Kathleen Thayne

Increased propensity for cell death in diabetic human heart is mediated by mitochondrial-dependent pathways

Progressive energy deficiency and loss of cardiomyocyte numbers are two prominent factors that lead to heart failure in experimental models. Signals that mediate cardiomyocyte cell death have been suggested to come from both extrinsic (e.g., cytokines) and intrinsic (e.g., mitochondria) sources, but the evidence supporting these mechanisms remains unclear, and virtually nonexistent in humans. In this study, we investigated the sensitivity of the mitochondrial permeability transition pore (mPTP) to calcium (Ca(2+)) using permeabilized myofibers of right atrium obtained from diabetic (n = 9) and nondiabetic (n = 12) patients with coronary artery disease undergoing nonemergent coronary revascularization surgery. Under conditions that mimic the energetic state of the heart in vivo (pyruvate, glutamate, malate, and 100 μM ADP), cardiac mitochondria from diabetic patients show an increased sensitivity to Ca(2+)-induced mPTP opening compared with nondiabetic patients. This increased mPTP Ca(2+) sensitivity in diabetic heart mitochondria is accompanied by a substantially greater rate of mitochondrial H(2)O(2) emission under identical conditions, despite no differences in respiratory capacity under these conditions or mitochondrial enzyme content. Activity of the intrinsic apoptosis pathway mediator caspase-9 was greater in diabetic atrial tissue, whereas activity of the extrinsic pathway mediator caspase-8 was unchanged between groups. Furthermore, caspase-3 activity was not significantly increased in diabetic atrial tissue. These data collectively suggest that the myocardium in diabetic patients has a greater overall propensity for mitochondrial-dependent cell death, possibly as a result of metabolic stress-imposed changes that have occurred within the mitochondria, rendering them more susceptible to insults such as Ca(2+) overload. In addition, they lend further support to the notion that mitochondria represent a viable target for future therapies directed at ameliorating heart failure and other comorbidities that come with diabetes.

Aldehyde stress and up-regulation of Nrf2-mediated antioxidant systems accompany functional adaptations in cardiac mitochondria from mice fed n−3 polyunsaturated fatty acids

Diets replete with n-3 PUFAs (polyunsaturated fatty acids) are known to have therapeutic potential for the heart, although a specifically defined duration of the n-3 PUFA diet required to achieve these effects remains unknown, as does their mechanism of action. The present study was undertaken to establish whether adaptations in mitochondrial function and stress tolerance in the heart is evident following short- (3xa0weeks) and long- (14xa0weeks) term dietary intervention of n-3 PUFAs, and to identify novel mechanisms by which these adaptations occur. Mitochondrial respiration [mO2 (mitochondrial O2)], H2O2 emission [mH2O2 (mitochondrial H2O2)] and Ca2+-retention capacity [mCa2+ (mitochondrial Ca2+)] were assessed in mouse hearts following dietary intervention. Mice fed n-3 PUFAs for 14xa0weeks showed significantly lower mH2O2 and greater mCa2+ compared with all other groups. However, no significant differences were observed after 3xa0weeks of the n-3 PUFA diet, or in mice fed on an HFC (high-fat control) diet enriched with vegetable shortening, containing almost no n-3 PUFAs, for 14xa0weeks. Interestingly, expression and activity of key enzymes involved in antioxidant and phase II detoxification pathways, all mediated by Nrf2 (nuclear factor E2-related factor 2), were elevated in hearts from mice fed the n-3 PUFA diet, but not hearts from mice fed the HFC diet, even at 3xa0weeks. This increase in antioxidant systems in hearts from mice fed the n-3 PUFA diet was paralleled by increased levels of 4-hydroxyhexenal protein adducts, an aldehyde formed from peroxidation of n-3 PUFAs. The findings of the present study demonstrate distinct time-dependent effects of n-3 PUFAs on mitochondrial function and antioxidant response systems in the heart. In addition, they are the first to provide direct evidence that non-enzymatic oxidation products of n-3 PUFAs may be driving mitochondrial and redox-mediated adaptations, thereby revealing a novel mechanism for n-3 PUFA action in the heart.

Monoamine Oxidase is a Major Determinant of Redox Balance in Human Atrial Myocardium and is Associated With Postoperative Atrial Fibrillation

Background Onset of postoperative atrial fibrillation (POAF) is a common and costly complication of heart surgery despite major improvements in surgical technique and quality of patient care. The etiology of POAF, and the ability of clinicians to identify and therapeutically target high‐risk patients, remains elusive. Methods and Results Myocardial tissue dissected from right atrial appendage (RAA) was obtained from 244 patients undergoing cardiac surgery. Reactive oxygen species (ROS) generation from multiple sources was assessed in this tissue, along with total glutathione (GSHt) and its related enzymes GSH‐peroxidase (GPx) and GSH‐reductase (GR). Monoamine oxidase (MAO) and NADPH oxidase were observed to generate ROS at rates 10‐fold greater than intact, coupled mitochondria. POAF risk was significantly associated with MAO activity (Quartile 1 [Q1]: adjusted relative risk [ARR]=1.0; Q2: ARR=1.8, 95% confidence interval [CI]=0.84 to 4.0; Q3: ARR=2.1, 95% CI=0.99 to 4.3; Q4: ARR=3.8, 95% CI=1.9 to 7.5; adjusted Ptrend=0.009). In contrast, myocardial GSHt was inversely associated with POAF (Quartile 1 [Q1]: adjusted relative risk [ARR]=1.0; Q2: ARR=0.93, 95% confidence interval [CI]=0.60 to 1.4; Q3: ARR=0.62, 95% CI=0.36 to 1.1; Q4: ARR=0.56, 95% CI=0.34 to 0.93; adjusted Ptrend=0.014). GPx also was significantly associated with POAF; however, a linear trend for risk was not observed across increasing levels of the enzyme. GR was not associated with POAF risk. Conclusions Our results show that MAO is an important determinant of redox balance in human atrial myocardium, and that this enzyme, in addition to GSHt and GPx, is associated with an increased risk for POAF. Further investigation is needed to validate MAO as a predictive biomarker for POAF, and to explore this enzymes potential role in arrhythmogenesis.

Novel role for thioredoxin reductase‐2 in mitochondrial redox adaptations to obesogenic diet and exercise in heart and skeletal muscle

•u2002 For reasons not completely understood, obesogenic high‐fat, high‐sucrose (HFHS) diets and exercise training both increase free fatty acid utilization and chronic oxidative stress, yet the former is deleterious to cardiovascular/metabolic health, whereas the latter is beneficial. •u2002 Here, we report that the heart shows decreased mitochondrial H2O2 (mH2O2) generation following HFHS diet, while skeletal muscle shows increased mH2O2, and uncover a novel role for thioredoxin reductase‐2 (TxnRd2) underlying these differences. •u2002 We also show that TxnRd2 is critical to controlling mH2O2 levels during mitochondrial fatty acid oxidation, especially following exercise training in skeletal muscle. •u2002 These findings are important in that they illustrate how the heart and skeletal muscle have contrasting adaptations in antioxidant capacity in response to HFHS diet, and uncover a new role for TxnRd2 in the overall control of mH2O2 in these organs with HFHS diet and exercise training.

Obesity in a model of gpx4 haploinsufficiency uncovers a causal role for lipid-derived aldehydes in human metabolic disease and cardiomyopathy

Objective Lipid peroxides and their reactive aldehyde derivatives (LPPs) have been linked to obesity-related pathologies, but whether they have a causal role has remained unclear. Glutathione peroxidase 4 (GPx4) is a selenoenzyme that selectively neutralizes lipid hydroperoxides, and human gpx4 gene variants have been associated with obesity and cardiovascular disease in epidemiological studies. This study tested the hypothesis that LPPs underlie cardio-metabolic derangements in obesity using a high fat, high sucrose (HFHS) diet in gpx4 haploinsufficient mice (GPx4+/−) and in samples of human myocardium. Methods Wild-type (WT) and GPx4+/− mice were fed either a standard chow (CNTL) or HFHS diet for 24 weeks, with metabolic and cardiovascular parameters measured throughout. Biochemical and immuno-histological analysis was performed in heart and liver at termination of study, and mitochondrial function was analyzed in heart. Biochemical analysis was also performed on samples of human atrial myocardium from a cohort of 103 patients undergoing elective heart surgery. Results Following HFHS diet, WT mice displayed moderate increases in 4-hydroxynonenal (HNE)-adducts and carbonyl stress, and a 1.5-fold increase in GPx4 enzyme in both liver and heart, while gpx4 haploinsufficient (GPx4+/−) mice had marked carbonyl stress in these organs accompanied by exacerbated glucose intolerance, dyslipidemia, and liver steatosis. Although normotensive, cardiac hypertrophy was evident with obesity, and cardiac fibrosis more pronounced in obese GPx4+/− mice. Mitochondrial dysfunction manifesting as decreased fat oxidation capacity and increased reactive oxygen species was also present in obese GPx4+/− but not WT hearts, along with up-regulation of pro-inflammatory and pro-fibrotic genes. Patients with diabetes and hyperglycemia exhibited significantly less GPx4 enzyme and greater HNE-adducts in their hearts, compared with age-matched non-diabetic patients. Conclusion These findings suggest LPPs are key factors underlying cardio-metabolic derangements that occur with obesity and that GPx4 serves a critical role as an adaptive countermeasure.

Na+, K+ ATPase α-subunit isoform distribution and abundance in guinea-pig longitudinal muscle/myenteric plexus after exposure to morphine

Previous work in the myenteric plexus has shown that the resting membrane potential of morphine-tolerant guinea-pig myenteric S neurons is significantly depolarized relative to placebo-implanted controls, and that this depolarization is associated with reduced electrogenic Na+, K+ pumping. Identification of the subunits of the sodium pump which are in the myenteric plexus was undertaken in order to facilitate direct qualitative and quantitative measurements of the abundance of sodium pump isoforms after morphine exposure, thereby confirming and extending the electrophysiological data to the molecular level. Seven days prior to the experiments, tolerance was induced by subcutaneous implantation of morphine pellets (one pellet, 75 mg/100 g body weight) while control guinea pigs received placebo pellets. Using immunohistochemistry and confocal microscopy, the distribution of the alpha subunit isoforms of the Na+/K+ -ATPase in placebo and morphine-tolerant guinea-pig ileum was determined. Only the alpha1 and alpha3 subunit isoforms were in sufficient abundance to be observed. The alpha1 subunit isoform was most highly concentrated in the mucosa and in neurons. In contrast, the alpha3 subunit isoform was uniquely localized to neurons. Western and slot blot analyses of longitudinal muscle/myenteric plexus homogenates identified a significant reduction of the alpha3 but not the alpha1 subunit isoform in tolerant preparations. It is concluded that the reduced electrogenic pumping in the S neurons after morphine exposure is associated with a reduction in the alpha3 subunit isoform.

Quantification of the α3 subunit of the Na+/K+-ATPase in developing rat cerebellum

Abstract Cerebellar Purkinje neurons of rats have been shown to exhibit a progressive increase in resting membrane potential as the animals develop postnatally. The magnitude of this increase was equivalent in magnitude to the increase in the depolarizing action of ouabain, consistent with a role for the Na+/K+-pump in the hyperpolarization. Ouabain binding sites in whole cerebellum also increased with age. The present study was undertaken to confirm that the increases in ouabain binding and the electrophysiological responses to ouabain were a consequence of increases in the sodium pump and to determine whether the changes seen at the whole organ level were reflective of changes taking place at the cellular level. Using antibodies directed against the α1, α2, and α3 subunits of the Na+/K+-ATPase, rats between 13 and 19 days of age exhibited a statistically significant increase in the relative amount of the α3 subunit at the level of the whole organ, as determined by Western and slot blot analyses, with no change in the levels of either the α1 or the α2 subunit. Using immunohistochemistry, the α3 subunit was shown to increase in both the Purkinje cell layer and the white matter during this postnatal time period, while the α1 subunit increased in the granular layer. These results support and extend previous work, which pointed to a role for the electrogenic sodium pump in the developmental increase in Purkinje cell membrane potential. Furthermore, the data provide a cellular mechanism underlying the increase in resting membrane potential, that is, by the specific modulation of the α3 subunit isoform.

Cardioprotection via preserved mitochondrial structure and function in the mPer2-mutant mouse myocardium.

We have previously shown that myocardial infarct size in nonreperfused hearts of mice with a functional deletion of the circadian rhythm gene mPer2 (mPer2-M) was reduced by 43%. We hypothesized that acute ischemia-reperfusion injury (I/R = 30 min I/2 h R) would also be reduced in these mice and that ischemic preconditioning (IPC) (3 × 5 min cycles) before I/R, which enhances protection in wild-type (WT) hearts, would provide further protection in mPer2-M hearts. We observed a 69 and 75% decrease in infarct size in mPer2-M mouse hearts compared with WT following I/R and IPC, respectively. This was coincident with 67% less neutrophil infiltration and 57% less apoptotic cardiomyocytes. IPC in mPer2-M mice before I/R had 48% less neutrophil density and 46% less apoptosis than their WT counterparts. Macrophage density was not different between WT and mPer2-M I/R, but it was 45% higher in mPer2-M IPC mouse hearts compared with WT IPC. There were no baseline differences in cardiac mitochondrial function between WT and mPer2-M mice, but, following I/R, WT exhibited a marked decrease in maximal O₂ consumption supported by complex I-mediated substrates, whereas mPer2-M did not, despite no difference in complex I content. Moreover, cardiac mitochondria from WT mice exhibited a very robust increase in ADP-stimulated O₂ consumption in response to exogenously added cytochrome c, along with a high rate of reactive oxygen species production, none of which was exhibited by cardiac mitochondria from mPer2-M following I/R. Taken together, these findings suggest that mPer2 deletion preserves mitochondrial membrane structure and functional integrity in heart following I/R injury, the consequence of which is preservation of myocardial viability. Understanding the mechanisms connecting cardiac events, mitochondrial function, and mPer2 could lead to preventative and therapeutic strategies for at risk populations.

Correlation of the time course of development and decay of tolerance to morphine with alterations in sodium pump protein isoform abundance.

Since the heterologous tolerance that develops after chronic morphine administration has been proposed to be an adaptive process, it follows that the time course of the change in the cellular components should coincide with the time course of the altered responsiveness. This study correlated the time course over which heterologous tolerance develops with changes in the abundance of selected proteins in the guinea-pig longitudinal muscle/myenteric plexus (LM/MP) preparation. Tissues were obtained at various times following a single surgical implantation procedure and heterologous tolerance confirmed by a significant reduction in the sensitivity of the LM/MP to inhibition of neurogenic twitches by morphine, DAMGO, and 2-CADO. Tolerance developed with a delayed onset (significant 2-5-fold reduction in sensitivity by day 4 after pellet implantation) that reached a maximum by 7 days (4-8-fold reduction in responsiveness) that was maintained through 14 days with normal sensitivity spontaneously returning by 21 days post-implantation. Dot blot analysis was used to examine the abundance of the alpha(1) and alpha(3) subunit isoforms of the Na(+)/K(+) ATPase and beta-actin over the same time course. The results showed significant decreases in abundance of the alpha(3) subunit at 4, 7, and 10 days following pellet implantation but no change in beta-actin or the alpha(1) subunit at any time period. These data support the idea that heterologous tolerance following chronic morphine exposure results from a cellular adaptive change that may involve a change in the abundance of the alpha(3) subunit isoform of the Na(+)/K(+) ATPase.

THE ROLE OF THE SODIUM PUMP IN THE DEVELOPMENTAL REGULATION OF MEMBRANE ELECTRICAL PROPERTIES OF CEREBELLAR PURKINJE NEURONS OF THE RAT

The postnatal development of the rat cerebellum has been well described morphologically and functionally. However, information regarding the electrical characteristics of Purkinje neurons during development is sparse. Using standard intracellular recording, the basic electrical properties of Purkinje neurons in cerebellar slices were compared at different postnatal ages. There was a significant, progressive increase in the resting membrane potential (RMP) of Purkinje neurons with age as well as a small but significant decrease in the cellular input resistance (Rin). The cardiac glycoside, ouabain (1 mM), an inhibitor of the sodium pump, depolarized Purkinje neurons significantly more with age. The magnitude of the increase in depolarizing activity of ouabain was equivalent to the magnitude of the increase in membrane potential. The number of ouabain binding sites was also found to increase with age suggesting an age related increase in the number of sodium pump sites. These results suggest that the predominant cellular mechanism which underlies the increase in membrane potential of Purkinje neurons during development is an increase in the density of Na+, K+ pump sites and in the contribution of the electrogenic sodium pump.