Anomalous peroxidase activity of cytochrome c is the primary pathogenic target in Barth syndrome.

Barth syndrome (BTHS) is a life-threatening genetic disorder with unknown pathogenicity caused by mutations in TAFAZZIN (TAZ) that affect remodeling of mitochondrial cardiolipin (CL). TAZ deficiency leads to accumulation of mono-lyso-CL (MLCL), which forms a peroxidase complex with cytochrome c (cyt c) capable of oxidizing polyunsaturated fatty acid-containing lipids. We hypothesized that accumulation of MLCL facilitates formation of anomalous MLCL-cyt c peroxidase complexes and peroxidation of polyunsaturated fatty acid phospholipids as the primary BTHS pathogenic mechanism. Using genetic, biochemical/biophysical, redox lipidomic and computational approaches, we reveal mechanisms of peroxidase-competent MLCL-cyt c complexation and increased phospholipid peroxidation in different TAZ-deficient cells and animal models and in pre-transplant biopsies from hearts of patients with BTHS. A specific mitochondria-targeted anti-peroxidase agent inhibited MLCL-cyt c peroxidase activity, prevented phospholipid peroxidation, improved mitochondrial respiration of TAZ-deficient C2C12 myoblasts and restored exercise endurance in a BTHS Drosophila model. Targeting MLCL-cyt c peroxidase offers therapeutic approaches to BTHS treatment.

Tafazzin deficiency attenuates anti-cluster of differentiation 40 and interleukin-4 activation of mouse B lymphocytes.

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.

Tafazzin deficiency in mouse mesenchymal stem cells promote reprogramming of activated B lymphocytes toward immunosuppressive phenotypes.

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.

Altered cardiolipin metabolism is associated with cardiac mitochondrial dysfunction in pulmonary vascular remodeled perinatal rat pups.

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.

Adiponectin deficiency induces hepatic steatosis during pregnancy and gestational diabetes in mice.

AIMS/HYPOTHESIS: Obesity and hepatic steatosis are risk factors for gestational diabetes mellitus (GDM), a common complication of pregnancy. Adiponectin is a fat-derived hormone that improves hepatic steatosis and insulin sensitivity. Low levels of circulating adiponectin are associated with GDM development. We hypothesised that adiponectin deficiency causes fatty liver during pregnancy, contributing to the development of GDM. METHODS: To determine the role of adiponectin in fatty liver development during pregnancy, we compared pregnant (third week of pregnancy) adiponectin knockout (KO) mice (strain B6;129-Adipoqtm1Chan/J) with wild-type mice and assessed several variables of hepatic lipid metabolism and glucose homeostasis. The impact of adiponectin supplementation was measured by administering adenovirus-mediated full-length adiponectin at the end of the second week of pregnancy and comparing with green fluorescent protein control. RESULTS: In the third week of pregnancy, fasted pregnant adiponectin KO mice were hyperglycaemic on a low-fat diet (9.2 mmol/l vs 7.7 mmol/l in controls, p<0.05) and were glucose and pyruvate intolerant relative to wild-type mice. Pregnant adiponectin KO mice developed hepatic steatosis and a threefold elevation in hepatic triacylglycerols (p<0.05) relative to wild-type mice. Gestational weight gain and food consumption were similar in KO and wild-type mice. Adenoviral-mediated adiponectin supplementation to pregnant adiponectin KO mice improved glucose tolerance, prevented fasting hyperglycaemia and attenuated fatty liver development. CONCLUSIONS/INTERPRETATION: Adiponectin deficiency increased hepatic lipid accumulation during the period of pregnancy associated with increased fat utilisation. Consequently, adiponectin deficiency contributed to glucose intolerance, dysregulated gluconeogenesis and hyperglycaemia, all of which are characteristic of GDM. Increasing adiponectin in the last week of pregnancy alleviated hepatic steatosis and restored normal glucose homeostasis during pregnancy.

Tafazzin deficiency impairs mitochondrial metabolism and function of lipopolysaccharide activated B lymphocytes in mice.

B lymphocytes are responsible for humoral immunity and play a key role in the immune response. Optimal mitochondrial function is required to support B cell activity during activation. We examined how deficiency of tafazzin, a cardiolipin remodeling enzyme required for mitochondrial function, alters the metabolic activity of B cells and their response to activation by lipopolysaccharide in mice. B cells were isolated from 3-month-old wild type or tafazzin knockdown mice and incubated for up to 72 h with lipopolysaccharide and cell proliferation, expression of cell surface markers, secretion of antibodies and chemokines, proteasome and immunoproteasome activities, and metabolic function determined. In addition, proteomic analysis was performed to identify altered levels of proteins involved in survival, immunogenic, proteasomal and mitochondrial processes. Compared to wild type lipopolysaccharide activated B cells, lipopolysaccharide activated tafazzin knockdown B cells exhibited significantly reduced proliferation, lowered expression of cluster of differentiation 86 and cluster of differentiation 69 surface markers, reduced secretion of immunoglobulin M antibody, reduced secretion of keratinocytes-derived chemokine and macrophage-inflammatory protein-2, reduced proteasome and immunoproteasome activities, and reduced mitochondrial respiration and glycolysis. Proteomic analysis revealed significant alterations in key protein targets that regulate cell survival, immunogenicity, proteasomal processing and mitochondrial function consistent with the findings of the above functional studies. The results indicate that the cardiolipin transacylase enzyme tafazzin plays a key role in regulating mouse B cell function and metabolic activity during activation through modulation of mitochondrial function.

Tafazzin deficiency in mouse mesenchymal stem cells potentiates their immunosuppression and impairs activated B lymphocyte immune 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.

Berberine elevates cardiolipin in heart of offspring from mouse dams with high fat diet-induced gestational diabetes mellitus.

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.

Tafazzin Deficiency Reduces Basal Insulin Secretion and Mitochondrial Function in Pancreatic Islets From Male Mice.

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 ß 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 ß-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 ß-cell function. These data indicate a novel role for TAZ in regulating pancreatic islet function, particularly under low-glucose conditions.

Supplemental Berberine in a High-Fat Diet Reduces Adiposity and Cardiac Dysfunction in Offspring of Mouse Dams with Gestational Diabetes Mellitus.

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.

Cardiolipin deficiency elevates susceptibility to a lipotoxic hypertrophic cardiomyopathy.

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.

A Phytosterolemic Mixture of Sterols Inhibits Cholesterol Synthesis, Esterification, and Low-Density Lipoprotein Receptor mRNA Abundance in HepG2 Cells.

HepG2 cells were incubated with a 16.5:1.7:1 ratio of cholesterol:sitosterol:campesterol (CSC), a ratio of the major sterols observed in the plasma of phytosterolemia patients, or with cholesterol alone in combination with [14 C]acetate for 24 h and the radioactivity incorporated into lipids determined. Cells incubated with CSC exhibited a 40% reduction in cholesterol esterification (p < 0.05) compared to cells incubated with cholesterol alone. In addition, a 17.5-fold reduction (p < 0.05) in total cholesterol (cholesterol plus cholesteryl ester) synthesis from [14 C]acetate was observed in cells incubated with CSC compared to cholesterol alone. Low-density lipoprotein receptor (LDLR) mRNA abundance was lower in cells incubated with CSC compared to cells incubated with cholesterol alone. Our results suggest that incubation of HepG2 cells with a ratio of sterols that mimic the plasma concentration seen in phytosterolemia patients reduces cholesterol esterification, total cholesterol synthesis, and inhibits LDLR mRNA abundance. We suggest that future cell and animal-based work on phytostosterolemia might employ this methodology to serve as a novel paradigm of the disease.

Simvastatin increases temozolomide-induced cell death by targeting the fusion of autophagosomes and lysosomes.

Temozolomide (TMZ) is a chemotherapy agent used to treat Grade IV astrocytoma, also known as glioblastoma (GBM). TMZ treatment causes DNA damage that results in tumor cell apoptosis and increases the survival rate of GBM patients. However, chemoresistance as a result of TMZ-induced autophagy significantly reduces this anticancer effects over time. Statins are competitive inhibitors of HMG-CoA reductase, the rate-limiting enzyme of the mevalonate (MEV) cascade. Statins are best known for their cholesterol (CH)-lowering effect. Long-term consumption of statins, prior to and in parallel with other cancer therapeutic approaches, has been reported to increase the survival rate of patients with various forms of cancers. In this study, we investigated the potentiation of TMZ-induced apoptosis by simvastatin (Simva) in human GBM cell lines and patient GBM cells, using cell monolayers and three-dimensional cell culture systems. The incubation of cells with a combination of Simva and TMZ resulted in a significant increase in apoptotic cells compared to cells treated with TMZ alone. Incubation of cells with CH or MEV cascade intermediates failed to compensate the decrease in cell viability induced by the combined Simva and TMZ treatment. Simva treatment inhibited the autophagy flux induced by TMZ by blocking autophago-lysosome formation. Our results suggest that Simva sensitizes GBM cells to TMZ-induced cell death in a MEV cascade-independent manner and identifies the inhibition of autophagosome-lysosome fusion as a promising therapeutic strategy in the treatment of GBM.

Correction to: Myocardin regulates mitochondrial calcium homeostasis and prevents permeability transition.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Maternal resveratrol administration protects against gestational diabetes-induced glucose intolerance and islet dysfunction in the rat offspring.

KEY POINTS: Maternal resveratrol (RESV) administration in gestational diabetes (GDM) restored normoglycaemia and insulin secretion. GDM-induced obesity was prevented in male GDM+RESV offspring but not in females. GDM+RESV offspring exhibited improved glucose tolerance and insulin sensitivity. GDM+RESV restored hepatic glucose homeostasis in offspring. Glucose-stimulated insulin secretion was enhanced in GDM+RESV offspring. ABSTRACT: Gestational diabetes (GDM), the most common complication of pregnancy, is associated with adverse metabolic health outcomes in offspring. Using a rat model of diet-induced GDM, we investigated whether maternal resveratrol (RESV) supplementation (147 mg kg-1  day-1 ) in the third week of pregnancy could improve maternal glycaemia and protect the offspring from developing metabolic dysfunction. Female Sprague-Dawley rats consumed a high-fat and sucrose (HFS) diet to induce GDM. Lean controls consumed a low-fat (LF) diet. In the third trimester, when maternal hyperglycaemia was observed, the HFS diet was supplemented with RESV. At weaning, offspring were randomly assigned a LF or HFS diet until 15 weeks of age. In pregnant dams, RESV restored glucose tolerance, normoglycaemia and improved insulin secretion. At 15 weeks of age, GDM+RESV-HFS male offspring were less obese than the GDM-HFS offspring. By contrast, the female GDM+RESV-HFS offspring were similarly as obese as the GDM-HFS group. Hepatic steatosis, insulin resistance, glucose intolerance and dysregulated gluconeogenesis were observed in the male GDM offspring and were attenuated in the offspring of GDM+RESV dams. The dysregulation of several metabolic genes (e.g. ppara, lpl, pepck and g6p) in the livers of GDM offspring was attenuated in the GDM+RESV offspring group. Glucose stimulated insulin secretion was also improved in the islets from offspring of GDM+RESV dams. Thus, maternal RESV supplementation during the third trimester of pregnancy and lactation induced several beneficial metabolic health outcomes for both mothers and offspring. Therefore, RESV could be an alternative to current GDM treatments.

Gestational Diabetes Adversely Affects Pancreatic Islet Architecture and Function in the Male Rat Offspring.

Fetal exposure to gestational diabetes mellitus (GDM) and poor postnatal diet are strong risk factors for type 2 diabetes development later in life, but the mechanisms connecting GDM exposure to offspring metabolic health remains unclear. In this study, we aimed to determine how GDM interacts with the postnatal diet to affect islet function in the offspring as well as characterize the gene expression changes in the islets. GDM was induced in female rats using a high-fat, high-sucrose (HFS) diet, and litters from lean or GDM dams were weaned onto a low-fat (LF) or HFS diet. Compared with the lean control offspring, GDM exposure reduced glucose-stimulated insulin secretion in islets isolated from 15-week-old offspring, which was additively worsened when GDM exposure was combined with postnatal HFS diet consumption. In the HFS diet-fed offspring of lean dams, islet size and number increased, an adaptation that was not observed in the HFS diet-fed offspring of GDM dams. Islet gene expression in the offspring of GDM dams was altered in such categories as inflammation (e.g., Il1b, Ccl2), mitochondrial function/oxidative stress resistance (e.g., Atp5f1, Sod2), and ribosomal proteins (e.g., Rps6, Rps14). These results demonstrate that GDM exposure induced marked changes in gene expression in the male young adult rat offspring that cumulatively interact to worsen islet function, whole-body glucose homeostasis, and adaptations to HFS diets.

Disentangling oxidation/hydrolysis reactions of brain mitochondrial cardiolipins in pathogenesis of traumatic injury.

Mechanical injury to the brain triggers multiple biochemical events whose specific contributions to the pathogenesis define clinical manifestations and the overall outcome. Among many factors, mitochondrial injury has recently attracted much attention due to the importance of the organelle for bioenergetics as well as intra- and extracellular signaling and cell death. Assuming the essentiality of a mitochondria-unique phospholipid, cardiolipin (CL), for the structural and functional organization of mitochondria, here we applied global (phospho) lipidomics and redox lipidomics to reveal and identify CL modifications during controlled cortical impact (CCI). We revealed 2 major pathways activated in the CCI-injured brain as time-specific responses: early accumulation of oxidized CL (CLox) products was followed by hydrolytic reactions yielding monolyso-CLs (mCLs) and free fatty acids. To quantitatively assess possible specific roles of peroxidation and hydrolysis of mitochondrial CL, we performed comparative studies of CL modifications using an animal model of Barth syndrome where deficiency of CL reacylation (Tafazzin [Taz] deficiency) was associated exclusively with the accumulation of mCLs (but not CLox). By comparing the in vitro and in vivo results with genetic manipulation of major CL-, CLox-, and mCL-metabolizing enzymes, calcium-independent phospholipase A2γ and Taz, we concluded that the 2 processes - CL oxidation and CL hydrolysis - act as mutually synergistically enhancing components of the pathogenic mechanism of mitochondrial injury in traumatic brain injury. This emphasizes the need for combined therapeutic approaches preventing the formation of both CLox and mCL.

Aberrant cardiolipin metabolism is associated with cognitive deficiency and hippocampal alteration in tafazzin knockdown mice.

Cardiolipin (CL) is a key mitochondrial phospholipid essential for mitochondrial energy production. CL is remodeled from monolysocardiolipin (MLCL) by the enzyme tafazzin (TAZ). Loss-of-function mutations in the gene which encodes TAZ results in a rare X-linked disorder called Barth Syndrome (BTHS). The mutated TAZ is unable to maintain the physiological CL:MLCL ratio, thus reducing CL levels and affecting mitochondrial function. BTHS is best known as a cardiac disease, but has been acknowledged as a multi-syndrome disorder, including cognitive deficits. Since reduced CL levels has also been reported in numerous neurodegenerative disorders, we examined how TAZ-deficiency impacts cognitive abilities, brain mitochondrial respiration and the function of hippocampal neurons and glia in TAZ knockdown (TAZ kd) mice. We have identified for the first time the profile of changes that occur in brain phospholipid content and composition of TAZ kd mice. The brain of TAZ kd mice exhibited reduced TAZ protein expression, reduced total CL levels and a 19-fold accumulation of MLCL compared to wild-type littermate controls. TAZ kd brain exhibited a markedly distinct profile of CL and MLCL molecular species. In mitochondria, the activity of complex I was significantly elevated in the monomeric and supercomplex forms with TAZ-deficiency. This corresponded with elevated mitochondrial state I respiration and attenuated spare capacity. Furthermore, the production of reactive oxygen species was significantly elevated in TAZ kd brain mitochondria. While motor function remained normal in TAZ kd mice, they showed significant memory deficiency based on novel object recognition test. These results correlated with reduced synaptophysin protein levels and derangement of the neuronal CA1 layer in hippocampus. Finally, TAZ kd mice had elevated activation of brain immune cells, microglia compared to littermate controls. Collectively, our findings demonstrate that TAZ-mediated remodeling of CL contributes significantly to the expansive distribution of CL molecular species in the brain, plays a key role in mitochondria respiratory activity, maintains normal cognitive function, and identifies the hippocampus as a potential therapeutic target for BTHS.

Phosphokinome Analysis of Barth Syndrome Lymphoblasts Identify Novel Targets in the Pathophysiology of the Disease.

Barth Syndrome (BTHS) is a rare X-linked genetic disease in which the specific biochemical deficit is a reduction in the mitochondrial phospholipid cardiolipin (CL) as a result of a mutation in the CL transacylase tafazzin. We compared the phosphokinome profile in Epstein-Barr-virus-transformed lymphoblasts prepared from a BTHS patient with that of an age-matched control individual. As expected, mass spectrometry analysis revealed a significant (>90%) reduction in CL in BTHS lymphoblasts compared to controls. In addition, increased oxidized phosphatidylcholine (oxPC) and phosphatidylethanolamine (PE) levels were observed in BTHS lymphoblasts compared to control. Given the broad shifts in metabolism associated with BTHS, we hypothesized that marked differences in posttranslational modifications such as phosphorylation would be present in the lymphoblast cells of a BTHS patient. Phosphokinome analysis revealed striking differences in the phosphorylation levels of phosphoproteins in BTHS lymphoblasts compared to control cells. Some phosphorylated proteins, for example, adenosine monophosphate kinase, have been previously validated as bonafide modified phosphorylation targets observed in tafazzin deficiency or under conditions of reduced cellular CL. Thus, we report multiple novel phosphokinome targets in BTHS lymphoblasts and hypothesize that alteration in the phosphokinome profile may provide insight into the pathophysiology of BTHS and potential therapeutic targets.

Myocardin regulates mitochondrial calcium homeostasis and prevents permeability transition.

Myocardin is a transcriptional co-activator required for cardiovascular development, but also promotes cardiomyocyte survival through an unclear molecular mechanism. Mitochondrial permeability transition is implicated in necrosis, while pore closure is required for mitochondrial maturation during cardiac development. We show that loss of myocardin function leads to subendocardial necrosis at E9.5, concurrent with elevated expression of the death gene Nix. Mechanistically, we demonstrate that myocardin knockdown reduces microRNA-133a levels to allow Nix accumulation, leading to mitochondrial permeability transition, reduced mitochondrial respiration, and necrosis. Myocardin knockdown elicits calcium release from the endo/sarcoplasmic reticulum with mitochondrial calcium accumulation, while restoration of microRNA-133a function, or knockdown of Nix rescues calcium perturbations. We observed reduced myocardin and elevated Nix expression within the infarct border-zone following coronary ligation. These findings identify a myocardin-regulated pathway that maintains calcium homeostasis and mitochondrial function during development, and is attenuated during ischemic heart disease. Given the diverse role of Nix and microRNA-133a, these findings may have broader implications to metabolic disease and cancer.