Running performance at high running velocities is impaired but V'O_{2max} and peripheral endothelial function are preserved in IL-6^{−/−} mice

It has been reported that IL-6 knockout mice (IL-6^{−/−}) possess lower endurance capacity than wild type mice (WT), however the underlying mechanism is poorly understood. The aim of the present work was to examine whether reduced endurance running capacity in IL-6^{−/−} mice is linked to impaired maximal oxygen uptake (V′O_{2max}), decreased glucose tolerance, endothelial dysfunction or other mechanisms. Maximal running velocity during incremental running to exhaustion was significantly lower in IL-6−/− mice than in WT mice (13.00±0.97 m.min^{-1} vs. 16.89±1.15 m.min^{-1}, P<0.02, respectively). Moreover, the time to exhaustion during running at 12 m.min^{-1} in IL-6^{−/−} mice was significantly shorter (P<0.05) than in WT mice. V′O_{2max} in IL-6^{−/−} (n = 20) amounting to 108.3±2.8 ml.kg^{-1}.min^{-1} was similar as in WT mice (n = 22) amounting to 113.0±1.8 ml.kg^{-1}.min^{-1}, (P = 0.16). No difference in maximal COX activity between the IL-6^{−/−} and WT mice in m. soleus and m. gastrocnemius was found. Moreover, no impairment of peripheral endothelial function or glucose tolerance was found in IL-6^{−/−} mice. Surprisingly, plasma lactate concentration during running at 8 m.min−1 as well at maximal running velocity in IL-6^{−/−} mice was significantly lower (P<0.01) than in WT mice. Interestingly, IL-6^{−/−} mice displayed important adaptive mechanisms including significantly lower oxygen cost of running at a given speed accompanied by lower expression of sarcoplasmic reticulum Ca^{2+}-ATPase and lower plasma lactate concentrations during running at submaximal and maximal running velocities. In conclusion, impaired endurance running capacity in IL-6^{−/−} mice could not be explained by reduced V′O_{2max}, endothelial dysfunction or impaired muscle oxidative capacity. Therefore, our results indicate that IL-6 cannot be regarded as a major regulator of exercise capacity but rather as a modulator of endurance performance. Furthermore, we identified important compensatory mechanism limiting reduced exercise performance in IL-6^{−/−} mice

Store-operated calcium entry contributes to abnormal Ca²⁺ signalling in dystrophic mdx mouse myoblasts

Sarcolemma damage and activation of various calcium channels are implicated in altered Ca2+ homeostasis in muscle fibres of both Duchenne muscular dystrophy (DMD) sufferers and in the mdx mouse model of DMD. Previously we have demonstrated that also in mdx myoblasts extracellular nucleotides trigger elevated cytoplasmic Ca2+ concentrations due to alterations of both ionotropic and metabotropic purinergic receptors. Here we extend these findings to show that the mdx mutation is associated with enhanced store-operated calcium entry (SOCE). Substantially increased rate of SOCE in mdx myoblasts in comparison to that in control cells correlated with significantly elevated STIM1 protein levels. These results reveal that mutation in the dystrophin-encoding Dmd gene may significantly impact cellular calcium response to metabotropic stimulation involving depletion of the intracellular calcium stores followed by activation of the store-operated calcium entry, as early as in undifferentiated myoblasts. These data are in agreement with the increasing number of reports showing that the dystrophic pathology resulting from dystrophin mutations may be developmentally regulated. Moreover, our results showing that aberrant responses to extracellular stimuli may contribute to DMD pathogenesis suggest that treatments inhibiting such responses might alter progression of this lethal disease

Control of PTH secretion by the TRPC1 ion channel

Familial Hypocalciuric Hypercalcemia (FHH) is a genetic condition associated with hypocalciuria, hypercalcemia and in some cases inappropriately high levels of circulating parathyroid hormone (PTH). FHH is associated with inactivating mutations in CaSR encoding the Ca2+ sensing receptor (CaSR), a G protein coupled receptor (GPCR) and GNA11 encoding G protein subunit alpha 11 (Gα11), implicating defective GPCR signaling as the root pathophysiology for FHH. However, the downstream mechanism by which CaSR activation inhibits PTH production/secretion is incompletely understood. Here, we show that mice lacking the transient receptor potential canonical channel 1 (TRPC1) develop chronic hypercalcemia, hypocalciuria, and elevated PTH levels mimicking human FHH. Ex vivo and in vitro studies reveal that TRPC1 serves a necessary and sufficient mediator to suppress PTH secretion from parathyroid glands (PTG) downstream of CaSR in response to high extracellular Ca2+ concentration. Gα11 physically interacts with both the N- and C-termini of TRPC1 and enhances CaSR-induced TRPC1 activity in transfected cells. These data identify TRPC1-mediated Ca2+ signaling as an essential component of the cellular apparatus controlling PTH secretion in the PTG downstream of CaSR

Skeletal muscle response to endurance training in IL-6^{-/-} mice

We examined effects of moderate-intensity endurance training on muscle COX/CS activities and V’O2max in control WT and IL-6−/− mice. Animals were exercised for 10 weeks on treadmill for 1 h, 5 days a week at velocity of 6 m·min−1 which was increased by 0.5 m·min−1 every 2 weeks up to 8 m·min−1 . Training triggered an increase of enzyme activities in soleus muscle of WT mice (COX: 480.3±8.9 U·g−1 in sedentary group vs. 773.3±62.6 U·g−1 in trained group, P&lt;0.05 and CS: 374.0±6.0 U·g−1 in sedentary group vs. 534.2±20.5 U·g−1 in trained group, P&lt;0.01, respectively) whereas no changes were observed in soleus of IL6−/− mice. Moreover, in mixed gastrocnemius muscle of trained IL-6−/− mice enzyme activities tended to be lower (COX: 410.7±48.4 U·g−1 for sedentary vs. 277.0±36.5 U·g−1 for trained group and CS: 343.8±24.6 U·g−1 for sedentary vs. 251.7±27.1 U·g−1 for trained group). No changes in V’O2max were observed in WT and IL-6−/− mice after training. Concluding, moderate-velocity endurance training-induced increase in COX and CS activities in muscles of WT mice only which suggests that IL-6 regulates training-induced skeletal muscle responses to exercise. Copyright © 2015, Georg Thieme Verlag KG. All rights reserved

Endurance time of WT mice and IL-6^−/− mice.

<p>WT age-matched littermates (–) and IL-6^−/− mice (–) were run on the treadmill at an inclination of 0° at either 10 (A) or 12 m^.min⁻¹ (B) for 2 hrs or 1 h, respectively, or until exhaustion as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088333#s2" target="_blank"><i>Materials and Methods</i></a>. The results were analyzed by the log-rank Mantel-Cox test in GraphPad Prism 5 and only P values lower than 0.05 were considered significant (n = 9–10).</p

The blood count and plasma lipid profile of WT control and IL-6^−/− mice.

<p>WBC (white blood cells: LYM% (% of lymphocytes), MON% (% of monocytes), GRA% (% of granulocytes)), RBC (red blood cells), HGB (hemoglobin), HCT (hematocrit), MCV ((red cell) mean corpuscular volume), MCH ((red cell) mean corpuscular hemoglobin), MCHC ((red cell) mean corpuscular hemoglobin concentration), RDW (red cell distribution width), PLT (platelets), MPV (mean platelets volume), LDL (low-density lipoprotein), HDL (high-density lipoprotein), TC (total cholesterol), TG (triglicerydes). Data are presented as means ± SEM.</p

Basal V′O₂, body weight, temperature, glucose tolerance and endothelial function of WT and IL-6^−/− mice.

<p>Basal oxygen consumption (A), body weight (B), resting body temperature (C), increase in body temperature after exercise (D), glucose tolerance of 10 month old mice (E), glucose tolerance of 12 month old mice (F), endothelium- dependent vasodilation of aortic rings (G), endothelium- independent vasodilation of aortic rings (H). (A) Basal oxygen consumption was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088333#s2" target="_blank"><i>Materials and Methods</i></a>. Glucose tolerance (E,F) of WT and IL-6^−/− mice at 10 and 12 month of age were compared. Temperature at rest was measured around 9 a.m. for all animals (C). Subsequently, the increase in body temperature after 1 hr run at 8 m^.min⁻¹ of WT and IL-6^−/− mice was compared (D). Peripheral endothelial function was assessed by the measurements of acetylcholine (Ach)- induced vasodilation (G). Subsequently, endothelium-independent SNP-induced vasodilation of the same aortic rings was record for comparison (H). Data are presented as the mean ± SEM. Statistical analysis was performed in Statistica 10 (A; ANCOVA, n = 10, P<0.0001) and in GraphPad Prism5 (two-sided T-test; n = 22–23 (B), n = 8–9 (C,D), n = 10–12 (E,F), n = 5 (G,H)).</p

Running performance during maximal incremental exercise test in WT and IL-6^−/− mice.

<p>Maximal velocity (v_max) (A), running intensity at 10 m^.min⁻¹ (B) and at 12 m^.min⁻¹ (C), maximal oxygen consumption (V′O_2max) (D), oxygen consumption during an incremental test with increasing speeds (E), oxygen consumption at maximal velocity (V′O₂ at v_max) (F) and oxygen consumption during 1-hour run at submaximal velocity of 6 m^.min⁻¹ (G). For determination of v_max (A), V′O_2max (D) and V′O₂ at v_max (F), WT control and IL-6^−/− mice were run at an inclination of 0° with the increasing speed and their oxygen consumption (V′O₂) was registered (E) whereas for measurement of V′O₂ during sub-maximal exercise, mice were run at velocity of 6 m^.min⁻¹ for 1 h (G). Data are presented as the mean ± SEM. The symbols * denote values significantly different: *(P<0.05), **(P<0.01), ***(P<0.001). Statistical analysis was performed in Statistica 10 (G; ANCOVA, n = 6, P<0.0001) or GraphPad Prism5 (two-sided T-test; n = 22-11).</p

Post-exercise plasma lactate and COX, CS activities in skeletal muscles in WT and IL-6^−/− mice.

<p>COX and CS activities in <i>soleus</i> (A, C) and <i>gastrocnemius</i> (B, D) skeletal muscles were measured in lysates from non-exercising WT control mice and IL-6^−/− mice. Post-exercise plasma lactate concentration (E) was assessed both in non-exercising mice (WT at rest and IL-6^−/− at rest) as well as in animals subjected to single bout of exercise (1-hour run at 8 m^.min⁻¹). (F) Plasma lactate concentration at V′O_2max during maximal incremental running test until exhaustion in WT mice and IL-6^−/− mice. Data are presented as the mean ± SEM and symbols * denote values significantly different: **(P<0.01), ***(P<0.001). Statistical analysis was performed in GraphPad Prism5 (two-sided T-test, n = 7–11).</p