(Received for publication, October 24, 1994; and in revised form, January 3, 1995)
From the
Two ryanodine receptor (RyR), sarcoplasmic reticulum
Ca release channels,
and
, co-exist in
chicken skeletal muscles. To investigate a two-RyR Ca
release system, we compared electrically evoked Ca
transients in Crooked Neck Dwarf (cn/cn) cultured muscle cells,
which do not make
RyR, and normal (+/?) cells. At day 3 in
culture, Ca
release in +/? cells required
extracellular Ca
(Ca
), and Ca
transients had slow kinetics. At day 5, Ca
release was Ca
-independent
in 40% of the cells, and transients were more rapid. By day 7, all
+/? cells had
Ca
-independent Ca
release. Contractions were observed in +/? cells on all
days. Ca
transients were observed in cn/cn cells on
days 3, 5, and 7, but in each case they were
Ca
-dependent and exhibited slow
kinetics. Localized vesiculations, not contractions, occurred in cn/cn
cells. By day 10, Ca
transients were no longer
observed in cn/cn cells even in
Ca
. Sarcoplasmic reticulum
Ca
was not depleted, as caffeine induced
Ca
transients. Thus, in the absence of
RyR there
is a failure to develop
Ca
-independent Ca
release and contractions and to sustain
Ca
-dependent release. Moreover,
contributions by the
RyR cannot be duplicated by the
RyR
alone.
Two RyR ()isoforms,
and
, are co-expressed in nonmammalian vertebrate skeletal muscles (1, 2, 3, 4, 5) . In
chicken skeletal muscle both proteins are localized to SR terminal
cisternae membranes at triad junctions (1) and embody
Ca
channels(6) . Thus, both RyRs may serve as
SR Ca
release channels. In vitro studies
indicate that these RyRs may be regulated differentially by
phosphorylation and calmodulin (7) and have different ion
channel properties(6) . In particular, the chicken
RyR is
activated by ATP to a relatively greater extent than by
Ca
, while the reverse is true for the
RyR
channel. Such differences have also been reported for the two RyRs
expressed in frog skeletal muscle(3, 8) . Nonmammalian
vertebrate
RyRs appear to be homologs of the mammalian skeletal
muscle RyR isoform, RyR1, while the
RyRs are more closely
related to the mammalian RyR, RyR3
(9). The latter RyR
was initially cloned from the brain (10) but appears to be
expressed in a number of tissues, including skeletal
muscle(10, 11, 28) .
Our studies describe
two aspects of the roles of the - and
RyRs in striated
muscles. The first concerns whether the RyRs are determinants of the
type of E-C coupling used in embryonic skeletal muscle. The second
aspect concerns the contributions made by each RyR in a two-RyR E-C
coupling system. At least two E-C coupling mechanisms exist in striated
muscles(12) . In cardiac muscle, Ca
entering
the cell as L-type Ca
currents through
dihydropyridine receptor (DHPR) channels activates the cardiac RyR
isoform (RyR2) channel resulting in a Ca
-induced
Ca
release (CICR). This type of coupling is defined
experimentally by its dependence on
Ca
(13) . In skeletal
muscle, DHPRs acting as voltage sensors are thought to interact with
the RyR1 isoform, either directly or indirectly via an intermediary
protein(s) in a voltage-dependent manner to cause a directly coupled SR
Ca
release (DCCR)(12) . Ca
influx is not required for DCCR and thus it is independent of
Ca
. Recently, both DCCR and CICR
mechanisms have been proposed to activate SR Ca
release in skeletal
muscle(12, 14, 15) . In this case the
activating Ca
involved in the CICR mechanism is
thought to be that released by DCCR rather than Ca
entering the cell. While the existence of CICR in skeletal muscle
is controversial(16) , it is tempting to hypothesize that the
RyR is activated by DCCR, while the
isoform subserves a CICR
mechanism.
We have investigated these issues within the context of
the recessive lethal cn mutation, in chickens, which results in an
embryonic skeletal muscle dysgenesis, the failure to maintain skeletal
muscles once they have formed, and ultimately the progressive
degeneration of all skeletal muscles(17, 18) .
Although we have not established the genetic nature of the cn mutation,
there is a failure to make normal RyR in cn/cn tissues, while
cultured cn/cn muscle cells express normal levels of the
RyR(18, 19) . Thus, the use of +/? cells,
which have a wild type phenotype, and cn/cn cells permits comparison of
muscle cells expressing both
and
RyRs with ones expressing
only the
RyR.
With two exceptions skeletal muscle cells from pectoral
muscles from day E10-E12 embryos were cultured as described
previously(19) . First, cells were plated at a total cell
density of 1 10
cells/cm
, and second,
no attempt was made to eliminate fibroblasts. These changes promoted
muscle cell differentiation. Intracellular calcium transients and
contractions were recorded as described previously(19) . Normal
Ca
was 2 mM, and the
absence of Ca
means no
Ca
was added. Contaminating Ca
was
3-8 µM. Electrical stimuli were applied for 4 ms via
a glass pipette placed close to the cell being studied, and caffeine
(10 mM) and acetylcholine (100 µM) were
administered locally via pipettes using a Picospritzer.
Figure 1:
SR
Ca release transients (indo-1 fluorescence) induced
by electrical stimulation for 4 ms (small arrows) in +/?
embryonic chick skeletal muscle cells maintained in culture for 3, 5,
7, and 10 days. The traces in panelsA and B show responses obtained in 2 mM Ca
and in the absence of
added Ca
, respectively.
Responses to transient applications of 10 mM caffeine (horizontalbar) in the absence of
Ca
are shown in panel
C.
Figure 3:
Percentage of normal (panel A)
and cn/cn cells (panel B) that responded with Ca transients to electrical stimuli applied either in the presence (open bars) or the absence (closed bars) of added
Ca
. At least 50 cells were tested for each time
and condition.
The Ca transients in cn/cn muscle
cells on day 3 were similar to those in +/? cells at this time in
that they were Ca
-dependent and had slow
on and off rates ( Fig. 2and Fig. 3). Unlike +/?
cells though, the Ca
transients observed in cn/cn
cells on days 5 and 7 did not have the more rapid kinetics and were not
Ca
-independent in any of the cells tested
( Fig. 2and Fig. 3). By day 10, many cn/cn cells no
longer responded with even Ca
-dependent
Ca
transients. This inability was not due to
depletion of SR Ca
stores, as caffeine elicited
ryanodine-sensitive transients in the absence of
Ca
in every cell tested. Similar results
were obtained when acetylcholine was used to activate the cells (data
not shown).
Figure 2:
SR
Ca release transients in cultured cn/cn embryonic
chick skeletal muscle cells. Details are the same as those given for Fig. 1.
Although we have not identified the primary defect associated
with the cn mutation, we have shown that cn/cn tissues fail to make
normal RyR, and several observations support the hypothesis that
this defect exists in the
RyR gene(18) . Moreover, the
phenotype described for other conditions that disrupt E-C coupling is
similar, if not identical, to that of the cn/cn muscle. Most notable of
these are the muscle dysgenesis (mdg) mutation in mice(20) ,
which involves a failure to make a normal skeletal muscle
DHPR subunit (21) , and the skrr
/skrr
mouse, which lacks a functional RyR1(22) . Regardless of
the basis of the cn mutation, we believe it reasonable to attribute
many, if not all, of the changes we have observed in the present
studies to the absence of
RyR in cn/cn muscle.
The switch from
a Ca-dependent to a
Ca
-independent E-C coupling process in
(+/?) muscle cells is similar to that observed for cultured
neonatal rat skeletal muscle cells(23) . The failure of cn/cn
muscle cells to make this transition suggests that either
RyR
protein or an activity associated with this isoform is required for
this event. The data of Tanabe et al.(13) obtained
with mdg/mdg muscle cells can be interpreted to indicate that the
isoform of the
DHPR subunit (cardiac or skeletal)
expressed is a determinant of whether
Ca
-dependent or
Ca
-independent coupling is used by
cultured mdg/mdg mouse skeletal muscle cells. Our results suggest that
the isoform of the RyR expressed is also important in this regard.
In preliminary studies ()using Western analyses we have
found that both the cardiac and skeletal muscle isoforms of the
-DHPR subunit are expressed at days 3 and 7 in
+/? and cn/cn chick muscle cells. The cardiac isoform is more
abundant at day 3 than it is at day 7, whereas the skeletal muscle
isoform is present at greater levels at day 7 than at day 3. Both the
and
RyRs are present at both days in +/? cells, while
the
RyR is present in cn/cn muscle cells at these times. Thus, in
+/? cells, each of the DHPR and RyR isoforms is present at
appreciable levels when only a single type of coupling is observed.
This suggests that while the DHPR and RyR isoform expressed may act as
determinants of the type of coupling used, they may not by themselves
be sufficient, and that other factors may also be necessary. Such
factors could be those that direct the formation of the junctional
structures required for DCCR and the localization of DHPR and RyR
isoforms to these structures, as well as the expression of other triad
junctional components. Studies to determine the relative distributions
of the DHPR and RyR isoforms and cell morphological features at
different times in culture are in progress.
The observation of
ryanodine-sensitive Ca-dependent
Ca
transients in response to electrical stimuli and
of Ca
-independent responses to caffeine
indicates that the
RyR expressed in cn/cn cells is functional as a
SR release channel and can be activated by external signals.
Importantly, these results also indicate that an E-C coupling system
consisting of only the
RyR is not sufficient to support
Ca
-independent E-C coupling. This
suggests that the
RyR may not be capable of the interactions
necessary for the Ca
-independent process
and that either the
RyR/skeletal muscle
-DHPR
subunit combination or both
- and
RyRs are necessary for
DCCR.
The on and off rates of
Ca-dependent Ca
transients are slower than those observed for the
Ca
-independent events. These differences
may be due to the activation mechanisms used, the channel properties of
the RyR isoforms involved, the isoforms and density of the SR
Ca
ATPase expressed, and the extent to which SR
membranes are organized as they are in mature muscles.
When indo-1
fluorescence signals were normalized using the fluorescence observed
prior to activation, the Ca transients observed in
+/? and cn/cn cells were comparable in magnitude. This is again
consistent with the results obtained with lower density
cultures(19) . Although indirect, these results suggest that
the same calcium stores may be accessed by both RyR isoforms in these
cells. They also suggest that if the
- and
RyRs operate in a
two-RyR Ca
release system, the
RyR may influence
the quantity of Ca
released by affecting the duration
rather than the amplitude of the SR Ca
release
transient.
A second defect in E-C coupling that precludes activation
of the RyR-mediated Ca
release events becomes
apparent in cn/cn cells by
day 10 in culture. It is unclear
whether this defect is related to the muscle cell degeneration that
becomes apparent by day 12. This failure of electrical stimuli to
activate the
RyR is not due to either an inoperative
RyR or
an inability to maintain SR calcium stores, as caffeine produces a
ryanodine-sensitive release of calcium that is undiminished in the
absence of Ca
.
As noted above, cn/cn
chick and skrr/skrr
mouse skeletal muscle
cells exhibit similar phenotypes. This is consistent with the absence
of homologous RyR isoforms,
RyR and RyR1, respectively, in these
cells. The sequence information available for bullfrog (9) and
chick
RyRs indicates that these proteins are most
closely related to RyR1. This relationship is also supported by
immunological data(5) . Of particular interest is that
electrical stimuli elicited low amplitude contractions and caffeine
produced significant contractures in skrr
/skrr
muscle cells (see Fig. 4 of (22) ) indicating the
presence of a non-RyR1-mediated Ca
release process.
Northern(11) , PCR (29) , and in situ hybridization studies (28) indicate the presence of RyR3
in mammalian skeletal muscles. Moreover, chick and bullfrog
RyRs
are most homologous to the mammalian RyR3
isoform
(9) , and thus RyR3 is a candidate as a
second RyR in mouse muscle cells. These results suggest that both chick
and mouse skeletal muscles may utilize two-RyR E-C coupling.
The
failure of cn/cn cells to develop coordinated whole cell contractions
is at least in part related to an inability to form normal sarcomeres.
A number of studies have indicated that some aspect of muscle cell
activation promotes myofibrillar
assembly(24, 25, 26) . The poorly organized
sarcomeres in cn/cn muscle cells suggest that Ca transients having specific characteristics may be important for
this event. The focal vesiculation-like contractions observed in cn/cn
cells result from a spontaneous, repetitive, and localized release of
SR Ca
. The latter may be due to the presence of only
a partial E-C coupling system and involve an inappropriate CICR. The
final consequence of the cn mutation, the degeneration and death of
cultured cn/cn muscle cells, is probably equivalent to the degenerative
loss of skeletal muscle seen in cn/cn embryos(17) . This
suggests that muscle cell activation and SR Ca
transients are important for maintaining skeletal muscle
integrity and again that the underlying Ca
transients
must have specific characteristics to be effective.
In conclusion, a
failure to make RyR is associated with an inability to develop
normal Ca
-independent E-C coupling and
indicates that the
RyR is one determinant of the type of E-C
coupling utilized in embryonic skeletal muscle cells. Moreover, the
altered E-C coupling in cn/cn muscle cells suggests that normally a
two-RyR system is utilized to couple excitation and contraction. The
presence of E-C coupling and caffeine-induced contractions in
skrr
/skrr
muscle cells suggests that a
two-RyR system may underlie E-C coupling in both nonmammalian and
mammalian skeletal muscles. At the molecular level such a coupling
system presents a new concept that may differ in a number of ways from
those envisioned previously. The use of a two-RyR system provides
additional complexities for the morphological, functional, and
regulatory properties of the processes that underlie skeletal muscle
activation. Two RyR isoforms are also co-expressed in avian cerebellar
Purkinje neurons(27) ; thus, a two-RyR system for generation of
cellular Ca
transients may be used in tissues other
than skeletal muscle.