Skeletal muscle Na currents in mice heterozygous for Six5 deficiency

DILAAWAR J. MISTRY1, J. RANDALL MOORMAN1,2,3, SITA REDDY4 and J. PAUL MOUNSEY1,3

1 Cardiovascular Division, Department of Internal Medicine
2 Department of Molecular Physiology and Biological Physics
3 Cardiovascular Research Center, University of Virginia Health System, Charlottesville, Virginia 22908
4 Institute for Genetic Medicine, University of Southern California School of Medicine, Los Angeles, California 90033


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Myotonic dystrophy results from a trinucleotide repeat expansion between the myotonic dystrophy protein kinase gene (Dmpk), which encodes a serine-threonine protein kinase, and the Six5 gene, which encodes a homeodomain protein. The disease is characterized by late bursts of skeletal muscle Na channel openings, and this is recapitulated in Dmpk -/- and Dmpk +/- murine skeletal muscle. To test whether deficiency of the nearby Six5 gene also affected Na channel gating in murine skeletal muscle, we measured Na currents from cell-attached patches in Six5 +/- mice and age-matched wild-type and Dmpk +/- mice. Late bursts of Na channel activity were defined as an opening probability >10% measured from 10 to 110 ms after depolarization. There was no significant difference in the occurrence of late Na channel bursts in wild-type and Six5 +/- muscle, whereas in Dmpk +/- muscle there was greater than fivefold increase in late bursts (P < 0.001). Compared with wild-type mice, Na current amplitude was unchanged in Six5 +/- muscle, whereas in Dmpk +/- muscle it was 36% reduced (P < 0.05). Thus, since Six5 +/- mice do not exhibit the Na channel gating abnormality of Dmpk deficiency, we conclude that Six5 deficiency does not contribute to the Na channel gating abnormality seen in dystrophia myotonica patients.

myotonic muscular dystrophy; myotonic dystrophy kinase; ion channels; protein kinases


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MYOTONIC MUSCULAR DYSTROPHY (dystrophia myotonica, DM) is an autosomal dominant multisystem disease with a prominent abnormality of membrane excitability. The symptoms are myotonia (the inability to relax a contracted muscle group), progressive weakness, cataracts, cardiac conduction disturbances, mild mental retardation, hypersomnia, sleep apnea, balding, and endocrinopathies (1, 13, 27, 32, 34). The genetic abnormality is amplification of a CTG repeat at the chromosome 19q13.3 site (2, 5, 11, 1618, 23, 24, 37). The mutation does not interrupt the coding sequence of any gene. The upstream gene Dmpk encodes a novel serine-threonine protein kinase, DMPK, that belongs to a new family of kinases (15, 19, 4143). The downstream gene Six5 encodes a transcription factor and is the vertebrate homolog of sine oculis, a gene essential for development of the eye in Drosophila melanogaster (12, 14, 20, 38).

Neither gene product is obviously responsible for all manifestations of the illness, and participation of multiple genes cannot be excluded. The CTG repeat mutation could lead to haploinsufficiency of the products of either of the proximate genes, or alternatively in the trans-dominant RNA model, sequestration of novel proteins binding to CUG RNA repeats could lead to abnormal expression of many proteins. There is evidence in support of all these possibilities. Expression of trinucleotide (CUG) repeats distant from the DM locus in a murine model reproduces the myotonia characteristic of the disease, although the cellular electrophysiological abnormality has not been reported, and these mice exhibit only a mild myopathy (25). Levels of DMPK mRNA and protein appear to be reduced in adult DM (4, 6, 10, 39), though the effect of muscle atrophy on the assays is unclear (35). Skeletal muscle Na channels are a substrate for DMPK (7, 29), and a mouse model with either complete or partial functional inactivation of Dmpk has a Na channel gating abnormality similar to human DM (28).

A deficiency of SIX5 mRNA in skeletal muscle from DM patients has also been demonstrated (20, 38; but see Ref. 12). Haploinsufficiency is a recognized cause of disorders resulting from mutations of homeodomain proteins (8, 21), and a reduced level of SIX5 may not be sufficient to fully complete pattern formation and tissue development, leading to dysmorphology. Six5-deficient mice develop cataracts, a feature of DM (36), and interestingly also show reduced levels of DMPK mRNA. Deletion of Six5 leads to a variable reduction of DMPK mRNA that approaches 50% in -/- mice and 25% in +/- mice. It is not known whether these changes in DMPK mRNA level are a direct effect of loss of Six5 function or are a cis effect of deletion of the Six5 locus.

The characteristic Na channel abnormality in DM is a plateau of noninactivating current caused by late Na channel reopenings (9). This abnormality is recapitulated in skeletal muscle from mice in which the Dmpk gene has been completely inactivated, and an identical Na channel lesion is present in mice heterozygous for Dmpk deficiency (28). We have attributed this Na channel abnormality to DMPK deficiency and have suggested that the channel gating defect is an all-or-none phenomenon occurring with a threshold DMPK mRNA level of <=50% of normal, the predicted level in Dmpk +/- mice.

There are other explanations, however. First, since SIX5 is a transcription factor, the reduced levels found in DM patients might indirectly lead to abnormal Na channel gating by altered expression of yet other genes. Second, the response to DMPK deficiency may be graded, with saturation of the effect on Na channels below 50% of normal levels of DMPK mRNA. In this regard, the Six5 +/- mouse model offers the opportunity to test the effect of an ~25% reduction in DMPK mRNA level.

We tested these alternatives by measuring Na channel gating in mice heterozygous for Six5 deficiency compared with mice heterozygous for Dmpk deficiency. This allows us to test whether partial loss of SIX5 function is sufficient to reproduce the Na channel gating abnormality of DM.


    METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

Isolation of mouse skeletal muscle cells.
The Six5 +/- and Dmpk +/- 129SV mouse models that we studied have been described previously (33, 36). All mice were >16 and <30 wk old. The mice were anesthetized with intraperitoneal pentobarbital (0.033 mg/g) and killed by cervical dislocation using a protocol approved by the University of Virginia Animal Care Committee. The hind limb was excised above the knee joint and transferred to oxygenated Tyrode solution at 37°C for 90 min. Tyrode solution contained (in mM) 120 NaCl, 5 KCl, 1 MgCl2, 2.5 CaCl2, 0.5 NaH2PO4, 10 HEPES, and 11 glucose, pH 7.4 (NaOH). The flexor digitorum brevis was isolated complete with tendons. Isolated cells were prepared by digestion of the entire muscle with collagenase (Worthington type 2, 2.6 mg/ml) in oxygenated Tyrode solution. The muscle was washed in enzyme-free Tyrode solution and stored until use at 4°C. The digested muscle was teased apart by careful sharp dissection, and myofibrils were transferred to an organ bath (volume, 2 ml) mounted on the stage of an inverting microscope. The cells were superfused at 3–4 ml/min with an oxygenated depolarizing solution containing (in mM) 160 KCl, 1 MgCl2, 0.5 EGTA, and 10 HEPES, pH 7.4 (KOH) and allowed to settle in the recording chamber for 30 min before patch clamp recordings were made. Segments of cells with intact membranes and clear cross-striations were selected for patch clamping.

Electrophysiological recording.
Cell-attached patch recordings were made at room temperature (20°C) by standard techniques using an Axopatch 200A (Axon Instruments, Foster City, CA) amplifier and pCLAMP (Axon) hardware and software. For macroscopic recordings, electrode resistance was 3–5 M{Omega}; for single-channel recording, 10- to 12-M{Omega} electrodes were used. Macroscopic currents were filtered at 2 kHz and sampled at 66 kHz; single-channel recordings were filtered at 2 kHz and sampled at 10 kHz. The electrodes were filled with the enzyme-free Tyrode solution containing also (in mM) 2 BaCl2, 2 CdCl2, 5 tetraethylammonium (TEA), 5 4-aminopyridine (4-AP), and 2 anthracene-9-carboxylic acid (9-AC) to block Ca, K, and Cl currents. The patch was held at -120 mV relative to the membrane potential, which was held near 0 mV by the depolarizing KCl solution. Currents were analyzed using pCLAMP (Axon), Transit (40), and Origin (MicroCal).

A single exponential decay function was fitted to the decay phases of macroscopic currents (Microsoft Excel) to yield the decay time constant. Conductance as a function of voltage was derived from the peak current-voltage relationship. Channel availability as a function of voltage was determined using a two pulse protocol, with a 100-ms step from the holding potential (-120 mV) to the test potential at 0.2 Hz. Currents were measured at -10 mV. Boltzmann functions were fitted to the raw data for each determination (Microsoft Excel).

For recordings from patches containing a few channels, the number of channels was estimated from the number of overlapping openings at strong depolarizations. Late openings and single-channel parameters were analyzed during a 100-ms period beginning 10 ms after the voltage step. Overlapping openings precluded analysis of single-channel parameters earlier in the clamp pulse. Bursts were identified using a critical closed time of 5 ms. Dwell-time histograms were described with sums of exponentials models using a maximum likelihood technique (40).

Statistical analysis.
The significance of the differences of between the peak current voltage relationships and the voltage dependence of current decay (Fig. 1, B and C) was tested using a nonparametric multivariate rank sum test (26, 30). It consists of assigning ranks to the measured parameters for each patch, summing the ranks for each patch, and comparing the sums using a one-way ANOVA. For groups that were significantly different from each other, a Tukey test was used for pairwise comparisons.



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Fig. 1. A: families of macroscopic mouse skeletal muscle Na currents recorded from cell-attached patches in wild-type (left), Dmpk +/- (middle), and Six5 +/- (right) muscle. Holding potential was -120 mV. B and C: pooled data for the peak current-voltage relationship and the time constants of macroscopic current decay. Data points are means ± SE. Open circles, wild-type muscle; solid circles, Dmpk +/- muscle; and solid triangles, Six5 +/- muscle. D: steady-state gating. The data points are means ± SE of individual determinations, and the smooth lines were derived from a least squares fit of the data points to the Boltzmann function. Both channel availability as a function of voltage and conductance as a function of voltage were superimposable in wild-type, Dmpk +/-, and Six5 +/- muscle. For channel availability as a function of voltage, the midpoints and slope factors, respectively, from fits of the data to the Boltzmann function were -95 mV and 6.6 in wild-type muscle, -94.2 mV and 7.2 in Dmpk +/- muscle, and -95.2 mV and 7.6 in Six5 +/- muscle. For conductance as a function of voltage, the corresponding values were -32.4 mV and -9.4 in wild-type muscle, -30.5 mV and -9.9 in Dmpk +/- muscle, and -31.2 mV and -10.5 in Six5 +/- muscle. Data were derived from 9–12 patches, in at least 3 mice. DMPK, myotonic dystrophy protein kinase; Dmpk, the gene encoding DMPK. SIX5, a homeodomain protein, the vertebrate equivalent of sine oculis; Six5, the gene encoding SIX5.

 
The significance of the differences of the proportion of traces with NPo > 0.1 (Fig. 2, CE) were tested using a z-test (SigmaStat). Differences in single-channel parameters were assessed using a one-way ANOVA on ranks because the data were not normally distributed. The Bonferroni correction for multiple comparisons was used. Where the difference between the groups was significant, the Dunn test was used to investigate the difference between the pairs. P < 0.05 was considered statistically significant. Data are given as means ± SE.



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Fig. 2. A: exemplary depolarizing clamp pulses from cell-attached patches containing one or a few Na channels in wild-type (left), Dmpk +/- (middle), and Six5 +/- (left) muscle. The vertical lines on the uppermost trace in each panel show the 100-ms epoch over which late channel activity was assessed. B: ensemble averages of idealized current recordings from multiple patches. The peaks of the currents have been normalized and are not shown, to emphasize the noninactivating persistent component of the current. Currents were recorded at 0 mV (holding potential -120 mV). A maximum of 100 depolarizing clamp pulses of 150-ms duration were studied from each patch. CE: frequency histograms of opening probability (NPo) derived from multiple patches derived from the three groups of mice tested. NPo was assessed over a 100-ms epoch beginning 10 ms after the start of the clamp pulse, i.e., after the macroscopic current had subsided. The left-most bar in each histogram (CD) shows traces with no openings over the epoch studied. The shaded portion of the histograms represents P(NPo > 0.1). In wild-type muscle we studied 18 patches (7 mice), in Dmpk +/- muscle we studied 7 patches (3 mice), and in Six5 +/- muscle we studied 14 patches (3 mice).

 

    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Current amplitude in cell-attached patches of Six5 +/- muscle was similar to wild type, but was reduced in cell-attached patches of Dmpk +/- muscle. This finding was apparent in the exemplary traces (Fig. 1A) and was confirmed in pooled data from multiple patches (Fig. 1B). Wild-type and Six5 +/- muscle were each significantly different from Dmpk +/- muscle (P < 0.05 for each comparison, multivariate rank sum test) but not different from each other [P = not significant (NS)]. Thus Six5 +/- muscle lacks one of the characteristic abnormalities of DMPK-deficient muscle.

Na current decay was significantly faster at potentials near threshold in Six5 +/- muscle compared with either wild-type or Dmpk +/- muscle (Fig. 1, A and C; P < 0.05 for each comparison). Wild-type and Dmpk +/- muscle were not different from each other (P = NS). There were no significant differences in equilibrium gating relationships in wild-type, Six5 +/-, and Dmpk +/- muscle. Figure 1D shows that channel availability and conductance as a function of voltage were essentially identical in the three types of muscle tested.

Figure 2 shows representative sweeps and ensemble average currents from cell-attached patches containing a few channels in wild-type and Dmpk +/- and Six5 +/- muscle. In wild-type muscle the expected finding is near-complete inactivation of Na currents within 5–10 ms and no persistent current. This was the case for the patches from wild-type muscle, where bursts of channel activity in the first few milliseconds after depolarization were only rarely followed by late channel opening (Fig. 1A, left). This was also the case in Six5 +/- muscle, where late Na channel reopenings were infrequent (Fig. 1A, right). In wild-type muscle, noninactivating current (i.e., persistent current at 30–40 ms after the step) accounted for only 0.5% of peak ensemble averaged current, and in Six5 +/- muscle the corresponding value was 0.7% (Fig. 2B). Dmpk +/- muscle, on the other hand, exhibited the characteristic abnormality of Dmpk deficiency. There were frequent late Na channel reopenings and more long bursts (Fig. 1A, middle). As a result, the ensemble average currents (Fig. 2B) displayed a larger noninactivating pedestal of current. This was, at 2.0% of peak current, similar to the magnitude of the effect that has been previously reported in young Dmpk -/- muscle (28).

Figure 2, C to E, shows frequency histograms of NPo, the product of the number of channels (N) and the opening probability (Po), for wild-type, Dmpk +/-, and Six5 +/- muscle. Opening probability was assessed over the 100-ms epoch starting 10 ms after depolarization, i.e., after macroscopic current had subsided. The majority of traces showed no activity (left-most bar in each panel). The shaded bars of the histogram represent traces where NPo exceeded 0.1, and we refer to this proportion of the total area as P(NPo > 0.1). This quantity was similar in wild-type (Fig. 2C) and Six5 +/- (Fig. 2D) muscle (1.3% vs. 3.0% of traces, P = NS, z test) but, in keeping with our previous reports of DMPK deficiency, was significantly increased to 8.9% in Dmpk +/- muscle (Fig. 2D, P < 0.001 for each comparison, z test).

Single-channel analysis confirmed similar properties of wild-type and Six5 +/- muscle and more long openings and more long bursts in Dmpk +/- muscle (Table 1). Open time and burst duration histograms were described by sums of two exponentials models. In all three groups there was a population of shorter openings lasting about 0.5 ms and a population of longer openings lasting 8–15 ms. The proportion of these longer openings was increased to 23% in Dmpk +/- muscle, which was significantly more than in either wild-type or Six5 +/- muscle (P < 0.05 for each comparison, ANOVA). There were also two populations of burst duration. The shorter bursts corresponded to single openings; the duration of bursts of multiple openings was 14 to 25 ms. Again, wild-type and Six5 +/- muscle were indistinguishable (P = NS, ANOVA), but in Dmpk +/- muscle there was an increase in the proportion of long bursts to 34% (P < 0.05 for comparisons with wild-type and Six5 +/- muscle, ANOVA).


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Table 1. Single Na channel analysis

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have measured macroscopic and microscopic Na currents from cell-attached patches of skeletal muscle from wild-type mice, mice heterozygous for Dmpk deficiency, and mice heterozygous for Six5 deficiency. Our major finding is that, although Dmpk +/- muscle recapitulates the Na channel gating abnormality of human myotonic muscular dystrophy, Six5 +/- muscle does not. Mice deficient in Six5 thus have cataracts similar to those encountered in human DM (36) but lack the Na channel abnormality characteristic of the disease. Our data thus concur with the idea that the varied manifestations of DM are the result of defects in the expression of multiple genes.

We have previously shown that Dmpk deficiency causes age-dependent but not gene dose-dependent effects on muscle Na channels (28). Macroscopic current amplitude is reduced, without any systematic effects of current decay or steady-state gating. There are multiple late Na channel reopenings after the macroscopic current has subsided, which is identical to the Na channel defect in human DM muscle (9). This results in membrane depolarization and may contribute to the weakness characteristic of the disease through partial inactivation of Ca channels (3). The single-channel defect deteriorates with increasing age, but among age-matched older mice, there is little difference between mice heterozygous or homozygous for Dmpk deficiency.

To investigate whether the Na channel abnormalities could also be due to Six5 deficiency, we compared Na currents in Dmpk +/- and Six5 +/- muscle. An examination of Six5 -/- muscle would not be expected to be rewarding, because Six5 -/- muscle exhibits a 50% reduction of DMPK mRNA levels, and we have previously shown that this is sufficient to reproduce the Na channel gating defect of the disease (28). In these young mice (<30 wk old), the reduced Na current amplitude and increased P(NPo > 0.1) in Dmpk +/- muscle was indistinguishable from our previously reported data from Dmpk -/- muscle (28). Six5 +/- muscle shared neither of these abnormalities. Na current amplitude and P(NPo > 0.1) were, in fact, indistinguishable from wild-type muscle. Our data show that a reduction in DMPK mRNA levels by <=25% (Six5 +/-) is not sufficient to produce the Na channel gating abnormality. Once the reduction is >=50% (Dmpk +/- and Dmpk -/-), the Na channel gating abnormality is present. The corresponding protein levels cannot yet be determined, because there is as yet no antibody that reliably recognizes the murine form of DMPK (22, 31).

Our previous data indicated no gradation in the effects of Dmpk deficiency on Na channel function between Dmpk +/- and Dmpk -/- muscle. This suggested a threshold for the effects of Dmpk deficiency on Na channel gating at 50% of normal or less. The current data extend this observation. Six5 +/- muscle exhibits a 25% reduction in DMPK mRNA (36). It is not clear whether this deficiency of DMPK mRNA was the direct result of loss of Six5, i.e., that Six5 controls Dmpk transcription, or whether there was a cis effect associated with the Six5 deletion. If the major result of the DM mutation is the inactivation of Six5, and if Six5 controls DMPK mRNA levels, then complete inactivation of Six5 in the disease would be sufficient to account for the Na channel gating defect through the secondary 50% reduction of DMPK mRNA. Patients afflicted with DM are, however, usually heterozygous, and we show here that partial loss of Six5, with a possible associated reduction of DMPK mRNA level by 25% (36), is insufficient to reproduce the Na channel gating abnormality of the disease. The indirect effects of partial Six5 deficiency on Dmpk levels might nonetheless be synergistic with any deficiency of Dmpk induced by the DM mutation. For example, a potential 50% reduction of DMPK induced by nuclear retention of the DMPK RNA in a patient heterozygous for the DM mutation could, in the extreme case, be increased to a 75% reduction by the effects of Six5 deficiency on Dmpk activity.

The sole direct effect of Six5 deficiency that we observed on muscle Na channels was an acceleration in Na current decay at potentials near threshold. The physiological significance and molecular mechanism of this change in current kinetics are not clear, nor are the possible consequences of combining this gating abnormality with those present in DMPK-deficient muscle. To test the potential relevance of the finding in conjunction with DMPK loss, we plan to study the doubly heterozygous Dmpk +/-/Six5 +/- strain.

In conclusion, we have demonstrated that, although Six5 deficiency does not lead to the characteristic Na channel gating seen in DM, partial Dmpk deficiency does recapitulate the abnormality. Our data thus support the idea that the Na channel defect in DM is the result of DMPK mRNA deficiency and suggest that the threshold for this effect is between 25% and 50% of normal levels, with saturation of the effect at a level less than 50% of normal DMPK mRNA levels.


    FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: J. P. Mounsey, Box 6012, MR4 Bldg., UVAHSC, Charlottesville, VA 22908 (E-mail: pmounsey{at}virginia.edu).


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 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
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