Muscle Of Wild-type Mice Research Articles

Insulin-like growth factor I slows the rate of denervation induced skeletal muscle atrophy

Loss of the nerve supply to skeletal muscle results in a relentless loss of muscle mass (atrophy) over time. The ability of insulin-like growth factor-1 to reduce atrophy resulting from denervation was examined after transection of the sciatic nerve in transgenic MLC/mIGF-1 mice that over-express mIGF-1 specifically in differentiated myofibres. The cross sectional area (CSA) of all types of myofibres and specifically type IIB myofibres was measured in tibialis anterior muscles from transgenic and wild-type mice at 28 days after denervation. There was a marked myofibre atrophy (∼60%) in the muscles of wild-type mice over this time with increased numbers of myofibres with small CSA. In the muscles of MLC/mIGF-1 mice, over-expression of mIGF-1 reduced the rate of denervation induced myofibre atrophy by ∼30% and preserved myofibres with larger CSA, compared to wild-type muscles. It is proposed that the protective effect of mIGF-1 on denervated myofibres might be due to reduced protein breakdown.

Alpha-enolase plasminogen receptor in myogenesis

Plasmin is a potent extracellular protease specialized in the degradation of fibrin (fibrinolysis). Active plasmin is generated by proteolytic activation of the zymogen plasminogen (Plg) by urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). Alpha-enolase, although traditionally considered a glycolytic enzyme, constitutes a receptor for plasminogen on several cell types, serving to localize and promote plasminogen activation pericellularly. Localization of plasmin activity on the cell surface plays a critical role in fibrinolysis and in physiopathological processes involving extracellular matrix remodelling. Previous studies have unambiguously demonstrated that uPA-dependent plasmin generation is necessary for myogenesis in vitro and for muscle regeneration in vivo. However, the implication of alpha-enolase plasminogen receptor in myogenesis had never been investigated. This review focuses on the recently reported expression and function of alpha-enolase plasminogen receptor during myogenesis. Skeletal myoblasts express alpha-enolase plasminogen receptor, being its expression greatly induced during the differentiation process in vitro. MAb 11G1, a monoclonal antibody against anti-alpha-enolase plasminogen receptor, that inhibits plasmin generation, was able to fully abrogate myoblast fusion and differentiation. Moreover, both plasmin activity and alpha-enolase plasminogen receptor expression were significantly augmented in injury-induced regenerating muscle of wild type mice and in the dystrophic muscle of mdx mice, an animal model of Duchenne muscular dystrophy (DMD). Altogether, these results indicate that the plasminogen activation (PA) system is an important component of skeletal myogenesis in vitro and in vivo. In particular, the expression of alpha-enolase plasminogen receptor may serve to concentrate and enhance plasmin generation on the cell surface of migratory myoblasts contributing to efficient muscle repair.

Cyclin-dependent kinase 5 phosphorylates signal transducer and activator of transcription 3 and regulates its transcriptional activity.

The activity of cyclin-dependent kinase 5 (Cdk5) depends on the association with one of its activators, p35 and p39, which are prominently expressed in the nervous system. Studies on the repertoire of protein substrates for Cdk5 have implicated the involvement of Cdk5 in neuronal migration and synaptic plasticity. Our recent analysis of the sequence of signal transducer and activator of transcription (STAT)3, a key transcription factor, reveals the presence of potential Cdk5 phosphorylation site. We report here that the Cdk5/p35 complex associates with STAT3 and phosphorylates STAT3 on the Ser-727 residue in vitro and in vivo. Intriguingly, whereas the Ser phosphorylation of STAT3 can be detected in embryonic and postnatal brain and muscle of wild-type mice, it is essentially absent from those of Cdk5-deficient embryos. In addition, treatment of cultured myotubes with neuregulin enhances the Ser phosphorylation of STAT3 and transcription of STAT3 target genes, such as c-fos and junB, in a Cdk5-dependent manner. Both the DNA-binding activity of STAT3 and the transcription of specific target genes, such as fibronectin, are reduced in Cdk5-deficient muscle. Taken together, these results reveal a physiological role of Cdk5 in regulating STAT3 phosphorylation and modulating its transcriptional activity.

Selenoprotein-Deficient Transgenic Mice Exhibit Enhanced Exercise-Induced Muscle Growth

Dietary intake of selenium has been implicated in a wide range of health issues, including aging, heart disease and cancer. Selenium deficiency, which can reduce selenoprotein levels, has been associated with several striated muscle pathologies. To investigate the role of selenoproteins in skeletal muscle biology, we used a transgenic mouse (referred to as i6A-) that has reduced levels of selenoproteins due to the introduction and expression of a dominantly acting mutant form of selenocysteine transfer RNA (tRNA[Ser]Sec). As a consequence, each organ contains reduced levels of most selenoproteins, yet these mice are normal with regard to fertility, overall health, behavior and blood chemistries. In the present study, although skeletal muscles from i6A- mice were phenotypically indistinguishable from those of wild-type mice, plantaris muscles were approximately 50% heavier after synergist ablation, a model of exercise overload. Like muscle in wild-type mice, the enhanced growth in the i6A- mice was completely blocked by inhibition of the mammalian target of rapamycin (mTOR) pathway. Muscles of transgenic mice exhibited increased site-specific phosphorylation on both Akt and p70 ribosomal S6 kinase (p70S6k) (P < 0.05) before ablation, perhaps accounting for the enhanced response to synergist ablation. Thus, a single genetic alteration resulted in enhanced skeletal muscle adaptation after exercise, and this is likely through subtle changes in the resting phosphorylation state of growth-related kinases.

Expression of mouse agrin in normal, denervated and dystrophic muscle

Agrin is a heparan sulfate proteoglycan that is required for the development of postsynaptic specializations at the neuromuscular junction. An alternatively spliced isoform of agrin that lacks this activity is found in basement membranes of several tissues including embryonic muscle. Overexpression of a miniaturized form of this agrin isoform ameliorates the severe muscle dystrophy of laminin α2-deficient mice, a mouse model for merosin-deficient congenital muscle dystrophy. Several lines of evidence indicate that this amelioration is based on the high-affinity binding of the mini-agrin to the laminins and to α-dystroglycan. Here, we used antibodies raised against mouse agrin to evaluate protein expression in adult muscle of normal and dystrophic mice. We find that expression of agrin in non-synaptic region varies greatly between different muscles in wild-type mice and that its levels are altered in dystrophic muscle.

Regeneration of Transgenic Skeletal Muscles with Altered Timing of Expression of the Basic Helix-Loop-Helix Muscle Regulatory Factor MRF4

In regenerating muscle cells, muscle regulatory factor (MRF) 4 is normally the last of the four MRFs to be expressed. To analyze how the timing of MRF4 expression affects muscle regeneration, we compared regeneration after local freeze injury of muscles from wild-type mice with muscles from transgenic mice in which MRF4 expression was under control of an approximately 1.6-kb fragment of the myogenin promoter. Three days after injury, masseter and tibialis anterior (TA) muscles in wild-type mice expressed little or no MRF4 mRNA; whereas these muscles in transgenic mice expressed abundant MRF4 mRNA from both the transgene and the endogenous gene. Thus, MRF4 up-regulation was accelerated in transgenic compared to wild-type regenerating muscles, and expression of the transgene appeared to activate, perhaps indirectly, expression of the endogenous MRF4 gene. At 11 days after injury, regeneration, as measured by cross-sectional area and density of regenerated fibers, was significantly impaired in transgenic TA compared to wild-type TA, whereas at 19 days after injury both transgenic and TA muscle fibers had fully recovered to preinjury values. Regeneration of masseter muscles, which normally regenerate much less completely than TA muscles, was unaffected by the transgene. Thus, the timing of MRF4 up-regulation, as well as additional muscle-specific factors, can determine the progress of muscle regeneration.

Knockout of PKC alpha enhances insulin signaling through PI3K.

Insulin stimulates glucose transport and certain other metabolic processes by activating atypical PKC isoforms (lambda, zeta, iota) and protein kinase B (PKB) through increases in D3-polyphosphoinositides derived from the action of PI3K. The role of diacylglycerol-sensitive PKC isoforms is less clear as they have been suggested to be both activated by insulin and yet inhibit insulin signaling to PI3K. Presently, we found that insulin signaling to insulin receptor substrate 1-dependent PI3K, PKB, and PKC lambda, and downstream processes, glucose transport and activation of ERK, were enhanced in skeletal muscles and adipocytes of mice in which the ubiquitous conventional diacylglycerol-sensitive PKC isoform, PKC alpha, was knocked out by homologous recombination. On the other hand, insulin provoked wortmannin-insensitive increases in immunoprecipitable PKC alpha activity in adipocytes and skeletal muscles of wild-type mice and rats. We conclude that 1) PKC alpha is not required for insulin-stimulated glucose transport, and 2) PKC alpha is activated by insulin at least partly independently of PI3K, and largely serves as a physiological feedback inhibitor of insulin signaling to the insulin receptor substrate 1/PI3K/PKB/PKC lambda/zeta/iota complex and dependent metabolic processes.

Prions in skeletal muscle.

Considerable evidence argues that consumption of beef products from cattle infected with bovine spongiform encephalopathy (BSE) prions causes new variant Creutzfeldt-Jakob disease. In an effort to prevent new variant Creutzfeldt-Jakob disease, certain "specified offals," including neural and lymphatic tissues, thought to contain high titers of prions have been excluded from foods destined for human consumption [Phillips, N. A., Bridgeman, J. & Ferguson-Smith, M. (2000) in The BSE Inquiry (Stationery Office, London), Vol. 6, pp. 413-451]. Here we report that mouse skeletal muscle can propagate prions and accumulate substantial titers of these pathogens. We found both high prion titers and the disease-causing isoform of the prion protein (PrP(Sc)) in the skeletal muscle of wild-type mice inoculated with either the Me7 or Rocky Mountain Laboratory strain of murine prions. Particular muscles accumulated distinct levels of PrP(Sc), with the highest levels observed in muscle from the hind limb. To determine whether prions are produced or merely accumulate intramuscularly, we established transgenic mice expressing either mouse or Syrian hamster PrP exclusively in muscle. Inoculating these mice intramuscularly with prions resulted in the formation of high titers of nascent prions in muscle. In contrast, inoculating mice in which PrP expression was targeted to hepatocytes resulted in low prion titers. Our data demonstrate that factors in addition to the amount of PrP expressed determine the tropism of prions for certain tissues. That some muscles are intrinsically capable of accumulating substantial titers of prions is of particular concern. Because significant dietary exposure to prions might occur through the consumption of meat, even if it is largely free of neural and lymphatic tissue, a comprehensive effort to map the distribution of prions in the muscle of infected livestock is needed. Furthermore, muscle may provide a readily biopsied tissue from which to diagnose prion disease in asymptomatic animals and even humans.

Pro- and Anti-apoptotic Members of the Bcl-2 Family in Skeletal Muscle: A Distinct Role for Bcl-2 in Later Stages of Myogenesis

Apoptotic myonuclei appear during myogenesis and in diseased muscles. To investigate cell death regulation in skeletal muscle, we examined how members of the Bcl-2 family of apoptosis regulators are expressed and function in the C2C12 muscle cell line and in primary muscle cells at different stages of development. Both anti-apoptotic (Bcl-W, Bcl-X(L)) and pro-apoptotic (Bad, Bak, Bax) members of the Bcl-2 family were expressed in developing skeletal muscle in vivo. Each was also expressed in embryonic (E11-12), fetal (E15-16), and neonatal muscle stem cells, myoblasts, and myotubes in vitro. In contrast, Bcl-2 expression was limited to a small group of mononucleate, desmin-positive, myogenin-negative muscle cells that were seen in fetal and neonatal, but not embryonic, muscle cell cultures. The cell surface protein Sca-1, which is associated with muscle and blood stem cells, was found on approximately 1/2 of these Bcl-2-positive cells. Loss of Bcl-2 did not affect expression of other family members, because neonatal muscles of wild-type and Bcl-2-null mice had similar amounts of Bcl-X(L), Bcl-W, Bad, Bak, and Bax mRNAs. Loss of Bcl-2 did have functional consequences; however, because neonatal muscles of Bcl-2-null mice had only approximately 2/3 as many fast muscle fibers as muscles in wild-type mice. Thus, Bcl-2 function is required for particular stages of fetal and postnatal myogenesis.

Postnatal Myosin Heavy Chain Isoform Expression in Normal Mice and Mice Null for IIb or IId Myosin Heavy Chains

The patterns of myosin heavy chain (MyHC) isoform expression in the embryo and in the adult mouse are reasonably well characterized and quite distinct. However, little is known about the transition between these two states, which involves major decreases and increases in the expression of several MyHC genes. In the present study, the expression of seven sarcomeric MyHCs was analyzed in the hindlimb muscles of wild-type mice and in mice null for the MyHC IIb or IId/x genes at several time points from 1 day of postnatal life (dpn) to 20 dpn. In early postnatal life, the developmental isoforms (embryonic and perinatal) comprise >90% of the total MyHC expression, while three adult fast isoforms (IIa, IIb, and IId) comprise <1% of the total MyHC protein. However, between 5 and 20 dpn their expression increases to comprise >90% of the total MyHC. Expression of each of the three adult fast isoforms occurs in a spatially and temporally distinct manner. We also show that α MyHC, which is almost exclusively expressed in the heart, is expressed in scattered fibers in all hindlimb muscles during postnatal development. Surprisingly, the timing and localization of expression of the MyHC isoforms is unchanged in IIb and IId/x null mice, although the magnitude of expression is altered for some isoforms. Together these data provide a comprehensive overview of the postnatal expression pattern of the sarcomeric MyHC isoforms in the mouse hindlimb.

Delayed onset of inflammation in protease-activated receptor-2-deficient mice.

Endothelial surface expression of P-selectin and subsequent leukocyte rolling in venules can be induced by mast cell-derived histamine and binding of thrombin to protease-activated receptor-1 (PAR1). We hypothesized that activation of endothelial PAR2 by mast cell tryptase or other proteases also contributes to inflammatory responses. Leukocyte rolling flux and rolling velocity were assessed by intravital microscopy of the cremaster muscles of wild-type mice following perivenular micropipette injections of a control (LSIGRL) or PAR2-activating (SLIGRL) oligopeptide. Injection of SLIGRL increased mean rolling leukocyte flux fraction from 34 +/- 11 to 71 +/- 24% (p < 0.05) and decreased mean rolling velocity from 63 +/- 29 to 32 +/- 2 micrometer/s (p < 0.05). No significant changes occurred with control peptide injection. To further evaluate the role of PAR2 in inflammatory responses, PAR2-deficient mice were generated by gene targeting and homologous recombination. Perivenular injections of SLIGRL resulted in only a small increase in rolling leukocyte flux fraction (from 21 +/- 8 to 30 +/- 2%) and no change in rolling velocity. Leukocyte rolling after surgical trauma was assessed in 9 PAR2-deficient and 12 wild-type mice. Early (0-15 min) after surgical trauma, the mean leukocyte rolling flux fraction was lower (10 +/- 3 vs 30 +/- 6%, p < 0.05) and mean rolling velocity was higher (67 +/- 46 vs 52 +/- 36 micrometer/s, p < 0.01) in PAR2-deficient compared with control mice. The defect in leukocyte rolling in PAR2-deficient mice did not persist past 30 min following surgical trauma. These results indicate that activation of PAR2 produces microvascular inflammation by rapid induction of P-selectin-mediated leukocyte rolling. In the absence of PAR2, the onset of inflammation is delayed.

Pro- and anti-apoptotic members of the Bcl-2 family in skeletal muscle: a distinct role for Bcl-2 in later stages of myogenesis.

Apoptotic myonuclei appear during myogenesis and in diseased muscles. To investigate cell death regulation in skeletal muscle, we examined how members of the Bcl-2 family of apoptosis regulators are expressed and function in the C2C12 muscle cell line and in primary muscle cells at different stages of development. Both anti-apoptotic (Bcl-W, Bcl-X(L)) and pro-apoptotic (Bad, Bak, Bax) members of the Bcl-2 family were expressed in developing skeletal muscle in vivo. Each was also expressed in embryonic (E11-12), fetal (E15-16), and neonatal muscle stem cells, myoblasts, and myotubes in vitro. In contrast, Bcl-2 expression was limited to a small group of mononucleate, desmin-positive, myogenin-negative muscle cells that were seen in fetal and neonatal, but not embryonic, muscle cell cultures. The cell surface protein Sca-1, which is associated with muscle and blood stem cells, was found on approximately 1/2 of these Bcl-2-positive cells. Loss of Bcl-2 did not affect expression of other family members, because neonatal muscles of wild-type and Bcl-2-null mice had similar amounts of Bcl-X(L), Bcl-W, Bad, Bak, and Bax mRNAs. Loss of Bcl-2 did have functional consequences; however, because neonatal muscles of Bcl-2-null mice had only approximately 2/3 as many fast muscle fibers as muscles in wild-type mice. Thus, Bcl-2 function is required for particular stages of fetal and postnatal myogenesis.

A K(ATP) channel deficiency affects resting tension, not contractile force, during fatigue in skeletal muscle.

The objective of this study was to determine how an ATP-sensitive K(+) (K(ATP)) channel deficiency affects the contractile and fatigue characteristics of extensor digitorum longus (EDL) and soleus muscle of 2- to 3-mo-old and 1-yr-old mice. K(ATP) channel-deficient mice were obtained by disrupting the Kir6.2 gene that encodes for the protein forming the pore of the channel. At 2-3 mo of age, the force-frequency curve, the twitch, and the tetanic force of EDL and soleus muscle of K(ATP) channel-deficient mice were not significantly different from those in wild-type mice. However, the tetanic force and maximum rate of force development decreased with aging to a greater extent in EDL and soleus muscle of K(ATP) channel-deficient mice (24-40%) than in muscle of wild-type mice (7-17%). During fatigue, the K(ATP) channel deficiency had no effect on the decrease in tetanic force in EDL and soleus muscle, whereas it caused a significantly greater increase in resting tension when compared with muscle of wild-type mice. The recovery of tetanic force after fatigue was not affected by the deficiency in 2- to 3-mo-old mice, whereas in 1-yr-old mice, force recovery was significantly less in muscle of K(ATP) channel-deficient than wild-type mice. It is suggested that the major function of the K(ATP) channel during fatigue is to reduce the development of a resting tension and not to contribute to the decrease in force. It is also suggested that the K(ATP) channel plays an important role in protecting muscle function in older mice.

1H and (31)P magnetization transfer studies of hindleg muscle in wild-type and creatine kinase-deficient mice.

The authors addressed the hypothesis that interactions with creatine kinase (CK) play a role in the off-resonance magnetization transfer (MT) effect of creatine in skeletal muscle. Toward that aim, (1)H MT studies were done on hindleg muscle in wild-type mice and in transgenic mice, lacking cytoplasmic CK and/or mitochondrial CK. The (1)H MT effect was essentially identical in wild-type muscle and the two single CK knock-out muscles, while moderately decreased in tissue lacking both CK isoforms. (31)P-NMR showed no off-resonance (31)P MT effect in skeletal muscle for PCr in any of the mice, while the enzymatic CK reaction flux was circa 0.2-0.3 sec(-1) in the wild-type muscle and in muscle deficient in mitochondrial CK. The CK enzyme flux was negligible in the other two CK knock-outs. These data suggest that CK plays a minor role in the (1)H MT effect of creatine. Irrespective of the underlying mechanism the creatine MT phenomenon probably has no significant consequences for the thermodynamic availability of total creatine to the CK reaction.

1H and 31P magnetization transfer studies of hindleg muscle in wild‐type and creatine kinase‐deficient mice

The authors addressed the hypothesis that interactions with creatine kinase (CK) play a role in the off-resonance magnetization transfer (MT) effect of creatine in skeletal muscle. Toward that aim, 1H MT studies were done on hindleg muscle in wild-type mice and in transgenic mice, lacking cytoplasmic CK and/or mitochondrial CK. The 1H MT effect was essentially identical in wild-type muscle and the two single CK knock-out muscles, while moderately decreased in tissue lacking both CK isoforms. 31P-NMR showed no off-resonance 31P MT effect in skeletal muscle for PCr in any of the mice, while the enzymatic CK reaction flux was circa 0.2–0.3 sec−1 in the wild-type muscle and in muscle deficient in mitochondrial CK. The CK enzyme flux was negligible in the other two CK knock-outs. These data suggest that CK plays a minor role in the 1H MT effect of creatine. Irrespective of the underlying mechanism the creatine MT phenomenon probably has no significant consequences for the thermodynamic availability of total creatine to the CK reaction. Magn Reson Med 43:657–664, 2000. © 2000 Wiley-Liss, Inc.

A High Fat Diet Impairs Stimulation of Glucose Transport in Muscle: FUNCTIONAL EVALUATION OF POTENTIAL MECHANISMS

A high fat diet causes resistance of skeletal muscle glucose transport to insulin and contractions. We tested the hypothesis that fat feeding causes a change in plasma membrane composition that interferes with functioning of glucose transporters and/or insulin receptors. Epitrochlearis muscles of rats fed a high (50% of calories) fat diet for 8 weeks showed approximately 50% decreases in insulin- and contraction-stimulated 3-O-methylglucose transport. Similar decreases in stimulated glucose transport activity occurred in muscles of wild-type mice with 4 weeks of fat feeding. In contrast, GLUT1 overexpressing muscles of transgenic mice fed a high fat diet showed no decreases in their high rates of glucose transport, providing evidence against impaired glucose transporter function. Insulin-stimulated system A amino acid transport, insulin receptor (IR) tyrosine kinase activity, and insulin-stimulated IR and IRS-1 tyrosine phosphorylation were all normal in muscles of rats fed the high fat diet for 8 weeks. However, after 30 weeks on the high fat diet, there was a significant reduction in insulin-stimulated tyrosine phosphorylation in muscle. The increases in GLUT4 at the cell surface induced by insulin or muscle contractions, measured with the 3H-labeled 2-N-4-(1-azi-2,2, 2-trifluoroethyl)-benzoyl-1,3-bis-(D-mannose-4-yloxy)-2-propyla min e photolabel, were 26-36% smaller in muscles of the 8-week high fat-fed rats as compared with control rats. Our findings provide evidence that (a) impairment of muscle glucose transport by 8 weeks of high fat feeding is not due to plasma membrane composition-related reductions in glucose transporter or insulin receptor function, (b) a defect in insulin receptor signaling is a late event, not a primary cause, of the muscle insulin resistance induced by fat feeding, and (c) impaired GLUT4 translocation to the cell surface plays a major role in the decrease in stimulated glucose transport.

CONTRACTION-INDUCED GLUCOSE TRANSPORT AND GLYCOGEN DEPLETION IN MUSCLES FROM MICE HETEROZYGOUS FOR IRS-1 PROTEIN

1398 Insulin receptor substrate-1 (IRS1) plays a pivotal role in insulin-stimulated glucose transport (GT). A role for IRS1 in contraction-induced GT has not been identified, but few studies have addressed this topic. Mice heterozygous for a null allele in the IRS1 gene (IRS1-HZ) have an ≈50% reduction in IRS-1 protein in skeletal muscle compared to wild-type (WT) mice (Brüning et al. Cell 88:561). Our initial goal was to characterize contraction-induced GT in the extensor digitorum longus (EDL) muscles of WT mice. We then compared WT to IRS1-HZ mice. After WT mice were anesthetized, the sciatic nerve was isolated and electrically stimulated (0.1 msec pulses at 100 Hz in 250 msec trains at a rate of 1/sec for 5 min) for 2 bouts (2X) with 1 min recovery between bouts. We also studied muscles stimulated identically for 4 bouts (4X) to determine if additional contractile activity would further increase GT. Muscles were rapidly dissected out and GT was determined in vitro using 2-deoxyglucose. Contractile activity led to an ≈3-fold higher GT in the 2X group compared to unstimulated, contralateral EDL muscles (p<0.01). The 2X and 4X protocols elicited similar values for GT. We also evaluated the influence of the 2X protocol in IRS1-HZ mice and found an increase above basal that was similar to that in WT mice (p<0.01). Glycogen in unstimulated muscles was not different for IRS1-HZ vs. WT mice, and contractile activity elicited a comparable decline in muscle glycogen, regardless of genotype. In conclusion, our results indicate that a substantial reduction in IRS-1 expression does not alter contraction-induced glycogen depletion or GT stimulation in skeletal muscle.

Dyspedic Mouse Skeletal Muscle Expresses Major Elements of the Triadic Junction but Lacks Detectable Ryanodine Receptor Protein and Function

The ry1(53) dyspedic mouse contains two disrupted alleles for ryanodine receptor type 1 (skeletal isoform of ryanodine receptor; Ry1R) resulting in perinatal death. In the present study, whole skeletal muscle homogenates and sucrose gradient-purified junctional sarcoplasmic reticulum from neonatal wild-type and dyspedic mice were assayed for biochemical and functional markers. Equilibrium binding experiments performed with 1-120 nM [3H]ryanodine reveal saturable high and low affinity binding to membrane preparations from wild-type mice, but not to preparations from dyspedic mice. Binding experiments performed with [3H]PN200 show a 2-fold reduction in [3H]PN200 binding capacity in dyspedic muscle, compared to age-matched wild-type muscle, with no change in receptor affinity. The presence or absence of proteins known to be critical for normal ryanodine receptor/Ca2+ channel complex function was assessed by Western blot analysis. Results indicate that FKBP-12, DHPRalpha1, triadin, calsequestrin, SERCA1 (sarco(endo)plasmic reticulum Ca2+ ATPase), and skeletal muscle myosin heavy chain are present in both dyspedic and wild-type muscle. Only wild-type membranes showed immunoreactivity toward Ry1R antibody. Neither dyspedic nor wild-type mouse muscle showed detectable immunoreactivity toward Ry2R or Ry3R antibodies, even after sucrose gradient purification of sarcoplasmic reticulum. These results indicate that proteins critical for ryanodine receptor function are expressed in dyspedic skeletal muscle in the absence of Ry1R. Ca2+ transport measurements show that membranes from wild-type controls, but not dyspedic mice, release Ca2+ upon exposure to ryanodine. Dyspedic mice and cells derived from them serve as excellent homologous expression systems in which to study how Ry1R structure relates to function.

Distinct phenotype of E-selectin-deficient mice. E-selectin is required for slow leukocyte rolling in vivo.

Leukocyte capture and rolling are mediated by calcium-dependent lectins expressed on most leukocytes (L-selectin) and the vascular endothelium (P- and E-selectin). To study the role of the selectins during inflammation, we have investigated leukocyte rolling in venules of tumor necrosis factor-alpha (TNF-alpha)-treated mouse cremaster muscles in wild-type mice and gene-targeted mice with homozygous deficiency for L-, P-, or E-selectin (L-/-, P-/-, or E-/-, respectively). TNF-alpha treatment induces expression of E-selectin and increases expression of P-selectin on endothelial cells. Consistent with previous reports of redundant P- and E-selectin function, a combination of monoclonal antibodies (mAbs) against P- and E-selectin (RB40.34 and 9A9, respectively) was necessary to block rolling in wild-type mice. The rolling leukocyte flux fraction (percent rolling cells) in L-/- mice was similar to that in wild-type mice, but rolling in these mice was blocked by a P-selectin mAb. The velocity of rolling leukocytes in TNF-alpha-treated wild-type, P-/-, or L-/- mice was 5 to 10 times slower (3 to 7 microns/s) than during trauma-induced rolling (20 to 50 microns/s). In contrast, leukocytes in venules of TNF-alpha-treated E-/- mice rolled significantly faster (12 to 20 microns/s): the rolling leukocyte flux fraction was more than doubled compared with wild-type, L-/-, or P-/- mice; and the number of adherent leukocytes was reduced. Addition of an E-selectin mAb, but not a P-selectin mAb, increased rolling flux fraction and rolling velocity in wild-type mice. Histological analysis revealed that 90% to 95% of all leukocytes interacting (rolling and adherent) with the venular endothelium in TNF-alpha-treated wild-type, L-/-, P-/-, and E-/- mice were granulocytes. These results identify a previously unrecognized phenotype of E-/- mice by establishing that at the site densities prevailing in vivo, E-selectin in responsible for slow (approximately 5 microns/s) granulocyte rolling. E-selectin-dependent slow rolling drastically increases the transit time of leukocytes rolling through an inflamed tissue and thus aids in targeting leukocytes activated by chemoattractants to the inflammatory microenvironment.

Sequential contribution of L- and P-selectin to leukocyte rolling in vivo.

Leukocyte recruitment into inflammatory sites is initiated by a reversible transient adhesive contact with the endothelium called leukocyte rolling, which is thought to be mediated by the selectin family of adhesion molecules. Selectin-mediated rolling precedes inflammatory cell emigration, which is significantly impaired in both P- and L-selectin gene-deficient mice. We report here that approximately 13% of all leukocytes passing venules of the cremaster muscle of wild-type mice roll along the endothelium at < 20 min after surgical dissection. Rolling leukocyte flux fraction reaches a maximum of 28% at 40-60 min and returns to 13% at 80-120 min. In P-selectin-deficient mice, rolling is absent initially and reaches 5% at 80-120 min. Rolling flux fraction in L-selectin-deficient mice is similar to wild type initially and declines to 5% at 80-120 min. In both wild-type and L-selectin-deficient mice, initial leukocyte rolling (0-60 min) is completely blocked by the P-selectin monoclonal antibody (mAb) RB40.34, but unaffected by L-selectin mAb MEL-14. Conversely, rolling at later time points (60-120 min) is inhibited by mAb MEL-14 but not by mAb RB40.34. After treatment with tumor necrosis factor (TNF)-alpha for 2 h, approximately 24% of all passing leukocytes roll in cremaster venules of wild-type and P-selectin gene-deficient mice. Rolling in TNF-alpha-treated mice is unaffected by P-selectin mAb or E-selectin mAb 10E9.6. By contrast, rolling in TNF-alpha-treated P-selectin-deficient mice is completely blocked by L-selectin mAb. These data show that P-selectin is important during the initial induction of leukocyte rolling after tissue trauma. At later time points and in TNF-alpha-treated preparations, rolling is largely L-selectin dependent. Under the conditions tested, we are unable to find evidence for involvement of E-selectin in leukocyte rolling in mice.