Respiration In Cardiomyocytes Research Articles

ISG15 blocks cardiac glycolysis and ensures sufficient mitochondrial energy production during Coxsackievirus B3 infection.

Virus infection triggers inflammation and, may impose nutrient shortage to the heart. Supported by type I interferon (IFN) signalling, cardiomyocytes counteract infection by various effector processes, with the IFN-stimulated gene of 15 kDa (ISG15) system being intensively regulated and protein modification with ISG15 protecting mice Coxsackievirus B3 (CVB3) infection. The underlying molecular aspects how the ISG15 system affects the functional properties of respective protein substrates in the heart are unknown. Based on the protective properties due to protein ISGylation, we set out a study investigating CVB3-infected mice in depth and found cardiac atrophy with lower cardiac output in ISG15-/- mice. By mass spectrometry, we identified the protein targets of the ISG15 conjugation machinery in heart tissue and explored how ISGylation affects their function. The cardiac ISGylome showed a strong enrichment of ISGylation substrates within glycolytic metabolic processes. Two control enzymes of the glycolytic pathway, hexokinase 2 (HK2) and phosphofructokinase muscle form (PFK1), were identified as bona fide ISGylation targets during infection. In an integrative approach complemented with enzymatic functional testing and structural modelling, we demonstrate that protein ISGylation obstructs the activity of HK2 and PFK1. Seahorse-based investigation of glycolysis in cardiomyocytes revealed that, by conjugating proteins, the ISG15 system prevents the infection-/IFN-induced up-regulation of glycolysis. We complemented our analysis with proteomics-based advanced computational modelling of cardiac energy metabolism. Our calculations revealed an ISG15-dependent preservation of the metabolic capacity in cardiac tissue during CVB3 infection. Functional profiling of mitochondrial respiration in cardiomyocytes and mouse heart tissue by Seahorse technology showed an enhanced oxidative activity in cells with a competent ISG15 system. Our study demonstrates that ISG15 controls critical nodes in cardiac metabolism. ISG15 reduces the glucose demand, supports higher ATP production capacity in the heart, despite nutrient shortage in infection, and counteracts cardiac atrophy and dysfunction.

Increased dosage of DYRK1A leads to congenital heart defects in a mouse model of Down syndrome.

Down syndrome (DS) is caused by trisomy of human chromosome 21 (Hsa21). DS is a gene dosage disorder that results in multiple phenotypes including congenital heart defects. This clinically important cardiac pathology is the result of a third copy of one or more of the approximately 230 genes on Hsa21, but the identity of the causative dosage-sensitive genes and hence mechanisms underlying this cardiac pathology remain unclear. Here, we show that hearts from human fetuses with DS and embryonic hearts from the Dp1Tyb mouse model of DS show reduced expression of mitochondrial respiration genes and cell proliferation genes. Using systematic genetic mapping, we determined that three copies of the dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1a) gene, encoding a serine/threonine protein kinase, are associated with congenital heart disease pathology. In embryos from Dp1Tyb mice, reducing Dyrk1a gene copy number from three to two reversed defects in cellular proliferation and mitochondrial respiration in cardiomyocytes and rescued heart septation defects. Increased dosage of DYRK1A protein resulted in impairment of mitochondrial function and congenital heart disease pathology in mice with DS, suggesting that DYRK1A may be a useful therapeutic target for treating this common human condition.

A potential adverse role for leptin and cardiac leptin receptor in the right ventricle in pulmonary arterial hypertension: effect of metformin is BMPR2 mutation-specific.

Pulmonary arterial hypertension is a fatal cardiopulmonary disease. Leptin, a neuroendocrine hormone released by adipose tissue, has a complex relationship with cardiovascular diseases, including PAH. Leptin is thought to be an important factor linking metabolic syndrome and cardiovascular disorders. Given the published association between metabolic syndrome and RV dysfunction in PAH, we sought to determine the association between leptin and RV dysfunction. We hypothesized that in PAH-RV, leptin influences metabolic changes via leptin receptors, which can be manipulated by metformin. Plasma leptin was measured in PAH patients and healthy controls from a published trial of metformin in PAH. Leptin receptor localization was detected in RV from PAH patients, healthy controls, animal models of PH with RV dysfunction before and after metformin treatment, and cultured cardiomyocytes with two different BMPR2 mutants by performing immunohistochemical and cell fractionation studies. Functional studies were conducted in cultured cardiomyocytes to examine the role of leptin and metformin in lipid-driven mitochondrial respiration. In human studies, we found that plasma leptin levels were higher in PAH patients and moderately correlated with higher BMI, but not in healthy controls. Circulating leptin levels were reduced by metformin treatment, and these findings were confirmed in an animal model of RV dysfunction. Leptin receptor expression was increased in PAH-RV cardiomyocytes. In animal models of RV dysfunction and cultured cardiomyocytes with BMPR2 mutation, we found increased expression and membrane localization of the leptin receptor. In cultured cardiomyocytes with BMPR2 mutation, leptin moderately influences palmitate uptake, possibly via CD36, in a mutation-specific manner. Furthermore, in cultured cardiomyocytes, the Seahorse XFe96 Extracellular Flux Analyzer and gene expression data indicate that leptin may not directly influence lipid-driven mitochondrial respiration in BMPR2 mutant cardiomyocytes. However, metformin alone or when supplemented with leptin can improve lipid-driven mitochondrial respiration in BMPR2 mutant cardiomyocytes. The effect of metformin on lipid-driven mitochondrial respiration in cardiomyocytes is BMPR2 mutation-specific. In PAH, increased circulating leptin can influence metabolic signaling in RV cardiomyocytes via the leptin receptor; in particular, it may alter lipid-dependent RV metabolism in combination with metformin in a mutation-specific manner and warrants further investigation.

Abstract P3194: Redd1 Is Required For Glucose Metabolism And Insulin Sensitivity In Cardiomyocytes

Type II diabetes (TIID) is characterized by insulin resistance (IR) and doubles a patient’s risk for cardiovascular disease (CVD). The pathogenesis of CVD in patients with TIID, or diabetic cardiomyopathy (DC), is unclear. Regulated in development and DNA damage (REDD)1 is a negative regulator of mammalian target of rapamycin complex (mTORC)1. Notably, deletion of REDD1 leads to systemic IR in mice. REDD1 is, therefore, necessary for insulin sensitivity, however the mechanisms required remain unclear. We hypothesized that REDD1 is required for glucose oxidation and, therefore, insulin sensitivity in cardiomyocytes. To examine the role of REDD1 in cardiomyocyte glucose metabolism and energetics, we cultured wild type (WT) AC16 cardiomyocytes or those with REDD1 deletion with 5.5 mM glucose and examined cellular respiration. The data demonstrate compromised respiration, as indicated by reduced maximal respiratory capacity in REDD1-null versus WT cells (1.5-fold±0.214). Importantly, western blotting confirmed the absence of REDD1 in cells with REDD1 deletion, as well as increased phosphorylation of eukaryotic translation initiation factor 4E-binding protein (p4E-BP)1 (Thr37/46) (1.3-fold±0.085), a direct target of mTORC1. Thus, REDD1 inhibits mTORC1 signaling and enhances glucose-driven respiration in cardiomyocytes. To examine the mechanisms by which REDD1 supports glucose utilization, we examined levels of phosphorylated pyruvate dehydrogenase (pPDH) (Ser293). Our data show that pPDH is increased in REDD1-null cells versus WT controls (1.2-fold±0.054), as well as in hearts from REDD1-deficient mice (1.2-fold±0.013), suggesting reduced glucose oxidation. We also observed enhanced extracellular acidification, an indirect measure of glycolysis, in our REDD1-null cells versus WT controls (1.7-fold±0.136). Pyruvate dehydrogenase kinases (PDKs) phosphorylate PDH. In our REDD1-null cells, we observe a significant increase in PDK4 mRNA versus WT controls (8-fold±0.593), as assessed by qPCR and consistent with the diabetic heart. Together, these data suggest that REDD1 inhibits mTORC1 activity and the expression of PDK4, activating PDH, and glucose oxidation as the mechanism by which cardiomyocytes remain insulin sensitive.

Respiration in isolated cardiomyocytes and microviscosity of their membranes in rats of different ages with heart failure

Background. According to the latest epidemiological data, heart failure (HF) is diagnosed in 10% of adult population over 70 years old. However, currently this diagnosis is being increasingly made in young and middleaged people. The pathogenesis of HF may be based on a decrease in respiration in cardiomyocytes with age, which affects the function of energy-dependent processes in cells.Aim. To study respiration in cardiomyocytes and microviscosity of their membranes in rats aged 2 and 15 months with heart failure.Materials and methods. The study was carried out on male Wistar rats. The animals were divided into 4 groups: 2 groups of intact rats aged 2 and 15 months (n = 12) and 2 groups of animals of similar ages (n = 10) with a model of HF. In the latter, HF formed by day 28 after a double subcutaneous injection of isoproterenol hydrochloride at a dose of 170 mg / kg with an interval of 24 hours. Isolated cardiomyocytes were obtained from enzyme-washed rat hearts. Cell respiration was studied in a thermostated chamber in an incubation medium supplemented with phosphorylation (ADP) and oxidation (succinate) substrates. The respiratory control (RC) ratio was calculated by dividing V3 respiration state to V4. Membrane microviscosity characteristics were assessed by the eximerization coefficient of pyrene fluorescence in the areas of protein – lipid and lipid – lipid interactions.Results. RC in cardiomyocyte membranes of intact animals did not change with age. In 2-month-old rats with HF, respiratory control ratio (RCR) did not change compared with the intact age-matched controls. In 15-month-old rats with HF, there was a significant decrease in RC of cardiomyocytes (CM) compared with the intact animals of this age and 2-month-old rats with HF. An age-dependent decrease in the microviscosity of CM membranes in the areas of lipid – lipid interactions and no significant changes in the parameter at the sites of protein – lipid interactions were noted. In 2-month-old animals with HF, the microviscosity of CM membranes in the areas of protein – lipid interactions significantly decreased, and in 15-month-old rats it increased, compared with the intact controls. When carrying out an intergroup comparison, an age-dependent increase in the microviscosity of CM membranes in the areas of protein – lipid interactions and no differences in the parameter in the areas of lipid – lipid interactions were revealed.Conclusion. In the intact rats, the absence of significant changes in respiration with age was revealed. In the 15-month-old animals with HF, respiration in CM was significantly lower than in the intact controls and 2-monthold animals with HF. These changes may be due to the differences in the membrane microviscosity characteristics in different periods of ontogenesis.

PGRMC1 Ablation Protects from Energy-Starved Heart Failure by Promoting Fatty Acid/Pyruvate Oxidation.

Heart failure (HF) is an emerging epidemic with a high mortality rate. Apart from conventional treatment methods, such as surgery or use of vasodilation drugs, metabolic therapy has been suggested as a new therapeutic strategy. The heart relies on fatty acid oxidation and glucose (pyruvate) oxidation for ATP-mediated contractility; the former meets most of the energy requirement, but the latter is more efficient. Inhibition of fatty acid oxidation leads to the induction of pyruvate oxidation and provides cardioprotection to failing energy-starved hearts. One of the non-canonical types of sex hormone receptors, progesterone receptor membrane component 1 (Pgrmc1), is a non-genomic progesterone receptor associated with reproduction and fertility. Recent studies revealed that Pgrmc1 regulates glucose and fatty acid synthesis. Notably, Pgrmc1 has also been associated with diabetic cardiomyopathy, as it reduces lipid-mediated toxicity and delays cardiac injury. However, the mechanism by which Pgrmc1 influences the energy-starved failing heart remains unknown. In this study, we found that loss of Pgrmc1 inhibited glycolysis and increased fatty acid/pyruvate oxidation, which is directly associated with ATP production, in starved hearts. Loss of Pgrmc1 during starvation activated the phosphorylation of AMP-activated protein kinase, which induced cardiac ATP production. Pgrmc1 loss increased the cellular respiration of cardiomyocytes under low-glucose conditions. In isoproterenol-induced cardiac injury, Pgrmc1 knockout resulted in less fibrosis and low heart failure marker expression. In summary, our results revealed that Pgrmc1 ablation in energy-deficit conditions increases fatty acid/pyruvate oxidation to protect against cardiac damage via energy starvation. Moreover, Pgrmc1 may be a regulator of cardiac metabolism that switches the dominance of glucose-fatty acid usage according to nutritional status and nutrient availability in the heart.

KDM8 epigenetically controls cardiac metabolism to prevent initiation of dilated cardiomyopathy

Cardiac metabolism is deranged in heart failure, but underlying mechanisms remain unclear. Here, we show that lysine demethylase 8 (Kdm8) maintains an active mitochondrial gene network by repressing Tbx15, thus preventing dilated cardiomyopathy leading to lethal heart failure. Deletion of Kdm8 in mouse cardiomyocytes increased H3K36me2 with activation of Tbx15 and repression of target genes in the NAD+ pathway before dilated cardiomyopathy initiated. NAD+ supplementation prevented dilated cardiomyopathy in Kdm8 mutant mice, and TBX15 overexpression blunted NAD+-activated cardiomyocyte respiration. Furthermore, KDM8 was downregulated in human hearts affected by dilated cardiomyopathy, and higher TBX15 expression defines a subgroup of affected hearts with the strongest downregulation of genes encoding mitochondrial proteins. Thus, KDM8 represses TBX15 to maintain cardiac metabolism. Our results suggest that epigenetic dysregulation of metabolic gene networks initiates myocardium deterioration toward heart failure and could underlie heterogeneity of dilated cardiomyopathy.

Reversible Thiol Oxidation Increases Mitochondrial Electron Transport Complex Enzyme Activity but Not Respiration in Cardiomyocytes from Patients with End-Stage Heart Failure.

Cardiomyocyte dysfunction in patients with end-stage heart failure with reduced ejection fraction (HFrEF) stems from mitochondrial dysfunction, which contributes to an energetic crisis. Mitochondrial dysfunction reportedly relates to increased markers of oxidative stress, but the impact of reversible thiol oxidation on myocardial mitochondrial function in patients with HFrEF has not been investigated. In the present study, we assessed mitochondrial function in ventricular biopsies from patients with end-stage HFrEF in the presence and absence of the thiol-reducing agent dithiothreitol (DTT). Isolated mitochondria exposed to DTT had increased enzyme activity of complexes I (p = 0.009) and III (p = 0.018) of the electron transport system, while complexes II (p = 0.630) and IV (p = 0.926) showed no changes. However, increased enzyme activity did not carry over to measurements of mitochondrial respiration in permeabilized bundles. Oxidative phosphorylation conductance (p = 0.439), maximal respiration (p = 0.312), and ADP sensitivity (p = 0.514) were unchanged by 5 mM DTT treatment. These results indicate that mitochondrial function can be modulated through reversible thiol oxidation, but other components of mitochondrial energy transfer are rate limiting in end-stage HFrEF. Optimal therapies to normalize cardiac mitochondrial respiration in patients with end-stage HFrEF may benefit from interventions to reverse thiol oxidation, which limits complex I and III activities.

The molecular role of Sigmar1 in regulating mitochondrial function through mitochondrial localization in cardiomyocytes

Sigmar1 is a widely expressed molecular chaperone protein in mammalian cell systems. Accumulating research demonstrated the cardioprotective roles of pharmacologic Sigmar1 activation by ligands in preclinical rodent models of cardiac injury. Extensive biochemical and immuno-electron microscopic research demonstrated Sigmar1′s sub-cellular localization largely depends on cell and organ types. Despite comprehensive studies, Sigmar1′s direct molecular role in cardiomyocytes remains elusive. In the present study, we determined Sigmar1′s subcellular localization, transmembrane topology, and function using complementary microscopy, biochemical, and functional assays in cardiomyocytes. Quantum dots in transmission electron microscopy showed Sigmar1 labeled quantum dots on the mitochondrial membranes, lysosomes, and sarcoplasmic reticulum-mitochondrial interface. Subcellular fractionation of heart cell lysates confirmed Sigmar1′s localization in purified mitochondria fraction and lysosome fraction. Immunocytochemistry confirmed Sigmar1 colocalization with mitochondrial proteins in isolated adult mouse cardiomyocytes. Sigmar1′s mitochondrial localization was further confirmed by Sigmar1 colocalization with Mito-Tracker in isolated mouse heart mitochondria. A series of biochemical experiments, including alkaline extraction and proteinase K treatment of purified heart mitochondria, demonstrated Sigmar1 as an integral mitochondrial membrane protein. Sigmar1′s structural requirement for mitochondrial localization was determined by expressing FLAG-tagged Sigmar1 fragments in cells. Full-length Sigmar1 and Sigmar1′s C terminal-deletion fragments were able to localize to the mitochondrial membrane, whereas N-terminal deletion fragment was unable to incorporate into the mitochondria. Finally, functional assays using extracellular flux analyzer and high-resolution respirometry showed Sigmar1 siRNA knockdown significantly altered mitochondrial respiration in cardiomyocytes. Overall, we found that Sigmar1 localizes to mitochondrial membranes and is indispensable for maintaining mitochondrial respiratory homeostasis in cardiomyocytes.

Anthelmintic niclosamide attenuates pressure-overload induced heart failure in mice

The heart is a high energy demand organ and enhancing mitochondrial function is proposed as the next-generation therapeutics for heart failure. Our previous study found that anthelmintic drug niclosamide enhanced mitochondrial respiration and increased adenosine triphosphate (ATP) production in cardiomyocytes, therefore, this study aimed to determine the effect of niclosamide on heart failure in mice and the potential molecular mechanisms. The heart failure model was induced by transverse aortic constriction (TAC) in mice. Oral administration of niclosamide improved TAC-induced cardiac hypertrophy, cardiac fibrosis, and cardiac dysfunction in mice. Oral administration of niclosamide reduced TAC-induced increase of serum IL-6 in heart failure mice. In vitro, niclosamide within 0.1 μM increased mitochondrial respiration and ATP production in mice heart tissues. At the concentrations more than 0.1 μM, niclosamide reduced the increased interleukin- 6 (IL-6) mRNA expression in lipopolysaccharide (LPS)-stimulated RAW264.7 and THP-1 derived macrophages. In cultured primary cardiomyocytes and cardiac fibroblasts, niclosamide (more than 0.1 μM) suppressed IL-6- and phenylephrine-induced cardiomyocyte hypertrophy, and inhibited collagen secretion from cardiac fibroblasts. In conclusion, niclosamide attenuates heart failure in mice and the underlying mechanisms include enhancing mitochondrial respiration of cardiomyocytes, inhibiting collagen secretion from cardiac fibroblasts, and reducing the elevated serum inflammatory mediator IL-6. The present study suggests that niclosamide might be therapeutic for heart failure.

Metabolic Alterations in Cardiomyocytes are Associated with Methamphetamine‐Induced Cardiomyopathy

IntroductionIllicit use of Methamphetamine (METH) in humans is associated with cardiac dysfunction known as METH‐induced cardiomyopathy. Specifically, the stimulant inhibits mitochondrial bioenergetics, resulting in energy deprivation, which may ultimately lead to the activation of the adaptive autophagy process.ObjectivesWe aim to determine the METH‐induced metabolic alterations in cardiomyocytes leading to the development of the METH‐associated cardiomyopathy.Methods and ResultsAutopsy heart samples from humans with a positive toxicology for METH use showed substantial fibrosis in the heart. ‘Binge’ METH‐administration in mice for four weeks developed cardiac hypertrophy and fibrosis in the heart. Mitochondria isolated from the METH‐treated heart showed defects in mitochondrial respiration. Western blot analysis of autophagy regulatory protein expression ‘Binge’ METH‐treated mice showed increased expression of LC3B‐II and p62 proteins. To determine the direct non‐catecholamine dependent effect of METH on cardiomyocytes, we treated primary cardiomyocytes with different doses of METH (100 and 250 μM). Western Blot analysis showed a dose and time‐dependent increase in the expression of LC3B‐II and autophagy‐related protein 7 (Atg7). We also observed increased phosphorylation of cellular metabolism‐related proteins, i.e., phosphorylated FOXO1 (at Ser 256) and FOXO3a (at Ser 253). We conducted autophagy flux assay to confirm the METH‐induced activation of autophagy exposing cardiomyocytes with METH (250 μM for 24 hours) followed by treatment with lysosomal inhibitor BafilomycinA1 (50nM for 3 hours). Western Blot analysis showed increased expression of LC3B‐II in BafA1 treated cardiomyocytes suggesting activation of autophagy following METH‐exposure.ConclusionsWe report that METH use causes adverse fibrotic remodeling in hearts from both human and pre‐clinical mouse model. We found that METH suppresses mitochondrial respiration in cardiomyocytes that elicit adaptive activation of autophagy and autophagy regulatory proteins as a response to METH‐induced energy deprivation.Support or Funding InformationThis project was performed as part of the CURIOUS program supported by grant R25HL147665 from the NIH/NHLBI. This work was further supported by NIH/NHLBI grants R00 HL122354, R01HL145753, R01HL145753‐01S1 and PGM121307A to Dr. Bhuiyan. American Heart Association postdoctoral fellowship to Dr. Alam; Malcolm Fiest post‐ and pre‐doctoral fellowships to Dr. Abdullah and Aishwarya by CCDS, LSUHSC‐S.

Linoleic acid improves assembly of the CII subunit and CIII2/CIV complex of the mitochondrial oxidative phosphorylation system in heart failure

BackgroundLinoleic acid is the major fatty acid moiety of cardiolipin, which is central to the assembly of components involved in mitochondrial oxidative phosphorylation (OXPHOS). Although linoleic acid is an essential nutrient, its excess intake is harmful to health. On the other hand, linoleic acid has been shown to prevent the reduction in cardiolipin content and to improve mitochondrial function in aged rats with spontaneous hypertensive heart failure (HF). In this study, we found that lower dietary intake of linoleic acid in HF patients statistically correlates with greater severity of HF, and we investigated the mechanisms therein involved.MethodsHF patients, who were classified as New York Heart Association (NYHA) functional class I (n = 45), II (n = 93), and III (n = 15), were analyzed regarding their dietary intakes of different fatty acids during the one month prior to the study. Then, using a mouse model of HF, we confirmed reduced cardiolipin levels in their cardiac myocytes, and then analyzed the mechanisms by which dietary supplementation of linoleic acid improves cardiac malfunction of mitochondria.ResultsThe dietary intake of linoleic acid was significantly lower in NYHA III patients, as compared to NYHA II patients. In HF model mice, both CI-based and CII-based OXPHOS activities were affected together with reduced cardiolipin levels. Silencing of CRLS1, which encodes cardiolipin synthetase, in cultured cardiomyocytes phenocopied these events. Feeding HF mice with linoleic acid improved both CI-based and CII-based respiration as well as left ventricular function, together with an increase in cardiolipin levels. However, although assembly of the respirasome (i.e., CI/CIII2/CIV complex), as well as assembly of CII subunits and the CIII2/CIV complex statistically correlated with cardiolipin levels in cultured cardiomyocytes, respirasome assembly was not notably restored by dietary linoleic acid in HF mice. Therefore, although linoleic acid may significantly improve both CI-based and CII-based respiration of cardiomyocytes, respirasomes impaired by HF were not easily repaired by the dietary intake of linoleic acid.ConclusionsDietary supplement of linoleic acid is beneficial for improving cardiac malfunction in HF, but is unable to completely cure HF.

Abstract 287: Altered Mitochondrial Flash Activity and mPTP Opening in the Aged Heart Are Reversed by Elamipretide Treatment

Rationale: The mitochondrial theory of aging pinpoints mitochondria as the major reactive oxygen species (ROS) production site and target of oxidative stress during aging. Mitochondrial flash (mitoflash) is recently discovered excitable event that is a manifestation of ROS production combined with pH change and coupled with mitochondrial permeability transition pore (mPTP) opening. Our preliminary data shown that 8-week SS-31 peptide (elamipretide) protects the functional performance of the aged mouse heart. This study aims to investigate mitoflash activity and mPTP opening in the aged heart, especially whether and how SS-31 protects the heart function involving modulation of these activities. Methods and Results: By using confocal microscopy imaging on the isolated rat cardiomyocytes with overexpression of mitochondrial targeted cpYFP, we found mitoflash activity in the cells from old (26 mo) was higher than that in young (5 mo) rat cells (2.2 ± 0.2 in old vs 1.3 ± 0.2 in young, /1000μm 2 /100s, n=27-64, p<0.05). SS-31 10 μM treatment normalized the mitoflash activity to the young level. Similarly, by photon-excitation induced mPTP opening, we found that aging increased the mPTP opening and 1 μM SS-31 stabilized the mPTP. Furthermore, by using Seahorse Assay on intact primary isolated cardiomyocytes, we found mitochondrial basal respiration in cells from old mice was higher than that in young mouse cells, however, the maximal respiration rate was without significant difference. The increased basal respiration was attributed to a higher proton leak in old cardiomyocytes (131 ± 14 in 24 month vs 94 ± 11 in young, pmol/min/800cells, n=17-35, p<0.05). Interestingly, acute (2 hrs) in vitro treatment of the old cardiomyocytes with SS-31 100 nm decreased proton leak. Moreover, SS-31 (100 nM) decreased the superoxide production as measured by ratio of MitoSOX Red to MitoTrackerGreen. Conclusion: These results indicate that SS-31 directly protects cardiac aging through rapid rejuvenation of mitochondrial respiration in cardiomyocytes, and in particular, by reducing proton leak, mitochondrial flash activity, and decreasing the mPTP opening. This study helps to uncover the mechanism of the cardiac aging and the protective effect from SS-31 treatment.

Влияние свободных жирных кислот на поглощение кислорода изолированными кардиомиоцитами крыс при ишемическом или диабетическом поражении сердца

Цель исследования - оценка влияния свободных полиненасыщенных и насыщенных жирных кислот (ЖК) на поглощение кислорода изолированными кардиомиоцитами интактных крыс и крыс с ишемическим или диабетическим поражением сердца. Методика. Опыты проведены на крысах-самцах Вистар (250-300 г). Инфаркт миокарда вызывали перевязкой левой коронарной артерии, сахарный диабет (СД) - однократной инъекцией стрептозотоцина в дозе 60 мг/кг, внутрибрюшинно. Изолированные кардиомиоциты получали ферментативным способом. Потребление кислорода оценивали полярографически при разной насыщенности инкубационной среды кислородом ([O] <8 мг/л и ([O] >16 мг/л). В качестве жирных кислот, добавляемых в инкубационную среду, использовали арахидоновую и пальмитиновую кислоты (20 мкМ/л). Результаты. Установлено, что в среде инкубации с высоким содержанием кислорода скорость его потребления изолированными кардиомиоцитами, из сердец крыс с ишемическим или диабетическим поражением сердца, в 2-2,5 раза превышает таковую в кардиомиоцитах из сердца интактных крыс. Введение в среду инкубации 20 мкМ арахидоновой или пальмитиновой ЖК статистически значимо увеличивало потребление кислорода кардиомиоцитами интактных крыс. При моделируемых патологиях был получен прямо противоположный результат. В среде инкубации с низким содержанием кислорода была зафиксирована более низкая исходная скорость дыхания кардиомиоцитов, независимо от того, из сердец крыс какой группы они были выделены. Присутствие жирных кислот приводило к еще большему снижению скорости потребления кислорода кардиомиоцитами интактных крыс. В кардиомиоцитах из сердец с ишемическим поражением жирные кислоты снижали скорость потребления кислорода практически до значений, полученных для кардиомиоцитов интактных крыс. Напротив, жирные кислоты не оказывали заметного влияния на дыхание кардиомиоцитов из сердца крыс с диабетическим поражением. Заключение. Полученные результаты свидетельствуют, что и насыщенные, и ненасыщенные свободные ЖК, на клеточном уровне, способны поддерживать баланс между поступлением и расходом кислорода. Действие ЖК определяется функциональным состоянием кардиомиоцитов и содержанием кислорода во внеклеточном пространстве. Это обстоятельство, вероятно, влияет на состояние энергетического метаболизма кардиомиоцитов и при реальном ишемическом или диабетическом поражении сердца пациентов.Aim. To evaluate the effect of free polyunsaturated and saturated fatty acids on oxygen uptake by isolated cardiomyocytes in intact rats and animals with ischemic or diabetic heart disease. Methods. This was a non-randomized, controlled study on three groups of male Wistar rats each containing 10 rats weighing 250-300 g. Myocardial infarction («heart attack») was produced by ligation of the left coronary artery; diabetes mellitus («diabetes») - by a single injection of streptozotocin 60 mg/kg, i.p. The control group consisted of intact animals. Cardomyocytes were isolated by an enzymatic method. Oxygen consumption was assessed polarographically at different saturation of the incubation medium with oxygen ([O] <8 mg/l and ([O] >16 mg/l). Arachidonic and palmitic acid were used as fatty acids to be added to the incubation medium. Results. In the high-oxygen incubation medium, the rate of O consumption by isolated cardiomyocytes from hearts of rats with ischemic or diabetic heart lesions was 2-2.5 times higher than in cardiomyocytes from heart of intact rats. Supplementing the incubation medium with 20 mkM arachidonic or palmitic acid statistically significantly increased oxygen consumption by cardiomyocytes of intact rats. An opposite result was obtained in models of diseases. In the low-oxygen incubation medium, the baseline rate of cardiomyocyte respiration was lower, no matter from which group of rats they had been isolated. The presence of fatty acids led to an even greater decrease in the rate of oxygen consumption by cardiomyocytes of intact rats. In cardiomyocytes from hearts with ischemic injury, fatty acids reduced the rate of oxygen consumption practically to the values observed for cardiomyocytes of intact rats. In contrast, fatty acids did not exert a noticeable effect on respiration of cardiomyocytes from the heart rats with diabetic lesions. Conclusion. The results suggested that saturated and unsaturated free fatty acids are able to maintain a balance between O supply and utilization. The effect of fatty acids is determined by functional state of cardiomyocytes and oxygen content in the extracellular space. This fact apparently influences cardiomyocyte energy metabolism in patients with ischemic or diabetic heart disease.

MiR-208 inhibits mitochondrial cardiomyocyte respiration in diabetic patients by inhibiting cytochrome C oxidase expression

Diabetes is a common disease that is characterized by myocardial mitochondrial dysfunction. MicroRNAs are small non-coding RNAs that participate in the post-transcriptional regulation of many signaling pathways, and are involved in diabetes and mitochondrial function. The aim of this study was to investigate whether cardiac mitochondrial dysfunction observed in diabetic patients could be related to alteration in non-coding RNAs as suggested in bench-side studies. A study of the non-coding transcriptome by microarrays was performed on 16 matched pairs of atrial myocardial samples from diabetic and non-diabetic patients who underwent aortic valve replacement and/or coronary artery bypass graft surgery at Lille University Hospital. Mitochondrial respiration in myocardial homogenates and expression of microRNAs identified by the transcriptomic analysis were studied in myocardial samples from a second population. Analysis of the non-coding transcriptome revealed four microRNAs significantly deregulated in diabetic patients and predicted to target mitochondrial oxidative phosphorylation gene transcripts by bioinformatic analysis, i.e. miR-208, miR-4738, miR-498 and miR-1. Interestingly, in line with the predicted interaction between miR-208 and cytochrome C oxidase transcript, a significant negative correlation was observed between this miR expression level and complex IV-dependent mitochondrial respiration and complex activity ( Fig. 1 ). miR-208 is likely linked to decreased cardiac expression of cytochrome c oxidase and subsequent depressed complex IV-dependent mitochondrial respiration in the myocardium of diabetic patients.

Telomere shortening and metabolic compromise underlie dystrophic cardiomyopathy

Duchenne muscular dystrophy (DMD) is an incurable X-linked genetic disease that is caused by a mutation in the dystrophin gene and affects one in every 3,600 boys. We previously showed that long telomeres protect mice from the lethal cardiac disease seen in humans with the same genetic defect, dystrophin deficiency. By generating the mdx4cv/mTRG2 mouse model with "humanized" telomere lengths, the devastating dilated cardiomyopathy phenotype seen in patients with DMD was recapitulated. Here, we analyze the degenerative sequelae that culminate in heart failure and death in this mouse model. We report progressive telomere shortening in developing mouse cardiomyocytes after postnatal week 1, a time when the cells are no longer dividing. This proliferation-independent telomere shortening is accompanied by an induction of a DNA damage response, evident by p53 activation and increased expression of its target gene p21 in isolated cardiomyocytes. The consequent repression of Pgc1α/β leads to impaired mitochondrial biogenesis, which, in conjunction with the high demands of contraction, leads to increased oxidative stress and decreased mitochondrial membrane potential. As a result, cardiomyocyte respiration and ATP output are severely compromised. Importantly, treatment with a mitochondrial-specific antioxidant before the onset of cardiac dysfunction rescues the metabolic defects. These findings provide evidence for a link between short telomere length and metabolic compromise in the etiology of dilated cardiomyopathy in DMD and identify a window of opportunity for preventive interventions.

Abstract 281: Sigma-1 Receptor Dependent Pathway for a Protective Endoplasmic Reticulum Stress Response in Cardiomyocytes

Rationale: We recently reported that Sigma 1 receptor (Sigmar1) is a molecular chaperone protein highly expressed in the heart. Studies involving different cancer and neuronal cell lines indicated Sigmar1 resides in the mitochondrion-associated ER membrane (MAM). However, the subcellular localization of Sigmar1 and the molecular function in ER-stress remains unknown in cardiomyocytes. Here we describe a function for Sigmar1 as an effector of an adaptive ER stress response. Objective: The objective of this study was to elucidate functional roles of Simgar1 in ER-stress in cardiomyocytes. Methods and Results: Subcellular fractionation of the mouse heart showed extensive localization of Sigmar1 in the MAM and mitochondrial fraction. To define the function in an ER-stress response, we used small interfering RNA-mediated Sigmar1 knockdown and adenovirus-mediated overexpression in cultured neonatal rat ventricular cardiomyocytes. We treated with tunicamycin to induce ER stress. In cardiomyocytes, tunicamycin increased C/EBP-homologous protein (CHOP) expression; Sigmar1 overexpression significantly decreased the CHOP expression. We found that Sigmar1 overexpression was sufficient to activate the nuclear transport of spliced X-box binding protein 1 (Xbp1s) with minimal effects in other adaptive ER stress proteins. Sigmar1 knockdown decreased the nuclear transport of Xbp1s, increased the expression of nuclear CHOP and significantly increased LDH release. We also observed significant Sigmar1 expression dependent increases in mitochondrial respiration in cardiomyocytes under ER stress. Hence, Sigmar1 can function inside the cell during disease remodeling to augment ER function and protect through a mechanism involving regulation of Xbp1s. Conclusions: Sigmar1 is an essential component of the adaptive ER stress response in cardiomyocytes. Sigmar1 can regulate ER-stress induced CHOP expression by activating XBP1s signaling in cardiomyocytes. Therefore, Sigmar1 residing at the ER-mitochondrion interface serves as an important subcellular entity in the regulation of cellular survival by enhancing the stress-response signaling between the ER and mitochondria.

Inhibition of NAPDH Oxidase 2 (NOX2) Prevents Oxidative Stress and Mitochondrial Abnormalities Caused by Saturated Fat in Cardiomyocytes.

Obesity and high saturated fat intake increase the risk of heart failure and arrhythmias. The molecular mechanisms are poorly understood. We hypothesized that physiologic levels of saturated fat could increase mitochondrial reactive oxygen species (ROS) in cardiomyocytes, leading to abnormalities of calcium homeostasis and mitochondrial function. We investigated the effect of saturated fat on mitochondrial function and calcium homeostasis in isolated ventricular myocytes. The saturated fatty acid palmitate causes a decrease in mitochondrial respiration in cardiomyocytes. Palmitate, but not the monounsaturated fatty acid oleate, causes an increase in both total cellular ROS and mitochondrial ROS. Palmitate depolarizes the mitochondrial inner membrane and causes mitochondrial calcium overload by increasing sarcoplasmic reticulum calcium leak. Inhibitors of PKC or NOX2 prevent mitochondrial dysfunction and the increase in ROS, demonstrating that PKC-NOX2 activation is also required for amplification of palmitate induced-ROS. Cardiomyocytes from mice with genetic deletion of NOX2 do not have palmitate-induced ROS or mitochondrial dysfunction. We conclude that palmitate induces mitochondrial ROS that is amplified by NOX2, causing greater mitochondrial ROS generation and partial depolarization of the mitochondrial inner membrane. The abnormal sarcoplasmic reticulum calcium leak caused by palmitate could promote arrhythmia and heart failure. NOX2 inhibition is a potential therapy for heart disease caused by diabetes or obesity.

Effect of Arachidonic Acid on the Rate of Oxygen Consumption in Isolated Cardiomyocytes from Intact Rats and Animals with Ischemic or Diabetic Injury to the Heart.

We studied the rate of oxygen consumption by isolated cardiomyocytes from intact rats and animals with experimental myocardial infarction or streptozotocin-induced diabetes mellitus. The measurements were performed in standard incubation medium under various conditions of oxygenation and after addition of arachidonic acid (20 μmol/liter). Under normoxic conditions, arachidonic acid improves respiration of cardiomyocytes from intact animals, but reduces this parameter in cells isolated from animals with pathologies. The intensity of O2 consumption by cardiomyocytes from intact rats and animals with pathologies was shown to decrease during hypoxia. Addition of arachidonic acid aggravated inhibition of respiration for cardiomyocytes from intact rats and specimens with myocardial infarction, but had no effect in diabetes mellitus. The effect of arachidonic acid on oxygen consumption rate is probably mediated by a nonspecific mechanism realized at the mitochondrial level.

GRK2 compromises cardiomyocyte mitochondrial function by diminishing fatty acid-mediated oxygen consumption and increasing superoxide levels

The G protein-coupled receptor kinase-2 (GRK2) is upregulated in the injured heart and contributes to heart failure pathogenesis. GRK2 was recently shown to associate with mitochondria but its functional impact in myocytes due to this localization is unclear. This study was undertaken to determine the effect of elevated GRK2 on mitochondrial respiration in cardiomyocytes. Sub-fractionation of purified cardiac mitochondria revealed that basally GRK2 is found in multiple compartments. Overexpression of GRK2 in mouse cardiomyocytes resulted in an increased amount of mitochondrial-based superoxide. Inhibition of GRK2 increased oxygen consumption rates and ATP production. Moreover, fatty acid oxidation was found to be significantly impaired when GRK2 was elevated and was dependent on the catalytic activity and mitochondrial localization of this kinase. Our study shows that independent of cardiac injury, GRK2 is localized in the mitochondria and its kinase activity negatively impacts the function of this organelle by increasing superoxide levels and altering substrate utilization for energy production.