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Background The transient receptor potential vanilloid 1 ( TRPV 1) mediates cellular responses to pain, heat, or noxious stimuli by calcium influx; however, the cellular localization and function of TRPV 1 in the cardiomyocyte is largely unknown. We studied whether myocardial injury is regulated by TRPV 1 and whether we could mitigate reperfusion injury by limiting the calcineurin interaction with TRPV 1. Methods and Results In primary cardiomyocytes, confocal and electron microscopy demonstrates that TRPV 1 is localized to the mitochondria. Capsaicin, the specific TRPV 1 agonist, dose‐dependently reduced mitochondrial membrane potential and was blocked by the TRPV 1 antagonist capsazepine or the calcineurin inhibitor cyclosporine. Using in silico analysis, we discovered an interaction site for TRPV 1 with calcineurin. We synthesized a peptide, V1‐cal, to inhibit the interaction between TRPV 1 and calcineurin. In an in vivo rat myocardial infarction model, V1‐cal given just prior to reperfusion substantially mitigated myocardial infarct size compared with vehicle, capsaicin, or cyclosporine (24±3% versus 61±2%, 45±1%, and 49±2%, respectively; n=6 per group; P TRPV 1 knockout rats. Conclusions TRPV 1 is localized at the mitochondria in cardiomyocytes and regulates mitochondrial membrane potential through an interaction with calcineurin. We developed a novel therapeutic, V1‐cal, that substantially reduces reperfusion injury by inhibiting the interaction of calcineurin with TRPV 1. These data suggest that TRPV 1 is an end‐effector of cardioprotection and that modulating the TRPV 1 protein interaction with calcineurin limits reperfusion injury.

Oxidative phosphorylation (OXPHOS) efficiency, defined as the ATP-to-O ratio, is a critical feature of mitochondrial function that has been implicated in health, aging, and disease. To date, however, the methods to measure ATP/O have primarily relied on indirect approaches or entail parallel rather than simultaneous determination of ATP synthesis and O2 consumption rates. The purpose of this project was to develop and validate an approach to determine the ATP/O ratio in permeabilized fiber bundles (PmFBs) from simultaneous measures of ATP synthesis (JATP) and O2 consumption (JO2 ) rates in real time using a custom-designed apparatus. JO2 was measured via a polarigraphic oxygen sensor and JATP via fluorescence using an enzyme-linked assay system (hexokinase II, glucose-6-phosphate dehydrogenase) linked to NADPH production. Within the dynamic linear range of the assay system, ADP-stimulated increases in steady-state JATP mirrored increases in steady-state JO2 (r(2) = 0.91, P0.0001, n = 57 data points). ATP/O ratio was less than one under low rates of respiration (15 μM ADP) but increased to more than two at moderate (200 μM ADP) and maximal (2,000 μM ADP) rates of respiration with an interassay coefficient of variation of 24.03, 16.72, and 11.99%, respectively. Absolute and relative (to mechanistic) ATP/O ratios were lower in PmFBs (2.09 ± 0.251, 84%) compared with isolated mitochondria (2.44 ± 0.124, 98%). ATP/O ratios in PmFBs were not affected by the activity of adenylate kinase or creatine kinase. These findings validate an enzyme-linked respiratory clamp system for measuring OXPHOS efficiency in PmFBs and provide evidence that OXPHOS efficiency increases as energy demand increases.

One proposed explanation for the V̇o2slow component is that lower-threshold motor units may fatigue and develop little or no tension but continue to use O2, thereby resulting in a dissociation of cellular respiration from force generation. The present study used intact isolated single myocytes with differing fatigue resistance profiles to investigate the relationship between fatigue, tension development, and aerobic metabolism. Single Xenopus skeletal muscle myofibers were allocated to a fast-fatiguing (FF) or a slow-fatiguing (SF) group, based on the contraction frequency required to elicit a fall in tension to 60% of peak. Phosphorescence quenching of a porphyrin compound was used to determine Δ intracellular Po2(PiO2; a proxy for V̇o2), and developed isometric tension was monitored to allow calculation of the time-integrated tension (TxT). Although peak ΔPiO2was not different between groups ( P = 0.36), peak tension was lower ( P < 0.05) in SF vs. FF (1.97 ± 0. 17 V vs. 2. 73 ± 0.30 V, respectively) and time to 60% of peak tension was significantly longer in SF vs. FF (242 ± 10 s vs. 203 ± 10 s, respectively). Before fatigue, both ΔPiO2and TxT rose proportionally with contraction frequency in SF and FF, resulting in ΔPiO2/TxT being identical between groups. At fatigue, TxT fell dramatically in both groups, but ΔPiO2decreased proportionately only in the FF group, resulting in an increase in ΔPiO2/TxT in the SF group relative to the prefatigue condition. These data show that more fatigue-resistant fibers maintain aerobic metabolism as they fatigue, resulting in an increased O2cost of contractions that could contribute to the V̇o2slow component seen in whole body exercise.

Intracellular pH (pHi) was measured in isolated Xenopus laevis single myofibres at the onset of contractions, with and without glycolytic blockade, to investigate the time course of glycolytic activation. Single myofibres (n= 8; CON) were incubated in 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoyxmethyl ester (10 μm; for fluorescence measurement of pHi) and stimulated for 15 s at 0.67 Hz in anoxia in the absence (control condition; CON) and presence of a glycolytic inhibitor (1 mm iodoacetic acid; IAA). Intracellular pHi and tension were continuously recorded, and the differences in pHi between conditions were used to estimate the activation time of glycolysis. An immediate and steady increase in pHi (initial alkalosis) at the onset of contractions was similar between CON and IAA trials for the first 9 s of the contractile bout. However, from six contractions (∼10 s) throughout the remainder of the bout, IAA demonstrated a continued rise in pHi, in contrast to a progressive decrease in pHi in CON (P < 0.05). These results demonstrate, with high temporal resolution, that glycolysis is activated within six contractions (10 s at 0.67 Hz) in single Xenopus skeletal muscle fibres.

The purpose of the present study was 1) to develop a stable model for measuring contraction-induced elevations in mRNA in single skeletal muscle fibers and 2) to utilize this model to investigate the response of heat shock protein 72 (HSP72) mRNA following an acute bout of fatiguing contractions. Living, intact skeletal muscle fibers were microdissected from lumbrical muscle of Xenopus laevis and either electrically stimulated for 15 min of tetanic contractions (EX; n = 26) or not stimulated to contract (REST; n = 14). The relative mean developed tension of EX fibers decreased to 29 ± 7% of initial peak tension at the stimulation end point. Following treatment, individual fibers were allowed to recover for 1 ( n = 9), 2 ( n = 8), or 4 h ( n = 9) prior to isolation of total cellular mRNA. HSP72, HSP60, and cardiac α-actin mRNA content were then assessed in individual fibers using quantitative PCR detection. Relative HSP72 mRNA content was significantly ( P < 0.05) elevated at the 2-h postcontraction time point relative to REST fibers when normalized to either HSP60 (18.5 ± 7.5-fold) or cardiac α-actin (14.7 ± 4.3-fold), although not at the 1- or 4-h time points. These data indicate that 1) extraction of RNA followed by relative quantification of mRNA of select genes in isolated single skeletal muscle fibers can be reliably performed, 2) HSP60 and cardiac α-actin are suitable endogenous normalizing genes in skeletal muscle following contractions, and 3) a significantly elevated content of HSP72 mRNA is detectable in skeletal muscle 2 h after a single bout of fatiguing contractions, despite minimal temperature changes and without influence from extracellular sources.

Cellular heterogeneity is a fundamental characteristic of many cancers. A lack of cellular homogeneity contributes to difficulty in designing targeted oncological therapies. Therefore, the development of novel methods to determine and characterize oncologic cellular heterogeneity is a critical next step in the development of novel cancer therapies. Single-cell sequencing (SCS) technology has been recently employed for analyzing the genetic polymorphisms of individual cells at the genome-wide level. SCS requires (1) precise isolation of the single cell of interest; (2) isolation and amplification of genetic material; and (3) descriptive analysis of genomic, transcriptomic, and epigenomic data. In addition to targeted analysis of single cells isolated from tumor biopsies, SCS technology may be applied to circulating tumor cells, which may aid in predicting tumor progression and metastasis. In this paper, we provide an overview of SCS technology and review the current literature on the potential application of SCS to clinical oncology and research.

The intrinsic activating factors that induce transcription of heat shock protein 72 (HSP72) in skeletal muscle following exercise remain unclear. We hypothesized that the cytosolic Ca2+ transient that occurs with depolarization is a determinant. We utilized intact, single skeletal muscle fibers from Xenopus laevis to test the role of the cytosolic Ca2+ transient and several other exercise-related factors (fatigue, hypoxia, AMP kinase, and cross-bridge cycling) on the activation of HSP72 transcription. HSP72 and HSP60 mRNA levels were assessed with real-time quantitative PCR; cytosolic Ca2+ concentration ([Ca2+]cyt) was assessed with fura-2. Both fatiguing and nonfatiguing contractions resulted in a significant increase in HSP72 mRNA. As expected, peak [Ca2+]cyt remained tightly coupled with peak developed tension in contracting fibers. Pretreatment with N-benzyl- p-toluene sulfonamide (BTS) resulted in depressed peak developed tension with stimulation, while peak [Ca2+]cyt remained largely unchanged from control values. Despite excitation-contraction uncoupling, BTS-treated fibers displayed a significant increase in HSP72 mRNA. Treatment of fibers with hypoxia (Po2: 2+ transient that occurs with skeletal muscle depolarization provides a sufficient activating stimulus for HSP72 transcription. Metabolic or mechanical factors associated with fatigue development and cross-bridge cycling likely play a more limited role.

Background: Isoflurane induces cell death in neurons undergoing synaptogenesis via increased production of pro-brain–derived neurotrophic factor (proBDNF) and activation of postsynaptic p75 neurotrophin receptor (p75NTR). Astrocytes express p75NTR, but their role in neuronal p75NTR-mediated cell death remains unclear. The authors investigated whether astrocytes have the capacity to buffer increases in proBDNF and protect against isoflurane/p75NTR neurotoxicity. Methods: Cell death was assessed in day in vitro (DIV) 7 mouse primary neuronal cultures alone or in co-culture with age-matched or DIV 21 astrocytes with propidium iodide 24 h after 1 h exposure to 2% isoflurane or recombinant proBDNF. Astrocyte-targeted knockdown of p75NTR in co-culture was achieved with small-interfering RNA and astrocyte-specific transfection reagent and verified with immunofluorescence microscopy. proBDNF levels were assessed by enzyme-linked immunosorbent assay. Each experiment used six to eight replicate cultures/condition and was repeated at least three times. Results: Exposure to isoflurane significantly (P < 0.05) increased neuronal cell death in primary neuronal cultures (1.5 ± 0.7 fold, mean ± SD) but not in co-culture with DIV 7 (1.0 ± 0.5 fold) or DIV 21 astrocytes (1.2 ± 1.2 fold). Exogenous proBDNF dose dependently induced neuronal cell death in both primary neuronal and co-cultures, an effect enhanced by astrocyte p75NTR inhibition. Astrocyte-targeted p75NTR knockdown in co-cultures increased media proBDNF (1.2 ± 0.1 fold) and augmented isoflurane-induced neuronal cell death (3.8 ± 3.1 fold). Conclusions: The presence of astrocytes provides protection to growing neurons by buffering increased levels of proBDNF induced by isoflurane. These findings may hold clinical significance for the neonatal and injured brain where increased levels of proBDNF impair neurogenesis.

The purpose of this investigation was to utilize 2,3-butanedione monoxime (BDM; an inhibitor of contractile activation) to dissociate cytosolic [Ca2+] ([Ca2+]c) from the putative respiratory regulators that arise from muscle contraction-induced ATP utilization in order to determine the relative contribution of [Ca2+]c on intracellular PO2 (PiO2) kinetics during the transition from rest to contractions in single skeletal myocytes isolated from Xenopus laevis lumbrical muscle. Myocytes were subjected to electrically induced isometric tetanic contractions (0.25 Hz; 2-min bouts) while peak tension and either [Ca2+]c (n= 7; ratiometric fluorescence microscopy) or PiO2 (n= 7; phosphorescence microscopy) was measured continuously. Cells were studied under both control and 3 mm BDM conditions in randomized order. Initial (control, 100 ± 0%; BDM, 72.6 ± 4.6%), midpoint (control, 86.7 ± 1.8%; BDM, 61.6 ± 4.1%) and end (control, 85.0 ± 2.8%; BDM, 57.5 ± 5.0%) peak tensions (normalized to initial control values) were significantly reduced (P 0.05) between control and BDM trials. Thus, the peak tension-to-peak [Ca2+]c ratio was reduced with BDM compared with control. The absolute fall in PiO2 with contractions, which is proportional to the rise in , was significantly reduced with BDM (13.2 ± 1.3 mmHg) compared with control (22.0 ± 2.0 mmHg). However, PiO2 onset kinetics (i.e. mean response time (MRT)) was not altered between BDM (66.8 ± 8.0 s) and control (64.9 ± 6.3 s) trials. Therefore, the initial rate of change (defined as the fall in PiO2/MRT) was significantly slower in BDM fibres compared with control. These data demonstrate in these isolated single skeletal muscle fibres that unchanged peak [Ca2+]c in the face of reduced metabolic feedback from the contractile sites evoked with BDM did not alter PiO2 onset kinetics in isolated single frog myocytes, suggesting that metabolic signals arising from the contractile sites play a more substantial role than [Ca2+]c in the signalling pathway to oxidative phosphorylation during the transition from rest to repeated tetanic contractions.

The purpose of the present study was to compare the individual fatigue characteristics of isolated single skeletal muscle fibres with their mitochondrial volume density (MVD), using direct histological morphometry. Single muscle fibres (n= 14) were microdissected from lumbrical muscle of adult female Xenopus laevis, and force was measured while fibres were stimulated (tetanic contractions of 200 ms trains with 70 Hz stimuli at 9 V) at progressively increasing frequencies (2 min each at 0.25, 0.33, 0.5 and 1 contractions s−1) until fatigue (

The purpose of this research was to develop a technique for rapid measurement of O2uptake (V˙o2) kinetics in single isolated skeletal muscle cells. Previous attempts to measure single cellV˙o2have utilized polarographic-style electrodes, thereby mandating large fluid volumes and relatively poor sensitivity. Thus our laboratory has developed an ∼100-μl, well-stirred chamber for the measurement ofV˙o2in isolated Xenopus laevismyocytes using a phosphorescence quenching technique [Ringer solution with 0.05 mM Pd- meso-tetra(4-carboxyphenyl)porphine] to monitor the fall in extracellular Po2(which is proportional to cellular V˙o2within the sealed chamber). V˙o2in single living myocytes dissected from Xenopus lumbrical muscles was measured from rest across a bout of repetitive tetanic contractions (0.33 Hz) and in response to a ramp protocol utilizing an increasing contraction frequency. In response to the square-wave contraction bout, the increase in V˙o2to steady state (SS) was 16.7 ± 1.3 ml · 100 g−1· min−1(range 13.0–21.9 ml · 100 g−1· min−1; n = 6). The rise in V˙o2at contractions onset ( n = 6) was fit with a time delay (2.1 ± 1.2 s, range 0.0–7.7 s) plus monoexponential rise to SS (time constant = 9.4 ± 1.5 s, range 5.2–14.9 s). Furthermore, in two additional myocytes,V˙o2increased progressively as contraction frequency increased (ramp protocol). This technique for measuring V˙o2in isolated, single skeletal myocytes represents a novel and powerful investigative tool for gaining mechanistic insight into mitochondrial function andV˙o2dynamics without potential complications of the circulation and other myocytes.

Recent studies have demonstrated that neural stem cell (NSC) culture at physiologically normoxic conditions (2-5% O2) is advantageous in terms of neuronal differentiation and survival. Neuronal differentiation is accompanied by a remarkable shift to mitochondrial oxidative metabolism compared with preferentially glycolytic metabolism of proliferating cells. However, metabolic changes induced by growth in a normoxic (5%) O2 culture environment in NSCs have been minimally explored. This study demonstrates that culturing under 5% O2 conditions results in higher levels of mitochondrial oxidative metabolism, decreased glycolysis, and reduced levels of reactive oxygen species in NSC cultures. Inflammation is one of the major environmental factors limiting postinjury NSC neuronal differentiation and survival. Our results show that NSCs differentiated under 5% O2 conditions possess better resistance to in vitro inflammatory injury compared with those exposed to 20% O2. The present work demonstrates that lower, more physiologically normal O2 levels support metabolic changes induced during NSC neuronal differentiation and provide increased resistance to inflammatory injury, thus highlighting O2 tension as an important determinant of cell fate and survival in various stem cell therapies.

Anti-NMDAR encephalitis is a recently identified autoimmune disease, described by an immune-mediated loss of NMDA glutamate receptors, resulting in progressive mental deterioration. To date, literature on anti-NMDAR encephalitis has been largely clinically oriented, including descriptions of the clinical presentation and course, diagnostic methods, and potential clinical treatments. However, the underlying molecular mechanisms contributing to the complex immunological cellular transformation that is associated with the progression of anti-NMDAR encephalitis remain to be adequately explored. This review will provide a summary of the current literature on anti-NMDAR encephalitis, including the immunologic molecular mechanisms contributing to disease progression. In particular this review will focus on the effect of anti-NMDAR on GluN2-NMDAR expression and the molecular transformation of B and T leukocytes in the loss of self-tolerance. Further research on the immunologic mechanisms contributing to anti-NMDAR encephalitis may provide an avenue for future novel diagnostic approaches, such as immunologic surveillance, as well as new therapeutic strategies for this recently identified autoimmune disease.

Anti-NMDAR encephalitis is a recently identified autoimmune disease, described by an immune-mediated loss of NMDA glutamate receptors, resulting in progressive mental deterioration. To date, literature on anti-NMDAR encephalitis has been largely clinically oriented, including descriptions of the clinical presentation and course, diagnostic methods, and potential clinical treatments. However, the underlying molecular mechanisms contributing to the complex immunological cellular transformation that is associated with the progression of anti-NMDAR encephalitis remain to be adequately explored. This review will provide a summary of the current literature on anti-NMDAR encephalitis, including the immunologic molecular mechanisms contributing to disease progression. In particular this review will focus on the effect of anti-NMDAR on GluN2-NMDAR expression and the molecular transformation of B and T leukocytes in the loss of self-tolerance. Further research on the immunologic mechanisms contributing to anti-NMDAR encephalitis may provide an avenue for future novel diagnostic approaches, such as immunologic surveillance, as well as new therapeutic strategies for this recently identified autoimmune disease.

miR-181 has deleterious effects on stroke outcome, and reducing miR-181a levels prior to middle cerebral artery occlusion (MCAO) was shown previously to be protective. Here we tested the effect of post-ischemic treatment with miR-181a antagomir by intracerebroventricular and intravenous routes of administration on infarct size, neurological outcome, inflammatory response and long term behavioral outcome. Post-treatment with miR-181a antagomir significantly reduced infarction size, improved neurological deficits and reduced NF-κB activation, numbers of infiltrating leukocytes and levels of Iba1. Targets affected by miR-181a antagomir administered after stroke onset include BCL2 and X-linked inhibitor of apoptosis protein (XIAP). Post-treatment with miR-181a antagomir significantly improved behavioral outcome assessed by rotarod at one month. These findings indicate that post-treatment with miR-181a antagomir has neuroprotective effects against ischemic neuronal damage and neurological impairment in mice, and the protection is long lasting including recovery of motor function and coordination over one month. The ability to protect the brain with post-treatment with miR-181a antagomir with long lasting effect makes this a promising therapeutic target and may be an innovative and effective new approach for stroke therapy.

This study examined the relationship between force and cytosolic free calcium concentration ([Ca2+]c) in different fiber types from Xenopus before, during, and after cells underwent postcontractile depression (PCD). During a standardized fatigue run, force in the two fast fatiguing (FF) fiber types (types 1 and 2, n = 10) fell more quickly (5.8 vs. 8.1 min) and to a greater degree [0.36 vs. 0.51 of initial (Po)] than in the slow fatiguing (SF) fiber type (type 3, n = 11). After the initial fatigue run, both FF and SF experienced a drop in force to o(PCD) at a similar time (20.6 vs. 21.4 min). A second stimulation period, undertaken during PCD, produced significant recovery of force in both groups, but significantly more so in SF than FF (64 ± 7 vs. 29 ± 2% Po). This force recovery during PCD was accompanied by a significant increase in peak [Ca2+]c, particularly in SF. However, despite the significant recovery of force during stimulation while in PCD, the amount of force produced for a given peak [Ca2+]cwas significantly lower in both groups during PCD than at any other point in the experiment. A final stimulation period, initiated when all fibers had recovered from PCD, demonstrated a recovery of both force and peak [Ca2+]cin both groups, but this recovery was significantly greater in SF vs. FF. These data demonstrate that with continuous electrical stimulation, it is possible to produce a significant recovery of force production during the normally quiescent period of PCD, but that it occurs with a decreased muscle force production for a given peak [Ca2+]c. This suggests that factors other than structural alterations of the sarcoplasmic reticulum are likely the cause of PCD in these fibers.

We tested the hypothesis that the mechanisms involved in the more rapid onset of fatigue when O2availability is reduced in contracting skeletal muscle are similar to those when O2availability is more sufficient. Two series of experiments were performed in isolated, single skeletal muscle fibers from Xenopus laevis. First, relative force and free cytosolic Ca2+concentrations ([Ca2+]c) were measured simultaneously in single fibers ( n = 6) stimulated at increasing frequencies (0.25, 0.33, 0.5, and 1 Hz) at an extracellular[Formula: see text] of either 22 or 159 Torr. Muscle fatigue (force = 50% of initial peak tension) occurred significantly sooner ( P < 0.05) during the low- (237 ± 40 s) vs. high-[Formula: see text]treatments (280 ± 38 s). Relative [Ca2+]cwas significantly decreased from maximal values at the fatigue time point during both the high- (72 ± 4%) and low-[Formula: see text] conditions (78 ± 4%), but no significant difference was observed between the treatments. In the second series of experiments, using the same stimulation regime as the first, fibers ( n = 6) exposed to 5 mM caffeine immediately after fatigue demonstrated an immediate but incomplete relative force recovery during both the low- (89 ± 4%) and high-[Formula: see text] treatments (82 ± 3%), with no significant difference between treatments. Additionally, there was no significant difference in relative [Ca2+]cbetween the high- (100 ± 12% of prefatigue values) and low-[Formula: see text] treatments (108 ± 12%) on application of caffeine. These results suggest that in isolated, single skeletal muscle fibers, the earlier onset of fatigue that occurred during the low-extracellular[Formula: see text] condition was modulated through similar pathways as the fatigue process during the high and involved a decrease in relative peak [Ca2+]c.

We tested the hypothesis that contracting skeletal muscle can rapidly restore force development during reperfusion after brief total ischemia and that this rapid recovery depends on O2 availability and not an alternate factor related to blood flow. Isolated canine gastrocnemius muscle ( n = 5) was stimulated to contract tetanically (isometric contraction elicited by 8 V, 0.2-ms duration, 200-ms trains, at 50-Hz stimulation) every 2 s until steady-state conditions of muscle blood flow (controlled by pump perfusion) and developed force were attained (3 min). While maintaining the same stimulation pattern, muscle blood flow was then reduced to zero (complete ischemia) for 2 min. Normal blood flow was then restored to the contracting muscle; however, two distinct conditions of oxygenation (at the same blood flow) were sequentially imposed: deoxygenated blood (30 s), blood with normal arterial O2content (30 s), a return to deoxygenated blood (30 s), and finally a return to normal arterial O2 content (90 s). During the ischemic period, force development fell to 39 ± 6 (SE)% of normal (from 460 ± 40 to 170 ± 20 N/100 g). When muscle blood flow was restored to normal by perfusion with deoxygenated blood, developed force continued to decline to 140 ± 20 N/100 g. Muscle force rapidly recovered to 310 ± 30 N/100 g ( P < 0.05) during the 30 s in which the contracting muscle was perfused with oxygenated blood and then fell again to 180 ± 30 N/100 g when perfused with blood with low[Formula: see text]. These findings demonstrate that contracting skeletal muscle has the capacity for rapid recovery of force development during reperfusion after a short period of complete ischemia and that this recovery depends on O2availability and not an alternate factor related to blood flow restoration.

The purpose of this study was to examine the development of fatigue in isolated, single skeletal muscle fibers when O2availability was reduced but not to levels considered rate limiting to mitochondrial respiration. Tetanic force was measured in single living muscle fibers ( n = 6) from Xenopus laevis while being stimulated at increasing contraction rates (0.25, 0.33, 0.5, and 1 Hz) in a sequential manner, with each stimulation frequency lasting 2 min. Muscle fatigue (determined as 75% of initial maximum force) was measured during three separate work bouts (with 45 min of rest between) as the perfusate [Formula: see text] was switched between values of 30 ± 1.9, 76 ± 3.0, or 159 Torr in a blocked-order design. No significant differences were found in the initial peak tensions between the high-, intermediate-, and low-[Formula: see text] treatments (323 ± 22, 298 ± 27, and 331 ± 24 kPa, respectively). The time to fatigue was reached significantly sooner ( P < 0.05) during the 30-Torr treatment (233 ± 39 s) compared with the 76- (385 ± 62 s) or 159-Torr (416 ± 65 s) treatments. The calculated critical extracellular [Formula: see text]necessary to develop an anoxic core within these fibers was 13 ± 1 Torr, indicating that the extracellular[Formula: see text] of 30 Torr should not have been rate limiting to mitochondrial respiration. The magnitude of an unstirred layer (243 ± 64 μm) or an intracellular O2diffusion coefficient (0.45 ± 0.04 × 10−5cm2/s) necessary to develop an anoxic core under the conditions of the study was unlikely. The earlier initiation of fatigue during the lowest extracellular[Formula: see text] condition, at physiologically high intracellular [Formula: see text] levels, suggests that muscle performance may be O2dependent even when mitochondrial respiration is not necessarily compromised.

Increased ventilation-perfusion (V˙a/Q˙) inequality is observed in ∼50% of humans during heavy exercise and contributes to the widening of the alveolar-arterial O2 difference (A-[Formula: see text]). Despite extensive investigation, the cause remains unknown. As a first step to more direct examination of this problem, we developed an animal model. Eight Yucatan miniswine were studied at rest and during treadmill exercise at ∼30, 50, and 85% of maximal O2 consumption (V˙o 2 max). Multiple inert-gas, blood-gas, and metabolic data were obtained. The A-[Formula: see text]increased from 0 ± 3 (SE) Torr at rest to 14 ± 2 Torr during the heaviest exercise level, but arterial[Formula: see text]([Formula: see text]) remained at resting levels during exercise. There was normalV˙a/Q˙inequality [log SD of the perfusion distribution (log[Formula: see text]) = 0.42 ± 0.04] at rest, and moderate increases (log[Formula: see text] = 0.68 ± 0.04, P < 0.0001) were observed with exercise. This result was reproducible on a separate day. TheV˙a/Q˙inequality changes are similar to those reported in highly trained humans. However, in swine, unlike in humans, there was no inert gas evidence for pulmonary end-capillary diffusion limitation during heavy exercise; there was no systematic difference in the measured[Formula: see text] and the[Formula: see text] as predicted from the inert gases. These data suggest that the pig animal model is well suited for studying the mechanism of exercise-inducedV˙a/Q˙inequality.

To test the hypothesis that muscle O2 uptake (V˙o 2) on-kinetics is limited, at least in part, by peripheral O2 diffusion, we determined theV˙o 2 on-kinetics in 1) normoxia (Control); 2) hyperoxic gas breathing (Hyperoxia); and 3) hyperoxia and the administration of a drug (RSR-13, Allos Therapeutics), which right-shifts the Hb-O2dissociation curve (Hyperoxia+RSR-13). The study was conducted in isolated canine gastrocnemius muscles ( n = 5) during transitions from rest to 3 min of electrically stimulated isometric tetanic contractions (200-ms trains, 50 Hz; 1 contraction/2 s; 60–70% peakV˙o 2). In all conditions, before and during contractions, muscle was pump perfused with constantly elevated blood flow (Q˙), at a level measured at steady state during contractions in preliminary trials with spontaneous Q˙. Adenosine was infused intra-arterially to prevent inordinate pressure increases with the elevated Q˙. Q˙ was measured continuously, arterial and popliteal venous O2 concentrations were determined at rest and at 5- to 7-s intervals during contractions, andV˙o 2 was calculated asQ˙ ⋅ arteriovenous O2 content difference.[Formula: see text] at 50% Hbo 2saturation (P50) was calculated. Mean capillary [Formula: see text](P [Formula: see text]) was estimated by numerical integration. P50 was higher in Hyperoxia+RSR-13 [40 ± 1 (SE) Torr] than in Control and in Hyperoxia (31 ± 1 Torr). After 15 s of contractions,P [Formula: see text]was higher in Hyperoxia (97 ± 9 Torr) vs. Control (53 ± 3 Torr) and in Hyperoxia+RSR-13 (197 ± 39 Torr) vs. Hyperoxia. The time to reach 63% of the difference between baseline and steady-stateV˙o 2 during contractions was 24.7 ± 2.7 s in Control, 26.3 ± 0.8 s in Hyperoxia, and 24.7 ± 1.1 s in Hyperoxia+RSR-13 (not significant). Enhancement of peripheral O2 diffusion (obtained by increasedP [Formula: see text]at constant O2 delivery) during the rest-to-contraction (60–70% of peakV˙o 2) transition did not affect muscle V˙o 2on-kinetics.

The purpose of this study was to examine the bioenergetics and regulation of O2uptake (V˙o2) and force production in contracting muscle when blood flow was moderately reduced during a steady-state contractile period. Canine gastrocnemius muscle ( n = 5) was isolated, and 3-min stimulation periods of isometric, tetanic contractions were elicited sequentially at rates of 0.25, 0.33, and 0.5 contractions/s (Hz) immediately followed by a reduction of blood flow [ischemic (I) condition] to 46 ± 3% of the value obtained at 0.5 Hz with normal blood flow. TheV˙o2of the contracting muscle was significantly ( P < 0.05) reduced during the I condition [6.5 ± 0.8 (SE) ml ⋅ 100 g−1⋅ min−1] compared with the same stimulation frequency with normal flow (11.2 ± 1.5 ml ⋅ 100 g−1⋅ min−1), as was the tension-time index (79 ± 12 vs. 123 ± 22 N ⋅ g−1⋅ min−1, respectively). The ratio ofV˙o2to tension-time index remained constant throughout all contraction periods. Muscle phosphocreatine concentration, ATP concentration, and lactate efflux were not significantly different during the I condition compared with the 0.5-Hz condition with normal blood flow. However, at comparable rates of V˙o2and tension-time index, muscle phosphocreatine concentration and ATP concentration were significantly less during the I condition compared with normal-flow conditions. These results demonstrate that, in this highly oxidative muscle, the normal balance of O2supply to force output was maintained during moderate ischemia by downregulation of force production. In addition, 1) the minimal disruption in intracellular homeostasis after the initiation of ischemia was likely a result of steady-state metabolic conditions having already been activated, and 2) the difference in intracellular conditions at comparable rates ofV˙o2and tension-time index between the normal flow and I condition may have been due to altered intracellular O2tension.

Intracellular pH (pHi) was measured in isolated Xenopus laevis single myofibres at the onset of contractions, with and without glycolytic blockade, to investigate the time course of glycolytic activation. Single myofibres (n=8; CON) were incubated in 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoyxmethyl ester (10 microM; for fluorescence measurement of pHi) and stimulated for 15 s at 0.67 Hz in anoxia in the absence (control condition; CON) and presence of a glycolytic inhibitor (1 mM iodoacetic acid; IAA). Intracellular pHi and tension were continuously recorded, and the differences in pHi between conditions were used to estimate the activation time of glycolysis. An immediate and steady increase in pHi (initial alkalosis) at the onset of contractions was similar between CON and IAA trials for the first 9 s of the contractile bout. However, from six contractions (approximately 10 s) throughout the remainder of the bout, IAA demonstrated a continued rise in pHi, in contrast to a progressive decrease in pHi in CON (P0.05). These results demonstrate, with high temporal resolution, that glycolysis is activated within six contractions (10 s at 0.67 Hz) in single Xenopus skeletal muscle fibres.

Hemoperitoneum may occur in various emergent conditions. In the trauma setting, evidence of intraperitoneal blood depicted at computed tomography (CT) should lead the radiologist to conduct a careful search of images for the injured visceral organ (the liver or spleen). Specific CT signs, such as a sentinel clot or extravasation of intravascular contrast material, may indicate the source of bleeding and help direct management. In addition, the configuration of accumulated blood may help identify the injured organ; for example, triangular fluid collections are observed in the mesentery most often in the setting of bowel or mesenteric injury. Less commonly, hemoperitoneum may have a nontraumatic origin. Iatrogenic hemoperitoneum may occur as a complication of surgery or other interventional procedures in the abdominal cavity or as a result of anticoagulation therapy. Hemoperitoneum also may be seen in the setting of blood dyscrasias such as hemophilia and polycythemia vera. Tumor-associated hemorrhage, which most often occurs in hepatocellular carcinoma, hepatic adenoma, or vascular metastatic disease, also may produce hemoperitoneum. Other potential causes of nontraumatic hemoperitoneum are gynecologic conditions such as hemorrhage or rupture of an ovarian cyst and rupture of the gestational sac in ectopic pregnancy, and hepatic hematoma in syndromic hemolysis with elevated liver enzymes and low platelet count (HELLP syndrome). Vascular lesions (visceral artery aneurysms and pseudoaneurysms) that occur in systemic vascular diseases such as Ehlers-Danlos syndrome or in pancreatitis are another less common source of hemoperitoneum.