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Defects in ryanodine receptor calcium release in skeletal muscle from post-myocardial infarct rats ¹

C. W. WARD^*, S. REIKEN, A. R. MARKS, I. MARTY, G. VASSORT and A. LACAMPAGNE²

INSERM U390/IFR3, Physiopathologie Cardiovasculaire, CHU Arnaud de Villeneuve, Montpellier, France;
^* Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, USA;
Center for Molecular Cardiology, Columbia University College of Physicians & Surgeons, New York, New York, USA; and
INSERM E9931 DBMS/CEA Grenoble, France

²Correspondence: INSERM U390/IFR3, Physiopathologie Cardiovasculaire, CHU Arnaud de Villeneuve, 34295 Montpellier CEDEX 05, France. E-mail: lacamp{at}montp.inserm.fr

Defective calcium (Ca²⁺) signaling and impaired contractile function have been observed in skeletal muscle during chronic myocardial overload (e.g. postmyocardial infarction), however the molecular basis for the muscle defects are not fully understood. In this study, we evaluated sarcoplasmic reticulum Ca²⁺ release mechanisms to further explore the determinants of altered Ca²⁺ signalling in skeletal muscle from rats with chronic myocardial overload.

SPECIFIC AIMS

To this end we examined the properties of 1) global (AP induced transient) and 2) local (i.e., spontaneous Ca²⁺ sparks) Ca²⁺ signaling mechanisms and 3) conducted biochemical analysis of the ryanodine-sensitive Ca²⁺ release channels (RyR1) in a rat post-myocardial infarction model (PMI) of myocardial overload.

PRINCIPAL FINDINGS

Functional and biochemical parameters were evaluated in the fast twitch muscle extensor digitorum longus (EDL) from a postmyocardial infarction rat model (PMI; n=11) or from sham (n=8) -operated control rats that did not receive an infarction. In both PMI and sham groups, the mass of the EDL muscles remained essentially unchanged when expressed as either total mass (332.12±20.79 vs. 290.75±41.90 mg) or mass corrected for body weight (0.57±0.03 vs. 0.58±0.04), consistent with previous reports by others using similar PMI models.

1. Global calcium signaling is altered in EDL muscle fibers during heart failure
Global Ca²⁺ transients were evaluated in single fibers in whole EDL muscle preparations equilibrated with the Ca²⁺ indicator Rhod-2 AM. Single action potential induced Ca²⁺ transients were imaged with multi-photon laser scanning fluorescence microscopy (Zeiss LSM 510, 25x NA=0.8, H₂O imm; Mira/Verdi Coherent 5W). Quantitative analysis revealed that PMI EDL fibers (n=12) had a significant reduction (40.3%; P<0.05) in the amplitude of the Ca²⁺ transient compared with sham fibers (n=17). Consistent with the reduction in amplitude, temporal kinetics were also reduced. The initial rate-of-rise of the Ca²⁺ transient in PMI fibers was depressed by 43% (1.45±0.18 vs. 0.82±0.15 ms^-1; P<0.05) and the total time of the rising phase was extended by 36.8% (7.6±0.4 vs. 10.4±1.7 ms; P<0.05) compared with control muscle. The decay time constant was significantly increased by 29.5% (22.3±1.5 vs. 28.9±2.0 ms; P<0.05).

These observations extend those of Perreault et al., who first described a reduction in the magnitude and a slowing of the uptake of SR Ca²⁺ in association with reductions in twitch and tetanic force in rat EDL muscle during PMI-induced heart failure (HF). These findings contradict those in recent reports by Lunde et al., who showed no differences in the twitch or tetanic forces in fast-type flexor digitorum brevis muscle fibers and no change in the force-Ca²⁺ relationship. However, they reported alterations in the contractile properties of slow twitch soleus muscle during fatigue, which they suggested to be consistent with reduced SR Ca²⁺ release and uptake. The reasons for the disparity of the results in fast-type muscle are not known, but differences in the duration of the underlying HF pathology (6 wk vs. 20 wk in the present study) could be a factor.

2. Local Ca²⁺ signaling is altered in EDL muscle fibers during heart failure
Spontaneous Ca²⁺ sparks were imaged with a laser scanning confocal microscope (Zeiss LSM 510, 63X NA=1.4, oil imm.) between sham and PMI groups on saponin skinned bundles of fibers manually dissected in a manner similar to that described previously. Spontaneous Ca²⁺ sparks are discrete, localized increases in myoplasmic fluorescence arising from a small cluster of RyR-Ca²⁺ release channels and are thought to form the basis for the global Ca²⁺ release behavior in mammalian cardiac and skeletal muscle as well as amphibian skeletal muscle.

Figure 1 A presents Ca²⁺ spark images constructed by synchronizing and averaging subpopulations of Ca²⁺ sparks observed in control (n=35) and PMI (n=27) fibers. This representation depicts all sparks in both conditions within 0.5 SD of the four measured parameters [amplitude, rise time (RT), full width at half-maximal (FWHM), and fall-tau] and synchronized at the start of the rising phase of the spark. This representation of sparks surrounding the mean values suggests that the sparks in PMI had lower amplitude; they also had slower temporal kinetics and greater spatial extent than those in the control conditions. The qualitative observation in Fig. 1A is confirmed when the population of Ca²⁺ sparks in each condition is compared. Figure 1B presents a box-plot of four independent variables [amplitude, rise time (RT), full width at half-maximal (FWHM), and fall-tau] measured in control (black) and PMI (red) fibers. All spatio-temporal properties of Ca²⁺ spark population of the PMI group were significantly altered (P<0.05) compared with sham controls. The mean amplitude was reduced from 0.66 ± 0.01 to 0.49 ± 0.01 F/F, the rise time was slowed from 3.80 ± 0.08 to 4.44 ± 0.17 ms, the decay time constant slowed from 6.21 ± 0.32 to 11.84 ± 0.10 ms and the spatial spread larger (FWHM, 1.74±0.02 vs. 2.05±0.03 µm).

View larger version (23K): [in this window] [in a new window]   Figure 1. Spatio-temporal properties of Ca2+ sparks in PMI. A) Identified Ca2+ sparks collected in sham and PMI animals were synchronized at their start and averaged as shown by the F/F line scan images in the left panel. Temporal and spatial profiles obtained from the images at location indicated by arrows are presented in the right panel. B) Distributions of the spatio-temporal properties of the total population of Ca2+ sparks in SHAM (black; nmuscles=4; nfibers=35; nsparks=314) and PMI (red; nmuscles=5; nfibers=27; nsparks=188) box plots indicate the 25, 50 (i.e., median), 75 percentiles; whiskers indicate the range between the 1st and 99th percentile values of the distribution (*P<0.05).

The relative SR content in the fiber bundle preparation used to measure Ca²⁺ sparks was estimated by evaluating the low-affinity Ca²⁺ indicator Fluo-3FF fluorescence signal after 50 µm 4-chloro-m-²cresol induced Ca²⁺ release. Peak F/F values measured in sham (23.45±3.61 F/F; n=3) and PMI (21.84±2.36 F/F; n=3) were not statistically different, indicating similar SR Ca²⁺ content in both conditions.

Consistent with other reports in mammalian muscle, long-lasting, "ember-like" (several hundred ms) fluorescent events can also be observed. In these experiments, we determined that the ratio of occurrence of sparks vs. embers fluorescence was quite low, but similar, in sham and PMI (2.4±1.1% vs. 3.8±2.3%). In this investigation, however, we limited the detailed analysis to Ca²⁺ spark activity (see on-line manuscript for details).

3. Biochemical properties of the skeletal muscle macromolecular Ca²⁺ release complex are altered during heart failure
It has been demonstrated that FKBP12 associates with the RyR1 channel, promoting a closed-state conformation and providing a mechanism for neighboring channels to operate synchronously (i.e., "coupled gating"). It has been suggested that this coordinated gating is responsible for the stereotypic spatio-temporal properties of Ca²⁺ spark. Recent evidence supports the role of loss of FKBP12.6-RyR association in contributing to abnormal RyR channel gating in cardiac muscle in HF.

Given the effect of FKBP loss on RyR2 function in HF, we examined the level of PKA phosphorylation of RyR1 and the relative amount of FKBP12 associated with the RyR1 macromolecular complex. We demonstrate that RyR1 from PMI EDL muscle was PKA hyperphosphorylated compared with RyR1 from sham EDL muscle (PMI= 1.3±0.3 (n=5) vs. sham = 2.3±0.6 mol (n=3) phosphate transferred per mole of RyR1 channel, P<0.05). The amount of FKBP12 in the RyR1 macromolecular complexes showed a 45% decrease in the PMI compared with sham skeletal muscles (P< 0.05).

CONCLUSION AND SIGNIFICANCE

In an intact muscle, the magnitude and kinetics of single fiber Ca²⁺ transients collectively determine the functional output of the muscle. Compared with control fibers, EDL fibers from PMI rats exhibited weaker and slower Ca²⁺ signaling with a 40% reduction in F/F amplitude and a 43% reduction in the initial rate of rise of transients. Ca²⁺ fluorescence decay was significantly slower and consequently the time-to-peak of transients were slightly increased. Thus, EDL muscle fibers from PMI rats exhibit alterations in both Ca²⁺ release and Ca²⁺ uptake properties of the SR.

In addition to the global Ca²⁺ signaling processes, we show alterations in the spatio-temporal properties of spontaneous Ca²⁺ sparks in EDL muscle from PMI rats. Populations of sparks from EDL fibers were significantly lower in amplitude and had slower temporal kinetics in CHF muscle, without evidence of alteration in SR Ca²⁺ load.

It has been suggested that this coordinated gating is responsible for the stereotypic spatio-temporal properties of Ca²⁺ sparks. By analogy to observations originally made in heart muscle, we demonstrated that RyR1 in PMI muscle is PKA hyperphosphorylated and depleted of FKBP12. Depletion of FKBP12 from the RyR1 macromolecular complex may uncouple channels from one another and allow stochastic as opposed to coupled gating, thus providing an attractive hypothesis for explaining the altered local RyR channel behavior as assayed by examining spontaneous Ca²⁺ spark properties.

In contrast to the Ca²⁺ sparks that arose spontaneously due to Ca²⁺-induced Ca²⁺ release mechanisms, the whole EDL fiber Ca²⁺ transients were elicited by triggered AP. Alterations in RyR1 function per se could be superimposed on specific altered EC coupling mechanisms at the level of DHPR-RyR1 interaction that governs voltage elicited SR Ca²⁺ release in skeletal muscle. It seems reasonable to conclude that any alteration in the gating of RyR may affect the global Ca²⁺ release. In addition, it has been demonstrated that the interaction of the DHPR II-III loop activating domain with the RyR1 channel is modulated by FKBP12. Taken together, we hypothesize that chronic hyperadrenergic activity at the level of the skeletal muscle triggers a disruption of the FKBP12 interaction with the RyR1 and RyR1-DHPR resulting in the altered global and local Ca²⁺ signaling seen in our PMI model.

Alterations in E-C coupling as seen here could play a significant role in the skeletal muscle specific force decrements and reduced exercise tolerance seen in humans and experimental models of HF. Moreover, since cellular Ca²⁺ homeostasis has been shown to play a crucial role in the determination of muscle fiber type, these alterations in RyR1-dependent Ca²⁺ release may play a determinant role in the remodeling of skeletal muscle that occurs during HF.

View larger version (76K): [in this window] [in a new window]   Figure 2. Schematic interpretation of the findings. Global and local measurements of SR Ca2+ release identified a reduction in the amplitude and increase in the spatio-temporal dynamics of the Ca2+ release wave form. We demonstrated that RyR1 in PMI muscle is PKA hyperphosphorylated and depleted of FKBP12. Depletion of FKBP12 from the RyR1 macromolecular complex may uncouple channels from one another and allow stochastic as opposed to coupled gating, providing an attractive hypothesis for explaining the altered local RyR channel behavior as assayed by examining spontaneous Ca2+ spark properties. Recent reports suggest a secondary effect of PKA hyperphosphorylation may alter DHPR-RyR interaction (denoted as ?), and this may also have played a role in the global Ca2+ release behavior elicited by single AP stimulation.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1083fje;

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