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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.


INTRODUCTION
The sarcolemma of normal skeletal muscle contains molecules that bind 1,4dihydropyridine (DHP) derivatives with high affinity (Fosset et al., 1983). These DHP receptors are hypothesized to perform two distinct functions, serving both as ion channels that mediate a slowly activating calcium current (Ica-s), and also as "voltage sensors" for excitation-contraction (E-C) coupling (Rios and Bruin, 1987;Tanabe et al., 1987Tanabe et al., , 1988. In their latter role, the DHP receptors are postulated to undergo a voltage-drive conformational change that is somehow communicated to the sarco-plasmic reticulum (SR), resulting in calcium release and contraction. An important feature of E-C coupling in skeletal muscle is that entry of external calcium is not required (Armstrong et al., 1972;Tanabe et al., 1988Tanabe et al., , 1990b. Thus, it is the voltage-sensing, and not the calcium-conducting property of the skeletal muscle DHP receptor that is responsible for triggering contraction.
In skeletal muscle from mice with muscular dysgenesis, depolarization of the sarcolemma does not normally cause contraction (Powell and Fambrough, 1973;Klaus et al., 1983;Tanabe et al., 1988). A variety of evidence indicates that the failure of E-C coupling in dysgenic muscle results from absence of the DHP receptors that predominate in normal skeletal muscle. For example, dysgenic skeletal muscle has a greatly reduced capacity to bind DHP compounds (Pincon-Raymond et al., 1985) and lacks the slowly activating, DHP-sensitive calcium current (/ca-s) associated with the normal skeletal muscle DHP receptor (Beam et al., 1986). In addition, genetic studies have shown that the gene encoding the skeletal muscle DHP-binding polypeptide is mutated in dysgenic mice, and the corresponding mRNA is strongly reduced or absent (Tanabe et al., 1988). Polyclonal antibodies directed against the DHP-binding polypeptide of skeletal muscle fail to detect this protein in dysgenic muscle (Knudson et al., 1989). Furthermore, expression of cDNA encoding the skeletal muscle DHP receptor stores both E-C coupling and Ica_~ in dysgenic skeletal muscle (Tanabe et al., 1988(Tanabe et al., , 1990b. We have recently described an endogenous, DHP-sensitive calcium current (Ic,-dy~) in dysgenic skeletal muscle, and have shown this current to be distinct from Ica_s of normal skeletal muscle (Adams and Beam, 1989). Because of the similarities between Ic,_dy s and other calcium currents associated with E-C coupling in cardiac and normal skeletal muscle, we performed experiments to test whether Ica-ays, or the DHP receptor underlying it, could ever couple depolarization to contraction. We have found that Ic~_ay~ can indeed mediate depolarization-induced contractions in dysgenic skeletal muscle, but only under conditions in which this inward calcium current is potentiated. Significantly, the contractions mediated by lca-ays are like those of cardiac muscle, and unlike those of normal skeletal muscle, in that they require the entry of external calcium.

METHODS
Primary cultures of myotubes were prepared from skeletal muscle of newborn dysgenic mice and their phenotypically normal littermates using methods previously described (Beam and Knudson, 1988;Adams and Beam, 1989). In all cases, the myotubes were grown on a substrate of killed and dessicated fibroblasts (Adams and Beam, 1989). This culture condition was used because it increases, via an unknown mechanism, the density at which lca-dys is expressed in dysgenic myotubes (Adams and Beam, 1989). 7-20 d after plating, myotubes were tested for the ability to contract in response to focal extraceUular stimulation via an agar/saline-filled micropipette (2-1xm tip diameter) placed within 100 I~m of the cell surface. The intensity of the stimulation pulse (60-80 V, 5 ms duration) was adjusted to be suprathreshold for evoking contractions of normal myotubes. Contractions were monitored optically and recorded as the voltage drop across a small photoresistor placed at the edge of each myotube's image on a video monitor. The output of the photoresistor was amplified, filtered at 5-10 Hz, and stored on computer.
Strength of contraction was evaluated qualitatively (i.e., weak, medium, or strong). Weak contractions were those that were just visibly detectible; intermediate contractions were pronounced but slow in onset and relaxation; strong contractions were forceful and brisk, resembling the twitches of normal myotubes. The location of each contracting myotube was recorded so that the same cells could later be relocated for voltage-clamping. Voltage clamp data were obtained using the whole-cell configuration of the patch clamp technique (Hamill et al., 1981) as previously described (Beam and Knudson, 1988;Adams and Beam, 1989). The pipettes had resistances of 1.3-2.3 M~. Electronic compensation was used to minimize the effective series resistance (generally to < 1 MI-/) and the time required to charge the cell capacitance (generally to < 1 ms). Whole-cell currents were measured during 200-ms depolarizing test pulses administered from a steady holding potential of -80 inV. For each cell, linear capacitance and leakage currents were measured for 8-10 "control" voltage steps between -80 and -100 mV; these control currents were averaged, scaled appropriately, and used to correct test currents for linear components of capacitative and leakage currents. To allow comparison of test currents recorded from different cells, test currents recorded from each cell were normalized by that cell's linear capacitance (current expressed as picoamperes per picofarad).
Evoked contractions were recorded while the myotubes were bathed in a solution containing (in mM) 145 NaC1, 10 CaCle, 10 HEPES, 5 KCI, and 0.001 Bay K 8644, pH 7.4 with NaOH. Calcium currents were measured while the myotubes were bathed in a solution containing (in mM) 145 TEAC1, 10 CaCI 2, 10 HEPES, 0.003 TI'X, and 0.001 Bay K 8644, pH 7.4 with CsOH. The patch pipettes were filled with a solution containing (in raM) 140 Cs-Aspartate, 5 MgC12, 10 Cs~-EGTA, and 10 HEPES, pH 7.4 with CsOH. The 1,4-DHP derivatives (_+)-Bay K 8644 (gift of Dr. A. Scriabine, Miles Laboratories, Inc., New Haven, CT) and (+)-PN 200-110 (gift of Dr. A. Lindenmann and Dr. E. Rossi, Sandoz Ltd., Basel, Switzerland) were added to the external solution just before use from stock solutions (10 mM in ethanol) which were stored in the dark at -20°C. All experiments were performed at room temperature (20-22°C). Values are presented in the text as mean ___ SEM with the number of observations in parentheses.

Depolarization-evoked Contractions of Dysgenic Skeletal Muscle
Previous studies (Powell and Fambrough, 1973;Tanabe et al., 1988) have shown that dysgenic myotubes bathed in physiological saline (containing ~ 2 mM calcium) are incapable of contracting either spontaneously or in response to electrical stimulation. However, the recent discovery of a DHP-sensitive calcium current (/ca-d,) in dysgenic skeletal muscle (Adams and Beam, 1989;Bournaud et al., 1989) led us to re-examine whether dysgenic myotubes could ever display E-C coupling under less physiological conditions. Specifically, we tested whether contractions could be triggered by calcium entry through the ICa-dys channels, or through a more direct interaction of the DHP receptor underlying Ica.dys with the SR.
To this end, we electrically stimulated dysgenic myotubes first while they were bathed in saline containing elevated (10 mM) calcium, and then again after the addition of 1 IxM Bay K 8644. No dysgenic myotubes were observed to contract in response to electrical stimulation while bathed in the high calcium saline that did not contain Bay K 8644 (> 1,000 myotubes from three different muscle cultures were individually tested). However, in the combined presence of 10 mM calcium and 1 IxM Bay K 8644 (which should potentiate Ica-ays approximately sixfold; Adams and Beam, 1989) many of these same myotubes produced twitchlike contractions. For example, in one culture of dysgenic muscle, 35 of 300 myotubes tested could contract after the addition of Bay K 8644. In a separate dysgenic muscle culture, 40 of 100 dysgenic myotubes tested could contract after the addition of Bay K 8644.
What mechanism accounts for the ability of dysgenic myotubes to contract in response to depolarization? One possibility is that in the combined presence of elevated external calcium and Bay K 8644, calcium entry through the Ic~-dy~ channels becomes sufficient to activate directly the contractile proteins, or to trigger calciuminduced calcium release from the SR as in cardiac-type E-C coupling. Another possibility is that under physiological conditions the DHP receptor underlying Ica~y ~ is in an "inactivated" state, but in the combined presence of high calcium and Bay K 8644 this DHP receptor becomes "activated," enabling it to mediate direct, skeletaltype E-C coupling. This latter possibility is suggested by results demonstrating that the voltage sensor for E-C coupling in normal skeletal muscle is influenced both by external calcium concentration (Brum et al., 1988a, b) and DHP compounds (Pizarro et al., 1988). To distinguish between these two possibilities, we perfbrmed the experiments described below.

The Role of Calcium Entry
Several experimental manipulations that block calcium currents were used to investigate the role of calcium entry in triggering the evoked contractions of dysgenic myotubes. These included removing calcium from the external solution (10 mM magnesium was substituted), adding 0.5 mM cadmium to the external solution, and replacing Bay K 8644 in the external solution with the DHP calcium channel antagonist (+)-PN 200-110. Fig. 1 illustrates the effects of these different manipulations. In three of three dysgenic myotubes tested, removal of external calcium abolished (reversibly) evoked contractions ( Fig. 1 A, top). In parallel with its effect on contraction, removing external calcium reduced the peak Ica-dr~ (measured during the test pulse) by 94 -+ 3% (n = 3; Fig. 1 B, top). The effect of calcium removal on Ica-dys was completely reversible (recovery not shown).
The evoked contractions of dysgenic myotubes were also abolished by the external application of 0.5 mM cadmium (n--4; Fig. 1 A, middle). As expected, 0.5 mM cadmium produced strong yet reversible block of Ica-dys, reducing the peak current during the test pulse by 92 + 1%, (n = 5; Fig. 1 B, middle). As described by Swandulla and Armstrong (1989) for chicken dorsal root ganglion cells, cadmium reduced but did not completely block the calcium channel tail current.
The effect of substituting the DHP calcium channel antagonist (+)-PN 200-110 (2 IxM) for Bay K 8644 (1 IxM) in the external solution was also examined. In three of three myotubes tested, this substitution diminished but did not completely abolish evoked contractions of dysgenic myotubes (Fig. 1 A, bottom). Parallel effects were seen on Ica-,ty~, where the peak calcium current was decreased by 46 _+ 6% (n = 3; Fig. 1 B, bottom). During these experiments, a large volume (~ 50 ml) of solution containing (+)-PN 200-110 (without Bay K 8644) was washed through the recording chamber (volume ~ 1 ml); the lack of complete blockade of contraction and current is possibly due to a higher affinity of the lca~lys channel for Bay K 8644 than for (+)-PN 200-110.
The present results clearly demonstrate that calcium entry is required for the evoked contractions of dysgenic myotubes. In contrast, calcium entry is not required for evoked contractions of normal myotubes (Tanabe et al., 1990b; Fig. 2A). Furthermore, the results obtained above with (+)-PN 200-110 strongly suggest that the calcium that triggers contraction of dysgenic myotubes enters through the DHP-sensitive calcium channels underlying Ica-dy,.

Dependence of Contraction Strength on the Density of lc~_ay,
If the contractions of dysgenic myotubes are triggered by calcium entry through the lca-dys channels, then the strength of contraction should be related to the density of Ica-dys. In support of this idea, we found that contracting dysgenic myotubes possessed substantially higher densities of ICa_dy s than noncontracting myotubes. In 19 dysgenic myotubes that produced evoked contractions, the peak density of Ica-dys (measured in the presence of 10 mM external calcium and 1 ixm Bay K 8644) was 15 -2 pA/pF (range 4-30 pA/pF). In contrast, in 12 dysgenic myotubes that could not contract (these cells were from the same culture and were tested at the same time), the peak density of Ica_dy s was only 0.7 ± 0.2 pA/pF (range 0-3 pA/pF). In addition, there was a positive correlation between the strength of contraction and the density of Ic,_dy~. For example, in five dysgenic myotubes that contracted weakly, average peak Ic~-dy~ was only 5.6 +--0.5 pA/pF (range 4-7 pA/pF), whereas in seven dysgenic myotubes that contracted strongly, average peak Ica-ays was 23 ---2 pA/pF (range 15-30 pA/pF).
In seven dysgenic myotubes that contracted with intermediate strength, average peak Ica~y~ was 14 _+ 0.6 pA/pF (range 12-17 pA/pF). This relationship between contraction strength and the density of Ic~y~ is illustrated in Fig. 3. Both normal and dysgenic myotubes can express a T-type calcium current (Ic~-ft) that is insensitive to DHP compounds (Cognard et al., 1986;Beam and Knudson, 1988). There was no correlation between the density of Ic~.,, and the strength of contraction (Fig. 3). In 19 contracting dysgenic myotubes, average peak Ica-f~ was 1.2 --+ 0.4 pA/pF (range 0-8.2 pA/pF), a value indistinguishable from that measured in 12 noncontracting dysgenic myotubes (1.1 + 0.6 pA/pF; range 0-7.4 pA/pF) from the same muscle culture. Interestingly, one noncontracting dysgenic myotube that lacked measurable Ic~_,~, did possess a relatively high density of Ica-ft (7.4 pA/pF); this density of Ica-f~ is higher than the minimum density of lc~y~ found in weakly contracting dysgenic myotubes (~ 4 pA/pF).

DISCUSSION
This paper describes the ability of an endogenous, DHP-sensitive calcium current, Ica-dys, to trigger contractions in dysgenic myotubes. Because the density of this calcium current is typically quite low (~ 1 pA pA/pF; Adams and Beam, 1989), contractions were not observed unless Icily ~ was first potentiated with Bay K 8644.
The evoked contractions of dysgenic myotubes were blocked or reduced by manipulations (removing external calcium, adding cadmium to the bath, or substituting (+)-PN 200-110 for Bay K 8644) that block or reduce Ica-dys" Furthermore, the intensity of contraction was positively correlated with the density of this calcium current. Thus, it seems clear that the evoked contractions of dysgenic myotubes depend upon calcium entry through the DHP-sensitive channels that underlie Ica-ays" Depolarization--contraction coupling in cardiac muscle also requires calcium entry (Niibauer et al., 1989). Because of this similarity, we suggest that potentiated Ic~-ay~ effects a "cardiac-type" of E-C coupling in dysgenic myotubes. By way of contrast, the contractions of normal myotubes (Fig. 2 A), or of dysgenic myotubes that express the normal skeletal muscle DHP receptor, as a result either of incorporation of normal fibroblast nuclei  or of cDNA injection (Tanabe et al., 1988(Tanabe et al., , 1990b do not require calcium entry. From these results we conclude that the type of E-C coupling mediated by the DHP receptor that underlies Ica-dy~ is fundamentally different from that mediated by the normal skeletal muscle DHP receptor. Thus, the DHP receptor underlying Ica~y~ appears unable to couple sarcolemmal depolarization directly to SR calcium release in the absence of calcium entry. This result further strengthens the findings of Tanabe et al. (1990a, b) that not all types of DHP receptors expressed in skeletal muscle are able to directly trigger calcium release from the SR.
As shown in Fig. 2 C, the evoked twitches of normal myotubes were unaffected when 2 IxM (+)-PN 200-110 was substituted for 1 p~M Bay K 8644 in the external solution. In other experiments (unpublished observations), we found that higher concentrations (up to 10 IxM) of (+)-PN 200-110 or nifedipine also had no significant effect on twitches of normal myotubes. In contrast, Cognard et al. (1990) reported that 5 txM nifedipine strongly reduced contractions of rat myoballs. However, it should be noted that they studied contractions elicited by depolarizations applied to cells under whole-cell voltage clamp, whereas we studied contractions elicited by focal, extracellular stimulation of intact myotubes. Moreover, the effects of dihydropyridines on contraction of skeletal muscle seem quite variable. For example, Gallant and Goettl (1985) and Dulhunty and Gage (1988) both found that nifedipine potentiated the twitches of small bundles of rodent muscle fibers.
It is interesting that dysgenic myotubes that do not contract under the conditions used here sometimes express Ica-ft at higher density than that density of Ica-dys (~4 pA/pF) needed to support contraction. This observation suggests that Ic,.f~ alone cannot mediate depolarization-contraction coupling in dysgenic myotubes. Perhaps Ica-ft is less effective in triggering contractions due to its kinetic properties (slower activation, faster inactivation than Ica-dys)" Another possibility is that the channels responsible for Ica.f, may be located more distantly from the SR calcium release channels than the channels responsible for lca.ay ~.
Recently, the cDNA encoding the cardiac DHP receptor was cloned and expressed in Xenopus oocytes (Mikami et al., 1989). This same cardiac DHP receptor was also expressed in dysgenic myotubes, where it produces an L-type calcium current and cardiac-type E-C coupling, which requires the entry of external calcium (Tanabe et al., 1990b). The inward current mediated by the cardiac DHP receptor expressed in dysgenic myotubes resembles Ica-dy~ in voltage dependence, kinetics, and pharmacology. However, a major difference between these two calcium currents is that the exogenous cardiac current is expressed at much higher densities (12-56 pA/pF, measured in the presence of 10 mM external calcium and in the absence of Bay K 8644; Tanabe et al., 1990b) than lca.dy ~ and can thus easily trigger contraction without further potentiation.
An interesting question raised by the present study is whether internal calcium stores participate in the contractions triggered by Ica_ay ~. In dysgenic myotubes expressing the cardiac DHP receptor cDNA, calcium-entry-induced release of internal calcimn does seem to be important. After caffeine treatment of such myotubes, contractions are initially small but then increase in size with repeated stimuli, even though the amplitude of the cardiac L-type calcium current remains constant (Tanabe et al., 1990b; see also N~ibauer et al., 1989). This result suggests that internal calcium stores initially depleted by the caffeine treatment become refilled and thus make an increasing contribution to contraction. It seems likely that the same internal calcium stores also participate in the contractions of dysgenic myotubes triggered by Ica_aw