©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Embryonic Chicken Skeletal Muscle Cells Fail to Develop Normal Excitation-Contraction Coupling in the Absence of the Ryanodine Receptor
IMPLICATIONS FOR A TWO-RYANODINE RECEPTOR SYSTEM (*)

(Received for publication, October 24, 1994; and in revised form, January 3, 1995)

Anna Ivanenko (§) David D. McKemy (§) James L. Kenyon (1) Judith A. Airey John L. Sutko (¶)

From the Departments of Pharmacology and Physiology, University of Nevada School of Medicine, Reno, Nevada 89557

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Two ryanodine receptor (RyR), sarcoplasmic reticulum Ca release channels, alpha and beta, 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 alphaRyR, 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 alphaRyR there is a failure to develop Ca-independent Ca release and contractions and to sustain Ca-dependent release. Moreover, contributions by the alphaRyR cannot be duplicated by the betaRyR alone.


INTRODUCTION

Two RyR (^1)isoforms, alpha and beta, 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 alphaRyR is activated by ATP to a relatively greater extent than by Ca, while the reverse is true for the betaRyR channel. Such differences have also been reported for the two RyRs expressed in frog skeletal muscle(3, 8) . Nonmammalian vertebrate alphaRyRs appear to be homologs of the mammalian skeletal muscle RyR isoform, RyR1, while the betaRyRs 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 alpha- and betaRyRs 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 alphaRyR is activated by DCCR, while the beta 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 alphaRyR in cn/cn tissues, while cultured cn/cn muscle cells express normal levels of the betaRyR(18, 19) . Thus, the use of +/? cells, which have a wild type phenotype, and cn/cn cells permits comparison of muscle cells expressing both alpha and betaRyRs with ones expressing only the betaRyR.


MATERIALS AND METHODS

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 times 10^5 cells/cm^2, 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 Cawas 2 mM, and the absence of Cameans 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.


RESULTS

Cell Growth and Myotube Formation

At the total cell densities used in these studies, both +/? and cn/cn skeletal muscle cells formed comparable numbers of similarly sized myotubes. The only difference discernible between these cells at the light microscope level was a failure of cn/cn cells to develop well defined cross-striations. This is consistent with similar observations made for cn/cn cells cultured at a lower cell density(19) .

E-C Coupling

Ca transients were elicited by electrical stimuli to assess the nature of the E-C coupling process in +/? and cn/cn muscle cells at days 3, 5, 7, and 10 in culture. On day 3, Ca transients having relatively slow on and off kinetics were observed in the presence but not the absence of Ca(o) in all 50 +/? cells tested ( Fig. 1and Fig. 3). In 10 responses that were quantitated, the time to the peak of the Ca transient was 1.39 ± 1.14 s. Caffeine (10 mM) produced similar Ca transients in +/? cells on both day 3 and day 10 (Fig. 1C) that were blocked by ryanodine. Thus, the requirement for Ca(o) observed at day 3 did not involve a depletion of releasable SR Ca stores. On day 5 the Ca transients in +/? cells had more rapid on and off rates and were observed in 40% of the cells in the absence of Ca(o) (Fig. 3). By day 7, the Ca transients in +/? cells appeared more robust in both amplitude and kinetics (time to the peak of the Ca transient = 0.09 ± 0.03 s, n = 10) and could be elicited in the absence of Ca(o) in 100% of the cells. Ca(o)-independent E-C coupling was also observed in 100% of the +/? cells tested on day 10 (Fig. 3).


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(o)-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(o)-independent in any of the cells tested ( Fig. 2and Fig. 3). By day 10, many cn/cn cells no longer responded with even Ca(o)-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(o) 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.



Contractile Activity

As observed previously for cells cultured at a lower total cell density(19) , there are differences in the contractile activity in +/? and cn/cn cells. At all days tested, electrical stimuli caused coordinated, whole cell contractions from +/? cells. Moreover, as they matured in culture, +/? cells developed spontaneous and vigorous contractions. In contrast, cn/cn cells did not exhibit whole cell contractions either spontaneously or in response to electrical stimuli at any time but did display very localized muscle cell vesiculations that appeared as repetitive ripples in the cell surface. These responses accompanied low level oscillations in intracellular Ca and could occur at different loci within the same cell, but the activities at different foci did not appear to be coordinated (data not shown).

Muscle Cell Viability

+/? and cn/cn muscle cells also differed in that cn/cn cells degenerated and died. The timing of this phenomenon was dependent on cell density. At the plating density used in the present studies, noticeable cell deterioration and cell loss occurred by days 12 and 14, respectively. When cells were plated at a 5-fold greater initial total cell density (5 times 10^5 cells/cm^2) degeneration and cell death were observed after 5 days in culture and became more precipitous, frequently requiring less than 10 h.


DISCUSSION

Although we have not identified the primary defect associated with the cn mutation, we have shown that cn/cn tissues fail to make normal alphaRyR, and several observations support the hypothesis that this defect exists in the alphaRyR 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 alpha(1) 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 alphaRyR in cn/cn muscle.

The switch from a Ca(o)-dependent to a Ca(o)-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 alphaRyR 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 alpha(1) DHPR subunit (cardiac or skeletal) expressed is a determinant of whether Ca(o)-dependent or Ca(o)-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 (^4)using Western analyses we have found that both the cardiac and skeletal muscle isoforms of the alpha(1)-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 alpha and betaRyRs are present at both days in +/? cells, while the betaRyR 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(o)-dependent Ca transients in response to electrical stimuli and of Ca(o)-independent responses to caffeine indicates that the betaRyR 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 betaRyR is not sufficient to support Ca(o)-independent E-C coupling. This suggests that the betaRyR may not be capable of the interactions necessary for the Ca(o)-independent process and that either the alphaRyR/skeletal muscle alpha(1)-DHPR subunit combination or both alpha- and betaRyRs are necessary for DCCR.

The on and off rates of Ca(o)-dependent Ca transients are slower than those observed for the Ca(o)-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 alpha- and betaRyRs operate in a two-RyR Ca release system, the alphaRyR 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 betaRyR-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 betaRyR is not due to either an inoperative betaRyR 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(o).

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, alphaRyR and RyR1, respectively, in these cells. The sequence information available for bullfrog (9) and chick^2 alphaRyRs 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 betaRyRs 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 alphaRyR is associated with an inability to develop normal Ca(o)-independent E-C coupling and indicates that the alphaRyR 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.


FOOTNOTES

*
This work was supported by National Science Foundation Grants DCB9108091 and IBN9306850 and National Institutes of Health Grant 27470. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
These authors contributed equally to this work.

To whom correspondence should be addressed: Dept. of Pharmacology/318, Howard Bldg., Rm. 214, University of Nevada School of Medicine, Reno, NV 89557. Tel.: 702-784-4121; Fax: 702-784-1620. sutko{at}scs.unr.edu.

(^1)
The abbreviations used are: RyR, ryanodine receptor; SR, sarcoplasmic reticulum; E-C, excitation-contraction; DHPR, dihydropyridine receptor; CICR, Ca-induced Ca release; DCCR, directly coupled SR Ca release.

(^2)
J. A. Airey, J. A. Hamerman, Y. Cheliah, and M. C. Bohlman, unpublished observations.

(^3)
V. Sorrentino, personal communication.

(^4)
D. D. McKemy, J. A. Airey, and J. L. Sutko, unpublished observations.


ACKNOWLEDGEMENTS

We thank Dr. Lucien Houenou for suggesting the cn mutation as a system that may be deficient in RyR function, Jean Haines and Drs. Lou Peirro and Ursula Abbott for helping to keep the cn mutation in existence, and Dr. Jon Lederer for reading the manuscript.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.