Diaphragm assessment in mice overexpressing phospholamban in slow‐twitch type I muscle fibers

Abstract Aims Phospholamban (PLN) and sarcolipin (SLN) are small inhibitory proteins that regulate the sarco(endo)plasmic reticulum Ca2+‐ATPase (SERCA) pump. Previous work from our laboratory revealed that in the soleus and gluteus minimus muscles of mice overexpressing PLN (Pln OE), SERCA function was impaired, dynamin 2 (3–5 fold) and SLN (7–9 fold) were upregulated, and features of human centronuclear myopathy (CNM) were observed. Here, we performed structural and functional experiments to evaluate whether the diaphragm muscles of the Pln OE mouse would exhibit CNM pathology and muscle weakness. Methods Diaphragm muscles from Pln OE and WT mice were subjected to histological/histochemical/immunofluorescent staining, Ca2+‐ATPase and Ca2+ uptake assays, Western blotting, and in vitro electrical stimulation. Results Our results demonstrate that PLN overexpression reduced SERCA's apparent affinity for Ca2+ but did not reduce maximal SERCA activity or rates of Ca2+ uptake. SLN was upregulated 2.5‐fold, whereas no changes in dynamin 2 expression were found. With respect to CNM, we did not observe type I fiber predominance, central nuclei, or central aggregation of oxidative activity in diaphragm, although type I fiber hypotrophy was present. Furthermore, in vitro contractility assessment of Pln OE diaphragm strips revealed no reductions in force‐generating capacity, maximal rates of relaxation or force development, but did indicate that ½ relaxation time was prolonged. Conclusions Therefore, the effects of PLN overexpression on skeletal muscle phenotype differ between diaphragm and the postural soleus and gluteus minimus muscles. Our findings here point to differences in SLN expression and type I fiber distribution as potential contributing factors.


Introduction
Centronuclear myopathies (CNM) are a group of congenital myopathies that, along with general muscle weakness, display increased central nuclei, type I fiber predominance and hypotrophy, and central aggregation of oxidative activity upon muscle biopsy (Sharma et al. 2009;Romero 2010). Phospholamban (PLN) is a small (52 amino acid) protein that physically interacts with and inhibits the sarco(endo)plasmic reticulum Ca 2+ -ATPase (SERCA) pump (Morita et al. 2008). Recently, we found that the soleus and gluteus minimus muscles from mice overexpressing PLN (Pln OE ) in their slow-twitch type I skeletal muscle fibers had impaired SERCA function and displayed the histopathological features associated with human CNM (Fajardo et al. 2015). Based on the appearance of radiating sarcoplasmic strands, type II fiber hypertrophy, and endomysial fibrosis, the CNM observed in the Pln OE mice resembles human autosomal dominant CNM and, to an extent, RYR-related and TTN-related CNM (Jungbluth and Gautel 2014).
A recently published review highlighted that although diaphragm and ventilatory function have been examined in several murine models of myopathy including Pompe disease and Duchenne muscular dystrophy, among other neuromuscular diseases, similar reports are not available for animal models of CNM (Smith et al. 2014). Compared with the other two major CNM variants, X-linked myotubular myopathy and autosomal-recessive CNM, respiratory function in patients with autosomal dominant CNM, for the most part, is intact and patients live a normal lifespan (Bitoun et al. 2005;Jungbluth et al. 2008;Romero 2010;Smith et al. 2014). Thus, it was of interest to determine whether or not the diaphragm muscles of the Pln OE mouse, which more closely resembles autosomal dominant CNM, would exhibit CNM pathology and muscle weakness. Since in this model, PLN is overexpressed specifically in the type I fibers and the murine diaphragm normally consists of around 10% type I fibers (Green et al. 1984;Talmadge et al. 2004); we initially hypothesized that the diaphragm from Pln OE mice would display impaired SERCA function, muscle weakness, and the appearance of the histopathological features associated with human CNM.

Animals and tissue collection
The Pln OE mice were resuscitated from cryopreserved embryos by the mmRRC (00067-MU, FVB/N background) to generate a breeding colony with WT FVB/N mice at the University of Waterloo. The Pln transgene was attached to the b-MHC promoter so that these mice exhibit type I fiber-specific PLN overexpression. A total of 15 male wild-type (WT; 30.9 AE 1.2 g) and 16 male Pln OE (30.7 AE 0.9 g) 4-6-month-old mice were used in the study. Animals were housed in an environmentally controlled room with a standard 12:12-h light-dark cycle and allowed access to food and water ad libitum. Mice were sacrificed by cervical dislocation and diaphragm, soleus, and gluteus minimus muscles were extracted. We restricted analyses to the anterior-lateral costal regions of the diaphragm muscles. Diaphragm strips were either used for the assessment of SERCA function, contractility, or histological/histochemical/immunofluorescence staining for the examination of central nuclei, central aggregation of oxidative activity and fiber type distribution and CSA. Nuclear and cytosolic fractions from the diaphragm, soleus, and gluteus minmus muscles were extracted as previously described (McMillan and Quadrilatero 2011). All animal procedures were reviewed and approved by the Animal Care Committee of the University of Waterloo and are consistent with the guidelines established by the Canadian Council on Animal Care.

SERCA activity and Ca 2+ uptake
Ca 2+ -dependent SERCA activity was assessed in homogenates prepared from mouse (WT and Pln OE ) diaphragm muscles over Ca 2+ concentrations ranging from pCa 7.0 to 4.8 at 37°C using a spectrophotometric plate reader assay that has been described previously (Duhamel et al. 2007). Briefly, 10 lL of crude diaphragm homogenate was added to 5 mL cocktail buffer containing the reaction buffer (200 mmol/L KCl, 20 mmol/L HEPES, 15 mmol/L MgCl 2 , 10 mmol/L NaN 3 , 10 mmol/L phosphoenolpyruvate, 5 mmol/L ATP, 1 mmol/L EGTA, pH 7.0), 18 U/mL lactate dehydrogenase, 18 U/mL pyruvate kinase, 0.3 mmol/L NADH, in the presence of ionophore A23187 (Sigma C7522, 4 lmol/L, St. Louis, MO). Total ATPase activity across the range of pCa was determined as the rate of NADH disappearance over 30 min measured at 340 nm using a spectrophotometric plate reader (SPEC-TRAMAX plus; Molecular Devices, Toronto, ON, Canada). SERCA activity was then calculated by subtracting ATPase activity in the presence of a SERCA-specific inhibitor, cyclopiazonic acid (40 lmol/L) (Seidler et al. 1989), and SERCA activity-pCa curves were generated with GraphPad Prism TM (version 6, GraphPah Software, Inc. La Jolla, CA) by nonlinear regression curve fitting using an equation for a general cooperative model for substrate activation. Ca 2+ uptake was measured in muscle homogenates at 37°C in the presence of the precipitating anion, oxalate, using the fluorescent dye Indo-1 (50042; Biotium, Hayward, CA) and a spectrofluorometer (RatiomasterTM System; Photon Technology International, Birmingham, NJ) equipped with a monochromator to control the excitation wavelength (355 nm) and two photomultiplier tubes to detect emitted light (405 and 485 nm) (Tupling and Green 2002). Rates of Ca 2+ uptake were assessed at a free Ca 2+ concentration of 1.0 lmol/L.

Histological, histochemical, and immunofluorescence staining
Diaphragm muscles from WT and Pln OE mice were removed and embedded in O.C.T. compound (Tissue-Tek, Sakura Finetek USA Inc., Torrance, CA), frozen in liquid nitrogen-cooled isopentane, stored at À80°C, and cut into 10-lm-thick cryosections with a cryostat (Thermo Fisher Scientific Inc., Grand Island, NY, United States) maintained at À20°C. To examine the percent of fibers containing centrally located nuclei and central aggregation of oxidative activity, hematoxylin and eosin (H&E) staining, and succinate dehydrogenase (SDH) activity were performed, respectively. Images were acquired with a brightfield Nikon microscope linked to a PixeLink digital camera and central nuclei counts were quantified with ImageJ software (National Institutes of Health, Bethesda, MD) using the cell counter plugin. One section of the diaphragm muscle from each mouse (n = 4 per genotype) was randomly selected and 793-1292 fibers were analyzed for central nuclei per section. For fiber type analysis (CSA and % distribution), immunofluorescence analysis of MHC expression with primary antibodies against MHCI, MHCIIa, and MHCIIb was performed. Details regarding the dilutions of the primary and secondary antibodies for fiber type analysis have been previously described (Bloemberg and Quadrilatero 2012). Slides were visualized with an Axio Observer Z1 fluorescent microscope equipped with standard red/green/blue filters, an AxioCam HRm camera, and AxioVision software (Carl Ziess, North York, ON, Canada). Details of the immunofluorescence procedures and analysis of MHC expression were previously described (McMillan and Quadrilatero 2011;Bloemberg and Quadrilatero 2012;Fajardo et al. 2013). Briefly, for fiber type distribution, one section of the diaphragm muscle from each mouse was randomly selected and all fiber types within that section were counted and sorted by fiber type (1126-3105 total fibers per section) using ImageJ's cell counter plugin. For analysis of fiber type area, 20 fibers of each type were randomly selected within a diaphragm section and the area of each fiber was determined and then averaged using ImageJ's area measurement tool after calibrating with the corresponding scale bar. This was repeated for five different WT and Pln OE mice, and the values reported represent the calculated averages from the five animals within the specific genotype.

In vitro diaphragm contractile assessment
Diaphragm strips were isolated and immediately placed into a bath of oxygenated Tyrode's solution (95% O 2 , 5% CO 2 ) containing 121 mmol/L NaCl 2 , 5 mmol/L KCl, 24 mmol/L NaHCO 3 , 1.8 mmol/L CaCl 2 , 0.4 mmol/L NaH 2 PO 4 , 5.5 mmol/L glucose, 0.1 mmol/L EDTA, and 0.5 mmol/L MgCl 2 , pH 7.3  and were maintained at 25°C. Muscle strips were situated between flanking platinum electrodes driven by a biphasic simulator (Model 710B; Aurora Scientific, Inc., Aurora, ON, Canada) and electrically evoked muscle force was assessed across a range of stimulation frequencies from 1 to 100 Hz at optimum length for force production. Data were analyzed using Dynamic Muscle Control Data Acquisition software (Aurora Scientific, Inc). Specifically, peak isometric force amplitude (mN) was determined across the range of stimulation frequencies and measures of twitch kinetics: maximal rates of force development (+dF/dt) and relaxation (ÀdF/dt), ½ relaxation time (1/2 RT), and time to peak tension (TPT) were assessed. Peak isometric force was then normalized to the calculated CSA of the muscle strip (m/l*d) where m is the muscle mass, l is the length, and d is mammalian skeletal muscle density (1.06 mg/mm 3 ) (Mendez 1960). A fatigue protocol (70 Hz for 350 ms every 2 sec for 5 min) was performed to determine the number of contractions required to reduce force to 60% of the force of the initial 70 Hz contraction.

Statistics
All values are presented as means AE standard error. Statistical significance was set to P < 0.05. Most comparisons between WT and Pln OE mice were performed using unpaired Student's t-tests; however, a two-way repeated measures ANOVA was used for force-frequency analysis of peak isometric force. A one-way ANOVA with a Dunnet's multiple comparison post hoc test was used to compare PLN expression levels in WT gluteus minimus and soleus muscles with diaphragm.

SERCA function and expression of Ca 2+ handling proteins
Phospholamban overexpression was evident in the diaphragm homogenates from Pln OE mice as only 2.5 lg of total protein was required to detect PLN compared to the 25 lg required in the WT diaphragm (Fig. 1A). Specifically, measurements of optical density relative to actin indicate a 13-fold and 22-fold overexpression of PLN monomer and pentamer, respectively, compared with WT. PLN overexpression in diaphragm muscles was associated with a rightward shift in the activity-pCa curves (Fig. 1B) and a significant increase in K Ca compared to WT (Table 1, P = 0.02). However, maximal SERCA activity was not significantly different between genotypes (Table 1, P = 0.45). We did observe lower rates of Ca 2+ uptake in the Pln OE diaphragm homogenates compared to WT (Fig. 1C), however, this only approached statistical significance (P = 0.08). Western blot analysis revealed no differences in the expression of SERCA1a (Fig. 1D) or SERCA2a (Fig. 1E) between WT and Pln OE mice. SLN expression was found to be upregulated nearly 2.5-fold in Figure 1. SERCA function in diaphragm muscles in Pln OE mice at 4-6 months of age. (A) Western blotting for PLN in WT and Pln OE mice from diaphragm muscle homogenates. For WT mice, 25 lg of total protein was loaded, whereas only 2.5 lg was required for Pln OE mice to detect PLN protein. (B) Ca 2+ -ATPase activity-pCa curves in WT (n = 6) and Pln OE mice (n = 6) in the presence of the Ca 2+ ionophore. (C) Ca 2+ uptake assessed in diaphragm muscles from WT (n = 4) and Pln OE mice (n = 5). Western blotting for SERCA1a (D), SERCA2a (E), and SLN (F) in soleus from WT and Pln OE mice (n = 4-5 per genotype). Total protein loaded was 1 lg, 4 lg, and 25 lg for SERCA1a, SERCA2a, and SLN, respectively. Actin was used as a loading control and all values are expressed relative to WT. For, A, D, E the two lanes for WT and Pln OE correspond to samples from two different WT and two different Pln OE mice, respectively. *P ≤ 0.05 versus WT using Student's t-test. All values are presented as mean AE standard error.  Fig. 2A and B). A significant reduction in the percentage of type I fibers was found ( Fig. 2C and D, P < 0.001) and although individually the type II fibers (IIA, IIX, IIB) were not different between genotypes, there was a significant increase in the collective percentage of all type II fibers in the Pln OE diaphragm (WT, 86.5 AE 0.7% vs. Pln OE , 95.0 AE 1.8%, P < 0.001). Furthermore, we observed a significant reduction (P < 0.001) in type I fiber CSA and a significant increase in type IIA (P = 0.01), type IIX (P < 0.001), and type IIB (P < 0.001) CSA in the Pln OE diaphragm (Fig. 2E). Western blotting for dynamin 2 expression in diaphragm revealed no significant differences between WT and Pln OE mice (Fig. 2F, P = 0.87).

Diaphragm contractility
Representative twitch and tetanic (100 Hz) force tracings illustrate no significant differences in force production between WT and Pln OE diaphragm strips ( Fig. 3A and B). In addition, there were no differences in maximal rates of relaxation (ÀdF/dt: WT, 0.73 AE 0.04 vs. Pln OE , 0.68 AE 0.07, mN/ms/mm 2 , P = 0.49) or maximal rates of force development (+dF/dt: WT, 5.04 AE 0.51 vs. Pln OE , 5.87 AE 0.68 mN/ms/mm 2 , P = 0.34). Correspondingly, there were no differences in TPT between genotypes (WT, 39. 2 AE 1.9 vs. Pln OE , 38.5 AE 1.7, ms, P = 0.78, n = 6 per genotype); however, there was a significant increase in ½ relaxation time in the Pln OE diaphragm compared with WT (WT, 78.0 AE 5.3 vs. Pln OE 95.8 AE 3.8, ms, P = 0.02, n = 6 per genotype). Interestingly, across submaximal and maximal frequencies, Pln OE diaphragm strips generated more specific force compared to WT but the difference was not significant (Fig. 3C). Finally, in response to a fatiguing stimulation protocol, Pln OE diaphragms required on average 100 AE 8 contractions to reach 60% maximum isometric force at 70 Hz compared to 132 AE 19 in the WT diaphragm; however, this difference was not statistically significant (Fig. 3D, P = 0.16).

Discussion
Studies examining diaphragm function in animal models of CNM are limited as recently highlighted in a review by Smith Goddard, and Childers (Smith et al. 2014). However, in a study published in the same year, Cowling et al. (2014) showed histopathological abnormalities with more atrophic fibers containing mislocalized nuclei in the diaphragm muscles from mtm1 À/y mice, a mouse model which accurately recapitulates human X-linked myotubular myopathy (Buj-Bello et al. 2002). Correspondingly, mtm1 À/y mice have a very short lifespan (6-14 weeks) (Buj-Bello et al. 2002) which is consistent with the severe respiratory complications often leading to early death reported in human cases of myotubular myopathy (Jungbluth et al. 2008). We have recently reported that the Pln OE mouse histopathologically resembles human autosomal dominant CNM (Fajardo et al. 2015), which, in contrast to myotubular myopathy, is generally less severe, and patients often have a normal lifespan with very few respiratory complications (Bitoun et al. 2005;Jungbluth et al. 2008;Romero 2010). Here, we have shown that the Pln OE diaphragm exhibits type I fiber hypotrophy but does not display type I fiber predominance, increased central nuclei, central aggregation of oxidative activity, or muscle weakness. In agreement with the differences in disease severity found in the diaphragm between mouse models, the Pln OE mice live much longer than mtm1 À/y mice, albeit, shorter than their WT littermates (Fig. S1).
Previous studies have shown that elevated dynamin 2 in skeletal muscle can lead to features of CNM (Cowling et al. 2011;Liu et al. 2011) while reducing its expression can improve muscle function and alleviate murine CNM pathology (Cowling et al. 2014). Since, dynamin 2 is upregulated 3-5 fold in the Pln OE soleus and gluteus minimus (Fajardo et al. 2015) but not diaphragm (Fig. 2F), it is possible that differences in dynamin 2 expression could explain the lack of CNM phenotype in the Pln OE diaphragm. How PLN overexpression and impaired SERCA function leads to increased dynamin 2 in the affected muscles is unclear. Furthermore, although dynamin 2 has been implicated in the pathology of murine CNM, its role in human CNM is less established since we have recently observed a 35% reduction in its expression in muscle biopsies from CNM patients (Fajardo et al. 2015) while others have seen a twofold increase in patients with myotubular myopathy (Cowling et al. 2014). Our findings of minimal histopathological defects within the Pln OE diaphragm despite PLN overexpression may argue against a potential role of elevated PLN in CNM pathology. An important difference between diaphragm and the postural limb muscles in the Pln OE model is that SERCA function was not impaired in the diaphragm to the extent we observed in the soleus and gluteus minimus muscles (Fajardo et al. 2015). Maximal SERCA activity was not reduced in the Pln OE diaphragm compared to WT and rates of Ca 2+ uptake were only trending to be lower in the Pln OE diaphragm compared to WT (À15%), whereas Ca 2+ uptake measured at the same pCa was significantly reduced in the Pln OE soleus (À75%) and gluteus minimus muscles (À25%) (Fig. S3). This may be surprising given the 13-fold overexpression of monomeric PLN in the Pln OE diaphragm compared with the previously observed 6.3-fold and 6.7-fold overexpression found in the soleus and gluteus miminus, respectively (Fajardo et al. 2015). However, since these measures are expressed relative to WT, it appears that the differences in PLN overexpression across muscle types can be accounted for by a fivefold lower monomeric PLN expression in the WT diaphragm compared with soleus and gluteus minimus muscles (Fig. S2). In our view, the simplest explanation for the muscle differences in this model is that WT diaphragm has the lowest proportion of type I fibers (~12%) compared with soleus and gluteus minimus, which is even lower in Pln OE (~6%); therefore, the inhibitory effects of PLN overexpression on SERCA function would be constrained to the few type I fibers present in diaphragm. Figure 2. Diaphragm muscles from Pln OE mice do not display CNM. Representative diaphragm cryosections from Pln OE and WT mice after H&E staining (A), SDH staining (B), immunofluorescence staining for fiber type analysis (C) (n = 5 per genotype for all staining protocols). Cross sections were stained with MHC antibodies to identify type I (blue), type IIA (green), type IIB (red), and type IIX (unstained). (D) Quantitative analysis of fiber type distribution in diaphragm muscles from WT and Pln OE mice at 4-6 months of age (n = 5 per genotype). (E) Quantitative analysis of fiber type cross-sectional area (CSA) in diaphragm muscles from WT and Pln OE mice at 4-6 months of age (n = 5 per genotype). (F) Dynamin 2 expression in diaphragm homogenates from WT and Pln OE mice (n = 6 per genotype). Total protein loaded for dynamin 2 was 7.5 lg. *P ≤ 0.05 versus WT using Student's t-test. All values are presented as mean AE standard error. Scale bars in (A-C) are set to 50 lm. Despite near normal maximal activity, SERCA's apparent affinity for Ca 2+ was reduced in the Pln OE diaphragm as indicated by a rightward shift in the activity-pCa curve and a significantly higher K Ca . Reduced Ca 2+ sensitivity of SERCA activity would be expected with PLN overexpression in type I fibers but could also be due to increased SLN expression. SLN is a well-known structural and functional homolog of PLN (Odermatt et al. 1998;Tupling et al. 2011;Fajardo et al. 2013;Gorski et al. 2013) and, together, they have been shown to form a superinhibitory ternary complex with SERCA that not only reduces SER-CA's affinity for Ca 2+ , but also the maximal rates of SERCA activity (Gorski et al. 2013) and Ca 2+ uptake (Asahi et al. 2002). Interestingly, compared to the soleus and gluteus minimus, where SLN protein was upregulated ninefold and sevenfold, respectively (Fajardo et al. 2015), SLN in the diaphragm was only elevated 2.5-fold, providing another possible mechanism which may contribute to the differences in severity of SERCA dysfunction across skeletal muscles in the Pln OE mouse. Finally, differences in SERCA's apparent affinity for Ca 2+ may be related to differences in SERCA isoform expression. In a previous study using human single fibers, it was shown that type I fibers, which express SERCA2, have greater Ca 2+ sensitivity of Ca 2+ uptake compared with type II fibers, which express SERCA1 (Lamboley et al. 2014). Corresponding with this, when we compare WT diaphragm, a fast-twitch muscle (our current study), with WT soleus, a slowtwitch muscle (Fajardo et al. 2015), we find a much lower Ca 2+ sensitivity of SERCA activity in diaphragm. While differences in SERCA isoform may contribute to the differences in K Ca we have observed between diaphragm and soleus, they cannot explain the reduction in SERCA's apparent affinity for Ca 2+ we observed in Pln OE diaphragm compared with WT since there were no differences in SERCA1 or SERCA2 between Pln OE and WT.
Upregulated SLN protein and/or mRNA is becoming widely known as a common feature in many myopathies (Nakagawa et al. 2005;Ottenheijm et al. 2008;Liu et al. 2011;Calvo et al. 2012) and findings in the mdx mouse and mdx-utrophin double knockout model suggest that its expression may be directly proportional to disease severity (Schneider et al. 2013). Similarly, SLN may follow disease severity in the Pln OE mouse since the myopathy in the diaphragm, where only abnormalities in fiber CSA were evident, is far less severe than that found in the Pln OE soleus and gluteus minimus, where all histopathological signs of CNM with additional endomysial fibrosis and core-like aspects were evident (Fajardo et al. 2015). However, the role that SLN plays in the Pln OE mouse model of CNM or any other animal model of myopathy remains unknown. Corresponding with the SERCA activity and Ca 2+ uptake results, the maximal rates of relaxation and rates of contraction were not different between genotypes in the diaphragm, which is in direct contrast to what we and others have observed previously in the soleus of these mice (Song et al. 2004;Fajardo et al. 2015). However, we did find that the Pln OE diaphragm displayed a prolonged ½ RT compared with WT, which is consistent with the significant and trending reductions in SERCA's apparent Brain and Behavior, doi: 10.1002/brb3.470 (7 of 10) affinity for Ca 2+ and rates of Ca 2+ uptake, respectively. Taken together, our results are suggestive of a relatively modest impairment in SERCA function in the Pln OE diaphragm, which, in part, may contribute to the lack of CNM histopathology in that muscle.
Analysis of hybrid fiber types further suggests that Pln overexpression in the diaphragm does not promote a type I fiber phenotype since the number of transitional type I/ IIA fibers was not different between WT and Pln OE (Fig. S4A). Moreover, there were increases in type IIA/IIX and IIX/IIB hybrid fibers in the Pln OE diaphragm compared with WT; however, these effects only approached statistical significance (P = 0.08 and 0.11, respectively; Fig. S4B and C). In any event, our data indicate that there was a general shift towards the faster fiber phenotype in the Pln OE diaphragm, which perhaps may explain the relatively small impairments in SERCA function and the lack in CNM phenotype. Analyses of central nuclei, fiber type distribution and CSA of 10-12-month-old mice produced similar findings to that of 4-6-month-old animals (Fig. S5), suggesting that the lack in CNM phenotype is not due to a delay in the disease progression of the diaphragm muscles.
To examine the underlying mechanisms behind these distinct effects on type I fiber proportions in the Pln OE diaphragm, soleus, and gluteus minimus muscles, we focused on the Ca 2+ -dependent serine/threonine phosphatase, calcineurin. We suspect that in compensation for the type I fiber hypotrophy, the type II fibers of the postural soleus and gluteus minimus muscles exhibit greater load-bearing activity thereby leading to myofiber hypertrophy and a fast-to-slow fiber type transition. A similar phenomenon is noted in functional overload studies whereby removal of the synergist muscles, soleus and gastrocnemius, causes the plantaris muscles to hypertrophy and transition towards a slow-oxidative phenotype (Dunn et al. 1999;Michel et al. 2004). Importantly, calcineurin is activated during functional overload and is known to promote both the slow-oxidative fiber phenotype (Timmerman et al. 1996;Dolmetsch et al. 1997;Chin et al. 1998) and myofiber hypertrophy (Dunn et al. 1999;Semsarian et al. 1999). Here, and similar to a previous study where calcineurin expression was found to be higher in the Pln OE soleus compared with WT (Song et al. 2004), our findings indicate greater calcineurin expression and NFAT nuclear content in the soleus and gluteus minimus muscles, but not in the Pln OE diaphragm (Fig. S6). Moreover, calsarcin-2, an endogenous calcineurin inhibitor, is known to be highly expressed in diaphragm muscles (Frey et al. 2008). Thus, the inability to promote calcineurin signaling may help to explain both the reduction in the percent of type I fibers and the apparent increased susceptibility to muscle fatigue in the Pln OE diaphragm com-pared with WT. Although we did not assess force recovery after our fatigue protocol, successful recovery has been shown with a similar protocol (Coirault et al. 1999), which suggests that the protocol used in this study likely does not induce damage to the muscle fibers. Finally, it should also be noted that respiratory muscles do not typically undergo transformation with training and inactivity in the same way as limb muscle fibers, particularly with respect to MHC isoform (Polla et al. 2004). Thus, the reduced type I and increased type II fiber population may also represent a selective loss or underdevelopment of type I fibers due to PLN overexpression. Nevertheless, this innate response found within the diaphragm to limit the type I fibers may represent a novel therapeutic strategy combatting CNM and other congenital myopathies such as central cores since these are all type I fiber-related myopathies (Sharma et al. 2009).
In summary, our results indicate that the Pln OE diaphragm, compared with soleus and gluteus minimus, is generally more resistant to the CNM phenotype, and that this lack in CNM pathology and weakness further supports the Pln OE mouse as a model of human autosomal dominant CNM where very little respiratory complications occur. Furthermore, we suggest that understanding the underlying mechanisms behind the diaphragm's apparent resistance to Pln OE -induced CNM and muscle weakness may lead to the generation of novel therapeutic strategies. Our findings here point to differences in SLN expression and type I fiber distribution as potential contributing factors, and future studies will determine their impact.

Supporting Information
Additional supporting information may be found online in the supporting information tab for this article: Figure S1. Lifespan of Pln OE (n = 23) and WT (n = 17) mice. Figure S2. Monomeric (m) PLN expression in WT diaphragm is lower than WT soleus and gluteus minimus. Figure S3. Ca 2+ uptake assays in the soleus (A) and gluteus minimus (B) muscles from Pln OE and WT mice. Figure S4. Percent distribution of hybrid I/IIA (A), IIA/ IIX (B), and IIX/IIB (C) fibers in the diaphragm muscles from WT and Pln OE mice (n = 5 per genotype). Figure S5. Analysis of central nuclei (A) and fiber type distribution (B) and cross-sectional area (CSA; C) in 10-12-month-old WT (n = 4) and Pln OE (n = 5) mice. Figure S6. Calcineurin (CnA) and nuclear factor of activated T-cell (NFAT) nuclear content in soleus (A, D), gluteus minimus (B, E), and diaphragm (C, F) muscles from Pln OE and WT mice. (G) Nuclear cell fraction purity demonstrated through Western blots from tibialis anterior muscles using Histone H2B and CuZnSOD as nuclear and cytosolic markers, respectively.