Brook L. Cathey

Inhibiting myosin-ATPase reveals a dynamic range of mitochondrial respiratory control in skeletal muscle

Assessment of mitochondrial ADP-stimulated respiratory kinetics in PmFBs (permeabilized fibre bundles) is increasingly used in clinical diagnostic and basic research settings. However, estimates of the Km for ADP vary considerably (~20-300 μM) and tend to overestimate respiration at rest. Noting that PmFBs spontaneously contract during respiration experiments, we systematically determined the impact of contraction, temperature and oxygenation on ADP-stimulated respiratory kinetics. BLEB (blebbistatin), a myosin II ATPase inhibitor, blocked contraction under all conditions and yielded high Km values for ADP of >~250 and ~80 μM in red and white rat PmFBs respectively. In the absence of BLEB, PmFBs contracted and the Km for ADP decreased ~2-10-fold in a temperature-dependent manner. PmFBs were sensitive to hyperoxia (increased Km) in the absence of BLEB (contracted) at 30 °C but not 37 °C. In PmFBs from humans, contraction elicited high sensitivity to ADP (Km<100 μM), whereas blocking contraction (+BLEB) and including a phosphocreatine/creatine ratio of 2:1 to mimic the resting energetic state yielded a Km for ADP of ~1560 μM, consistent with estimates of in vivo resting respiratory rates of <1% maximum. These results demonstrate that the sensitivity of muscle to ADP varies over a wide range in relation to contractile state and cellular energy charge, providing evidence that enzymatic coupling of energy transfer within skeletal muscle becomes more efficient in the working state.

Mast cells contribute to altered vascular reactivity and ischemia-reperfusion injury following cerium oxide nanoparticle instillation

Abstract Cerium oxide (CeO2) represents an important nanomaterial with wide ranging applications. However, little is known regarding how CeO2 exposure may influence pulmonary or systemic inflammation. Furthermore, how mast cells would influence inflammatory responses to a nanoparticle exposure is unknown. We thus compared pulmonary and cardiovascular responses between C57BL/6 and B6.Cg-KitW-sh mast cell deficient mice following CeO2 nanoparticle instillation. C57BL/6 mice instilled with CeO2 exhibited mild pulmonary inflammation. However, B6.Cg-KitW-sh mice did not display a similar degree of inflammation following CeO2 instillation. Moreover, C57BL/6 mice instilled with CeO2 exhibited altered aortic vascular responses to adenosine and an increase in myocardial ischemia/reperfusion injury which was absent in B6.Cg-KitW-sh mice. In vitro CeO2 exposure resulted in increased production of PGD2, TNF-α, IL-6 and osteopontin by cultured mast cells. These findings demonstrate that CeO2 nanoparticles activate mast cells contributing to pulmonary inflammation, impairment of vascular relaxation and exacerbation of myocardial ischemia/reperfusion injury.

Pyruvate dehydrogenase complex and nicotinamide nucleotide transhydrogenase constitute an energy-consuming redox circuit

Cellular proteins rely on reversible redox reactions to establish and maintain biological structure and function. How redox catabolic (NAD+/NADH) and anabolic (NADP+/NADPH) processes integrate during metabolism to maintain cellular redox homoeostasis, however, is unknown. The present work identifies a continuously cycling mitochondrial membrane potential (ΔΨm)-dependent redox circuit between the pyruvate dehydrogenase complex (PDHC) and nicotinamide nucleotide transhydrogenase (NNT). PDHC is shown to produce H2O2 in relation to reducing pressure within the complex. The H2O2 produced, however, is effectively masked by a continuously cycling redox circuit that links, via glutathione/thioredoxin, to NNT, which catalyses the regeneration of NADPH from NADH at the expense of ΔΨm. The net effect is an automatic fine-tuning of NNT-mediated energy expenditure to metabolic balance at the level of PDHC. In mitochondria, genetic or pharmacological disruptions in the PDHC-NNT redox circuit negate counterbalance changes in energy expenditure. At the whole animal level, mice lacking functional NNT (C57BL/6J) are characterized by lower energy-expenditure rates, consistent with their well-known susceptibility to diet-induced obesity. These findings suggest the integration of redox sensing of metabolic balance with compensatory changes in energy expenditure provides a potential mechanism by which cellular redox homoeostasis is maintained and body weight is defended during periods of positive and negative energy balance.

Mitochondrial Respiratory Capacity and Content Are Normal in Young Insulin-Resistant Obese Humans

Considerable debate exists about whether alterations in mitochondrial respiratory capacity and/or content play a causal role in the development of insulin resistance during obesity. The current study was undertaken to determine whether such alterations are present during the initial stages of insulin resistance in humans. Young (∼23 years) insulin-sensitive lean and insulin-resistant obese men and women were studied. Insulin resistance was confirmed through an intravenous glucose tolerance test. Measures of mitochondrial respiratory capacity and content as well as H2O2 emitting potential and the cellular redox environment were performed in permeabilized myofibers and primary myotubes prepared from vastus lateralis muscle biopsy specimens. No differences in mitochondrial respiratory function or content were observed between lean and obese subjects, despite elevations in H2O2 emission rates and reductions in cellular glutathione. These findings were apparent in permeabilized myofibers as well as in primary myotubes. The results suggest that reductions in mitochondrial respiratory capacity and content are not required for the initial manifestation of peripheral insulin resistance.

The anticancer agent doxorubicin disrupts mitochondrial energy metabolism and redox balance in skeletal muscle

The combined loss of muscle strength and constant fatigue are disabling symptoms for cancer patients undergoing chemotherapy. Doxorubicin, a standard chemotherapy drug used in the clinic, causes skeletal muscle dysfunction and premature fatigue along with an increase in reactive oxygen species (ROS). As mitochondria represent a primary source of oxidant generation in muscle, we hypothesized that doxorubicin could negatively affect mitochondria by inhibiting respiratory capacity, leading to an increase in H2O2-emitting potential. Here we demonstrate a biphasic response of skeletal muscle mitochondria to a single doxorubicin injection (20mg/kg). Initially at 2h doxorubicin inhibits both complex I- and II-supported respiration and increases H2O2 emission, both of which are partially restored after 24h. The relationship between oxygen consumption and membrane potential (ΔΨ) is shifted to the right at 24h, indicating elevated reducing pressure within the electron transport system (ETS). Respiratory capacity is further decreased at a later time point (72 h) along with H2O2-emitting potential and an increased sensitivity to mitochondrial permeability transition pore (mPTP) opening. These novel findings suggest a role for skeletal muscle mitochondria as a potential underlying cause of doxorubicin-induced muscle dysfunction.

Reversal of Voltage-Dependent Erectile Responses in the Zucker Obese-Diabetic Rat by Rosuvastatin-Altered RhoA/Rho-kinase Signaling

INTRODUCTION The combination of independent risk factors for erectile dysfunction, obesity, hypertension, and diabetes are collectively manifested in a condition known as metabolic syndrome X (MSX). However, the regulatory mechanisms responsible for the erectile dysfunction (ED) are not fully understood. Clinical studies suggest that a pleiotropic effect of statins ability to enhance vascular relaxation might be through an impact on nitric oxide signaling or through a regulation of RhoA activation. AIM We hypothesized that regulatory aspects of short-term statin therapy involve the alteration of the RhoA/Rho-kinase signaling cascade and will reverse the ED seen in a rat model of MSX. MAIN OUTCOME MEASURES The magnitude and sensitivity of the voltage-dependent maintenance of intracavernosal blood pressure and mean arterial blood pressure. These responses were correlated with tissue protein and mRNA expression levels of RhoA and Rho kinases. METHODS Erectile function was evaluated by assessing voltage-dependent stimulation of the cavernosal nerve in 16-20 weeks old lean and obese-diabetic Zucker rats treated with 5 mg/kg/day of rosuvastatin intraperitoneally for 3 days. Cavernosal tissue RhoA and Rho-kinases expression levels were evaluated by real-time reverse transcriptase-polymerase chain reaction, Western blot. RESULTS The voltage-dependent erectile responses were suppressed by >30% in the obese-diabetic Zucker rat. The 3-day treatment with rosuvastatin partially restored the erectile response. The Rho-kinase inhibitor, H-1152, dose dependently increased the erectile responses and shifted the voltage sensitivity with statin treatment. Analysis of protein expression levels suggested elevation of RhoA and Rho kinases in obese-diabetics and statin treatment lowering Rho-kinase II. The RhoA and Rho-kinase II mRNA levels were significantly reduced in the rosuvastatin-treated obese-diabetic animals. CONCLUSIONS These results support a hypothesis that short-term statin therapy may lower RhoA/Rho-kinase expression levels and improve cavernosal blood pressure response to Rho-kinase inhibition and voltage-stimulation, and reversing an augmented vasoconstricted state associated with diabetes and/or hypertension in MSX.

Targeted overexpression of mitochondrial catalase protects against cancer chemotherapy-induced skeletal muscle dysfunction

The loss of strength in combination with constant fatigue is a burden on cancer patients undergoing chemotherapy. Doxorubicin, a standard chemotherapy drug used in the clinic, causes skeletal muscle dysfunction and increases mitochondrial H2O2 We hypothesized that the combined effect of cancer and chemotherapy in an immunocompetent breast cancer mouse model (E0771) would compromise skeletal muscle mitochondrial respiratory function, leading to an increase in H2O2-emitting potential and impaired muscle function. Here, we demonstrate that cancer chemotherapy decreases mitochondrial respiratory capacity supported with complex I (pyruvate/glutamate/malate) and complex II (succinate) substrates. Mitochondrial H2O2-emitting potential was altered in skeletal muscle, and global protein oxidation was elevated with cancer chemotherapy. Muscle contractile function was impaired following exposure to cancer chemotherapy. Genetically engineering the overexpression of catalase in mitochondria of muscle attenuated mitochondrial H2O2 emission and protein oxidation, preserving mitochondrial and whole muscle function despite cancer chemotherapy. These findings suggest mitochondrial oxidants as a mediator of cancer chemotherapy-induced skeletal muscle dysfunction.