John M. Hollander

Overexpression of Wild-Type Heat Shock Protein 27 and a Nonphosphorylatable Heat Shock Protein 27 Mutant Protects Against Ischemia/Reperfusion Injury in a Transgenic Mouse Model

Background—The small heat shock protein 27 (hsp27) increases in expression with ischemia/reperfusion (I/R) insult in the heart. One feature of the small hsps is their ability to oligomerize and form intracellular aggregates. Oligomerization pattern is governed by the phosphorylation state of the protein that may influence their ability to protect against cellular stresses. Methods and Results—We generated transgenic (tg) mice that overexpress a wild-type human hsp27 (hsp27tg) protein or a mutant hsp27 protein (mut-hsp27tg), in which serine residues (aa15, aa78, and aa82) were replaced by alanine residues, rendering them incapable of phosphorylation. Using a Langendorff perfusion model and an intraventricular balloon, we subjected hearts to 20 minutes of ischemia followed by 1 hour of reperfusion. During reperfusion, negative and positive pressure derivatives as well as developed pressures were significantly higher in both hsp27tg and mut-hsp27tg compared with control (P<0.01) mice, with no significant difference between hsp27tg and mut-hsp27tg. Creatine kinase release during reperfusion was higher in control compared with both hsp27tg and mut-hsp27tg (P<0.05). Malondialdehyde content as well as protein oxidation products were lower in mut-hsp27tg compared with control (P<0.05). hsp27tg hearts possessed oligomers that ranged in size from small to large, whereas mut-hsp27tg hearts contained no small oligomers. Conclusions—These results indicate that in a tg mouse model, overexpression of either wild-type hsp27 or a nonphosphorylatable hsp27 mutant was equally capable of protecting the heart from I/R injury. Furthermore, the phosphorylation status of hsp27 may influence its ability to decrease oxidative stress.

Oxidative Stress and Aging: Role of Exercise and Its Influences on Antioxidant Systems

Strenuous exercise is characterized by an increased oxygen consumption and disturbance of intracellular prooxidant‐antioxidant homeostasis. At least three biochemical pathways, that is, mitochondrial electron transport chain, xanthine oxidase, and polymorphoneutrophil have been identified as potential sources of intracellular free radical generation during exercise. These deleterious reactive oxygen species pose a serious threat to the cellular antioxidant defense system, such as diminished reserve of antioxidant vitamins and glutathione, and have been shown to cause oxidative damage in exercising and/or exercised muscle and other tissues. However, enzymatic and nonenzymatic antioxidants have demonstrated great versatility and adaptability in response to acute and chronic exercise. The delicate balance between prooxidants and antioxidants during exercise may be altered with aging. Study of the complicated interaction between aging and exercise under the influence of reactive oxygen species would provide more definitive information as to how much aged individuals should be involved in physical activity and whether supplementation of nutritional antioxidants would be desirable.

Diabetic cardiomyopathy-associated dysfunction in spatially distinct mitochondrial subpopulations

Diabetic cardiomyopathy is the leading cause of heart failure among diabetic patients, and mitochondrial dysfunction has been implicated as an underlying cause in the pathogenesis. Cardiac mitochondria consist of two spatially, functionally, and morphologically distinct subpopulations, termed subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM). SSM are situated beneath the plasma membrane, whereas IFM are embedded between myofibrils. The goal of this study was to determine whether spatially distinct cardiac mitochondrial subpopulations respond differently to a diabetic phenotype. Swiss-Webster mice were subjected to intraperitoneal injections of streptozotocin or citrate saline vehicle. Five weeks after injections, diabetic hearts displayed decreased rates of contraction, relaxation, and left ventricular developed pressures (P < 0.05 for all three). Both mitochondrial size (forward scatter, P < 0.01) and complexity (side scatter, P < 0.01) were decreased in diabetic IFM but not diabetic SSM. Electron transport chain complex II respiration was decreased in diabetic SSM (P < 0.05) and diabetic IFM (P < 0.01), with the decrease being greater in IFM. Furthermore, IFM complex I respiration and complex III activity were decreased with diabetes (P < 0.01) but were unchanged in SSM. Superoxide production was increased only in diabetic IFM (P < 0.01). Oxidative damage to proteins and lipids, indexed through nitrotyrosine residues and lipid peroxidation, were higher in diabetic IFM (P < 0.05 and P < 0.01, respectively). The mitochondria-specific phospholipid cardiolipin was decreased in diabetic IFM (P < 0.01) but not SSM. These results indicate that diabetes mellitus imposes a greater stress on the IFM subpopulation, which is associated, in part, with increased superoxide generation and oxidative damage, resulting in morphological and functional abnormalities that may contribute to the pathogenesis of diabetic cardiomyopathy.

Superoxide dismutase gene expression in skeletal muscle: fiber-specific adaptation to endurance training.

The effects of endurance training on the enzyme activity, protein content, and mRNA abundance of Mn and CuZn superoxide dismutase (SOD) were studied in various phenotypes of rat skeletal muscle. Female Sprague-Dawley rats were randomly divided into trained (T, n = 8) and untrained (U, n = 8) groups. Training, consisting of treadmill running at 27 m/min and 12% grade for 2 h/day, 5 days/wk for 10 wk, significantly increased citrate synthase activity ( P < 0.01) in the type I (soleus), type IIa (deep vastus lateralis, DVL), and mixed type II (plantaris) muscles but not in type IIb (superficial vastus lateralis, SVL) muscle. Mitochondrial (Mn) SOD activity was elevated by 80% ( P < 0.05) with training in DVL. SVL and plantaris muscle in T rats showed 54 and 42% higher pooled immunoreactive Mn SOD protein content, respectively, than those in U rats. However, no change in Mn SOD mRNA level was found in any of the muscles. CuZn SOD activity, protein content, and mRNA level in general were not affected by training, except for a 160% increase in pooled CuZn SOD protein in SVL. Training also significantly increased glutathione peroxidase and catalase activities ( P < 0.05), but only in DVL muscle. These data indicate that training adaptations of Mn SOD and other antioxidant enzymes occur primarily in type IIa fibers, probably as a result of enhanced free radical generation and modest antioxidant capacity. Differential training responses of mRNA, enzyme protein, and activity suggest that separate cellular signals may control pre- and posttranslational regulation of SOD.

Mitochondrial dysfunction in the type 2 diabetic heart is associated with alterations in spatially distinct mitochondrial proteomes.

Cardiac complications and heart failure are the leading cause of death in type 2 diabetic patients. Mitochondrial dysfunction is central in the pathogenesis of the type 2 diabetic heart. However, it is unclear whether this dysfunction is specific for a particular subcellular region. The purpose of this study was to determine whether mitochondrial dysfunction in the type 2 diabetic heart is specific to a spatially distinct subset of mitochondria. We investigated mitochondrial morphology, function, and proteomic composition of subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM) in 18-wk-old db/db mice. Oxidative damage was assessed in subpopulations through the measurement of lipid peroxidation byproducts and nitrotyrosine residues. Proteomic profiles and posttranslational modifications were assessed in mitochondrial subpopulations using iTRAQ and multi-dimensional protein identification technologies, respectively. SSM from db/db hearts had altered morphology, including a decrease in size and internal complexity, whereas db/db IFM were increased in internal complexity. Db/db SSM displayed decreased state 3 respiration rates, electron transport chain activities, ATP synthase activities, and mitochondrial membrane potential and increased oxidative damage, with no change in IFM. Proteomic assessment revealed a greater impact on db/db SSM compared with db/db IFM. Inner mitochondrial membrane proteins, including electron transport chain, ATP synthesis, and mitochondrial protein import machinery, were predominantly decreased. We provide evidence that mitochondrial dysfunction in the type 2 diabetic heart is associated with a specific subcellular locale. Furthermore, mitochondrial morphological and functional indexes are impacted differently during type 2 diabetic insult and may result from the modulation of spatially distinct mitochondrial proteomes.

Proteomic alterations of distinct mitochondrial subpopulations in the type 1 diabetic heart: contribution of protein import dysfunction.

Diabetic cardiomyopathy is associated with increased risk of heart failure in type 1 diabetic patients. Mitochondrial dysfunction is suggested as an underlying contributor to diabetic cardiomyopathy. Cardiac mitochondria are characterized by subcellular spatial locale, including mitochondria located beneath the sarcolemma, subsarcolemmal mitochondria (SSM), and mitochondria situated between the myofibrils, interfibrillar mitochondria (IFM). The goal of this study was to determine whether type 1 diabetic insult in the heart influences proteomic make-up of spatially distinct mitochondrial subpopulations and to evaluate the role of nuclear encoded mitochondrial protein import. Utilizing multiple proteomic approaches (iTRAQ and two-dimensional-differential in-gel electrophoresis), IFM proteomic make-up was impacted by type 1 diabetes mellitus to a greater extent than SSM, as evidenced by decreased abundance of fatty acid oxidation and electron transport chain proteins. Mitochondrial phosphate carrier and adenine nucleotide translocator, as well as inner membrane translocases, were decreased in the diabetic IFM (P < 0.05 for both). Mitofilin, a protein involved in cristae morphology, was diminished in the diabetic IFM (P < 0.05). Posttranslational modifications, including oxidations and deamidations, were most prevalent in the diabetic IFM. Mitochondrial heat shock protein 70 (mtHsp70) was significantly decreased in diabetic IFM (P < 0.05). Mitochondrial protein import was decreased in the diabetic IFM with no change in the diabetic SSM (P < 0.05). Taken together, these results indicate that mitochondrial proteomic alterations in the type 1 diabetic heart are more pronounced in the IFM. Further, proteomic alterations are associated with nuclear encoded mitochondrial protein import dysfunction and loss of an essential mitochondrial protein import constituent, mtHsp70, implicating this process in the pathogenesis of the diabetic heart.

Enhanced apoptotic propensity in diabetic cardiac mitochondria: influence of subcellular spatial location

Cardiovascular complications, such as diabetic cardiomyopathy, account for the majority of deaths associated with diabetes mellitus. Mitochondria are particularly susceptible to the damaging effects of diabetes mellitus and have been implicated in the pathogenesis of diabetic cardiomyopathy. Cardiac mitochondria consist of two spatially distinct subpopulations, termed subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM). The goal of this study was to determine whether subcellular spatial location is associated with apoptotic propensity of cardiac mitochondrial subpopulations during diabetic insult. Swiss Webster mice were subjected to intraperitoneal injection of streptozotocin or citrate saline vehicle. Ten weeks following injection, diabetic hearts displayed increased caspase-3 and caspase-9 activities, indicating enhanced apoptotic signaling (P < 0.05, for both). Mitochondrial size (forward scatter) and internal complexity (side scatter) were decreased in diabetic IFM (P < 0.05, for both) but not in diabetic SSM. Mitochondrial membrane potential (Delta(Psim)) was lower in diabetic IFM (P < 0.01) but not in diabetic SSM. Mitochondrial permeability transition pore (mPTP) opening was increased in diabetic compared with control IFM (P < 0.05), whereas no differences were observed in diabetic compared with control SSM. Examination of mPTP constituents revealed increases in cyclophilin D in diabetic IFM. Furthermore, diabetic IFM possessed lower cytochrome c and BcL-2 levels and increased Bax levels (P < 0.05, for all 3). No significant changes in these proteins were observed in diabetic SSM compared with control. These results indicate that diabetes mellitus is associated with an enhanced apoptotic propensity in IFM, suggesting a differential apoptotic susceptibility of distinct mitochondrial subpopulations based upon subcellular location.

Superoxide dismutase gene expression in skeletal muscle: fiber-specific effect of age

The influence of ageing on the expression of two superoxide dismutase (SOD) isozymes was examined in three different skeletal muscle fiber types of young (Y, 8 mo) and old (O, 25 mo) rats. Total SOD activity was increased with age in the gastrocnemius (Gas, type II(mix)) and superficial vastus lateralis (SVL, type IIb) but unchanged in the soleus (Sol, type I). The increased SOD activity in SVL was due to increased cytosolic SOD (CuZn SOD), whereas both mitochondrial (Mn SOD) and CuZn SOD activities were increased in Gas. In Sol, Mn SOD activity was significantly increased in aged rats. Mn SOD mRNA level was significantly decreased with age in all three muscles examined, while Mn SOD protein content was not altered. Ageing did not affect CuZn SOD mRNA abundance in any of the muscles, but significantly increased CuZn SOD protein content in aged Gas and Sol. Binding of two redox-sensitive transcription factors, nuclear factor-kappaB (NFkappaB) and activator protein-1 (AP-1) was significantly decreased with age in all three muscle types. These results indicate that increased SOD activity in aged skeletal muscle is not associated with higher levels of gene transcription. Increases in Mn SOD activity seen in aged Gas and Sol are the result of post-translational modification of the enzyme, whereas increases in CuZn SOD activity during ageing may be due to both translational and post-translational control.

Suppression of Oxidative Stress by Resveratrol After Isometric Contractions in Gastrocnemius Muscles of Aged Mice

This study tested the hypothesis that resveratrol supplementation would lower oxidative stress in exercised muscles of aged mice. Young (3 months) and aged (27 months) C57BL/6 mice received a control or a 0.05% trans-resveratrol-supplemented diet for 10 days. After 7 days of dietary intervention, 20 maximal electrically evoked isometric contractions were obtained from the plantar flexors of one limb in anesthetized mice. Exercise was conducted for three consecutive days. Resveratrol supplementation blunted the exercise-induced increase in xanthine oxidase activity in muscles from young (25%) and aged (53%) mice. Resveratrol lowered H(2)O(2) levels in control (13%) and exercised (38%) muscles from aged animals, reduced Nox4 protein in both control and exercised muscles of young (30%) and aged mice (40%), and increased the ratio of reduced glutathione to oxidized glutathione in exercised muscles from young (38%) and aged (135%) mice. Resveratrol prevented the increase in lipid oxidation, increased catalase activity, and increased MnSOD activity in exercised muscles from aged mice. These data show that dietary resveratrol suppresses muscle indicators of oxidative stress in response to isometric contractions in aged mice.

Mitochondria-specific transgenic overexpression of phospholipid hydroperoxide glutathione peroxidase (GPx4) attenuates ischemia/reperfusion-associated cardiac dysfunction.

Ischemia/reperfusion (I/R) injury elicits damage to mitochondria. Antioxidants provide protection from I/R-induced mitochondrial damage. The goal of this study was to determine the impact of mitochondria-specific overexpression of GPx4 (PHGPx) on cardiac function following I/R. Transgenic mice were created in which PHGPx was overexpressed solely in the mitochondrion (mPHGPx). MPHGPx and littermate control hearts were subjected to global no-flow ischemia (20 min) followed by reflow reperfusion (30, 60, and 90 min). Following I/R, mPHGPx hearts possessed significantly better rates of contraction, developed pressures, and peak-systolic pressures as compared to controls (P<0.05). No differences were observed in rates of relaxation or end-diastolic pressures. Lipid peroxidation was significantly lower in mitochondria from mPHGPx hearts as compared to controls, following I/R (P<0.05). Electron transport chain (ETC) complex I, III, and IV activities were significantly higher in mPHGPx hearts as compared to controls, following I/R (P<0.05). MPHGPx overexpression enhanced ETC complex I, III, and IV activities in subsarcolemmal mitochondria (SSM; P<0.05), and ETC complex I and III activities in interfibrillar mitochondria (IFM; P<0.05) following I/R. These results indicate that mitochondria-specific GPx4 overexpression protects cardiac contractile function and preserves ETC complex activities following I/R. These results provide further rationale for the use of mPHGPx as a therapeutic protectant.