Impaired mitochondrial energy supply coupled to increased H2O2 emission under energy/redox stress leads to myocardial dysfunction during Type I diabetes.

In Type I diabetic (T1DM) patients, both peaks of hyperglycaemia and increased sympathetic tone probably contribute to impair systolic and diastolic function. However, how these stressors eventually alter cardiac function during T1DM is not fully understood. In the present study, we hypothesized that impaired mitochondrial energy supply and excess reactive oxygen species (ROS) emission is centrally involved in T1DM cardiac dysfunction due to metabolic/redox stress and aimed to determine the mitochondrial sites implicated in these alterations. To this end, we used isolated myocytes and mitochondria from Sham and streptozotocin (STZ)-induced T1DM guinea pigs (GPs), untreated or treated with insulin. Relative to controls, T1DM myocytes exhibited higher oxidative stress when challenged with high glucose (HG) combined with ß-adrenergic stimulation [via isoprenaline (isoproterenol) (ISO)], leading to contraction/relaxation deficits. T1DM mitochondria had decreased respiration with complex II and IV substrates and markedly lower ADP phosphorylation rates and higher H2O2 emission when challenged with oxidants to mimic the more oxidized redox milieu present in HG + ISO-treated cardiomyocytes. Since in T1DM hearts insulin-sensitivity is preserved and a glucose-to-fatty acid (FA) shift occurs, we next tested whether insulin therapy or acute palmitate (Palm) infusion prevents HG + ISO-induced cardiac dysfunction. We found that insulin rescued proper cardiac redox balance, but not mitochondrial respiration or contractile performance. Conversely, Palm restored redox balance and preserved myocyte function. Thus, stressors such as peaks of HG and adrenergic hyperactivity impair mitochondrial respiration, hampering energy supply while exacerbating ROS emission. Our study suggests that an ideal therapeutic measure to treat metabolically/redox-challenged T1DM hearts should concomitantly correct energetic and redox abnormalities to fully maintain cardiac function.

HNO enhances SERCA2a activity and cardiomyocyte function by promoting redox-dependent phospholamban oligomerization.

AIMS: Nitroxyl (HNO) interacts with thiols to act as a redox-sensitive modulator of protein function. It enhances sarcoplasmic reticular Ca(2+) uptake and myofilament Ca(2+) sensitivity, improving cardiac contractility. This activity has led to clinical testing of HNO donors for heart failure. Here we tested whether HNO alters the inhibitory interaction between phospholamban (PLN) and the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) in a redox-dependent manner, improving Ca(2+) handling in isolated myocytes/hearts. RESULTS: Ventriculocytes, sarcoplasmic reticulum (SR) vesicles, and whole hearts were isolated from control (wildtype [WT]) or PLN knockout (pln(-/-)) mice. Compared to WT, pln(-/-) myocytes displayed enhanced resting sarcomere shortening, peak Ca(2+) transient, and blunted ß-adrenergic responsiveness. HNO stimulated shortening, relaxation, and Ca(2+) transient in WT cardiomyocytes, and evoked positive inotropy/lusitropy in intact hearts. These changes were markedly blunted in pln(-/-) cells/hearts. HNO enhanced SR Ca(2+) uptake in WT but not pln(-/-) SR-vesicles. Spectroscopic studies in insect cell microsomes expressing SERCA2a±PLN showed that HNO increased Ca(2+)-dependent SERCA2a conformational flexibility but only when PLN was present. In cardiomyocytes, HNO achieved this effect by stabilizing PLN in an oligomeric disulfide bond-dependent configuration, decreasing the amount of free inhibitory monomeric PLN available. INNOVATION: HNO-dependent redox changes in myocyte PLN oligomerization relieve PLN inhibition of SERCA2a. CONCLUSIONS: PLN plays a central role in HNO-induced enhancement of SERCA2a activity, leading to increased inotropy/lusitropy in intact myocytes and hearts. PLN remains physically associated with SERCA2a; however, less monomeric PLN is available resulting in decreased inhibition of the enzyme. These findings offer new avenues to improve Ca(2+) handling in failing hearts.

Acyloxy nitroso compounds inhibit LIF signaling in endothelial cells and cardiac myocytes: evidence that STAT3 signaling is redox-sensitive.

We previously showed that oxidative stress inhibits leukemia inhibitory factor (LIF) signaling by targeting JAK1, and the catalytic domains of JAK 1 and 2 have a cysteine-based redox switch. Thus, we postulated that the NO sibling and thiophylic compound, nitroxyl (HNO), would inhibit LIF-induced JAK-STAT3 activation. Pretreatment of human microvascular endothelial cells (HMEC-1) or neonatal rat cardiomyocytes with the HNO donors Angeli's salt or nitrosocyclohexyl acetate (NCA) inhibited LIF-induced STAT3 activation. NCA pretreatment also blocked the induction of downstream inflammatory genes (e.g. intercellular adhesion molecule 1, CCAAT/enhancer binding protein delta). The related 1-nitrosocyclohexyl pivalate (NCP; not a nitroxyl donor) was equally effective in inhibiting STAT3 activation, suggesting that these compounds act as thiolate targeting electrophiles. The JAK1 redox switch is likely not a target of acyloxy nitroso compounds, as NCA had no effect on JAK1 catalytic activity and only modestly affected JAK1-induced phosphorylation of the LIF receptor. However, pretreatment of recombinant human STAT3 with NCA or NCP reduced labeling of free sulfhydryl residues. We show that NCP in the presence of diamide enhanced STAT3 glutathionylation and dimerization in adult mouse cardiac myocytes and altered STAT3 under non-reducing conditions. Finally, we show that monomeric STAT3 levels are decreased in the Gαq model of heart failure in a redox-sensitive manner. Altogether, our evidence indicates that STAT3 has redox-sensitive cysteines that regulate its activation and are targeted by HNO donors and acyloxy nitroso compounds. These findings raise the possibility of new therapeutic strategies to target STAT3 signaling via a redox-dependent manner, particularly in the context of cardiac and non-cardiac diseases with prominent pro-inflammatory signaling.

Nitroxyl-mediated disulfide bond formation between cardiac myofilament cysteines enhances contractile function.

RATIONALE: In the myocardium, redox/cysteine modification of proteins regulating Ca(2+) cycling can affect contraction and may have therapeutic value. Nitroxyl (HNO), the one-electron-reduced form of nitric oxide, enhances cardiac function in a manner that suggests reversible cysteine modifications of the contractile machinery. OBJECTIVE: To determine the effects of HNO modification in cardiac myofilament proteins. METHODS AND RESULTS: The HNO-donor, 1-nitrosocyclohexyl acetate, was found to act directly on the myofilament proteins, increasing maximum force (F(max)) and reducing the concentration of Ca(2+) for 50% activation (Ca(50)) in intact and skinned cardiac muscles. The effects of 1-nitrosocyclohexyl acetate are reversible by reducing agents and distinct from those of another HNO donor, Angeli salt, which was previously reported to increase F(max) without affecting Ca50. Using a new mass spectrometry capture technique based on the biotin switch assay, we identified and characterized the formation by HNO of a disulfide-linked actin-tropomyosin and myosin heavy chain-myosin light chain 1. Comparison of the 1-nitrosocyclohexyl acetate and Angeli salt effects with the modifications induced by each donor indicated the actin-tropomyosin and myosin heavy chain-myosin light chain 1 interactions independently correlated with increased Ca(2+) sensitivity and force generation, respectively. CONCLUSIONS: HNO exerts a direct effect on cardiac myofilament proteins increasing myofilament Ca(2+) responsiveness by promoting disulfide bond formation between critical cysteine residues. These findings indicate a novel, redox-based modulation of the contractile apparatus, which positively impacts myocardial function, providing further mechanistic insight for HNO as a therapeutic agent.

GSH or palmitate preserves mitochondrial energetic/redox balance, preventing mechanical dysfunction in metabolically challenged myocytes/hearts from type 2 diabetic mice.

In type 2 diabetes, hyperglycemia and increased sympathetic drive may alter mitochondria energetic/redox properties, decreasing the organelle's functionality. These perturbations may prompt or sustain basal low-cardiac performance and limited exercise capacity. Yet the precise steps involved in this mitochondrial failure remain elusive. Here, we have identified dysfunctional mitochondrial respiration with substrates of complex I, II, and IV and lowered thioredoxin-2/glutathione (GSH) pools as the main processes accounting for impaired state 4→3 energetic transition shown by mitochondria from hearts of type 2 diabetic db/db mice upon challenge with high glucose (HG) and the ß-agonist isoproterenol (ISO). By mimicking clinically relevant conditions in type 2 diabetic patients, this regimen triggers a major overflow of reactive oxygen species (ROS) from mitochondria that directly perturbs cardiac electro-contraction coupling, ultimately leading to heart dysfunction. Exogenous GSH or, even more so, the fatty acid palmitate rescues basal and ß-stimulated function in db/db myocyte/heart preparations exposed to HG/ISO. This occurs because both interventions provide the reducing equivalents necessary to counter mitochondrial ROS outburst and energetic failure. Thus, in the presence of poor glycemic control, the diabetic patient's inability to cope with increased cardiac work demand largely stems from mitochondrial redox/energetic disarrangements that mutually influence each other, leading to myocyte or whole-heart mechanical dysfunction.

Thioredoxin reductase-2 is essential for keeping low levels of H(2)O(2) emission from isolated heart mitochondria.

Respiring mitochondria produce H(2)O(2) continuously. When production exceeds scavenging, H(2)O(2) emission occurs, endangering cell functions. The mitochondrial peroxidase peroxiredoxin-3 reduces H(2)O(2) to water using reducing equivalents from NADPH supplied by thioredoxin-2 (Trx2) and, ultimately, thioredoxin reductase-2 (TrxR2). Here, the contribution of this mitochondrial thioredoxin system to the control of H(2)O(2) emission was studied in isolated mitochondria and cardiomyocytes from mouse or guinea pig heart. Energization of mitochondria by the addition of glutamate/malate resulted in a 10-fold decrease in the ratio of oxidized to reduced Trx2. This shift in redox state was accompanied by an increase in NAD(P)H and was dependent on TrxR2 activity. Inhibition of TrxR2 in isolated mitochondria by auranofin resulted in increased H(2)O(2) emission, an effect that was seen under both forward and reverse electron transport. This effect was independent of changes in NAD(P)H or membrane potential. The effects of auranofin were reproduced in cardiomyocytes; superoxide and H(2)O(2) levels increased, but similarly, there was no effect on NAD(P)H or membrane potential. These data show that energization of mitochondria increases the antioxidant potential of the TrxR2/Trx2 system and that inhibition of TrxR2 results in increased H(2)O(2) emission through a mechanism that is independent of changes in other redox couples.

Altered myofilament stoichiometry in response to heart failure in a cardioprotective α-myosin heavy chain transgenic rabbit model.

PURPOSE: Decreases in α myosin heavy chain (α-MHC) is a common feature of human heart failure (HF), whereas α-MHC overexpression in transgenic (TG) rabbits is cardioprotective against tachycardia-induced cardiomyopathy (TIC). Hypothesizing that MHC isoform content alterations would impact sarcomere and mitochondrial energetics protein complement, we investigated the impact of α-MHC overexpression on global cardiac protein expression. EXPERIMENTAL DESIGN: Protein expression was assessed by two-dimensional gel electrophoresis and MS on the extracts from TG and nontransgenic (NTG) rabbits under TIC or sham-operated conditions. RESULTS: We observed significant changes in the levels of actin, myosin light chain 2, and desmin between the left ventricular (LV) tissue of TG and NTG animals. The proteome was broadly impacted, with significant changes in mitochondrial energetics and chaperone protein families. No changes were observed in total cellular MHC or in myofibril-associated MHC. In myofibrils isolated from TG(sham) animals, only actin levels were altered in TG(sham) compared with NTG(sham) animals, suggesting careful myofibril assembly regulation. CONCLUSIONS AND CLINICAL RELEVANCE: These data suggest that myofibril protein composition may protect against TIC, emphasizing protein interconnectivity and demonstrating the need for broad-based proteomic studies in understanding targeted genetic manipulations. This study identifies the targets for future development of cardioprotective agents and elucidates tachycardia-induced heart failure pathways.

Playing with cardiac "redox switches": the "HNO way" to modulate cardiac function.

The nitric oxide (NO(•)) sibling, nitroxyl or nitrosyl hydride (HNO), is emerging as a molecule whose pharmacological properties include providing functional support to failing hearts. HNO also preconditions myocardial tissue, protecting it against ischemia-reperfusion injury while exerting vascular antiproliferative actions. In this review, HNO's peculiar cardiovascular assets are discussed in light of its unique chemistry that distinguish HNO from NO(•) as well as from reactive oxygen and nitrogen species such as the hydroxyl radical and peroxynitrite. Included here is a discussion of the possible routes of HNO formation in the myocardium and its chemical targets in the heart. HNO has been shown to have positive inotropic/lusitropic effects under normal and congestive heart failure conditions in animal models. The mechanistic intricacies of the beneficial cardiac effects of HNO are examined in cellular models. In contrast to ß-receptor/cyclic adenosine monophosphate/protein kinase A-dependent enhancers of myocardial performance, HNO uses its "thiophylic" nature as a vehicle to interact with redox switches such as cysteines, which are located in key components of the cardiac electromechanical machinery ruling myocardial function. Here, we will briefly review new features of HNO's cardiovascular effects that when combined with its positive inotropic/lusitropic action may render HNO donors an attractive addition to the current therapeutic armamentarium for treating patients with acutely decompensated congestive heart failure.

Preparation of proteins and peptides for mass spectrometry analysis in a bottom-up proteomics workflow.

This unit outlines the steps required to prepare a sample for MS analysis following protein separation or enrichment by gel electrophoresis, liquid chromatography, and affinity capture within the context of a bottom-up proteomics workflow in which the protein is first broken up into peptides, either by chemical or enzymatic digestion, prior to MS analysis. Also included are protocols for enrichment at the peptide level, including phosphopeptide enrichment and reversed-phase chromatography for sample purification immediately prior to MS analysis. Finally, there is a discussion regarding the types of MS technologies commonly used to analyze proteomics samples, as well as important parameters that should be considered when analyzing the MS data to ensure stringent and robust protein identifications and characterization.

A solubility optimization protocol for two-dimensional gel electrophoresis of cardiac tissue.

We outline a strategy for the optimization of buffer conditions for the solubilization, extraction, and isoelectric focusing (IEF) of proteins from cardiac tissue for two-dimensional gel electrophoresis (2DE). This strategy, which involves altering both the extraction and IEF buffers, allows one to ensure representation of the proteome that is as complete as possible. Initial buffer choices are given, as well as basic protocols for modifications. Although these conditions have been effectively demonstrated for human myocardium, in principle this procedure can be used for the initial screen of any new sample of tissue or cultured cells.

A method for the effective depletion of albumin from cellular extracts: application to human myocardium.

Proteomic analysis of large numbers of proteins is assisted if each protein species is present at approximately equal concentrations. As such, the extraction of proteins from tissue samples should be designed to maintain a limited dynamic range in the concentration of proteins present. However, in many tissue extracts a high concentration of serum albumin exists from tissue perfusion and/or an inability to effectively rinse the tissue owing to surgical limitations. The analysis of these tissues would be assisted if contaminating serum albumin could be reduced. This chapter outlines a protocol for the effective reduction of serum albumin levels from human myocardium extracts enriched for soluble cytoplasmic proteins.

Optimization of cardiac troponin I pull-down by IDM affinity beads and SELDI.

Cardiac troponin I (cTnI) is a key regulator of cardiac muscle contraction. Upon myocardial cell injury, cTnI is lost from the cardiac myocyte and can be detected in serum, in some cases with specific disease-induced modifications, making it an important diagnostic marker for acute myocardial injury. Presently, hospital laboratories use enzyme-linked immunosorbent assays to detect cTnI, but this type of analysis lacks information about modified forms of protein (degradation or phosphorylation) that may give a more specific diagnosis from either serum or biopsies. Because cardiac and serum tissues are widely used for proteomic analysis, it is important to detect these cTnI posttranslational modifications. Therefore, we have chosen to optimize the enrichment and detection of cTnI protein by IDM Affinity Bead pull-down and surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS or SELDI) analyses. By adjusting the chemical compositions of the buffers, we have retained antibody specificity and enriched for different forms of cTnI and its associated proteins.

Heart disease, clinical proteomics and mass spectrometry.

Heart disease is the leading cause of mortality and morbidity in the world. As such, biomarkers are needed for the diagnosis, prognosis, therapeutic monitoring and risk stratification of acute injury (acute myocardial infarction (AMI)) and chronic disease (heart failure). The procedure for biomarker development involves the discovery, validation, and translation into clinical practice of a panel of candidate proteins to monitor risk of heart disease. Two types of biomarkers are possible; heart-specific and cardiovascular pulmonary system monitoring markers. Here we review the use of MS in the process of cardiac biomarker discovery and validation by proteomic analysis of cardiac myocytes/tissue or serum/plasma. An example of the use of MS in biomarker discovery is given in which the albumin binding protein sub-proteome was examined using MALDI-TOF MS/MS. Additionally, an example of MS in protein validation is given using affinity surface enhanced laser desorption ionization (SELDI) to monitor the disease-induced post-translational modification and the ternary status of myocyte-originating protein, cardiac troponin I in serum.

Optimizing protein solubility for two-dimensional gel electrophoresis analysis of human myocardium.

In order to maximize the myocardial proteome observed by two-dimensional gel electrophoresis (2-DE), the effect of (1) either an ionic or different zwitterionic detergents during tissue homogenization and (2) altering the "standard" detergent for isoelectric focusing (3-[(3-cholamidopropyl)dimethylamino]-1-propane sulfonate (CHAPS)) to either the zwitterionic detergent amidosulfobetaine-14 (ASB-14) or N-decyl-N-N'-dimethyl-3-ammonio-1-propane sulfonate (SB3-10) was investigated. Sodium dodecyl sulfate was shown to be a superior detergent for extraction of proteins during homogenization of cardiac tissue compared to the detergents ASB-14, SB3-10 or CHAPS. Additionally, both ASB-14 and SB3-10 exhibited better extraction than CHAPS for distinct regions of two-dimensional gels. In most cases, the best combination of homogenization and focusing conditions did not involve the use of the same detergent. Specifically, it was found that the ability to mix homogenization and focusing conditions can allow one to obtain an optimum balance between the resolution and number of protein spots obtained in 2-DE analysis of cardiac tissue. An excellent initial combination of buffers to utilize for the general examination of cardiac proteins was determined to be initial homogenization in a buffer containing ASB-14 followed by focusing in a buffer containing CHAPS.