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Overexpression of phospholamban in slow-twitch skeletal muscle is associated with depressed contractile function and muscle remodeling¹

QIUJING SONG^*,2, KAREN B. YOUNG^*,2, GUOXIANG CHU^*, JAMES GULICK, MICHAEL GERST^*, INGRID L. GRUPP^*, JEFFREY ROBBINS and EVANGELIA G. KRANIAS^*,3

^* Department of Pharmacology and Cell Biophysics, University of Cincinnati, College of Medicine, Cincinnati, Ohio, USA; and
Division of Molecular Cardiovascular Biology, Children’s Hospital Research Foundation, Cincinnati, Ohio, USA

³Correspondence: Department of Pharmacology and Cell Biophysics, University of Cincinnati, College of Medicine, 231 Albert B. Sabin Way, P.O. Box 670575, Cincinnati, OH 45267-0575, USA. E-mail: Litsa.Kranias{at}uc.edu

SPECIFIC AIMS

Although the functional importance of phospholamban (PLN), the crucial regulator of sarcoplasmic reticulum (SR) Ca²⁺-ATPase (SERCA2a), has been intensively investigated in cardiac muscle, little is known about the role of PLN in the slow-twitch skeletal muscle. To define the physiological significance of PLN in the slow-twitch skeletal muscle, we generated transgenic mice with PLN specific overexpression in soleus muscle and analyzed the resulting functional and structural alterations.

PRINCIPAL FINDINGS

1. PLN protein levels and the PLN/SERCA2a ratio in transgenic soleus are comparable to those in cardiac muscle
In wild-type mice (WT), there was no detectable PLN expression observed in soleus by immunoblotting of muscle homogenates or immunostaining of muscle cryostat sections. However, in the transgenic mice (TG), generated using the ß-myosin heavy chain (ß-MHC) promoter to drive PLN expression in the slow-twitch skeletal muscle, protein levels of PLN in soleus were similar to those of WT cardiac muscle. Overexpressed PLN was codistributed with SERCA2a in the soleus muscle determined by immunocolocalization assessment. The abundance of SERCA2a in transgenic soleus muscle was comparable to that of WT cardiac muscle, indicating that the PLN/SERCA2a ratio in transgenic soleus was similar to that found in normal murine cardiac muscle. However, examination of other important Ca²⁺ handling proteins in PLN-overexpressing soleus revealed significant decreases in the levels of L-type Ca²⁺ channel (WT: 1±0.06-fold; TG: 0.56±0.09-fold; P<0.05), ryanodine receptor (WT: 1±0.04-fold; TG: 0.51±0.03-fold; P<0.05), and its associated protein FKBP-12 (WT: 1±0.05-fold; TG: 0.65±0.06-fold; P<0.05) compared with WT.

2. Isometric twitch contraction is depressed in PLN-overexpressing soleus and can be relieved by isoproterenol stimulation
To evaluate whether the increased PLN expression was associated with alterations in muscle function, isometric twitch contractions at L_max were assessed in soleus from WT and transgenic animals. The transgenic soleus exhibited a significantly altered isometric twitch contraction profile compared with WT. The force developed by PLN-overexpressing soleus was significantly reduced (WT: 4091±344 mg; TG: 2480±154 mg; P<0.05). However, the developed force was similar to that of WT after normalization to the cross-sectional area of the muscle (WT: 600±97 mg/mm²; TG: 592±92 mg/mm²; P>0.05), which was significantly smaller in transgenic soleus. The maximal twitch rate of contraction (+dF/dt) was reduced by 40% (WT: 107.4±8.8 mg/ms; TG: 65.7±2.9 mg/ms; P<0.05) and the maximal twitch rate of relaxation (–dF/dt) was decreased by 45% (WT: 62.9±3.8 mg/ms; TG: 34.8±1.5 mg/ms; P<0.05) in transgenic soleus. There was significant prolongation of both the contraction time (WT: 33±0.9 ms; TG: 65±1.0 ms; P<0.05) and half-relaxation time (WT: 31±0.8 ms; TG: 69±1.4 ms; P<0.05) in PLN-overexpressing soleus compared with WT.

Since PLN has been shown to be a major regulator of the cardiac ß-adrenergic response, we determined whether the prolonged contraction and half-relaxation times in the transgenic soleus could be reversed by ß-adrenergic stimulation using cumulative doses of isoproterenol. Isoproterenol stimulated force production by 33% in WT and 30% in transgenic soleus muscles, respectively. However, isoproterenol stimulation had no significant effect on the contraction time or half-relaxation time in WT soleus whereas it reversed the depressed contraction time and half-relaxation time in transgenic soleus (Fig. 1 ). The maximally stimulated half-relaxation time by isoproterenol was similar in WT and transgenic soleus (Fig. 1b ). Nevertheless, the maximally stimulated contraction time upon isoproterenol in transgenic soleus muscle remained prolonged relative to that in WT (Fig. 1a ).

View larger version (18K): [in this window] [in a new window]   Figure 1. Responses of contraction time and half-relaxation time to the isoproterenol stimulation in slow-twitch skeletal muscles from WT (, n=3) and transgenic animals (TG, , n=4). Data represent the mean ± SE. *P < 0.05 vs. WT, Student’s t test.

3. PLN-overexpressing soleus exhibits a significant diminished muscle mass with smaller cross-sectional area compared with WT
Although the body weight of the transgenic mice was not different from WT, the PLN-overexpressing soleus had a significantly diminished wet weight after normalization to body weight compared with wild-type (WT: 0.24±0.02 mg/g; TG: 0.16±0.01 mg/g; P<0.05). The cross-sectional area of the transgenic soleus muscle was remarkably smaller (WT: 1.0±0.01-fold; TG: 0.76±0.06-fold; P<0.05) although no differences were observed in fiber numbers between the transgenic and WT soleus muscles. There were no pathological alterations in the hematoxylin/eosin stained transgenic soleus. The protein level of calpain, a calcium-activated protease, was significantly increased in the transgenic soleus (2.15±0.09-fold) compared with WT (1.0±0.06-fold).

4. The percentage of slow-type muscle fibers in PLN-overexpressing soleus is significantly increased compared with WT
To verify whether the gross morphological changes in the soleus muscle were related to muscle fiber-type alterations in transgenic mice, the proportion of slow and fast fibers was determined. An average of 176 fibers per subject (n=3 for WT; n=3 for transgenic mice) were classified as slow (type I) or fast (type II) fibers according to the nomenclature of Brooke and Kaiser (Fig. 2 a–f). The transgenic soleus contained a similar number of total muscle fibers per section as WT; however, the percentage of slow fibers was 25% higher in transgenics compared with WT (Fig. 2g ).

View larger version (56K): [in this window] [in a new window]   Figure 2. Myofiber type specification in wild-type (WT, n=3) and transgenic soleus muscles (TG, n=3). Cryostat cross-sections of soleus muscles were stained for myosin ATPase after preincubation at different pH. a, d) pH = 4.2; b, e) pH = 4.5; c, f) pH = 10.2. Slow fibers (type I): stained heavily when pH = 4.2 and 4.5, lightly when pH = 10.2; fast fibers (type II): stained lightly when pH = 4.2 and 4.5, heavily when pH = 10.2. Original magnification x40. g) Quantitation of muscle fiber type specification. Data represent the mean ± SE. *P < 0.05 vs. WT, Student’s t test.

Several studies have suggested the involvement of Ca²⁺/calcineurin (Cn) signaling in controlling the skeletal muscle fiber type. The CnA ß protein levels were significantly increased in the transgenic soleus (WT: 1±0.17; TG: 1.58±0.13; P<0.05).

CONCLUSIONS

This study presents the first evidence that PLN overexpression driven by the ß-MHC promoter is associated with significant alterations of isometric twitch performance and muscle remodeling in slow-twitch skeletal muscles. Protein levels of PLN and the ratio of PLN/SERCA2a were similar to those in normal mouse cardiac muscle, indicating that 40% of the SERCA2a molecules in transgenic soleus were in the "inhibited state" by PLN. This inhibition resulted in prolongation (2-fold) of the half-relaxation and contraction times in transgenic soleus. However, isoproterenol stimulation could relieve the inhibitory effects of PLN in the transgenic soleus, whereas no alterations in contraction or relaxation time upon isoproterenol stimulation were observed in WT soleus, reflecting the low levels of endogenous PLN in this muscle. Isoproterenol stimulation relieved the depression of half-relaxation time in the PLN-overexpressing soleus but did not fully reverse the prolonged contraction time, implying involvement of other players in the ß-adrenergic response of slow-twitch skeletal muscles. Thus, our findings suggest that the alterations in basal twitch dynamics of transgenic soleus were mostly due to overexpression of PLN, which acts as an inhibitor of SERCA2a in slow-twitch skeletal muscle. Phosphorylation of PLN during isoproterenol stimulation relieved the PLN inhibitory effects. Detailed biochemical studies of SR Ca²⁺ transport in WT and PLN-overexpressing soleus may allow us to better define the functional role of PLN in transgenic SR membranes, but the small size of this muscle will present technical limitations.

The PLN-overexpressing soleus exhibited significantly diminished muscle mass and increased percentage of slow-type fibers, which may contribute to its depressed contractile function. Muscle mass is principally maintained by the balance of muscle protein synthesis and degradation. The calcium-dependent protease calpain has been demonstrated to play a prominent role in muscle degradation. Indeed, calpain levels were significantly increased in the transgenic soleus. Elevated levels of calpain coupled with chronic accumulation of cytosolic Ca²⁺ due to the inhibited SR Ca²⁺ cycling by PLN overexpression may lead to enhanced activation of calpain, resulting in increased degradation of skeletal muscle protein. However, the primary cause responsible for the smaller soleus muscle in the transgenic mice remains elusive due to potential involvement of embryonic overexpression of PLN in slow-twitch skeletal muscles.

Another finding of our study is the increased number of slow-type fibers in PLN-overexpressing soleus muscles. Previous studies using cross-innervation or electrical pacing approaches revealed that changes in intracellular Ca²⁺ concentration can promote fiber-type switch due to the activation of calcineurin. Recent transgenic studies revealed that overexpression of calcineurin leads to increase of slow-type fibers in vivo. In contrast, CnA ß-deficient mice showed a significant decrease of slow-type fibers in skeletal muscles. CnA ß protein levels were elevated in the PLN-overexpressing soleus muscle, accompanied by increased proportion of slow type fibers. More studies are required to dissect the molecular mechanisms underlying the association of elevated CnA ß levels with increased proportion of slow muscle fibers in the PLN-overexpressing soleus.

In summary, our findings indicate that the slow-twitch skeletal muscle mechanics may be attenuated by increased PLN expression in vivo and this is associated with muscle remodeling. Thus, induction of PLN expression in slow-twitch skeletal muscle would result in functional and structural alterations compromising the physiological responsiveness of slow-twitch fibers especially in "fight or flight" situations.

View larger version (72K): [in this window] [in a new window]   Figure 3. Schematic diagram describing proposed molecular mechanisms underlying the functional and structural alterations in slow-twitch skeletal muscles induced by overexpression of PLN. Transgenic expression of PLN in soleus results in significant inhibition of Ca2+ uptake into SR by SERCA2a. With the decreases in the SR Ca2+-releasing channel RyR1 and sarcolemmal L-type Ca2+ channel, less Ca2+ is available during contraction, which is responsible for the depressed contractility in the PLN-overexpressing soleus. However, phosphorylation of PLN and other regulatory Ca2+ handling proteins by cAMP-dependent protein kinase A (PKA) upon ß-adrenergic stimulation can improve the impaired muscle function. Elevation of the cytosolic Ca2+ due to depressed SR cycling by PLN overexpression may activate calcineurin and its downstream targets to promote increases in slow-type fibers. Accumulation of the cytosolic Ca2+ may also lead to activation of calpain, a principal protease involved in skeletal muscle protein degradation and diminished muscle mass. In turn, these structural changes may contribute to the depressed contractile function in the PLN-overexpressing soleus.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1058fje;

² Both authors contributed equally to this work.

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This Article Full Text (PDF) All Versions of this Article: 18/9/974 03-1058fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by SONG, Q. Articles by KRANIAS, E. G. Search for Related Content PubMed PubMed Citation Articles by SONG, Q. Articles by KRANIAS, E. G.