Skip to main content
Download PDF

Transcriptional changes of genes encoding sarcoplasmic reticulum calcium binding and up-taking proteins in normal and Duchenne muscular dystrophy dogs

BMC Musculoskeletal Disorders volume 25, Article number: 811 (2024) Cite this article

Abstract

Background

Cytosolic calcium overload contributes to muscle degradation in Duchenne muscular dystrophy (DMD). The sarcoplasmic reticulum (SR) is the primary calcium storage organelle in muscle. The sarco-endoplasmic reticulum ATPase (SERCA) pumps cytosolic calcium to the SR during muscle relaxation. Calcium is kept in the SR by calcium-binding proteins.

Methods

Given the importance of the canine DMD model in translational studies, we examined transcriptional changes of SERCA (SERCA1 and SERCA2a), SERCA regulators (phospholamban, sarcolipin, myoregulin, and dwarf open reading frame), and SR calcium-binding proteins (calreticulin, calsequestrin 1, calsequestrin 2, and sarcalumenin) in skeletal muscle (diaphragm and extensor carpi ulnaris) and heart (left ventricle) in normal and affected male dogs by droplet digital PCR before the onset (≤ 2-m-old), at the active stage (8 to 16-m-old), and at the terminal stage (30 to 50-m-old) of the disease. Since many of these proteins are expressed in a fiber type-specific manner, we also evaluated fiber type composition in skeletal muscle.

Results

In affected dog skeletal muscle, SERCA and its regulators were down-regulated at the active stage, but calcium-binding proteins (except for calsequestrin 1) were upregulated at the terminal stage. Surprisingly, nominal differences were detected in the heart. We also examined whether there exists sex-biased expression in 8 to 16-m-old dogs. Multiple transcripts were significantly downregulated in the heart and extensor carpi ulnaris muscle of female dogs. In fiber type analysis, we found significantly more type I fiber in the diaphragm of 8 to 16-m-old affected dogs, and significantly more type II fibers in the extensor carpi ulnaris of 30 to 50-m-old affected dogs. However, no difference was detected between male and female dogs.

Conclusions

Our study adds new knowledge to the understanding of muscle calcium regulation in normal and dystrophic canines.

Peer Review reports

Background

Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy in children [1]. DMD is caused by the loss of dystrophin, a protein important for muscle integrity. Absence of dystrophin leads to muscle degeneration. Consequently, patients show progressive muscle weakness, losing the ability to walk in their early teenage years, and dying in early adulthood. It has long been recognized that aberrant cytosolic calcium elevation is a pivotal pathogenic mechanism underlying muscle degeneration in DMD [2, 3]. High levels of cytosolic calcium induce protein degradation by activating calcium-dependent proteases and induce lipid membrane degradation by activating calcium-dependent phospholipases [4, 5].

The sarcoplasmic reticulum (SR) is the primary calcium storage house in muscle [6]. It plays a critical role in regulating cytosolic calcium levels. During contraction, calcium is released from the SR. During relaxation, calcium is returned to the SR by the sarco-endoplasmic reticulum ATPase (SERCA) [7, 8]. The activity of SERCA is negatively regulated by inhibitory micropeptides including phospholamban (PLB), sarcolipin (SLN), and myoregulin [9]. SERCA function can also be enhanced by the activating micropeptide dwarf open reading frame (DWORF) [10]. Inside the SR, calcium is sequestered by calreticulin, calsequestrin, and sarcalumenin. These calcium-binding proteins provide another layer of regulation.

In DMD, an increased amount of calcium is released from the SR while a decreased amount returns to the SR, leading to cytosolic calcium buildup [3]. Changes in the expression of SR calcium-binding proteins, SERCA, and SERCA regulators have been implicated in cytosolic calcium overloading in DMD [11,12,13,14,15,16,17,18].

Dystrophin-deficient canines are excellent models for translational studies. Unlike the mouse model, the canine model has a similar dystrophic phenotype to human patients [19,20,21]. Unfortunately, little is known about transcriptional changes of the genes encoding SR calcium handling proteins in normal and affected dogs. To address this issue, we quantified transcript levels of four calcium-binding proteins (calreticulin, calsequestrin 1, calsequestrin 2, and sarcalumenin), two SERCA isoforms (SERCA1 and SERCA2a), and four SERCA regulators (PLB, SLN, myoregulin, and DWORF) using droplet digital PCR (ddPCR) in the heart (left ventricle), diaphragm, and extensor carpi ulnaris (ECU) muscle of age-matched male normal and affected dogs before the onset of muscle disease (≤ 2-m-old) [19, 21], at the symptomatic stage (8 to 16-m-old), and the terminal stage (30 to 50-m-old). Significant differences were detected in skeletal muscles but not in the heart. We further compared 8 to 16-m-old male and female dogs and found significant differences in the heart and ECU muscle but not the diaphragm. Interestingly, we did not detect a correlation between mRNA expression and skeletal muscle fiber type composition.

Methods

Tissues

This study used curated tissues previously collected at the University of Missouri. Tissue collection was approved by the Animal Care and Use Committee of the University of Missouri and was performed in accordance with National Institutes of Health guidelines. Specifically, tissues were from 12 normal males, 3 normal females, 12 affected males, and 3 affected females. In males, one-third were less than 2 months, one-third were 8 to 16 months, and one-third were 30 to 50 months. All females were 8 to 16 months. Only male dogs were used to evaluate disease-stage (pre-onset, symptomatic, and terminal) associated changes. Dog information and sample size are provided in Table 1. No animals were euthanized for the purpose of this study.

Table 1 Dog information
Full size table

Myofiber type evaluation

Fiber type was examined by myosin heavy-chain isoform immunofluorescence staining on ten-micron cryosections using our published protocol [22, 23]. Fiber type-specific monoclonal antibodies were purchased from the University of Iowa Hybridoma Bank (https://dshb.biology.uiowa.edu/monoclonal/mouse). Specifically, type I fibers were detected with antibody BA-D5 (1:20 dilution). Type IIa/IIx fibers were detected with antibody SC-71 (1:100 dilution). Type IIb fibers were detected with antibody BF-F3 (1:40 dilution). Embryonic myosin heavy chain was detected with antibody F1.652. Myofiber outlines were recognized using a goat anti-rabbit antibody against laminin (1:200 dilution; Sigma-Aldrich, St. Louis, MO). Myofiber type composition was quantified from 6 to 9 random 20 × field images for each muscle.

Transcript quantification

Tissue collection, RNA extraction and quantification, cDNA preparation and quantification, and ddPCR were performed according to our published protocols [24]. Briefly, the fresh left ventricle, diaphragm, and ECU samples were snap-frozen in liquid nitrogen. Approximately 30 mg tissues were used for RNA extraction using the RNeasy Fibrous Tissue Mini kit (Qiagen, Hilder, Germany; Cat No: 74704). Reverse transcription was performed using the SuperScript IV VILO Master Mix with ezDNase Enzyme (Thermo Fisher Scientific, Hampton, NH, USA; Cat No: 11766050). cDNA concentrations were determined using the Qubit ssDNA assay kit (Thermo Fisher Scientific, Cat No: Q10212). cDNA was diluted in the range of 0.5 to 50 ng/ul for ddPCR. Loading mass (ng) per reaction for detection of each transcript was empirically determined. ddPCR was performed using the QX200 ddPCR system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using ddPCR supermix for probes (no dUTP) (Bio-Rad, Cat No: 186–3024). Primers and probes used in the study are listed in Table 2. All probes contained a 5′ 6-carboxyfluorescein, an internal ZEN quencher, and a 3’ Iowa black quencher. Results are presented as absolute copies of transcript per ng of cDNA used in the reaction.

Table 2 TaqMan PCR primers and probes
Full size table

Statistical analysis

Data are presented as mean ± standard error of the mean. The normality of the data was determined by the Shapiro–Wilk test. Parametric data were analyzed by the unpaired Student t-test between two groups. Non-parametric data were analyzed by the Mann-Whitey test between the two groups. Analyses were performed using GraphPad PRISM software version 9.1.2 (GraphPad Software, La Jolla, California). A p < 0.05 was considered statistically significant.

Results

Characterization of fiber type composition of the diaphragm and ECU muscle

In this study, we evaluated the diaphragm and ECU muscle. We chose the diaphragm because it is the most severely affected muscle in DMD [1]. We chose the ECU muscle because its histopathology and function have been extensively characterized [22, 25], and this muscle has also been used to test genetic therapeutics in the canine model [26,27,28]. We quantified the fiber type distribution (Fig. 1). In the diaphragm, statistically significant differences were detected in 8 to 16-m-old dogs. In this age range, affected dogs had significantly more type I fibers, while normal dogs had significantly more type II fibers [29]. In the ECU muscle, statistically significant differences were detected in 30 to 50-m-old dogs. In this age range, affected dogs had significantly more type II fibers, while normal dogs had significantly more type I fibers [22]. We also compared the fiber type composition of 8 to 16-m-old male and female dogs. No significant difference was detected (Fig. 1B).

Fig. 1
figure 1

Evaluation of myofiber type composition. A Fiber type composition in the diaphragm (top panels) and the extensor carpi ulnaris (ECU) muscle of ≤ 2-m-old, 8 to 16-m-old, and 30 to 50-m-old normal and affected dogs. Fiber types are classified as type I (slow), type II (fast), and other. Other refers to hybrid myofibers that expressed both type I and type II myosin heavy chain and myofibers that expressed embryonic myosin heavy chain. B Representative photomicrographs of fiber type immunofluorescence staining. Type I fibers are in blue. Type II fibers are in red. Myofibers are outlined by laminin (green) C Comparison of fiber type composition of 8 to 16-m-old male and female normal and affected dogs

Full size image

Before the onset of muscle disease

Dystrophin-deficient dogs show no dystrophic symptoms before weaning (≤ 2-m-old) [19, 21]. At this stage, no significant differences were detected between normal and affected puppies in the transcript levels of SR calcium-binding proteins (calreticulin, calsequestrin 1, calsequestrin 2, and sarcalumenin) or SERCAs and their regulators (SERCA1, SERCA2a, PLB, SLN, myoregulin, and DWORF) (Fig. 2). Interestingly, the transcript levels of SERCA1, SLN, myoregulin, and DWORF in the left ventricles were several thousand times lower than those in the diaphragm and ECU muscle (Fig. 2).

Fig. 2
figure 2

Quantification of the cDNA copy number in genes encoding SR calcium-binding protein, SERCAs, and SERCA regulators in pre-symptomatic dogs. A Diaphragm. B extensor carpi ulnaris. C Heart. CSQ1, calsequestrin 1; CSQ2, calsequestrin 2; DWORF, dwarf open reading frame; ns, not statistically significant; PLB, phospholamban; SERCA, sarcoendoplasmic reticulum calcium ATPase; SLN, sarcolipin; SR, sarcoplasmic reticulum

Full size image

At the symptomatic stage

Between 8 and 16 months, affected dogs display clinical signs and symptoms of muscular dystrophy. In the diaphragm of affected dogs, the transcript levels of SERCA1 and DWORF were significantly reduced (Fig. 3A). In the ECU muscle of affected dogs, the transcript levels of sarcalumenin, SERCA1, SERCA2a, PLB, myoregulin, and DWORF were significantly reduced (Fig. 3B). No statistically significant differences were detected between normal and affected dogs for any transcripts in the heart (Fig. 3C). The transcript levels of SERCA1, SLN, myoregulin, and DWORF remained drastically reduced in cardiac muscle compared to skeletal muscle (Fig. 3).

Fig. 3
figure 3

Quantification of the cDNA copy number in genes encoding SR calcium-binding protein, SERCAs, and SERCA regulators in 8 to 16-m-old dogs. A Diaphragm. B Extensor carpi ulnaris. C Heart. CSQ1, calsequestrin 1; CSQ2, calsequestrin 2; DWORF, dwarf open reading frame; ns, not statistically significant; PLB, phospholamban; SERCA, sarcoendoplasmic reticulum calcium ATPase; SLN, sarcolipin; SR, sarcoplasmic reticulum. *, p < 0.05; **, P < 0.01; ***, p < 0.001

Full size image

At the terminal stage

The lifespan of affected dogs is approximately 3 years [19]. To examine transcript changes at the end stage, we collected tissues from 30 to 50-m-old dogs. In the diaphragm of affected dogs, the transcript level of calsequestrin 2 was significantly upregulated but the transcript levels of calsequestrin 1 and SERCA1 and DWORF were significantly downregulated at this stage (Fig. 4A). In the ECU muscle of affected dogs, the transcript levels of calreticulin, sarcalumenin, and myoregulin were significantly upregulated (Fig. 4B). In the heart of affected dogs, the transcript level of sarcalumenin was significantly upregulated (Fig. 4C).

Fig. 4
figure 4

Quantification of the cDNA copy number in genes encoding SR calcium-binding protein, SERCAs, and SERCA regulators in 30 to 50-m-old dogs. A Diaphragm. B Extensor carpi ulnaris. C Heart. CSQ1, calsequestrin 1; CSQ2, calsequestrin 2; DWORF, dwarf open reading frame; ns, not statistically significant; PLB, phospholamban; SERCA, sarcoendoplasmic reticulum calcium ATPase; SLN, sarcolipin; SR, sarcoplasmic reticulum. *, p < 0.05

Full size image

Differences between male and female dogs

We compared transcript levels in 8 to 16-m-old male and female dogs. In the diaphragm, the only significant change is the upregulation of calsequestrin 1 in normal female dogs (Figs. 5A and 6A). In the ECU muscle, the transcript levels of calreticulin, calsequestrin 2, sarcalumenin, PLB, and DWORF were significantly higher in normal males (Fig. 5B). The transcript level of SERCA2a was significantly upregulated in both normal and affected males (Figs. 5B and 6B). In the heart, the transcript levels of calreticulin, calsequestrin 2, sarcalumenin, and SERCA2a were significantly higher in male dogs irrespective of muscle disease (Figs. 5C and 6C). The PLB transcript was only significantly upregulated in affected male dogs although a trend was detected in normal male dogs (Fig. 6C).

Fig. 5
figure 5

Quantification of the cDNA copy number in genes encoding SR calcium-binding protein, SERCAs, and SERCA regulators in 8 to 16-m-old normal dogs. A Diaphragm. B Extensor carpi ulnaris. C Heart. CSQ1, calsequestrin 1; CSQ2, calsequestrin 2; DWORF, dwarf open reading frame; ns, not statistically significant; PLB, phospholamban; SERCA, sarcoendoplasmic reticulum calcium ATPase; SLN, sarcolipin; SR, sarcoplasmic reticulum. *, p < 0.05; **, P < 0.01; ***, p < 0.001

Full size image
Fig. 6
figure 6

Quantification of the cDNA copy number in genes encoding SR calcium-binding protein, SERCAs, and SERCA regulators in 8 to 16-m-old affected dogs. A Diaphragm. B Extensor carpi ulnaris. C Heart. CSQ1, calsequestrin 1; CSQ2, calsequestrin 2; DWORF, dwarf open reading frame; ns, not statistically significant; PLB, phospholamban; SERCA, sarcoendoplasmic reticulum calcium ATPase; SLN, sarcolipin; SR, sarcoplasmic reticulum. *, p < 0.05; **, P < 0.01; ***, p < 0.001

Full size image

Discussion

In this study, we examined the expression of genes encoding SR calcium-binding proteins (calreticulin, calsequestrin 1, calsequestrin 2, and sarcalumenin), SR calcium pumps (SERCA1 and SERCA2a), and SERCA regulators (PLB, SLN, myoregulin, and DWORF) in the heart, diaphragm, and limb muscle of the canine DMD model and age/sex-matched normal dogs by ddPCR. No statistically significant differences between normal and affected dogs were detected in the heart (Figs. 2, 3, and 4). However, disease stage-associated changes were observed in the diaphragm and extensor carpi ulnaris muscle (Figs. 2, 3, and 4). We also compared 8 to 16-m-old male and female dogs and found significant downregulation of multiple transcripts in the heart and extensor carpi ulnaris muscle in female dogs (Figs. 5, and 6). Since many of these proteins are expressed in a fiber type-specific manner in skeletal muscle, we also evaluated fiber type composition (Fig. 1). Interestingly, we did not observe a consistent correlation between the transcript level and fiber type composition.

Calcium homeostasis is pivotal to muscle health and function. Calcium mishandling in DMD leads to pathological calcium accumulation in the cytosol [3]. Aberrant cytosolic calcium overloading is in part due to changes in SR calcium-binding proteins, SERCA, and SERCA regulators. These changes may occur at the mRNA level, the protein level, or through post-translational protein modifications (such as nitrosylation and phosphorylation). Here, we focused on transcript changes in the clinically relevant canine model. The most striking observation is the differences between cardiac and skeletal muscles. Nominal changes were detected between normal and affected dogs in the heart (Figs. 2, 3, and 4), suggesting transcription is not a primary regulatory mechanism in the cardiac muscle of the canine DMD model. Interestingly, an earlier Northern blot study showed a down-regulation of SERCA2a mRNA in the heart of mdx mice [30]. We recently found that DWORF transcript is significantly reduced in the heart of mdx mice [18]. These results suggest the existence of important differences in transcriptional regulation between canine and murine models.

Another interesting difference between cardiac and skeletal muscle is the extremely low transcription of SERCA1, sarcolipin, myoregulin, and DWORF in the left ventricle. The copy numbers of these transcripts were hundred times lower than those in skeletal muscle in both normal and affected dogs (Figs. 2, 3, and 4). These results highlight tissue-specific calcium regulation at the transcriptional level. It is also worth noting that the RNA levels of SERCA1, sarcolipin, and myoregulin were also very low in the mouse ventricles [31, 32]. However, the DWORF RNA is highly expressed in the mouse ventricles, suggesting a species-specific regulation mechanism for DWORF RNA expression [10, 18].

In contrast to the heart, skeletal muscle showed distinctive gene expression patterns at different stages of the disease course. Before the onset of muscle disease, there was no difference between dystrophin-null puppies and normal puppies (Fig. 2). This is consistent with the notion that calcium mishandling is a secondary, rather than the primary, pathogenic mechanism in DMD. In other words, dystrophin deficiency alone cannot induce transcriptional changes in the absence of muscle damage. Our results are different from a microarray study in pre-symptomatic DMD patients [33]. Pescatori et al. examined gene expression profile of the quadriceps from 1.5 to 22-month-old DMD patients using the Affymetrix technology. The authors found a significant down-regulation of PLB and up-regulation of calreticulin. PLB is a negative regulator of SERCA. Reduction in PLB expression would increase SERCA activity. Calreticulin is a calcium-binding protein in the SR. Increased calreticulin expression would favor calcium retention in the SR. Collectively, these changes would prevent, rather than induce cytosolic calcium overloading. We do not have a good explanation for why the muscles of pre-symptomatic DMD patients, but not affected puppies, show counterintuitive changes in PLB and calreticulin expression.

At the active stage of the disease, the most prominent changes are the reduced expression of SERCA and DWORF (Fig. 3). Statistically significant differences were detected in the diaphragm and ECU muscle for SERCA1 and DWORF, and the ECU muscle for SERCA2a. DWORF is a positive regulator of SERCA [10]. Downregulation of SERCA and DWORF would reduce the active transportation of cytosolic calcium to the SR and contribute to cytosolic calcium overload. DWORF is a recently discovered protein. DWORF gene expression has not been studied in dystrophin-deficient skeletal muscle. However, SERCA gene expression has been extensively profiled in transcriptomic studies in rodent DMD models, patient-derived pluripotent stem cells, and patient muscles. SERCA1 mRNA is significantly reduced in 2 to 4-m-old mdx mouse limb muscle [34]. SERCA1 mRNA and SERCA2a mRNA are significantly reduced in the tibialis anterior muscle of the rat model at 9 months of age [35]. Morera et al. and Mournetas et al. showed significant reduction of SERCA1 mRNA and SERCA2a mRNA, respectively, in patient cell-derived cellular models [36, 37]. Haslett et al. and Nieves-Rodriguez et al. found significant reduction of SERCA2a mRNA in the quadriceps of 5 to 7-year-old patients and the tibialis anterior muscle of 2 to 7-year-old patients, respectively [38, 39]. In summary, our ddPCR results from the canine model and findings from transcriptomic studies in rodent models, patient cells, and patient muscles suggest that transcriptional downregulation of SERCA and DWORF contributes to calcium mishandling in DMD skeletal muscle.

At the terminal stage of the disease, the most consistent finding is the upregulation of genes encoding SR calcium-binding proteins calreticulin, calsequestrin-2, and sarcalumenin. Statistical significance was reached for calsequestrin-2 in the diaphragm and for calreticulin and sarcalumenin in the ECU muscle (Fig. 4). Increased calcium binding in the SR would reduce free calcium for cytosolic release. Consequently, this would help to reduce the cytosolic calcium level. We speculate that the transcriptional changes in SR calcium-binding proteins may reflect a compensatory mechanism in the advanced stage of DMD. Future studies are needed to determine whether this is unique to the canine DMD model or a common finding in rodent models and human patients.

Given sex is a biological variable, we compared male and female dogs (Figs. 5 and 6). Most transcriptional differences were observed in the heart. Female hearts exhibited significantly reduced transcript levels of calreticulin, calsequestrin 2, sarcalumenin, and SERCA2a, regardless of whether the canines were normal or affected (Figs. 5C and 6C). These findings suggest major differences between male and female calcium regulation in the heart. Statistically significant differences between male and female dogs were also detected in the ECU muscle of normal dogs. Specifically, the female ECU muscle exhibited significantly reduced transcript levels of calreticulin, calsequestrin 2, sarcalumenin, SERCA2a, PLB, and DWORF (Fig. 5B). Interestingly, except for SERCA2a, these differences were lost in the affected ECU muscle, suggesting muscle disease may differentially impact transcription in male and female dogs (Fig. 6B). Except for the upregulation of calsequestrin 1 in normal female dogs, nominal transcriptional differences were found in the diaphragm (Figs. 5A and 6A). This is unexpected given sex is an important regulator of gene expression in mammalian organ development and evolution [40]. It would be interesting to examine gene expression in other cellular processes in the diaphragm of male and female dogs to determine whether only calcium-handling proteins are the exceptions to sex-biased expression.

Many proteins involved in SR calcium binding and uptake show fiber type-specific expression. For example, SERCA1 and calsequestrin 1 are mainly expressed in type II fibers, while SERCA2a, calsequestrin 2, and DWORF are highly expressed in type I fibers [3, 10, 32, 41, 42]. In the diaphragm of 8 to 16-m-old affected dogs, there were significantly more type I fibers (Fig. 1A) [29]. However, in transcript analysis, not only the SERCA1 level was significantly reduced, but the DWORF level was also significantly reduced (Fig. 3A). In the ECU muscle of 8 to 16-m-old affected dogs, we detected significant downregulation of sarcalumenin, SERCA1, SERCA2a, PLB, myoregulin, and DWORF (Fig. 3B). However, there were no significant changes in the fiber type composition (Fig. 1A). Collectively, our data suggest that changes in the transcription level of calcium-handling proteins did not directly correlate with fiber type composition in the diaphragm and ECU muscle of the canine DMD model.

Conclusions

Calcium mishandling is an important pathogenic mechanism and therapeutic target in DMD. Most of the knowledge on calcium handling comes from studies conducted in mice. Given the importance of the canine model in translational studies, we quantified transcriptional changes of genes encoding SR calcium-binding proteins, SERCAs, and SERCA regulators in normal and dystrophin-deficient dogs at different stages of the disease. Consistent with transcriptomic studies performed in rodents and patients, we found mRNA expression represents an important mechanism in regulating cytosolic calcium levels in skeletal (but not cardiac) muscle of the canine DMD model. We also compared expression in male and female dogs and found sex-biased downregulation in female dogs. We further showed no direct correlation between fiber type composition and mRNA level changes. These findings expand knowledge on calcium regulation in canine muscles and muscular dystrophy.

Data availability

The dataset analyzed in the current study is available from the corresponding author upon reasonable request.

Abbreviations

CSQ1:

Calsequestrin 1

CSQ2:

Calsequestrin 2

ddPCR:

Droplet digital PCR

DMD:

Duchenne muscular dystrophy

DWORF:

Dwarf open reading frame

ECU:

Extensor carpi ulnaris

PLB:

Phospholamban

SERCA:

Sarcoendoplasmic reticulum calcium ATPase

SLN:

Sarcolipin

SR:

Sarcoplasmic reticulum

References

  1. Duan D, Goemans N, Takeda S, Mercuri E, Aartsma-Rus A. Duchenne muscular dystrophy. Nat Rev Dis Primers. 2021;7(1):13.

    Article  PubMed  Google Scholar 

  2. Burr AR, Molkentin JD. Genetic evidence in the mouse solidifies the calcium hypothesis of myofiber death in muscular dystrophy. Cell Death Differ. 2015;22(9):1402–12.

    Article  PubMed  Google Scholar 

  3. Mareedu S, Million ED, Duan D, Babu GJ. Abnormal calcium handling in Duchenne muscular dystrophy: mechanisms and potential therapies. Front Physiol. 2021;12: 647010.

    Article  PubMed  Google Scholar 

  4. Turner PR, Westwood T, Regen CM, Steinhardt RA. Increased protein degradation results from elevated free calcium levels found in muscle from mdx mice. Nature. 1988;335(6192):735–8.

    Article  PubMed  Google Scholar 

  5. Lindahl M, Backman E, Henriksson KG, Gorospe JR, Hoffman EP. Phospholipase A2 activity in dystrophinopathies. Neuromuscul Disord. 1995;5(3):193–9.

    Article  PubMed  Google Scholar 

  6. Rossi AE, Dirksen RT. Sarcoplasmic reticulum: the dynamic calcium governor of muscle. Muscle Nerve. 2006;33(6):715–31.

    Article  PubMed  Google Scholar 

  7. Primeau JO, Armanious GP, Fisher ME, Young HS. The SarcoEndoplasmic Reticulum Calcium ATPase. Subcell Biochem. 2018;87:229–58.

    Article  PubMed  Google Scholar 

  8. Xu H, Van Remmen H. The SarcoEndoplasmic Reticulum Calcium ATPase (SERCA) pump: a potential target for intervention in aging and skeletal muscle pathologies. Skelet Muscle. 2021;11(1):25.

    Article  PubMed  Google Scholar 

  9. Rathod N, Bak JJ, Primeau JO, Fisher ME, Espinoza-Fonseca LM, Lemieux MJ, Young HS: Nothing Regular about the Regulins: Distinct Functional Properties of SERCA Transmembrane Peptide Regulatory Subunits. Int J Mol Sci. 2021;22(16):8891.

  10. Nelson BR, Makarewich CA, Anderson DM, Winders BR, Troupes CD, Wu F, Reese AL, McAnally JR, Chen X, Kavalali ET, et al. A peptide encoded by a transcript annotated as long noncoding RNA enhances SERCA activity in muscle. Science. 2016;351(6270):271–5.

    Article  PubMed  Google Scholar 

  11. Culligan K, Banville N, Dowling P, Ohlendieck K. Drastic reduction of calsequestrin-like proteins and impaired calcium binding in dystrophic mdx muscle. J Appl Physiol. 2002;92(2):435–45.

    Article  PubMed  Google Scholar 

  12. Dowling P, Doran P, Ohlendieck K. Drastic reduction of sarcalumenin in Dp427 (dystrophin of 427 kDa)-deficient fibres indicates that abnormal calcium handling plays a key role in muscular dystrophy. Biochem J. 2004;379(Pt 2):479–88.

    Article  PubMed  Google Scholar 

  13. Lohan J, Ohlendieck K. Drastic reduction in the luminal Ca2+ -binding proteins calsequestrin and sarcalumenin in dystrophin-deficient cardiac muscle. Biochim Biophys Acta. 2004;1689(3):252–8.

    Article  PubMed  Google Scholar 

  14. Pertille A, de Carvalho CL, Matsumura CY, Neto HS, Marques MJ. Calcium-binding proteins in skeletal muscles of the mdx mice: potential role in the pathogenesis of Duchenne muscular dystrophy. Int J Exp Pathol. 2010;91(1):63–71.

    Article  PubMed  Google Scholar 

  15. Schneider JS, Shanmugam M, Gonzalez JP, Lopez H, Gordan R, Fraidenraich D, Babu GJ. Increased sarcolipin expression and decreased sarco(endo)plasmic reticulum Ca uptake in skeletal muscles of mouse models of Duchenne muscular dystrophy. J Muscle Res Cell Motil. 2013;34(5–6):349–56.

    Article  PubMed  Google Scholar 

  16. Lai Y, Zhao J, Yue Y, Wasala NB, Duan D. Partial restoration of cardiac function with ∆PDZ nNOS in aged mdx model of Duchenne cardiomyopathy. Hum Mol Genet. 2014;23(12):3189–99.

    Article  PubMed  Google Scholar 

  17. Voit A, Patel V, Pachon R, Shah V, Bakhutma M, Kohlbrenner E, McArdle JJ, Dell’Italia LJ, Mendell JR, Xie LH, et al. Reducing sarcolipin expression mitigates Duchenne muscular dystrophy and associated cardiomyopathy in mice. Nat Commun. 2017;8(1):1068.

    Article  PubMed  Google Scholar 

  18. Morales ED, Yue Y, Watkins TB, Han J, Pan X, Gibson AM, Hu B, Brito-Estrada O, Yao G, Makarewich CA et al: Dwarf Open Reading Frame (DWORF) Gene Therapy Ameliorated Duchenne Muscular Dystrophy Cardiomyopathy in Aged mdx Mice. J American Heart Assoc. 2023;12(3):e027480.

  19. McGreevy JW, Hakim CH, McIntosh MA, Duan D. Animal models of Duchenne muscular dystrophy: from basic mechanisms to gene therapy. Dis Model Mech. 2015;8(3):195–213.

    Article  PubMed  Google Scholar 

  20. Duan D. Duchenne muscular dystrophy gene therapy in the canine model. Hum Gene Ther Clin Dev. 2015;26(1):57–69.

    Article  PubMed  Google Scholar 

  21. Kornegay JN. The golden retriever model of Duchenne muscular dystrophy. Skelet Muscle. 2017;7(1):9.

    Article  PubMed  Google Scholar 

  22. Hakim CH, Yang HT, Burke MJ, Teixeira J, Jenkins GJ, Yang NN, Yao G, Duan D. Extensor carpi ulnaris muscle shows unexpected slow-to-fast fiber type switch in Duchenne muscular dystrophy dogs. Dis Model Mech. 2021;14(12):dmm049006.

    Article  PubMed  Google Scholar 

  23. Hakim CH, Burke MJ, Teixeira J, Duan D. Histological Assessment of Gene Therapy in the Canine DMD Model. Methods Mol Biol. 2023;2587:303–38.

    Article  PubMed  Google Scholar 

  24. Hakim CH, Perez-Lopez D, Burke MJ, Teixeira J, Duan D. Molecular and Biochemical Assessment of Gene Therapy in the Canine Model of Duchenne Muscular Dystrophy. Methods Mol Biol. 2023;2587:255–301.

    Article  PubMed  Google Scholar 

  25. Yang HT, Shin J-H, Hakim CH, Pan X, Terjung RL, Duan D. Dystrophin deficiency compromises force production of the extensor carpi ulnaris muscle in the canine model of Duchenne muscular dystrophy. PLoS ONE. 2012;7(9): e44438.

    Article  PubMed  Google Scholar 

  26. Shin J-H, Pan X, Hakim CH, Yang HT, Yue Y, Zhang K, Terjung RL, Duan D. Microdystrophin ameliorates muscular dystrophy in the canine model of Duchenne muscular dystrophy. Mol Ther. 2013;21(4):750–7.

    Article  PubMed  Google Scholar 

  27. Kodippili K, Hakim CH, Pan X, Yang HT, Yue Y, Zhang Y, Shin JH, Yang NN, Duan D. Dual AAV gene therapy for Duchenne muscular dystrophy with a 7-kb mini-dystrophin gene in the canine model. Hum Gene Ther. 2018;29(3):299–311.

    Article  PubMed  Google Scholar 

  28. Kodippili K, Hakim CH, Burke MJ, Yue Y, Teixeira JA, Zhang K, Yao G, Babu GJ, Herzog RW, Duan D. SERCA2a overexpression improves muscle function in a canine Duchenne muscular dystrophy model. Molecular therapy Methods & clinical development. 2024;32(2): 101268.

    Article  Google Scholar 

  29. Yuasa K, Nakamura A, Hijikata T, Takeda S. Dystrophin deficiency in canine X-linked muscular dystrophy in Japan (CXMDJ) alters myosin heavy chain expression profiles in the diaphragm more markedly than in the tibialis cranialis muscle. BMC Musculoskelet Disord. 2008;9:1.

    Article  PubMed  Google Scholar 

  30. Rohman MS, Emoto N, Takeshima Y, Yokoyama M, Matsuo M. Decreased mAKAP, ryanodine receptor, and SERCA2a gene expression in mdx hearts. Biochem Biophys Res Commun. 2003;310(1):228–35.

    Article  PubMed  Google Scholar 

  31. Anderson DM, Makarewich CA, Anderson KM, Shelton JM, Bezprozvannaya S, Bassel-Duby R, Olson EN. Widespread control of calcium signaling by a family of SERCA-inhibiting micropeptides. Sci Signal. 2016;9(457):ra119.

    Article  PubMed  Google Scholar 

  32. Anderson DM, Anderson KM, Chang CL, Makarewich CA, Nelson BR, McAnally JR, Kasaragod P, Shelton JM, Liou J, Bassel-Duby R, et al. A micropeptide encoded by a putative long noncoding RNA regulates muscle performance. Cell. 2015;160(4):595–606.

    Article  PubMed  Google Scholar 

  33. Pescatori M, Broccolini A, Minetti C, Bertini E, Bruno C, D’Amico A, Bernardini C, Mirabella M, Silvestri G, Giglio V, et al. Gene expression profiling in the early phases of DMD: a constant molecular signature characterizes DMD muscle from early postnatal life throughout disease progression. Faseb J. 2007;21(4):1210–26.

    Article  PubMed  Google Scholar 

  34. Camerino GM, Cannone M, Giustino A, Massari AM, Capogrosso RF, Cozzoli A, De Luca A. Gene expression in mdx mouse muscle in relation to age and exercise: aberrant mechanical-metabolic coupling and implications for pre-clinical studies in Duchenne muscular dystrophy. Hum Mol Genet. 2014;23(21):5720–32.

    Article  PubMed  Google Scholar 

  35. Fujikura Y, Yamanouchi K, Sugihara H, Hatakeyama M, Abe T, Ato S, Oishi K. Ketogenic diet containing medium-chain triglyceride ameliorates transcriptome disruption in skeletal muscles of rat models of duchenne muscular dystrophy. Biochem Biophys Rep. 2022;32: 101378.

    PubMed  Google Scholar 

  36. Morera C, Kim J, Paredes-Redondo A, Nobles M, Rybin D, Moccia R, Kowala A, Meng J, Garren S, Liu P, et al. CRISPR-mediated correction of skeletal muscle Ca(2+) handling in a novel DMD patient-derived pluripotent stem cell model. Neuromuscul Disord. 2022;32(11–12):908–22.

    Article  PubMed  Google Scholar 

  37. Mournetas V, Massourides E, Dupont JB, Kornobis E, Polveche H, Jarrige M, Dorval ARL, Gosselin MRF, Manousopoulou A, Garbis SD, et al. Myogenesis modelled by human pluripotent stem cells: a multi-omic study of Duchenne myopathy early onset. J Cachexia Sarcopenia Muscle. 2021;12(1):209–32.

    Article  PubMed  Google Scholar 

  38. Haslett JN, Sanoudou D, Kho AT, Bennett RR, Greenberg SA, Kohane IS, Beggs AH, Kunkel LM. Gene expression comparison of biopsies from Duchenne muscular dystrophy (DMD) and normal skeletal muscle. Proc Natl Acad Sci U S A. 2002;99(23):15000–5.

    Article  PubMed  Google Scholar 

  39. Nieves-Rodriguez S, Barthelemy F, Woods JD, Douine ED, Wang RT, Scripture-Adams DD, Chesmore KN, Galasso F, Miceli MC, Nelson SF. Transcriptomic analysis of paired healthy human skeletal muscles to identify modulators of disease severity in DMD. Front Genet. 2023;14:1216066.

    Article  PubMed  Google Scholar 

  40. Rodriguez-Montes L, Ovchinnikova S, Yuan X, Studer T, Sarropoulos I, Anders S, Kaessmann H, Cardoso-Moreira M. Sex-biased gene expression across mammalian organ development and evolution. Science. 2023;382(6670):eadf1046.

    Article  PubMed  Google Scholar 

  41. Periasamy M, Kalyanasundaram A. SERCA pump isoforms: their role in calcium transport and disease. Muscle Nerve. 2007;35(4):430–42.

    Article  PubMed  Google Scholar 

  42. Murphy RM, Larkins NT, Mollica JP, Beard NA, Lamb GD. Calsequestrin content and SERCA determine normal and maximal Ca2+ storage levels in sarcoplasmic reticulum of fast- and slow-twitch fibres of rat. J Physiol. 2009;587(2):443–60.

    Article  PubMed  Google Scholar 

Download references

Funding

This study was supported by grants from the National Institutes of Health (AR70517, to DD), Department of Defense (MD210064, to DD), The Jesse Davidson Foundation-Defeat Duchenne Canada (to DD), Jackson Freel DMD Research Fund (to DD), the University of Missouri Life Science Fellowship (to EDM), and the University of Missouri Research Excellence Program (to DW).

Author information

Author notes

  1. Emily D. Morales, Dongxin Wang and Matthew J. Burke contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Microbiology and Immunology, School of Medicine, The University of Missouri, Columbia, MO, 65212, USA

    Emily D. Morales, Dongxin Wang, Matthew J. Burke, Jin Han, Drake D. Devine, Keqing Zhang & Dongsheng Duan

  2. Department of Neurology, School of Medicine, The University of Missouri, Columbia, MO, 65212, USA

    Dongsheng Duan

  3. Department of Chemical and Biomedical Engineering, College of Engineering, The University of Missouri, Columbia, MO, 65212, USA

    Dongsheng Duan

  4. Department of Biomedical Sciences, College of Veterinary Medicine, The University of Missouri, Columbia, MO, 65212, USA

    Dongsheng Duan

Authors
  1. Emily D. Morales
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dongxin Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Matthew J. Burke
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jin Han
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Drake D. Devine
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Keqing Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Dongsheng Duan
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Design the study (EDM, DW, MJB, and DD), data acquisition (EDM, DW, MJB, JH, DDD, and KZ), data analysis (EDM, DW, MJB, and DD), data interpretation (EDM, DW, MJB, and DD), funding acquisition (DD), supervision (DW, MJB, and DD), writing and editing (EDM, DW, MJB, and DD).

Corresponding author

Correspondence to Dongsheng Duan.

Ethics declarations

Ethics approval and consent to participate

This study used curated tissues previously collected at the University of Missouri. Tissue collection was approved by the Animal Care and Use Committee of the University of Missouri. No animals were euthanized for the purpose of this study. All the study protocols were followed according to ARRIVE guidelines.

Consent for publication

Not applicable.

Competing interests

DD is a member of the scientific advisory board for Solid Biosciences and an equity holder of Solid Biosciences, a member of the scientific advisory board for Sardocor Corp, and an inventor of several issued and filed patents on DMD gene therapy and AAV vectors. All other authors have nothing to disclose.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Morales, E.D., Wang, D., Burke, M.J. et al. Transcriptional changes of genes encoding sarcoplasmic reticulum calcium binding and up-taking proteins in normal and Duchenne muscular dystrophy dogs. BMC Musculoskelet Disord 25, 811 (2024). https://doi.org/10.1186/s12891-024-07927-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12891-024-07927-8

Keywords

BMC Musculoskeletal Disorders

ISSN: 1471-2474

Contact us