Inhibitory control over Ca(2+) sparks via mechanosensitive channels is disrupted in dystrophin deficient muscle but restored by mini-dystrophin expression

Abstract

Background: In dystrophic skeletal muscle, osmotic stimuli somehow relieve inhibitory control of dihydropyridine receptors (DHPR) on spontaneous sarcoplasmic reticulum elementary Ca(2+) release events (ECRE) in high Ca(2+) external environments. Such 'uncontrolled' Ca(2+) sparks were suggested to act as dystrophic signals. They may be related to mechanosensitive pathways but the mechanisms are elusive. Also, it is not known whether truncated dystrophins can correct the dystrophic disinhibition.

Methodology/principal findings: We recorded ECRE activity in single intact fibers from adult wt, mdx and mini-dystrophin expressing mice (MinD) under resting isotonic conditions and following hyper-/hypo-osmolar external shock using confocal microscopy and imaging techniques. Isotonic ECRE frequencies were small in wt and MinD fibers, but were markedly increased in mdx fibers. Osmotic challenge dramatically increased ECRE activity in mdx fibers. Sustained osmotic challenge induced marked exponential ECRE activity adaptation that was three times faster in mdx compared to wt and MinD fibers. Rising external Ca(2+) concentrations amplified osmotic ECRE responses. The eliminated ECRE suppression in intact osmotically stressed mdx fibers was completely and reversibly resuscitated by streptomycine (200 microM), spider peptide GsMTx-4 (5 microM) and Gd(3+) (20 microM) that block unspecific, specific cationic and Ca(2+) selective mechanosensitive channels (MsC), respectively. ECRE morphology was not substantially altered by membrane stress. During hyperosmotic challenge, membrane potentials were polarised and a putative depolarisation through aberrant MsC negligible excluding direct activation of ECRE through tubular depolarisation.

Conclusions/significance: Dystrophin suppresses spontaneous ECRE activity by control of mechanosensitive pathways which are suggested to interact with the inhibitory DHPR loop to the ryanodine receptor. MsC-related disinhibition prevails in dystrophic muscle and can be resuscitated by transgenic mini-dystrophin expression. Our results have important implications for the pathophysiology of DMD where abnormal MsC in dystrophic muscle confer disruption of microdomain Ca(2+) homeostasis. MsC blockers should have considerable therapeutic potential if more muscle specific compounds can be found.

Figure 1. ECREs in intact single muscle fibers from wt, mdx and MinD mice under isotonic conditions.

A, image sequence from a time series of ECRE (arrows) recordings in a resting intact wt, mdx and MinD fiber bathed in isotonic external solution. B, ECRE are more frequent in mdx fibers than in wt or MinD fibers. SF: spark frequency. SSF: spatial spark frequency. *: P<0.05 vs. wt. Scale bar: 40 µm.

Figure 2. ECRE activity following hyperosmolar or hypoosmolar challenge.

Image series from a single intact mdx fiber either stressed with hypertonic (A) or hypotonic external solution (B). Compared with the initial isotonic condition, osmotic challenge markedly increases apparent SF (C). The response time for the SF increase in hypotonic solution is larger than in hypertonic solution. Apparent SF shows adaptation to ongoing osmotic challenge with a rapid decline before the washout of hyper-/hypotonic medium. Scale bar: 20 µm.

Figure 3. ECRE activity increase and kinetics by hypertonic and hypotonic stress.

A, SSF evaluated from XYT recordings in several hundred (n) single wt, mdx and MinD fibers under isotonic, hypertonic or hypotonic external conditions. Average SSF values are similar in wt and MinD fibers but significantly increased in mdx fibers under all conditions. The kinetics of the apparent SSF based on a frame-by-frame analysis is shown in Fig. 2C for a random selection of 15 fibers for each condition and strain. (B), (C) and (D), time-to-peak (TTP), peak SSF and time constants τ_dec of the exponential SSF decline under maintained osmotic challenge. *: P<0.05 vs. both wt and MinD, or as indicated by the appropriate bar. #: P<0.05 hypotonic vs. hypertonic, same strain.

Figure 4. The increase in ECRE activity following osmotic challenge is independent of external Ca²⁺.

A, representative recording of ECRE in a single mdx fiber under resting condition in isotonic solution and following osmotic stress in hypertonic Ca²⁺ free external solution at the time points indicated. B, summary of SF and SSF values from up to ∼100 single mdx fibers following hypertonic or hypotonic challenge without external Ca²⁺. The approximately fivefold increase in SSF under both conditions is similar to the increase observed in the presence of 2 mM external Ca²⁺ (Fig. 3A). *: P<0.001 vs. isotonic solution. Scale bar: 25 µm.

Figure 5. Osmotic-induced increase in ECRE activity is mediated by mechanosensitive channels.

A, mean apparent ECRE frequencies (SSF_max) on a frame-by-frame analysis from ∼40 single mdx fibers following osmotic challenge (hypertonic: filled symbols, hypotonic: open symbols) and application of either 200 µM streptomycine (circles) or 20 µM Gd³⁺ (triangles), respectively. The presence of the blockers is indicated by a black line. Image sequence above: ECRE response in a representative single mdx fiber during hypertonic shock and streptomycine (str.) application. Right two bar panels show averaged SSF values during the time intervals prior to osmotic shock (isotonic), during osmotic shock, after blocker application and during washout. B, SSF_max values in at least twelve single mdx fibers subjected to osmotic shock after pre-incubation with the MsC blockers. Pre-incubation with blockers failed to induce the marked response of osmotic shock seen in (A). Discontinuities in SSF_max are due to the fact that data points were omitted for clarity when no ECREs were present in all fibers analysed for this time point (SSF_max = 0). All experiments performed in 2 mM Ca²⁺ containing external solution. Image sequence above: fiber pre-incubated with Gd³⁺ followed by hypotonic shock. *: P<0.01 blockers compared to the previous condition, #: P<0.001 osmotic shock compared to the previous condition, §: P<0.01 washout is different from all previous conditions. Scale bars: 20 µm.

Figure 6. Ca²⁺ dependent and Ca²⁺-independent component of MsC-mediated osmotically induced ECRE activity.

A, apparent SSF_max values during hypertonic challenge in ∼35 single mdx fibers following hypertonic challenge in either 2 mM Ca²⁺ containing (filled circles) or Ca²⁺-free external solution (open squares) and block of ECRE responses by application of 5 µM of the specific MsC blocker GsMTx-4. The inset shows the averaged SSF values during the time intervals in isotonic solution, during hypertonic shock, after blocker application and during washout. Note the different scale for both external Ca²⁺ conditions. B, shows the averaged SSF values from similar experiments performed in ∼35 other single fibers during hypotonic shock. *: P<0.001 compared to the previous conditions. #: P<0.001 Ca²⁺-free vs. 2 mM Ca²⁺ under the same condition (isotonic, shock, block or washout).

Figure 7. ECRE morphology in single mdx fibers following osmotic challenge.

A, line-scan (XT) recordings in a single mdx fiber under isotonic, hypertonic or hypotonic external conditions. Upper row: noisy raw data; lower row: wavelet-denoised data. ECRE time course is shown by intensity profiles (red lines). Morphology parameter histograms from several hundred ECREs: ECRE amplitudes (B), rise-times RT (C), full-width at half-maximum FWHM (D), full-duration at half-maximum FDHM (E). Apart from a right-shift of the amplitude and RT distributions, there were no major alterations in ECRE morphology following osmotic challenge. Horizontal scale bar: 200 ms, vertical scale bars: 20 µm.

Figure 8. Ca²⁺ waves induced by osmotic challenge in single mdx fibers.

A, single mdx fiber with global Ca²⁺ waves rather than increased ECRE activity following osmotic challenge (hypertonic solution, 2 mM Ca²⁺). Numbers indicate the frame number during a 50 frame XYT series. Scale bar: 20 µm. White line: ROI from which spatial profiles (B) were obtained. Ca²⁺ waves were never observed in wt or MinD fibers.

Figure 9. Mechanosensitive channel activity during osmotic challenge does not induce marked membrane depolarisations in mdx fibres.

Resting membrane potentials in many intact fibres were recorded by repetitive impalement of whole interossei muscles from wt (black) and mdx (white) mice without enzymatic treatment under isotonic and hypertonic conditions. The contribution of cation influx through mechanosensitive channels to the resting potential when muscles were immersed in either isotonic or hypertonic Ringer solution was assessed by pre-incubation with 5 µM of the spider peptide GsMTx-4, a selective MsC blocker. Although potentials were more depolarised in mdx fibres already under isotonic conditions, this did not increase under hypertonic conditions, nor was there a marked contribution from MsC to depolarisation. n: number of individual potential recording.

Figure 10. Proposed model of interactions of mechanosensitive channels (MsC/MsCa) with spontaneous ECRE inhibition in wt muscle and relieved inhibition in mdx muscle during osmotic stress.

In wt muscle, dystrophin expression is linked to suppressed MsC/MsCa activity probably by directly stabilising them to the membrane scaffold under isotonic and hypertonic conditions. The DHPR α1_S subunit exerts an inhibitory effect on the RyR1 Mg²⁺ site via the II–III loop. A direct modulation of this inhibition by mechanosensitive channels is postulated that would not play a major factor in the wt. As a result, under both resting and membrane stress conditions, spontaneous ECRE would be largely suppressed. In the mdx phenotype, stabilisation of MsC/MsCa to the tubule membrane is insufficient in the absence of dystrophin, thus mechanically opening mechanosensitive channels in an aberrantly transducting mode. The putative direct interaction of the latter with the DHPR loop (or other sites) would relief inhibition of the RyR under resting and, more importantly, under membrane stress conditions. ECRE frequency is expected to increase via disinhibited Ca²⁺ release. Depending on additional Ca²⁺ influx through mechanosensitive channels under different external Ca²⁺ containing conditions, peak ECRE frequencies are modulated by Ca²⁺ dependent downstream activation of NOX/ROS pathways that may directly activate ECRE . SR: sarcoplasmic reticulum. DAG: dystrophin-associated glycoprotein complex.