Atherosclerosis and valvular heart disease often require treatment with corrective surgery to prevent future myocardial infarction, ischemic heart disease, and heart failure. Mechanisms underlying the development of the associated complications of surgery are multifactorial and have been linked to inflammation and oxidative stress, classically as measured in the blood or plasma of patients. Post-operative pericardial fluid (PO-PCF) has not been investigated in depth with respect to the potential to induce oxidative stress. This is important since cardiac surgery disrupts the integrity of the pericardial membrane surrounding the heart, and causes significant alterations in the composition of the pericardial fluid (PCF). This includes contamination with hemolyzed blood and high concentrations of oxidized hemoglobin, which suggests that cardiac surgery results in oxidative stress within the pericardial space. Accordingly, we tested the hypothesis that PO-PCF is highly pro-oxidant and that the potential interaction between inflammatory cell-derived hydrogen peroxide with hemoglobin is associated with oxidative stress. Blood and PCF were collected from 31 patients at the time of surgery and postoperatively from 4 to 48 hours after coronary artery bypass grafting, valve replacement, or valve repair (mitral or aortic). PO-PCF contained high concentrations of neutrophils and monocytes which are capable of generating elevated amounts of superoxide and hydrogen peroxide through the oxidative burst. In addition, PO-PCF primed naïve neutrophils resulting in an enhanced oxidative burst upon stimulation. The PO-PCF also contained increased concentrations of cell-free oxidized hemoglobin which was associated with elevated levels of F2α-isoprostanes and prostaglandins, consistent with both oxidative stress and activation of cyclooxygenase. Lastly, protein analysis of the PO-PCF revealed evidence of protein thiol oxidation and protein carbonylation. We conclude that PO-PCF is highly pro-oxidant and speculate that it may contribute to the risk of post-operative complications.
The mitochondrion plays a crucial role in the immune system particularly in regulating the responses of monocytes and macrophages to tissue injury, pathogens, and inflammation. In systemic diseases such as atherosclerosis and chronic kidney disease (CKD) it has been established that disruption to monocyte and macrophage function can lead to chronic inflammation. Polarization of macrophages into the pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes results in distinct metabolic reprograming which corresponds to the progression and resolution of inflammation. In this review, we will discuss the role of the mitochondrion in monocyte and macrophage function and how these cells specifically influence the pathophysiology of atherosclerosis and CKD. We propose that assessing monocyte bioenergetics in different disease states could (1) enhance our understanding of the energetic perturbations occurring in systemic inflammatory conditions and (2) aid in identifying therapeutic interventions to mitigate these disorders in patients.
monocyte; macrophage; atherosclerosis; chronic kidney disease; oxidative stress; metabolic shift; biomarker
Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging. Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis. This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry. An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.
Immunology; Issue 85; bioenergetics; translational; mitochondria; oxidative stress; reserve capacity; leukocytes
Bioenergetics has become central to our understanding of pathological mechanisms, the
development of new therapeutic strategies and as a biomarker for disease progression
in neurodegeneration, diabetes, cancer and cardiovascular disease. A key concept is
that the mitochondrion can act as the ‘canary in the coal mine’ by
serving as an early warning of bioenergetic crisis in patient populations. We propose
that new clinical tests to monitor changes in bioenergetics in patient populations
are needed to take advantage of the early and sensitive ability of bioenergetics to
determine severity and progression in complex and multifactorial diseases. With the
recent development of high-throughput assays to measure cellular energetic function
in the small number of cells that can be isolated from human blood these clinical
tests are now feasible. We have shown that the sequential addition of
well-characterized inhibitors of oxidative phosphorylation allows a bioenergetic
profile to be measured in cells isolated from normal or pathological samples. From
these data we propose that a single value–the Bioenergetic Health Index
(BHI)–can be calculated to represent the patient's composite mitochondrial
profile for a selected cell type. In the present Hypothesis paper, we discuss how BHI
could serve as a dynamic index of bioenergetic health and how it can be measured in
platelets and leucocytes. We propose that, ultimately, BHI has the potential to be a
new biomarker for assessing patient health with both prognostic and diagnostic
aging; cardiovascular disease; haplotype; hepatotoxicity; neurodegenerative disease; oxidative stress; reserve capacity; BHI, Bioenergetic Health Index; ETC, electron transport chain; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; HNE, hydroxynonenal; LDA, linear discriminant analysis; mtDNA, mitochondrial DNA; OCR, oxygen consumption rate; RNS, reactive nitrogen species; ROS, reactive oxygen species
Mitochondrial dysfunction is associated with a broad range of pathologies including diabetes, ethanol toxicity, metabolic syndrome, and cardiac failure. It is now becoming clear that maintaining mitochondrial quality through a balance between biogenesis, reserve capacity, and mitophagy is critical in determining the response to metabolic or xenobiotic stress. In diseases associated with metabolic stress, such as type II diabetes, non-alcoholic and alcoholic steatosis, the mitochondria are subjected to multiple “hits” such as hypoxia and oxidative and nitrative stress, which can overwhelm mitochondrial quality control pathways. In addition, the underlying mitochondrial genetics which evolved to accommodate high energy demand, low calorie supply environments may now be maladapted to modern lifestyles (low energy demand, high calorie environments). The pro-oxidant and pro-inflammatory environment of a sedentary western lifestyle has been associated with modified redox cell signaling pathways such as steatosis, hypoxic signaling, inflammation, and fibrosis. These data suggest that loss of mitochondrial quality control is intimately associated with the aberrant activation of redox cell signaling pathways under pathological conditions. In this short review, we will discuss evidence from alcoholic liver disease supporting this concept, the insights obtained from experimental models, and the application of bioenergetic based therapeutics in the context of maintaining mitochondrial quality.
reserve respiratory capacity; mitochondria; cellular bioenergetics; steatosis; alcoholic liver disease; diabetes; metabolic syndrome; MitoQ
Mitochondria are recognized as critical sites of localized injury in a number of chronic pathologies which has led to the development of organelle directed therapeutics. One of the approaches employed to target molecules to the mitochondrion is to conjugate a delocalized cation such as triphenylphosphonium (TPP+) to various redox active compounds. Mitochondrially targeted antioxidants have also been used in numerous cell culture based studies as probes of the contribution of the mitochondrial generation of reactive oxygen species on cell signaling events. However, concentrations used in vitro are typically 10–100 times greater than those generated from oral dosing in a wide range of animal models and in humans. In the present study, we determined the effects of mitochondrial targeted antioxidants, MitoQ, MitoTempol, and MitoE on cellular bioenergetics of mesangial cells in culture and compared these to TPP+ conjugated compounds which lack the antioxidant functional group. We found that all TPP+ compounds inhibited oxidative phosphorylation to different extents independent of the antioxidant functional groups. These findings show that the TPP+ moiety can disrupt mitochondrial function at concentrations frequently observed in cell culture and this behavior is dependent on the linker group and independent of antioxidant properties. Moreover, TPP+ moiety alone is unlikely to achieve the concentrations needed to contribute to the protective mechanisms of the mitochondrially targeted compounds that have been reported in vivo.
Mitochondrial targeted compounds; mitochondria; respiration; extracellular flux; MitoQ; TPP+ derivatives; redox
Investigators in the redox biology field have long recognized the unique role mitochondrial superoxide generation plays in physiological signaling and in dysregulated bioenergetic dysfunction. Pharmacological manipulation has been challenging and in this issue of Chemistry & Biology, Kelso et al. (2012) present synthesis and characterization of a novel mitochondrial-targeted SOD mimetic, MitoSOD.
Cold storage (CS) is regarded as a necessary procedure during donation of a deceased donor kidney that helps to optimize organ viability. Increased oxidant generation during both CS as well as during the reperfusion (or rewarming/CS.RW) phase have been suggested to be a major contributor to renal injury; although the source and/or biochemical pathways involved with oxidant production remain unclear. The purpose of this study was to determine if renal tubular mitochondrial superoxide is capable of inducing oxidant production and mitochondrial damage in response to a CS.RW insult. To test the role of mitochondrial superoxide in CS.RW injury, we used rat renal proximal tubular (NRK) cells overexpressing manganese superoxide dismutase (MnSOD), the major mitochondrial antioxidant. Oxidant production, mitochondrial membrane potential, respiratory complex function, and cell death were all altered following exposure of NRK cells to CS.RW. MnSOD overexpression or inhibition of nitric oxide synthase (NOS) provided significant protection against oxidant generation, respiratory complex inactivation, and cell death. These findings implicate mitochondrial superoxide, nitric oxide, and their reaction product, peroxynitrite, as key signaling molecules involved in CS.RW injury of renal tubular cells, and suggest that therapeutic inhibition of these pathways may protect the donor kidney.
Cold preservation; cold storage; superoxide; nitric oxide; peroxynitrite; mitochondria; respiratory complexes
Mitochondria are recognized as critical sites of localized injury in a number of chronic pathologies which has led to the development of organelle directed therapeutics. One of the approaches employed to target molecules to the mitochondrion is to conjugate a delocalized cation such as triphenylphosphonium (TPP+) to various redox active compounds. Mitochondrially targeted antioxidants have also been used in numerous cell culture based studies as probes of the contribution of the mitochondrial generation of reactive oxygen species on cell signaling events. However, concentrations used in vitro are typically 10–100 times greater than those generated from oral dosing in a wide range of animal models and in humans. In the present study, we determined the effects of mitochondrial targeted antioxidants, MitoQ, MitoTempol, and MitoE on cellular bioenergetics of mesangial cells in culture and compared these to TPP+ conjugated compounds which lack the antioxidant functional group. We found that all TPP+ compounds inhibited oxidative phosphorylation to different extents independent of the antioxidant functional groups. These findings show that the TPP+ moiety can disrupt mitochondrial function at concentrations frequently observed in cell culture and this behavior is dependent on the linker group and independent of antioxidant properties. Moreover, the TPP+ moiety alone is unlikely to achieve the concentrations needed to contribute to the protective mechanisms of the mitochondrially targeted compounds that have been reported in vivo.
► Mitochondrial targeted antioxidants are conjugated to a triphenylphosphonium cation (TPP+) moiety that allows the compound to accumulate within the mitochondria. ► The effect of MitoQ, MitoTempol, and MitoE and their TPP+ carrier groups on oxidative phosphorylation was examined. ► Higher concentrations of TPP+ conjugated compounds alter cellular bioenergetics independent of the functional antioxidant group. ► Many of the effects ascribed to mitochondrial oxidant scavenging by these compounds in cell culture may be nonspecific effects of the carrier molecule.
AA, Antimycin A; BTPP, butyl triphenylphosphonium; DTPP, decyl triphenylphosphonium; ECAR, extracellular acidification rate; FCCP, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; MES-13, murine mesangial cells; MitoE, Mito Vitamin E; MitoQ, Mitoquinone; OCR, oxygen consumption rate; Oligo, Oligomycin; RNS, reactive nitrogen species; ROS, reactive oxygen species; TPP+, triphenylphosphonium; TPMP, triphenylmethylphosphonium; Mitochondrial targeted compounds; Mitochondria; Respiration; Extracellular flux; MitoQ; TPP+ derivatives; Redox
Inactivation of manganese superoxide dismutase (MnSOD), a mitochondrial antioxidant, has been associated with renal disorders and often results in detrimental downstream events that are mechanistically not clear. Development of an animal model that exhibits kidney-specific deficiency of MnSOD would be extremely beneficial in exploring the downstream events that occur following MnSOD inactivation. Using Cre-Lox recombination technology, kidney-specific MnSOD deficient mice (both 100% and 50%) were generated that exhibited low expression of MnSOD in discrete renal cell types and reduced enzymatic activity within the kidney. These kidney-specific 100% KO mice possessed a normal life-span, although it was interesting that the mice were smaller. Consistent with the important role in scavenging superoxide radicals, the kidney-specific KO mice showed a significant increase in oxidative stress (tyrosine nitration) in a gene-dose dependent manner. In addition, loss of MnSOD resulted in mild renal damage (tubular dilation and cell swelling). Hence, this novel mouse model will aid in determining the specific role (local and/or systemic) governed by MnSOD within certain kidney cells. Moreover, these mice will serve as a powerful tool to explore molecular mechanisms that occur downstream of MnSOD inactivation in renal disorders or possibly in other pathologies that rely on normal renal function.
Cre-Lox technology; Kidney; MnSOD; Cre recombinase; Superoxide; Nitrotyrosine
Diabetes has become the single most common cause for end-stage renal disease in the United States. It has been established that mitochondrial damage occurs during diabetes; however, little is known about what initiates mitochondrial injury and oxidant production during the early stages of diabetes. Inactivation of mitochondrial respiratory complexes or alteration of their critical subunits can lead to generation of mitochondrial oxidants, mitochondrial damage, and organ injury. Thus, one goal of this study was to determine the status of mitochondrial respiratory complexes in the rat kidney during the early stages of diabetes (5-weeks post streptozotocin injection).
Mitochondrial complex activity assays, blue native gel electrophoresis (BN-PAGE), Complex III immunoprecipitation, and an ATP assay were performed to examine the effects of diabetes on the status of respiratory complexes and energy levels in renal mitochondria. Creatinine clearance and urine albumin excretion were measured to assess the status of renal function in our model.
Interestingly, of all four respiratory complexes only cytochrome c reductase (Complex-III) activity was significantly decreased, whereas two Complex III subunits, Core 2 protein and Rieske protein, were up regulated in the diabetic renal mitochondria. The BN-PAGE data suggested that Complex III failed to assemble correctly, which could also explain the compensatory upregulation of specific Complex III subunits. In addition, the renal F0F1-ATPase activity and ATP levels were increased during diabetes.
In summary, these findings show for the first time that early (and selective) inactivation of Complex-III may contribute to the mitochondrial oxidant production which occurs in the early stages of diabetes.
Renal ischemia/reperfusion (I/R) injury often occurs as a result of vascular surgery, organ procurement, or transplantation. We previously showed that renal I/R results in ATP depletion, oxidant production, and manganese superoxide dismutase (MnSOD) inactivation. There have been several reports that overexpression of MnSOD protects tissues/organs from I/R related damage, thus a loss of MnSOD activity during I/R likely contributes to tissue injury. The present study examined the therapeutic benefit of a catalytic antioxidant Mn(III) meso-tetrakis(N-hexylpyridinium-2-yl)porphyrin, (MnTnHex-2-PyP5+) using the rat renal I/R model. This was the first study to examine the effects of MnTnHex-2-PyP5+ in an animal model of oxidative stress injury. Our results showed that porphyrin pretreatment of rats for 24 hr protected against ATP depletion, MnSOD inactivation, nitrotyrosine formation, and renal dysfunction. The dose (50 μg/kg) used in this study is lower than doses of various types of antioxidants commonly used in animal models of oxidative stress injuries. In addition, using novel proteomic techniques, we identified ATP synthase- beta subunit as a key protein induced by MnTnHex-2-PyP5+ treatment alone, and complex V (ATP synthase) as a target of injury during renal I/R. These results showed that MnTnHex-2-PyP5+ protected against renal I/R injury via induction of key mitochondrial proteins that may be capable of blunting oxidative injury.
kidney; ischemia/reperfusion; metalloporphyrin; proteomics; MnSOD; mitochondria; oxidants; nitrotyrosine; blue native polyacrylamide gel electrophoresis BN-PAGE; two dimensional fluorescence differential in gel electrophoresis (2D-DIGE)