Hemodynamic testing to guide vasodilator therapy in cardiac amyloidosis.

Cardiac amyloidosis is a restrictive cardiomyopathy for which diuretics are frequently used, but vasodilators have classically been relatively contraindicated due to side effects of hypotension. In the setting of decompensated heart failure, this may not be the case. We report a man with advanced cardiac amyloidosis who presented to the hospital with decompensated heart failure, in part, due to elevated systemic vascular resistance. Through the use of invasive hemodynamic testing, we were able to demonstrate an increase in cardiac output in response to a nitroprusside challenge. In turn, the patient had an improvement in his symptoms and was sent home on afterload reducing medications. This discerns a subpopulation of cardiac amyloidosis patients in decompensated heart failure who benefit from medications that reduce systemic vascular resistance, and can benefit from hemodynamic testing, especially when diuretics fail to control symptoms. Learning objective: Medications that cause peripheral vasodilation are standard therapy for patients with reduced ejection fraction, however, they are seldom used for patients with cardiac amyloidosis due to adverse effects. In some cases, there may be value in using hemodynamic measurements in patients with advanced cardiac amyloidosis to guide management as some patients may have hemodynamics that resemble those of systolic heart failure. This may offer a novel approach to symptomatic treatment of advanced cardiac amyloidosis.

SGLT2 inhibitor ertugliflozin decreases elevated intracellular sodium, and improves energetics and contractile function in diabetic cardiomyopathy.

BACKGROUND: Elevated myocardial intracellular sodium ([Na+]i) was shown to decrease mitochondrial calcium ([Ca2+]MITO) via mitochondrial sodium/calcium exchanger (NCXMITO), resulting in decreased mitochondrial ATP synthesis. The sodium-glucose co-transporter 2 inhibitor (SGLT2i) ertugliflozin (ERTU) improved energetic deficit and contractile dysfunction in a mouse model of high fat, high sucrose (HFHS) diet-induced diabetic cardiomyopathy (DCMP). As SGLT2is were shown to lower [Na+]i in isolated cardiomyocytes, we hypothesized that energetic improvement in DCMP is at least partially mediated by a decrease in abnormally elevated myocardial [Na+]i. METHODS: Forty-two eight-week-old male C57BL/6J mice were fed a control or HFHS diet for six months. In the last month, a subgroup of HFHS-fed mice was treated with ERTU. At the end of the study, left ventricular contractile function and energetics were measured simultaneously in isolated beating hearts by 31P NMR (Nuclear Magnetic Resonance) spectroscopy. A subset of untreated HFHS hearts was perfused with vehicle vs. CGP 37157, an NCXMITO inhibitor. Myocardial [Na+]i was measured by 23Na NMR spectroscopy. RESULTS: HFHS hearts showed diastolic dysfunction, decreased contractile reserve, and impaired energetics as reflected by decreased phosphocreatine (PCr) and PCr/ATP ratio. Myocardial [Na+]i was elevated > 2-fold in HFHS (vs. control diet). ERTU reversed the impairments in HFHS hearts to levels similar to or better than control diet and decreased myocardial [Na+]i to control levels. CGP 37157 normalized the PCr/ATP ratio in HFHS hearts. CONCLUSIONS: Elevated myocardial [Na+]i contributes to mitochondrial and contractile dysfunction in DCMP. Targeting myocardial [Na+]i and/or NCXMITO may be an effective strategy in DCMP and other forms of heart disease associated with elevated myocardial [Na+]i.

Oxidized GAPDH transfers S-glutathionylation to a nuclear protein Sirtuin-1 leading to apoptosis.

AIMS: S-glutathionylation is a reversible oxidative modification of protein cysteines that plays a critical role in redox signaling. Glutaredoxin-1 (Glrx), a glutathione-specific thioltransferase, removes protein S-glutathionylation. Glrx, though a cytosolic protein, can activate a nuclear protein Sirtuin-1 (SirT1) by removing its S-glutathionylation. Glrx ablation causes metabolic abnormalities and promotes controlled cell death and fibrosis in mice. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a key enzyme of glycolysis, is sensitive to oxidative modifications and involved in apoptotic signaling via the SirT1/p53 pathway in the nucleus. We aimed to elucidate the extent to which S-glutathionylation of GAPDH and glutaredoxin-1 contribute to GAPDH/SirT1/p53 apoptosis pathway. RESULTS: Exposure of HEK 293T cells to hydrogen peroxide (H2O2) caused rapid S-glutathionylation and nuclear translocation of GAPDH. Nuclear GAPDH peaked 10-15 min after the addition of H2O2. Overexpression of Glrx or redox dead mutant GAPDH inhibited S-glutathionylation and nuclear translocation. Nuclear GAPDH formed a protein complex with SirT1 and exchanged S-glutathionylation to SirT1 and inhibited its deacetylase activity. Inactivated SirT1 remained stably bound to acetylated-p53 and initiated apoptotic signaling resulting in cleavage of caspase-3. We observed similar effects in human primary aortic endothelial cells suggesting the GAPDH/SirT1/p53 pathway as a common apoptotic mechanism. CONCLUSIONS: Abundant GAPDH with its highly reactive-cysteine thiolate may function as a cytoplasmic rheostat to sense oxidative stress. S-glutathionylation of GAPDH may relay the signal to the nucleus where GAPDH trans-glutathionylates nuclear proteins such as SirT1 to initiate apoptosis. Glrx reverses GAPDH S-glutathionylation and prevents its nuclear translocation and cytoplasmic-nuclear redox signaling leading to apoptosis. Our data suggest that trans-glutathionylation is a critical step in apoptotic signaling and a potential mechanism that cytosolic Glrx controls nuclear transcription factors.

Effects of Sodium-Glucose Linked Transporter 2 Inhibition With Ertugliflozin on Mitochondrial Function, Energetics, and Metabolic Gene Expression in the Presence and Absence of Diabetes Mellitus in Mice.

Background Inhibitors of the sodium-glucose linked transporter 2 improve cardiovascular outcomes in patients with or without type 2 diabetes mellitus, but the effects on cardiac energetics and mitochondrial function are unknown. We assessed the effects of sodium-glucose linked transporter 2 inhibition on mitochondrial function, high-energy phosphates, and genes encoding mitochondrial proteins in hearts of mice with and without diet-induced diabetic cardiomyopathy. Methods and Results Mice fed a control diet or a high-fat, high-sucrose diet received ertugliflozin mixed with the diet (0.5 mg/g of diet) for 4 months. Isolated mitochondria were assessed for functional capacity. High-energy phosphates were assessed by 31P nuclear magnetic resonance spectroscopy concurrently with contractile performance in isolated beating hearts. The high-fat, high-sucrose diet caused myocardial hypertrophy, diastolic dysfunction, mitochondrial dysfunction, and impaired energetic response, all of which were prevented by ertugliflozin. With both diets, ertugliflozin caused supernormalization of contractile reserve, as measured by rate×pressure product at high work demand. Likewise, the myocardial gene sets most enriched by ertugliflozin were for oxidative phosphorylation and fatty acid metabolism, both of which were enriched independent of diet. Conclusions Ertugliflozin not only prevented high-fat, high-sucrose-induced pathological cardiac remodeling, but improved contractile reserve and induced the expression of oxidative phosphorylation and fatty acid metabolism gene sets independent of diabetic status. These effects of sodium-glucose linked transporter 2 inhibition on cardiac energetics and metabolism may contribute to improved structure and function in cardiac diseases associated with mitochondrial dysfunction, such as heart failure.

Differential Effects of Sacubitril/Valsartan on Diastolic Function in Mice With Obesity-Related Metabolic Heart Disease.

Mice with obesity and metabolic heart disease (MHD) due to a high-fat, high-sucrose diet were treated with placebo, a clinically relevant dose of sacubitril (SAC)/valsartan (VAL), or an equivalent dose of VAL for 4 months. There were striking differences between SAC/VAL and VAL with regard to: 1) diastolic dysfunction; 2) interstitial fibrosis; and to a lesser degree; 3) oxidative stress-all of which were more favorably affected by SAC/VAL. SAC/VAL and VAL similarly attenuated myocardial hypertrophy and improved myocardial energetics. In mice with obesity-related MHD, neprilysin inhibition exerts favorable effects on diastolic function.

Redox-Resistant SERCA [Sarco(endo)plasmic Reticulum Calcium ATPase] Attenuates Oxidant-Stimulated Mitochondrial Calcium and Apoptosis in Cardiac Myocytes and Pressure Overload-Induced Myocardial Failure in Mice.

BACKGROUND: SERCA [sarco(endo)plasmic reticulum calcium ATPase] is regulated by oxidative posttranslational modifications at cysteine 674 (C674). Because sarcoplasmic reticulum (SR) calcium has been shown to play a critical role in mediating mitochondrial dysfunction in response to reactive oxygen species, we hypothesized that SERCA oxidation at C674 would modulate the effects of reactive oxygen species on mitochondrial calcium and mitochondria-dependent apoptosis in cardiac myocytes. METHODS: Adult rat ventricular myocytes expressing wild-type SERCA2b or a redox-insensitive mutant in which C674 is replaced by serine (C674S) were exposed to H2O2 (100 µmol/Lµ). Free mitochondrial calcium concentration was measured in adult rat ventricular myocytes with a genetically targeted fluorescent probe, and SR calcium content was assessed by measuring caffeine-stimulated release. Mice with heterozygous knock-in of the SERCA C674S mutation were subjected to chronic ascending aortic constriction. RESULTS: In adult rat ventricular myocytes expressing wild-type SERCA, H2O2 caused a 25% increase in mitochondrial calcium concentration that was associated with a 50% decrease in SR calcium content, both of which were prevented by the ryanodine receptor inhibitor tetracaine. In cells expressing the C674S mutant, basal SR calcium content was decreased by 31% and the H2O2-stimulated rise in mitochondrial calcium concentration was attenuated by 40%. In wild-type cells, H2O2 caused cytochrome c release and apoptosis, both of which were prevented in C674S-expressing cells. In myocytes from SERCA knock-in mice, basal SERCA activity and SR calcium content were decreased. To test the effect of C674 oxidation on apoptosis in vivo, SERCA knock-in mice were subjected to chronic ascending aortic constriction. In wild-type mice, ascending aortic constriction caused myocyte apoptosis, LV dilation, and systolic failure, all of which were inhibited in SERCA knock-in mice. CONCLUSIONS: Redox activation of SERCA C674 regulates basal SR calcium content, thereby mediating the pathologic reactive oxygen species-stimulated rise in mitochondrial calcium required for myocyte apoptosis and myocardial failure.

Role of Glutaredoxin-1 and Glutathionylation in Cardiovascular Diseases.

Cardiovascular diseases are the leading cause of death worldwide, and as rates continue to increase, discovering mechanisms and therapeutic targets become increasingly important. An underlying cause of most cardiovascular diseases is believed to be excess reactive oxygen or nitrogen species. Glutathione, the most abundant cellular antioxidant, plays an important role in the body's reaction to oxidative stress by forming reversible disulfide bridges with a variety of proteins, termed glutathionylation (GSylation). GSylation can alter the activity, function, and structure of proteins, making it a major regulator of cellular processes. Glutathione-protein mixed disulfide bonds are regulated by glutaredoxins (Glrxs), thioltransferase members of the thioredoxin family. Glrxs reduce GSylated proteins and make them available for another redox signaling cycle. Glrxs and GSylation play an important role in cardiovascular diseases, such as myocardial ischemia and reperfusion, cardiac hypertrophy, peripheral arterial disease, and atherosclerosis. This review primarily concerns the role of GSylation and Glrxs, particularly glutaredoxin-1 (Glrx), in cardiovascular diseases and the potential of Glrx as therapeutic agents.

A novel tracer for in vivo optical imaging of fatty acid metabolism in the heart and brown adipose tissue.

Multiplexed imaging is essential for the evaluation of substrate utilization in metabolically active organs, such as the heart and brown adipose tissue (BAT), where substrate preference changes in pathophysiologic states. Optical imaging provides a useful platform because of its low cost, high throughput and intrinsic ability to perform composite readouts. However, the paucity of probes available for in vivo use has limited optical methods to image substrate metabolism. Here, we present a novel near-infrared (NIR) free fatty acid (FFA) tracer suitable for in vivo imaging of deep tissues such as the heart. Using click chemistry, Alexa Fluor 647 DIBO Alkyne was conjugated to palmitic acid. Mice injected with 0.05 nmol/g bodyweight of the conjugate (AlexaFFA) were subjected to conditions known to increase FFA uptake in the heart (fasting) and BAT [cold exposure and injection with the ß3 adrenergic agonist CL 316, 243(CL)]. Organs were subsequently imaged both ex vivo and in vivo to quantify AlexaFFA uptake. The blood kinetics of AlexaFFA followed a two-compartment model with an initial fast compartment half-life of 0.14 h and a subsequent slow compartment half-life of 5.2 h, consistent with reversible protein binding. Ex vivo fluorescence imaging after overnight cold exposure and fasting produced a significant increase in AlexaFFA uptake in the heart (58 ± 12%) and BAT (278 ± 19%) compared to warm/fed animals. In vivo imaging of the heart and BAT after exposure to CL and fasting showed a significant increase in AlexaFFA uptake in the heart (48 ± 20%) and BAT (40 ± 10%) compared to saline-injected/fed mice. We present a novel near-infrared FFA tracer, AlexaFFA, that is suitable for in vivo quantification of FFA metabolism and can be applied in the context of a low cost, high throughput, and multiplexed optical imaging platform.

Cross organelle stress response disruption promotes gentamicin-induced proteotoxicity.

Gentamicin is a nephrotoxic antibiotic that causes acute kidney injury (AKI) primarily by targeting the proximal tubule epithelial cell. The development of an effective therapy for gentamicin-induced renal cell injury is limited by incomplete mechanistic insight. To address this challenge, we propose that RNAi signal pathway screening could identify a unifying mechanism of gentamicin-induced cell injury and suggest a therapeutic strategy to ameliorate it. Computational analysis of RNAi signal screens in gentamicin-exposed human proximal tubule cells suggested the cross-organelle stress response (CORE), the unfolded protein response (UPR), and cell chaperones as key targets of gentamicin-induced injury. To test this hypothesis, we assessed the effect of gentamicin on the CORE, UPR, and cell chaperone function, and tested the therapeutic efficacy of enhancing cell chaperone content. Early gentamicin exposure disrupted the CORE, evidenced by a rise in the ATP:ADP ratio, mitochondrial-specific H2O2 accumulation, Drp-1-mediated mitochondrial fragmentation, and endoplasmic reticulum-mitochondrial dissociation. CORE disruption preceded measurable increases in whole-cell oxidative stress, misfolded protein content, transcriptional UPR activation, and its untoward downstream effects: CHOP expression, PARP cleavage, and cell death. Geranylgeranylacetone, a therapeutic that increases cell chaperone content, prevented mitochondrial H2O2 accumulation, preserved the CORE, reduced the burden of misfolded proteins and CHOP expression, and significantly improved survival in gentamicin-exposed cells. We identify CORE disruption as an early and remediable cause of gentamicin proteotoxicity that precedes downstream UPR activation and cell death. Preserving the CORE significantly improves renal cell survival likely by reducing organelle-specific proteotoxicity during gentamicin exposure.

Increasing mitochondrial ATP synthesis with butyrate normalizes ADP and contractile function in metabolic heart disease.

Metabolic heart disease (MHD), which is strongly associated with heart failure with preserved ejection fraction, is characterized by reduced mitochondrial energy production and contractile performance. In this study, we tested the hypothesis that an acute increase in ATP synthesis, via short chain fatty acid (butyrate) perfusion, restores contractile function in MHD. Isolated hearts of mice with MHD due to consumption of a high fat high sucrose (HFHS) diet or on a control diet (CD) for 4 months were studied using 31 P NMR spectroscopy to measure high energy phosphates and ATP synthesis rates during increased work demand. At baseline, HFHS hearts had increased ADP and decreased free energy of ATP hydrolysis (ΔG~ATP ), although contractile function was similar between the two groups. At high work demand, the ATP synthesis rate in HFHS hearts was reduced by over 50%. Unlike CD hearts, HFHS hearts did not increase contractile function at high work demand, indicating a lack of contractile reserve. However, acutely supplementing HFHS hearts with 4mM butyrate normalized ATP synthesis, ADP, ΔG~ATP and contractile reserve. Thus, acute reversal of depressed mitochondrial ATP production improves contractile dysfunction in MHD. These findings suggest that energy starvation may be a reversible cause of myocardial dysfunction in MHD, and opens new therapeutic opportunities.

Redox Regulation via Glutaredoxin-1 and Protein S-Glutathionylation.

Significance: Over the past several years, oxidative post-translational modifications of protein cysteines have been recognized for their critical roles in physiology and pathophysiology. Cells have harnessed thiol modifications involving both oxidative and reductive steps for signaling and protein processing. One of these stages requires oxidation of cysteine to sulfenic acid, followed by two reduction reactions. First, glutathione (reduced glutathione [GSH]) forms a S-glutathionylated protein, and second, enzymatic or chemical reduction removes the modification. Under physiological conditions, these steps confer redox signaling and protect cysteines from irreversible oxidation. However, oxidative stress can overwhelm protein S-glutathionylation and irreversibly modify cysteine residues, disrupting redox signaling. Critical Issues: Glutaredoxins mainly catalyze the removal of protein-bound GSH and help maintain protein thiols in a highly reduced state without exerting direct antioxidant properties. Conversely, glutathione S-transferase (GST), peroxiredoxins, and occasionally glutaredoxins can also catalyze protein S-glutathionylation, thus promoting a dynamic redox environment. Recent Advances: The latest studies of glutaredoxin-1 (Glrx) transgenic or knockout mice demonstrate important distinct roles of Glrx in a variety of pathologies. Endogenous Glrx is essential to maintain normal hepatic lipid homeostasis and prevent fatty liver disease. Further, in vivo deletion of Glrx protects lungs from inflammation and bacterial pneumonia-induced damage, attenuates angiotensin II-induced cardiovascular hypertrophy, and improves ischemic limb vascularization. Meanwhile, exogenous Glrx administration can reverse pathological lung fibrosis. Future Directions: Although S-glutathionylation modifies many proteins, these studies suggest that S-glutathionylation and Glrx regulate specific pathways in vivo, and they implicate Glrx as a potential novel therapeutic target to treat diverse disease conditions. Antioxid. Redox Signal. 32, 677-700.

Production of adeno-associated virus vectors for in vitro and in vivo applications.

Delivering and expressing a gene of interest in cells or living animals has become a pivotal technique in biomedical research and gene therapy. Among viral delivery systems, adeno-associated viruses (AAVs) are relatively safe and demonstrate high gene transfer efficiency, low immunogenicity, stable long-term expression, and selective tissue tropism. Combined with modern gene technologies, such as cell-specific promoters, the Cre/lox system, and genome editing, AAVs represent a practical, rapid, and economical alternative to conditional knockout and transgenic mouse models. However, major obstacles remain for widespread AAV utilization, such as impractical purification strategies and low viral quantities. Here, we report an improved protocol to produce serotype-independent purified AAVs economically. Using a helper-free AAV system, we purified AAVs from HEK293T cell lysates and medium by polyethylene glycol precipitation with subsequent aqueous two-phase partitioning. Furthermore, we then implemented an iodixanol gradient purification, which resulted in preparations with purities adequate for in vivo use. Of note, we achieved titers of 1010-1011 viral genome copies per µl with a typical production volume of up to 1 ml while requiring five times less than the usual number of HEK293T cells used in standard protocols. For proof of concept, we verified in vivo transduction via Western blot, qPCR, luminescence, and immunohistochemistry. AAVs coding for glutaredoxin-1 (Glrx) shRNA successfully inhibited Glrx expression by ~66% in the liver and skeletal muscle. Our study provides an improved protocol for a more economical and efficient purified AAV preparation.

Genetically targeted fluorescent probes reveal dynamic calcium responses to adrenergic signaling in multiple cardiomyocyte compartments.

Calcium (Ca2+), an important second messenger, regulates many cellular activities and varies spatiotemporally within the cell. Conventional methods to monitor Ca2+ changes, such as synthetic Ca2+ indicators, are not targetable, while genetically encoded Ca2+ indicators (GECI) can be precisely directed to cellular compartments. GECIs are chimeric proteins composed of calmodulin (or other proteins that change conformation on Ca2+ binding) coupled with two fluorescent proteins that come closer together after an increase in [Ca2+], and enhance Förster resonance energy transfer (FRET) that allows for ratiometric [Ca2+] assessment. Here, adult rat ventricular myocytes were transfected with specifically targeted calmodulin-based GECIs and Ca2+ responses to a physiological stimulus, norepinephrine (NE, 10 µM), were observed in a) sarcoplasmic reticulum (SR), b) mitochondria, c) the space between the mitochondria and SR, termed the Mitochondria Associated Membrane space (MAM) and d) cytosol for 10 min after stimulation. In SR and mitochondria, NE increased the [Ca2+] ratio by 17% and by 8%, respectively. In the MAM the [Ca2+] ratio decreased by 16%, while in cytosol [Ca2+] remained unchanged. In conclusion, adrenergic stimulation causes distinct responses in the cardiomyocyte SR, mitochondria and MAM. Additionally, our work provides a toolkit-update for targeted [Ca2+] measurements in multiple cellular compartments.

Energetic Dysfunction Is Mediated by Mitochondrial Reactive Oxygen Species and Precedes Structural Remodeling in Metabolic Heart Disease.

Aims: Metabolic syndrome is associated with metabolic heart disease (MHD) that is characterized by left ventricular (LV) hypertrophy, interstitial fibrosis, contractile dysfunction, and mitochondrial dysfunction. Overexpression of catalase in mitochondria (transgenic expression of catalase targeted to the mitochondria [mCAT]) prevents the structural and functional features of MHD caused by a high-fat, high-sucrose (HFHS) diet for ≥4 months. However, it is unclear whether the effect of mCAT is due to prevention of reactive oxygen species (ROS)-mediated cardiac remodeling, a direct effect on mitochondrial function, or both. To address this question, we measured myocardial function and energetics in mice, with or without mCAT, after 1 month of HFHS, before the development of cardiac structural remodeling. Results: HFHS diet for 1 month had no effect on body weight, heart weight, LV structure, myocyte size, or interstitial fibrosis. Isolated cardiac mitochondria from HFHS-fed mice produced 2.2- to 3.8-fold more H2O2, and 16%-29% less adenosine triphosphate (ATP). In isolated beating hearts from HFHS-fed mice, [phosphocreatine (PCr)] and the free energy available for ATP hydrolysis (ΔG∼ATP) were decreased, and they failed to increase with work demands. Overexpression of mCAT normalized ROS and ATP production in isolated mitochondria, and it corrected myocardial [PCr] and ΔG∼ATP in the beating heart. Innovation: This is the first demonstration that in MHD, mitochondrial ROS mediate energetic dysfunction that is sufficient to impair contractile function. Conclusion: ROS produced and acting in the mitochondria impair myocardial energetics, leading to slowed relaxation and decreased contractile reserve. These effects precede structural remodeling and are corrected by mCAT, indicating that ROS-mediated energetic impairment, per se, is sufficient to cause contractile dysfunction in MHD.

Decreased ATP production and myocardial contractile reserve in metabolic heart disease.

Metabolic syndrome is a cluster of obesity-related metabolic abnormalities that lead to metabolic heart disease (MHD) with left ventricular pump dysfunction. Although MHD is thought to be associated with myocardial energetic deficiency, two key questions have not been answered. First, it is not known whether there is a sufficient energy deficit to contribute to pump dysfunction. Second, the basis for the energy deficit is not clear. To address these questions, mice were fed a high fat, high sucrose (HFHS) 'Western' diet to recapitulate the MHD phenotype. In isolated beating hearts, we used 31P NMR spectroscopy with magnetization transfer to determine a) the concentrations of high energy phosphates ([ATP], [ADP], [PCr]), b) the free energy of ATP hydrolysis (∆G~ATP), c) the rate of ATP production and d) flux through the creatine kinase (CK) reaction. At the lowest workload, the diastolic pressure-volume relationship was shifted upward in HFHS hearts, indicative of diastolic dysfunction, whereas systolic function was preserved. At this workload, the rate of ATP synthesis was decreased in HFHS hearts, and was associated with decreases in both [PCr] and ∆G~ATP. Higher work demands unmasked the inability of HFHS hearts to increase systolic function and led to a further decrease in ∆G~ATP to a level that is not sufficient to maintain normal function of sarcoplasmic Ca2+-ATPase (SERCA). While [ATP] was preserved at all work demands in HFHS hearts, the progressive increase in [ADP] led to a decrease in ∆G~ATP with increased work demands. Surprisingly, CK flux, CK activity and total creatine were normal in HFHS hearts. These findings differ from dilated cardiomyopathy, in which the energetic deficiency is associated with decreases in CK flux, CK activity and total creatine. Thus, in HFHS-fed mice with MHD there is a distinct metabolic phenotype of the heart characterized by a decrease in ATP production that leads to a functionally-important energetic deficiency and an elevation of [ADP], with preservation of CK flux.

Mitochondrial Reactive Oxygen Species Mediate Cardiac Structural, Functional, and Mitochondrial Consequences of Diet-Induced Metabolic Heart Disease.

BACKGROUND: Mitochondrial reactive oxygen species (ROS) are associated with metabolic heart disease (MHD). However, the mechanism by which ROS cause MHD is unknown. We tested the hypothesis that mitochondrial ROS are a key mediator of MHD. METHODS AND RESULTS: Mice fed a high-fat high-sucrose (HFHS) diet develop MHD with cardiac diastolic and mitochondrial dysfunction that is associated with oxidative posttranslational modifications of cardiac mitochondrial proteins. Transgenic mice that express catalase in mitochondria and wild-type mice were fed an HFHS or control diet for 4 months. Cardiac mitochondria from HFHS-fed wild-type mice had a 3-fold greater rate of H2O2 production (P=0.001 versus control diet fed), a 30% decrease in complex II substrate-driven oxygen consumption (P=0.006), 21% to 23% decreases in complex I and II substrate-driven ATP synthesis (P=0.01), and a 62% decrease in complex II activity (P=0.002). In transgenic mice that express catalase in mitochondria, all HFHS diet-induced mitochondrial abnormalities were ameliorated, as were left ventricular hypertrophy and diastolic dysfunction. In HFHS-fed wild-type mice complex II substrate-driven ATP synthesis and activity were restored ex vivo by dithiothreitol (5 mmol/L), suggesting a role for reversible cysteine oxidative posttranslational modifications. In vitro site-directed mutation of complex II subunit B Cys100 or Cys103 to redox-insensitive serines prevented complex II dysfunction induced by ROS or high glucose/high palmitate in the medium. CONCLUSION: Mitochondrial ROS are pathogenic in MHD and contribute to mitochondrial dysfunction, at least in part, by causing oxidative posttranslational modifications of complex I and II proteins including reversible oxidative posttranslational modifications of complex II subunit B Cys100 and Cys103.

High fat, high sucrose diet causes cardiac mitochondrial dysfunction due in part to oxidative post-translational modification of mitochondrial complex II.

BACKGROUND: Diet-induced obesity leads to metabolic heart disease (MHD) characterized by increased oxidative stress that may cause oxidative post-translational modifications (OPTM) of cardiac mitochondrial proteins. The functional consequences of OPTM of cardiac mitochondrial proteins in MHD are unknown. Our objective was to determine whether cardiac mitochondrial dysfunction in MHD due to diet-induced obesity is associated with cysteine OPTM. METHODS AND RESULTS: Male C57BL/6J mice were fed either a high-fat, high-sucrose (HFHS) or control diet for 8months. Cardiac mitochondria from HFHS-fed mice (vs. control diet) had an increased rate of H2O2 production, a decreased GSH/GSSG ratio, a decreased rate of complex II substrate-driven ATP synthesis and decreased complex II activity. Complex II substrate-driven ATP synthesis and complex II activity were partially restored ex-vivo by reducing conditions. A biotin switch assay showed that HFHS feeding increased cysteine OPTM in complex II subunits A (SDHA) and B (SDHB). Using iodo-TMT multiplex tags we found that HFHS feeding is associated with reversible oxidation of cysteines 89 and 231 in SDHA, and 100, 103 and 115 in SDHB. CONCLUSIONS: MHD due to consumption of a HFHS "Western" diet causes increased H2O2 production and oxidative stress in cardiac mitochondria associated with decreased ATP synthesis and decreased complex II activity. Impaired complex II activity and ATP production are associated with reversible cysteine OPTM of complex II. Possible sites of reversible cysteine OPTM in SDHA and SDHB were identified by iodo-TMT tag labeling. Mitochondrial ROS may contribute to the pathophysiology of MHD by impairing the function of complex II. This article is part of a Special Issue entitled "Mitochondria: From Basic Mitochondrial Biology to Cardiovascular Disease".

Cytosolic H2O2 mediates hypertrophy, apoptosis, and decreased SERCA activity in mice with chronic hemodynamic overload.

Oxidative stress in the myocardium plays an important role in the pathophysiology of hemodynamic overload. The mechanism by which reactive oxygen species (ROS) in the cardiac myocyte mediate myocardial failure in hemodynamic overload is not known. Accordingly, our goals were to test whether myocyte-specific overexpression of peroxisomal catalase (pCAT) that localizes in the sarcoplasm protects mice from hemodynamic overload-induced failure and prevents oxidation and inhibition of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), an important sarcoplasmic protein. Chronic hemodynamic overload was caused by ascending aortic constriction (AAC) for 12 wk in mice with myocyte-specific transgenic expression of pCAT. AAC caused left ventricular hypertrophy and failure associated with a generalized increase in myocardial oxidative stress and specific oxidative modifications of SERCA at cysteine 674 and tyrosine 294/5. pCAT overexpression ameliorated myocardial hypertrophy and apoptosis, decreased pathological remodeling, and prevented the progression to heart failure. Likewise, pCAT prevented oxidative modifications of SERCA and increased SERCA activity without changing SERCA expression. Thus cardiac myocyte-restricted expression of pCAT effectively ameliorated the structural and functional consequences of chronic hemodynamic overload and increased SERCA activity via a post-translational mechanism, most likely by decreasing inhibitory oxidative modifications. In pressure overload-induced heart failure cardiac myocyte cytosolic ROS play a pivotal role in mediating key pathophysiologic events including hypertrophy, apoptosis, and decreased SERCA activity.

A redox-resistant sirtuin-1 mutant protects against hepatic metabolic and oxidant stress.

Sirtuin-1 (SirT1), a member of the NAD(+)-dependent class III histone deacetylase family, is inactivated in vitro by oxidation of critical cysteine thiols. In a model of metabolic syndrome, SirT1 activation attenuated apoptosis of hepatocytes and improved liver function including lipid metabolism. We show in SirT1-overexpressing HepG2 cells that oxidants (nitrosocysteine and hydrogen peroxide) or metabolic stress (high palmitate and high glucose) inactivated SirT1 by reversible oxidative post-translational modifications (OPTMs) on three cysteines. Mutating these oxidation-sensitive cysteines to serine preserved SirT1 activity and abolished reversible OPTMs. Overexpressed mutant SirT1 maintained deacetylase activity and attenuated proapoptotic signaling, whereas overexpressed wild type SirT1 was less protective in metabolically or oxidant-stressed cells. To prove that OPTMs of SirT1 are glutathione (GSH) adducts, glutaredoxin-1 was overexpressed to remove this modification. Glutaredoxin-1 overexpression maintained endogenous SirT1 activity and prevented proapoptotic signaling in metabolically stressed HepG2 cells. The in vivo significance of oxidative inactivation of SirT1 was investigated in livers of high fat diet-fed C57/B6J mice. SirT1 deacetylase activity was decreased in the absence of changes in SirT1 expression and associated with a marked increase in OPTMs. These results indicate that glutathione adducts on specific SirT1 thiols may be responsible for dysfunctional SirT1 associated with liver disease in metabolic syndrome.