INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

ASIDE FROM USING CHRONIC electrostimulation to counteract deleterious effects in denervated and immobilized muscle fibers (20) and employing transformed muscle as sphincter assist devices (59), dynamic cardiomyoplasty is a promising new form of biomechanical ventricular support in the treatment of congestive heart failure (16, 35). However, most physiological and biochemical factors affecting the integrity of transformed muscle grafts and the influence of activity-rest regimens of electrostimulation are mostly unknown (4, 47). Two of the best understood model systems of fast-to-slow muscle transition processes are the chronic low-frequency-stimulated rabbit extensor digitorum longus and tibialis anterior muscles (53). Extensive studies into the effect of electrostimulation of rabbit hind leg muscle fibers through the peroneal nerve have demonstrated that skeletal muscle belongs to the class of highly plastic and adaptable tissues (52). Aside from motorneuron activity, other exogenous factors, such as fuel supply, hormonal influences, and patterns of recruitment, may also cause adaptive changes in adult skeletal muscle fibers (54). Hence, differentiated motor units do not represent invariable physiological entities but exhibit a relatively high capacity to adapt to altered functional demands.

After low-frequency stimulation of fast-twitch fibers, not only cellular destruction and regeneration are observed (37, 38) but also the recruitment of satellite cells and true transdifferentation from a fast to a predominantly slow muscle phenotype (52-54). From a cell biological, biochemical, and physiological viewpoint, long-term, low-frequency stimulation produces muscle cells of decreased caliber, an elevation of the aerobic-oxidative capacity in transformed cells, and fibers that are more resistant to fatigue and that exhibit an increase in the time to peak twitch tension and half relaxation time (22). During the fast-to-slow transition process, drastic biochemical changes are represented by a gradual replacement of myosin heavy chains (MHC) from the MHC IIb isoform to MHC IId(x) to MHC IIa to MHC I (31). In addition, changes in the abundance and/or isoform expression pattern of Ca²⁺ regulatory membrane proteins, including central regulatory elements of the excitation-contraction-relaxation cycle, also occur during muscle conditioning (49). This encompasses the voltage-sensing dihydropyridine receptor (DHPR) of the transverse tubules (where the _1S-DHPR switches to the _1C-DHPR) (18), the ryanodine receptor (RyR) Ca²⁺-release channel of the sarcoplasmic reticulum (where the RyR1 isoform switches to the RyR2 isoform) (18), the luminal Ca²⁺-reservoir complex calsequestrin (CSQ) of the terminal cisternae (where the fCSQ isoform switches to the sCSQ isoform) (51), and the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) representing the Ca²⁺-uptake apparatus of the longitudinal tubules (where the SERCA 1 isoform switches to the SERCA 2 isoform) (10, 23, 50). Although many studies have previously investigated the reciprocal changes in muscle isoforms in response to the initiation and cessation of various electrostimulation protocols (11, 15, 26, 52-54), relatively little is known about the effect of reversal of chronic conditioning on key regulatory components of the excitation-contraction-relaxation cycle. Our laboratory has recently shown that stimulation-induced changes in the expression of Ca²⁺ regulatory membrane proteins were partially or totally reversed during a 30-day recovery period (18). In analogy to these studies, we have determined here whether daily recovery phases significantly change the overall effect of electrostimulation. Thus to study how continuous vs. discontinuous low-frequency stimulation affects the relative density and oligomerization of a key Ca²⁺ regulatory protein, we investigated the well-characterized Ca²⁺ pump of the sarcoplasmic reticulum.

As reviewed by Martonosi (41, 42), in mammalian tissues the SERCA are encoded by three distinct genes. The SERCA1 gene produces the mature fast-twitch skeletal muscle isoform SERCA 1a and its alternatively spliced neonatal form, which is termed SERCA 1b. The SERCA2 gene produces the adult slow-twitch skeletal muscle/cardiac muscle isoform SERCA 2a and the alternatively spliced SERCA 2b isoform, which is predominantly present in nonmuscle cells. The third main type of this class of ion pumps is represented by SERCA3, a relatively broadly distributed Ca²⁺- ATPase. Sarcoplasmic reticulum Ca²⁺ pumps belong to the class of P-type ATPases (34) and exhibit an apparent monomeric molecular mass of 110 kDa (9). The Ca²⁺-ATPase is predominantly located in the longitudinal tubule region (13). On the basis of extensive structural and biochemical investigations, a model for the Ca²⁺-transporting ATPase has been suggested that predicts that a distinct molecular cavity leads to the Ca²⁺ binding site, providing the path for calcium ions into the lumen of the sarcoplasmic reticulum (61), and large domain movements were shown to take place during active transport (58). Individual domains of SERCA monomers are represented by a stalk domain, a transmembrane domain, and a cytoplasmic head piece, which contains the functionally important nucleotide binding site and phosphorylation site (32, 33). Ca²⁺ pumping against a step gradient is mediated by a complex reaction cycle that includes the formation of intermediate and conformational phosphoprotein changes during ATP hydrolysis (2). Oligomerization of the Ca²⁺-ATPase appears to be important for cooperative kinetics and protection against proteolytic degradation (41, 42). We, therefore, performed a chemical cross-linking analysis of this sarcoplasmic reticulum protein after muscle conditioning to determine whether stimulation-induced changes in the abundance of this ion pump also influence protein-protein interactions.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Chronic low-frequency stimulation. To investigate the effect of continuous vs. discontinuous low-frequency electrostimulation on the expression and oligomerization of the sarcoplasmic reticulum Ca²⁺-ATPase, the left hindlimbs of adult male New Zealand White rabbits were telestimulated through the peroneal nerve (55). All experimental procedures were approved by the Regierungspräsidium, Freiburg, Germany. Muscles were continuously (24 h daily) or discontinuously (12-h stimulation and 12-h rest daily) stimulated at 10 Hz, and the stimulation voltage was individually adjusted to yield maximal contractions of the ankle dorsiflexors. After chronic low-frequency stimulation for 4 days, the extensor digitorum longus, the tibialis anterior, and the soleus muscles were excised, cut into several longitudinal sections, and then quick frozen in liquid nitrogen. Muscle specimens were transported on dry ice and stored at 70°C before further use.

Reagents. The cross-linker dithiobis(succinimidyl propionate) (DSP) and chemiluminescence substrates were obtained from Pierce and Warriner (Chester, UK). Peroxidase-conjugated secondary antibodies, protease inhibitors, and acrylamide stock solutions were purchased from Boehringer-Mannheim (Lewis, UK). Immobilon-NC nitrocellulose membranes were from Millipore (Bedford, MA). Monoclonal antibodies IIH11 and IID8 to the fast SERCA 1 and slow SERCA 2 isoform , respectively, of the sarcoplasmic reticulum Ca²⁺-ATPase, as well as monoclonal antibodies IIG12 to triadin, VIIID1₂ to CSQ, and 20A against the ₂-subunit of the DHPR were from Affinity BioReagents (Golden, CO). The monoclonal antibody IIID5 to the ₁-subunit of the DHPR was a generous gift from Dr. Kevin P. Campbell (Howard Hughes Medical Institute, University of Iowa, Iowa City, IA). All other chemicals were of analytic grade and purchased from Sigma Chemical (Dorset, UK).

Preparation of microsomes and chemical cross-linking. Microsomal membrane vesicles were isolated from muscle homogenates by an established protocol at 0-4°C in the presence of a protease inhibitor cocktail [0.2 mM Pefabloc, 1.4 µM pepstatin A, 0.3 µM trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, 1 µM leupeptin, 1 mM EDTA, and 0.5 µM soybean trypsin inhibitor] (46). With the use of myofibrillar proteins as a standard, the protein concentration of isolated membrane vesicles was determined by the method of Bradford (7). To stabilize native complex formation of the sarcoplasmic reticulum Ca²⁺-ATPase, freshly prepared membrane vesicles were incubated with the hydrophobic 1.2-nm cross-linker DSP. With the use of established cross-linking protocols (19), microsomes were treated at room temperature in 50 mM HEPES, pH 8.0, and 0.9% (wt/vol) NaCl for 30 min with 0, 12.5, 25, 37.5, 50, and 100 µg cross-linker/mg membrane protein. The cross-linking reaction was terminated by the addition of 50 µl of 1 M ammonium acetate/ml reaction medium, and then the cross-linker-stabilized complexes were solubilized in 4% (wt/vol) SDS-containing electrophoresis sample buffer (29).

Gel electrophoresis and immunoblot analysis. Proteins were electrophoretically separated under nonreducing conditions according to Laemmli (29). With the use of a Mini-Protean II gel system from Bio-Rad Laboratories (Hemel, Hempstead, UK), SDS-PAGE was performed with 7% (wt/vol) separation gels at a constant voltage of 280 V/h with 10 µg protein per lane. After electrophoretic separation, proteins were transferred from the gel onto Immobilon-NC membranes, according to the method of Towbin et al. (57), using a Bio-Rad Mini-Protean II blotting system (Bio-Rad Laboratories). Subsequently, nitrocellulose sheets were blocked and incubated with primary and peroxidase-conjugated secondary antibodies, as previously described in detail (46). To visualize immunodecorated protein bands, enhanced chemiluminescence was employed (6). Densitometric scanning was performed on a Molecular Dynamics 300S computing densitometer (Sunnyvale, CA) with ImageQuant V3.0 software.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Chronic low-frequency stimulation. To determine whether daily recovery phases significantly change the overall effect of low-frequency electrostimulation on the expression of the relaxation-inducing Ca²⁺ pump of the sarcoplasmic reticulum, two different stimulation protocols were employed. Predominantly fast-twitch rabbit skeletal muscles underwent chronic stimulation or 12-h conditioning followed by a 12-h rest period per day. Before the analysis of the Ca²⁺-ATPase, changes in the relative expression of Ca²⁺ regulatory marker proteins during the early stage of the fast-to-slow transition process were established. As illustrated by immunoblotting, the 94-kDa junctional protein triadin was clearly downregulated (Fig. 1B), whereas the Ca²⁺ binding protein calreticulin increased (Fig. 1D) after chronic electrostimulation. This effect was less prominent for both proteins in muscle fibers stimulated by the alternating activity-rest regimen (Fig. 1, A and C). The increased expression of calreticulin might be due to invading mononucleated cells and/or the appearance of satellite cell-derived myotubules during degeneration-regeneration processes (37, 38). The relative density of the fast isoform of the terminal cisternae Ca²⁺ binding protein CSQ (Fig. 1, E and F) and the ₁- and ₂-subunits of the transverse-tubular DHPR (Fig. 1, G-J) were not drastically affected during the early stages of muscle conditioning. These findings agree with previous reports on the effect of electrostimulation on fast muscle (10, 18, 22, 23, 49-51) and show that the stimulation protocols employed in this study cause a successful, early fast-to-slow transition stage. After 4 days of stimulation, the early markers triadin and calreticulin exhibited drastic changes in their relative expression, whereas the late markers DHPR and CSQ were unchanged; therefore, these transformed muscle specimens could be employed in analyzing the early effect of muscle conditioning on the expression and oligomerization of the Ca²⁺-ATPase.

View larger version (36K): [in this window] [in a new window]   Fig. 1.   Immunoblot analysis of the early fast-to-slow transition phase in continuously and discontinuously low-frequency-electrostimulated rabbit fast muscle. Shown are immunoblots decorated with antibodies to the muscle-specific isoform of the 94-kDa junctional face membrane component triadin (TRI; A and B), the ubiquitous Ca2+ binding protein calreticulin (CAL; C and D), the fast isoform of the terminal cisternae Ca2+ binding protein calsequestrin (CSQ; E and F), the 1-subunit of the voltage-sensing dihydropyridine receptor (1-DHPR; G and H), and the 2-subunit of the transverse-tubular DHPR (2-DHPR; I and J). Lanes 1-4 (A, C, E, G, I) represent 4 days of discontinuously low-frequency (10 Hz)-stimulated muscles (12-h activity and 12-h rest per day) (4d-DS), and lanes 5-8 (B, D, F, H, J) represent continuously low-frequency (10 Hz)-stimulated muscles (24 h/day) (4d-CS). Analyzed microsomal membranes were derived from unstimulated control tibialis anterior muscle (TA/C; lanes 1 and 5), electrostimulated tibialis anterior muscle (TA/S; lanes 2 and 6), unstimulated control extensor digitorum longus muscle (EDL/C; lanes 3 and 7), and electrostimulated extensor digitorum longus muscle (EDL/S; lanes 4 and 8). The position of immunodecorated protein bands is marked by arrows. Sizes of molecular mass standards (in kDa), as deduced from rat myofibrillar proteins, are indicated on the left.

Effect of activity-rest regimens on Ca²⁺-ATPase expression. The immunoblot analysis illustrated in Fig. 2 was performed with monoclonal antibodies IIH11 and IID8, which highly specifically recognize the fast SERCA 1 isoform and the slow SERCA 2 isoform, respectively, of the sarcoplasmic reticulum Ca²⁺- ATPase (50). The chronic low-frequency stimulation protocol (24 h/day) clearly induced a decrease in the relative abundance of the SERCA 1 isoform in both muscles studied (Fig. 2A) and an upregulation of the SERCA 2 isoform (Fig. 2B). In discontinuously conditioned extensor digitorum longus and tibialis anterior muscle (12-h stimulation, 12-h rest per day), the recovery phase almost completely reversed this effect (Fig. 2, C and D). For control purposes, the predominantly slow-twitching soleus muscle was also analyzed. No major changes in the relative density of SERCA 1 and SERCA 2 species were observed in the control soleus in discontinuously stimulated muscles (Fig. 2, C and D). The apparent decrease of SERCA 1 units in control soleus in chronic low-frequency-stimulated muscles may be an artifact or a compensatory mechanism. However, the relatively low density of the fast isoform in this predominantly slow-twitch muscle does not allow for a proper comparison of immunodecoration. These data demonstrate that clear differences exist between the two stimulation regimens with respect to changing the overall expression levels of both Ca²⁺-ATPase isoforms in the extensor digitorum longus and tibialis anterior muscle.

View larger version (48K): [in this window] [in a new window]   Fig. 2.   Expression of the fast and slow sarcoplasmic reticulum Ca2+-ATPase in electrostimulated rabbit skeletal muscle. Shown are representative immunoblots labeled with antibodies to the fast Ca2+-ATPase isoform sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA 1; A and C) and the slow Ca2+-ATPase isoform SERCA 2 (B and D). Individual lanes represent microsomal membrane preparations derived from contralateral TA/C and EDL/C or EDL/S and TA/S muscles, which had been exposed to 4d-CS (A and B) or 4d-DS (C and D). After continuous stimulation, a significant decrease of 41 ± 7% (n = 4; P < 0.05) was found for SERCA 1 expression, and a significant increase of 62 ± 8% (n = 4; P < 0.05) was found for SERCA 2 expression. In contrast, no significant differences were found in the expression levels of SERCA 1 or SERCA 2 in CS vs. DS muscle samples. For control purposes, immunoblots also show labeling of microsomal membranes isolated from soleus muscles of the unstimulated left control leg (Sol/C) and stimulated right leg (Sol/S) of rabbits. Immunodecorated bands are marked by arrows. Sizes of molecular mass standards (in kDa), as deduced from rat myofibrillar proteins, are indicated on the left.

Chemical cross-linking analysis of SERCA 1. To evaluate whether electrostimulation has an effect on the oligomeric status of the fast-twitch isoform of the sarcoplasmic reticulum Ca²⁺-ATPase, an established chemical cross-linking protocol was applied to determine the oligomeric status of this protein. Immunodecoration of the Ca²⁺-ATPase resulted in a much clearer distinction between monomeric and oligomeric species in membrane samples isolated from the extensor digitorum longus muscle compared with the tibialis anterior muscle (not shown); we, therefore, performed the immunoblot analysis after chemical cross-linking with the microsomal preparation derived from homogenates of the extensor digitorum longus muscle. Incubation of native vesicles with the hydrophobic 1.2-nm cross-linker DSP clearly resulted in the appearance of a high-molecular-mass band in normal control samples (Fig. 3, A and C). As demonstrated by the representative immunoblots in Fig. 3, B and D, the appearance of this cross-linker-stabilized oligomeric complex of reduced electrophoretic mobility was not affected by continuous or discontinuous electrostimulation. Consequently, during the early phase of the low-frequency-stimulation-induced transition process, the sarcoplasmic reticulum Ca²⁺-ATPase retains its oligomeric status, probably representing the physiologically active homodimeric and homotetrameric Ca²⁺ pump units.

View larger version (51K): [in this window] [in a new window]   Fig. 3.   Chemical cross-linking analysis of the fast sarcoplasmic reticulum Ca2+- ATPase in electrostimulated rabbit skeletal muscle. Shown are representative immunoblots labeled with an antibody to the fast Ca2+-ATPase isoform SERCA 1. Microsomal membrane preparations were derived from unstimulated contralateral control (A and C) or electrostimulated (B and D) EDL muscles, which had been exposed to 4d-CS (B) or 4d-DS (D). Lanes 1-6 represent 0, 12.5, 25, 37.5, 50, and 100 µg dithiobis(succinimidyl propionate) (DSP)/mg protein, respectively. No significant differences were found in the oligomerizatiuon pattern of SERCA 1 in control vs. stimulated muscle samples (n = 4). Solid arrows indicate the position of monomers, and open arrows mark immunodecorated high-molecular-mass complexes. Sizes of molecular mass standards (in kDa), as deduced from rat myofibrillar proteins, are indicated on the left.

Chemical cross-linking analysis of SERCA 2. In analogy to our immunoblot analysis of the SERCA 1 isoform of the sarcoplasmic reticulum Ca²⁺-ATPase, we investigated the oligomeric status of its slow-twitch counterpart. After 4 days of stimulation, chemical cross-linking stabilized a protein species of reduced electrophoretic mobility (Fig. 4). No apparent difference was observed for the oligomeric status of SERCA 2 between unstimulated control muscle and discontinuously stimulated muscles (Fig. 4, A and B), as well as between chronically stimulated and discontinuously stimulated fast muscle fibers (not shown). Thus stimulation-induced changes in the isoform expression pattern of sarcoplasmic reticulum Ca²⁺-ATPases did not trigger a modification of protein-protein interactions within Ca²⁺ pump units. For control purposes, the cross-linking analysis of SERCA 2 was also performed with membrane vesicles isolated from the soleus muscle from the stimulated and unstimulated hind leg. In both cases, incubation of microsomes with DSP increased the intensity of immunodecoration of the high-molecular-mass complexes recognized by monoclonal antibody IID8 (Fig. 4, C and D). Therefore, any stimulation-induced artifacts and/or compensatory changes in the expression levels of the Ca²⁺-ATPases in the soleus muscle had no effect on the oligomeric status of this sarcoplasmic reticulum component.

View larger version (52K): [in this window] [in a new window]   Fig. 4.   Chemical cross-linking analysis of the slow sarcoplasmic reticulum Ca2+- ATPase in electrostimulated rabbit skeletal muscle. Shown are representative immunoblots labeled with an antibody to the slow Ca2+-ATPase isoform SERCA 2. Microsomal membrane preparations were derived from unstimulated contralateral control (A) or electrostimulated EDL muscles, which had been exposed to 4d-DS (B). For control purposes, immunoblots in C and D show labeling of microsomal membranes isolated from Sol muscles of the unstimulated left control leg and the stimulated right leg of rabbits, respectively. Lanes 1-6 represent 0, 12.5, 25, 37.5, 50, and 100 µg DSP/mg protein, respectively. Solid arrows indicate the position of monomers, and open arrows mark immunodecorated high-molecular-mass complexes. Sizes of molecular mass standards (in kDa), as deduced from rat myofibrillar proteins, are indicated on the left.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The major reason for physiological differences between predominantly fast- and slow-twitch fibers with respect to Ca²⁺-ATPase activities and Ca²⁺-uptake rates is believed to be a differential isoform expression pattern of the Ca²⁺ pump and fiber-type-specific variations in the abundance of sarcoplasmic reticulum membranes (8, 60). Low-frequency electrostimulation is an established model system in investigating the effect of motorneuron-specific impulse patterns on the expression of muscle proteins (52-54) and was applied in this study to determine the fate of SERCA isoforms in the early phase of the fast-to-slow transition process. The finding that both the fast SERCA 1 and the slow SERCA 2 isoform of the sarcoplasmic reticulum Ca²⁺-ATPase form high-molecular-mass complexes in native membrane vesicles isolated from both normal and transformed fast muscles agrees with a variety of previous studies on the subunit composition of this ion pump (1, 28, 36). Electron microscopic studies using comparative freeze-fracture analysis identified 8.5-nm intramembranous particles as dimeric and tetrameric clusters of the sarcoplasmic reticulum Ca²⁺ pump (41, 42). This idea is supported by biochemical studies, i.e., exclusion chromatography of detergent-solubilized sarcoplasmic reticulum preparations, fluorescence energy transfer measurements of native and reconstituted membranes, kinetic measurements on Ca²⁺ translocation, and radiation inactivation studies of the Ca²⁺-ATPase (17, 24, 25, 41). On the other hand, the detailed analysis of the secondary structure and topology of a single Ca²⁺-ATPase molecule predicts a sufficient number of transmembrane helices to constitute a cation channel (9, 33, 34). Furthermore, several studies with monomeric SERCA molecules have demonstrated that the detergent-solubilized enzyme is capable of performing all steps of the complex reaction cycle (3, 21, 40). Despite this experimental evidence on the isolated protein, it is believed that, under native conditions, the physiologically active ion pump has a strong tendency to oligomerize. Although, under in vitro conditions, the Ca²⁺-ATPase may be able to function as a monomer, subunit interactions probably play an important role under in vivo conditions in enzyme stabilization, protection against proteolytic degradation, and/or cooperative kinetics (41, 42).

Previous studies on the effect of chronic low-frequency stimulation on rabbit fast-twitch muscle have shown that the enzyme activity of the sarcoplasmic reticulum Ca²⁺-ATPase is drastically reduced during the early phase of the fast-to-slow transition process (52-54). On the other hand, the relative expression of the fast SERCA 1 isoform does not decrease until 3-4 days after chronic stimulation (56), whereas the Ca²⁺ pump activity is already 50% decreased after only 1-2 days (30). The discrepancy between these two findings could possibly be explained by impaired oligomerization of the Ca²⁺ pump, because protein-protein interactions play a role in cooperative kinetics (41). However, the experimental evidence presented in this report demonstrates that this molecular scenario does not seem to trigger the inactivation of this ion pump. Although the abundance of the enzyme was shown to be reduced, the oligomeric status of SERCA 1 is not affected during the early phase of muscle transformation. Alternatively, chemical modifications in the nucleotide binding site of the Ca²⁺ pump (14, 45), inactivation of the enzyme due to protein oxidation and peroxynitrite-mediated tyrosine nitration (27), a restricted inactivation of only a subset of Ca²⁺-ATPase units (44), or a reduced expression of the SERCA 1 regulatory component sarcolipin (48) may be the reason why the rapid decline in Ca²⁺ pump activity during the very early stages of skeletal muscle transformation does not coincide with a drastic reduction in the amount of sarcoplasmic reticulum Ca²⁺ pumps. Thus oligomerization appears to be of central importance in the proper physiological functioning in both slow and fast isoforms of the Ca²⁺-ATPase. Although no differences could be detected in the oligomeric status of Ca²⁺ pumps from continuously vs. discontinuously stimulated muscle specimens, the activity-rest stimulation regimen had a less pronounced effect on changes in the abundance of SERCA units compared with the chronic stimulation protocol. This suggests that skeletal muscle fibers are extremely plastic and that stimulation-induced changes are reversed in a relatively short period of time. Thus the motoneuron-specific impulse pattern has a profound influence on protein expression levels in the postsynaptic muscle membrane system.

Although chronic electrostimulated muscles are presently employed as ventricular assist devices in dynamic cardiomyoplasty (12, 16, 39), as sphincter assist devices (59), and in the treatment of immobilized and denervated motor units (20), the optimum activity-rest regimens of electrostimulation and the full complexity of interacting biological factors affecting the integrity of the transformed muscle grafts still have to be determined (4, 47). Because the prognosis for chronic heart failure, characterized by left ventricular dysfunction, is usually poor and sudden death is a constant threat even with pharmacological intervention (43), the usage of a transformed, fatigue-resistant latissimus dorsi muscle wrapped around the failing heart is a valid alternative to cardiac transplantation (16). The advantages of dynamic cardiomyoplasty are that autografting does not trigger an immune response and that the transformed skeletal muscle represents a biological cardiac assist device that can generate large forces without being dependent on an external energy supply (39). However, sustained ventricular tachycardia and acute atrial fibrillation are a high risk even after dynamic cardiomyoplasty (5), making biochemical studies into the molecular mechanisms involved in producing chronic low-frequency-stimulated fast fibers an important issue for modern cardiomyoplasty research. In this respect, this report clearly shows that, even after short-term electrostimulation, profound changes occur in muscle fibers. These molecular variations reflect physiological adaptations to changed functional demands, and they appear to be a basic biological property of differentiated skeletal muscle fibers.