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Project No. and Title:
NC1184: Molecular Mechanisms Regulating Skeletal Muscle Growth and Differentiation
Period Covered: 10-2010 to 09-2011
Date of Report: 3 Jan 2012
Annual Meeting Dates: 28-Oct-2011 to 29-Oct-2009
Participants:
Yong Soo Kim, University of Hawaii
Anna Dilger, University of Illinois
William Dayton, University of Minnesota
Mike White, University of Minnesota
Paul Mozdziak, North Carolina State
Shihuan Kuang, Purdue University
David Gerrard, Virginia Polytechnic Institute and State University
Honglin Jiang, Virginia Polytechnic Institute and State University
Robert Rhoads, Virginia Polytechnic Institute and State University
Gale Strasburg, Michigan State University
Marion Greaser, University of Wisconsin
Sandy Velleman, The Ohio State University
Michael Zeece, University of Nebraska
Deb Hamernik – Administrative Advisor
Mark Mirando – NIFA Representative
Brief Summary of Minutes of Annual Meeting: Members not attending: Rod Hill,
Idaho; J.E. Minton, Kansas; Michael Dodson, Washington State; Gordon Murdoch,
Washington State; Ronald Allen, University of Arizona; Joshua Selsby, Iowa State
University; Catherine Ernst, Michigan State University; Marcia Hathaway, University of
Minnesota; Jun Liao, Mississippi State University; Rajkumar Prabhu, Mississippi State
University; Doug McFarland, South Dakota State University; Penny Riggs, Texas A&M.
The annual meeting of the NC-1184 technical committee meeting was held at Virginia
Polytechnic Institute and State University, on October 28-29, 2011, and was hosted by
Drs. Gerrard, Jiang and Rhoads from the Department of Animal and Poultry Science.
On October 28th the group was welcomed by Dr. Alan Grant, Dean of the College of
Agricultural Sciences. NIFA Representative, Dr. Mark Mirando, made brief remarks via
teleconference to the group regarding changes at NIFA, the USDA funding outlook, and
status of funding RFA revisions. The business meeting was chaired by the
Administrative Advisor, Dr. Deb Hamernik. Next year's annual meeting of the NC1184 committee will be held at the University of Florida. The group decided that the
2013 meeting will be held at North Carolina State University, Paul Modziak, chair. The
remainder of the day was filled by oral station reports summarizing each station's
contributions to the objectives of the NC-1184 project. The meeting adjourned, and the
group met for dinner at the Gerrard residence. The meeting reconvened on October 29
and the remaining stations reported on their activities. Following completion of the
oral reports, the meeting adjourned for the year.
Accomplishments
Objective 1. Characterize the signal transduction pathways that regulate skeletal
muscle growth and metabolism including the influence of endogenous growth
factors and various production practices.
The Indiana Station provided insight into the involvement of nuclear receptors and
notch signaling as mediators of muscle growth and regeneration. Peroxisome
proliferator-activated receptors (PPARs) are a class of nuclear receptors that play
important roles in development and energy metabolism. Whereas PPARδ has been
shown to regulate mitochondria biosynthesis and slow muscle fiber types, its function
in skeletal muscle progenitors (satellite cells) is unknown. As constitutive mutation of
Pparδ leads to embryonic lethality, we address this question by conditional knockout
(cKO) using Myf5-Cre/Pparδflox/flox alleles to ablate PPARδ in myogenic progenitor cells.
Although Pparδ cKO mice were born normally and initially displayed no difference in
body weight, muscle size or muscle composition, they later developed metabolic
syndrome manifested by increased body weight and reduced response to glucose
challenge at 9 months old. Compared to wild type littermates, Pparδ cKO mice had 40%
fewer satellite cells and these satellite cells exhibited reduced growth kinetics and
proliferation in vitro. Furthermore, regeneration of Pparδ cKO muscles was impaired
after cardiotoxin-induced injury. Gene expression analysis showed reduced expression
of FoxO1 in Pparδ cKO muscles under both quiescent and regenerating conditions,
results support a function of PPARδ in regulating skeletal muscle metabolism and
insulin sensitivity, and establish a novel role of PPARδ in muscle progenitor cells and
postnatal muscle regeneration.
Notch signaling is a conserved cell fate regulator during development and postnatal
tissue regeneration. Using skeletal muscle satellite cells as a model and through
conditional over-expression of constitutively activated Notch1intracellular domain
(NICDOE), here we investigate how Notch signaling regulates adult stem cell fate
choice. Myogenic lineage-specific NICDOE results in severely reduced muscle mass and
perinatal lethality. At the cellular level, NICDOE increased satellite cell number but
decreased the number of myonuclei/myofiber and myofibers/muscle. At the molecular
level, NICDOE increases Pax7 but decreases Ki67 and MyoD expression, resulting in
enhanced self-renewal but reduced proliferation and differentiation. Overexpression of
canonical Notch target genes mimics the effect of NICDOE on Ki67 and MyoD, but not
Pax7. Instead, NICD regulates Pax7 through interaction with RBPconsensus sites upstream of the Pax7 gene. Finally, NICDOE in postnatal satellite cells
leads to impaired regeneration of skeletal muscles along with increased Pax7+
mononuclear cells. Therefore, besides Notch’s well-known inhibitory function in
myogenic differentiation, our results establish a novel role of Notch signaling in
promoting the self-renewal of muscle stem cells through Pax7.
The Hawaii Station detailed the production and purification of myostatin propeptide
and follistatin from bacteria. Mstn propeptide inhibits the activity of Mstn, indicating
its potential as an agent to improve the efficiency of meat production by enhancing
skeletal muscle growth. Follistatin inhibits the activity of Mstn, indicating its potential
as an agent to improve the efficiency of meat production by enhancing skeletal muscle
growth. The station demonstrated that affinity-purified pMSTNprod possesses Mstninhibitory activity in an in vitro reporter gene assay. The MSTN inhibitory capacity of
pMSTNProM was not significantly different from that of pMSTNProM. By contrast,
SDS-PAGE analysis of E. coli culture after induction showed insoluble expression of the
follistatin proteins, indicating that the proteins cannot be expressed in soluble forms
using the periplasmic E. coli expression system.
The Minnesota Station reported on the underlying mechanisms by which estrogen and
IGF-I alter bovine satellite cell (BSC) myogenesis. Although androgenic and estrogenic
steroids are widely used to enhance muscle growth and increase feed efficiency in
feedlot cattle, their mechanism of action is not well understood.
Treatment of cultured BSC with ER-a siRNA suppresses ER-a mRNA expression by
approximately 70-80% (p < 0.05) and has no significant effect on expression of either
IGFR-1b or EGFR mRNA; thus, establishing the specificity of the silencing. ER-a
protein levels are reduced to non-detectable levels by treatment with ER-a siRNA.
Silencing ER-a suppresses the ability of E2 to stimulate proliferation but has no effect on
IGF-1 or hbEGF stimulated proliferation of BSC.
Treatment of cultured BSC with IGFR-1b siRNA suppresses IGFR-1b mRNA expression
by approximately 70-80% (p < 0.05) and has no significant effect on expression of either
ER-a or EGFR mRNA; thus, establishing the specificity of the silencing. IGFR-1b protein
levels are substantially reduced by treatment with IGFR-1β siRNA. Silencing IGFR-1b
suppresses the ability of both IGF-1 and E2 to stimulate proliferation but has no effect
on hbEGF stimulated proliferation of BSC.
Treatment of cultured BSC with EGFR siRNA suppresses EGFR mRNA expression by
70-80% (p < 0.05) and has no significant effect on expression of either ER-a or IGFR-1b
mRNA; thus, establishing the specificity of the silencing. EGFR protein levels are
substantially reduced by treatment with EGFR siRNA. Silencing of EGFR suppresses
the ability of IGF-1, E2 and EGF to stimulate proliferation of BSC but has no effect on
the ability of fetal bovine serum to stimulate proliferation.
These data establish that we can silence expression of IGFR-1b, EGFR and ER-a in
cultured BSC and support our hypothesis that IGFR-1 and EGFR are required for
stimulation of cultured BSC proliferation by E2. Additionally, or data show that EGFR
also is required for stimulation of BSC proliferation by IGF-1.
The Virginia Station reported that fiber hypertrophy and increased oxidative capacity
can occur simultaneously. An inverse relationship exists between skeletal muscle fiber
cross-sectional area (CSA) and oxidative capacity. This suggests muscle fibers increase
in size or hypertrophy at the expense of endurance or oxidative capacity. Therefore, our
objective was to utilize pigs possessing mutations associated with increased oxidative
capacity (AMP-activated protein kinase, AMPKγ3R200Q) or muscle fiber size (ryanodine
receptor, RyRR615C) to determine if these events could occur in parallel. Pigs
heterozygous for both mutations were mated to generate offspring with all genotype
combinations. At approximately 120 kg, wild type, γ3R200Q, RyRR615C, and γ3R200QRyRR615C pigs were harvested. Immediately after exsanguination, longissimus muscle
samples were collected and processed for histology, biochemical, or mitochondrial
function assays. Mean fiber CSA was determined from at least 100 fibers (range 100-400)
per pig; RyRR615C increased (P<0.05) mean fiber CSA by 35% (6830 vs. 5050 µm2). In
contrast, γ3R200Q pig muscle exhibited greater (P<0.05) citrate synthase and bhydroxyacyl coA dehydrogenase activity, indicative of increased oxidative capacity by
the TCA cycle and fatty acid b-oxidation, respectively. Increased oxidative capacity of
γ3R200Q muscle was consistent with increased (P<0.05) mitochondrial DNA copy number
relative to nuclear DNA, suggesting greater mitochondrial biogenesis. Moreover,
isolated mitochondria from γ3R200Q muscle exhibited the highest state 3 (maximal, ADPstimulated) oxygen consumption rate in the presence of either succinate/rotenone
(P<0.05) or pyruvate/malate (P<0.05). No interaction (γ3R200Q x RyRR615C) was detected
for any of the aforementioned response variables. Thus, pigs that possess both γ3R200Q
and RyRR615C exhibit increased muscle fiber CSA as well as greater oxidative enzyme
activity, mitochondrial DNA content, and mitochondrial oxidative phosphorylation
capacity. Altogether, this supports that hypertrophy and enhanced oxidative capacity
can occur simultaneously in skeletal muscle, suggesting that the signaling mechanisms
controlling these events can be independently regulated.
Lean growth rate in most domestic animals differs with muscle type. Satellite cells (SC)
potentiate muscle growth but little is known regarding how populations of SC differ
with muscle type, especially in pigs. Therefore, the Virginia Station characterized SC
from red (RST) and white (WST) portions of the semitendinosus muscle of 6-week-old
piglets and determined their capacity to proliferate, differentiate and express various
myosin heavy chain (MyHC) isoforms in vitro. RST yielded 20% more (P<0.001) SC per
gram muscle compared to WST. Satellite cells from RST proliferated faster (P<0.001)
than those from WST, as indicated by a shorter cell doubling time, 18.6 h versus 21.3 h,
respectfully. Consequently, SC from RST formed myotubes quicker than those from
WST. Once differentiated, however, SC from WST differentiated faster (P<0.05) than
those from RST in the first 24 h, 41.6% versus 34.3%, respectively; but reached similar
ultimate fusion percentages by 48 h. Over 90% of MyHC expressed in fully
differentiated SC from both RST and WST was restricted to the embryonic isoform.
Type IIA MyHC mRNA was detected at low levels, while type IIX MyHC mRNA was
not detectable. Even so however, myotube cultures from RST expressed more (P<0.01)
type I MyHC isoform mRNA than those from WST, whereas those cultures from WST
expressed more (P<0.05) type IIB MyHC transcripts. These data show SC from red and
white muscles differ and suggest intrinsic characteristics of these cells may be partially
restricted to a particular muscle type prior to birth.
The AMP-activated protein kinase (AMPK) is activated by upstream kinases and
negatively regulated by protein phosphatases. Intracellular calcium mediates protein
phosphatase 2A, which is in a heterotrimeric complex with the PR72 subunit. The PR72
subunit contains two calcium binding sites formed by EF hands. A previous study from
the Virigina Station has shown that chronic calcium exposure decreases AMPK
activity. To define the specific molecular mechanism whereby calcium can deactivate
AMPK, activities of AMPK and PP2A were analyzed in C2C12 muscle cell cultures and
skeletal muscle tissues from mutant pigs possessing the AMPK mutation or the RyR1
(calcium gating) mutation, or both. C2C12 myotubes treated with calcium releasing
agent (caffeine) for 10 h decreased (P<0.05) AICAR-induced AMPK activity to control
levels and this negative effect was eliminated by the ryanodine receptor stabilizer,
dantrolene. Interestingly, muscle from pigs with ryanodine receptor (RyR1) mutation
and C2C12 cells administered with 10 h caffeine showed higher (P<0.05) PP2A activity
compared to controls. More importantly, the inhibitory effect of caffeine on AMPK
activity was attenuated by the PP2A inhibitor, okadaic acid or siRNA induced
knockdown of PP2A. These data show the inhibitory effect of chronic calcium on
AMPK activity is exerted through the activation of PP2A.
The Virginia Station further identified the signaling pathways that mediate the effects
of IGF-I on proliferation, fusion, protein synthesis, and protein degradation in bovine
muscle cells. Satellite cells were isolated from adult cattle skeletal muscle and were
allowed to activate and proliferate or were induced to form myotubes. Cell proliferation
was determined by measuring the numbers of viable cells at different times. Protein
synthesis and degradation were determined by measuring the accumulation of 3Hphenylalanine in cellular protein and the release of 3H-phenylalanine to the medium,
respectively. The signaling pathway involved was identified by including in the
medium rapamycin, LY294002, or PD98095, which are specific inhibitors of the IGF-I
receptor signaling molecules mTOR, AKT (PKB), and ERK (MAPK), respectively.
Western blotting confirmed that IGF-I action caused phosphorylations of p70S6K (a
signaling molecule immediately downstream of mTOR), AKT, and ERK, and that these
phosphorylations were completely or near completely blocked by their corresponding
inhibitors. Proliferation of bovine myoblasts was stimulated by 500 ng/mL IGF-I
(P<0.01), and this stimulation was partially blocked by PD98059 (P<0.05), and was
completely blocked by rapamycin or LY294002 (P<0.01). Protein degradation in
myotubes was inhibited by approximately 20% by 500 ng/mL IGF-I (P<0.05), and this
inhibition was completely relieved by LY294002 (P<0.01), but not at all by rapamycin or
PD98095. Protein synthesis in myotubes was increased by 30% by 500 ng/mL IGF-I
(P<0.01), and this increase was completely blocked by rapamycin, LY294002, or
PD98059 (P<0.01). Addition of IGF-I to the culture medium had no effect on fusion of
myoblasts into myotubes. These data suggest that IGF-I stimulates proliferation of
bovine myoblasts and protein synthesis in bovine myotubes through both the
PI3K/AKT and the MAPK signaling pathways, and that IGF-I inhibits protein
degradation in bovine myotubes through the PI3K/AKT pathway.
Objective 2. Characterize the cellular and molecular basis of myogenesis.
Current research efforts at the Ohio Station are focused on understanding the
mechanism of how the heparan sulfate family of proteoglycans may be involved in the
regulation of muscle growth properties. Fibroblast growth factor 2 (FGF2) is a potent
stimulator of muscle cell proliferation and a strong inhibitor of muscle cell
differentiation. Heparan sulfate proteoglycans function as a low affinity receptor for
FGF2 thus permitting a high affinity interaction of FGF2 with its receptor. Our research
is focused on the heparan sulfate proteoglycan syndecan and glypican families. There
are 6 glypican family members with only glypican-1 found in skeletal muscle. The
report this year progresses research completed on glypican-1 and syndecan-4.
Glypican-1 is a cell membrane heparan sulfate proteoglycan that is composed of a core
protein and covalently attached glycosaminoglycan (GAG) chains and N-linked
glycosylated (N-glycosylated) chains. The glypican-1 GAG chains are required for cell
differentiation and responsiveness to fibroblast growth factor 2 (FGF2). The role of
glypican-1 N-glycosylated chains in regulating cell activities has not been reported. The
objective of the current study was to investigate the role of glypican-1 N-glycosylated
chains and the interaction between N-glycosylated and GAG chains in turkey myogenic
satellite cell proliferation, differentiation, and FGF2 responsiveness. The wild type
turkey glypican-1 and turkey glypican-1 with mutated GAG chain attachment sites
were cloned into the pCMS-EGFP mammalian expression vector, and used as templates
to generate glypican-1 N-glycosylated one-chain and no-chain mutants with or without
GAG chains by site-directed mutagenesis. The wild type glypican-1, all glypican-1 Nglycosylated one-chain and no-chain mutants with or without GAG chains were
transfected into turkey myogenic satellite cells. Cell proliferation, differentiation, and
FGF2 responsiveness were measured. The over-expression of glypican-1 Nglycosylated one-chain and no-chain mutants without GAG chains increased cell
proliferation and differentiation compared to the wild type glypican-1, but not the
glypican-1 N-glycosylated mutants with GAG chains attached. Cells over-expressing
glypican-1 N-glycosylated mutants with or without GAG chains increased cell
responsiveness to FGF2 compared to wild type glypican-1. These data suggest that
glypican-1 N-glycosylated chains and GAG chains are critical in regulating turkey
myogenic satellite cell proliferation, differentiation, and responsivness to FGF2.
Syndecan-4 core protein is composed of extracellular, transmembrane, and cytoplasmic
domains. The cytoplasmic domain functions in transmitting signals into the cell
through the protein kinase C alpha (PKCα) pathway. The glycosaminoglycan (GAG)
and N-linked glycosylated (N-glycosylated) chains attached to the extracellular domain
influence cell proliferation. The current study investigated the function of syndecan-4
cytoplasmic domain in combination with GAG and N-glycosylated chains in turkey
muscle cell proliferation, differentiation, fibroblast growth factor 2 (FGF2)
responsiveness, and PKCα membrane localization. Syndecan-4 construct or syndecan-4
construct without the cytoplasmic domain and with or without the GAG and Nglycosylated chains were transfected or co-transfected with a small interfering RNA
targeting syndecan-4 cytoplasmic domain into turkey muscle satellite cells. The
overexpression of syndecan-4 mutants increased cell proliferation but did not change
cell differentiation compared to syndecan-4. Syndecan-4 cytoplasmic domain and GAG
and N-glycosylated chain mutants had increased cellular responsiveness to FGF2
during proliferation. Syndecan-4 increased PKCα cell membrane localization, whereas
the syndecan-4 mutants had decreased PKCα cell membrane localization compared to
syndecan-4. However, compared to the cells without transfection, syndecan-4 mutants
increased cell membrane localization of PKCα. These data indicated that the
syndecan‐ 4 cytoplasmic domain and the GAG and N-glycosylated chains are critical
in syndecan-4 regulating satellite cell proliferation, responsiveness to FGF2, and PKCα
cell membrane localization.
Previous work by the Michigan Station demonstrated a differential gene expression
during turkey muscle growth. The station further clarified gene expression changes
using a turkey skeletal-muscle-specific oligonucleotide microarray and revealed that
more than 3,000 genes were differentially expressed as a function of three critical stages
of muscle development: hyperplasia (18 d embryo), hypertrophy (1 d post-hatch), and
mature muscle (16 wk). The genes versican, matrix Gla protein (MGP), and deathassociated protein 1 (DAP1) were selected for further study for their potential effects on
modulation of muscle satellite cell proliferation and differentiation. Moreover, these
genes exhibited large fold-changes in expression as a function of muscle development
in the turkey. Small interfering RNA was used to knock down expression of these genes
during proliferation and differentiation of cultured turkey muscle satellite cells; DNA
content and creatine kinase activity were quantified as markers of proliferation and
differentiation, respectively. Knockdown of each of the genes was associated with
altered rates of proliferation and differentiation. Versican and MGP predominantly
affected proliferation, but later stages of differentiation were also affected by the
knockdown of versican and MGP. The knockdown of DAP1 dramatically inhibited
satellite cell differentiation to form myotubes, with reduction in creatine kinase activity
of up to 90% compared to the control. Microarray and pathway analysis of the
proliferating and differentiating DAP1 knockdown cells indicated that several genes
associated with calcium signaling were differentially expressed. This is the first report
that these genes, with no previously documented functions in regulation of muscle
development, may play critical roles in muscle cell proliferation and differentiation.
Skeletal muscle fiber formation is under genetic control, but little is known about the
specific genes involved or how their expression patterns are coordinated. The aim of
this study was to identify differentially expressed genes in longissimus dorsi muscle of
Yorkshire x Landrace pigs at 40 and 70 d of gestation (encompassing the transition from
primary to secondary fibers). Total RNA was pooled from 3 fetuses from gilts at each
gestational age (n = 3). Transcriptional profiling was performed by direct sequencing
(RNAseq) with an Illumina GAIIx revealing 6,299 differentially expressed tags (FDR <
0.10). The same samples were previously evaluated using the Pigoligoarray microarray
comprised of 20,400 70-mers, and qPCR was completed for a subset of genes. To
perform a cross-platform comparison between RNAseq and microarray analyses,
microarray results were expressed as log-fold change (FC) with associated p-value and
q-value (FDR), and comparisons filtered with FDR < 0.10 (n=1,218). Microarray
oligonucleotides were matched to RNAseq tags based on HGNC annotation resulting in
1,410 matching pairs of oligonucleotides and sequenced transcripts (with multiple
transcripts mapping to the same oligonucleotide). Correlation of log-FC between the
technologies was 0.72. Expression patterns obtained with RNAseq for 11 genes assayed
by qPCR (annotated in Build 9) were validated. These included 4 non-differentially
expressed genes (FDR > 0.10 in both assays; CTNNB1, HPRT1, STAT1, TIMP3), 6 genes
more highly expressed at 70 d (FDR < 0.10; CA3, DLK1, FBXO32, MYOZ1, NRAP,
USP13) and 1 gene more highly expressed at 40 d (FDR < 0.10; TNC). Relative FC from
RNAseq and qPCR for all genes agreed in both direction and magnitude. As expected,
RNAseq identified additional differentially expressed transcripts over the microarray
results. However, this analysis demonstrated that the microarray results were
repeatable, and results of both technologies were comparable to qPCR. Thus, both
microarrays and RNAseq are reliable and RNAseq may complement and extend
microarray studies.
The Illinois Station reported that organ and muscle growth exhibit a coordinated
down-regulation of genes important for cell proliferation. During early post-natal
development, cells are rapidly proliferating as animals grow quickly. This proliferation
slows as animals near their adult size. Furthermore, this proliferation slows in several
tissues simultaneously but does not seem to be controlled by systemic or circulating
growth factors. A set of genes which are all similarly down-regulated with growth in
several organs and thought to be important in proliferation was identified (Lui et al.,
2010). Our hypothesis was that, given the nature of skeletal muscle growth, these genes
would not be similarly expressed or regulated in skeletal muscle. Furthermore, we
believed that these genes would be expressed differently between male and female mice
in skeletal muscle and organs.
Using quantitative PCR, expression of 7 previously identified genes (Gpc3, Mest, Peg3,
Plag1, Ezh2, Mdk, and Mycn) was evaluated in skeletal muscle, heart and liver samples
from male and female wild-type mice at 0, 1 and 3 weeks of age. All expression was
normalized to 18s ribosomal RNA, standardized to a common sample used on each
plate and expressed as a fold-change compared to newborn males of each tissue.
Body, liver, heart and muscle weight increased as expected from 0 to 3 weeks of age,
and male body and organ weights were heavier than females. Similar to previous
reports, the expression of Gpc3, Mest, Peg3, and Plag1 were all reduced with growth in
heart and liver tissue. However, the expression of Ezh2 and Mdk were down-regulated
in heart tissue but not in liver. In our study, the expression of Mycn was not downregulated with age in either organ, contrary to previous reports. In these organs,
differences in expression between males and females were only noted in the liver and
for only for Mest and Plag1.
In skeletal muscle, the expression of Gpc3, Mest, Peg3, and Plag1 were all downregulated with growth similar to organs and, with the exception of Peg3, were all also
affected by sex with expression in females being less than in males. The expression of
Ezh2, Mdk, and Mycn remained constant or increased slightly between 0 and 1 weeks of
age but was down-regulated at 3 weeks of age. The expression of these three genes was
not affected by sex. Interestingly, the expression levels of all genes studied were similar
in skeletal muscle when compared with liver and heart. These data suggest that
similarities may exist between organs and skeletal muscle with regards to the regulation
of ultimate tissue size.
The North Carolina Station presented new information on the molecular basis for
muscle fiber heterogeneity. Skeletal muscle is composed of metabolically
heterogeneous myofibres that exhibit high plasticity at both the morphological and
transcriptional levels. The objective of this study was to employ microarray analysis to
elucidate the differential gene expression between the tonic-‘red’ anterior latissimus
dorsi (ALD) muscle, the phasic-‘white’ posterior latissimus dorsi (PLD) and ‘mixed’phenotype biceps femoris (BF) in 1-week-and 19-week-old male turkeys. A total of 170
differentially expressed genes were identified in the muscle samples analysed (P < 0.05).
Gene GO analysis software was utilized to identify top gene networks and metabolic
pathways involving differentially expressed genes. Quantitative real-time PCR for
selected genes (BAT2D, CLU, EGFR and LEPROT) was utilized to validate the
microarray data. The largest differences were observed between ALD and PLD muscles,
in which 32 genes were over-expressed and 82 genes were under-expressed in ALD1-
PLD1 comparison, and 70 genes were over-expressed and 70 under-expressed in
ALD19-PLD19 comparison. The largest number of genes over-expressed in ALD
muscles, as compared to other muscles, code for extracellular matrix proteins such as
dystroglycan and collagen. The gene analysis revealed that phenotypically ‘red’ BF
muscle has high expression of glycolytic genes usually associated with the ‘white’
muscle phenotype. Muscle-specific differences were observed in expression levels of
genes coding for proteins involved in mRNA processing and translation regulation,
proteosomal degradation, apoptosis and insulin resistance. The current findings may
have large implications in muscle-type-related disorders and improvement of muscle
quality in agricultural species.
Coenzyme Q(10) (CoQ(10)) plays an essential role in determination of mitochondrial
membrane potential and substrate utilization in all metabolically important tissues. The
objective of the present study was to investigate the effect of Coenzyme Q analog
(MitoQ(10)) on oxidative phenotype and adipogenesis in myotubes derived from fastglycolytic Pectoralis major (PM) and slow-oxidative Anterior latissimus dorsi (ALD)
muscles of the turkey (Meleagris gallopavo). The myotubes were subjected to the
following treatments: fusion media alone, fusion media +125 nM MitoQ(10), and 500
nM MitoQ(10). Lipid accumulation was visualized by Oil Red 0 staining and quantified
by measuring optical density of extracted lipid at 500 nm. Quantitative Real-Time PCR
was utilized to quantify the expression levels of peroxisome proliferator-activated
receptor (PPAR gamma) and PPAR gamma co-activator-1 alpha (PGC-1 alpha).
MitoQ(10) treatment resulted in the highest (P<0.05) lipid accumulation in PM
myotubes. MitoQ(10) up-regulated genes controlling oxidative mitochondrial
biogenesis and adipogenesis in PM myotube cultures. In contrast, MitoQ(10) had a
limited effect on adipogenesis and down-regulated oxidative metabolism in ALD
myotube cultures. Differential response to MitoQ(10) treatment may be dependent on
the cellular redox state. MitoQ(10) likely controls a range of metabolic pathways
through its differential regulation of gene expression levels in myotubes derived from
fast-glycolytic and slow-oxidative muscles.
The Indiana Station reported on the cellular origins of intramuscular adipocytes in
mice. Ectopic accumulation of adipose in the skeletal muscle is associated with muscle
wasting, insulin resistance and diabetes. However, the developmental origin of
postnatal intramuscular adipose and its interaction with muscle tissue are unclear. We
report here that compared to the fast EDL muscles, slow SOL muscles are more
enriched with adipogenic progenitors and have higher propensity to form adipose.
Using Cre/LoxP mediated lineage tracing in mice, we show that intramuscular adipose
in both EDL and SOL muscles is exclusively derived from a Pax3- non-myogenic
lineage. In contrast, inter-scapular brown adipose is derived from the Pax3+ lineage. To
dissect the interaction between adipose and skeletal muscle tissues, we used Myf5-Cre
and aP2-Cre mice in combination with ROSA26-iDTR mice to genetically ablate
myogenic and adipogenic cell lineages, respectively. Whereas ablation of the myogenic
cell lineage facilitated adipogenic differentiation, ablation of the adipogenic cell lineage
surprisingly impaired the regeneration of acutely injured skeletal muscles. These results
reveal striking heterogeneity of tissue-specific adipose and a previously unappreciated
role of intramuscular adipose in skeletal muscle regeneration.
Titin is a giant protein with multiple functions in skeletal and cardiac muscles. In a
study conducted by the Wisconsin Station, titin from eight skeletal muscles: tibialis
anterior (TA), longissimus dorsi (LD) and gastrocnemius (GA), extensor digitorum
longus (ED), soleus (SO), ssoas (PS), extensor oblique (EO) and diaphram (DI) were
characterized in wild type and homozygous mutant (Hm) rats. The developmental titin
isoform transitions in protein were investigated in TA, LD and GA, and alternative
splicing of cDNAs were analyzed in TA, LD and left ventricle (LV) for both genotypes.
Titin isoforms in TA undergo the largest size change from a 3.66 MDa isoform in the
neonatal to 3.43/3.29 MDa isoforms in the adult. In contrast LD titin size only changes
from 3.70 MDa in the neonatal to 3.65 MDa in the adult. Homozygote mutant titins in
all skeletal muscles do not change with development; neonatal 3.75 MDa titin remains
dominant through 180 days of age. cDNA analysis show that adult wild type TA titin
mRNA undergoes large scale exon skipping within the middle Ig and PEVK regions. In
contrast exons in the homozygous TA, wild type LD and homozygous LD are
constitutively spliced in the coding regions of most of the middle Ig and the 5’ end of
the PEVK. Titin alternative splicing is initiated at different times in different tissues, and
within each tissue, different regions of titin mRNA were alternatively spliced at
different times. Intron retention in the cDNA near the N2B unique region reveals
possible mechanisms for the absence of N2B titin protein in skeletal muscles.
Objective 3. Characterize mechanisms of protein assembly and degradation in
skeletal muscle
The Nebraska Station reported on the degradation of myofibril proteins. Bovine
skeletal muscle (stearnomandibularis) was used to investigate the easily releasable
myofilament (ERM) fraction of myofibrils. The ERM isolated in this work were
subsequently characterized by 2-dimentional electrophoresis and mass spectrometry.
Two dimensional electrophoresis of ERM filaments resulted in a considerably different
separation pattern when compared to that of myofibrils. Several additional spots were
observed in region of the 2-D separation corresponding to actin, tropomyosin, and
troponin. Mass spectroscopy was used to determine the identity of predominant spots
in the ERM separation. Additional analysis of the MS/MS data revealed that
polypeptides in these spots contained post translational modifications that resulted in
differential migration. For example, six spots of the same Mr were identified as posttranslationally modified alpha actin. These spots contained varying degrees of
phosphorylation, glycosylation, biotinylation, and even Lys-linked polyglutamate.
Examination of additional spots revealed similar post-translational modifications in
tropomyosin, troponin, and other myofibrillar polypeptides. Ubiquitin was also
identified in the peptide profiles of several spots. These findings are significant in that
they provide evidence for the proteasome pathway for degradation of myofibrillar
proteins.
The mechanisms controlling thin filament length in muscle remain controversial. It was
recently reported that thin filament length was related to titin size, and that the latter
might be involved in thin filament length determination. Titin plays several crucial
roles in the sarcomere, but its function as it pertains to the thin filament has not been
explored. The Wisconsin Station tested this relationship using several muscles from
wild type rats and from a mutant rat model, which results in increased titin size.
Myofibrils were isolated from skeletal muscles (extensor digitorum longus, external
oblique, gastrocnemius, longissimus dorsi, psoas major, and tibialis anterior) using both
adult wild type (WT) and homozygous mutant (HM) rats (n=6 each). Phalloidin and
antibodies against tropomodulin-4 and nebulin’s N-terminus were used to determine
thin filament length. The WT rats studied express skeletal muscle titin sizes ranging
from 3.2 to 3.7 MDa, while the HM rats express a giant titin isoform sized at 3.8 MDa.
No differences in phalloidin-based thin filament length, nebulin distance, or
tropomodulin distance were observed across genotypes. However, the HM rats
demonstrated a significantly increased (p<0.01) rest sarcomere length relative to the WT
phenotype. It appears that the increased titin size, predominantly observed in HM rats’
middle Ig domain, allows for increased extensibility. The data indicates that, although
titin performs many sarcomeric functions, its correlation with thin filament length and
structure could not be demonstrated in the rat.
Although in vivo studies have indicated that androgens affect protein synthesis and
protein degradation rate in muscle, results from in vitro studies have been inconsistent.
The Minnesota Station examined the effects of trenbolone acetate (TBA), a synthetic
androgen, on protein synthesis and degradation rates in fused bovine satellite cell (BSC)
cultures. Additionally, we have examined the effects of compounds that interfere with
binding of TBA or insulin-like growth factor-1 (IGF-1) to their respective receptors on
TBA-induced alterations in protein synthesis and degradation rates in BSC cultures.
Treatment of fused BSC cultures with TBA results in a concentration-dependent
increase (P < 0.05) in protein synthesis rate and a decrease (P < 0.05) in degradation rate,
establishing that TBA directly affects these parameters. Flutamide, a compound that
prevents androgen binding to the androgen receptor, suppresses (P < 0.05) TBAinduced alterations in protein synthesis and degradation in fused BSC cultures,
indicating the androgen receptor is involved. JB1, a competitive inhibitor of IGF-1
binding to the type 1 IGF receptor (IGF1R), suppresses (P < 0.05) TBA-induced
alterations in protein synthesis and degradation, indicating that this receptor also is
involved in the actions of TBA on both synthesis and degradation. In summary, our
data show that TBA acts directly to alter both protein synthesis and degradation rates in
fused BSC cultures via mechanisms involving both the androgen receptor and IGF1R.
Impact Statements
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New knowledge that will be disseminated by publication in peer reviewed
journals.
Graduate students and post-doctoral fellows are being educated..
Inhibition of myostatin activity by the prodomain represents a possible means of
increasing muscle growth in pigs and chickens.
Redox signals contribute to muscle fiber types.
Intramuscular fat cells differ based upon location in the body.
N-glycosylation of matrix proteins affects satellite cell activities.
Trenbolone acetate, a common androgenic implant in beef cattle, activities are
mediated by the androgen receptor and IGFI receptor.
Alternative mRNA splicing in skeletal muscles will provide new clues about the
efficiency of protein synthesis and its control.
Identification of genes limiting organ growth.
RNAseq and microarray technologies complement each other as gene discovery
tools.
Calcium alters muscle metabolism through both kinase and phosphatase specific
pathways.
IGF-I inhibits muscle degradation through activation of protein kinase B.
Publications
Published
Boler DD, Holmer SF, Duncan DA, Carr SM, Ritter MJ, Petry DB, Hinson RB, Allee GL,
McKeith FK and J Killfer. 2010. Fresh meat and further processing characteristics of ham
muscles from finishing pigs fed Ractopamine hydrochloride (Paylean®). Journal of
Animal Science, 89: 210-220.
Clark DL, Boler DD, Kutzler LW, Jones KA, McKeith FK, Killefer J, Carr TR, and AC
Dilger. 2011. Muscle Gene Expression Associated with Increased Marbling in Beef
Cattle. Anim Biotechnology, 22:51-63.
Dahiya S, Bhatnagar S, Jiang C, Paul PK, Kuang S, Kumar A. 2011. Elevated levels of
active matrix metalloproteinase-9 cause hypertrophy and limit fibrosis in skeletal
muscle of normal and dystrophin-deficient mdx mice. Human Molecular Genetics.
20:4345-59.
Dilger AC, Spurlock ME, Grant AL, and DE Gerrard. 2010. Myostatin null mice respond
differently to dietary-induced and genetic obesity. Anim Sci Journal, 81:586-593.
Dodson, M.V., G.J. Hausman, L.L. Guan, M. Du, T.P. Rasmussen, S.P Poulos, P. Mir,
W.G. Bergen, M.E. Fernyhough, D.C. McFarland, R.P. Rhoads, B. Soret, J.M. Reecy, S.G.
Velleman and Z. Jiang. 2010. Lipid metabolism, adipocyte depot physiology and
utilization of meat animals as experimental models for metabolic research. International
Journal of Biological Science 6(7):682-690
Dodson, M.V. 2010. Publication and citation analysis: Needed academic partners.
NACTA Journal 54(3):55
Dodson, M.V and M.E. Benson. 2010. Where have our animal science graduates gone?
NACTA Journal 54(3):9-13
Dodson, M.V., Z. Jiang, M. Du and G.J Hausman. 2011. Adipogenesis: It is not just lipid
that comprises adipose tissue. Journal of Genomics 1(1):1-4
Dodson, M.V., P. Mir, G.J. Hausman, L.L. Guan, M. Du, M.E. Fernyhough and W.G.
Bergen. 2011. Obesity, metabolic syndrome and adipocytes. Journal of Lipids 721686:1-5
Dodson, M.V., G.J. Hausman, L.L. Guan, M. Du, and Z. Jiang. 2011. Potential impact of
mature adipocyte dedifferentiation in terms of cell numbers. International Journal of
Stem Cells 4:76-78
Dodson, M.V. 2011. Outside of class reading of scientific journal articles is invaluable,
and helps the instructor just as much as the student. NACTA Journal 55(3):98-99
Dodson, M.V. 2011. Is our next generation of scholars going to be capable of affording
us? NACTA Journal 55(3):101
Jin, W.W., J.M. Romao, M.V. Dodson, G.J. Hausman, S.S. Moore and L.L. Guan. 2011.
MicroRNA regulation of mammalian adipogenesis. Experimental Biology and Medicine
236:997-1004
Kamanga-Sollo, E., M. S. Pampusch, M. E. White, M. R. Hathaway, and W. R. Dayton.
2011. Effects of heat stress on proliferation, protein turnover, and abundance of heat
shock protein messenger ribonucleic acid in cultured porcine muscle satellite cells. J.
Anim Sci. 89:3473-3480.
Kamanga-Sollo, E., M. E. White, M. R. Hathaway, W. J. Weber, and W. R. Dayton. 2011.
Effect of trenbolone acetate on protein synthesis and degradation rates in fused bovine
satellite cell cultures. Domest. Anim Endocrinol. 40:60-66.
Kim, K.H., Y.S. Kim and J.Z. Yang. 2011. Skeletal muscle hypertrophic effect of
clenbuterol is additive to the hypertrophic effect of myostatin suppression. Muscle &
Nerve 43:700-707.
Lee, S.B., M.J. Cho, J.H. Kim, Y.S. Kim, H.J. Jin. 2011. Production of bioactive rockfish
(Sebastes schlegeli) myostatin-1 prodomain in an Escherichia coli system. Protein
Journal 30:52-58.
Lowe BK, Clark DL, Boler DD, Dilger AC, McKeith FK, Eggert JM, Newcom DW, and J.
Killefer. 2011. Characterization of loin shape from Duroc and Duroc composite finishing
gilts. Meat Science, 87:146-150.
Michelizzi, V.N., X. Wu, M.V. Dodson, J.J. Michal, J. Jorge-Zambrano-Varon, D.J.
McLean and Z. Jiang. 2011. A global view of 54,001 single nucleotide polymorphisms
(SNPs) on the Illumina Bovine SNP-50 Bead Chip and their transferability to water
buffalo. International of Journal Biological Science 7(1):18-27
Michelizzi, V.N., M.V. Dodson, Z. Pan, J.J. Michal, D.J. McLean, J.E. Womack and Z.
Jiang. 2010. Water buffalo genome science comes of age. International Journal of
Biological Science 6:333-349
Nierobisz, L.S., D.C. McFarland and P.E. Mozdziak. 2011. MitoQ(10) induces
adipogenesis and oxidative metabolism in myotube cultures. Comp. Biochem. Physiol.
158:125-131.
Pampusch, M. S., E. Kamanga-Sollo, M. R. Hathaway, M. E. White, and W. R. Dayton.
2011. Low-density lipoprotein-related receptor protein 1 (LRP-1) is not required for
insulin-like growth factor binding protein 3 (IGFBP-3) to suppress L6 myogenic cell
proliferation. Domest. Anim Endocrinol. 40:197-204.
Park S, T.L. Scheffler, D.E. Gerrard. 2011. Chronic high cytosolic calcium decreases
AICAR-induced AMPK activity via calcium/calmodulin activated protein kinase II
signaling cascade. Cell Calcium. 50(1):73-83.
Rybakova, I.N., M.L. Greaser, and R.L. Moss. 2011. Myosin binding protein C
interaction with actin: characterization and mapping of the binding site. J. Biol. Chem.
286:2008-2016.
Scheffler T.L., Park S., and Gerrard D.E. 2011. Lessons to learn about postmortem
metabolism using the AMPK3(R200Q) mutation in the pig. Meat Sci. 89(3):244-250.
Sporer KR, Chiang W, Tempelman RJ, Ernst CW, Reed KM, Velleman SG, Strasburg
GM. 2011. Characterization of a 6K oligonucleotide turkey skeletal
muscle
microarray. Anim. Genet. 42(1):75-82
Sporer KR, Tempelman RJ, Ernst CW, Reed KM, Velleman SG, Strasburg GM.
2011.
Transcriptional profiling identifies differentially expressed genes in developing turkey
skeletal muscle. BMC Genomics 12:143 – 156.
Tavarez MA, Boler DD, Bess KN, Zhao J, Yan F, Dilger AC, McKeith FK, and J Killefer.
2011. Effect of antioxidant inclusion and oil quality on broiler performance, meat
quality, and lipid oxidation. Poultry Science, 90:922-930.
In press, accepted or submitted
Angione A, Jiang C, Pan D, Wang Y, Kuang S. PPARg regulates satellite cell
proliferation and skeletal muscle regeneration. Skeletal Muscle. In press.
Boler DD, Killefer J, Meeuwse DM, King VL, McKeith FK, and AC Dilger. 2011. Effects
of harvest time post-second injection on carcass cutting yields and bacon characteristics
of immunologically castrated male pigs. Journal of Animal Science (in press).
Chakkalakal JV, Kuang S, Buffelli M, Lichtman JW, Sanes JR. 2011. Mouse transgenic
lines that selectively label slow (type I), intermediate (Type IIa) and fast (types IIx and
IIb) skeletal muscle fibers. Genesis. In press. DOI: 10.1002/dvg.2079
England E.M., K.D. Fisher, S.J. Wells, D.A. Mohrhauser, D.E. Gerrard, and A.D. Weaver.
2011. Postmortem titin proteolysis is influenced by sarcomere length in bovine muscle.
J. Anim. Sci. In press.
Guo, W., S. Bharmal, K. Esbona, and M. Greaser. 2010. Titin diversity - alternative
splicing gone wild. J. Biomed. Biotechnol. 2010: ID 753675 (8 pg). PMCID: PMC2843904.
He, M.L., P.S. Mir, R. Sharma, K. Schwartzkopf-Genswein, T. Entz, G. Travis, M.E.R.
Dugan, D. Rolland, E.K. Okine and M.V. Dodson. 2011. Effect of supplementation of
beef cattle diets with oil containing n6 and n3 fatty acids and subjecting the steers to 48h
feed withdrawal treatments on animal productivity, carcass characteristics and fatty
acid composition. Livestock Science doi:10.1016/j.livsci.2011.08.002
Liu W*, Liu Y*, Lai X, Kuang S. Intramuscular adipose is derived from a non-Pax3
lineage and required for efficient muscle regeneration. Developmental Biology. In press.
10.1016/j.ydbio.2011.10.011.
Nierobisz, L.S., K.R. Sporer, G.M. Strasburg, K.M. Reed, S.G. Velleman, C.M. Ashwell,
J.V. Felts and P.E. Mozdziak. 2011. Differential expression of genes characterizing
myofiber phenotype. Animal Genetics doi: 10:1111/j.1365-2052.2011.02249.x
Sollero, B.P., S.E.F. Guimarães, V.D. Rilington, R.J. Tempelman, N.E. Raney, J.P. Steibel,
J.D. Guimarães, P.S. Lopes, M.S. Lopes and C.W. Ernst. 2011. Transcriptional profiling
during foetal skeletal muscle development of Piau and Yorkshire-Landrace crossbred
pigs. Anim. Genet. doi:10.1111/j.1365-2052.2011.02186.x.
Wen Y, Bi P, Keller C, Kuang S. Constitutive Notch activation upregulates Pax7 and
promotes the self-renewal of skeletal muscle satellite cells. Submitted.
Invited presentations and abstracts
England E.M., Scheffler J.M., Park S., Kasten S.C., Scheffler T.L., Zhu H., Fisher K.D.,
Reinholt B.M., Van Eyk G.R., Stevenson J.M., Roberson R.C., Gerrard D.E. 2011.
Proteolysis may be controlled by postmortem energy metabolism. 56th Annual
International Congress of Meat Science and Technology. Ghent, Belgium. 005.
Ernst, C.W., J.P. Steibel, B.P. Sollero, G.M. Strasburg, S.E.F. Guimarães and N.E. highthroughput sequencing and cross-platform comparison with gene expression
microarrays. J. Anim. Sci. 89(E-Suppl. 1):ii. Late-breaking Original Research Selected
Abstract.
Fisher, K.D., T.L. Scheffler, S.C. Kasten, B.M. Reinholt, G.R. van Eyk, J.E. Escobar, J.M.
Scheffler, and D.E. Gerrard. 2011. Pre-pubertal pigs as a model for childhood obesity.
Experimental Biology. Washington D.C. 109.1.
Guo, Wei, M. Greaser, S. Li, H. Schulz, K. Saar, M. Radke, Michael T. Hacker, K. Saupe,
and M. Gotthardt. 2011. A novel splicing factor that affects titin alternative splicing.
Biophys. J., 100 (3 Supplement 1), 287a.
Haq, W.Y., H.C. Kang, S.K. Kang, J.A. Park, Y.J. Choi, C.N. Lee and Y.S. Kim. 2011.
Soluble expression of porcine myostatin propeptide in an Escherichia coli expression
system. Asian Congress on Biotechnology 2011, May 11-15, Shanghai, China.
Kamanga-Sollo, E., M. S. Pampusch, M. E. White, M. R. Hathaway, and W. R. Dayton.
2011.Effects of heat stress on proliferation, protein turnover, and levels of heat shock
protein mRNAs in cultured porcine muscle satellite cells. J. Anim. Sci. 89-E-supplement
1:71.
McCann, M.A., J. M. Scheffler, S.P. Greiner, M.D. Hanigan, G.A. Bridges, S.L. Lake, J.M.
Stevenson, H. Jiang, T.L. Scheffler and D.E. Gerrard. 2011. Early metabolic imprinting
events increase marbling scores in fed cattle. ASAS. M59.
Nayananjalie, W.A.D., M. Bell, J.M. Scheffler, H. Jiang, M. A. McCann, D. E. Gerrard, J.
Escobar and M. D. Hanigan. 2011. Effect of early grain feeding on ADG and signaling
proteins for protein synthesis in the muscle tissues of beef animals. ASAS. M307.
Nayananjalie, W. A. D., T. R. Wiles, S. Arriola, M. Aguliar, J. Escobar, M. A. McCann, D.
E. Gerrard, M. L. McGilliard, and M. D. Hanigan. 2011 Acetate clearance rates and
postabsorptive capacity to utilize acetate by beef steers. ASAS. M309
Park, S., S.S. Rossie, E. England, H. Zhu, J.M. Scheffler, S.C. Kasten, T.L. Scheffler, K.D.
Fisher, and D.E. Gerrard. 2011. Long-term high calcium decreases AMPK-activated
protein kinase activity via activating protein phosphatase 2A. Experimental Biology.
Washington D.C. LB136.
Park S., H. Zhu, S.C. Kasten, E.M. England, B.M. Reinholt, G.R. VanEyk, R.C. Roberson,
K.D. Fisher, T.L. Scheffler, J.M. Scheffler, and D.E. Gerrard. 2011. Myogenic progenitor
cells in runt pigs. 56th Annual International Congress of Meat Science and Technology.
Ghent, Belgium. P094.
Yarlagadda, S., C.N. Lee, Y.S. Kim, J.Y. Yang and W.Y. Ho. 2011. Effect of transgenic
myostatin depression on reproductive parameters and placental superoxide dismutases
in mice. 2011 Joint ADSA and ASAS Annual Meeting.
Zhu, H., S. Park, J.M. Scheffler, E.M. England, S.C. Kasten, T.L. Scheffler, K.D. Fisher,
B.M. Reinholt, G.R. VanEyk, J.M. Stevenson, R.C. Roberson, and D.E. Gerrard. 2011.
Characterization of porcine satellite cells. 56th Annual International Congress of Meat
Science and Technology. Ghent, Belgium. P095.
Book chapters
Greaser, M.L. and W. Guo. 2011. “Post-mortem Muscle Chemistry” In: “Handbook of
Meat and Meat Processing, Second Edition”, Taylor and Francis (in press).
Greaser, M.L. and C.M. Warren. 2011. “Protein electrophoresis in agarose gels for
separating high molecular weight proteins” In: Protein Electrophoresis: Methods and
Protocols, Humana Press (in press).