Effect of Diabetes on Microvascular Morphology and Permeability of Rat Skeletal Muscle: In Vivo Imaging Using Two-Photon Laser Scanning Microscopy.

This investigation evaluated the microvascular permeability and ultrastructure of skeletal muscle capillaries in skeletal muscle of diabetic (DIA) rats using two-photon laser scanning microscopy (TPLSM) and transmission electron microscopy (TEM). Microvascular permeability was assessed in the tibialis anterior muscle of control (CON) and DIA (streptozocin) male Wistar rats (n = 20, 10-14 wk) by in vivo imaging using TPLSM after fluorescent dye intravenous infusion. Fluorescent dye leakage was quantified to determine microvascular permeability. The ultrastructure was imaged by TEM ex vivo to calculate the size and number of intercellular clefts between capillary endothelial cells and also intracellular vesicles. Compared with control, the volumetrically determined interstitial fluorescent dye leakage, the endothelial cell thickness, and the number of intercellular clefts per capillary perimeter were significantly higher, and the cleft width was significantly narrower in TA of DIA (interstitial fluorescent dye leakage, 2.88 ± 1.40 vs. 10.95 ± 1.41 µm3 x min x 106; endothelial thickness 0.28 ± 0.02 vs. 0.45 ± 0.03 µm; number of intercellular clefts per capillary perimeter 6.3 ± 0.80 vs. 13.6 ± 1.7 /100 µm; cleft width 11.92 ± 0.95 vs. 8.40 ± 1.03 nm, CON vs. DIA respectively, all p <0.05). The size of intracellular vesicles in the vascular endothelium showed an increased proportion of large vesicles in the DIA group compared to the CON group (p < 0.05). Diabetes mellitus enhances the microvascular permeability of skeletal muscle microvessels, due, in part, to a higher density and narrowing of the endothelial intercellular clefts, and larger intracellular vesicles.

Eccentric contraction increases hydrogen peroxide levels and alters gene expression through Nox2 in skeletal muscle of male mice.

Hydrogen peroxide (H2O2) is one of the key signaling factors regulating skeletal muscle adaptation to muscle contractions. Eccentric (ECC) and concentric (CONC) contractions drive different muscle adaptations with ECC resulting in greater changes. The present investigation tested the hypothesis that ECC produces higher cytosolic and mitochondrial H2O2 concentrations [H2O2] and alters gene expression more than CONC. Cytosolic and mitochondrial H2O2-sensitive fluorescent proteins, HyPer7 and MLS-HyPer7, were expressed in the anterior tibialis muscle of C57BL6J male mice. Before and for 60 min after either CONC or ECC (100 Hz, 50 contractions), [H2O2]cyto and [H2O2]mito were measured by in vivo fluorescence microscopy. RNA sequencing was performed in control (non-contracted), CONC and ECC muscles to identify genes impacted by the contractions. [H2O2]cyto immediately after ECC was greater than after CONC (CONC: + 6%, ECC: + 11% vs rest, p < 0.05) and remained higher for at least 60 min into recovery. In contrast, the elevation of [H2O2]mito was independent of the contraction modes (Time; p < 0.0042, contraction mode; p = 0.4965). The impact of ECC on [H2O2]cyto were abolished by NADPH oxidase 2 (Nox2) inhibition (GSK2795039). Differentially expressed genes were not present after CONC or ECC+GSK but were found after ECC and were enriched for vascular development and apoptosis-related genes, among others. In conclusion, in mouse anterior tibialis ECC, but not CONC, evoke a pronounced cytosolic H2O2 response, caused by Nox2, that is mechanistically linked to gene expression modifications.

Effects of aging on diaphragm hyperemia and blood flow distribution in male and female Fischer 344 rats.

Aging is associated with inspiratory muscle dysfunction; however, the impact of aging on diaphragm blood flow (BF) regulation, and whether sex differences exist, is unknown. We tested the hypotheses in young animals that diaphragm BF and vascular conductance (VC) would be greater in females and that aging would decrease the diaphragm's ability to increase BF with contractions. Young (4-6 mo) and old (22-24 mo) Fischer 344 rats were divided into four groups: young female (YF, n = 7), young male (YM, n = 8), old female (OF, n = 9), and old male (OM, n = 9). Diaphragm BF (mL/min/100 g) and VC (mL/mmHg/min/100 g) were determined, via fluorescent microspheres, at rest and during 1 Hz contractions. In YF versus OF, aging blunted the increase in medial costal diaphragm BF (44 ± 5% vs. 16 ± 12%; P < 0.05) and VC (43 ± 7% vs. 21 ± 12%; P < 0.05). Similarly, in YM versus OM, aging blunted the increase in medial costal diaphragm BF (43 ± 6% vs. 24 ± 12%; P < 0.05) and VC (50 ± 6% vs. 34 ± 10%; P < 0.05). In female rats, age increased dorsal costal diaphragm BF, whereas in male rats, age increased crural diaphragm BF (P < 0.05). Compared with age-matched females, dorsal costal diaphragm BF was lower in YM and OM (P < 0.05). In conclusion, aging results in an inability to augment medial costal diaphragm BF and alters regional diaphragm BF distribution in response to muscular contractions. Furthermore, sex differences in regional diaphragm BF are present in young and old animals.NEW & NOTEWORTHY This is the first study, to our knowledge, to demonstrate that old age impairs the hyperemic response and alters blood flow distribution in the diaphragm of both female and male rats. In addition, this investigation provides novel evidence of sex differences in regional diaphragm blood flow distribution with contractions. The data presented herein suggest that aging compromises diaphragm vascular function and provides a potential mechanism for the diaphragm contractile dysfunction associated with old age.

Pulmonary hypertension impairs vasomotor function in rat diaphragm arterioles.

Pulmonary hypertension (PH) is a chronic, progressive condition in which respiratory muscle dysfunction is a primary contributor to exercise intolerance and dyspnea in patients. Contractile function, blood flow distribution, and the hyperemic response are altered in the diaphragm with PH, and we sought to determine whether this may be attributed, in part, to impaired vasoreactivity of the resistance vasculature. We hypothesized that there would be blunted endothelium-dependent vasodilation and impaired myogenic responsiveness in arterioles from the diaphragm of PH rats. Female Sprague-Dawley rats were randomized into healthy control (HC, n = 9) and monocrotaline-induced PH rats (MCT, n = 9). Endothelium-dependent and -independent vasodilation and myogenic responses were assessed in first-order arterioles (1As) from the medial costal diaphragm in vitro. There was a significant reduction in endothelium-dependent (via acetylcholine; HC, 78 ± 15% vs. MCT, 47 ± 17%; P < 0.05) and -independent (via sodium nitroprusside; HC, 89 ± 10% vs. MCT, 66 ± 10%; P < 0.05) vasodilation in 1As from MCT rats. MCT-induced PH also diminished myogenic constriction (P < 0.05) but did not alter passive pressure responses. The diaphragmatic weakness, impaired hyperemia, and blood flow redistribution associated with PH may be due, in part, to diaphragm vascular dysfunction and thus compromised oxygen delivery which occurs through both endothelium-dependent and -independent mechanisms.

In vivo heat production dynamics during a contraction-relaxation cycle in rat single skeletal muscle fibers.

Skeletal muscle generates heat via contraction-dependent (shivering) and independent (nonshivering) mechanisms. While this thermogenic capacity of skeletal muscle undoubtedly contributes to the body temperature homeostasis of animals and impacts various cellular functions, the intracellular temperature and its dynamics in skeletal muscle in vivo remain elusive. We aimed to determine the intracellular temperature and its changes within skeletal muscle in vivo during contraction and following relaxation. In addition, we tested the hypothesis that sarcoplasmic reticulum Ca2+ ATPase (SERCA) generates heat and increases the myocyte temperature during a transitory Ca2+-induced contraction-relaxation cycle. The intact spinotrapezius muscle of anesthetized adult male Wistar rats (n = 18) was exteriorized and loaded with the fluorescent probe Cellular Thermoprobe for Fluorescence Ratio (49.3 µM) by microinjection over 1 s. The fluorescence ratio (i.e., 580 nm/515 nm) was measured in vivo during 1) temperature increases induced by means of an external heater, and 2) Ca2+ injection (3.9 nL, 2.0 mM). The fluorescence ratio increased as a linear function of muscle surface temperature from 25 °C to 40 °C (r2 = 0.97, P < 0.01). Ca2+ injection (3.9 nL, 2.0 mM) significantly increased myocyte intracellular temperature: An effect that was suppressed by SERCA inhibition with cyclopiazonic acid (CPA, Ca2+: 38.3 ± 1.4 °C vs Ca2++CPA: 28.3 ± 2.8 °C, P < 0.01 at 1 min following injection). While muscle shortening occurred immediately after the Ca2+ injection, the increased muscle temperature was maintained during the relaxation phase. In this investigation, we demonstrated a novel model for measuring the intracellular temperature of skeletal muscle in vivo and further that heat generation occurs concomitant principally with SERCA functioning and muscle relaxation.

In vivo cytosolic H2O2 changes and Ca2+ homeostasis in mouse skeletal muscle.

Hydrogen peroxide (H2O2) and calcium ions (Ca2+) are functional regulators of skeletal muscle contraction and metabolism. Although H2O2 is one of the activators of the type-1 ryanodine receptor (RyR1) in the Ca2+ release channel, the interdependence between H2O2 and Ca2+ dynamics remains unclear. This study tested the following hypotheses using an in vivo model of mouse tibialis anterior (TA) skeletal muscle. 1) Under resting conditions, elevated cytosolic H2O2 concentration ([H2O2]cyto) leads to a concentration-dependent increase in cytosolic Ca2+ concentration ([Ca2+]cyto) through its effect on RyR1; and 2) in hypoxia (cardiac arrest) and muscle contractions (electrical stimulation), increased [H2O2]cyto induces Ca2+ accumulation. Cytosolic H2O2 (HyPer7) and Ca2+ (Fura-2) dynamics were resolved by TA bioimaging in young C57BL/6J male mice under four conditions: 1) elevated exogenous H2O2; 2) cardiac arrest; 3) twitch (1 Hz, 60 s) contractions; and 4) tetanic (30 s) contractions. Exogenous H2O2 (0.1-100 mM) induced a concentration-dependent increase in [H2O2]cyto (+55% at 0.1 mM; +280% at 100 mM) and an increase in [Ca2+]cyto (+3% at 1.0 mM; +8% at 10 mM). This increase in [Ca2+]cyto was inhibited by pharmacological inhibition of RyR1 by dantrolene. Cardiac arrest-induced hypoxia increased [H2O2]cyto (+33%) and [Ca2+]cyto (+20%) 50 min postcardiac arrest. Compared with the exogenous 1.0 mM H2O2 condition, [H2O2]cyto after tetanic muscle contractions rose less than one-tenth as much, whereas [Ca2+]cyto was 4.7-fold higher. In conclusion, substantial increases in [H2O2]cyto levels evoke only modest Ca2+ accumulation via their effect on the sarcoplasmic reticulum RyR1. On the other hand, contrary to hypoxia secondary to cardiac arrest, increases in [H2O2]cyto from muscle contractions are small, indicating that H2O2 generation is unlikely to be a primary factor driving the significant Ca2+ accumulation after, especially tetanic, muscle contractions.NEW & NOTEWORTHY We developed an in vivo mouse myocyte H2O2 imaging model during exogenous H2O2 loading, ischemic hypoxia induced by cardiac arrest, and muscle contractions. In this study, the interrelationship between cytosolic H2O2 levels and Ca2+ homeostasis during muscle contraction and hypoxic conditions was revealed. These results contribute to the elucidation of the mechanisms of muscle fatigue and exercise adaptation.

Chronic repetitive cooling and caffeine-induced intracellular Ca2+ elevation differentially impact adaptations in slow- and fast-twitch rat skeletal muscles.

Intracellular Ca2+ concentration ([Ca2+]i) is considered important in the regulation of skeletal muscle mass. This study tested the hypothesis that chronic repeated cooling and/or caffeine ingestion would acutely increase [Ca2+]i and hypertrophy muscles potentially in a fiber-type-dependent manner. Control rats and those fed caffeine were subjected to repeated bidiurnal treatments of percutaneous icing, under anesthesia, to reduce the muscle temperature below ∼5°C. The predominantly fast-twitch tibialis anterior (TA) and slow-twitch soleus (SOL) muscles were evaluated after 28 days of intervention. The [Ca2+]i elevating response to icing was enhanced by caffeine loading only in the SOL muscle, with the response present across a significantly higher temperature range than in the TA muscle under caffeine-loading conditions. In both the TA and SOL muscles, myofiber cross-sectional area (CSA) was decreased by chronic caffeine treatment (mean reductions of 10.5% and 20.4%, respectively). However, in the TA, but not the SOL, CSA was restored by icing (+15.4 ± 4.3% vs. noniced, P < 0.01). In the SOL, but not TA, icing + caffeine increased myofiber number (20.5 ± 6.7%, P < 0.05) and satellite cell density (2.5 ± 0.3-fold) in cross sections. These contrasting muscle responses to cooling and caffeine may reflect fiber-type-specific [Ca2+]i responses and/or differential responses to elevated [Ca2+]i.

Capillary-Mitochondrial Oxygen Transport in Muscle: Paradigm Shifts.

When exercising humans increase their oxygen uptake (V̇O2) 20-fold above rest the numbers are staggering: Each minute the O2 transport system - lungs, cardiovascular, active muscles - transports and utilizes 161 sextillion (10 21) O2 molecules. Leg extension exercise increases the quadriceps muscles' blood flow 100-times; transporting 17 sextillion O2 molecules per kilogram per minute from microcirculation (capillaries) to mitochondria powering their cellular energetics. Within these muscles, the capillary network constitutes a prodigious blood-tissue interface essential to exchange O2 and carbon dioxide requisite for muscle function. In disease, microcirculatory dysfunction underlies the pathophysiology of heart failure, diabetes, hypertension, pulmonary disease, sepsis, stroke and senile dementia. Effective therapeutic countermeasure design demands knowledge of microvascular/capillary function in health to recognize and combat pathological dysfunction. Dated concepts of skeletal muscle capillary (from the Latin capillus meaning 'hair') function prevail despite rigorous data-supported contemporary models; hindering progress in the field for future and current students, researchers and clinicians. Following closely the 100th anniversary of August Krogh's 1920 Nobel Prize for capillary function this Evidence Review presents an anatomical and physiological development of this dynamic field: Constructing a scientifically defensible platform for our current understanding of microcirculatory physiological function in supporting blood-mitochondrial O2 transport. New developments include: 1. Putative roles of red blood cell aquaporin and rhesus channels in determining tissue O2 diffusion. 2. Recent discoveries regarding intramyocyte O2 transport. 3. Developing a comprehensive capillary functional model for muscle O2 delivery-to-V̇O2 matching. 4. Use of kinetics analysis to discriminate control mechanisms from collateral or pathological phenomena.

Assessing cardiorespiratory fitness relative to sex improves surgical risk stratification.

BACKGROUND: To what extent sex-related differences in cardiorespiratory fitness (CRF) impact postoperative patient mortality and corresponding implications for surgical risk stratification remains to be established. METHODS: To examine this, we recruited 640 patients (366 males vs. 274 females) who underwent cardiopulmonary exercise testing prior to elective colorectal surgery. Patients were defined high risk if peak oxygen uptake was <14.3 mL kg-1  min-1 and ventilatory equivalent for carbon dioxide at 'anaerobic threshold' >34. Between-sex CRF and mortality was assessed, and sex-specific CRF thresholds predictive of mortality was calculated. RESULTS: Seventeen percent of deaths were attributed to sub-threshold CRF, which was higher than established risk factors for cardiovascular disease (CVD). The group (independent of sex) exhibited a 5-fold higher mortality (high vs. low risk patients hazard ratio = 4.80, 95% confidence interval 2.73-8.45, p < 0.001). Females exhibited 39% lower CRF (p < 0.001) with more classified high risk than males (36 vs. 23%, p = 0.001), yet mortality was not different (p = 0.544). Upon reformulation of sex-specific CRF thresholds, lower cut-offs for mortality were observed in females, and consequently, fewer (20%) were stratified with sub-threshold CRF compared to the original 36% (p < 0.001). CONCLUSIONS: Low CRF accounted for more deaths than traditional CVD risk factors, and when CRF was considered relative to sex, the disproportionate number of females stratified unfit was corrected. These findings support clinical consideration of 'sex-specific' CRF thresholds to better inform postoperative mortality and improve surgical risk stratification.

Effects of comorbid type II diabetes mellitus and heart failure on rat hindlimb and respiratory muscle blood flow during treadmill exercise.

In rats with type II diabetes mellitus (T2DM) compared with nondiabetic healthy controls, muscle blood flow (Q̇m) to primarily glycolytic hindlimb muscles and the diaphragm muscle are elevated during submaximal treadmill running consequent to lower skeletal muscle mass, a finding that held even when muscle mass was normalized to body mass. In rats with heart failure with reduced ejection fraction (HF-rEF) compared with healthy controls, hindlimb Q̇m was lower, whereas diaphragm Q̇m is elevated during submaximal treadmill running. Importantly, T2DM is the most common comorbidity present in patients with HF-rEF, but the effect of concurrent T2DM and HF-rEF on limb and respiratory Q̇m during exercise is unknown. We hypothesized that during treadmill running (20 m·min-1; 10% incline), hindlimb and diaphragm Q̇m would be higher in T2DM Goto-Kakizaki rats with HF-rEF (i.e., HF-rEF + T2DM) compared with nondiabetic Wistar rats with HF-rEF. Ejection fractions were not different between groups (HF-rEF: 30 ± 5; HF-rEF + T2DM: 28 ± 8%; P = 0.617), whereas blood glucose was higher in HF-rEF + T2DM (209 ± 150 mg/dL) compared with HF-rEF rats (113 ± 28 mg/dL; P = 0.040). Hindlimb muscle mass normalized to body mass was lower in rats with HF-rEF + T2DM (36.3 ± 1.6 mg/g) than in nondiabetic HF-rEF counterparts (40.3 ± 2.7 mg/g; P < 0.001). During exercise, Q̇m was elevated in rats with HF-rEF + T2DM compared with nondiabetic counterparts to the hindlimb (HF-rEF: 100 ± 28; HF-rEF + T2DM: 139 ± 23 mL·min-1·100 g-1; P < 0.001) and diaphragm (HF-rEF: 177 ± 66; HF-rEF + T2DM: 215 ± 93 mL·min-1·100g-1; P = 0.035). These data suggest that the pathophysiological consequences of T2DM on hindlimb and diaphragm Q̇m during treadmill running in the GK rat persist even in the presence of HF-rEF.NEW & NOTEWORTHY Herein, we demonstrate that rats comorbid with heart failure with reduced ejection fraction (HF-rEF) and type II diabetes mellitus (T2DM) have a higher hindlimb and respiratory muscle blood flow during submaximal treadmill running (20 m·min-1; 10% incline) compared with nondiabetic HF-rEF counterparts. These data may carry important clinical implications for roughly half of all patients with HF-rEF who present with T2DM.

A straightforward graphical/statistical approach to help substantiate cheating on multiple-choice examinations.

Academic dishonesty is prevalent in universities in the form of cheating on examinations, with the problem being much greater in classes that have a large number of students that require close seating arrangements for in-class exams. The scenario described below was experienced during an in-class exam that included the possibility of an Honor Code violation between two students that was observed independently by three different faculty proctors. Herein we detail an objective, statistical approach taken to maintain exam and academic integrity that is compelling and transparent to students and the University Honor Council. Using the established error-similarity analysis for multiple-choice exams, it was determined that the number of identical incorrect answers found on the exams of the two individuals in question was sufficiently greater than the number expected by chance (probability of P < 0.00001). The number of total identical incorrect answers found on the remaining exams (across 65 students, n = 89 comparisons) was plotted as function of the number of total incorrect answers found on these exams (incorrect answers ranged from 1 to 22) and clearly supported that there was an Honor Code violation between the two students in question. The techniques used herein established, beyond a reasonable doubt, that a form of cheating had occurred between these students. However, caution must be taken as further investigation is requisite to establish whether the Honor Code violation was unidirectional (one student copying off the other) or bidirectional (collusion between the two students) in nature.NEW & NOTEWORTHY Academic dishonesty is prevalent in universities, especially on examinations with a large number of students in close seating arrangements. Cheating on a multiple-choice exam was suspected by observations from proctors of the examination. Application of error-similarity analysis associated with identical incorrect answers demonstrated that the probability of cheating was confirmed (P < 0.00001) between two examinees. Further comparisons with the remaining exams provided graphic evidence that a violation of the University's Honor Code had occurred.