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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 non-failing (NF) 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. This article is protected by copyright. All rights reserved.

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

Summary 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., Graphical Abstract, Highlights • Standard (glucose) cultured hiPSC-CMs demonstrate a blunted hypertrophic response • A maturation method induces hiPSC-CM maturation and suppresses HIF1A expression • Mature hiPSC-CMs demonstrate improved sarcomeric morphology and contractility • Mature hiPSC-CMs respond to agonist- or mutation-induced hypertrophy, In this article, Song and colleagues show that a combination of fatty acid medium and micropatterned surfaces induces maturation in human induced pluripotent stem cell-derived cardiomyocytes. Matured cells display improved sarcomere morphology, metabolic maturation, and contractility. These cells also show increased sensitivity to hypertrophic stimuli, including hypertrophic agonist and genetic mutations, representing an ideal system to model cardiovascular disease.

BackgroundPulmonary vascular remodeling (PVR)in uteroresults in the development of heart failure (HF). The alterations that occur in cardiac lipid and mitochondrial bioenergetics during the development ofin uteroPVR was unknown.MethodsPVR was induced in pupsin uteroby exposure of pregnant dams to indomethacin and hypoxia. Cardiac lipids, echocardiographic function and cardiomyocyte mitochondrial function were subsequently examined.ResultsPerinatal 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 mitochondrial dysfunction. Although abundance of mitochondrial respiratory complexes were unaltered, increased trilinoleoyl-lysocardiolipin levels in these pups was observed. A compensatory increase in both cardiolipin (CL) and phosphatidylethanolamine (PE) content were observed due to increased synthesis of these phospholipids.ConclusionAlterations in cardiac cardiolipin and phospholipid metabolism in PVR rat pups is associated with the mitochondrial bioenergetic and cardiac functional defects observed in their hearts.Impact statement- Phospholipid metabolism was examined in pulmonary vascular remodeling in perinatal rat pups.- Pulmonary vascular remodeling was inducedin uteroby treating pregnant dams with hypoxia and indomethacin at 19-21 days of gestation.- The offspring exhibited altered pulmonary arterial remodeling with subsequent cardiac hypertrophy, ventricular dysfunction, cardiac myocyte mitochondrial dysfunction with altered fatty acid utilization.- In addition, the offspring exhibited elevated cardiolipin, lysocardiolipin and phosphatidylethanolamine content which may potentially contribute to the cardiac mitochondrial dysfunction.

Visual Abstract, Highlights • Mitochondrial function is impaired in explanted failing pediatric and adult human hearts. • Elamipretide is a novel mitochondria-targeted drug that is targeted to cardiolipin on the inner mitochondrial membrane and improves coupling of the electron transport chain. • Treatment of explanted human hearts with elamipretide improves human cardiac mitochondrial function. • The study provides novel methods to evaluate the influence of compounds on mitochondria in the human heart and provides proof of principle for the use of elamipretide to improve mitochondrial energetics in failing myocardium due to multiple etiologies and irrespective of age., Summary 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.

Barth Syndrome (BTHS) is a rare X-linked genetic disorder caused by mutation in the TAFAZZIN gene which encodes the cardiolipin (CL) transacylase tafazzin (Taz). Taz deficiency in BTHS patients results in reduced CL in their tissues and a neutropenia which contributes to the risk of infections. However, the impact of Taz deficiency in other cells of the immune system is poorly understood. Mesenchymal stem cells (MSCs) are well known for their immune inhibitory function. We examined whether Taz-deficiency in murine MSCs impacted their ability to modulate lipopolysaccharide (LPS)-activated wild type (WT) murine B lymphocytes. MSCs from tafazzin knockdown (TazKD) mice exhibited a 50% reduction in CL compared to wild type (WT) MSCs. However, mitochondrial oxygen consumption rate and membrane potential were unaltered. In contrast, TazKD MSCs exhibited increased glycolysis compared to WT MSCs and this was associated with 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. When co-cultured with LPS-activated WT B cells, TazKD MSCs inhibited B cell proliferation and growth rate and reduced B cell secretion of IgM to a greater extent than B cells co-cultured with WT MSCs. In addition, co-culture of LPS-activated WT B cells with TazKD MSCs induced B cell differentiation toward potent immunosuppressive phenotypes including interleukin-10 secreting plasma cells and B regulatory cells compared to activated B cells co-cultured with WT MSCs. These results indicate that Taz deficiency in MSCs enhances MSCs-mediated immunosuppression of activated B lymphocytes.

Pediatric dilated cardiomyopathy (DCM) is a devastating and poorly understood disease with most clinical treatment paradigms extrapolated from the adult population. Our studies have demonstrated that aspects of metabolism and mitochondrial function are dysregulated in pediatric DCM hearts. Cardiolipin (CL), a unique phospholipid in the inner mitochondrial membrane, is essential for optimal mitochondrial function and was shown to be dysregulated in both the failing adult and pediatric human heart. The objective of this study is to investigate if serum circulating factors from pediatric DCM patients can remodel CL resulting in mitochondrial dysfunction in vitro , similar to what is observed in the failing pediatric heart. Using a novel in vitro model that consists of treating neonatal rat ventricular myocytes (NRVMs) with serum from pediatric DCM patients or from non-failing (NF) healthy controls, mitochondrial respiration was assessed using the Agilent Seahorse, and reactive oxygen species (ROS) was assessed using Electron Paramagnetic Resonance Spectroscopy. Relative mitochondrial DNA (mtDNA) copy number was determined by qPCR and expression of enzymes involved in CL biosynthesis and remodeling were analyzed using RT-qPCR. Mass-spectrometry was used to quantitate total and specific CL species and to investigate the metabolite composition of NRVMs treated with NF or DCM serum. While mitochondrial ROS and mtDNA copy number were not significantly altered, we show that DCM serum decreases mitochondrial function, which is associated with alterations in CL content and composition and the downregulation of enzymes implicated in CL biosynthesis and remodeling. Analysis of metabolite content showed an alteration of pathways involved in fatty acid metabolism, mitochondrial biogenesis and regulation of β-oxidation by the transcription factor PPARα. In conclusion, pediatric DCM serum circulating factors can promote CL remodeling resulting mitochondrial dysfunction in primary cardiomyocytes. These findings suggest that CL could be a novel therapeutic target for this particular population.

The protein kinase C delta (PKCδ) signalosome exists as a high molecular weight complex in mitochondria and controls mitochondrial oxidative phosphorylation. Barth Syndrome (BTHS) is a rare X-linked genetic disease in which mitochondrial oxidative phosphorylation is impaired due to a mutation in the gene TAFAZZIN which results in reduction in the phospholipid cardiolipin and an accumulation of monolysocardiolipin. Here we examined if PKCδ association with a higher molecular weight complex was altered in mitochondria of BTHS lymphoblasts. Immunoblot analysis of blue native-polyacrylamide gel electrophoresis mitochondrial fractions revealed that PKCδ associated with a higher molecular weight complex in control lymphoblasts but this was markedly reduced in BTHS patient B lymphoblasts in spite of an increase in PKCδ protein expression. We hypothesize that the lack of PKCδ within this higher molecular weight complex may contribute to defective mitochondrial PKCδ signaling and thus to the bioenergetic defects observed in BTHS.

OBJECTIVES: Anthracyclines damage the heart by binding to the inner mitochondrial membrane phospholipid cardiolipin and promoting mitochondrial dysfunction. Our goal is to characterize how anthracylcines change the fatty acid profile of cardiolipin, and the genes controlling cardiolipin synthesis and remodeling in order to develop dietary strategies to target cardiolipin within damaged hearts. METHODS: Male C57BL/6J mice were randomized to receive seven IP, anthracycline injections (one week apart) or saline control injections. Six hours after the final injection, mice were euthanized under isoflurane and hearts were collected for mRNA and cardiolipin species analysis. Cardiolipin was measured using liquid chromatography coupled to electrospray ionization mass spectrometry in an API 4000 mass spectrometer (Sciex, Framingham, MA). A student's t-test was used to determine significance between the groups. RESULTS: While the amount of tetralinolylcardiolipin (the major cardiolipin species in the heart) was not altered with anthracycline treatment, several other cardiolipin species were significantly increased, including cardiolipin species that contain arachidonic acid and docosahexaenoic acid. In addition we observed a significant decrease in the proportion of monolysocardiolipin species in the heart and a trend towards increased oxidized cardiolipin species given anthracycline treatment. CONCLUSIONS: Previous studies have shown the fatty acid composition of cardiolipin effects mitochondrial structure and function. Administration of anthracyclines alters cardiolipin species in the heart. Knowing that anthracyclines influences the fatty acid composition of cardiolipin, we can explore dietary interventions that favorably change cardiolipin fatty acid composition, with the goal of preventing anthracycline-induced heart damage by improving mitochondrial function. FUNDING SOURCES: Funding was provided by NIH R21CA185140, Ohio Agriculture Research and Development Center, the Carol S. Kennedy Professorship, and the Ohio State University Education and Human Ecology Dissertation Research Fellowship.

OBJECTIVES: Linoleic acid (LA) is the major fatty acid in cardiolipin (CL), a phospholipid important for mitochondria function. Increasing dietary LA elevates tetralinoleoyl CL (LA(4) CL) in cardiac muscle of animals; however, it is not clear if dietary LA increases LA(4) CL in humans. The aim of this study was to determine if daily consumption of a cookie made with a LA-rich oil for two weeks could alter LA(4) CL in peripheral blood mononuclear cells (PBMC) in healthy adults. METHODS: In a double-masked placebo-controlled study, 84 healthy adults were randomly assigned to consume one cookie made with LA-rich grapeseed oil (LA-cookie) or one cookie made with oleic acid-rich (OA) safflower oil (OA-cookie), every day for two weeks. Fasting blood samples were collected for analysis of PBMC cardiolipin species. RESULTS: Daily consumption of the LA-cookie for 2 weeks increased LA(4) CL in PBMC, while consuming the OA-cookie did not alter LA(4) CL (group x week interaction p

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.

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.

ObjectiveTafazzin (TAZ) is a cardiolipin (CL) biosynthetic enzyme important for maintaining mitochondrial function. TAZ impacts 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.MethodsMale 4-month-old doxycycline-inducible TAZ knock-down (TAZ KD) mice and wild-type littermate controls were utilized. Immunohistochemistry was used to assess β-cell morphology in whole pancreas sections, while ex vivo insulin secretion, CL content, RNA-Seq analysis and mitochondrial oxygen consumption were measured from isolated islet preparations.ResultsEx vivo insulin secretion under non-stimulatory 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 reduced oxidized CL content. In addition, RNA-Seq 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.ConclusionThese data indicate a novel role for TAZ in regulating normal β-cell function, particularly under low-glucose conditions.

The most robust risk factor for type 2 diabetes in childhood is prior exposure to diabetes during gestation. Currently, there are few evidence-based strategies to attenuate the of 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. It has been used safely for centuries in humans. Our objective was to determine whether BBR treatment improves health outcomes in the mouse offspring of GDM dams. Dams were fed either a lean low-fat diet (Lean, LF,10% kcal fat) or a GDM-inducing high-fat/high sucrose diet (GDM, HF, 45% kcal fat) prior to breeding and throughout pregnancy. The resulting Lean and GDM-exposed offspring were randomly assigned a LF, HF or HF diet containing BBR (160 mg/kg/d) for 12 weeks. We determined that BBR treatment significantly reduced body weight (∼20%), % body fat (∼40%) and gonadal fat pad mass (∼60%) compared to HF-fed GDM offspring. Furthermore, BBR treatment of HF-fed GDM offspring normalized insulin levels in the plasma and isolated pancreatic islets. Differences in food consumption did not contribute to altered body composition in BBR treated mice, as levels remained similar between experimental groups. Alternatively, BBR-treatment was associated with increased whole-body oxygen consumption (VO2), activity and heat production. Additionally, we determined that HF-fed GDM offspring developed a cardiomyopathy, characterized by increased isovolumetric contraction (∼150%, IVCT), relaxation time (∼70%, IVRT), elevated cardiac triglyceride (∼120%) and reduced mitochondrial function (30%, spare capacity) compared to LF fed Lean controls. BBR treatment normalized heart function, reduced triglyceride levels and maintained mitochondrial function. Our data supports BBR as a potential pharmacotherapeutic approach to improve health outcomes in individuals exposed to GDM.Key Points SummaryGestational diabetes mellitus is a common metabolic complication of pregnancy which is increasing worldwide due to the prevalence of obesity.It is known that individuals exposed to gestational diabetes have elevated risk of developing metabolic syndrome however there are few evidence-based strategies which provide protection.Berberine is a natural compound found in Chinese herbs which has been safely used for centuries to treat type 2 diabetes mellitus.We determined that berberine treatment of offspring exposed to gestational diabetes attenuated weight gain, reduced insulin levels and normalized both heart and pancreatic function.Our data supports berberine as a potential pharmacotherapeutic approach to improve health outcomes in individuals exposed to gestational diabetes.

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 an 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.

RationaleHuman 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.ObjectiveWe sought to develop a simple method which could drive cultured hiPSC-CMs towards maturity across a number of phenotypes.Methods and resultsCells were cultured in fatty acid-based media and plated on micropatterned surfaces to promote alignment and elongation. These cells display many characteristics of adult human cardiomyocytes, including elongated cell morphology, enhanced maturity of sarcomeric structures, metabolic behavior, and increased myofibril contractile force. Most notably, hiPSC-CMs cultured under optimal maturity-inducing conditions recapitulate the pathological hypertrophy caused by either a pro-hypertrophic agent or genetic mutations.ConclusionsThe more mature hiPSC-CMs produced by the methods described here will serve as a usefulin vitroplatform for characterizing cardiovascular disease.

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.

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.

OBJECTIVES: Compare the effects of high-fat diets containing linoleic-acid (LA)-rich oil and lard on measures of whole-body energy metabolism and skeletal muscle function in a mouse model of diet-induced obesity. METHODS: 9-week old male C57BL6/J mice (n = 24) were assigned to high fat isocaloric diets (24% fat by wt) containing LA-rich safflower oil (SO) or lard (LD) for 18 weeks. Food intake and body weight were measured every 2 days. EchoMRI was used at Days 0, 65, and 100 to determine the effects of each diet on body composition. Grip strength was measured to assess muscle strength at Day 113. Fasting blood glucose was measured and an insulin tolerance test (ITT) was performed on Day 85 to measure insulin sensitivity and glucose uptake. After 130 days, quadriceps muscle mitochondrial and cardiolipin remodeling gene expression was measured by qRT-PCR. Cardiolipin (CL) speciation of muscle was measured by normal phase liquid chromatography coupled to electrospray ionization mass spectrometry. RESULTS: Although LD-fed mice gained body mass at a faster rate, there was no difference in body mass or cumulative food intake after 130 days. Similarly, % adiposity of LD-fed mice increased at Day 65 compared to those fed the SO diet, but not at Day 100. Mice consuming the SO diet exhibited increased hindlimb grip strength. SO-fed mice exhibited lower blood glucose levels at 90 and 120 minutes post-insulin injection, demonstrating improved glucose clearance over time. Significant differences in CL speciation, particularly that of LA-containing species, were observed. CONCLUSIONS: When compared to the LD diet, mice consuming the SO diet exhibited early reductions in adiposity that were undistinguishable after 130 days of dietary intervention. However, mice fed SO diet exhibited improved grip strength, insulin-stimulated glucose clearance and significant differences in skeletal muscle cardiolipin speciation. Future studies will address the relationship between LA-derived metabolites, CL speciation, and improved muscle function. FUNDING SOURCES: Funding was provided by NIH, Ohio Agriculture Research and Development Center and the Carol S. Kennedy Professorship. DBS received support from the AOCS Thomas H. Smouse Memorial Fellowship.

Introduction Pediatric dilated cardiomyopathy (DCM) is a devastating and poorly understood disease with most clinical treatment paradigms extrapolated from the adult population. Our studies showed that aspects of metabolism and mitochondria function are dysregulated in pediatric hearts. Cardiolipin (CL), a unique phospholipid in the inner mitochondrial membrane, is essential for optimal mitochondrial function and was shown to be dysregulated in failing adult hearts. Objective We investigate if serum circulating factors from pediatric DCM patients can remodel CL resulting in mitochondrial dysfunction similar to what is observed in the failing pediatric heart. Method Using a novel in vitro model that consists of treating neonatal rat ventricular myocytes (NRVMs) with serum from pediatric DCM patients or from non-failing (NF) healthy controls, mitochondrial respiration was assessed using Seahorse assay, and reactive oxygen species (ROS) was assessed using Electron Paramagnetic Resonance Spectroscopy. Expression of enzymes involved in CL biosynthesis and remodeling were analyzed using RT-qPCR and relative mtDNA copy number determined by qPCR. Mass spectroscopy was used to quantitate total and specific CL species and to investigate the metabolite composition of NRVMs treated with NF or DCM serum. Results While mitochondrial ROS and copy number were not significantly altered, we showed that DCM serum decreases mitochondrial function, which is associated with alterations in CL content and composition and gene expression of enzymes implicated in CL biosynthesis and remodeling. Analysis of metabolite content showed an alteration of pathways involved in fatty acid metabolism, mitochondrial biogenesis and regulation of β-oxidation by PPARα. Conclusion Pediatric DCM serum circulating factors can promote CL remodeling resulting in mitochondrial dysfunction in cardiomyocytes. These findings suggest that CL could be a therapeutic target for this particular population.

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.

OBJECTIVES: Investigate the impact of LA-rich oil (LO) on measures of energy metabolism in a mouse model of metabolic syndrome. METHODS: Ob/ob mice were fed diets containing 6% wt LO, oleic acid-rich (OO) or palmitic acid-rich (PO) for 6 weeks. Body composition was measured at weeks 0 and 6. Plasma was collected at necropsy to measure adiponectin, insulin, and glucose. Grip strength and muscle fiber cross-sectional area (CSA) of total and succinate dehydrogenase-positive (SDH) fibers were quantified in quadriceps. In white adipose tissue, mRNA was measured for markers of beiging and lipid storage. RESULTS: Mice fed OO and LO diets (vs. PO diet) had reduced % adipose. There was no difference of oils on plasma adiponectin or HOMA-IR. Decreases in grip strength were observed in PO-fed mice, while OO and LO-fed mice maintained strength throughout the study. LO-fed mice exhibited smaller skeletal muscle fibers compared to the PO-fed mice. OO-fed mice had fewer intermediate-sized SDH fibers. In white adipose tissue, LO-fed mice exhibited increased PGC1a, and decreased PPARy and LPL mRNA compared to PO-fed mice. CONCLUSIONS: These findings suggest that dietary LA may alter lipid mobilization and metabolism in obese mice. These preliminary results showcase the importance of future investigation of lipid storage and mitochondrial phospholipid biology in skeletal muscle. FUNDING SOURCES: Funding was provided by NIH R21CA185140, Ohio Agriculture Research and Development Center and the Carol S. Kennedy Professorship. DBS received support from the AOCS Thomas H. Smouse Memorial Fellowship.

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.

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.

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.

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/O2consumption) 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.

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 (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% O2) environment for 3 days. Cardiolipin biosynthetic and remodeling enzymes, mitochondrial complex II + III activity, incorporation of [1-14C]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.

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.

Cardiolipin (CL) is responsible for modulation of activities of various enzymes involved in oxidative phosphorylation. Although energy production decreases in heart failure (HF), regulation of cardiolipin during HF development is unknown. Enzymes involved in cardiac cardiolipin synthesis and remodeling were studied in spontaneously hypertensive HF (SHHF) rats, explanted hearts from human HF patients, and nonfailing Sprague Dawley (SD) rats. The biosynthetic enzymes cytidinediphosphatediacylglycerol synthetase (CDS), phosphatidylglycerolphosphate synthase (PGPS) and cardiolipin synthase (CLS) were investigated. Mitochondrial CDS activity and CDS-1 mRNA increased in HF whereas CDS-2 mRNA in SHHF and humans, not in SD rats, decreased. PGPS activity, but not mRNA, increased in SHHF. CLS activity and mRNA decreased in SHHF, but mRNA was not significantly altered in humans. Cardiolipin remodeling enzymes, monolysocardiolipin acyltransferase (MLCL AT) and tafazzin, showed variable changes during HF. MLCL AT activity increased in SHHF. Tafazzin mRNA decreased in SHHF and human HF, but not in SD rats. The gene expression of acyl-CoA: lysocardiolipin acyltransferase-1, an endoplasmic reticulum MLCL AT, remained unaltered in SHHF rats. The results provide mechanisms whereby both cardiolipin biosynthesis and remodeling are altered during HF. Increases in CDS-1, PGPS, and MLCL AT suggest compensatory mechanisms during the development of HF. Human and SD data imply that similar trends may occur in human HF, but not during nonpathological aging, consistent with previous cardiolipin studies.

The mitochondrial phospholipid cardiolipin is required for optimal mitochondrial respiration. In this study, cardiolipin molecular species and cytochrome oxidase (COx) activity were studied in interfibrillar (IF) and subsarcolemmal (SSL) cardiac mitochondria from Spontaneously Hypertensive Heart Failure (SHHF) and Sprague-Dawley (SD) rats throughout their natural life span. Fisher Brown Norway (FBN) and young aortic-constricted SHHF rats were also studied to investigate cardiolipin alterations in aging versus pathology. Additionally, cardiolipin was analyzed in human hearts explanted from patients with dilated cardiomyopathy. A loss of tetralinoleoyl cardiolipin (L(4)CL), the predominant species in the healthy mammalian heart, occurred during the natural or accelerated development of heart failure in SHHF rats and humans. L(4)CL decreases correlated with reduced COx activity (no decrease in protein levels) in SHHF cardiac mitochondria, but with no change in citrate synthase (a matrix enzyme) activity. The fraction of cardiac cardiolipin containing L(4)CL became much lower with age in SHHF than in SD or FBN mitochondria. In summary, a progressive loss of cardiac L(4)CL, possibly attributable to decreased remodeling, occurs in response to chronic cardiac overload, but not aging alone, in both IF and SSL mitochondria. This may contribute to mitochondrial respiratory dysfunction during the pathogenesis of heart failure.

Data regarding the effectiveness of chronic exercise training in improving survival in patients with congestive heart failure (CHF) are inconclusive. Therefore, we conducted a study to determine the effect of exercise training on survival in a well-defined animal model of heart failure (HF), using the lean male spontaneously hypertensive HF (SHHF) rat. In this model, animals typically present with decompensated, dilated HF between ∼18 and 23 mo of age. SHHF rats were assigned to sedentary or exercise-trained groups at 9 and 16 mo of age. Exercise training consisted of 6 mo of low-intensity treadmill running. Exercise training delayed the onset of overt HF and improved survival ( P < 0.01), independent of any effects on the hypertensive status of the rats. Training delayed the myosin heavy chain (MyHC) isoform shift from α- to β-MyHC that was seen in sedentary animals that developed HF. Exercise was associated with a concurrent increase in cardiomyocyte length (≈6%), width, and area and prevented the increase in the length-to-width ratio seen in sedentary animals in HF. The increases in proteinuria, plasma atrial natriuretic peptide, and serum leptin levels observed in rats with HF were suppressed by low-intensity exercise training. No significant alterations in sarco(endo)plasmic reticulum Ca2+ ATPase, phospholamban, or Na+/Ca2+ exchanger protein expression were found in response to training. Our results indicate that 6 mo of low-intensity exercise training delays the onset of decompensated HF and improves survival in the male SHHF rat. Similarly, exercise intervention prevented or suppressed alterations in several key variables that normally occur with the development of overt CHF. These data support the idea that exercise may be a useful and inexpensive intervention in the treatment of HF.

Electrospray ionization mass spectrometry has previously been used to probe qualitative changes in the phospholipid cardiolipin (CL), but it has rarely been used in a quantitative manner. We assessed changes in the amount of individual molecular species of cardiac CL present in a model of congestive heart failure using 1,1′,2,2′-tetramyristoyl cardiolipin as an internal standard. There was a linear relationship between the ratio of the negative molecular ion ([M-H]−) current from four different CL reference standards and the [M-H]− from the internal standard, as a function of the concentration of CL molecular species. Therefore, this internal standard can be used to quantitate many naturally occurring CL molecular species over a wide range of CL concentrations. Using this method, changes to individual molecular species of CL in failing hearts from male spontaneously hypertensive heart failure rats were examined. CL isolated from cardiac mitochondria was compared with left ventricular tissue to demonstrate the feasibility of extracting and quantitating CL from either mitochondrial or tissue samples. The acyl chain composition of individual CL molecular species was identified using tandem mass spectrometry. In animals with heart failure, the major cardiac CL species (tetralinoloyl) decreased significantly, whereas other minor CL species were significantly increased.

Moderate alcohol consumption has been shown to reduce the morbidity and mortality from coronary heart disease. Ethanol elicits its protective effects via mechanisms that include activation of protein kinases linked to growth and survival. Our results in isolated neonatal rat cardiomyocytes demonstrate that repeated short-term, low-dose exposure to ethanol is sufficient to activate the growth and/or survival pathways that involve PKC-ε, Akt, and AMP-activated kinase. In addition, we are able to induce apoptosis in these cardiomyocytes using the saturated fatty acid palmitate. Pretreatment with multiple low-dose ethanol exposures attenuates the apoptotic response to palmitate. This protection is manifested by a reduction in caspase-3-like activity, decreased mitochondrial loss of cytochrome c, and decreased loss of the mitochondrial lipid cardiolipin. We previously reported that incubation of cardiomyocytes with palmitate results in decreased production of reactive oxygen species compared with cells incubated with the nonapoptotic fatty acid oleate. In the present study, we observed an increase in the production of superoxide and the rates of fatty acid oxidation in cardiomyocytes pretreated with ethanol and then exposed to fatty acids. The level of superoxide production in palmitate-treated cells returns to the levels observed in oleate-treated cells after ethanol exposure. Taken together with our observed increase in AMP-activated kinase activity, we propose that ethanol pretreatments stimulate oxidative metabolism and electron transport within cardiomyocytes. We postulate that stimulation of palmitate metabolism may protect cardiomyocytes by preventing accumulation of unsaturated precursor molecules of cardiolipin synthesis. Maintaining cardiolipin levels may be sufficient to prevent the mitochondrial loss of cytochrome c and the downstream activation of caspases.

The effect of endurance training on the resistance of the heart to left ventricular (LV) functional deficit and infarction after a transient regional ischemia and subsequent reperfusion was examined. Female Sprague-Dawley rats were randomly assigned to an endurance exercise training (Tr) group or a sedentary (Sed) control group. After 20 wk of training, hearts were excised, perfused, and instrumented for assessment of LV mechanical function, and the left anterior descending coronary artery was occluded to induce a transient regional ischemia (1 h) that was followed by 2 h of reperfusion. Throughout much of the regional ischemia-reperfusion protocol, coronary flow rates, diastolic function, and LV developed pressure were better preserved in hearts from Tr animals. During the regional ischemia, coronary flow to myocardium outside the ischemic zone at risk (ZAR) was maintained in Tr hearts, whereas it progressively fell in Sed hearts. On release of the coronary artery ligature, flow to the ZAR was greater in Tr than in Sed hearts. Infarct size, expressed as a percentage of the ischemic ZAR, was significantly smaller in hearts from Tr rats (24 ± 3 vs. 32 ± 2% of ZAR, P < 0.05). Mn- and CuZn-SOD protein expression were higher in the LV myocardium of Tr animals ( P < 0.05 for both isoforms). Our data indicate that long-term exercise training leads to infarct sparing and better maintenance of coronary flow and mechanical function after ischemia-reperfusion.

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.

Fatty acids are the primary source of energy in the adult heart. Recently, however, it was discovered that certain saturated fatty acids, such as palmitate and stearate, cause cardiac and other types of cells to undergo programmed cell death (apoptosis). In cardiac ischemia/reperfusion injury, where blood flow is blocked and then restored to the heart, recovery of cardiac cells is inversely proportional to the concentration of fatty acids (largely composed of palmitate and stearate) in the reperfusate. The aim of this review is to summarize what is known about fatty acid induction of heart disease, the role of fatty acids in apoptosis, and apoptosis in the heart, including the role that mitochondria play in this process.

Cardiolipin (CL) is a unique phospholipid that is an essential component of the inner-mitochondrial membrane critical for normal energy metabolism. Biosynthesis of CL occurs via an enzymatic pathway or through remodeling of existing CLs. The content of total CL and (18:2) 4CL (tetralinoleic form

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.

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 (L4CL). A selective loss of L4CL 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 L4CL 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 L4CL 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.

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 ≈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 supplemention. Fish oil limited LV hypertrophy on the standard diet, and prevented upregulation of fetal genes associated with heart failure (myosin heavy chain-β 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.

Cardiolipin (CL) is a mitochondrial phospholipid that fundamentally contributes to the function of many proteins in the inner mitochondrial membrane, where it is actively involved in the integrity and flux of the electron transport chain. In the heart, functional CL is linoleic acid rich, and the loss of linoleic acid content is associated with cardiac disorders including ischemia and reperfusion, heart failure, and diabetes, as well as the X-linked recessive disease, Barth syndrome. To attain its high levels of linoleic acid, newly synthesized CL must initially undergo remodeling. CL modification and depletion by pathological processes may represent a failure of this remodeling pathway or activation of an alternative "pathological remodeling" pathway that causes the substitution of higher molecular weight, polyunsaturated fatty acyl side chains such as arachidonic or docosahexaenoic acid onto CL. This remodeling may occur in response to the alteration of CL by oxidative mechanisms, but the substituted side chains can also provoke further oxidation of CL. It is increasingly thought that cardiac pathology may result, at least partially, due to changes in CL resulting from the pathological remodeling process. Dietary interventions may restore CL to its linoleic acid-rich form and improve cardiac function by redirecting the remodeling process.

Retention of bile acids within the liver is a primary factor in the pathogenesis of cholestatic liver disorders, which are more common in human infants. The objective of this study was to evaluate developmental changes in mitochondrial factors involved in bile acid-induced hepatocyte injury. Hepatic mitochondria from adult rats (aged 9 weeks) underwent a mitochondrial permeability transition (MPT) and release of cytochrome c upon exposure to glycochenodeoxycholic acid (GCDC). In contrast, mitochondria from young rats (age 6–36 days) were resistant to MPT induction and cytochrome c release. Neither mitochondrial levels of MPT-associated proteins (voltage-dependent anion channel, cyclophilin D, or adenine nucleotide translocase), Bcl-2 family proteins, nor antioxidant enzymes explained this resistance. Mitochondria from young rats contained 2–3-fold higher α-tocopherol (α-TH). In vivo α-TH enrichment of adult hepatic mitochondria increased their MPT resistance. Tetra-linoleoyl cardiolipin (TL-CL), the primary molecular species of cardiolipin (CL), was reduced in mitochondria of the young rat; however, enrichment with CL and TL-CL only modestly increased their MPT susceptibility. In conclusion, we observed an unexpected resistance in young rats to bile acid induction of mitochondrial cell death pathways, which may be related to developmental differences in membrane composition.

Potential regulation of two factors linked to physiological outcomes with left ventricular (LV) hypertrophy, resistance to apoptosis, and matching of metabolic capacity, by the transcription factor cyclic-nucleotide regulatory element binding protein (CREB), was examined in the two models of physiological LV hypertrophy: involuntary treadmill running of female Sprague-Dawley rats and voluntary exercise wheel running in female C57Bl/6 mice. Comparative studies were performed in the models of pathological LV hypertrophy and failure: the spontaneously hypertension heart failure (SHHF) rat and the hypertrophic cardiomyopathy (HCM) transgenic mouse, a model of familial idiopathic cardiomyopathy. Activating CREB serine-133 phosphorylation was decreased early in remodeling in response to both physiological (decreased 50–80%) and pathological (decreased 60–80%) hypertrophic stimuli. Restoration of LV CREB phosphorylation occurred concurrent with completion of physiological hypertrophy (94% of sedentary control), but remained decreased (by 90%) during pathological hypertrophy. In all models of hypertrophy, CREB phosphorylation/activation demonstrated strong positive correlations with 1) expression of the anti-apoptotic protein bcl-2 (a CREB-dependent gene) and subsequent reductions in the activation of caspase 9 and caspase 3; 2) expression of peroxisome proliferator-activated receptor-γ coactivator-1 (PGC-1; a major regulator of mitochondrial content and respiratory capacity), and 3) LV mitochondrial respiratory rates and mitochondrial protein content. Exercise-induced increases in LV mitochondrial respiratory capacity were commensurate with increases observed in LV mass, as previously reported in the literature. Exercise training of SHHF rats and HCM mice in LV failure improved cardiac phenotype, increased CREB activation (31 and 118%, respectively), increased bcl-2 content, improved apoptotic status, and enhanced PGC-1 content and mitochondrial gene expression. Adenovirus-mediated expression of constitutively active CREB in neonatal rat cardiac recapitulated exercise-induced upregulation of PGC-1 content and mitochondrial oxidative gene expression. These data support a model wherein CREB contributes to physiological hypertrophy by enhancing expression of genes important for efficient oxidative capacity and resistance to apoptosis.

Cardiolipin (CL) is a structurally unique dimeric phospholipid localized in the inner mitochondrial membrane where it is required for optimal mitochondrial function. In addition to its role in maintaining membrane potential and architecture, CL is known to provide essential structural and functional support to several proteins involved in mitochondrial bioenergetics. A loss of CL content, alterations in its acyl chain composition, and/or CL peroxidation have been associated with mitochondrial dysfunction in multiple tissues in a variety of pathological conditions, including ischemia, hypothyroidism, aging, and heart failure. Recently, aberrations in CL metabolism have been implicated as a primary causative factor in the cardioskeletal myopathy known as Barth syndrome, underscoring an important role of CL in human health and disease. The purpose of this review is to provide an overview of evidence that has linked changes in the CL profile to mitochondrial dysfunction in various pathological conditions. In addition, a brief overview of CL function and biosynthesis, and a discussion of methods used to examine CL in biological tissues are provided.