenase, cytotoxic T-lymphocyte-associated protein 4, interleukin-10, and cluster of differentiation 59 compared to controls. Inhibition of glycolysis with 2-deoxyglucose attenuated the TazKD-mediated increased expression of cytotoxic T-lymphocyte-associated protein 4 and interleukin-10. When co-cultured with LPS-activated WT B cells, TazKD MSCs inhibited B cell proliferation and growth rate and reduced B cell secretion of immunoglobulin M compared to controls. In addition, co-culture of LPS-activated WT B cells with TazKD MSCs promoted B cell differentiation toward interleukin-10 secreting plasma cells and B regulatory cells compared to controls. The results indicate that Taz deficiency in MSCs promote reprogramming of activated B lymphocytes toward immunosuppressive phenotypes., (© 2022 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Scope: Higher circulating linoleic acid (LA) and muscle-derived tetralinoleoyl-cardiolipin (LA 4 CL) are each associated with decreased cardiometabolic disease risk. Mitochondrial dysfunction occurs with low LA 4 CL. Whether LA-rich oil fortification can increase LA 4 CL in humans is unknown. The aims of this study are to determine whether dietary fortification with LA-rich oil for 2 weeks increases: 1) LA in plasma, erythrocytes, and peripheral blood mononuclear cells (PBMC); and 2) LA 4 CL in PBMC in adults., Methods and Results: In this randomized controlled trial, adults are instructed to consume one cookie per day delivering 10 g grapeseed (LA-cookie, N = 42) or high oleate (OA) safflower (OA-cookie, N = 42) oil. In the LA-cookie group, LA increases in plasma, erythrocyte, and PBMC by 6%, 7%, and 10% respectively. PBMC and erythrocyte OA increase by 7% and 4% in the OA-cookie group but is unchanged in the plasma. PBMC LA 4 CL increases (5%) while LA 3 OA 1 CL decreases (7%) in the LA-cookie group but are unaltered in the OA-cookie group., Conclusions: LA-rich oil fortification increases while OA-oil has no effect on LA 4 CL in adults. Because LA-rich oil fortification reduces cardiometabolic disease risk and increases LA 4 CL, determining whether mitochondrial dysfunction is repaired through dietary fortification is warranted., (© 2022 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH GmbH.)

Extracellular superoxide dismutase (EC-SOD) is highly expressed in the lung and vasculature. A common human single nucleotide polymorphism (SNP) in the matrix binding region of EC-SOD leads to a single amino acid substitution, R213G, and alters EC-SOD tissue binding affinity. The change in tissue binding affinity redistributes EC-SOD from tissue to extracellular fluids. Mice (R213G mice) expressing a knock-in of this EC-SOD SNP exhibit elevated plasma and reduced lung EC-SOD content and activity and are protected against bleomycin-induced lung injury and inflammation. It is unknown how the redistribution of EC-SOD alters site-specific redox-regulated molecules relevant for protection. In this study, we tested the hypothesis that the change in the local EC-SOD content would influence not only the extracellular redox microenvironment where EC-SOD is localized but also protect the intracellular redox status of the lung. Mice were treated with bleomycin and harvested 7 days post-treatment. Superoxide levels, measured by electron paramagnetic resonance (EPR), were lower in plasma and Bronchoalveolar lavage fluid (BALF) cells in R213G mice compared to wild-type (WT) mice, while lung cellular superoxide levels in R213G mice were not elevated post-bleomycin compared to WT mice despite low lung EC-SOD levels. Lung glutathione redox potential (E h GSSG), determined by HPLC and fluorescence, was more oxidized in WT compared to R213G mice. In R213G mice, lung mitochondrial oxidative stress was reduced shown by mitochondrial superoxide level measured by EPR in lung and the resistance to bleomycin-induced cardiolipin oxidation. Bleomycin treatment suppressed mitochondrial respiration in WT mice. Mitochondrial function was impaired at baseline in R213G mice but did not exhibit further suppression in respiration post-bleomycin. Collectively, the results indicate that R213G variant preserves intracellular redox state and protects mitochondrial function in the setting of bleomycin-induced inflammation.

Introduction: Lactate is an important signaling molecule with autocrine, paracrine and endocrine properties involved in multiple biological processes including regulation of gene expression and metabolism. Levels of lactate are increased chronically in diseases associated with cardiometabolic disease such as heart failure, type 2 diabetes, and cancer. Using neonatal ventricular myocytes, we tested the hypothesis that chronic lactate exposure could decrease the activity of cardiac mitochondria that could lead to metabolic inflexibility in the heart and other tissues., Methods: Neonatal rat ventricular myocytes (NRVMs) were treated for 48 h with 5, 10, or 20 mM lactate and CPT I and II activities were tested using radiolabelled assays. The molecular species profile of the major mitochondrial phospholipid, cardiolipin, was determined using electrospray ionization mass spectrometry along with reactive oxygen species (ROS) levels measured by Amplex Red and mitochondrial oxygen consumption using the Seahorse analyzer., Results: CPT I activity trended downward ( p = 0.07) and CPT II activity significantly decreased with lactate exposure ( p < 0.001). Cardiolipin molecular species containing four 18 carbon chains (72 carbons total) increased with lactate exposure, but species of other sizes decreased significantly. Furthermore, ROS production was strongly enhanced with lactate ( p < 0.001) and mitochondrial ATP production and maximal respiration were both significantly down regulated with lactate exposure ( p < 0.05 and p < 0.01 respectively)., Conclusions: Chronic lactate exposure in cardiomyocytes leads to a decrease in fatty acid transport, alterations of cardiolipin remodeling, increases in ROS production and decreases in mitochondrial oxygen consumption that could have implications for both metabolic health and flexibility. The possibility that both intra-, or extracellular lactate levels play roles in cardiometabolic disease, heart failure, and other forms of metabolic inflexibility needs to be assessed in vivo ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 San-Millan, Sparagna, Chapman, Warkins, Chatfield, Shuff, Martinez and Brooks.)

Pulmonary vascular remodeling (PVR) in utero results in the development of heart failure. The alterations that occur in cardiac lipid and mitochondrial bioenergetics during the development of in utero PVR was unknown. In this study, PVR was induced in pups in utero by exposure of pregnant dams to indomethacin and hypoxia and cardiac lipids, echocardiographic function and cardiomyocyte mitochondrial function were subsequently examined. Perinatal rat pups with PVR exhibited elevated left and right cardiac ventricular internal dimensions and reduced ejection fraction and fractional shortening compared to controls. Cardiac myocytes from these pups exhibited increased glycolytic capacity and glycolytic reserve compared to controls. However, respiration with glucose as substrate was unaltered. Fatty acid oxidation and ATP-insensitive respiration were increased in isolated cardiac myocytes from these pups compared to controls indicating a mitochondrial dysfunction. Although abundance of mitochondrial respiratory chain complexes was unaltered, increased trilinoleoyl-lysocardiolipin levels in these pups was observed. A compensatory increase in both cardiolipin and phosphatidylethanolamine content were observed due to increased synthesis of these phospholipids. These data indicate that alterations in cardiac cardiolipin and phospholipid metabolism in PVR rat pups is associated with the mitochondrial bioenergetic and cardiac functional defects observed in their hearts., Competing Interests: The authors have declared that no competing interests exist.

Barth syndrome (BTHS) is an X-linked disorder that results from mutations in the TAFAZZIN gene, which encodes a phospholipid transacylase responsible for generating the mature form of cardiolipin in inner mitochondrial membranes. BTHS patients develop early onset cardiomyopathy and a derangement of intermediary metabolism consistent with mitochondrial disease, but the precise alterations in cardiac metabolism that distinguish BTHS from idiopathic forms of cardiomyopathy are unknown. We performed the first metabolic analysis of myocardial tissue from BTHS cardiomyopathy patients compared to age- and sex-matched patients with idiopathic dilated cardiomyopathy (DCM) and nonfailing controls. Results corroborate previous evidence for deficiencies in cardiolipin content and its linoleoyl enrichment as defining features of BTHS cardiomyopathy, and reveal a dramatic accumulation of hydrolyzed (monolyso-) cardiolipin molecular species. Respiratory chain protein deficiencies were observed in both BTHS and DCM, but a selective depletion of complex I was seen only in BTHS after controlling for an apparent loss of mitochondrial density in cardiomyopathic hearts. Distinct shifts in the expression of long-chain fatty acid oxidation enzymes and the tissue acyl-CoA profile of BTHS hearts suggest a specific block in mitochondrial fatty acid oxidation upstream of the conventional matrix beta-oxidation cycle, which may be compensated for by a greater reliance upon peroxisomal fatty acid oxidation and the catabolism of ketones, amino acids, and pyruvate to meet cardiac energy demands. These results provide a comprehensive foundation for exploring novel therapeutic strategies that target the adaptive and maladaptive metabolic features of BTHS cardiomyopathy., (© 2021 SSIEM.)

Berberine (BBR) is an isoquinoline alkaloid from plants known to improve cardiac mitochondrial function in gestational diabetes mellitus (GDM) offspring but the mechanism is poorly understood. We examined the role of the mitochondrial phospholipid cardiolipin (CL) in mediating this cardiac improvement. C57BL/6 female mice were fed either a Lean-inducing low-fat diet or a GDM-inducing high-fat diet for 6 weeks prior to breeding. Lean and GDM-exposed male offspring were randomly assigned a low-fat, high-fat, or high-fat diet containing BBR at weaning for 12 weeks. The content of CL was elevated in the heart of GDM offspring fed a high fat diet containing BBR. The increase in total cardiac CL was due to significant increases in the most abundant and functionally important CL species, tetralinoleoyl-CL and this correlated with an increase in the expression of the CL remodeling enzyme tafazzin. Additionally, BBR treatment increased expression of cardiac enzymes involved in fatty acid uptake and oxidation and electron transport chain subunits in high fat diet fed GDM offspring. Thus, dietary BBR protection from cardiac dysfunction in GDM exposed offspring involves improvement in mitochondrial function mediated through increased synthesis of CL., (© 2021. The Author(s).)

Tafazzin (TAZ) is a cardiolipin (CL) biosynthetic enzyme important for maintaining mitochondrial function. TAZ affects both the species and content of CL in the inner mitochondrial membrane, which are essential for normal cellular respiration. In pancreatic β cells, mitochondrial function is closely associated with insulin secretion. However, the role of TAZ and CL in the secretion of insulin from pancreatic islets remains unknown. Male 4-month-old doxycycline-inducible TAZ knock-down (KD) mice and wild-type littermate controls were used. Immunohistochemistry was used to assess β-cell morphology in whole pancreas sections, whereas ex vivo insulin secretion, CL content, RNA-sequencing analysis, and mitochondrial oxygen consumption were measured from isolated islet preparations. Ex vivo insulin secretion under nonstimulatory low-glucose concentrations was reduced ~52% from islets isolated from TAZ KD mice. Mitochondrial oxygen consumption under low-glucose conditions was also reduced ~58% in islets from TAZ KD animals. TAZ deficiency in pancreatic islets was associated with significant alteration in CL molecular species and elevated polyunsaturated fatty acid CL content. In addition, RNA-sequencing of isolated islets showed that TAZ KD increased expression of extracellular matrix genes, which are linked to pancreatic fibrosis, activated stellate cells, and impaired β-cell function. These data indicate a novel role for TAZ in regulating pancreatic islet function, particularly under low-glucose conditions., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: There are few evidence-based strategies to attenuate the risk of metabolic syndrome in offspring exposed to gestational diabetes mellitus (GDM). Berberine (BBR) is an isoquinoline alkaloid extracted from Chinese herbs and exhibits glucose lowering properties., Objectives: We hypothesized that dietary BBR would improve health outcomes in the mouse offspring of GDM dams., Methods: Wild-type C57BL/6 female mice were fed either a Lean-inducing low-fat diet (L-LF,10% kcal fat, 35% kcal sucrose) or a GDM-inducing high-fat diet (GDM-HF, 45% kcal fat, 17.5% sucrose) for 6 wk prior to breeding with wild-type C57BL/6 male mice throughout pregnancy and the suckling period. The resulting Lean and GDM-exposed male and female offspring were randomly assigned an LF (10% kcal fat, 35% kcal sucrose), HF (45% kcal fat, 17.5% sucrose), or high-fat berberine (HFB) (45% kcal fat, 17.5% sucrose diet) containing BBR (160 mg/kg/d, HFB) at weaning for 12 wk. The main outcome was to evaluate the effects of BBR on obesity, pancreatic islet function, and cardiac contractility in GDM-exposed HF-fed offspring. Significance between measurements was determined using a 2 (gestational exposure) × 3 (diet) factorial design by a 2- way ANOVA using Tukey post-hoc analysis., Results: In the GDM-HF group, body weights were significantly increased (16%) compared with those in baseline (L-LF) animals (P < 0.05). Compared with the L-LF animals, the GDM-HF group had a reduction in pancreatic insulin glucose-stimulated insulin secretion (74%) and increased cardiac isovolumetric contraction time (IVCT; ∼150%) (P < 0.05). Compared with GDM-HF animals, the GDM-HFB group with the dietary addition of BBR had significantly reduced body weight (16%), increased glucose-stimulated insulin secretion from pancreatic islets (254%), and reduced systolic heart function (46% IVCT) (P < 0.05)., Conclusions: In a mouse model of GDM, dietary BBR treatment provided protection from obesity and the development of pancreatic islet and cardiac dysfunction., (© The Author(s) 2021. Published by Oxford University Press on behalf of the American Society for Nutrition.)

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a powerful platform for biomedical research. However, they are immature, which is a barrier to modeling adult-onset cardiovascular disease. Here, we sought to develop a simple method that could drive cultured hiPSC-CMs toward maturity across a number of phenotypes, with the aim of utilizing mature hiPSC-CMs to model human cardiovascular disease. hiPSC-CMs were cultured in fatty acid-based medium and plated on micropatterned surfaces. These cells display many characteristics of adult human cardiomyocytes, including elongated cell morphology, sarcomeric maturity, and increased myofibril contractile force. In addition, mature hiPSC-CMs develop pathological hypertrophy, with associated myofibril relaxation defects, in response to either a pro-hypertrophic agent or genetic mutations. The more mature hiPSC-CMs produced by these methods could serve as a useful in vitro platform for characterizing cardiovascular disease., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Barth syndrome is a mitochondrial myopathy resulting from mutations in the tafazzin ( TAZ ) gene encoding a phospholipid transacylase required for cardiolipin remodeling. Cardiolipin is a phospholipid of the inner mitochondrial membrane essential for the function of numerous mitochondrial proteins and processes. However, it is unclear how tafazzin deficiency impacts cardiac mitochondrial metabolism. To address this question while avoiding confounding effects of cardiomyopathy on mitochondrial phenotype, we utilized Taz -shRNA knockdown ( Taz KD ) mice, which exhibit defective cardiolipin remodeling and respiratory supercomplex instability characteristic of human Barth syndrome but normal cardiac function into adulthood. Consistent with previous reports from other models, mitochondrial H 2 O 2 emission and oxidative damage were greater in Taz KD than in wild-type (WT) hearts, but there were no differences in oxidative phosphorylation coupling efficiency or membrane potential. Fatty acid and pyruvate oxidation capacities were 40-60% lower in Taz KD mitochondria, but an up-regulation of glutamate oxidation supported respiration rates approximating those with pyruvate and palmitoylcarnitine in WT. Deficiencies in mitochondrial CoA and shifts in the cardiac acyl-CoA profile paralleled changes in fatty acid oxidation enzymes and acyl-CoA thioesterases, suggesting limitations of CoA availability or "trapping" in Taz KD mitochondrial metabolism. Incubation of Taz KD mitochondria with exogenous CoA partially rescued pyruvate and palmitoylcarnitine oxidation capacities, implicating dysregulation of CoA-dependent intermediary metabolism rather than respiratory chain defects in the bioenergetic impacts of tafazzin deficiency. These findings support links among cardiolipin abnormalities, respiratory supercomplex instability, and mitochondrial oxidant production and shed new light on the distinct metabolic consequences of tafazzin deficiency in the mammalian heart., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Le et al.)

Cardiolipin (CL) is a unique tetra-acyl phospholipid localized to the inner mitochondrial membrane and essential for normal respiratory function. It has been previously reported that the failing human heart and several rodent models of cardiac pathology have a selective loss of CL. A rare genetic disease, Barth syndrome (BTHS), is similarly characterized by a cardiomyopathy due to reduced levels of cardiolipin. A mouse model of cardiolipin deficiency was recently developed by knocking-down the cardiolipin biosynthetic enzyme tafazzin (TAZ KD). These mice develop an age-dependent cardiomyopathy due to mitochondrial dysfunction. Since reduced mitochondrial capacity in the heart may promote the accumulation of lipids, we examined whether cardiolipin deficiency in the TAZ KD mice promotes the development of a lipotoxic cardiomyopathy. In addition, we investigated whether treatment with resveratrol, a small cardioprotective nutraceutical, attenuated the aberrant lipid accumulation and associated cardiomyopathy. Mice deficient in tafazzin and the wildtype littermate controls were fed a low-fat diet, or a high-fat diet with or without resveratrol for 16 weeks. In the absence of obesity, TAZ KD mice developed a hypertrophic cardiomyopathy characterized by reduced left-ventricle (LV) volume (~36%) and 30-50% increases in isovolumetric contraction (IVCT) and relaxation times (IVRT). The progression of cardiac hypertrophy with tafazzin-deficiency was associated with several underlying pathological processes including altered mitochondrial complex I mediated respiration, elevated oxidative damage (~50% increase in reactive oxygen species, ROS), the accumulation of triglyceride (~250%) as well as lipids associated with lipotoxicity (diacylglyceride ~70%, free-cholesterol ~44%, ceramide N:16-35%) compared to the low-fat fed controls. Treatment of TAZ KD mice with resveratrol maintained normal LV volumes and preserved systolic function of the heart. The beneficial effect of resveratrol on cardiac function was accompanied by a significant improvement in mitochondrial respiration, ROS production and oxidative damage to the myocardium. Resveratrol treatment also attenuated the development of cardiac steatosis in tafazzin-deficient mice through reduced de novo fatty acid synthesis. These results indicate for the first time that cardiolipin deficiency promotes the development of a hypertrophic lipotoxic cardiomyopathy. Furthermore, we determined that dietary resveratrol attenuates the cardiomyopathy by reducing ROS, cardiac steatosis and maintaining mitochondrial function., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Despite advances in both medical and surgical therapies, individuals with single ventricle heart disease (SV) remain at high risk for the development of heart failure (HF). However, the molecular mechanisms underlying remodeling and eventual HF in patients with SV are poorly characterized. Cardiolipin (CL), an inner mitochondrial membrane phospholipid, is critical for proper mitochondrial function, and abnormalities in CL content and composition are known in various cardiovascular disease etiologies. The purpose of this study was to investigate myocardial CL content and composition in failing and nonfailing single right ventricle (RV) samples compared with normal control RV samples, to assess mRNA expression of CL biosynthetic and remodeling enzymes, and to quantitate relative mitochondrial copy number. A cross-sectional analysis of RV myocardial tissue from 22 failing SV (SVHF), 9 nonfailing SV (SVNF), and 10 biventricular control samples (BVNF) was performed. Expression of enzymes involved in CL biosynthesis and remodeling were analyzed using RT-qPCR and relative mitochondrial DNA copy number determined by qPCR. Normal phase high-pressure liquid chromatography coupled to electrospray ionization mass spectrometry was used to quantitate total and specific CL species. While mitochondrial copy number was not significantly different between groups, total CL content was significantly lower in SVHF myocardium compared with BVNF controls. Despite having lower total CL content however, the relative percentage of the major tetralinoleoyl CL species is preserved in SVHF samples relative to BVNF controls. Correspondingly, expression of enzymes involved in CL biosynthesis and remodeling were upregulated in SVHF samples when compared with both SVNF samples and BVNF controls. NEW & NOTEWORTHY The mechanisms underlying heart failure in the single ventricle (SV) congenital heart disease population are largely unknown. In this study we identify alterations in cardiac cardiolipin metabolism, composition, and content in children with SV heart disease. These findings suggest that cardiolipin could be a novel therapeutic target in this unique population of patients.

Negative alterations of mitochondria are known to occur in heart failure (HF). This study investigated the novel mitochondrial-targeted therapeutic agent elamipretide on mitochondrial and supercomplex function in failing human hearts ex vivo. Freshly explanted failing and nonfailing ventricular tissue from children and adults was treated with elamipretide. Mitochondrial oxygen flux, complex (C) I and CIV activities, and in-gel activity of supercomplex assembly were measured. Mitochondrial function was impaired in the failing human heart, and mitochondrial oxygen flux, CI and CIV activities, and supercomplex-associated CIV activity significantly improved in response to elamipretide treatment. Elamipretide significantly improved failing human mitochondrial function.

The most severe manifestation of peripheral arterial disease (PAD) is critical limb ischemia (CLI). CLI patients suffer high rates of amputation and mortality; accordingly, there remains a clear need both to better understand CLI and to develop more effective treatments. Gastrocnemius muscle was obtained from 32 older (51-84 years) non-PAD controls, 27 claudicating PAD patients (ankle-brachial index [ABI] 0.65 ± 0.21 SD), and 19 CLI patients (ABI 0.35 ± 0.30 SD) for whole transcriptome sequencing and comprehensive mitochondrial phenotyping. Comparable permeabilized myofiber mitochondrial function was paralleled by both similar mitochondrial content and related mRNA expression profiles in non-PAD control and claudicating patient tissues. Tissues from CLI patients, despite being histologically intact and harboring equivalent mitochondrial content, presented a unique bioenergetic signature. This signature was defined by deficits in permeabilized myofiber mitochondrial function and a unique pattern of both nuclear and mitochondrial encoded gene suppression. Moreover, isolated muscle progenitor cells retained both mitochondrial functional deficits and gene suppression observed in the tissue. These findings indicate that muscle tissues from claudicating patients and non-PAD controls were similar in both their bioenergetics profile and mitochondrial phenotypes. In contrast, CLI patient limb skeletal muscles harbor a unique skeletal muscle mitochondriopathy that represents a potentially novel therapeutic site for intervention.

Life history strategies, physiological traits, and behavior are thought to covary along a "pace of life" axis, with organisms at the fast end of this continuum having higher fecundity, shorter lifespan, and more rapid development, growth, and metabolic rates. Countergradient variation represents a special case of pace of life variation, in which high-latitude organisms occupy the fast end of the continuum relative to low-latitude conspecifics when compared at a common temperature. Here, we use Atlantic killifish (Fundulus heteroclitus) to explore the role of mitochondrial properties as a mechanism underlying countergradient variation, and thus variation in the pace of life. This species is found along the Atlantic coast of North America, through a steep latitudinal thermal gradient. The northern subspecies has faster development, more rapid growth, higher routine metabolic rate, and higher activity than the southern subspecies when compared at a common temperature. The northern subspecies also has greater mitochondrial respiratory capacity in the liver, although these differences are not evident in other tissues. The increased respiratory capacity of liver mitochondria in northern fish is associated with increases in the activity of multiple electron transport complexes, which largely reflects an increase in the amount of inner mitochondrial membrane per mitochondrion in the northern fish. There are also differences in the lipid composition of liver mitochondrial membranes, including differences in cardiolipin species, which could also influence respiratory capacity. These data suggest that variation in mitochondrial properties could, at least in part, underlie variation in the pace of life in Atlantic killifish.

The effect of temperature on mitochondrial performance is thought to be partly due to its effect on mitochondrial membranes. Numerous studies have shown that thermal acclimation and adaptation can alter the amount of inner-mitochondrial membrane (IMM), but little is known about the capacity of organisms to modulate mitochondrial membrane composition. Using northern and southern subspecies of Atlantic killifish ( Fundulus heteroclitus ) that are locally adapted to different environmental temperatures, we assessed whether thermal acclimation altered liver mitochondrial respiratory capacity or the composition and amount of IMM. We measured changes in phospholipid headgroups and headgroup-specific fatty acid (FA) remodeling, and used respirometry to assess mitochondrial respiratory capacity. Acclimation to 5°C and 33°C altered mitochondrial respiratory capacity in both subspecies. Northern F. heteroclitus exhibited greater mitochondrial respiratory capacity across acclimation temperatures, consistent with previously observed subspecies differences in whole-organism aerobic metabolism. Mitochondrial phospholipids were altered following thermal acclimation, and the direction of these changes was largely consistent between subspecies. These effects were primarily driven by remodeling of specific phospholipid classes and were associated with shifts in metabolic phenotypes. There were also differences in membrane composition between subspecies that were driven largely by differences in phospholipid classes. Changes in respiratory capacity between subspecies and with acclimation were largely but not completely accounted for by alterations in the amount of IMM. Taken together, these results support a role for changes in liver mitochondrial function in the ectothermic response to thermal stress during both acclimation and adaptation, and implicate lipid remodeling as a mechanism contributing to these changes., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Cardiac mitochondrial phospholipid acyl chains regulate respiratory enzymatic activity. In several diseases, the rodent cardiac phospholipidome is extensively rearranged; however, whether specific acyl chains impair respiratory enzyme function is unknown. One unique remodeling event in the myocardium of obese and diabetic rodents is an increase in docosahexaenoic acid (DHA) levels. Here, we first confirmed that cardiac DHA levels are elevated in diabetic humans relative to controls. We then used dietary supplementation of a Western diet with DHA as a tool to promote cardiac acyl chain remodeling and to study its influence on respiratory enzyme function. DHA extensively remodeled the acyl chains of cardiolipin (CL), mono-lyso CL, phosphatidylcholine, and phosphatidylethanolamine. Moreover, DHA lowered enzyme activities of respiratory complexes I, IV, V, and I+III. Mechanistically, the reduction in enzymatic activities were not driven by a dramatic reduction in the abundance of supercomplexes. Instead, replacement of tetralinoleoyl-CL with tetradocosahexaenoyl-CL in biomimetic membranes prevented formation of phospholipid domains that regulate enzyme activity. Tetradocosahexaenoyl-CL inhibited domain organization due to favorable Gibbs free energy of phospholipid mixing. Furthermore, in vitro substitution of tetralinoleoyl-CL with tetradocosahexaenoyl-CL blocked complex-IV binding. Finally, reintroduction of linoleic acid, via fusion of phospholipid vesicles to mitochondria isolated from DHA-fed mice, rescued the major losses in the mitochondrial phospholipidome and complexes I, IV, and V activities. Altogether, our results show that replacing linoleic acid with DHA lowers select cardiac enzyme activities by potentially targeting domain organization and phospholipid-protein binding, which has implications for the ongoing debate about polyunsaturated fatty acids and cardiac health., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Rationale: Cardiac lipotoxicity, characterized by increased uptake, oxidation, and accumulation of lipid intermediates, contributes to cardiac dysfunction in obesity and diabetes mellitus. However, mechanisms linking lipid overload and mitochondrial dysfunction are incompletely understood., Objective: To elucidate the mechanisms for mitochondrial adaptations to lipid overload in postnatal hearts in vivo., Methods and Results: Using a transgenic mouse model of cardiac lipotoxicity overexpressing ACSL1 (long-chain acyl-CoA synthetase 1) in cardiomyocytes, we show that modestly increased myocardial fatty acid uptake leads to mitochondrial structural remodeling with significant reduction in minimum diameter. This is associated with increased palmitoyl-carnitine oxidation and increased reactive oxygen species (ROS) generation in isolated mitochondria. Mitochondrial morphological changes and elevated ROS generation are also observed in palmitate-treated neonatal rat ventricular cardiomyocytes. Palmitate exposure to neonatal rat ventricular cardiomyocytes initially activates mitochondrial respiration, coupled with increased mitochondrial polarization and ATP synthesis. However, long-term exposure to palmitate (>8 hours) enhances ROS generation, which is accompanied by loss of the mitochondrial reticulum and a pattern suggesting increased mitochondrial fission. Mechanistically, lipid-induced changes in mitochondrial redox status increased mitochondrial fission by increased ubiquitination of AKAP121 (A-kinase anchor protein 121) leading to reduced phosphorylation of DRP1 (dynamin-related protein 1) at Ser637 and altered proteolytic processing of OPA1 (optic atrophy 1). Scavenging mitochondrial ROS restored mitochondrial morphology in vivo and in vitro., Conclusions: Our results reveal a molecular mechanism by which lipid overload-induced mitochondrial ROS generation causes mitochondrial dysfunction by inducing post-translational modifications of mitochondrial proteins that regulate mitochondrial dynamics. These findings provide a novel mechanism for mitochondrial dysfunction in lipotoxic cardiomyopathy., (© 2017 American Heart Association, Inc.)

Cardiac phospholipids, notably cardiolipin, undergo acyl chain remodeling and/or loss of content in aging and cardiovascular diseases, which is postulated to mechanistically impair mitochondrial function. Less is known about how diet-induced obesity influences cardiac phospholipid acyl chain composition and thus mitochondrial responses. Here we first tested if a high fat diet remodeled murine cardiac mitochondrial phospholipid acyl chain composition and consequently disrupted membrane packing, supercomplex formation and respiratory enzyme activity. Mass spectrometry analyses revealed that mice consuming a high fat diet displayed 0.8-3.3 fold changes in cardiac acyl chain remodeling of cardiolipin, phosphatidylcholine, and phosphatidylethanolamine. Biophysical analysis of monolayers constructed from mitochondrial phospholipids of obese mice showed impairment in the packing properties of the membrane compared to lean mice. However, the high fat diet, relative to the lean controls, had no influence on cardiac mitochondrial supercomplex formation, respiratory enzyme activity, and even respiration. To determine if the effects were tissue specific, we subsequently conducted select studies with liver tissue. Compared to the control diet, the high fat diet remodeled liver mitochondrial phospholipid acyl chain composition by 0.6-5.3-fold with notable increases in n-6 and n-3 polyunsaturation. The remodeling in the liver was accompanied by diminished complex I to III respiratory enzyme activity by 3.5-fold. Finally, qRT-PCR analyses demonstrated an upregulation of liver mRNA levels of tafazzin, which contributes to cardiolipin remodeling. Altogether, these results demonstrate that diet-induced obesity remodels acyl chains in the mitochondrial phospholipidome and exerts tissue specific impairments of respiratory enzyme activity., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Mitochondria are the nexus of energy metabolism, and consequently their dysfunction has been implicated in the development of metabolic complications and progression to insulin resistance and type 2 diabetes. The unique tetra-acyl phospholipid cardiolipin (CL) is located in the inner mitochondrial membrane, where it maintains mitochondrial integrity. Here we show that knockdown of Tafazzin (TAZ kd), a CL transacylase, in mice results in protection against the development of obesity, insulin resistance, and hepatic steatosis. We determined that hypermetabolism protected TAZ kd mice from weight gain. Unexpectedly, the large reduction of CL in the heart and skeletal muscle of TAZ kd mice was not mirrored in the liver. As a result, TAZ kd mice exhibited normal hepatic mitochondrial supercomplex formation and elevated hepatic fatty acid oxidation. Collectively, these studies identify a key role for hepatic CL remodeling in regulating susceptibility to insulin resistance and as a novel therapeutic target for diet-induced obesity., (© 2016 by the American Diabetes Association.)

Mitochondrial oxidation of fatty acids accounts for the majority of cardiac ATP production in the heart. Fatty acid utilization by cardiac mitochondria is controlled at the level of fatty acid uptake, lipid synthesis, mobilization and mitochondrial import and oxidation. Consequently defective mitochondrial function appears to be central to the development of heart failure. Cardiolipin is a key mitochondrial phospholipid required for the activity of the electron transport chain. In heart failure, loss of cardiolipin and tetralinoleoylcardiolipin helps to fuel the generation of excessive reactive oxygen species that are a by-product of inefficient mitochondrial electron transport chain complexes I and III. In this vicious cycle, reactive oxygen species generate lipid peroxides and may, in turn, cause oxidation of cardiolipin catalyzed by cytochrome c leading to cardiomyocyte apoptosis. Hence, preservation of cardiolipin and mitochondrial function may be keys to the prevention of heart failure development. In this review, we summarize cardiac energy metabolism and the important role that fatty acid uptake and metabolism play in this process and how defects in these result in heart failure. We highlight the key role that cardiolipin and sirtuins play in cardiac mitochondrial β-oxidation. In addition, we review the potential of pharmacological modulation of cardiolipin through the polyphenolic molecule resveratrol as a sirtuin-activator in attenuating mitochondrial dysfunction. Finally, we provide novel experimental evidence that resveratrol treatment increases cardiolipin in isolated H9c2 cardiac myocytes and tetralinoleoylcardiolipin in the heart of the spontaneously hypertensive rat and hypothesize that this leads to improvement in mitochondrial function. This article is part of a Special Issue entitled: Heart Lipid Metabolism edited by G.D. Lopaschuk., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Huntington's Disease (HD) is an autosomal dominant disease that occurs as a result of expansion of the trinucleotide repeat CAG (glutamine) on the HTT gene. HD patients exhibit various forms of mitochondrial dysfunction within neurons and peripheral tissues. Cardiolipin (Ptd2Gro) is a polyglycerophospholipid found exclusively in mitochondria and is important for maintaining mitochondrial function. We examined if altered Ptd2Gro metabolism was involved in the mitochondrial dysfunction associated with HD. Mitochondrial basal respiration, spare respiratory capacity, ATP coupling efficiency and rate of glycolysis were markedly diminished in Epstein-Barr virus transformed HD lymphoblasts compared to controls (CTRL). Mitochondrial supercomplex formation and Complex I activity within these supercomplexes did not vary between HD patients with different length of CAG repeats and appeared unaltered compared to CTRL. In contrast, in vitro Complex I enzyme activity in mitochondrial enriched samples was reduced in HD lymphoblasts compared to CTRL. The total cellular pool size of Ptd2Gro and its synthesis/remodeling from [(3)H]acetate/[(14)C]oleate were unaltered in HD lymphoblasts compared to CTRL. In addition, the molecular species of Ptd2Gro were essentially unaltered in HD lymphoblasts compared to CTRL. We conclude that compared to CTRL lymphoblasts, HD lymphoblasts display impaired mitochondrial basal respiration, spare respiratory capacity, ATP coupling efficiency and rate of glycolysis with any pathological CAG repeat length, but this is not due to alterations in Ptd2Gro metabolism. We suggest that HD patient lymphoblasts may be a useful model to study defective energy metabolism that does not involve alterations in Ptd2Gro metabolism.

The contribution of α-subunit of trifunctional protein (αTFP) to cardiolipin (CL) (diphosphatidylglycerol) remodelling and mitochondrial supercomplex formation was examined in heart and liver mitochondria from wild-type (WT) and αTFP heterozygous knockout [Mtpa(+/-)] mice. Mtpa(+/-) mouse heart and liver exhibited an approximate 55% and 50% reduction in αTFP protein expression compared with WT respectively. Monolysocardiolipin (MLCL) acyltransferase (MLCL AT)-1 protein derived from αTFP was reduced by 30% in Mtpa(+/-) mouse heart but not in liver compared with WT. In vitro acylation of MLCL was significantly reduced in heart but not in liver mitochondria of Mtpa(+/-) mice compared with WT. CL mass was reduced and significant reductions in linoleate-containing CL species, in particular tetralinoleoyl-CL (L4-CL) and trilinoleoyl-CL (L3-MLCL) species, were observed in heart and liver mitochondria of Mtpa(+/-) mice compared with WT. Cardiac and liver mitochondrial supercomplex assembly and NADH dehydrogenase (complex I) activity within these supercomplexes were unaltered in both Mtpa(+/-) mouse heart and Mtpa(+/-) mouse liver compared with WT. The results indicate that αTFP may modulate CL molecular species composition in murine heart and liver. In addition, L4-CL might not be an essential requirement for mitochondrial supercomplex assembly., (© 2015 Authors; published by Portland Press Limited.)

Cardiolipin, a unique phospholipid in the inner mitochondrial membrane, is critical for optimal mitochondrial function. CL abnormalities have been demonstrated in the failing rodent and adult human heart. The aim of this study was to determine whether abnormalities in CL content and the CL biosynthesis and remodeling pathways are present in pediatric idiopathic dilated cardiomyopathy (IDC). A cross-sectional analysis of myocardial tissue from 119 IDC and non-failing (NF) control samples was performed. Electrospray ionizing mass spectrometry was used to measure total CL and CL species content in LV tissue. RT-PCR was employed to measure gene expression of the enzymes in the CL biosynthesis and remodeling pathways in both the adult and pediatric heart. Significantly lower total and (18:2)4CL (the beneficial species) content was demonstrated in myocardium from pediatric patients with IDC compared to NF controls. Analysis of mitochondrial gene transcripts was used to demonstrate that there is no decrease in mitochondrial content. Expression of two biosynthesis enzymes and one remodeling enzyme was significantly lower in pediatric IDC compared to NF controls. Expression of two phospholipases involved in CL degradation were also altered, one up- and one down-regulated. Except for one remodeling enzyme, these changes are unique from those in the failing adult heart. Similar to what has been seen in adults and in a rat model of IDC, total and (18:2)4CL are lower in pediatric IDC. Unique CL species profiles are seen in heart tissue from children with IDC compared to adults. Differences in CL biosynthesis and remodeling enzyme expression likely explain the differences in CL profiles observed in IDC and implicate unique age-related mechanisms of disease., (Published by Elsevier Ltd.)

Previous studies from our laboratory have shown positive benefits of linoleic acid (LA) feeding for attenuation of rat heart failure (HF). However, another research group concluded LA feeding was detrimental to cardiac function, using the American Institute of Nutrition 76A (AIN) diet as a background diet for the experimental animals only. To reconcile these conflicting results and determine whether (i) AIN has effects on cardiovascular function, and (ii) AIN reverses the positive effects of LA feeding, studies were performed using spontaneously hypertensive heart failure (SHHF) rats in both a survival study with lifetime feeding of AIN (control: Purina 5001) and a 2 × 2 factorial design for 6 weeks in young male SHHF rats with background diet and LA as variables. During a lifetime of AIN feeding, mortality from heart failure is significantly accelerated, cardiolipin altered and triglycerides increased. In young rats, 6 weeks on the AIN diet promoted increased systolic and diastolic blood pressure, increased fed and fasting blood glucose, increased serum inflammatory eicosanoids, decreased docosahexanoic acid, increased posterior wall thickness in diastole and an altered cardiolipin subspecies profile. The addition of LA to the AIN diet was able to rescue blood pressure. However, the combination increased retroperitoneal fat mass, body weight and fed blood glucose beyond the levels with the AIN diet alone. Because the AIN diet has wide ranging effects on cardiovascular parameters, our results suggest that it should not be used in animal studies involving the cardiovascular system unless induction of cardiac dysfunction is the desired outcome., (© 2013 Blackwell Verlag GmbH.)

Background: Remodeling of myocardial phospholipids has been reported in various forms of heart failure for decades, but the mechanism and pathophysiological relevance of this phenomenon have remained unclear. We examined the hypothesis that δ-6 desaturase (D6D), the rate-limiting enzyme in long-chain polyunsaturated fatty acid biosynthesis, mediates the signature pattern of fatty acid redistribution observed in myocardial phospholipids after chronic pressure overload and explored plausible links between this process and disease pathogenesis., Methods and Results: Compositional analysis of phospholipids from hearts explanted from patients with dilated cardiomyopathy revealed elevated polyunsaturated fatty acid product/precursor ratios reflective of D6D hyperactivity, manifesting primarily as lower levels of linoleic acid with reciprocally higher levels of arachidonic and docosahexaenoic acids. This pattern of remodeling was attenuated in failing hearts chronically unloaded with a left ventricular assist device. Chronic inhibition of D6D in vivo reversed similar patterns of myocardial polyunsaturated fatty acid redistribution in rat models of pressure overload and hypertensive heart disease and significantly attenuated cardiac hypertrophy, fibrosis, and contractile dysfunction in both models. D6D inhibition also attenuated myocardial elevations in pathogenic eicosanoid species, lipid peroxidation, and extracellular receptor kinase 1/2 activation; normalized cardiolipin composition in mitochondria; reduced circulating levels of inflammatory cytokines; and elicited model-specific effects on cardiac mitochondrial respiratory efficiency, nuclear factor κ B activation, and caspase activities., Conclusions: These studies demonstrate a pivotal role of essential fatty acid metabolism in myocardial phospholipid remodeling induced by hemodynamic stress and reveal novel links between this phenomenon and the propagation of multiple pathogenic systems involved in maladaptive cardiac remodeling and contractile dysfunction [corrected].

Clinical metabolic syndrome conveys a poor prognosis in patients with acute coronary syndrome, not fully accounted for by the extent of coronary atherosclerosis. To explain this observation, we determined whether postischemic myocardial contractile and metabolic function are impaired in a porcine dietary model of metabolic syndrome without atherosclerosis. Micropigs (n = 28) were assigned to a control diet (low fat, no added sugars) or an intervention diet (high saturated fat and simple sugars, no added cholesterol) for 7 mo. The intervention diet produced obesity, hypertension, dyslipidemia, and impaired glucose tolerance, but not atherosclerosis. Under open-chest, anesthetized conditions, pigs underwent 45 min of low-flow myocardial ischemia and 120 min of reperfusion. In both diet groups, contractile function was similar at baseline and declined similarly during ischemia. However, after 120 min of reperfusion, regional work recovered to 21 ± 12% of baseline in metabolic syndrome pigs compared with 61 ± 13% in control pigs (P = 0.01). Ischemia-reperfusion caused a progressive decline in mechanical/metabolic efficiency (regional work/O2 consumption) in metabolic syndrome hearts, but not in control hearts. Metabolic syndrome hearts demonstrated altered fatty acyl composition of cardiolipin and increased Akt phosphorylation in both ischemic and nonischemic regions, suggesting tonic activation. Metabolic syndrome hearts used more fatty acid than control hearts (P = 0.03). When fatty acid availability was restricted by prior insulin exposure, differences between groups in postischemic contractile recovery and mechanical/metabolic efficiency were eliminated. In conclusion, pigs with characteristics of metabolic syndrome demonstrate impaired contractile and metabolic recovery after low-flow myocardial ischemia. Contributory mechanisms may include remodeling of cardiolipin, abnormal activation of Akt, and excessive utilization of fatty acid substrates.

Here, we report for the first time in vitro reconstitution of the respiratory supercomplexes from individual complexes III and IV. Complexes III and IV were purified from Saccharomyces cerevisiae mitochondria. Complex III contained eight molecules of cardiolipin, and complex IV contained two molecules of cardiolipin, as determined by electrospray ionization-mass spectrometry. Complex IV also contained Rcf1p. No supercomplexes were formed upon mixing of the purified complexes, and low amounts of the supercomplex trimer III(2)IV(1) were formed after reconstitution into proteoliposomes containing only phosphatidylcholine and phosphatidylethanolamine. Further addition of cardiolipin to the proteoliposome reconstitution mixture resulted in distinct formation of both the III(2)IV(1) supercomplex trimer and III(2)IV(2) supercomplex tetramer. No other anionic phospholipid was as effective as cardiolipin in supporting tetramer formation. Phospholipase treatment of complex IV prevented trimer formation in the absence of cardiolipin. Both trimer and tetramer formations were restored by cardiolipin. Analysis of the reconstituted tetramer by single particle electron microscopy confirmed native organization of individual complexes within the supercomplex. In conclusion, although some trimer formation occurred dependent only on tightly bound cardiolipin, tetramer formation required additional cardiolipin. This is consistent with the high cardiolipin content in the native tetramer. The dependence on cardiolipin for supercomplex formation suggests that changes in cardiolipin levels resulting from changes in physiological conditions may control the equilibrium between individual respiratory complexes and supercomplexes in vivo.

Here we present for the first time a three-dimensional cryo-EM map of the Saccharomyces cerevisiae respiratory supercomplex composed of dimeric complex III flanked on each side by one monomeric complex IV. A precise fit of the existing atomic x-ray structures of complex III from yeast and complex IV from bovine heart into the cryo-EM map resulted in a pseudo-atomic model of the three-dimensional structure for the supercomplex. The distance between cytochrome c binding sites of complexes III and IV is about 6 nm, which supports proposed channeling of cytochrome c between the individual complexes. The opposing surfaces of complexes III and IV differ considerably from those reported for the bovine heart supercomplex as determined by cryo-EM. A closer association between the individual complex domains at the aqueous membrane interface and larger spaces between the membrane-embedded domains where lipid molecules may reside are also demonstrated. The supercomplex contains about 50 molecules of cardiolipin (CL) with a fatty acid composition identical to that of the inner membrane CL pool, consistent with CL-dependent stabilization of the supercomplex.

Aims: Cardiolipin (CL) is a tetra-acyl phospholipid that provides structural and functional support to several proteins in the inner mitochondrial membrane. The majority of CL in the healthy mammalian heart contains four linoleic acid acyl chains (L(4)CL). A selective loss of L(4)CL is associated with mitochondrial dysfunction and heart failure in humans and animal models. We examined whether supplementing the diet with linoleic acid would preserve cardiac L(4)CL and attenuate mitochondrial dysfunction and contractile failure in rats with hypertensive heart failure., Methods and Results: Male spontaneously hypertensive heart failure rats (21 months of age) were administered diets supplemented with high-linoleate safflower oil (HLSO) or lard (10% w/w; 28% kilocalorie fat) or without supplemental fat (control) for 4 weeks. HLSO preserved L(4)CL and total CL to 90% of non-failing levels (vs. 61-75% in control and lard groups), and attenuated 17-22% decreases in state 3 mitochondrial respiration observed in the control and lard groups (P < 0.05). Left ventricular fractional shortening was significantly higher in HLSO vs. control (33 ± 2 vs. 29 ± 2%, P < 0.05), while plasma insulin levels were lower (5.4 ± 1.1 vs. 9.1 ± 2.3 ng/mL; P < 0.05), with no significant effect of lard supplementation. HLSO also increased serum concentrations of several eicosanoid species compared with control and lard diets, but had no effect on plasma glucose or blood pressure., Conclusion: Moderate consumption of HLSO preserves CL and mitochondrial function in the failing heart and may be a useful adjuvant therapy for this condition.

Cardiolipin (CL) is a mitochondrial membrane phospholipid which plays a key role in apoptosis and supports mitochondrial respiratory chain complexes involved in the generation of ATP. In order to facilitate its role CL must be remodeled with appropriate fatty acids. We previously identified a human monolysocardiolipin acyltransferase activity which remodels CL via acylation of monolysocardiolipin (MLCL) to CL and was identical to the alpha subunit of trifunctional protein (αTFP) lacking the first 227 amino acids. Full length αTFP is an enzyme that plays a prominent role in mitochondrial β-oxidation, and in this study we assessed the role, if any, which this metabolic enzyme plays in the remodeling of CL. Purified human recombinant αTFP exhibited acyl-CoA acyltransferase activity in the acylation of MLCL to CL with linoleoyl-CoA, oleoyl-CoA and palmitoyl-CoA as substrates. Expression of αTFP increased radioactive linoleate or oleate or palmitate incorporation into CL in HeLa cells. Expression of αTFP in Barth Syndrome lymphoblasts, which exhibit reduced tetralinoleoyl-CL, elevated linoleoyl-CoA acylation of MLCL to CL in vitro, increased mitochondrial respiratory Complex proteins and increased linoleate-containing species of CL. Knock down of αTFP in Barth Syndrome lymphoblasts resulted in greater accumulation of MLCL than those with normal αTFP levels. The results clearly indicate that the human αTFP exhibits MLCL acyltransferase activity for the resynthesis of CL from MLCL and directly links an enzyme of mitochondrial β-oxidation to CL remodeling.

Persistent pulmonary hypertension of the newborn (PPHN) results in right ventricular (RV) hypertrophy followed by right heart failure and an associated mitochondrial dysfunction. The phospholipid cardiolipin plays a key role in maintaining mitochondrial respiratory and cardiac function via modulation of the activities of enzymes involved in oxidative phosphorylation. In this study, changes in cardiolipin and cardiolipin metabolism were investigated during the development of right heart failure. Newborn piglets (<24 h old) were exposed to a hypoxic (10% O(2)) environment for 3 days, resulting in the induction of PPHN. Two sets of control piglets were used: 1) newborn or 2) exposed to a normoxic (21% O(2)) environment for 3 days. Cardiolipin biosynthetic and remodeling enzymes, mitochondrial complex II + III activity, incorporation of [1-(14)C]linoleoyl-CoA into cardiolipin precursors, and the tetralinoleoyl-cardiolipin pool size were determined in both the RV and left ventricle (LV). PPHN resulted in an increased heart-to-body weight ratio, RV-to-LV plus septum weight ratio, and expression of brain naturetic peptide in RV. In addition, PPHN reduced cardiolipin biosynthesis and remodeling in the RV and LV, which resulted in decreased tetralinoleoyl-cardiolipin levels and reduced complex II + III activity and protein levels of mitochondrial complexes II, III, and IV in the RV. This is the first study to examine the pattern of cardiolipin metabolism during the early development of both the RV and LV of the newborn piglet and to demonstrate that PPHN-induced alterations in cardiolipin biosynthetic and remodeling enzymes contribute to reduced tetralinoleoyl-cardiolipin and mitochondrial respiratory chain function during the development of RV hypertrophy. These defects in cardiolipin may play an important role in the rapid development of RV dysfunction and right heart failure in PPHN.

Treatment with the omega-3 polyunsaturated fatty acids (PUFAs) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) exerts cardioprotective effects, and suppresses Ca2+-induced opening of the mitochondrial permeability transition pore (MPTP). These effects are associated with increased DHA and EPA, and lower arachidonic acid (ARA) in cardiac phospholipids. While clinical studies suggest the triglyceride lowering effects of DHA and EPA are equivalent, little is known about the independent effects of DHA and EPA on mitochondria function. We compared the effects of dietary supplementation with the omega-3 PUFAs DHA and EPA on cardiac mitochondrial phospholipid fatty acid composition and Ca2+-induced MPTP opening. Rats were fed a standard lab diet with either normal low levels of omega-3 PUFA, or DHA or EPA at 2.5% of energy intake for 8 weeks, and cardiac mitochondria were isolated and analyzed for Ca2+-induced MPTP opening and phospholipid fatty acyl composition. DHA supplementation increased both DHA and EPA and decreased ARA in mitochondrial phospholipid, and significantly delayed MPTP opening as assessed by increased Ca2+ retention capacity and decreased Ca2+-induced mitochondria swelling. EPA supplementation increased EPA in mitochondrial phospholipids, but did not affect DHA, only modestly lowered ARA, and did not affect MPTP opening. In summary, dietary supplementation with DHA but not EPA, profoundly altered mitochondrial phospholipid fatty acid composition and delayed Ca2+-induced MPTP opening., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Cardiolipin (CL) is an essential phospholipid component of the inner mitochondrial membrane. In the mammalian heart, the functional form of CL is tetralinoleoyl CL [(18:2)(4)CL]. A decrease in (18:2)(4)CL content, which is believed to negatively impact mitochondrial energetics, occurs in heart failure (HF) and other mitochondrial diseases. Presumably, (18:2)(4)CL is generated by remodeling nascent CL in a series of deacylation-reacylation cycles; however, our overall understanding of CL remodeling is not yet complete. Herein, we present a novel cell culture method for investigating CL remodeling in myocytes isolated from Spontaneously Hypertensive HF rat hearts. Further, we use this method to examine the role of calcium-independent phospholipase A(2) (iPLA(2)) in CL remodeling in both HF and nonHF cardiomyocytes. Our results show that 18:2 incorporation into (18:2)(4)CL is: a) performed singly with respect to each fatty acyl moiety, b) attenuated in HF relative to nonHF, and c) partially sensitive to iPLA(2) inhibition by bromoenol lactone. These results suggest that CL remodeling occurs in a step-wise manner, that compromised 18:2 incorporation contributes to a reduction in (18:2)(4)CL in the failing rat heart, and that mitochondrial iPLA(2) plays a role in the remodeling of CL's acyl composition.

Consumption of omega-3 fatty acids from fish oil, specifically eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), decreases risk for heart failure and attenuates pathologic cardiac remodeling in response to pressure overload. Dietary supplementation with EPA + DHA may also impact cardiac mitochondrial function and energetics through alteration of membrane phospholipids. We assessed the role of EPA + DHA supplementation on left ventricular (LV) function, cardiac mitochondrial membrane phospholipid composition, respiration, and sensitivity to mitochondrial permeability transition pore (MPTP) opening in normal and infarcted myocardium. Rats were subjected to sham surgery or myocardial infarction by coronary artery ligation (n=10-14), and fed a standard diet, or supplemented with EPA + DHA (2.3% of energy intake) for 12 weeks. EPA + DHA altered fatty acid composition of total mitochondrial phospholipids and cardiolipin by reducing arachidonic acid content and increasing DHA incorporation. EPA + DHA significantly increased calcium uptake capacity in both subsarcolemmal and intrafibrillar mitochondria from sham rats. This treatment effect persisted with the addition of cyclosporin A, and was not accompanied by changes in mitochondrial respiration or coupling, or cyclophilin D protein expression. Myocardial infarction resulted in heart failure as evidenced by LV dilation and contractile dysfunction. Infarcted LV myocardium had decreased mitochondrial protein yield and activity of mitochondrial marker enzymes, however respiratory function of isolated mitochondria was normal. EPA + DHA had no effect on LV function, mitochondrial respiration, or MPTP opening in rats with heart failure. In conclusion, dietary supplementation with EPA + DHA altered mitochondrial membrane phospholipid fatty acid composition in normal and infarcted hearts, but delayed MPTP opening only in normal hearts.

Supplementation with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from fish oil may prevent development of heart failure through alterations in cardiac phospholipids that favorably impact inflammation and energy metabolism. A high-fat diet may block these effects in chronically stressed myocardium. Pathological left ventricle (LV) hypertrophy was generated by subjecting rats to pressure overload by constriction of the abdominal aorta. Animals were fed: (1) standard diet (10% of energy from fat), (2) standard diet with EPA+DHA (2.3% of energy intake as EPA+DHA), (3) high fat (60% fat); or (4) high fat with EPA+DHA. Pressure overload increased LV mass by approximately 40% in both standard and high-fat diets without fish oil. Supplementation with fish oil increased their incorporation into cardiac phospholipids, and decreased the proinflammatory fatty acid arachidonic acid and urine thromboxane B(2) with both the standard and high-fat diet. Linoleic acid and tetralinoloyl cardiolipin (an essential mitochondrial phospholipid) were decreased with pressure overload on standard diet, which was prevented by fish oil. Animals fed high-fat diet had decreased linoleic acid and tetralinoloyl cardiolipin regardless of fish oil supplementation. Fish oil limited LV hypertrophy on the standard diet, and prevented upregulation of fetal genes associated with heart failure (myosin heavy chain-beta and atrial natriuetic factor). These beneficial effects of fish oil were absent in animals on the high-fat diet. In conclusion, whereas treatment with EPA+DHA prevented tetralinoloyl cardiolipin depletion, LV hypertrophy, and abnormal genes expression with pressure overload, these effects were absent with a high-fat diet.