Muscular dysgenesis in mice: a model system for studying excitation-contraction coupling

Muscular dysgenesis (mdg) is a lethal autosomal, recessive mutation of mice. Skeletal muscle from dysgenic mice is paralyzed due to the failure of excitation-contraction (E-C) coupling. Considerable evidence indicates that this failure results from the absence of a specific gene product, the alpha 1 subunit of the skeletal muscle receptor for dihydropyridine calcium channel modifiers. This dihydropyridine receptor is hypothesized to function in E-C coupling of normal skeletal muscle as the voltage sensor that triggers calcium release from the sarcoplasmic reticulum and thereby causes contraction. The skeletal muscle dihydropyridine receptor is also postulated to function as the ion channel responsible for a slowly activating, dihydropyridine-sensitive calcium current (Islow). Dysgenic skeletal muscle lacks Islow but expresses, at low levels, a distinctly different dihydropyridine-sensitive calcium current (Idys). The channel protein underlying Idys is incapable of serving as a voltage sensor for E-C coupling. Studies using dysgenic skeletal muscle have provided significant insight into the role of dihydropyridine receptors in E-C coupling.     

Specific absence of the alpha 1 subunit of the dihydropyridine receptor in mice with muscular dysgenesis

Muscular dysgenesis is a lethal mutation in mice that results in a complete absence of skeletal muscle contraction due to the failure of depolarization of the transverse tubular membrane to trigger calcium release from the sarcoplasmic reticulum. In order to determine whether the defect in muscular dysgenesis leads to a specific loss of one of the components of excitation-contraction coupling or to a generalized loss of all components of excitation-contraction coupling, we have analyzed skeletal muscle from control and dysgenic mice for the sarcoplasmic reticulum and transverse tubular proteins which are believe to function in excitation-contraction coupling. We report that the proteins involved in sarcoplasmic reticulum calcium transport, storage, and release [Ca2+ + Mg2+)-ATPase, calsequestrin, and calcium release channel) are present in dysgenic muscle. Also present in dysgenic muscle is the 175/150-kDa glycoprotein subunit (alpha 2) of the dihydropyridine receptor. However, the 170-kDa dihydropyridine binding subunit (alpha 1) of the dihydropyridine receptor is absent in dysgenic muscle. These results suggest that the specific absence of the alpha 1 subunit of the dihydropyridine receptor is responsible for the defects in muscular dysgenesis and that the alpha 1 subunit of the dihydropyridine receptor is essential for excitation-contraction coupling in skeletal muscle.     

Restoration of dysgenic murine (mdg) myotube contraction after addition of Schwann cells from normal mice in vitro

Muscular dysgenesis in mutant mice is characterised by failure of excitation-contraction (E-C) coupling and consequent loss of skeletal muscle contraction. Contractile activity is restored in vitro by the addition of normal mice cells (11) (18) (7). In the present study we show a new model: contraction and ultrastructural organization of dysgenic myotubes are restored by coculture with Schwann cells from normal mice.     

Rescue of excitation-contraction coupling in dysgenic muscle by addition of fibroblasts in vitro

Muscular dysgenesis (mdg) in mice causes the failure of excitation-contraction (E-C) coupling in skeletal muscle. Cultured dysgenic muscle fails to contract upon depolarization, lacks typical muscle ultrastructure, including normal triads, and lacks functional voltage-dependent slow calcium channels. We show that normal rodent fibroblasts and 3T3 fibroblasts "rescue" dysgenic myotubes, reestablishing contractions (i.e., E-C coupling), normal ultrastructure, and functional slow calcium channels. These results support the finding that the expression of the slow calcium channel is affected in the mdg mutation and that this protein is essential for E-C coupling. Additionally, fibroblast rescue provides a system for examining the mechanisms of heterotypic cellular influence on cell function.     

Contractions of dysgenic skeletal muscle triggered by a potentiated, endogenous calcium current

The dihydropyridine (DHP) receptor of normal skeletal muscle is hypothesized to function as the voltage sensor for excitation-contraction (E-C) coupling, and also as the calcium channel underlying a slowly activating, DHP-sensitive current (termed ICa-s). Skeletal muscle from mice with muscular dysgenesis lacks both E-C coupling and ICa-s. However, dysgenic skeletal muscle does express a small DHP-sensitive calcium current (termed ICa-dvs) which is kinetically and pharmacologically distinct from ICa-s. We have examined the ability of ICa-dys, or the DHP receptor underlying it, to couple depolarization and contraction. Under most conditions ICa-dys is small (approximately 1 pA/pF) and dysgenic myotubes do not contract in response to sarcolemmal depolarization. However, in the combined presence of the DHP agonist Bay K 8644 (1 microM) and elevated external calcium (10 mM), ICa-dys is strongly potentiated and some dysgenic myotubes contract in response to direct electrical stimulation. These contractions are blocked by removing external calcium, by adding 0.5 mM cadmium to the bath, or by replacing Bay K 8644 with the DHP antagonist (+)-PN 200-110. Only myotubes having a density of ICa-dys greater than approximately 4 pA/pF produce detectible contractions, and the strength of contraction is positively correlated with the density of ICa-dys. Thus, unlike the contractions of normal myotubes, the contractions of dysgenic myotubes require calcium entry. These results demonstrate that the DHP receptor underlying ICa-dys is unable to function as a "voltage sensor" that directly couples membrane depolarization to calcium release from the sarcoplasmic reticulum.     

The muscular dysgenesis mutation in mice leads to arrest of the genetic program for muscle differentiation

Muscular dysgenesis (mdg) is a mutation in mice which causes the failure of excitation-contraction coupling in skeletal muscle. Although the sarcolemma, the sarcoplasmic reticulum, and the contractile apparatus all maintain nearly normal function, sarcolemmal depolarization fails to cause calcium release from the sarcoplasmic reticulum. Recently, the primary genetic defect in this mutation was shown to be located in the structural gene for the dihydropyridine receptor. We have examined the developmental expression from Fetal Day 15 onward, in normal and mutant muscle, of several unidentified genes as well as genes which are known markers of muscle differentiation. We find that the majority of mRNA sequences are found at similar concentrations in normal and dysgenic muscles at birth. Many differentiation-related genes also are expressed at normal levels early during myogenesis in mutant mice. However, as late fetal development progresses in dysgenic muscle, the mRNA concentrations for these genes fail to undergo the rapid rise which is characteristic of normal muscle. Several additional, unidentified genes, which normally would be down-regulated during development, remain expressed at a high level in dysgenic muscle. Thus, the primary absence of a functional dihydropyridine receptor appears to prevent the changes in gene expression which are necessary for maturation of skeletal muscle.     

A single nucleotide deletion in the skeletal muscle-specific calcium channel transcript of muscular dysgenesis (mdg) mice.

The skeletal muscle-specific dihydropyridine-sensitive calcium channel is a critical component of excitation-contraction coupling in skeletal muscle. A recessive mutation in mice, muscular dysgenesis (mdg), has previously been described as resulting in defective excitation-contraction coupling. Although the channel-forming subunit (alpha 1) of the skeletal calcium channel is not detectable immunologically, specific mRNA of normal size is present in dysgenic muscle. cDNA for this calcium channel alpha 1 subunit has now been cloned from dysgenic muscle and sequenced in its entirety. A single nucleotide deletion occurs at nucleotide 4010 of the cDNA, resulting in a shift of the translational reading frame. The mutation has been confirmed by direct sequencing of PCR products from homozygous mutant and normal muscle. The mutant polypeptide is predicted to contain the first three repeating domains, five of the normal six transmembrane helices of the last repeating domain, and an altered and truncated C terminus. The mature mRNA encoding the dysgenic alpha 1 subunit appears to be labile. It is possible that premature termination of translation renders the mutant mRNA subject to degradation by nucleases. This work resolves a long-standing controversy on the nature of the primary genetic defect in muscular dysgenesis.     

Formation of triads without the dihydropyridine receptor alpha subunits in cell lines from dysgenic skeletal muscle

Muscular dysgenesis (mdg/mdg), a mutation of the skeletal muscle dihydropyridine receptor (DHPR) alpha 1 subunit, has served as a model to study the functions of the DHPR in excitation-contraction coupling and its role in triad formation. We have investigated the question of whether the lack of the DHPR in dysgenic skeletal muscle results in a failure of triad formation, using cell lines (GLT and NLT) derived from dysgenic (mdg/mdg) and normal (+/+) muscle, respectively. The lines were generated by transfection of myoblasts with a plasmid encoding a Large T antigen. Both cell lines express muscle-specific proteins and begin organization of sarcomeres as demonstrated by immunocytochemistry. Similar to primary cultures, dysgenic (GLT) myoblasts show a higher incidence of cell fusion than their normal counterparts (NLT). NLT myotubes develop spontaneous contractile activity, and fluorescent Ca2+ recordings show Ca2+ release in response to depolarization. In contrast, GLTs show neither spontaneous nor depolarization-induced Ca2+ transients, but do release Ca2+ from the sarcoplasmic reticulum (SR) in response to caffeine. Despite normal transverse tubule (T-tubule) formation, GLT myotubes lack the alpha 1 subunit of the skeletal muscle DHPR, and the alpha 2 subunit is mistargeted. Nevertheless, the ryanodine receptor (RyR) frequently develops its normal, clustered organization in the absence of both DHPR alpha subunits in the T-tubules. In EM, these RyR clusters correspond to T-tubule/SR junctions with regularly spaced feet. These findings provide conclusive evidence that interactions between the DHPR and RyR are not involved in the formation of triad junctions or in the normal organization of the RyR in the junctional SR.     

Author index for volume 81

Muscular dysgenesis (mdg) is an autosomal recessive mutation in which skeletal muscle excitation and contraction are uncoupled. In vitro, action potential generation triggers the morphological deterioration of dysgenic myotubes. Active dysgenic muscle is vacuolated and atrophied. Cellular degeneration is accompanied by a decrease in the amount of myosin heavy chain (MHC) accumulated in active mdg muscle compared to that accumulated in quiescent mdg muscle. A marked increase in the rate of MHC degradation primarily accounts for the relatively low quantities of MHC in cells generating action potentials. MHC synthesis is only slightly reduced in active cells.     

Functional and structural recovery of myotubes from mice with muscular dysgenesis after co-culture with normal,non-myoblastic cells

Muscular dysgenesis is a mutation which is characterized by paralysis of skeletal muscle cells. Excitation-contraction coupling is deficient and muscle cells display atypical ultrastructure. In vitro, mutant myotubes recover a normal phenotype when cocultured with spinal cord cells from normal animals or with normal fibroblasts. We have shown that other types of cells, eg certain glial cells present in the spinal cord or in other tissues, have this capacity. In contrast, intervention of neurons in the recovery does not appear likely. Very different types of non-myoblastic cells, then, are capable of restoring contractile activity of dysgenic myotubes in vitro, suggesting that a non-specific mechanism is involved in the phenotypic reversion of affected muscle cells. The restoration process seems to imply a close relationship between myotubes and normal glial cells.     

Restoration of junctional tetrads in dysgenic myotubes by dihydropyridine receptor cDNA.

Excitation-contraction coupling was restored in primary cultures of dysgenic myotubes by transfecting the cells with an expression plasmid encoding the rabbit skeletal muscle dihydropyridine receptor. Dishes containing normal, dysgenic, and transfected myotubes were fixed, freeze-fractured, and replicated for electron microscopy. Numerous small domains in the surface membrane of normal myotubes contain ordered arrays of intramembrane particles in groups of four (tetrads). The disposition of tetrads in the arrays is consistent with alternate positioning of tetrads relative to the underlying feet of the sarcoplasmic reticulum. Dysgenic myotubes have no arrays of tetrads. Some myotubes from successfully transfected cultures have arrays of tetrads with spacings equal to those found in normal myotubes. Thus the dihydropyridine receptor appears to be needed for the formation of tetrads and their association with the sarcoplasmic reticulum feet. This result is consistent with the hypothesis that each tetrad is composed of four dihydropyridine receptors.     

Distribution and quantification of ACh receptors and innervation in diaphragm muscle of normal and mdg mouse embryos

Muscular dysgenesis (mdg) in the mouse is an autosomal recessive mutation, expressed in the homozygous state (in vivo and in vitro) as an absence of skeletal muscle contraction. The distribution of acetylcholine receptors (ACh R) in the diaphragms of phenotypically normal and dysgenic (mdg/mdg) embryos was studied from the 14th to 19th day of gestation by binding of 125I-alpha-bungarotoxin to the muscle, followed by autoradiography of longitudinally sectioned hemidiaphragms and/or of isolated muscle fibers. Localization of ACh R at putative motor end-plate regions begins 14 to 15 days in utero in both normal and dysgenic diaphragms. The distribution of high ACh R density patches is aberrantly scattered beyond the normal innervation pattern in dysgenic diaphragms. Isolated mutant fibers possess (1) multiple ACh R clusters, up to five per single fiber, (2) larger clusters of more variable morphology and variable receptor density than normal clusters, and (3) higher levels of extrajunctional receptors than normal fibers. These autoradiographic results correlate well with higher total level of toxin binding sites per diaphragm and per milligram protein in dysgenic vs normal muscle, as quantified from gamma counting of sucrose density gradient isolation of 125I-toxin-ACh R complexes. The dispersed distribution of ACh R patches on dysgenic muscle may be correlated with extensive phrenic nerve branching as demonstrated by silver impregnation technique. We suggest that the aberrant ACh R cluster distribution is a result of multiple innervation of single fibers from the branched nerve terminals. Possible causes of the excessive nerve branching in the mutant are discussed in light of generalized nerve sprouting found in paralyzed muscle.     

Modification of mitochondrial metabolism in fibroblasts from mice with a skeletal muscle mutation (muscular dysgenesis). Evidence of embryonic communication between myoblasts and fibroblasts

Muscle development during embryogenesis is a complex process involving many mechanisms. It requires a close communication among the different cellular types of the muscle, especially the fibroblasts and myoblasts. Indeed, any abnormality in one cell type might influence the differentiation of the other. Thus, any disturbance altering the metabolism of the myoblasts might lead to modifications in the fibroblasts. To study this phenomenon, we used the dysgenic mouse (mdg-"muscular dysgenesis") carrying a homozygous recessive lethal mutation expressed only in skeletal muscle cells. First, we found that fibroblasts isolated from such mutant muscle (and not from mutant skin tissue) and grown in culture exhibited an altered metabolism. Secondly, muscle fibroblasts showed a lower capacity for proliferation. We also observed that respiration and ATP synthesis of dysgenic muscle fibroblasts were deficient, while respiratory chain enzymatic activities were normal. Finally, intracellular [Ca2+] levels of dysgenic fibroblasts are 50% of those of normal fibroblasts. These results support the hypothesis that certain characteristics of fibroblasts are determined by the surrounding cellular environment during embryonic organogenesis, and that such modifications are stable when the fibroblasts are isolated in vitro. Since fibroblast differentiation was disrupted permanently, this suggests, in the case of myopathies, that the modified cells, surrounding the muscle tissue, could contribute to the muscle pathology. Synergistic activities of this type should be considered when studying the course of pathologies in different types of muscle diseases.     

A genetic model for the study of abnormal nerve-muscle interactions at the level of excitation-contraction coupling: the mutation muscular dysgenesis

Excitation-contraction in muscle fibers are coupled through a complex mechanism involving multiproteic components located at a specialized cellular site, the triadic junction. Triads in normal muscle fiber result from the apposition of sarcoplasmic reticulum citernae and T-tubule and possess strikingly organized ultrastructural elements, bridging both types of membranes, the "junctional feet". Muscular dysgenesis in the mouse is characterized by total muscle inactivity in the developing skeletal muscles due to excitation-contraction uncoupling. Triads have been found to be disorganized with no "junctional feet" and dihydropyridine (DHP) binding sites are decreased with no slow Ca2+ currents, suggesting a basic defect in the excitation-contraction coupling machinery itself. We may hypothesize that muscular dysgenesis results in a marked defect in a functional protein involved in the morphogenesis of the triad and/or directly involved in Ca2+ release for contraction.     

Heterologous expression of BI Ca2+ channels in dysgenic skeletal muscle

We have examined the ability of BI (class A) Ca2+ channels, cloned from rabbit brain, to mediate excitation-contraction (E-C) coupling in skeletal muscle. Expression plasmids carrying cDNA encoding BI channels were microinjected into the nuclei of dysgenic mouse myotubes grown in primary culture. Ionic currents and intramembrane charge movements produced by the BI channels were recorded using the whole-cell patch- clamp technique. Injected myotubes expressed high densities of ionic BI Ca2+ channel current (average 31 pA/pF) but did not display spontaneous contractions, and only very rarely displayed evoked contractions. The expressed ionic current was pharmacologically distinguished from the endogenous L-type current of dysgenic skeletal muscle (Idys) by its insensitivity to the dihydropyridine antagonist (+)-PN 200-110. Peak BI Ca2+ currents activated with a time constant (tau a) of approximately 2 ms and inactivated with a time constant (tau h) of approximately 260 ms (20-23 degrees C). The time constant of inactivation (tau h) was not increased by substituting Ba2+ for Ca2+ as charge carrier, demonstrating that BI channels expressed in dysgenic myotubes do not undergo Ca(2+)-dependent inactivation. The average maximal Ca2+ conductance (Gmax) produced by the BI channels was quite large (approximately 534 S/F). In contrast, the average maximal charge movement (Qmax) produced in the same myotubes (approximately 2.7 nC/microF) was quite small, being barely larger than Qmax in control dysgenic myotubes (approximately 2.3 nC/microF). Thus, the ratio Gmax/Qmax for the BI channels was considerably higher than previously found for cardiac or skeletal muscle L-type Ca2+ channels expressed in the same system, indicating that neuronal BI Ca2+ channels exhibit a much higher open probability than these L-type Ca2+ channels.     

The ryanodine receptor/junctional channel complex is regulated by growth factors in a myogenic cell line

The ryanodine receptor/junctional channel complex (JCC) forms the calcium release channel and foot structures of the sarcoplasmic reticulum. The JCC and the dihydropyridine (DHP) receptor in the transverse tubule are two of the major components involved in excitation-contraction (E-C) coupling in skeletal muscle. The DHP receptor is believed to serve as the voltage sensor in E-C coupling. Both the JCC and DHP receptor, as well as many skeletal muscle-specific contractile protein genes, are expressed in the BC3H1 muscle cell line. In the present study, we find that during differentiation of BC3H1 cells, induced by mitogen withdrawal, induction of the JCC and DHP receptor mRNAs is temporally similar to that of the skeletal muscle contractile protein genes alpha-tropomyosin and alpha-actin. Our data suggest that there is coordinate regulation of both the contractile protein genes (which have been studied in detail previously) and the genes encoding the calcium channels involved in E-C coupling. Induction of both calcium channels is accompanied by profound changes in BC3H1 cell morphology including the development of many components of mature skeletal muscle cells, despite lack of myoblast fusion. Visualized by electron microscopy, the JCC appears as "foot structures" located in the dyad junction between the plasmalemma and the sarcoplasmic reticulum of the BC3H1 cells. Development of foot structures is concomitant with JCC mRNA expression. Expression of the JCC and DHP receptor mRNAs and formation of the foot structures are inhibited specifically by fibroblast growth factor.     

Relationship of calcium transients to calcium currents and charge movements in myotubes expressing skeletal and cardiac dihydropyridine receptors

In both skeletal and cardiac muscle, the dihydropyridine (DHP) receptor is a critical element in excitation-contraction (e-c) coupling. However, the mechanism for calcium release is completely different in these muscles. In cardiac muscle the DHP receptor functions as a rapidly-activated calcium channel and the influx of calcium through this channel induces calcium release from the sarcoplasmic reticulum (SR). In contrast, in skeletal muscle the DHP receptor functions as a voltage sensor and as a slowly-activating calcium channel; in this case, the voltage sensor controls SR calcium release. It has been previously demonstrated that injection of dysgenic myotubes with cDNA (pCAC6) encoding the skeletal muscle DHP receptor restores the slow calcium current and skeletal type e-c coupling that does not require entry of external calcium (Tanabe, Beam, Powell, and Numa. 1988. Nature. 336:134-139). Furthermore, injection of cDNA (pCARD1) encoding the cardiac DHP receptor produces rapidly activating calcium current and cardiac type e-c coupling that does require calcium entry (Tanabe, Mikami, Numa, and Beam. 1990. Nature. 344:451-453). In this paper, we have studied the voltage dependence of, and the relationship between, charge movement, calcium transients, and calcium current in normal skeletal muscle cells in culture. In addition, we injected pCAC6 or pCARD1 into the nuclei of dysgenic myotubes and studied the relationship between the restored events and compared them with those of the normal cells. Charge movement and calcium currents were recorded with the whole cell patch-clamp technique. Calcium transients were measured with Fluo-3 introduced through the patch pipette. The kinetics and voltage dependence of the charge movement, calcium transients, and calcium current in dysgenic myotubes expressing pCAC6 were qualitatively similar to the ones elicited in normal myotubes: the calcium transient displayed a sigmoidal dependence on voltage and was still present after the addition of 0.5 mM Cd2+ + 0.1 mM La3+. In contrast, the calcium transient in dysgenic myotubes expressing pCARD1 followed the amplitude of the calcium current and thus showed a bell shaped dependence on voltage. In addition, the transient had a slower rate of rise than in pCAC6-injected myotubes and was abolished completely by the addition of Cd2+ + La3+.     

Coordinated development of myofibrils, sarcoplasmic reticulum and transverse tubules in normal and dysgenic mouse skeletal muscle, in vivo and in vitro

We studied the development of transverse (T)-tubules and sarcoplasmic reticulum (SR) in relationship to myofibrillogenesis in normal and dysgenic (mdg/mdg) mouse skeletal muscle by immunofluorescent labeling of specific membrane and myofibrillar proteins. At E16 the development of the myofibrils and membranes in dysgenic and normal diaphragm was indistinguishable, including well developed myofibrils, a delicate network of T-tubules, and a prominent SR which was not yet cross-striated. In diaphragms of E18 dysgenic mice, both the number and size of muscle fibers and myofibrillar organization were deficient in comparison to normal diaphragms, as previously reported. T-tubule labeling was abnormal, showing only scattered tubules and fragments. However, many muscle fibers displayed cross striation of sarcomeric proteins and SR comparable to normal muscle. In cultured myotubes, cross-striated organization of sarcomeric proteins proceeded essentially in two stages: first around the Z-line and later in the A-band. Sarcomeric organization of the SR coincided with the first stage, while the appearance of T-tubules in the mature transverse orientation occurred infrequently, only after A-band maturation. In culture, myofibrillar and membrane organization was equivalent in normal and dysgenic muscle at the earlier stage of development, but half as many dysgenic myotubes reached the later stage as compared to normal. We conclude that the mdg mutation has little effect on the initial stage of membrane and myofibril development and that the deficiencies often seen at later stages result indirectly from the previously described absence of dihydropyridine receptor function in the mutant.     

A novel calcium current in dysgenic skeletal muscle

The whole-cell patch-clamp technique was used to study voltage-dependent calcium currents in primary cultures of myotubes and in freshly dissociated skeletal muscle from normal and dysgenic mice. In addition to the transient, dihydropyridine (DHP)-insensitive calcium current previously described, a maintained DHP-sensitive calcium current was found in dysgenic skeletal muscle. This current, here termed ICa-dys, is largest in acutely dissociated fetal or neonatal dysgenic muscle and also in dysgenic myotubes grown on a substrate of killed fibroblasts. In dysgenic myotubes grown on untreated plastic culture dishes, ICa-dys is usually so small that it cannot be detected. In addition, ICa-dys is apparently absent from normal skeletal muscle. From a holding potential of -80 mV. ICa-dys becomes apparent for test pulses to approximately -20 mV and peaks at approximately +20 mV. The current activates rapidly (rise time approximately 5 ms at 20 degrees C) and with 10 mM Ca as charge carrier inactivates little or not at all during a 200-ms test pulse. Thus, ICa-dys activates much faster than the slowly activating calcium current of normal skeletal muscle and does not display Ca-dependent inactivation like the cardiac L-type calcium current. Substituting Ba for Ca as the charge carrier doubles the size of ICa-dys without altering its kinetics. ICa-dys is approximately 75% blocked by 100 nM (+)-PN 200-110 and is increased about threefold by 500 nM racemic Bay K 8644. The very high sensitivity of ICa-dys to these DHP compounds distinguishes it from neuronal L-type calcium current and from the calcium currents of normal skeletal muscle. ICa-dys may represent a calcium channel that is normally not expressed in skeletal muscle, or a mutated form of the skeletal muscle slow calcium channel.