duchenne muscular dystrophy (DMD) is a fatal X-linked disease that affects 1 in 3,500 male births (19). It results in progressive skeletal muscle wasting leading to respiratory failure, which is the main cause of death. In addition, DMD results in a dilated cardiac myopathy (DCM). Heart failure is the cause of death in 30% of patients, although up to 90% of patients with DMD exhibit subclinical or clinical cardiac involvement (22). The low rate of cardiac failure in patients has been attributed to the reduced cardiac workload of the wheelchair-bound boys and may increase as respiratory failure is delayed by the use of ventilatory support devices (22).

DMD is caused by the absence of the protein dystrophin, which forms part of the dystroglycan complex. The complex is a group of tightly associated transmembrane and cytoskeletal proteins that form a molecular bridge between dystrophin and the extracellular matrix (20). One hypothesis to explain the DMD phenotype is that dystrophin is involved in regulating sarcolemmal channels, and, in its absence, some channels are abnormally regulated, such as the L-type Ca²⁺ channel and aquaporin (24, 64). One channel of particular interest is the stretch-activated channel (SAC). SACs are nonselective cation channels that respond to mechanical stress with an increase in open probability (27). It is believed that the canonical transient receptor potential channel 1 (TRPC1) gene encodes the mammalian SAC (44). In both the mdx mouse and DMD, the lack of dystrophin results in increased activity of SACs in skeletal muscle (23, 59). The resulting increase in resting intracellular Ca²⁺ concentration ([Ca²⁺]_i) is thought to activate proteases and has been implicated in the pathogenesis of skeletal muscle damage in DMD (65). SACs have been reported in ventricular cells (16) and are proposed to have a role in tachycardia-induced chronic heart failure (14). However, the role of SACs in the DCM associated with DMD is unknown.

The mdx mouse also lacks the protein dystrophin and has been extensively used to study the skeletal muscle manifestations of DMD. A growing body of evidence suggests that the mdx mouse is also an appropriate model in which to study the DCM associated with DMD. In DMD, heart failure develops with age; the number of patients exhibiting DCM increases from one third at 14 years of age to all by 18 years of age (46). In mdx mice the cardiac phenotype also progresses with age, with old animals displaying cardiac dilation, reduced fractional shortening (51), conduction defects (5), and fibrosis (62).

It is widely recognized that failing hearts have defects in Ca²⁺ handling and excitation-contraction coupling (40). It is believed that these findings are due to changes in the expression levels and phosphorylation status of Ca²⁺-handling proteins (40, 55), in addition to alterations in the pathways that regulate these proteins (6, 7). Mdx mouse hearts have been shown to have defects in Ca²⁺-handling proteins, such as decreased levels of sarcoplasmic reticulum (SR) luminal Ca²⁺-binding proteins (42), decreased cardiac SR Ca²⁺-ATPase 2 (SERCA2) mRNA (52), and an increase in resting [Ca²⁺]_i (2). However, no studies have investigated Ca²⁺ transients in mdx mice.

The aim of the current study was to investigate the role of SACs and Ca²⁺ handling in mdx mice hearts, with a focus on age-related changes. Our hypothesis was that SACs may have a role in the development of excitation-contraction coupling changes and heart failure.

METHODS

Animals.

Male wild-type (WT; C57BL/10ScSn) and mdx mice of either 2–3 mo or 9–12 mo of age (young and old, respectively) were obtained from the Animal Resource Center (Perth, Australia). All experiments were approved by the Animal Ethics Committee of the University of Sydney, Australia.

Isolation of cardiac myocytes.

Isolation of single ventricular myocytes was based on the methods of Ju et al. (34). Briefly, mice were anesthetized, the hearts were isolated and Langendorff perfused, and cardiomyocytes were released by collagenase and protease. All myocytes used in this study had well-defined striations and did not spontaneously contract when perfused at 1.5 ml/min at room temperature with a physiological salt solution (PSS, in mM: 140 NaCl, 5 HEPES, 1 MgCl₂, 5.4 KCl, 0.33 NaH₂PO₄, 5.5 glucose, and 1 CaCl₂; pH 7.4).

Measurement of [Ca²⁺]_i.

[Ca²⁺]_i was measured using the fluorescent indicator fluo-4 (Molecular Probes). Single ventricular myocytes were incubated with 1.4 μM fluo-4 AM for 25 min at room temperature and then stimulated at 10% above threshold and at 1 Hz. The experimental protocol was performed on the stage of an inverted confocal microscope (Leica TCS SL). Ca²⁺ transient decay constants were calculated by fitting exponential decay curves to data.

Calibration of fluo-4.

Calibration of fluo-4 was based on the method of Ward et al. (60). At the end of an experiment, cells were exposed to a Ca²⁺-free PSS that contained (in mM) 10 caffeine, 10 2,3-butanedione monoxime (BDM), 10 EGTA, 0.1 2,5-di(tert-butyl)-1,4-benzohydroquinone (TBQ), 1 ouabain, and 0.005 ionomycin. The resulting fluorescence was designated F_min. Cells were then exposed to a NaCl-free, high-Ca²⁺ PSS solution that contained (in mM) 140 LiCl, 2 CaCl₂, 10 caffeine, 10 BDM, 0.1 TBQ, 1 ouabain, and 0.005 ionomycin. The resulting fluorescence was designated F_max. The following equation was then used to convert fluo-4 fluorescence readings into [Ca²⁺]_i concentrations:

Preparation of heart homogenate.

The relative levels of SERCA2, cardiac ryanodine receptor (RyR2), phospholamban (PLN), serine-16 phosphorylated PLN (Ser¹⁶ p-PLN), TRPC1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined by quantitative immunoblotting. Preparation of cardiac tissue homogenates followed the protocol on the Sigma Mammalian Cell product information sheet (Sigma) and was based on the method of Meyer el al. (45). In addition to a protease inhibitor cocktail (1%; Sigma), a phosphatase inhibitor (0.5%; Sigma) was added to the lysis buffer.

Immunoblotting.

Heart sample homogenates were solubilized in Laemmli sample buffer and heated before being separated via SDS-PAGE (Bio-Rad). After electrophoresis, protein was transferred to a nitrocellulose membrane in a Mini Trans-Blot Transfer Cell (Bio-Rad). Membranes were blocked with 5% skim milk powder dissolved in PBS-Tween (PBST; 0.1% Tween; PBS contained in mM: 137 NaCl, 2 KCl, and 10 phosphate buffer) for a minimum of 4 h before being incubated at room temperature for 1 h with primary antibodies (SERCA2 1:20,000, Affinity Bioreagents; RyR2 1:100, SantaCruz; PLN 1:5000 Affinity Bioreagents; Ser¹⁶ p-PLN 1:5000, Badrilla; TRPC1 1:800, Alomone; GAPDH 1:60,000, Biogenesis) and 1 h with secondary antibodies (diluted in PBST). Blots were developed using an enhanced chemiluminescence detection system (Amersham BioSciences). Molecular mass standards were used to calculate the molecular mass of proteins of interest (Invitrogen and Amersham Bioscience). Each individual value represents the mean of two different bands. Total protein loading was normalized by using a Bradford assay (Bio-Rad). All bands were normalized to the protein GAPDH.

Immunohistochemistry.

Immunohistochemistry was based on the methods of Yeung et al. (65). Cross-sections of hearts (10 μm) were blocked in PBS containing 2% bovine serum albumin (PBS-BSA) for 30 min. Sections were incubated for 2 h at room temperature with collagen type-3 primary antibody (1:80; Chemicon) and 1 h at room temperature with a Cy3-conjugated secondary antibody (1:400; Jackson Laboratories).

Statistical analysis.

Data are expressed as means ± SE; n is the number of cells, and N is the number of animals. Differences between means were analyzed using ANOVA or with the Student’s t-test.

RESULTS

Resting [Ca²⁺]_i and Ca²⁺ transients in ventricular myocytes.

It is well established that in heart failure changes occur in the Ca²⁺-handling properties of ventricular myocytes (40). To determine whether similar changes occurred in ventricular myocytes from mdx mice, resting [Ca²⁺]_i was recorded. Resting [Ca²⁺]_i was found to be higher in old mdx mice compared with that of age-matched WTs (old mdx, 67 ± 6 nM; old WT 42 ± 3 nM; P < 0.05; Fig. 1A). There was no difference in the resting [Ca²⁺]_i of young mdx and WT mice.

Ca²⁺ transients produced by electrical stimulation were measured in isolated ventricular myocytes from WT and mdx mice. Myocytes were allowed to rest for 5 min before a stimulation series. The first Ca²⁺ transient in a series had the largest amplitude, with subsequent transients becoming smaller until a steady state was reached (Fig. 1B). We analyzed both the first Ca²⁺ transient in a series and the steady-state Ca²⁺ transients. Here we present results only for the first Ca²⁺ transient, though similar changes were also observed in the steady-state transients. In both young and old mdx mice, the amplitude of the first Ca²⁺ transient was greater than that of age-matched WT mice (young WT, 1,003 ± 11 nM; young mdx, 1,358 ± 18 nM; old WT, 1,167 ± 6 nM; old mdx, 1,700 ± 18 nM; P < 0.05 for both young and old mdx mice; Fig. 1, C and D). The time to peak was shorter in mdx compared with that of WT mice of both ages (Fig. 1E). The decay constant of the Ca²⁺ transient was greater in mdx mice compared with that of age-matched WT (Fig. 1F), indicating that mdx cardiomyocytes took longer to lower [Ca²⁺]_i following a Ca²⁺ transient. These results demonstrate that even in cardiomyocytes of young mdx mice, significant changes have already occurred in Ca²⁺ handling.

Alterations in Ca²⁺-handling protein expression levels.

Immunoblotting experiments were performed on left ventricular tissue to investigate whether the observed changes in the Ca²⁺ transients of mdx mice (Fig. 1) were accompanied by alterations in the protein expression levels of Ca²⁺-handling proteins. RyR2 protein expression levels in young mdx mice showed a twofold increase compared with young WT mice (P < 0.05; Fig. 2, A and B), whereas old mdx hearts showed a threefold increase over old WT hearts (P < 0.001). These results suggest that the increased amplitude and the shorter time to peak of the Ca²⁺ transients in mdx mice could be due to increased RyR2 expression, which would allow for more rapid release of Ca²⁺ from the SR store.

SERCA2 and the Na⁺/Ca²⁺ exchanger (NCX) are the two primary mechanisms for returning [Ca²⁺]_i to resting levels following a Ca²⁺ transient. Thus a decrease in either the expression and/or function of SERCA2 and/or NCX could be responsible for the increased decay constant of Ca²⁺ transients in mdx mice. SERCA2 can be reversibly inhibited by PLN. In the dephosphorylated state, PLN inhibits SERCA2 function (56), whereas phosphorylation of PLN at Ser¹⁶ and Thr¹⁷ following β-adrenergic stimulation reverses this inhibition. It is also believed that a dynamic equilibrium exists between PLN monomers and pentamers (15) and that the PLN monomer is responsible for inhibiting SERCA2 (3, 35).

To test whether decreased levels of SERCA2 and/or altered PLN or Ser¹⁶ p-PLN protein expression were responsible for the increased decay constant of the Ca²⁺ transients in mdx mice, additional immunoblotting experiments were performed. In young and old mdx hearts, SERCA2 protein expression was not significantly different from that of age-matched WT hearts (Fig. 2, C and D). Thus a change in expression levels of SERCA2 cannot explain the increased decay constants of Ca²⁺ transients in mdx mice. However, expression levels of Ser¹⁶ p-PLN monomer were found to be lower in mdx hearts compared with age-matched WT hearts (Fig. 2, G and H). This suggests that the increased decay constants of Ca²⁺ transients in mdx mice is due, at least in part, to increased inhibition of SERCA2 by dephosphorylated PLN monomer. With regard to protein expression of total PLN monomer (Fig. 2, E and F) and total and Ser¹⁶ phosphorylated PLN pentamer (data not shown), no difference was found between mdx and age-matched WT mice.

Caffeine-induced Ca²⁺ transients.

To evaluate whether the increase in the decay constant of Ca²⁺ transients in mdx mice was due to decreased removal of Ca²⁺ via the NCX, caffeine-induced (CI) Ca²⁺ transients were measured. The peak of the Ca²⁺ transient produced by rapid application of caffeine (10 mM) provides an estimate of SR Ca²⁺ content (4), whereas the decay constant is indicative of NCX function (10). To achieve steady-state SR Ca²⁺ loading, cells were stimulated at 1 Hz for 15 s, allowed to rest for 15 s, and then exposed to caffeine.

In young and old mdx mice, the amplitude of the CI Ca²⁺ transient was increased compared with that of age-matched WTs (P < 0.05; Fig. 3, A–C), suggesting that SR Ca²⁺ content is greater in mdx mice of both ages. In old mdx mice the CI Ca²⁺ transient decay constant was decreased compared with age-matched WT mice (P < 0.01; Fig. 3D), suggesting that NCX function is increased. As decreased NCX function does not appear to be involved in the increased decay constant of Ca²⁺ transients in mdx mice, decreased SERCA2 function is probably responsible for the increased decay constant.

Role of stretch-activated channels on intracellular Ca²⁺ handling.

To investigate the role of SACs on the observed Ca²⁺-handling abnormalities in mdx mice, the effect of two SAC inhibitors on resting [Ca²⁺]_i levels in quiescent, unstretched cardiomyocytes was investigated. The two SAC inhibitors used were streptomycin (100 μM) (28) and the spider venom toxin GsMTx-4 (10 μM) (57). Streptomycin had no effect on resting [Ca²⁺]_i in young mice of both strains (Fig. 4A). However, streptomycin reduced resting [Ca²⁺]_i in old mdx mice by 15 ± 4% (P < 0.001) but had no effect on old WT mice (Fig. 4B). To confirm that the action of streptomycin was through blocking SACs, the more potent and specific SAC inhibitor GsMTx-4 (57) was also used. GsMTx-4 reduced resting [Ca²⁺]_i in old mdx mice by 35 ± 4% (P < 0.001) from 159 ± 13% to 107 ± 8% of the old WT value (Fig. 4C). GsMTx-4 had no effect on old WT mice. These results support the hypothesis that SACs are involved in the increased resting [Ca²⁺]_i in old mdx mice.

To further explore this finding, immunoblotting experiments were performed to evaluate TRPC1 protein expression levels, which has been shown to form the mammalian SAC (44). TRPC1 was found to be present in all groups studied. Control experiments using the TRPC1-blocking peptide (antigen) prevented TRPC1 detection (data not shown). There was no difference in the expression levels of TRPC1 between young mdx and WT mice. However, old mdx mice displayed a threefold increase (P < 0.001) in protein expression levels compared with that of old WT mice (Fig. 5, A and B). The above findings suggest that increased protein expression of TRPC1 could play a role in the observed increase in resting [Ca²⁺]_i in old mdx mice.

Old mdx mice have extensive fibrosis.

Fibrosis frequently accompanies heart failure and has been proposed to limit cardiomyocyte motion and increases extracellular electrical resistance resulting in a decrease in conduction velocity (61). The degree of cardiac fibrosis in mdx mice increased with age. Young mdx mice showed no difference in collagen type 3 levels compared with young WT. However, old mdx mice had over a fourfold increase (P < 0.05) in collagen type 3 compared with that of old WT mice (Fig. 6). Increased cardiac fibrosis has been shown to occur in mdx mice that are in heart failure (51). Thus the presence of cardiac fibrosis in the current study suggests that the old mdx mice are in heart failure.

DISCUSSION

It is currently unknown why the absence of the protein dystrophin leads to heart failure. In skeletal muscle it is generally accepted that changes in Ca²⁺ handling are involved in the pathogenic process, although the underlying mechanism leading to raised resting [Ca²⁺]_i is still disputed (1, 11, 29). The aim of the present study was to explore the possibility that absence of dystrophin leads to dysfunction of SACs and intracellular Ca²⁺ handling in cardiac muscle. This study is the first to measure Ca²⁺ transients and Ca²⁺ handling in mdx mice and to determine TRPC1 expression levels in hearts from mdx mice. The data suggests that SACs, and in particular TRPC1, may play a role in the pathogenesis of the cardiomyopathy associated with DMD.

Changed resting [Ca²⁺]_i and Ca²⁺ transients in mdx ventricular myocytes.

The finding that resting [Ca²⁺]_i is raised in mdx mice corresponds well with previous findings from cardiac muscle (2, 18). Increasing resting [Ca²⁺]_i would be expected to lead to an increase in SR Ca²⁺ content, which in turn results in an increase in the amplitude of Ca²⁺ transients (4), as found in the present study. Interestingly, previous work has shown that luminal SR Ca²⁺ binding proteins are reduced in mdx mice (42), suggesting that the increased SR Ca²⁺ content found in the current study may be influenced in a complex manner by the resting [Ca²⁺]_i, the rate of Ca²⁺ uptake by the SR, and a reduction in bound SR Ca²⁺ due to changes in SR Ca²⁺ binding proteins.

The observed increase in the amplitude of Ca²⁺ transients in mdx cardiomyocytes was unexpected, as other models of heart failure have typically found a decrease (47). However, an increase in the amplitude of Ca²⁺ transients has been reported in trabeculas from failing rat and human hearts (58, 60). The time to the peak of the Ca²⁺ transient was shorter in mdx mice, which again is not usually found in established DCM (37). However, the finding correlates well with the observation that RyR2 protein expression was increased in mdx mice. The increased decay constant in mdx mice and protein expression levels of SERCA2 are similar to previous findings (54). In mdx mice of both ages, reduced SERCA2 function appears to be responsible for the increased decay constant of Ca²⁺ transients, as NCX function is not decreased. In fact, NCX function was increased in old mdx mice, as found in other models of heart failure (4). As SERCA2 expression was unchanged in mdx mice, inhibition of SERCA2 by PLN monomer appears to be responsible for the increased decay constants of Ca²⁺ transients in mdx mice. Although total PLN monomer was unchanged, Ser¹⁶ p-PLN monomer was decreased in mdx compared with age-matched WT mice, suggesting that reduced SERCA2 function is at least partly the result of increased inhibition via dephosphorylated PLN monomer. This finding correlates well with findings from patients with DCM (55) and from rat models of myocardial infarction (31, 53). A reduction in p-PLN monomer may result from β-adrenoceptor downregulation, which is thought to occur in human heart failure (6) and mdx mice (43).

It is disputed whether aging of adult hearts affects the decay constant of Ca²⁺ transients (30, 32, 41); in the current study no change was found in mdx or WT hearts. On first inspection, this observation is surprising, because one might predict the decay constant to be reduced due to the finding that SERCA2 expression was increased in mdx and WT old hearts. This conflict can be reconciled by the knowledge that SERCA activity is decreased in isolated SR vesicles extracted from aged animals (25, 36). Importantly, the ability of PLN to be phosphorylated and the responsiveness of SERCA to PLN phosphorylation are not affected by age (33). Thus, in the current study, the increase in SERCA2 expression may be offset by a decrease in SERCA2 activity; the net result being no change in the decay constants in young versus old mice.

It is interesting to note that although young mdx mice display abnormal Ca²⁺ homeostasis and Ca²⁺ dynamics, they do not show signs of heart failure (51). This observation suggests that abnormalities in myocardial Ca²⁺ handling could play a role in the development of the cardiac damage, which precedes heart failure.

What causes the changes in intracellular Ca²⁺ handling?

Two main hypotheses are proposed to explain the raised resting [Ca²⁺]_i in mdx muscle. The first hypothesis proposes that lack of dystrophin renders the sarcolemma more susceptible to damage and, as a result, Ca²⁺ leaks into cells via membrane ruptures (29, 50). However, there is little direct evidence that increased membrane fragility causes the increased membrane permeability to Ca²⁺.

The second hypothesis suggests that dystrophin, directly or indirectly, regulates channels involved in Ca²⁺ homeostasis. In the absence of dystrophin, the activity of these channels becomes greater and causes increased Ca²⁺ entry into mdx myocytes. One particular group of channels that may be regulated via such a mechanism are SACs.

SACs are more active in the skeletal muscle of mdx mice (23) and in humans with DMD (59). Moreover, increased influx of Ca²⁺ through these channels has been demonstrated in mdx skeletal muscle (65). The results from the present study in dystrophic cardiac muscle suggest they might be involved in the DCM found in DMD. As mdx mice age, it is known that they develop cardiac fibrosis (62) and decreased cardiac function (51). The present study demonstrated that, in parallel with the disease onset in mdx mice, as shown by increased cardiac fibrosis, TRPC1 protein levels also increase. Furthermore, in quiescent, unstretched old mdx cardiomyocytes, the two SAC inhibitors streptomycin and GsMTx-4 both reduced resting [Ca²⁺]_i toward that seen in old WT mice; this finding correlates well with the effect of streptomycin on unstretched mdx skeletal muscle (65). Interestingly, this observation implies that in the absence of stretch, SAC activity is increased in mdx cardiomyocytes. This has been shown previously by Franco-Obregon and Lansman (23) who demonstrated that SAC has an increased opening probability in mdx skeletal muscle. It is hypothesized that in healthy animals, when cardiomyocytes become stretched during ventricular filling, SACs open appropriately due to regulation by dystrophin. However, in dystrophin-deficient mdx mice, myocardial stretch results in inappropriate opening of SACs and as a result, an increase in [Ca²⁺]_i.

Increased influx of Ca²⁺ through SACs in mdx muscle could arise from increased expression and/or increased activity of SACs. In the present study, the changed Ca²⁺-handling properties of young mdx cardiomyocytes may have been the result of increased activity of TRPC1, since TRPC1 levels were found to be normal. However, in old mdx mice, increased TRPC1 expression could explain the observed changes in Ca²⁺ homeostasis and dynamics. This raises the question: how are TRPC1 levels increased in old mdx mice? One possibility is that a small increase in resting [Ca²⁺]_i in young mdx mice leads to an increase in reactive oxygen species (ROS Ref. 8), consistent with increased ROS in mdx and DMD skeletal muscle (9, 17). An increase in ROS activates the transcription factor NF-κB (38), which has been shown to increase TRPC1 protein expression (49).

How does absence of dystrophin and raised resting [Ca²⁺]_i lead to heart failure?

Absence of dystrophin appears to lead to increased SAC expression and activity, which allows increased Ca²⁺ influx and an increase in resting [Ca²⁺]_i. How does raised resting [Ca²⁺]_i lead to heart failure? One possible explanation is that Ca²⁺-activated calpains degrade troponin I (21, 26), which produces contractile dysfunction due to decreased maximal Ca²⁺-activated force and decreased Ca²⁺ sensitivity (12, 39, 48).

If this is the case, why are no signs of heart failure observed in young mdx mice? One explanation is that, although dysfunction of SACs and the resulting degradation of troponin I start to occur in young cardiomyocytes, compensatory mechanisms offset any expected fall in force production. Compensatory mechanisms include increased sympathetic stimulation (43) and increased heart rate (13), in addition to a change in Ca²⁺ regulatory proteins and an increase in the amplitude of Ca²⁺ transients. However, in the final stages of DMD, following further degradation of troponin I and extensive fibrosis, force production is eventually compromised and animals progress to heart failure.

In conclusion, SACs could play a role in the pathogenesis of the DCM associated with DMD. Early in the disease process, and before the onset of clinical symptoms, increased SAC activity could explain the observed alterations in Ca²⁺ handling and the increased SR Ca²⁺ content in young mdx mice. Thus early interventions that are able to prevent these changes may have a therapeutic role in DMD.

GRANTS

This work was supported by the National Health and Medical Research Council of Australia. I. A. Williams was the recipient of a Northcote Graduate Scholarship, United Kingdom.

FOOTNOTES

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