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DEVELOPMENTAL DYNAMICS 216:177–189 (1999)
Protein Kinase C Activity Regulates Slow Myosin Heavy
Chain 2 Gene Expression in Slow Lineage
Skeletal Muscle Fibers
JOSEPH X. DIMARIO* AND PHILLIP E. FUNK
Department of Cell Biology and Anatomy, The Chicago Medical School, North Chicago, Illinois
ABSTRACT
Expression of the slow myosin
heavy chain (MyHC) 2 gene defines slow versus
fast avian skeletal muscle fiber types. Fetal, or
secondary, skeletal muscle fibers express slow
MyHC isoform genes in developmentally regulated patterns within the embryo, and this patterning is at least partly dependent on innervation in vivo. We have previously shown that slow
MyHC 2 gene expression in vitro is regulated by a
combination of innervation and cell lineage. This
pattern of gene expression was indistinguishable
from the pattern observed in vivo in that it was
restricted to innervated muscle fibers of slow
muscle origin. We show here that slow MyHC 2
gene expression in the slow muscle fiber lineage
is regulated by protein kinase C (PKC) activity.
Inhibition of PKC activity induced slow MyHC 2
gene expression, and the capacity to express the
slow MyHC 2 gene was restricted to muscle fibers
of slow muscle (medial adductor) origin. Fast
muscle fibers derived from the pectoralis major
did not express significant levels of slow MyHC 2
with or without inhibitors of PKC activity. This
differential expression pattern coincided with
different inherent PKC activities in fast versus
slow muscle fiber types. Furthermore, overexpression of an unregulated PKC␣ mutant suppressed slow MyHC 2 gene expression in muscle
fibers of the slow lineage. Lastly, denervation of
skeletal muscles caused an increase in PKC activity, particularly in the slow medial adductor
muscle. This increase in PKC activity was associated with lack of slow MyHC 2 gene expression in
vivo. These results provide a mechanistic link
between innervation, an intracellular signaling
pathway mediated by PKC, and expression of a
muscle fiber type-specific contractile protein
gene. Dev Dyn 1999;216:177–189.
r 1999 Wiley-Liss, Inc.
genes expressed, and in particular, the myosin heavy
chain (MyHC) genes expressed, affect and directly
correlate with the contraction/relaxation characteristics of individual muscle fibers (Reiser et al., 1985,
1988). The numerous MyHC isoform genes identified in
mammalian and avian genomes encode protein isoforms with either relatively fast or slow ATPase activities (Wydro et al., 1983; Saez et al., 1986; Robbins et al.,
1986). Expression of distinct subsets of these MyHC
genes results in an individual muscle fiber being classified physiologically, biochemically, and molecularly as
fast, slow, or mixed (fast and slow).
Central unresolved questions regarding the phenotypic differences among skeletal muscle fibers include
the mechanisms by which differences arise during
myogenesis and the means by which these differences
are maintained after myogenic differentiation. Previous work in avian model systems has demonstrated
that diversity among early muscle fiber types is present
as they initially form in the limb (Crow and Stockdale,
1986a; Page et al., 1992). These primary muscle fiber
types exhibit differences in expression of the slow
MyHC 2 gene in vivo and in clonal cultures of embryonic myoblasts (Miller and Stockdale, 1986a,b). The
commitment of particular embryonic myoblast cell types
to the formation of different primary muscle fiber types
is intrinsic to the myoblasts and is stable in vivo
(DiMario et al., 1993). Diversification of muscle fiber
types in a homogeneous in vitro environment and in
vivo, before functional innervation of primary muscle
fibers, has led to consideration of distinct myogenic cell
lineages with intrinsically restricted developmental
potentials as contributors to initial muscle fiber diversity (Stockdale, 1992; 1997).
Although the significance of myogenic cell lineages
with respect to primary muscle fiber types in avian
muscle is well established, and while concepts of myogenic cell lineage are becoming increasingly clear in
mammalian skeletal muscle as well (Cusella-DeAngelis
Key words: muscle; protein kinase C; myosin
heavy chain; phosphorylation
INTRODUCTION
Vertebrate skeletal muscle is composed of muscle
fibers with diverse arrays of expression of musclespecific contractile protein genes. The repertoire of
r 1999 WILEY-LISS, INC.
Abbreviations used: Protein kinase C (PKC); protein kinase A
(PKA); myosin heavy chain (MyHC); medial adductor (MA); pectoralis
major (PM); basic helix-loop-helix (bHLH); myogenic regulatory factor
(MRF).
*Correspondence to: J.X. DiMario, The Chicago Medical School,
Department of Cell Biology and Anatomy, 3333 Green Bay Road,
North Chicago, IL 60064. E-mail: dimarioj@finchcms.edu
Received 7 May 1999; Accepted 9 July 1999
178
DIMARIO AND FUNK
et al., 1994; Pin and Merrifield, 1993; Rosenblatt et al.,
1996), less clear are the mechanisms that initially
regulate and maintain muscle fiber type diversity and
MyHC isoform gene expression in fetal or secondary
muscle fibers. Earlier studies of fetal chicken myoblasts
revealed no differences in MyHC gene expression among
the fibers formed in vitro (Schafer et al., 1987). This
was in contrast to secondary muscle fibers in vivo which
can be easily distinguished as ‘‘fast’’ or ‘‘slow’’ based on
monoclonal antibody immunostaining (Page et al.,
1992).
The discrepancy between in vitro and in vivo expression of the slow MyHC 2 gene in secondary muscle
fibers suggested that extrinsic factors control slow
MyHC 2 gene expression in vivo and was at first in
agreement with the hypothesis that extrinsic factors
such as innervation were solely responsible for modulation of secondary fiber type. The significant role of
innervation and patterns of neuronal firing on expression of contractile protein genes has been well established by denervation and cross-reinnervation studies
that showed transition of muscle fiber type based on
neural activity (Pette and Vrbova, 1985; Gauthier et al.,
1983; Crow and Stockdale, 1986a; Condon et al., 1990).
In an attempt to recapitulate neuronal regulation of
secondary muscle fiber type diversity observed in vivo,
we have previously shown that this fiber type diversity
in vitro can be induced by functional innervation of
muscle fibers by neurons from embryonic spinal cords
(DiMario and Stockdale, 1997). This diversity was
manifested by expression of the slow MyHC 2 gene.
Interestingly, expression of the slow MyHC 2 gene
occurred only in innervated muscle fibers formed from
fetal myoblasts of the slow medial adductor (MA)
muscle. Secondary muscle fibers derived from fast
pectoralis major (PM) muscle myoblasts did not express
the slow MyHC 2 gene in vitro in the absence or
presence of functional innervation. These results demonstrated that slow MyHC 2 gene expression in secondary muscle fibers is regulated by both intrinsic and
extrinsic mechanisms. Differentiation of slow and fast
myoblasts in the presence of innervation in vitro invokes both of these mechanisms to recapitulate the
pattern of slow MyHC 2 gene expression observed in
vivo.
The mechanism by which innervation regulates genes
that define muscle fiber type is only recently beginning
to be unraveled. One component in this mechanism
may be the calcium-regulated serine/threonine phosphatase, calcineurin. Constitutively active calcineurin increases expression of the slow troponin I (TnIs) gene in
muscle fibers (Chin et al., 1998). This regulatory pathway may be mediated by interactions of calcineurin
with NFAT and MEF2 transcription factors leading to
slow fiber type-specific expression of contractile protein
genes. Calcineurin-regulated expression of TnIs probably occurs through reduction of transcription factor
phosphorylation. This follows previous findings that
the activity of protein kinases can reduce transcrip-
tional activity of muscle-specific promoters. Reduced
capacity of myogenic regulatory factors (MRFs) to
trans-activate muscle-specific genes may be due to a
direct change in the phosphorylation state of a MRF(s)
(Li et al., 1992). Interestingly, other studies have shown
altered muscle-specific gene trans-activation without
clear correlation of the phosphorylation state of individual MRFs, suggesting combinatorial interactions of
MRFs with other phosphorylated factors (Mitsui et al.,
1993; Winter et al., 1993; Hardy et al., 1993; Johnson et
al., 1996).
To understand the cellular mechanism regulating
MyHC gene expression under the influence of innervation, we have begun to investigate potential regulatory
pathways mediated by protein kinase C (PKC). PKC is
a serine/threonine kinase with multiple isoforms in a
protein family. PKC isoforms comprise subgroups based
on dependence of calcium for activity. Of the known
PKC isoforms, PKC␣ and PKC␪ are the predominant
isoforms in skeletal muscle (Osada et al., 1992; Hilgenberg and Miles, 1995; Donnelly et al., 1994). PKC␣ is a
member of the conventional PKC (cPKC) subgroup that
is activated by phorbol esters and calcium. PKC␪ is a
member of the novel PKC (nPKC) subgroup of PKC
isoforms that are activated by phorbol esters but are
not dependent on calcium for activation. PKC is preferentially localized to neuromuscular junctions in innervated muscle fibers, suggesting that a signaling pathway, initiated by functional innervation, may be
mediated by PKC in the immediate cellular context of
the neuromuscular junction (Hilgenberg and Miles,
1995; Hilgenberg et al., 1996).
In this study, we demonstrate that slow MyHC gene
expression in fetal muscle fibers in vitro can for the first
time be elicited without functional innervation. Expression of the slow MyHC 2 gene in slow muscle fibers is
dependent on activity of protein kinase C. Furthermore, although PKC activity can regulate slow MyHC 2
gene expression, its effect is restricted by intrinsic
qualities resident in different types of secondary muscle
fibers. The pattern of slow MyHC gene expression
reflects the diversity of fiber types observed with innervation in vitro and in vivo. These results provide
evidence for a signaling pathway mediated by PKC that
unites functional innervation with the intrinsic and
extrinsic qualities of secondary muscle fiber type lineages ultimately leading to expression of muscle fiber
type-specific genes.
RESULTS
Inhibition of PKC Induces Slow MyHC 2
Expression in MA Muscle Fibers
Previously, we reported that avian secondary muscle
fibers formed from myoblasts of slow muscle origin
expressed the slow MyHC 2 gene in vitro only when
innervated (DiMario and Stockdale, 1997). To unravel
the cellular mechanism of innervation-induced slow
MyHC 2 gene expression in these muscle fibers, the
potential involvement of protein kinase activities was
PKC ACTIVITY REGULATES SLOW MyHC 2 GENE EXPRESSION
179
Fig. 1. Inhibition of protein kinase
activity by staurosporine induced slow
MyHC gene expression in vitro in
medial adductor muscle fibers. ED13
pectoralis major (PM) (A, C, E, G)
and medial adductor (MA) (B, D, F, H)
myoblasts were isolated and cultured
for 7 days. Well-differentiated muscle
fibers were present by day 3 of incubation. At that time, medium was
replaced with control medium (A–D)
or medium containing the protein kinase inhibitor, staurosporine, (E–H)
at a concentration of 4 ng/ml. Cultures were immunostained with the
anti-fast MyHC mAb, F59, (A, B, E, F)
and the anti-slow MyHC specific mAb,
S58, (C, D, G, H) followed by Texas
Red and fluorescein-conjugated secondary antibodies, respectively. All
PM and MA muscle fibers immunostained for fast MyHC with F59. PM
and MA muscle fibers incubated in
control medium did not express detectable amounts of slow MyHC. However, in medium containing staurosporine, slow MyHC was detected by
S58 immunostaining in MA muscle
fibers (H). Slow MyHC expressing
muscle fibers were rarely present in
PM muscle fiber cultures incubated in
medium containing staurosporine (G).
examined. In initial experiments, the protein kinase
inhibitor, staurosporine, was used at several concentrations. Staurosporine is a potent inhibitor of PKC activity (IC50 ⫽ 0.7 nM) but is also a cell permeable inhibitor
of protein kinase A (PKA) (IC50 ⫽ 7 nM) and protein
kinase G (PKG) (IC50 ⫽ 8.5 nM). Staurosporine was
added to cell culture medium at concentrations of 1, 4,
and 10 ng/ml on day 3 of incubation of differentiated
muscle fibers in vitro. This range of staurosporine
concentrations has been shown to alter expression of
NCAM and acetylcholine receptor ␣ subunit genes
(Klarsfeld et al., 1989; Rafuse and Landmesser, 1996).
Muscle fibers were formed from myoblasts isolated
from embryonic day 13 pectoralis major (PM) and
medial adductor (MA) muscles. In vivo, the PM contains muscle fibers that express exclusively fast MyHC
genes, whereas the medial adductor contains muscle
fibers that express fast MyHCs and slow MyHCs 1 and
2 (Page et al., 1992). After 4 days of incubation in the
presence of staurosporine, muscle fiber cultures were
immunostained with monoclonal antibodies F59 and
S58 that specifically recognize fast MyHCs and slow
MyHCs 2 and 3, respectively (Crow and Stockdale,
1986a,b).
Figure 1 illustrates the effect of staurosporine on
slow MyHC gene expression in PM and MA muscle
180
DIMARIO AND FUNK
fibers in vitro. In the absence of staurosporine, both
cultures of PM and MA muscle fibers expressed only
fast MyHCs (Fig. 1A and B). Slow MyHC expression
was not detected in these cultures (Fig. 1C and D).
Similarly, all PM muscle fibers cultured in the presence
of 4 ng/ml staurosporine expressed fast MyHCs and
expressed slow MyHCs only infrequently (less than
10% of muscle fibers) (Fig. 1E and G). However, the
majority (⬇80%) of MA muscle fibers incubated in
medium containing staurosporine expressed a slow
MyHC gene(s) (Fig. 1F and H). Fewer muscle fibers in
both PM and MA muscle fiber cultures immunostained
with S58 when cultures contained 1 ng/ml staurosporine. Conversely, more S58 immunostaining was apparent in cultures that contained 10 ng/ml staurosporine
(not shown). However, muscle fiber morphology was
compromised at this higher concentration. All further
experiments using staurosporine were performed at a
concentration of 4 ng/ml. Therefore, addition of staurosporine to the cell culture medium suggested that a
serine/threonine protein kinase was involved in the
expression of slow MyHC. Furthermore, this expression
was preferentially restricted to muscle fibers derived
from myoblasts of a slow muscle.
To better define the serine/threonine kinase activity
that regulates slow MyHC gene expression, compounds
that specifically inhibit PKA, PKG, and PKC were
tested for their ability to mimic the effect of staurosporine and induce slow MyHC gene expression in MA
muscle fibers in vitro. The compound, N-[2-((p-Bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, is a
cell-permeable, highly selective inhibitor of protein
kinase A (Ki ⫽ 48.0 nM). Protein kinase C activity is
inhibited only at substantially higher (660-fold) concentrations (Ki ⫽ 31.7 µM). The highly selective PKA
inhibitory peptide 14–22, myristoylated to increase its
cell permeability, also inhibits PKA (Ki ⫽ 36 nM)
activity with no detectable inhibition of PKC activity.
These PKA inhibitory compounds in normal culture
medium were added separately to differentiated muscle
fiber cultures on day 3 of incubation and cultured for an
additional 4 days. The concentrations used for both
inhibitors were 10, 20, 50, 100, 200, and 500nM and 1,
2, 5, and 10 µM — up to 210 times the PKA Ki value. In
both PM and MA cultures, with or without either PKA
inhibitor at any concentration, no slow MyHC gene
expression was detected by immunostaining with monoclonal antibody (mAb) S58. Muscle fibers with normal
morphology were prominent in all cultures and immunostained for fast MyHC with mAb F59. These results
strongly suggest that PKA activity is not directly
involved in slow MyHC gene expression, but that some
other protein kinase activity, inhibited by staurosporine, is involved.
To test the possible role of protein kinase G (cGMPdependent protein kinase) activity in slow MyHC gene
expression in the culture system, guanosine 38,58-cyclic
monophosphorothioate, 8-(4-chlorophenylthio)-, Rp-
isomer, was used. This compound is highly potent (Ki ⫽
0.5 µM), cell permeable, and selective for PKG with no
detectable inhibitory activity for PKA or PKC. PM and
MA muscle fiber cultures were incubated in medium
containing this PKG inhibitor at concentrations of 0.5,
1.0, 2.0, 5.0, 10, 20, and 50 µM for 4 days. Cultures were
immunostained with mAbs F59 and S58. All cultures
contained muscle fibers that immunostained with F59
for fast MyHCs. However, no muscle fiber also immunostained with S58 for the presence of slow MyHC.
To determine the possible role of PKC activity in the
regulation of slow MyHC gene expression, the compound, bisindolylmaleimide I (GF109203X), was added
to cultures of PM and MA muscle fibers. GF109203X is
a highly potent (Ki ⫽ 10 nM), cell permeable, and
specific inhibitor of PKC activity. GF109203X is, furthermore, a selective inhibitor of calcium-dependent PKC
isoforms such as PKC␣ (Altiok et al., 1995; Toullec et
al., 1991). It inhibits PKA activity only at 200-fold (2
µM) higher concentrations. Incubation of cultures in 10
and 20 nM GF109203X resulted in slow MyHC gene
expression in MA muscle fibers as determined by
immunostaining with mAb S58 (Fig. 2). PM cultures
had no detectable slow MyHC at either 10 or 20 nM
GF109203X.
Studies were also performed to examine the possible
role of the MAP kinase signaling pathway as an alternative to a mechanism involving PKC in the regulation of
slow MyHC gene expression. The compound, PD98059,
prevents phosphorylation of MEK (IC50 ⫽ 2 µM) and
thereby inactivates the Ras/MAP kinase signaling pathway (Pang et al., 1995). PM and MA muscle fiber
cultures were incubated in 2 to 100 µM PD98059 for 4
to 7 days and then immunostained with F59 and S58.
All muscle fibers expressed fast MyHCs but none
expressed a slow MyHC. Therefore, inhibition of the
Ras/MAP kinase signaling mechanism by PD98059 had
no significant effect on the regulation of the slow MyHC
gene in both PM and MA muscle fiber cultures.
In total, these results suggest that a serine/threonine
kinase regulates slow MyHC gene expression in slow
MA muscle fibers and that this kinase activity resides
in protein kinase C. Furthermore, lack of slow MyHC
gene expression in PM muscle fibers cultured in the
presence of PKC inhibitors suggests that slow MyHC
gene expression in these fibers is regulated by a different, fiber type-specific, mechanism.
Monoclonal antibody S58 recognizes both slow MyHC
2 and MyHC 3 (Page et al., 1992). To determine whether
slow MyHC 2 gene expression was induced by inhibition of protein kinase C activity, northern blot analysis
was performed (Fig. 3). Total RNAs were isolated from
separate day 7 cultures of PM and MA muscle fibers
only, separate day 7 co-cultures of PM and MA muscle
fibers plus embryonic spinal cord explants, as well as
from individual PM and MA muscle fiber cultures
incubated in medium containing 4 ng/ml staurosporine,
or medium containing 10 nM GF109203X. RNA blots
PKC ACTIVITY REGULATES SLOW MyHC 2 GENE EXPRESSION
181
Fig. 2. The protein kinase C specific inhibitor, bisindolylmaleimide I
(GF109203X), induced slow MyHC
gene expression in vitro in MA muscle
fibers only. ED13 PM (A, C) and MA
(B, D) muscle fibers were cultured for
7 days. Bisindolylmaleimide I was
added to the culture medium on days
3 and 5 of incubation at a concentration of 20 nM. Cultures were immunostained with anti-fast MyHC mAb,
F59 (A, B) and anti-slow MyHC 2
mAb S58 (C, D) followed by Texas
red and fluorescein-conjugated secondary antibodies, respectively. All
PM and MA muscle fibers expressed
a fast MyHC gene(s) (A, B) but only
MA muscle fibers expressed slow
MyHC in the presence of the PKC
inhibitor, bisindolylmaleimide I (D).
Fig. 3. Northern blot analysis of PM and MA muscle fiber cultures
shows slow MyHC 2 gene expression in MA cultures only. PM and MA
muscle fiber cultures were established and incubated as before without
embryonic spinal cord explants (PM and MA), with embryonic spinal cord
explants from day 3–7 of incubation (⫹ Nerves), or in medium containing
either 4 ng/ml staurosporine (⫹ Stauro) or 20 nM GF109203X (⫹ GF). (A)
RNA samples were probed with a slow MyHC 2 specific oligonucleotide
(See Methods.) Slow MyHC 2 mRNA was not detected in PM RNA
samples from any experimental condition. Slow MyHC 2 mRNA was also
not detected in MA RNA samples, but it was detected when MA muscle
fibers were incubated in the presence of innervation as previously
described (DiMario and Stockdale, 1997) and when culture medium
contained staurosporine or GF109203X. (B) Ethidium bromide staining of
aliquots of each RNA sample in an agarose gel shows approximate equal
loading of rRNA.
were probed with a slow MyHC 2 oligonucleotide that
does not cross-hybridize with slow MyHC 1 or MyHC 3
mRNAs (DiMario and Stockdale, 1997). Slow MyHC 2
mRNA was not detected in extracts of PM muscle fibers
in any experimental condition. It was also not detected
in extracts of MA muscle fibers in vitro without innervation or PKC inhibitors. However, slow MyHC 2 mRNA
was abundant in extracts from MA muscle fibers cocultured with embryonic spinal cord explants and in
MA muscle fibers cultured in the presence of either
staurosporine or GF109203X. Therefore, inhibition of
PKC activity resulted in expression of the slow MyHC 2
gene in MA muscle fibers in vitro and resulted in no
significant expression of the slow MyHC 2 gene in PM
muscle fibers in the same culture conditions.
PKC Activity Differs Between MA and PM Muscle
Fibers In Vitro
Addition of staurosporine to culture medium resulted
in slow MyHC 2 gene expression in MA muscle fibers
and in few PM muscle fibers (Fig. 1). Furthermore,
decreased staurosporine concentrations resulted in
fewer S58 immunostained MA and PM muscle fibers. A
possible explanation of these results is that the difference between slow MyHC 2 gene expression in MA and
PM muscle fibers cultured in the presence of PKC
182
DIMARIO AND FUNK
TABLE 1. PKC Activity in PM and MA Muscle
Fibers In Vitroa
PM
MA
(pmol/min/mg) (pmol/min/mg)
Control muscle fibers
64.84 ⫾ 8.42
25.45 ⫾ 11.51**
Control plus staurosporine
7.16 ⫾ 2.14*
6.54 ⫾ 1.71*
PKC-7-muscle fibers
3,186 ⫾ 226
2854 ⫾ 197
aPKC activities were measured in whole cell extracts of PM
and MA muscle fibers in vitro, some of which were transfected
with PKC-7. MA PKC activity in muscle fibers in vitro was
significantly less than PKC activity in cultured PM muscle
fibers (**p ⫽ 0.026). Some PKC assays included 4 ng/ml
staurosporine (control plus staurosporine). PKC activities in
both PM and MA muscle fiber extracts were significantly
reduced compared to control reactions without staurosporine
(*p ⫽ 0.005). PKC activities between PM and MA extracts in
the presence of staurosporine were not significantly different.
PKC-7 transfected PM and MA muscle fibers contained significantly greater PKC activity than nontransfected muscle fibers. PKC activities between PKC-7 transfected PM and MA
cultures were not significantly different. Assays were performed in triplicate in three independent experiments. Values
are the mean ⫾ SD. PKC activities were normalized to total
protein in cell extracts.
inhibitors is due to differences in total PKC activities
between slow and fast muscle fibers. To determine
whether differences in PKC activities correlate with
slow MyHC 2 gene expression in MA and PM muscle
fibers, PKC activities in these muscle fibers in vitro
were measured. ED13 MA and PM myoblasts were
isolated and placed into culture. Cultures contained
well-formed muscle fibers on day 4 of incubation. At
that time, cytosine arabinoside (AraC) was added to the
culture medium at a concentration of 10µg/ml to decrease the number of proliferating, nondifferentiated
cells that may have PKC activities different from
muscle fibers. PKC activities were measured in extracts
from day 7 cultures. PKC activity in cultured fast PM
muscle fibers was significantly greater (2.5-fold) than
PKC activity in cultured slow MA muscle fibers (Table 1
and Fig. 4).
PKC activities from MA and PM in vitro muscle fiber
extracts were also assayed for inhibition by staurosporine. Addition of 0.1 to 10 nM (0.05 to 4.67 ng/ml)
staurosporine resulted in equal inhibition of PKC activities relative to noninhibited activities in extracts from
MA and PM muscle fibers (Table 1 and Fig. 4). The
equal relative inhibition of PKC activities in these
assays suggests that PKC inhibition occurred to the
same relative extent in MA and PM muscle fibers in the
presence of staurosporine in vitro. Therefore, PM and
MA muscle fibers cultured in the absence of innervation
have different inherent PKC activities. Furthermore,
the capacity to express the slow MyHC 2 gene is fiber
type specific. Inhibition of PKC activity in slow MA
muscle fibers induced slow MyHC 2 gene expression.
However, equal inhibition of PKC activity in fast PM
muscle fibers did not induce slow MyHC 2 gene expression suggesting that other intrinsic differences between
Fig. 4. PM muscle fibers contain higher PKC activity relative to MA
muscle fibers in vitro. PKC activities were determined in protein extracts
from normal (with and without staurosporine; ⫹ST, -ST) and PKC-7
transfected ED13 PM and MA muscle fiber cultures. Data are derived from
three independent experiments with samples run in triplicate. Bars show
the average-fold difference in PKC activity in PM muscle fiber extracts
versus MA muscle fiber extracts in the three experiments. Averages are
different than those in Table 1 because Table 1 shows average net PKC
activities and values here show average-fold difference in PKC activities
from individual experiments. PKC activities in PM and MA muscle fiber
extracts were equally inhibited by 4 ng/ml staurosporine (⫹ST). Overexpression of PKC resulting from transfection of PKC-7 showed no
significant difference between PM and MA muscle fibers in vitro (PKC-7).
fast and slow muscle fiber types suppress slow MyHC
genes in fast muscle fibers.
Increased PKC Activity Suppresses Slow MyHC 2
Expression in MA Muscle Fibers
In addition to correlating low PKC activity with
activation of slow MyHC 2 gene expression in MA
muscle fibers, experiments were performed to increase
PKC activity and assess changes in slow MyHC 2 gene
expression. To increase PKC activity in differentiated
MA muscle fibers, the expression construct PKC-7
containing the constitutively expressed PKC␣ catalytic
subunit coupled to the hemagglutinin antigen (HA)
epitope tag was transfected into MA muscle fibers in
vitro on the third day of incubation. Some of the
transfected cultures were incubated in medium containing 4 ng/ml staurosporine immediately after transfection and were maintained in this medium for 2 days.
Cells were immunostained with an anti-HA tag antibody to identify transfected cells and with mAb S58 to
locate cells expressing the slow MyHC 2 gene.
PKC ACTIVITY REGULATES SLOW MyHC 2 GENE EXPRESSION
183
Fig. 5. Increased PKC␣ expression suppresses slow MyHC 2 gene
expression in MA muscle fibers. MA
myogenic cultures were transfected
with PKC-7. After transfection, cultures were incubated in control medium (A, B) or medium containing 4
ng/ml staurosporine (C, D). After an
additional 2 days incubation, muscle
fibers were immunostained with an
anti-HA antibody to detect transfected cells (A, C) and with mAb S58
to detect slow MyHC 2 (B, D) followed by Texas red and fluoresceinconjugated secondary antibodies, respectively. MA muscle fibers did not
express slow MyHC 2 without staurosporine (A, B). In the presence of
staurosporine, nontransfected MA
muscle fibers expressed slow MyHC
2 (arrowhead in C, D), but transfected MA muscle fibers (arrow in C,
D) never expressed slow MyHC 2.
As shown in Figure 5, transfected and nontransfected
MA muscle fibers cultured in the absence of staurosporine did not express the slow MyHC 2 gene — in
agreement with results shown in Figure 1. Nontransfected MA muscle fibers cultured with staurosporine
did express the slow MyHC 2 gene. However, no PKC-7
transfected MA muscle fiber also expressed the slow
MyHC 2 gene in the presence of staurosporine.
PKC activities in control and PKC-7 transfected MA
muscle fiber cultures were measured (Table 1 and Fig.
4). Extracts from PKC-7 transfected cultures had PKC
activities significantly greater than extracts from control, nontransfected, and pCDM8 transfected cultures.
These results indicate that increased PKC activity
suppresses slow MyHC 2 gene expression in slow
muscle fibers.
MA PKC Activity In Vivo Is Reduced Compared
to PM PKC Activity
PM and MA muscle extracts were assayed to determine whether fully innervated fast versus slow muscles
in vivo contained different PKC activities. PM and MA
muscles were dissected from ED13 chick embryos, and
PKC activities were determined as before. PKC activity
in the MA muscle extract was 38% that of PM PKC
activity (Table 2). This difference was similar to the
difference measured between PM and MA muscle fibers
in vitro. Therefore, the reduced PKC activity in MA
muscle corresponds to expression of the slow MyHC 2
gene in this muscle.
PKC Activity Is Increased in Denervated
Slow MA Muscle
Innervation and inhibition of PKC activity in MA
muscle fibers in vitro resulted in slow MyHC 2 gene
TABLE 2. PKC Activity in Normal, Control and
Denervated PM and MA Muscle Fibers In Vivoa
PM
MA
(pmol/min/mg) (pmol/min/mg)
Normal muscle
60.82 ⫾ 5.73* 22.91 ⫾ 6.27
Control PBS-treated muscle 64.78 ⫾ 7.08 21.32 ⫾ 7.54
Curare-treated muscle
75.41 ⫾ 11.30 43.07 ⫾ 10.45**
aPKC activities were measured in extracts of normal PM and
MA muscles, and control (PBS-treated) and curare-treated
PM and MA muscles. PM PKC activity in whole muscle
extracts was significantly greater than PKC activity in MA
muscles in vivo (*p ⫽ 0.0015). PKC activity in curare-treated
MA extracts was significantly greater than in control extracts
(**p ⫽ 0.020). Activities in control and curare-treated PM
muscle extracts were not significantly different. Assays were
performed in triplicate in three independent experiments.
Values are the mean ⫾ SD. PKC activities were normalized to
total protein in cell extracts.
expression. To determine whether innervation causes a
reduction of PKC activity in vivo, muscles were paralyzed by functional denervation with curare. Developing chick embryos were administered curare from
ED9-ED12. This time course was used to allow initiation of fetal, secondary fiber formation on ED8 (Page et
al., 1992). To assess the effects of curare on slow MyHC
2 gene expression in PM and MA muscles, cryosections
of normal and curare-treated ED13 PM muscle and
thigh were prepared. Sections were immunostained
with mAbs F59 and S58 (Fig. 6). Muscle fibers in
normal and curare-treated PM contained fast MyHC(s)
exclusively. No slow MyHC 2 was detected in the PM. In
the normal ED13 MA, all muscle fibers, including fetal,
secondary muscle fibers, immunostained with F59 and
S58 indicating expression of a fast MyHC gene(s) and
the slow MyHC 2 gene. However, in the curare-treated
184
DIMARIO AND FUNK
Fig. 6. Expression of fast and slow MyHC 2 in control (PBS-treated)
and denervated (curare-treated) PM and MA muscle fibers. Cryosections
of control (A–D) and curare-treated (E–H) ED13 PM (A,B,E,F) and MA
(C,D,G,H) muscles were immunostained with mAbs F59 (A,C,E,G) and
S58 (B,D,F,H) for fast and slow MyHCs, respectively. Fibers in control and
curare-treated PM muscle immunostained with F59 exclusively. All fibers
in control MA immunostained with both F59 and S58 whereas some fibers
(arrows) in curare-treated MA muscle did not immunostain with S58.
MA, some muscle fibers immunostained exclusively
with F59. No S58 immunostaining was observed in
these fibers indicating lack of slow MyHC 2 gene
expression.
PKC activities in protein extracts from control and
curare-treated ED13 PM and MA muscles were assayed
(Table 2). Activities in control PM versus MA extracts
were again significantly different. Activities in extracts
from control versus curare-treated PM were not significantly different. However, PKC activity in extracts from
curare-treated MA muscles was significantly greater
than activity in extracts from control, innervated MA.
PKC ACTIVITY REGULATES SLOW MyHC 2 GENE EXPRESSION
These results indicate that innervation reduces PKC
activity.
DISCUSSION
Skeletal muscle fibers express a diverse array of
contractile protein isoform genes. We have previously
shown that both innervation and intrinsic properties
within secondary muscle fibers of fast and slow muscle
origin contribute to muscle fiber type-specific expression of slow MyHC genes (DiMario and Stockdale,
1997). Expression of the slow MyHC 2 gene in muscle
fiber cultures was restricted to innervated MA muscle
fibers. PM muscle fibers, whether innervated or not, did
not express the slow MyHC 2 gene.
In the results reported here, the same innervationinduced pattern of slow MyHC 2 gene expression was
induced by inhibition of protein kinase C activity.
Significant levels of slow MyHC 2 gene expression were
detected only in MA muscle fibers in vitro incubated in
medium containing PKC inhibitors. This response was
also concentration-dependent since lower (1ng/ml) concentrations of staurosporine induced fewer MA muscle
fibers to express the slow MyHC 2 gene. PM muscle
fibers were generally refractory to induction of the slow
MyHC 2 gene in the presence of PKC inhibitors.
Northern blot analysis further indicated that the level
of slow MyHC 2 gene expression in PM muscle fibers
was significantly less than that of MA muscle fibers
incubated in PKC inhibitors.
The similar pattern of slow MyHC 2 gene expression
in MA muscle fibers innervated by motor neurons or
incubated with PKC inhibitors suggested that PKC
activity in MA muscle fibers may be regulated by
innervation. To address this possibility, in vivo denervation studies were performed to correlate PKC activity
with innervation status. PKC activity in curaretreated, functionally denervated MA muscle extracts
was significantly greater than activity in control extracts. The activity in denervated MA extracts approached that of normal and curare-treated PM extracts. Also denervated fetal MA muscle contained
muscle fibers that lacked slow MyHC 2 gene expression.
Therefore, innervation reduces PKC activity in MA
muscle fibers, and this reduction in PKC activity is
correlated with slow MyHC 2 gene expression, further
strengthening a link between PKC activity and slow
MyHC 2 gene expression in slow muscle fibers.
Causative evidence for the regulation of slow MyHC 2
gene expression by PKC activity comes from the transfection of MA muscle fibers with the constitutively
expressed PKC␣ catalytic subunit. MA muscle fibers
that normally expressed the slow MyHC 2 gene when
incubated in medium containing PKC inhibitor, did not
express it when transfected with PKC-7. It is likely that
the PKC inhibitors did not significantly reduce the
greatly increased PKC activity in PKC-7 transfected
cultures. These results are significant because they not
only correlate high PKC activity with suppression of
slow MyHC 2 gene expression, but also show a caus-
185
ative role for PKC activity in the downstream regulation of fiber type-specific gene expression. The fiber
type-specific reduction of PKC activity in slow MA
muscle fibers may provide a link between signals from
innervation such as depolarization and intracellular
calcium concentrations to patterns of fiber type-specific
gene expression.
Recently, the serine/threonine phosphatase, calcineurin, was shown to affect expression of slow muscle fiber
type-specific genes (Chin et al., 1998). The effects of
calcineurin activity were observed in NFAT and MEF2
transcription factors, but may also affect other musclespecific and ubiquitous transcription factors. Transcription of muscle fiber type-specific genes is controlled by
combinatorial interactions among transcription factors, and therefore the activities of calcineurin and PKC
may act on one or several different factors involved in
the transcription of a particular gene. The fact that
calcineurin activity is calcium-dependent and is regulated by patterns of neuronal firing suggests that a
unifying mechanism employing both calcineurin and
PKC is plausible. The calcium-dependent activities of
calcineurin and PKC isoforms may provide a reciprocating regulatory mechanism that controls fiber typespecific transcriptional activity in response to patterns
of innervation. Detailed analysis of the response of PKC
isoform activity to transient and sustained intracellular calcium concentrations due to patterns of neuronal
firing will more clearly define any reciprocal role of
calcineurin and PKC activities in the regulation of fiber
type-specific genes.
Other possible PKC substrates that may govern fiber
type-specific gene expression are members of the myogenic regulatory factor (MRF) family of basic helix-loophelix (bHLH) transcription factors. Although no clear
correlation between the expression of individual MRFs
and muscle fiber type has been consistently shown,
kinase activity does affect transcriptional activity of
MRF-dependent gene promoters. MyoD and myogenin
are substrates for PKC, and phosphorylation in a
conserved DNA binding domain may directly decrease
the ability of these factors to trans-activate musclespecific promoters (Li et al., 1992). Alternatively, phosphorylation may affect the ability of myogenic bHLH
monomers to form homodimers or to form heterodimers
with ubiquitous HLH factors, such as E12 and E47
(Mitsui et al., 1993). A mechanism in which phosphorylation indirectly affects myogenic bHLH transcriptional
activity is supported by studies showing altered MRF4
transcriptional activity due to protein kinase activity,
but with no direct correlation to the phosphorylation
state of MRF4 (Hardy et al., 1993; Johnson et al., 1996).
Therefore, the mechanism of MRF modulation by phosphorylation may be specific to each MRF resulting in
specific patterns of muscle-specific gene expression.
Further analysis of phosphorylation of the other myogenic bHLH factors and the degree to which specific
potential phosphorylation sites are phosphorylated by
186
DIMARIO AND FUNK
PKC will provide further insight into potential regulatory pathways.
Numerous isoforms of PKC exist, and it is not known
which isoform(s) is involved in the regulation of slow
MyHC 2 gene expression. Some indications of the
isoform(s) involved can be seen in these studies. The
PKC inhibitor, staurosporine, has known specificities
for PKC␣, ␤, ␥, ␦, ⑀, and ␨. The specificities for PKC␪ and
µ are not known. Of the known PKC isoforms, PKC␣
and PKC␪ are the predominant isoforms in skeletal
muscle (Osada et al., 1992; Hilgenberg and Miles, 1995;
Donnelly et al., 1994). It is potentially significant that
slow MyHC 2 gene expression in MA muscle fibers was
also induced by the PKC inhibitor, bisindolylmaleimide
I (GF109203X), which is specific for calcium-dependent
PKC isoforms such as PKC␣ (Toullec et al., 1991; Altiok
et al., 1995). In addition, over-expression of PKC␣ in
MA muscle fibers caused the suppression of slow MyHC
2 gene expression. These results raise the possibility
that a calcium-dependent PKC isoform has a critical
role in regulation of slow MyHC 2 gene expression,
although further studies are currently underway to
determine which specific PKC isoform is involved in the
regulation of this gene.
Previous work demonstrated that slow MyHC 2 gene
expression in fetal muscle fibers was regulated by both
extrinsic (innervation) and intrinsic, lineage-based
mechanisms (DiMario and Stockdale, 1997). This was
evident by restriction of slow MyHC 2 gene expression
in innervated muscle fibers derived from slow muscle
myoblasts. Innervated fast PM muscle fibers did not
express slow MyHC 2. The same lineage-restricted
pattern of slow MyHC 2 gene expression occurred in
muscle fibers in which PKC activity was inhibited. In
agreement with this pattern of gene expression and
corresponding PKC activities, intrinsic differences in
the regulation of PKC activity were detected between
slow MA and fast PM muscle fibers. Uninnervated MA
muscle fibers had significantly less PKC activity than
PM muscle fibers in vitro. This was also true in
innervated and denervated MA and PM muscles in
vivo. Therefore, intrinsic differences between slow MA
and fast PM muscle fibers appear to establish different
basal PKC activities in the two fiber types.
In addition to intrinsic regulation of PKC activity in
slow MA muscle fibers, PKC activity is also regulated
by innervation. Extracts of denervated MA muscle in
vivo had almost twice as much PKC activity indicating
that innervation reduces PKC activity in these fibers.
Interestingly, denervated fast PM muscle had only a
slight, nonsignificant increase in PKC activity. This
again suggests that PKC activity in fast versus slow
muscle fibers is partly regulated by intrinsic lineagebased mechanisms.
These studies show that suppression of slow MyHC 2
gene expression in fast PM muscle fibers occurs by at
least two means. The first was evident by the infrequent (⬍10%) PM muscle fibers that did express slow
MyHC 2 when cultured in staurosporine. Although
Northern blots could not detect significant amounts of
slow MyHC 2 mRNA in these cultures, even the low
frequency of slow MyHC 2-expressing fibers was greater
than in cultures without staurosporine. These cultures
contain virtually no slow MyHC 2. Therefore, the
persistently high PKC activity in innervated and denervated PM muscle fibers may suppress the limited
amount of slow MyHC 2 gene expression observed in
these cultures. Secondly, other lineage-based differences between fast and slow muscle fibers suppress
slow MyHC 2 gene expression in PM muscle fibers.
Addition of staurosporine to PM muscle extracts effectively inhibited PKC activity. If PKC activity were the
sole regulator of slow MyHC 2 gene expression in PM
muscle fibers, then all PM muscle fibers would express
it in culture with staurosporine. Yet only a small
percentage of them did so. Therefore, the lack of slow
MyHC 2 gene expression in these fibers cultured with
staurosporine suggests that other intrinsic qualities of
fast PM muscle fibers suppress the slow fiber phenotype.
In summary, slow MyHC 2 gene expression in slow
muscle fiber types is regulated by PKC activity and this
activity in turn is controlled by innervation. In addition
to innervation, intrinsic differences between fast and
slow muscle fiber lineages regulate PKC activities in
innervated and denervated muscle. These lineagebased mechanisms significantly contribute to suppression of slow MyHC 2 gene expression in innervated fast
muscle fibers and allow slow MyHC 2 gene expression
in innervated slow muscle fibers.
EXPERIMENTAL PROCEDURES
Cell Culture and Transfections
Myoblasts were isolated from the medial adductor
(MA) and PM muscles of fetal embryonic day (ED) 13
chick embryos and cultured on collagen-coated dishes
as previously described (O’Neill and Stockdale, 1972;
Miller and Stockdale, 1986b). Cells were cultured in
10% horse serum (Hyclone Laboratories, Logan, UT),
5% chick embryo extract, in Ham’s F-10 basal medium
supplemented with 1.1mM CaCl2, 2 mM glutamine,
and antibiotics (GIBCO/BRL; penicillin/streptomycin/
Fungizone). Medium was changed every other day. To
inhibit protein kinase activities in muscle fibers, some
cultures were incubated for 4 days in medium containing 1 to 10 ng/ml staurosporine (Sigma Chemical Co.,
St. Louis, MO), 1 to 20 µM bisindolylmaleimide I
(Sigma), 10 nM to 10 µM myristoylated protein kinase
A inhibitory peptide 14–22 (Calbiochem, San Diego,
CA) (Glass et al., 1989), 10 nM to 10 µM {N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide,
HCl} (Calbiochem) (Findik et al., 1995), 0.5 to 50 µM
protein kinase G inhibitor, guanosine 38, 58-cyclic monophosphorothioate, 8-(4-chlorophenylthio)-, Rp-Isomer,
(Calbiochem) (Butt et al., 1994), or 20 µM MEK1/2
inhibitor, PD98059 (New England Biolabs, Beverly,
MA).
PKC ACTIVITY REGULATES SLOW MyHC 2 GENE EXPRESSION
187
Some muscle fiber cultures included explants of ED5
chick spinal cords. These were obtained from the thoracic region of chick embryos, minced into small pieces,
and placed into muscle fiber cultures on the third day of
incubation as previously described (DiMario and Stockdale, 1997).
For cell transfection, myoblasts isolated from ED13
medial adductor muscles were cultured for 3 days as
described above. On day 3 of culture, 4 µg of the plasmid
PKC-7 were transfected into the muscle fiber cultures
using Lipofectamine Plus reagent (GIBCO/BRL). PKC-7
encodes amino acids 302–672 of the catalytic domain of
bovine PKC␣ and lacks the regulatory domain. Transcription of the sequence encoding the unregulated
catalytic domain of PKC␣ is constitutively driven by the
cytomegalovirus promoter and enhancer within the
expression vector pCDM8 (James and Olson, 1992).
After transfection, some of the cultures were incubated
in normal culture medium, and other cultures were
incubated in medium containing 4 ng/ml staurosporine.
Cells were immunostained 24 hr after transfection.
Northern Blot Analysis
Immunocytochemistry
Curare Administration
Muscle fibers were immunostained for fast and slow
MyHCs with monoclonal antibodies F59 and S58. The
specificities of these antibodies have been previously
characterized (Crow and Stockdale, 1986b; Page et al.,
1992). Briefly, F59 is an IgG1 that recognizes multiple
fast avian MyHC isoforms, and S58 is an IgA that
recognizes slow MyHC 2 and MyHC 3. Cultures were
washed twice with phosphate buffered saline (PBS) and
then fixed for 5 min with 100% ethanol. The cultures
were washed three times with PBS. The cells were
incubated in blocking solution containing 5% horse
serum and 2% bovine serum albumin in PBS for 1 hr at
room temperature. Cells were then incubated in MyHCspecific monoclonal supernatants diluted 1 : 10 in blocking solution for 1 hr at room temperature. Cultures
transfected with PKC-7 were immunostained with a
hemagglutinin antigen (HA) -specific antibody (Santa
Cruz Biotechnology, Santa Cruz, CA) diluted 1 : 100 in
blocking solution along with anti-MyHC antibodies for
1 hr at room temperature. Cells were washed three
times with PBS and then incubated in fluorochromeconjugated secondary antibodies (Vector Laboratories,
Burlingame, CA) diluted 1 : 100 in blocking solution for
1 hr at room temperature. Cells were again washed
three times with PBS and a drop of 2.5% diazabicyclooctane in 90% glycerol was added before coverslips
were applied. Cells were viewed by epifluorescence.
For cryosectioning, ED13 PM and MA muscles were
embedded in Tissue-Tek O.C.T. compound and frozen
using liquid nitrogen. Muscles were sectioned at a
thickness of 14 µm and placed onto gelatin-coated
slides. Sections were dehydrated in 100% ethanol for 5
min and then rehydrated in PBS for 5 min. Muscle fiber
cross sections were immunostained with mAbs F59 and
S58 as described above.
Chick embryos in ovo were administered d-tubocurarine (Sigma) as previously described (Crow and Stockdale, 1986a). Briefly, a small hole was bored through
the eggshell into the air sac with a 20-gauge needle.
Curare at a concentration of 1.5mg curare/0.1ml sterile
PBS was injected onto the chorioallantoic membrane
twice daily from ED9 to ED12. Control embryos received injections of PBS only. The hole was covered with
tape and embryos were returned to the 38°C incubator
between injections. On ED13, control and curaretreated PM and MA muscles were isolated and processed for immunocytochemistry or PKC activity assays.
Total RNA was extracted from muscle fiber cultures
(Tel-TEST ‘‘B’’; RNA Stat-60) and electrophoresed in a
1% agarose/formaldehyde gel. RNA was transferred to
nitrocellulose (BA-85, Schleicher and Schuell, Keene,
NH) by capillary action (Sambrook et al., 1989) and the
blot was baked at 80°C for 2 hr in a vacuum. Prehybridization was done in 20 ml of 6⫻ standard saline citrate
(SSC), 5⫻ Denhardt’s solution, 0.5% sodium dodecyl
sulfate (SDS), 0.05% sodium pyrophosphate, and
0.1mg/ml salmon sperm DNA at 42°C for 4 hr. The blot
was hybridized with a 32P-end labeled oligonucleotide
(107 cpm/10 ml prehybridization solution) at 42°C overnight. The oligonucleotide (58-GGGCTGCAGCTCATCCTCCTTC- 38) specifically hybridizes to sequence
encoding slow MyHC 2 and not to sequences encoding
slow MyHC 1 or MyHC 3 (DiMario and Stockdale,
1997). The blot was washed four times in 250 ml of 1⫻
SSC, 0.05% sodium pyrophosphate at 50°C. Kodak
X-OMAT film was exposed overnight with intensification.
PKC Activity Assay
PKC activity was determined in PM and MA muscles
dissected from ED13 chick fetuses and in PM and MA
muscle fibers formed from ED13 myoblasts after 7 days
in culture. Medium for PM and MA muscle fiber cultures was supplemented with 10 µg/ml cytosine arabinoside (AraC) from day 4 to day 7 of incubation. Protein
extracts of dissected muscles were made by disruption
in a dounce homogenizer using an extraction buffer
consisting of 20 mM Tris, pH 7.5, 0.5 mM ethylenediaminetetraacetic acid (EDTA), 0.5 mM ethyleneglycoltetraacetic acid (EGTA), 0.5% Triton X-100, 25
µg/ml aprotinin and leupeptin. Protein extracts of
cultured cells were prepared by scraping cells from
100-mm plates in extraction buffer. Cells were homogenized by repeated passage through a 22-gauge needle.
Suspensions were incubated on ice for 30 min and spun
in a microcentrifuge for 2 min. PKC activities in the
resulting supernatants were determined by use of a
protein kinase C assay system (GIBCO/BRL) with a
synthetic peptide from myelin basic protein as a sub-
188
DIMARIO AND FUNK
strate according to manufacturer’s instructions. PKC
was purified using DE-52 (Whatman, Fairfield, NJ)
column chromatography according to manufacturer’s
instructions (Gibco/BRL). Assays included control reactions with the PKC pseudosubstrate inhibitor peptide
PKC (19–36) to confirm PKC specificity in the phosphorylation reactions. In addition, to assess the effectiveness of the PKC inhibitor, staurosporine, on inhibition
of PKC activities in extracts of PM and MA muscle
fibers, staurosporine at concentrations ranging from
0.1 to 10 nM (0.05–4.7 ng/ml; Ki ⫽ 0.7 nM) was added to
some control reactions. PKC activities falling within a
linear range of phosphorylation of substrate relative to
amount of protein extract were used for comparison of
PKC activities. Total protein in extracts of whole muscles
and muscle fiber cultures was determined using a BCA
protein assay reagent (Pierce, Rockford, IL).
ACKNOWLEDGMENT
We thank Dr. J. Staudinger for providing the PKC-7
expression construct.
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