Type 2 Diabetes Related Mitochondrial Defects in Peripheral Mononucleated Blood Cells from Overweight Postmenopausal Women.

Type 2 diabetes (T2D) is a multisystem disease that is the subject of many studies, but the earliest cause of the disease has yet to be elucidated. Mitochondrial impairment has been associated with diabetes in several tissues. To extend the association between T2D and mitochondrial impairment to blood cells, we investigated T2D-related changes in peripheral mononucleated blood cells' (PBMCs) mitochondrial function in two groups of women (CTRL vs. T2D; mean age: 54.1 ± 3.8 vs. 60.9 ± 4.8; mean BMI 25.6 ± 5.2 vs. 30.0 ± 5), together with a panel of blood biomarkers, anthropometric measurements and physiological parameters (VO2max and strength tests). Dual-energy X-ray absorptiometry (DXA) scan analysis, cardio-pulmonary exercise test and blood biomarkers confirmed hallmarks of diabetes in the T2D group. Mitochondrial function assays performed with high resolution respirometry highlighted a significant reduction of mitochondrial respiration in the ADP-stimulated state (OXPHOS; −30%, p = 0.006) and maximal non-coupled respiration (ET; −30%, p = 0.004) in PBMCs samples from the T2D group. The total glutathione antioxidant pool (GSHt) was significantly reduced (−38%: p = 0.04) in plasma samples from the T2D group. The fraction of glycated hemoglobin (Hb1Ac) was positively associated with markers of inflammation (C-reactive protein-CRP r = 0.618; p = 0.006) and of dyslipidemia (triglycerides-TG r = 0.815; p < 0.0001). The same marker (Hb1Ac) was negatively associated with mitochondrial activity levels (OXPHOS r = −0.502; p = 0.034; ET r = −0.529; p = 0.024). The results obtained in overweight postmenopausal women from analysis of PBMCs mitochondrial respiration and their association with anthropometric and physiological parameters indicate that PBMC could represent a reliable model for studying T2D-related metabolic impairment and could be useful for testing the effectiveness of interventions targeting mitochondria.

Effects of nociceptive and mechanosensitive afferents sensitization on central and peripheral hemodynamics following exercise-induced muscle damage.

This study aims to test the separated and combined effects of mechanoreflex activation and nociception through exercise-induced muscle damage (EIMD) on central and peripheral hemodynamics before and during single passive leg movement (sPLM). Eight healthy young males undertook four experimental sessions, in which a sPLM was performed on the dominant limb while in each specific session the contralateral was: 1) in a resting condition (CTRL), 2) stretched (ST), 3) resting after EIMD called delayed onset muscle soreness (DOMS) condition, or 4) stretched after EIMD (DOMS + ST). EIMD was used to induce DOMS in the following 24-48 h. Femoral blood flow (FBF) was assessed using Doppler ultrasound whereas central hemodynamics were assessed via finger photoplethysmography. Leg vascular conductance (LVC) was calculated as FBF/mean arterial pressure (MAP). RR-intervals were analyzed in the time (root mean squared of successive intervals; RMSSD) and frequency domain [low frequency (LF)/high frequency (HF)]. Blood samples were collected before each condition and gene expression analysis showed increased fold changes for P2X4 and IL1ß in DOMS and DOMS + ST compared with baseline. Resting FBF and LVC were decreased only in the DOMS + ST condition (-26 mL/min and -50 mL/mmHg/min respectively) with decreased RMSSD and increased LF/HF ratio. MAP, HR, CO, and SV were increased in ST and DOMS + ST compared with CTRL. Marked decreases of Δpeaks and AUC were observed for FBF (Δ: -146 mL/min and -265 mL respectively) and LVC (Δ: -8.66 mL/mmHg/min and ±1.7 mL/mmHg/min respectively) all P < 0.05. These results suggest that the combination of mechanoreflex and nociception resulted in decreased vagal tone and concomitant rise in sympathetic drive that led to increases in resting central hemodynamics with reduced limb blood flow before and during sPLM.NEW & NOTEWORTHY Exercise-induced muscle damage (EIMD) is a well-known model to study mechanical hyperalgesia and muscle peripheral nerve sensitizations. The combination of static stretching protocol on the damaged limb extensively increases resting central hemodynamics with reduction in resting limb blood flow and passive leg movement-induced hyperemia. The mechanism underlining these results may be linked to reduction of vagal tone with concomitant increase in sympathetic activity following mechano- and nociceptive activation.

Human Platelet Mitochondrial Function Reflects Systemic Mitochondrial Alterations: A Protocol for Application in Field Studies.

Human blood cells may offer a minimally invasive strategy to study systemic alterations of mitochondrial function. Here we tested the reliability of a protocol designed to study mitochondrial respiratory control in human platelets (PLTs) in field studies, using high-resolution respirometry (HRR). Several factors may trigger PLT aggregation during the assay, altering the homogeneity of the cell suspension and distorting the number of cells added to the two chambers (A, B) of the Oroboros Oxygraph-2k (O2k). Thus, inter-chamber variability (∆ab) was calculated by normalizing oxygen consumption to chamber volume (JO2) or to a specific respiratory control state (flux control ratio, FCR) as a reliable parameter of experimental quality. The method's reliability was tested by comparing the ∆ab of laboratory-performed experiments (LAB, N = 9) to those of an ultramarathon field study (three sampling time-points: before competition (PRE, N = 7), immediately after (POST, N = 10) and 24 h after competition (REC; N = 10)). Our results show that ∆ab JO2 changed PRE-POST, but also for LAB-POST and LAB-REC, while all ∆ab FCR remained unchanged. Thus, we conclude that our method is reliable for assessing PLT mitochondrial function in LAB and field studies and after systemic stress conditions.

Interferon regulatory factor 7 impairs cellular metabolism in aging adipose-derived stromal cells.

Dysregulated immunity and widespread metabolic dysfunctions are the most relevant hallmarks of the passing of time over the course of adult life, and their combination at midlife is strongly related to increased vulnerability to diseases; however, the causal connection between them remains largely unclear. By combining multi-omics and functional analyses of adipose-derived stromal cells established from young (1 month) and midlife (12 months) mice, we show that an increase in expression of interferon regulatory factor 7 (IRF7) during adult life drives major metabolic changes, which include impaired mitochondrial function, altered amino acid biogenesis and reduced expression of genes involved in branched-chain amino acid (BCAA) degradation. Our results draw a new paradigm of aging as the 'sterile' activation of a cell-autonomous pathway of self-defense and identify a crucial mediator of this pathway, IRF7, as driver of metabolic dysfunction with age.

Effects of Ultramarathon Running on Mitochondrial Function of Platelets and Oxidative Stress Parameters: A Pilot Study.

Only a few studies have evaluated changes in mitochondrial function and oxidative stress associated with ultramarathon running. Invasive biopsies are needed to assess mitochondrial function of skeletal muscle, which may not be well tolerated by some individuals. Platelets (PLTs) as a metabolically highly active and homogenous cell population were suggested as a potentially valuable surrogate to investigate mitochondrial function. Thus, this study was aimed to evaluate mitochondrial function of PLTs and its association with individual race performance and markers of oxidative stress, muscle damage and renal dysfunction. Race performance and mitochondrial function (high-resolution respirometry, HRR) of PLTs using different substrates inducing ROUTINE, LEAK, N-pathway control state (Complex I linked oxidative phosphorylation; CI, OXPHOS), NS-pathway control state (CI + II linked OXPHOS and electron transfer pathway; ET), S-pathway control state (CII linked ET) as well as parameters of oxidative stress and antioxidant capacity, and markers of muscle and renal injury were assessed in eight male ultramarathon runners (26-45 years) before, immediately after and 24 h after an ultramarathon race (PRE, POST, and REC). Ultramarathon running induced an increase in LEAK O2 flux of PLT mitochondria and slight, largely non-significant changes in the oxidant/antioxidant balance. Levels of creatine kinase (CK), lactate dehydrogenase (LDH), blood urea nitrogen, and creatinine were all significantly elevated POST and remained high in REC. There were inverse correlations between race time and N-linked substrate state PRE-POST, and changes in CK and LDH levels were significantly related to PLT mitochondrial LEAK and N-linked respiration PRE. Although race-related changes in respirometry parameters of PLT mitochondria were rather small, a somewhat more pronounced increase in the relative N-linked respiration in faster runners might suggest PLT CI as indicator of physical fitness. The higher PLT LEAK PRE and diminished increase of CK during the race may represent a prophylactic preconditioning and the slight but non-significant elevation of the antioxidant potential post-race as a protective consequence of the race-related oxidative stress and potential threat to the kidney. Our findings point toward an interrelationship between mitochondrial function of PLTs, individual fitness levels and extreme physical and metal stresses, which stimulates further research.

High Intensity Interval Training Does Not Have Compensatory Effects on Physical Activity Levels in Older Adults.

BACKGROUND: Exercise has beneficial effects on older adults, but controversy surrounds the purported "compensatory effects" that training may have on total daily physical activity and energy expenditure in the elderly. We wanted to determine whether 8 weeks of high-intensity interval training (HIIT) induced such effects on physical activity and energy expenditure in healthy, active older adult men. METHODS: Twenty-four healthy elderly male volunteers were randomized to two groups. The experimental group performed HIIT (7 × 2 min cycling repetitions, 3 d/w); the control group performed continuous moderate-intensity training (20-30 min cycling, 3 d/w). Physical activity and energy expenditure were measured with a multisensor activity monitor SenseWear Armband Mini. RESULTS: During HIIT, significant changes were observed in moderate and vigorous physical activity, average daily metabolic equivalents (METs), physical activity level, and activity energy expenditure (p < 0.05) but not in total energy expenditure. Sleep and sedentary time, and levels of light physical activity remained constant during the training period. CONCLUSIONS: The findings suggest that HIIT induced no compensatory effect: HIIT does not adversely affect lifestyle, as it does not reduce daily energy expenditure and/or increase sedentary time.

ASCs-Exosomes Recover Coupling Efficiency and Mitochondrial Membrane Potential in an in vitro Model of ALS.

The amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by motoneurons death. Mutations in the superoxide dismutase 1 (SOD1) protein have been identified to be related to the disease. Beyond the different altered pathways, the mitochondrial dysfunction is one of the major features that leads to the selective death of motoneurons in ALS. The NSC-34 cell line, overexpressing human SOD1(G93A) mutant protein [NSC-34(G93A)], is considered an optimal in vitro model to study ALS. Here we investigated the energy metabolism in NSC-34(G93A) cells and in particular the effect of the mutated SOD1(G93A) protein on the mitochondrial respiratory capacity (complexes I-IV) by high resolution respirometry (HRR) and cytofluorimetry. We demonstrated that NSC-34(G93A) cells show a reduced mitochondrial oxidative capacity. In particular, we found significant impairment of the complex I-linked oxidative phosphorylation, reduced efficiency of the electron transfer system (ETS) associated with a higher rate of dissipative respiration, and a lower membrane potential. In order to rescue the effect of the mutated SOD1 gene on mitochondria impairment, we evaluated the efficacy of the exosomes, isolated from adipose-derived stem cells, administrated on the NSC-34(G93A) cells. These data show that ASCs-exosomes are able to restore complex I activity, coupling efficiency and mitochondrial membrane potential. Our results improve the knowledge about mitochondrial bioenergetic defects directly associated with the SOD1(G93A) mutation, and prove the efficacy of adipose-derived stem cells exosomes to rescue the function of mitochondria, indicating that these vesicles could represent a valuable approach to target mitochondrial dysfunction in ALS.

Physiological and Pathophysiological Responses to Ultramarathon Running in Non-elite Runners.

Ultramarathon running represents a major physical challenge even for elite athletes. Runners wellbeing may be challenged by fluid and electrolyte disturbances, hemolysis and skeletal muscle damage, decline in hepatic function and kidney injury. We hypothesized that these effects may even be exacerbated in non-elite runners. Physiological, hematological and biochemical parameters of ten males (26-45 years, weekly training time 8.5 h), participating in a mountain ultramarathon (67 km; approximately 4,500 m of total ascent), were determined before (PRE), immediately after finishing the ultramarathon (POST), and 24 h after the individual finish (REC). Race times of the 8 finishers (2 drop-outs due to hot ambient temperature) varied between 10.4 and 16.1 h, which almost represents the range of the entire starter field (8.82 h-17.47 h). The following changes in mean values of selected markers for skeletal muscle damage and kidney injury were observed from PRE to POST: creatine kinase (CK) + 1289%, lactate dehydrogenase (LDH) + 87%, serum creatinine (CR) + 72%, blood urea nitrogen (BUN) + 96%, and estimated glomerular filtration rate (eGFR) - 45%. Values of CK + 1447%, LDH + 56%, and BUN + 71% remained elevated at REC. White blood cells were increased (+ 137%) only POST. In conclusion, CK and LDH levels and leucocytosis may be considered to be relatively harmless "side-effects" of prolonged running in this group of male subjects with rather moderate ultramarathon experience and training status. However, acute kidney injury may become clinically relevant in this population under the certain conditions, which should be considered by responsible race managers and medical advisors.

Effects of High-Intensity Interval Training and Isoinertial Training on Leg Extensors Muscle Function, Structure, and Intermuscular Adipose Tissue in Older Adults.

We compared the effects of aerobic high-intensity training (HIT) and isoinertial resistance training (IRT) on the strength, mass, architecture, intermuscular adipose tissue (IMAT) quality, and neuromuscular activation of the quadriceps in elderly subjects. Twelve healthy men (69.3 ± 4.2 years; 77.8 ± 10.4 kg; 1.72 ± 0.05 m) were exposed to 8 weeks of HIT (7 × 2-min cycling repetitions at 90% of V . O2 peak, 3 times/week) and, after 4 months (detraining), to IRT (4 × 7 maximal concentric-eccentric knee extensions, 3 times/week). Before and after trainings, we measured knee extension isometric (T MVC) and dynamic (T C) maximal concentric torque, anatomical cross-sectional area (ACSA) at 25, 50, and 75% of femur length, quadriceps volume (Vol), IMAT, pennation angle (θ p ) of the fibers from the vastus lateralis, and voluntary activation (%Act). T MVC and T C were significantly larger only after IRT (P = 0.008); IRT was able to elicit a greater increase of ACSA than HIT; Vol increases similarly and significantly after HIT and IRT (P = 0.003-0.001); IMAT at 50% of femur length decreased after both HIT and IRT (P = 0.001-0.003); physiological cross-sectional area (PCSA) was larger after IRT than before (P = 0.025); specific torque did not change throughout the study (45.5 N cm-2 ± 12.0); %Act of the quadriceps was significantly affected only by IRT (P = 0.011). Both HIT and IRT are able to elicit beneficial modifications of muscular mass, architecture, and quality (reducing IMAT) in elderly subjects in connection with an amelioration of strength. HIT and IRT caused a homogeneous increase of ACSA and of Vol of the quadriceps. PCSA increases, but specific strength per unit of PCSA did not change. The increases of functional torque seemed to be attributed to a parallel increase of %Act and muscle hypertrophy only after IRT. Data suggest that IMAT may be a prominent indicator to track metabolic-dependent activity and skeletal muscle quality.

Neuromuscular and Muscle Metabolic Functions in MELAS Before and After Resistance Training: A Case Study.

Mitochondrial encephalomyopathy, lactic acidosis, and recurrent stroke-like episodes syndrome (MELAS) is a rare degenerative disease. Recent studies have shown that resistant training (RT) can ameliorate muscular force in mitochondrial diseases. However, the effects of RT in MELAS are unknown. The aim of this case report was to investigate the effects of RT on skeletal muscle and mitochondrial function in a 21-years old patient with MELAS. RT included 12 weeks of RT at 85% of 1 repetition maximum. Body composition (DXA), in vivo mitochondrial respiration capacity (mVO2) utilizing Near-infrared spectroscopy on the right plantar-flexor muscles, maximal voluntary torque (MVC), electrically evoked resting twitch (EET) and maximal voluntary activation (VMA) of the right leg extensors (LE) muscles were measured with the interpolated twitch technique. The participant with MELAS exhibited a marked increase in body mass (1.4 kg) and thigh muscle mass (0.3 kg). After the training period MVC (+5.5 Nm), EET (+2.1 N⋅m) and VMA (+13.1%) were ameliorated. Data of mVO2 revealed negligible changes in the end-exercise mVO2 (0.02 mM min-1), Δ mVO2 (0.09 mM min-1), while there was a marked amelioration in the kinetics of mVO2 (τ mVO2; Δ70.2 s). This is the first report of RT-induced ameliorations on skeletal muscle and mitochondrial function in MELAS. This case study suggests a preserved plasticity in the skeletal muscle of a patient with MELAS. RT appears to be an effective method to increase skeletal muscle function, and this effect is mediated by both neuromuscular and mitochondrial adaptations.

Effect of Endurance and Strength Training on the Slow Component of O 2 Kinetics in Elderly Humans.

We compared the effects of 8 weeks of high intensity, aerobic interval training (HIT) and isoinertial resistance training (IRT) on: (i) O 2 kinetics during heavy (HiEx) intensity exercise and; (ii) work economy during moderate (ModEx) intensity exercise in 12 healthy elderly men (69.3 ± 4.2 years). Breath-by-breath O 2 and muscle deoxygenation ([HHb] by means of NIRS) were measured in HiEx and ModEx at identical workloads before and after trainings. In HiEx, O 2 and HHb responses were modeled as tri-exponential and mono-exponential increasing functions, respectively. A two-way ANOVA for repeated measures analysis was made; Effect size (η2) was also evaluated. After HIT the amplitude and the time delay of the slow component of O2 uptake (O 2sc) during HiEx were smaller (-32%; P = 0.045) and longer (+19.5%; P = 0.001), respectively. At Post IRT: (i) during ModEx, gain was lower (-5%; P = 0.050); (ii) during HiEx, τ2 (+14.4%; P = 0.050), d3 (+8.6%; P = 0.050), and τ3 (+17.2%; P = 0.050) were longer than at Pre IRT. After HIT, the decrease of the O 2sc amplitude was likely induced by the beneficial effects of training on a more responsive O 2 delivery and consumption cascade leading to a better muscle metabolic stability. IRT training was able to increase exercise economy during ModEx and to reduce the amplitude and delay the onset of O 2sc during HiEx. These effects should be due to the reduction and the delayed recruitment of Type II muscle fibers. The better exercise economy and the delayed appearance of O 2sc induced by IRT suggests that strength training might be included in endurance training programs to improve exercise economy and resistance to fatigue in this population of old subjects.

Skeletal muscle mass is controlled by the MRF4-MEF2 axis.

PURPOSE OF REVIEW: The review is focused on the unexpected role of myogenic regulatory factor 4 (MRF4) in controlling muscle mass by repressing myocyte enhancer binding factor 2 (MEF2) activity in adult skeletal muscle, and on the emerging role of MEF2 in skeletal muscle growth. RECENT FINDINGS: The MRF4s of the MyoD family (MyoD, MYF5, MRF4, myogenin) and the MEF2 factors are known to play a major role in embryonic myogenesis. However, their function in adult muscle tissue is not known. A recent study shows that MRF4 loss in adult skeletal muscle causes muscle hypertrophy and prevents denervation atrophy. This effect is mediated by MEF2 factors that promote muscle growth, with MRF4 acting as a repressor of MEF2 activity. The role of MEF2 in skeletal muscle growth is supported by the finding that muscle regeneration is impaired by muscle-specific triple knockout of Mef2a, c, and d genes. SUMMARY: The finding that the MRF4-MEF2 axis controls muscle growth opens a new perspective for preventing muscle wasting. A unique feature of this pathway is that MRF4 is exclusively expressed in skeletal muscle, thus reducing the risk that interventions aimed at down-regulating MRF4 or interfering with the interaction between MRF4 and MEF2 may have off-target effects in other tissues.

MRF4 negatively regulates adult skeletal muscle growth by repressing MEF2 activity.

The myogenic regulatory factor MRF4 is highly expressed in adult skeletal muscle but its function is unknown. Here we show that Mrf4 knockdown in adult muscle induces hypertrophy and prevents denervation-induced atrophy. This effect is accompanied by increased protein synthesis and widespread activation of muscle-specific genes, many of which are targets of MEF2 transcription factors. MEF2-dependent genes represent the top-ranking gene set enriched after Mrf4 RNAi and a MEF2 reporter is inhibited by co-transfected MRF4 and activated by Mrf4 RNAi. The Mrf4 RNAi-dependent increase in fibre size is prevented by dominant negative MEF2, while constitutively active MEF2 is able to induce myofibre hypertrophy. The nuclear localization of the MEF2 corepressor HDAC4 is impaired by Mrf4 knockdown, suggesting that MRF4 acts by stabilizing a repressor complex that controls MEF2 activity. These findings open new perspectives in the search for therapeutic targets to prevent muscle wasting, in particular sarcopenia and cachexia.

Aging: a portrait from gene expression profile in blood cells.

The availability of reliable biomarkers of aging is important not only to monitor the effect of interventions and predict the timing of pathologies associated with aging but also to understand the mechanisms and devise appropriate countermeasures. Blood cells provide an easily available tissue and gene expression profiles from whole blood samples appear to mirror disease states and some aspects of the aging process itself. We report here a microarray analysis of whole blood samples from two cohorts of healthy adult and elderly subjects, aged 43±3 and 68±4 years, respectively, to monitor gene expression changes in the initial phase of the senescence process. A number of significant changes were found in the elderly compared to the adult group, including decreased levels of transcripts coding for components of the mitochondrial respiratory chain, which correlate with a parallel decline in the maximum rate of oxygen consumption (VO2max), as monitored in the same subjects. In addition, blood cells show age-related changes in the expression of several markers of immunosenescence, inflammation and oxidative stress. These findings support the notion that the immune system has a major role in tissue homeostasis and repair, which appears to be impaired since early stages of the aging process.

Effects of eight weeks of aerobic interval training and of isoinertial resistance training on risk factors of cardiometabolic diseases and exercise capacity in healthy elderly subjects.

We investigated the effect of 8 weeks of high intensity interval training (HIT) and isoinertial resistance training (IRT) on cardiovascular fitness, muscle mass-strength and risk factors of metabolic syndrome in 12 healthy older adults (68 yy ± 4). HIT consisted in 7 two-minute repetitions at 80%-90% of V˙O2max, 3 times/w. After 4 months of recovery, subjects were treated with IRT, which included 4 sets of 7 maximal, bilateral knee extensions/flexions 3 times/w on a leg-press flywheel ergometer. HIT elicited significant: i) modifications of selected anthropometrical features; ii) improvements of cardiovascular fitness and; iii) decrease of systolic pressure. HIT and IRT induced hypertrophy of the quadriceps muscle, which, however, was paralleled by significant increases in strength only after IRT. Neither HIT nor IRT induced relevant changes in blood lipid profile, with the exception of a decrease of LDL and CHO after IRT. Physiological parameters related with aerobic fitness and selected body composition values predicting cardiovascular risk remained stable during detraining and, after IRT, they were complemented by substantial increase of muscle strength, leading to further improvements of quality of life of the subjects.

Running economy during a simulated 60-km trial.

The effect of a prolonged running trial on the energy cost of running (C(r)) during a 60-km ultramarathon simulation at the pace of a 100-km competition was investigated in 13 men (40.8 ± 5.6 y, 70.7 ± 5.5 kg, 177.5 ± 4.5 cm) and 5 women (40.4 ± 2.3 y, 53.7 ± 4.4 kg, 162.4 ± 4.8 cm) who participated in a 60-km trial consisting of 3 consecutive 20-km laps. Oxygen uptake (VO(2)) at steady state was determined at constant speed before the test and at the end of each lap; stride length (SL) and frequency and contact time were measured at the same time points; serum creatine kinase (S-CPK) was measured before and at the end of the test. C(r) in J · kg(-1) · m(-1), as calculated from VO(2ss) and respiratory-exchange ratio, did not increase with distance. SL significantly decreased with distance. The net increase in S-CPK was linearly related with the percentage increase of C(r) observed during the trial. It is concluded that, in spite of increased S-CPK, this effort was not able to elicit any peripheral or central fatigue or biomechanical adaptation leading to any modification of C(r).

Determination of maximal lactate steady state in healthy adults: can NIRS help?

PURPOSE: We tested the hypothesis that the maximal lactate steady state (MLSS) can be accurately determined in healthy subjects based on measures of deoxygenated hemoglobin (deoxyHb), an index of oxygen extraction measured noninvasively by near-infrared spectroscopy (NIRS). METHODS: Thirty-two healthy men (mean ± SD age = 48 ± 17 yr, range = 23-74 yr) performed an incremental cycling test to exhaustion and square wave tests for MLSS determination. Cardiorespiratory variables were measured bbb and deoxyHb was monitored noninvasively on the right vastus lateralis with a quantitative NIRS device. The individual values of V˙O2 and HR corresponding to the MLSS were calculated and compared to the NIRS-derived MLSS (NIRSMLSS) that was, in turn, determined by double linear function fitting of deoxyHb during the incremental exercise. RESULTS: V˙O2 and HR at MLSS were 2.25 ± 0.54 L·min (76% ± 9% V˙O2max) and 133 ± 14 bpm (81% ± 7% HRmax), respectively. Muscle O2 extraction increased as a function of exercise intensity up to a deflection point, NIRSMLSS, at which V˙O2 and HR were 2.23 ± 0.59 L·min (76% ± 9% V˙O2max) and 136 ± 17 bpm (82% ± 8% HRmax), respectively. For both V˙O2 and HR, the difference of NIRSMLSS from MLSS values was not significant and the measures were highly correlated (r = 0.81 and r = 0.76). The Bland-Altman analysis confirmed a nonsignificant bias for V˙O2 and HR (-0.015 L·min and 3 bpm, respectively) and a small imprecision of 0.26 L·min and 8 bpm. CONCLUSIONS: A plateau in muscle O2 extraction was demonstrated in coincidence with MLSS during an incremental cycling exercise, confirming the hypothesis that this functional parameter can be accurately estimated with a quantitative NIRS device. The main advantages of NIRSMLSS over lactate-based techniques are the noninvasiveness and the time/cost efficiency.

NFAT isoforms control activity-dependent muscle fiber type specification.

The intracellular signals that convert fast and slow motor neuron activity into muscle fiber type specific transcriptional programs have only been partially defined. The calcium/calmodulin-dependent phosphatase calcineurin (Cn) has been shown to mediate the transcriptional effects of motor neuron activity, but precisely how 4 distinct muscle fiber types are composed and maintained in response to activity is largely unknown. Here, we show that 4 nuclear factor of activated T cell (NFAT) family members act coordinately downstream of Cn in the specification of muscle fiber types. We analyzed the role of NFAT family members in vivo by transient transfection in skeletal muscle using a loss-of-function approach by RNAi. Our results show that, depending on the applied activity pattern, different combinations of NFAT family members translocate to the nucleus contributing to the transcription of fiber type specific genes. We provide evidence that the transcription of slow and fast myosin heavy chain (MyHC) genes uses different combinations of NFAT family members, ranging from MyHC-slow, which uses all 4 NFAT isoforms, to MyHC-2B, which only uses NFATc4. Our data contribute to the elucidation of the mechanisms whereby activity can modulate the phenotype and performance of skeletal muscle.

The LGI1/epitempin gene encodes two protein isoforms differentially expressed in human brain.

The leucine-rich, glioma inactivated 1 (LGI1)/Epitempin gene has been linked to two phenotypes as different as gliomagenesis and autosomal dominant lateral temporal epilepsy. Its function and the biochemical features of the encoded protein are unknown. We characterized the LGI1/Epitempin protein product by western blot analysis of mouse and human brain tissues. Two proteins of about 60 and 65 kDa were detected by an anti-LGI1 antibody within the expected molecular mass range. The two proteins appeared to reside in different subcellular compartments, as they were fractionated by differential centrifugation. The specificity of both polypeptides was validated by cell transfection assay and mass spectrometry analysis. Immunoblot analysis of protein distribution in various zones of the human brain revealed variable amounts of both proteins. Notably, these proteins were more abundant in the temporal neocortex than in the hippocampus, the difference in abundance of the 65-kDa product being particularly pronounced. These results suggest that the two protein isoforms encoded by LGI1/Epitempin are differentially expressed in the human brain, and that higher expression levels of these proteins in the lateral temporal cortex may underlie the susceptibility of this brain region to the epileptogenic effects of LGI1/Epitempin mutations.