Contribution of stretch to the change of activation properties of muscle fibers in the diaphragm at the transition from fetal to neonatal life

The transition from fetal to postnatal life involves clearance of liquid from the lung and airways, and rapid formation of a functional residual capacity. Despite the importance of the diaphragm in this process, the impact of birth on the mechanical and functional activity of its muscle fibers is not known. This study determined the contractile characteristics of individual “skinned” diaphragm fibers from 70 days (0.47) gestation to after birth in sheep. Based on differential sensitivity to the divalent ions calcium (Ca²⁺) and strontium (Sr²⁺), all fibers in the fetal diaphragm were classified as “fast,” whereas fibers from the adult sheep diaphragm exhibited a “hybrid” phenotype where both “fast” and “slow” characteristics were present within each single fiber. Transition to the hybrid phenotype occurred at birth, was evident after only 40 min of spontaneous breathing, and could be induced by simple mechanical stretch of diaphragm fibers from near-term fetuses (∼147 days gestation). Both physical stretch of isolated fibers, and mechanical ventilation of the fetal diaphragm in situ, significantly increased sensitivity to Ca²⁺ and Sr²⁺, maximum force generating capacity, and decreased passive tension in near-term and preterm fetuses; however, only fibers from near-term fetuses showed a complete transition to a “hybrid” activation profile. These findings suggest that stretch associated with the transition from a liquid to air-filled lung at birth induces physical changes of proteins determining the activation and elastic properties of the diaphragm. These changes may allow the diaphragm to meet the increased mechanical demands of breathing immediately after birth.

Differential effects of arginine, glutamate and phosphoarginine on Ca2+-activation properties of muscle fibres from crayfish and rat

The effects of two amino acids, arginine which has a positively charged side-chain and glutamate which has a negatively charged side-chain on the Ca²⁺-activation properties of the contractile apparatus were examined in four structurally and functionally different types of skeletal muscle; long- and short-sarcomere fibres from the claw muscle of the yabby (a freshwater decapod crustacean), and fast- and slow-twitch fibres from limb muscles of the rat. Single skinned fibres were activated in carefully balanced solutions of different pCa (-log₁₀[Ca²⁺]) that either contained the test solute (“test”) or not (“control”). The effect of phosphoarginine, a phosphagen that bears a nett negative charge, was also compared to the effects of arginine. Results show that (i) arginine (33-36 mmol l^-1) significantly shifted the force–pCa curve by 0.08–0.13 pCa units in the direction of increased sensitivity to Ca²⁺-activated contraction in all fibre types; (ii) phosphoarginine (9–10 mmol l^-1) induced a significant shift of the force–pCa curve by 0.18–0.24 pCa units in the direction of increased sensitivity to Ca²⁺ in mammalian fast- and slow-twitch fibres, but had no significant effects on the force–pCa relation in either long- or short-sarcomere crustacean fibres; (iii) glutamate (36–40 mmol l^-1), like arginine affected the force–pCa relation of all fibre types investigated, but in the opposite direction, causing a significant decrease in the sensitivity to Ca²⁺-activated contraction by 0.08–0.19 pCa units; (iv) arginine, phosphoarginine and glutamate had little or no effect on the maximum Ca²⁺-activated force of crustacean and mammalian fibres. The results suggest that the opposing effects of glutamate and arginine are not related to simply their charge structure, but must involve complex interactions between these molecules, Ca²⁺ and the regulatory and other myofibrillar proteins.

Slow skeletal troponin I gene transfer, expression, and myofilament incorporation enhances adult cardiac myocyte contractile function

The functional significance of the developmental transition from slow skeletal troponin I (ssTnI) to cardiac TnI (cTnI) isoform expression in cardiac myocytes remains unclear. We show here the effects of adenovirus-mediated ssTnI gene transfer on myofilament structure and function in adult cardiac myocytes in primary culture. Gene transfer resulted in the rapid, uniform, and nearly complete replacement of endogenous cTnI with the ssTnI isoform with no detected changes in sarcomeric ultrastructure, or in the isoforms and stoichiometry of other myofilament proteins compared with control myocytes over 7 days in primary culture. In functional studies on permeabilized single cardiac myocytes, the threshold for Ca2+-activated contraction was significantly lowered in adult cardiac myocytes expressing ssTnI relative to control values. The tension–Ca2+ relationship was unchanged from controls in primary cultures of cardiac myocytes treated with adenovirus containing the adult cardiac troponin T (TnT) or cTnI cDNAs. These results indicate that changes in Ca2+ activation of tension in ssTnI-expressing cardiac myocytes were isoform-specific, and not due to nonspecific functional changes resulting from overexpression of a myofilament protein. Further, Ca2+-activated tension development was enhanced in cardiac myocytes expressing ssTnI compared with control values under conditions mimicking the acidosis found during myocardial ischemia. These results show that ssTnI enhances contractile sensitivity to Ca2+ activation under physiological and acidic pH conditions in adult rat cardiac myocytes, and demonstrate the utility of adenovirus vectors for rapid and efficient genetic modification of the cardiac myofilament for structure/function studies in cardiac myocytes.

Activated calcineurin ameliorates contraction-induced injury to skeletal muscles of mdx dystrophic mice

Utrophin expression is regulated by calcineurin and up-regulating utrophin can decrease the susceptibility of dystrophic skeletal muscle to contraction-induced injury. We overexpressed the constitutively active calcineurin-A α in skeletal muscle of mdx dystrophic mice (mdx CnA*) and examined the tibialis anterior muscle to determine whether the presence of activated calcineurin promotes resistance to muscle damage after lengthening contractions. Two stretches (10 s apart) of 40% strain relative to muscle fibre length were initiated from the plateau of a maximal isometric tetanic contraction. Muscle damage was assessed 1, 5 and 15 min later by the deficit in maximum isometric force and by quantifying the proportion of muscle fibres staining positive for intracytoplasmic albumin. The force deficit at all time points after the lengthening contractions was approximately 80% in mdx muscles and 30% in mdxCnA* muscles. The proportion of albumin-positive fibres was significantly less in control and injured muscles from mdxCnA* mice than from mdx mice. Compared with mdx mice, mean fibre cross-sectional area was 50% less in muscles from mdxCnA* mice. Furthermore, muscles frommdxCnA* mice exhibited a higher proportion of fibres expressing the slow(er) myosin heavy chain (MyHC) I and IIa isoforms, prolonged contraction and relaxation times, lower absolute and normalized maximum forces, and a clear leftward shift of the frequency–force relationship with greater force production at lower stimulation frequencies. These are structural and functional markers of a slower muscle phenotype. Taken together, our findings show that muscles from mdxCnA* mice have a smaller mean fibre cross-sectional area, a greater sarcolemmal to cytoplasmic volume ratio, and an increase in utrophin expression, promoting an attenuated susceptibility to contraction-induced injury. We conclude that increased calcineurin activity may confer functional benefits to dystrophic skeletal muscles.

Deletion of TRPC4 and TRPC6 in Mice Impairs Smooth Muscle Contraction and Intestinal Motility In Vivo.

BACKGROUND & AIMS: Downstream effects of muscarinic receptor stimulation in intestinal smooth muscle include contraction and intestinal transit. We thought to determine whether classic transient receptor potential (TRPC) channels integrate the intracellular signaling cascades evoked by the stimulated receptors and thereby contribute to the control of the membrane potential, Ca-influx, and cell responses. METHODS: We created trpc4-, trpc6-, and trpc4/trpc6-gene-deficient mice and analyzed them for intestinal smooth muscle function in vitro and in vivo. RESULTS: In intestinal smooth muscle cells TRPC4 forms a 55 pS cation channel and underlies more than 80% of the muscarinic receptor-induced cation current (mI(CAT)). The residual mI(CAT) depends on the expression of TRPC6, indicating that TRPC6 and TRPC4 determine mI(CAT) channel activity independent of other channel subunits. In TRPC4-deficient ileal myocytes the carbachol-induced membrane depolarizations are diminished greatly and the atropine-sensitive contraction elicited by acetylcholine release from excitatory motor neurons is reduced greatly. Additional deletion of TRPC6 aggravates these effects. Intestinal transit is slowed down in mice lacking TRPC4 and TRPC6. CONCLUSIONS: In intestinal smooth muscle cells TRPC4 and TRPC6 channels are gated by muscarinic receptors and are responsible for mI(CAT). They couple muscarinic receptors to depolarization of intestinal smooth muscle cells and voltage-activated Ca(2+)-influx and contraction, and thereby accelerate small intestinal motility in vivo.

Expression and function of proteinase-activated receptor 2 in human bronchial smooth muscle

Trypsin and mast cell tryptase cleave proteinase-activated receptor 2 (PAR2) to induce alterations in contraction of airway smooth muscle that have been implicated in asthma in experimental animals. Although tryptase inhibitors are under development for treatment of asthma, little is known about the localization and function of PAR2 in human airways. We detected PAR2 expression in primary cultures of human airway smooth muscle cells using reverse transcriptase/polymerase chain reaction (RT-PCR) and immunofluorescence. The PAR2 agonists trypsin, tryptase, and an activating peptide (SLIGKV-NH2) stimulated calcium mobilization in these cells. PAR2 agonists strongly desensitized responses to a second challenge of trypsin and SLIGKV-NH2, but not to thrombin, indicating that they activate a receptor distinct from the thrombin receptors. Immunoreactive PAR2 was detected in smooth muscle, epithelium, glands, and endothelium of human bronchi. Trypsin, SLIGKV-NH2, and tryptase stimulated contraction of isolated human bronchi. Contraction was increased by removal of the epithelium and diminished by indomethacin. Thus, PAR2 is expressed by human bronchial smooth muscle where its activation mobilizes intracellular Ca2+ and induces contraction. These results are consistent with the hypothesis that PAR2 agonists, including tryptase, induce bronchoconstriction of human airway by stimulating smooth muscle contraction. PAR2 antagonists may be useful drugs to prevent bronchoconstriction.

Presence and bronchomotor activity of protease-activated receptor-2 in guinea pig airways

The protease activated receptor-2 (PAR-2) belongs to a family of G-protein-coupled receptors that are activated by proteolysis. Trypsin cleaves PAR-2, exposing an N-terminal tethered ligand (SLIGRL) that activates the receptor. Messenger RNA (mRNA) for PAR-2 was found in guinea pig airway tissue by reverse transcription-polymerase chain reaction, and PAR-2 was found by immunohistochemistry in airway epithelial and smooth-muscle cells. In anesthetized guinea pigs, trypsin and SLIGRL-NH(2) (given intratracheally or intravenously) caused a bronchoconstriction that was inhibited by the combination of tachykinin-NK(1) and -NK(2) receptor antagonists and was potentiated by inhibition of nitric oxide synthase (NOS). Trypsin and SLIGRL-NH(2) relaxed isolated trachea and main bronchi, and contracted intrapulmonary bronchi. Relaxation of main bronchi was abolished or reversed to contraction by removal of epithelium, administration of indomethacin, and NOS inhibition. PAR-1, PAR-3, and PAR-4 were not involved in the bronchomotor action of either trypsin or SLIGRL-NH(2), because ligands of these receptors were inactive either in vitro or in vivo, and because thrombin (a PAR-1 and PAR-3 agonist) did not show cross-desensitization with PAR-2 agonists in vivo. Thus, we have localized PAR-2 to the guinea-pig airways, and have shown that activation of PAR-2 causes multiple motor effects in these airways, including in vivo bronchoconstriction, which is in part mediated by a neural mechanism.

Extracellular Signal-Regulated Kinase 1/2 Activation, via Downregulation of Mitogen-Activated Protein Kinase Phosphatase 1, Mediates Sex Differences in Desoxycorticosterone Acetate-Salt Hypertension Vascular Reactivity

Extracellular signal-regulated kinase (ERK) 1/2 has been reported to play a role in vascular dysfunction associated with mineralocorticoid hypertension. We hypothesized that, compared with female rats, an upregulation of ERK1/2 signaling in the vasculature of male rats contributes to augmented contractile responses in mineralocorticoid hypertension. Uninephrectomized male and female Sprague-Dawley rats received desoxycorticosterone acetate (DOCA) pellets (200 mg per animal) and saline to drink for 3 weeks. Control uninephrectomized rats received tap water to drink. Blood pressure, measured by telemetry, was significantly higher in male DOCA rats (191 +/- 3 mm Hg) compared with female DOCA rats (172 +/- 7 mm Hg; n=5). DOCA treatment resulted in augmented contractile responses to phenylephrine in aorta (22 +/- 3 mN; n=6) and small mesenteric arteries (13 +/- 2 mN; n=6) from male DOCA rats versus uninephrectomized male rats (16 +/- 3 and 10 +/- 2 mN, respectively; P<0.05) and female DOCA rats (15 +/- 1 and 11 +/- 1 mN, respectively). ERK1/2 inhibition with PD-98059 (10 mu mol/L) abrogated increased contraction to phenylephrine in aorta (14 +/- 2 mN) and small mesenteric arteries (10 +/- 2 mN) from male DOCA rats, without any effects in arteries from male uninephrectomized or female animals. Compared with the other groups, phosphorylated ERK1/2 levels were increased in the aorta from male DOCA rats, whereas mitogen-activated protein kinase phosphatase 1 expression was decreased. Interleukin-10 plasma levels, which positively regulate mitogen-activated protein kinase phosphatase 1 activity, were reduced in male DOCA-salt rats. We speculate that augmented vascular reactivity in male hypertensive rats is mediated via activation of the ERK1/2 pathway. In addition, mitogen-activated protein kinase phosphatase 1 and interleukin 10 play regulatory roles in this process. (Hypertension. 2010; 55: 172-179.)

5`-aminoimidazole-4-carboxyamide-ribonucleoside- activated glucose transport is not prevented by nitric oxide synthase inhibition in rat isolated skeletal muscle

1. The nucleoside intermediate 5'-aminoimidazole-4-carboxyamide-ribonucleoside (AICAR) activates skeletal muscle AMP-activated protein kinase (AMPK) and increases glucose uptake. The AMPK phosphorylates neuronal nitric oxide synthase (nNOS)µ in skeletal muscle fibres. There is evidence that both AMPK and nNOSµ may be involved in the regulation of contraction-stimulated glucose uptake.
2. We examined whether both AICAR- and contraction-stimulated glucose uptake were mediated by NOS in rat skeletal muscle.
3. Rat isolated epitrochlearis muscles were subjected in vitro to electrically stimulated contractions for 10 min and/or incubated in the presence or absence of AICAR (2 mmol/L) or the NOS inhibitor NG-monomethyl-l-arginine (l-NMMA; 100 µmol/L).
4. Muscle contraction significantly (P < 0.05) altered the metabolic profile of the muscle. In contrast, AICAR and l-NMMA had no effect on the metabolic profile of the muscle, except that AICAR increased muscle 5'-aminoimidazole-4-carboxyamide-ribonucleotide (ZMP) and AICAR content. Nitric oxide synthase inhibition caused a small but significant (P < 0.05) reduction in basal 3-O-methylglucose transport, which was observed in all treatments. 5'-Aminoimidazole-4-carboxyamide-ribonucleoside significantly increased (P < 0.05) glucose transport above basal, with NOS inhibition decreasing this slightly (increased by 209% above basal compared with 184% above basal with NOS inhibition). Contraction significantly increased glucose transport above basal, with NOS inhibition substantially reducing this (107% increase vs 31% increase). 5'-Aminoimidazole-4-carboxyamide-ribonucleoside plus contraction in combination were not additive on glucose transport.
5. These results suggest that NO plays a role in basal glucose uptake and may regulate contraction-stimulated glucose uptake. However, NOS/nitric oxide do not appear to be signalling intermediates in AICAR-stimulated skeletal muscle glucose uptake.

Altering dietary nutrient intake that reduces glycogen content leads to phosphorylation of nuclear p38 MAP kinase in human skeletal muscle: association with IL-6 gene transcription during contraction

To determine the effect of glycogen availability and contraction on intracellular signaling and IL-6 gene transcription, eight males performed 60 min of exercise on two occasions: either with prior ingestion of a normal (Con) or low carbohydrate (LCHO) diet that reduced pre-exercise muscle glycogen content. Muscle biopsies were obtained and analyzed for IL-6 mRNA. In addition, nuclear proteins were isolated from the samples and analyzed for the mitogen- activated protein kinases (MAPK) c-jun amino-terminal kinase (JNK) 1 and 2 and p38 MAPK. Nuclear fractions were also analyzed for the phosphorylated forms of JNK (p-JNK) and p38 MAPK (p-p38 MAPK) and the abundance of the nuclear transcription factors nuclear factor of activated T cells (NFAT) and nuclear factor kappa-β (NF-κβ). No differences were observed in the protein abundance of total JNK 1/2, p38 MAPK, NFAT, or NF-κβ before exercise, but the nuclear abundance of p-p38 MAPK was higher (P<0.05) in LCHO. Contraction resulted in an increase (P<0.05) in nuclear p-JNK 1/2, but there were no differences when comparing CON with LCHO. The fold increase in IL-6 mRNA with contraction was potentiated (P<0.05) in LCHO. A correlation between pre-exercise nuclear phosphorylated p38 MAPK and contraction-induced fold increase in IL-6 mRNA was performed, revealing a highly significant correlation (r=0.96; P<0.01). We next incubated L6 myotubes in ionomycin (a compound known to induce IL-6 mRNA) with or without the pyridinylimidazole p38 MAPK inhibitor SB203580. Treatments did not affect total nuclear p38 MAPK, but ionomycin increased (P<0.05) both nuclear p-p38 MAPK and IL-6 mRNA. The addition of SB203580 to ionomycin decreased (P<0.05) nuclear p-p38 MAPK and totally abolished (P<0.05) the ionomycin- induced increase in IL-6 mRNA. These data suggest that reduced carbohydrate intake that results in low intramuscular glycogen leads to phosphorylation of p38 MAPK at the nucleus. Furthermore, phosphorylation of p38 MAPK in the nucleus appears to be an upstream target for IL-6, providing new insights into the regulation of IL-6 gene transcription.

Potential role of nitric oxide in contraction-stimulated glucose uptake and mitochondrial biogenesis in skeletal muscle

• 1. The present review discusses the potential role of nitric oxide (NO) in the: (i) regulation of skeletal muscle glucose uptake during exercise; and (ii) activation of mitochondrial biogenesis after exercise.
• 2. We have shown in humans that local infusion of an NO synthase inhibitor during exercise attenuates increases in skeletal muscle glucose uptake without affecting blood flow. Recent studies from our laboratory in rodents support these findings in humans, although rodent studies from other laboratories have yielded conflicting results.
• 3. There is clear evidence that NO increases mitochondrial biogenesis in non-contracting cells and that NO influences basal skeletal muscle mitochondrial biogenesis. However, there have been few studies examining the potential role of NO in the activation of mitochondrial biogenesis following an acute bout of exercise or in response to exercise training. Early indications are that NO is not involved in regulating the increase in mitochondrial biogenesis that occurs in response to exercise.
• 4. Exercise is considered the best prevention and treatment option for diabetes, but unfortunately many people with diabetes do not or cannot exercise regularly. Alternative therapies are therefore critical to effectively manage diabetes. If skeletal muscle NO is found to play an important role in regulating glucose uptake and/or mitochondrial biogenesis, pharmaceutical agents designed to mimic these effects of exercise may improve glycaemic control.

Local nitric oxide synthase inhibition reduces skeletal muscle glucose uptake but not capillary blood flow during in situ muscle contraction in rats

OBJECTIVE: We have previously shown in humans that local infusion of a nitric oxide synthase (NOS) inhibitor into the femoral artery attenuates the increase in leg glucose uptake during exercise without influencing total leg blood flow. However, rodent studies examining the effect of NOS inhibition on contraction-stimulated skeletal muscle glucose uptake have yielded contradictory results. This study examined the effect of local infusion of an NOS inhibitor on skeletal muscle glucose uptake (2-deoxyglucose) and capillary blood flow (contrast-enhanced ultrasound) during in situ contractions in rats.

RESEARCH DESIGN AND METHODS: Male hooded Wistar rats were anesthetized and one hindleg electrically stimulated to contract (2 Hz, 0.1 ms) for 30 min while the other leg rested. After 10 min, the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) (arterial concentration of 5 µmol/l) or saline was infused into the epigastric artery of the contracting leg.

RESULTS: Local NOS inhibition had no effect on blood pressure, heart rate, or muscle contraction force. Contractions increased (P < 0.05) skeletal muscle NOS activity, and this was prevented by L-NAME infusion. NOS inhibition caused a modest significant (P < 0.05) attenuation of the increase in femoral blood flow during contractions, but importantly there was no effect on capillary recruitment. NOS inhibition attenuated (P < 0.05) the increase in contraction-stimulated skeletal muscle glucose uptake by ~35%, without affecting AMP-activated protein kinase (AMPK) activation.

CONCLUSIONS: NOS inhibition attenuated increases in skeletal muscle glucose uptake during contraction without influencing capillary recruitment, suggesting that NO is critical for part of the normal increase in skeletal muscle fiber glucose uptake during contraction.

Differential phosphorylation, desensitization, and internalization of α1A-adrenoceptors activated by norepinephrine and oxymetazoline

Loss of response on repetitive drug exposure (i.e., tachyphylaxis) is a particular problem for the vasoconstrictor effects of medications containing oxymetazoline (OXY), an α1-adrenoceptor (AR) agonist of the imidazoline class. One cause of tachyphylaxis is receptor desensitization, usually accompanied by phosphorylation and internalization. It is well established that a1A-ARs are less phosphorylated, desensitized, and internalized on exposure to the phenethylamines norepinephrine (NE), epinephrine, or phenylephrine (PE) than are the a1B and a1D subtypes. However, here we show in human embryonic kidney-293 cells that the low-efficacy agonist OXY induces G protein-coupled receptor kinase 2-dependent a1A-AR phosphorylation, followed by rapid desensitization and internalization (∼40% internalization after 5 minutes of stimulation), whereas phosphorylation of α1A-ARs exposed to NE depends to a large extent on protein kinase C activity and is not followed by desensitization, and the receptors undergo delayed internalization (∼35% after 60 minutes of stimulation). Native α1A-ARs from rat tail artery and vas deferens are also desensitized by OXY, but not by NE or PE, indicating that thisproperty of OXY is not limited to recombinant receptors expressed in cell systems. The results of the present study are clearly indicative of agonist-directed a1A-AR regulation. OXY shows functional selectivity relative to NE and PE at a1A-ARs, leading to significant receptor desensitization and internalization, which is important in view of the therapeutic vasoconstrictor effects of this drug and the varied biologic process regulated by α1A-ARs. Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics.

Degree of conversion of nanofilled and microhybrid composite resins photo-activated by different generations of LEDs

Objective: This study aimed at evaluating the degree of conversion (DC) of four composite resins, being one nanofilled and 3 microhybrid resins, photo-activated with second- and third-generation light-emitting diodes (LEDs). Material and methods: Filtek (TM) Z350 nanofilled composite resins and Amelogen (R) Plus, Vit-l-escence (TM) and Opallis microhybrid resins were photo-activated with two second-generation LEDs (Radii-cal and Elipar Free Light (TM) 2) and one third-generation LED (Ultra-Lume LED 5) by continuous light mode, and a quartz halogen-tungsten bulb (QHT, control). After 24 h of storage, the samples were pulverized into fine powder and 5 mg of each material were mixed with 100 mg of potassium bromide (KBr). After homogenization, they were pressed, which resulted in a pellet that was evaluated using an infrared spectromer (Nexus 470, Thermo Nicolet) equipped with TGS detector using diffuse reflectance (32 scans, resolution of 4 cm(-1)) coupled to a computer. The percentage of unreacted carbon-carbon double bonds (% C=C) was determined from the ratio of absorbance intensities of aliphatic C=C (peak at 1637 cm-1) against internal standard before and after curing of the specimen: aromatic C-C (peak at 1610 cm-1). Results: The ANOVA showed a significant effect on the interaction between the light-curing units (LCUs) and the composite resins (p<0.001). The Tukey's test showed that the nanofilled resin (Filtek (TM) Z350) and Opallis when photo-activated by the halogen lamp (QTH) had the lowest DC compared with the other microhybrid composite resins. The DC of the nanofilled resin (Filtek (TM) Z350) was also lower using LEDs. The highest degrees of conversion were obtained using the third-generation LED and one of second-generation LEDs (Elipar Free Light (TM) 2). Conclusions: The nanofilled resin showed the lowest DC, and the Vit-l-escence (TM) microhybrid composite resin showed the highest DC. Among the LCUs, it was not possible to establish an order, even though the second-generation LED Radii-cal provided the lowest DC.