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. 2020 Nov 2;23(11):101760. doi: 10.1016/j.isci.2020.101760

Radiation-Induced Damage to Prepubertal Pax7+ Skeletal Muscle Stem Cells Drives Lifelong Deficits in Myofiber Size and Nuclear Number

John F Bachman 1,2, Roméo S Blanc 2, Nicole D Paris 2, Jacob G Kallenbach 2,3, Carl J Johnston 4, Eric Hernady 5, Jacqueline P Williams 5,6, Joe V Chakkalakal 2,3,6,7,8,9,∗
PMCID: PMC7674517  PMID: 33241204

Summary

During prepubertal development, muscle stem cells (satellite cells, SCs) actively contribute to myofiber growth. Because some SCs are active during this time, they may be particularly susceptible to damage. Using a Small Animal Radiation Research Platform (SARRP), we investigated the effects of local fractionated radiation treatment on prepubertal SCs. Immediately after this regimen, there was a reduction in SC number. Although surviving SCs had deficiencies in function, some myogenic potential remained. Indeed, some muscle regenerative capacity persisted immediately after irradiation. Lastly, we assessed the long-term consequences of radiation-induced SC loss during prepuberty. We observed a reduction of myofiber size and corresponding loss of nuclei in both fast- and slow-contracting muscles 14 months post-irradiation. Notably, prepubertal SC depletion mimicked these lifelong deficits. This work highlights the susceptibility of prepubertal SCs to radiation exposure. We also reveal the importance of prepubertal SC contribution to the lifelong maintenance of skeletal muscle.

Subject Areas: Cell Biology, Stem Cell Research, Cancer

Graphical Abstract

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Highlights

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    Increased sensitivity of satellite cells to irradiation during prepubertal growth

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    Prepubertal irradiation leads to lifelong deficits in skeletal muscle regenerative capacity

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    Lifelong reduction in myofiber size and nuclear number is a consequence of prepubertal irradiation

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    Satellite cell ablation mimics the lifelong effects of prepubertal irradiation on myofiber size and nuclear number

Cell Biology; Stem Cell Research; Cancer

Introduction

Radiation is a mainstay therapy of many cancer treatments. Approximately 50% of all patients with cancer will receive radiation during the course of their treatments (Baskar et al., 2012). Although effective at killing highly proliferative cancer cells, radiation treatment has the potential to damage off-target normal tissues, negatively impacting tissue growth, maintenance, and regeneration (Bentzen, 2006; Williams et al., 2016). This is especially true in children, as the damage to actively dividing cells and progenitors responsible for tissue growth is a known consequence of radiotherapy. Long-term follow-up of cancer survivors, who had received radiation therapy as children, revealed indices of tissue decline suggestive of accelerated aging (Ness et al., 2005, 2007, 2015; Paulino, 2004; Rayar et al., 2013). Consistent with these findings, juvenile cancer survivors suffer from physical limitations associated with skeletal muscle decline, including weakness and exercise intolerance (Ness et al., 2007; Paulino, 2004; Rayar et al., 2013). Notably, ∼80% of pediatric sarcoma survivors demonstrated atrophy of the irradiated limb (∼20 years post-treatment) (Paulino, 2004). Thus, understanding the mechanisms of both the immediate and late effects of pediatric irradiation on skeletal muscle regeneration, growth, and maintenance are of great concern.

Early skeletal muscle growth is contingent upon a population of Pax7-expressing muscle stem cells known as satellite cells (SCs) (Chal and Pourquie, 2017; Hutcheson et al., 2009; Seale et al., 2000; Tajbakhsh, 2009). As skeletal muscle growth proceeds, some SCs escape terminal commitment, maintain Pax7 expression, and reside in a quiescent state at the interface of multinucleated skeletal muscle fibers (myofibers) and the overlaying basal lamina (Chakkalakal et al., 2012, 2014; Gros et al., 2005; Kassar-Duchossoy et al., 2005; Relaix et al., 2005). In adults, SCs endow skeletal muscle with extensive regenerative potential. In response to injury, SCs activate, divide, and derived progenitors differentiate and fuse to form regenerated myofibers (Crist, 2017; Tajbakhsh, 2009; Yin et al., 2013). Depletion studies have revealed that Pax7-expressing SCs are essential for late embryonic growth and adult regeneration of skeletal muscle, with additional roles in adult skeletal muscle adaptation and repair (Fry et al., 2014, 2016; Hutcheson et al., 2009; Klose et al., 2018; Liu et al., 2015, 2017; Murach et al., 2017; Murphy et al., 2011). Moreover, a lingering contribution of SCs to late juvenile/prepubertal myofiber growth has been observed (Bachman et al., 2018; Chakkalakal et al., 2014; Gattazzo et al., 2020; Huh et al., 2017; Kim et al., 2016; Pawlikowski et al., 2015). The relevance of the sustained loss of this residual postnatal SC activity to lifelong myofiber maintenance is currently unknown.

Experimentally, the impact of irradiation on skeletal muscle regeneration and early development has been examined (Heslop et al., 2000; Mozdiak et al., 1996; Pagel and Partridge, 1998). However, dose and age of the mouse are key determinants of experimental outcomes. For example, mouse pups irradiated at 16 days of age and examined 6 weeks later had loss of muscle weight and myofiber cross-sectional area (CSA) following a single high 18-Gy dose, whereas irradiating adult mice at 15 weeks elicited little effect in terms of muscle size change (Pagel and Partridge, 1998). Radiation exposure has also been shown to have a profound impact on skeletal muscle regenerative capacity. A single 18-Gy dose was sufficient to induce regenerative delays in wild-type and mdx mice, the latter being a model of muscular dystrophy (Heslop et al., 2000). Recently a subpopulation of SCs capable of contributing to muscle regeneration, albeit with delayed kinetics, after 9-Gy whole-body irradiation of adult mice was identified (Scaramozza et al., 2019). This subpopulation possessed the ability to withstand radiation-induced stressors, such as reactive oxygen species production, through a coordinated program involving the expression of stress and developmental-associated genes. To date, there has yet to be a study investigating the impact of a clinically relevant radiation regimen, delivered during the critical late juvenile/prepubertal period, on SC function and lifelong skeletal muscle maintenance.

In this study, we examined the consequences of a local fractionated radiation regimen on prepubertal SC function and skeletal muscle regenerative potential. Through the use of RNA sequencing, enumeration of SC number, and lineage tracing of indelibly labeled SCs and derived cell fate, we identified a significant reduction in SC number and function, 3 weeks after prepubertal irradiation. Assessing SCs ∼2–4 hrs following the last radiation fractionation, we found an immediate reduction in prepubertal SC number, confirming their sensitivity to ionizing radiation. Once isolated and cultured, surviving SCs displayed deficits in expansion and differentiation. This was associated with severely delayed and incomplete muscle regeneration following degenerative muscle injury. To assess the long-term consequences of prepubertal radiation exposure, we aged mice to 14 months post-irradiation. Aged irradiated mice exhibited significant deficits in myofiber size and nuclear number in both fast-contracting extensor digitorum longus (EDL) and slow-contracting soleus (SOL) muscles. Last, these lifelong deficits were mimicked in a mouse model of targeted inducible SC depletion (Bachman et al., 2018; Klose et al., 2018; Liu et al., 2015, 2017). Collectively, we confirm the potential for radiation-induced loss of SCs during prepubertal growth, a critical time of postnatal skeletal muscle development.

Results

Radiation Treatment Decreases Prepubertal SC Pool Size and Induces Gene Signatures Consistent with Radiation-Induced Damage

To assess the effects of a clinically relevant radiation exposure on the prepubertal SC pool, we performed fractionated radiation treatments. Utilizing the Small Animal Research Radiation Platform's (SARRP) CT capabilities, we delivered a targeted dose to the lower right limb (knee to ankle) of mice at ∼4 weeks of age using an 8.2 Gy 3X (MWF) protocol (Wong et al., 2008) (Figures 1A and S1A). This hyper-fractionated protocol allowed us to compensate for the relatively low sensitivity of the murine model, to work within the time constraints of the SARRP core, and to limit irradiation to the condensed prepubertal mouse period.

Figure 1.

Figure 1

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Loss of SC Number and Derived Contribution Is a Consequence of Prepubertal Irradiated Skeletal Muscle

(A) Scheme representing radiation treatment paradigm. Mice were irradiated at ∼4 weeks of age with an 8.2 Gy dose 3X (MWF) to the lower right limb. Mice were sacrificed ∼3 weeks following the last radiation dosage. Control (CTL) animals were age-matched and received no radiation.

(B) Heatmap displaying significantly differentially expressed (DE) genes (FDR <.05) from RNA-seq of CTL, CL (contralateral limb), and RL (irradiated limb) gastrocnemius muscles 3 weeks post-radiation. N = 3 mice per group; 361 DE genes (CTL versus RL). Red is upregulated and blue is downregulated.

(C) Heatmap displaying CTL versus RL DE genes related to Muscle Adaptation (GO:0043502) and Muscle Regeneration (GO:0043403).

(D) Scheme representing radiation experiments of P7nTnG. Mice were injected at ∼3.5 weeks of age with tamoxifen (Tmx) to label SCs and derived myonuclei during prepubertal muscle development. (E) Representative cross sections of CTL and RL P7nTnG EDL and SOL muscles. Sections were stained with GFP (green), Dapi (blue), and laminin (white).

(F and G) Quantification of nGFP+ myonuclei per 100 fibers in CTL, CL, and RL EDL (F) and SOL (G) muscles. One-way ANOVA, Tukey. Significance is depicted relative to RL.

To assess global changes in muscle gene expression due to irradiation, we performed genome-wide RNA sequencing (RNA-seq) analysis on age-matched 0-Gy control animals (CTL), contralateral (non-irradiated) limb (CL), and irradiated limb (RL) gastrocnemius muscles (Figure 1A) 3 weeks post-radiation treatment. When comparing CTL with RL samples, we identified 361 differentially expressed genes (FDR <0.05) (Figure 1B). Assessing gene changes within the GO Terms “Muscle Adaptation” and “Muscle Regeneration” revealed expression of the paired box transcription factor Pax7, a marker for SCs, to be significantly decreased in RL muscles (Figure 1C). In addition, we observed minimal changes (12 genes, FDR <0.05) in gene expression when comparing CL with CTL, indicating the negligible difference between the two conditions. As a relatively rare population in skeletal muscle, SCs make up only 2%–7% of all sublaminal nuclei in adult muscles (Yin et al., 2013). To determine if SC loss is a feature of prepubertal radiation, we counted SC number in EDL and SOL lower limb skeletal muscles 3 weeks after the last radiation fractionation. Consistent with the RNA-seq results, we observed a reduction of SC number in both RL muscles (Figures S1B–S1D).

To understand other radiation-induced changes, we examined significant differentially expressed genes within the GO Term: Cellular Response to Radiation (Table S1 and Figure S2A). Three weeks post-radiation, we observed an induction of apoptotic mediators Bax and Bmf. Additionally, there was still strong expression of the cell cycle inhibitor Cdkn1a. To obtain a more complete look as to the pathways affected within our RNA-seq, we employed Ingenuity Pathway Analysis (IPA) (Table S1). This analysis revealed that all of the top 12 most significantly modified pathways were related to an inflammatory response (Table S1). This was further confirmed by utilizing the upstream regulators function (Table S1 and Figure S2B). IPA predicted potent cytokines IFNγ (z = 6.472), TNF (z = 5.289), and IL-1β (z = 4.535) to be activated 3 weeks post-irradiation. Assessing the commonalities between these cytokines, we identified 18 potential nodal points (Figure S2C). Among these was the chemokine receptor Ccr2, the top significant gene within our Seq dataset, which is a critical regulator of inflammatory cell recruitment after injury that becomes disrupted with aging (Blanc et al., 2020; Tidball, 2017). Last, we assessed changes in the Diseases & Functions IPA category (Figure S2D). Confirming our work, the heatmap was indicative of an induction of inflammation and cell death. This analysis demonstrates that gene signatures indicative of prolonged inflammation, cell death, and cell-cycle arrest are hallmarks of fractionated irradiation during prepuberty.

Radiation Treatment Reduces SC-Derived Contribution during Prepubertal Development

During prepubertal muscle growth, some SCs are actively dividing and derived progenitors are fusing into growing myofibers (Bachman et al., 2018; Chakkalakal et al., 2014; Gattazzo et al., 2020; Huh et al., 2017; Kim et al., 2016; Pawlikowski et al., 2015). To understand if prepubertal irradiation interferes with SC-derived contribution, we utilized a previously characterized lineage tracing mouse model, Pax7CreERT2/+; Rosa26nTnG/+ (P7nTnG) (Bachman et al., 2018; Liu et al., 2015, 2017). Upon tamoxifen injection, this mouse enables indelible labeling of SCs with a nuclear GFP (nGFP), allowing for an assessment of SC-derived contribution to myofibers. Tamoxifen was administered to juvenile P7nTnG mice at ∼3.5 weeks of age (Figure 1D). Mice were subsequently irradiated at 4 weeks of age with the previously described dosage and sacrificed at 8 weeks of age (Figure 1D). We found that SC-derived nGFP+ contribution was significantly reduced in RL when compared with CTL EDL (∼48 nGFP+/100 fibers in CTL versus ∼7 nGFP+/100 fibers in RL) and SOL (∼105 nGFP+/100 fibers in CTL versus ∼32 nGFP+/100 fibers in RL) muscles (Figures 1E–1G). Because our P7nTnG model indelibly labels SC and derived myonuclei, we compared our nGFP+ counts to our SC counts (Figures S1C and S1D). In CTL conditions, we only counted ∼3 and 10 SCs/100 fibers in 8-week-old EDL and SOL sections, respectively, indicating that an overwhelming proportion of nGFP+ cells are not Pax7-expressing SCs. The loss of juvenile SC-derived nGFP+ contribution was also observed in other irradiated lower limb muscles, including the tibialis anterior, plantaris, and gastrocnemius (Figure S3A). There was no significant change when comparing nGFP+ in CTL and CL conditions. Therefore, a principal feature of prepubertal irradiated skeletal muscle is loss of SC number and contribution to growing myofibers.

Loss of SC Number Is an Immediate Consequence of Prepubertal Irradiation

An immediate consequence of irradiation is the induction of DNA damage leading to cell loss (Eriksson and Stigbrand, 2010). To determine if SC loss is an immediate consequence of prepubertal skeletal muscle irradiation, we assessed SC number in irradiated EDL and SOL muscles harvested ∼2–4 hrs after the final fractionated radiation dose (I-RL) (Figure 2A). Consistent with the instantaneous cytotoxic effects of radiation exposure, we observed a significant loss of SCs in both EDL and SOL muscles (4.8 SCs/100 fibers in CTL EDL, 2.78 SCs/100 fibers in I-RL EDL) (14.69 SCs/100 fibers in CTL SOL, 8.3 SCs/100 fibers in I-RL SOL) (Figures 2B–2D). Thus, radiation-induced SC loss is an immediate consequence of prepubertal irradiation.

Figure 2.

Figure 2

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Irradiation of Prepubertal Skeletal Muscle Results in Immediate SC Loss

(A) Scheme representing immediate radiation treatment paradigms. Animals were sacrificed ∼2–4 hrs after receiving the final 8.2-Gy dose.

(B) Representative cross sections of CTL and immediately irradiated (I-RL) EDL and SOL muscles stained to assess Pax7 number. Sections were stained with Pax7 (red), DAPI (blue), and laminin (white). Example SCs are identified with white arrows. (C and D) Quantification of Pax7 number per 100 fibers in CTL, CL, and I-RL EDL (C) and SOL (D) muscles. One-way ANOVA, Tukey. Significance is depicted relative to I-RL.

To understand if the extent of SC loss immediately after fractionated irradiation is unique to the prepubertal period, we assessed the effect of our paradigm on adult SCs. Adult mouse lower limbs were irradiated at 3 months of age and SC number was assessed ∼2–4 hrs after the final fractionation (Figure S4A). Comparing adult CL to I-RL, we observed no difference in EDL SC number (Figures S4B and S4C). In juveniles a 44% reduction in SC number was observed in I-RL SOL muscles; there was only a ∼27% loss of SC number in I-RL adult SOL muscles (Figures S4B and S4E). Comparing juvenile with adult SC numbers, there was no statistical difference between the adult CL and I-RL conditions; however, juvenile CL was significant to juvenile I-RL, adult CL, and adult I-RL (Figures S4D and S4F). Thus, SCs are more sensitive to the effects of radiation, before adolescence when they are relatively more active (Bachman et al., 2018; Chakkalakal et al., 2014; Gattazzo et al., 2020).

Remaining I-RL SCs Are Deficient in Proliferative Expansion In Vitro

As we observed that prepubertal irradiation resulted in an immediate ∼44% loss in SC number, we next wanted to assess the function of the remaining SCs. SCs were isolated from I-RL, CL, and CTL muscles and cultured for 24, 48, 72, and 96 hrs (DMEM, 10% HS) (Figure 3A). To understand if irradiation reduced the ability of SCs to grow and expand, we counted cell density at each time point and assessed for expression of Ki67 (a marker of active cells) (Figures 3B–3E). CL and CTL SCs were able to expand exponentially in culture, whereas I-RL SCs barely increased in number between 24 and 96 hrs (Figure 3C). We observed a high percentage of Ki67+ cells between 24 and 72 hrs in CTL and CL SC-derived cultures with a peak of ∼80% at 48 and 72 hrs (Figures 3B and 3D). Indeed, nearly 80% of CTL and CL Ki67+ cells were equally positive for MyoD (myogenic progenitor marker) at 72 hrs (Figure 3E). In contrast to CTL and CL, the proportion of Ki67+ cells in I-RL SC-derived cultures never got above ∼40% (Figure 3D). We did observe a decrease in Ki67+ cells in CTL and CL cells at 96 hrs consistent with increased terminal myogenic commitment (evident from increased Myogenin expression) at that time point (Figure 3D).

Figure 3.

Figure 3

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I-RL SCs Have Deficits in Proliferative Capacity

(A) Scheme representing immediate radiation culture experiments. SCs were isolated from CTL, CL, and I-RL by magnetic sort 2–4 hrs after the last fractionated dose. SCs were plated at a density of 5,000 cells/well and cultured for 24, 48, 72, and 96 hrs.

(B) Representative images of 72-hr CTL and I-RL cultures. Cells were stained with Ki67 (green), MyoD (red), and DAPI (blue). (C) Quantification of cell density at 24, 48, 72, and 96 hrs in CTL, CL, and I-RL cultures. One-way ANOVA, Tukey. Significance is depicted relative to I-RL. (n=3 per time point).

(D) Quantification of Ki67+ cells at 24, 48, 72, and 96 hrs. One-way ANOVA, Tukey. Significance is depicted relative to I-RL.

(E) Quantification of Ki67+/MyoD+ and Ki67+/MyoD− at 72 hrs. One-way ANOVA, Tukey.

(F) Representative images of 72-h CL and I-RL SA-β-Gal staining. (G) Quantification of the percentage of SA-β-Gal+ cells in CL and I-RL. Unpaired t test.

Senescence is a state of permanent cell-cycle arrest, phenotypically characterized by enlarged flattened cells and positive staining for senescence-associated β-galactosidase (Dimri et al., 1995; Hayflick, 1965; Wang and Dreesen, 2018). Due to the observed deficit in I-RL growth capacity, we assessed cellular senescence of irradiated SCs and derived progenitors through an SA-β-Gal stain after 72 hrs in culture. We observed an increase (∼21% in I-RL, ∼9% in CL) in the amount of SA-β-Gal+ cells in our I-RL conditions (Figures 3F and 3G). Together, these data demonstrate that the remaining I-RL SCs have severe deficits in their ability to expand in number, and some are prone to senescence.

I-RL SCs Have Deficits in Myogenic Commitment

Upon activation, SCs and derived myogenic progenitors undergo a coordinated program of proliferation and differentiation through the expression of myogenic determination factors (Yin et al., 2013). Ultimately the SC-derived progenitors are able to fuse together to form immature multinucleated myofibers (myotubes). We next tested the myogenic potential of CTL, CL, and I-RL SCs based on expression of the myogenic regulatory factors MyoD and Myogenin (terminal myogenic differentiation marker) (Figure 4A). As expected, ∼95% of cells in CTL and CL SC-derived cultures expressed MyoD after 72 and 96 hrs of culture (Figures 4B and 4C). In contrast, only ∼50% of cells in I-RL SC-derived cultures expressed MyoD (Figure 4C). At 72 hrs, the proportion of Myogenin+ (MyoG) cells in CTL, CL, and I-RL cultures was negligible (∼1–2%) (Figure 4D). However, at 96 hrs, there was a substantial increase in the proportion of MyoG+ cells in CTL and CL cultures (∼34–40%), whereas, in I-RL cultures, the proportion of MyoG+ cells remained low (∼8%) (Figures 4B and 4D). Although not to the same extent as CTL and CL cultures, these data indicate that some I-RL SCs are able to commit to the myogenic lineage.

Figure 4.

Figure 4

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I-RL SCs Have Deficits in Myogenic Commitment

(A) Scheme representing culture experiments performed immediately following irradiation.

(B) Representative images of 96-h CTL and I-RL cultures. Cultures were stained with Myogenin (green), MyoD (red), and DAPI (blue).

(C and D) Quantification of MyoD (C) and Myogenin (D) at 72 and 96 hrs in CTL, CL, and I-RL samples. One-way ANOVA, Tukey. Significance is depicted relative to I-RL.

(E) Representative images of 72-h I-RL cells with morphology characterized as normal, nuclear aberrations, or pyknotic/karyolytic. Cells are stained with MyoD (red) and DAPI (blue). Each frame is one individual cell.

(F) Quantification of the % of MyoD+ and MyoD− I-RL cells that were morphologically normal, had nuclear aberrations, or were pyknotic/karyolytic. More than 100 cells were assessed per condition (n = 3). Unpaired t test. (G) Quantification of nuclear area of 72-h CTL MyoD+, CL MyoD+, I-RL MyoD+, and I-RL MyoD− cells. More than 100 nuclei sized per n. One-way ANOVA, Tukey. Significance is depicted relative to I-RL MyoD−.

Next, isolated CTL, CL, and I-RL SCs were cultured for 96 hrs, followed by a 24-hr treatment with low-serum media (DMEM, 3% HS) to induce myotube formation (Figure S5A). Consistent with reduced cell density and impaired myogenic commitment, I-RL cultures displayed an extremely low capacity to form myotubes (Figure S5B–S5F). To understand if I-RL SCs could form myotubes if plated at a higher density, we plated them at 15,000 and 30,000 per well and induced myotube formation after 96 hrs in culture (Figure S5G). At the 30,000 condition, I-RL cells were able to fuse together more efficiently with a significant increase in the number of myotubes (Figures S5I and S5J). However, these myotubes were still rather small in terms of the number of myonuclei per myotube (Figure S5K). Thus, even though these I-RL SCs were plated at six times the density of CTL and CL SCs, they still demonstrated deficits in terminal myogenic fusion and myotube formation.

Cultured I-RL SCs Demonstrate Phenotypes Consistent with Mitotic Damage

An indicator of cellular fitness and radiation-induced injury is DNA morphology (Suzuki et al., 2003). As 50% of the cells in I-RL SC-derived cultures expressed MyoD, we questioned if these cells were morphologically unique from MyoD-cells. We defined cell morphology based on three different categories: (1) normal, (2) nuclear aberrations, and (3) pyknotic/karyolytic (Figure 4E). Pyknosis (shrunken nuclei) and karyolysis (swollen dissolution of the nuclei) are hallmarks of a dying cell (Majno and Joris, 1995). Biomarkers of radiation-induced chromosomal damage include nuclear aberrations, such as micronuclei, nucleoplasmic bridges, binucleated cells, and nuclear buds (Fenech et al., 2011; Pernot et al., 2012; Ronald et al., 2016) (Figure 4E). Within the MyoD+ I-RL SC-derived cell population, we found ∼26.7% of cells to have nuclear aberrations (compared with only 1.7% in MyoD− cells) (Figure 4F). This indicates that although those MyoD+ cells were able to proliferate, a portion of them still had significant phenotypes consistent with chromosomal damage. Interestingly, we did not observe many pyknotic or karyolytic MyoD+ I-RL cells (4.93%); however, more than half (55.5%) of MyoD- I-RL cells were of this appearance. Overall, ∼69% of the MyoD+ cells appeared to be phenotypically normal, whereas this number was reduced to ∼42.8% in the MyoD− population (Figure 4F). Finally, we quantified the size of CTL MyoD+, CL MyoD+, I-RL MyoD+, and I-RL MyoD− cells with intact nuclei (excluding nuclear aberrations) (Figure 4G). The I-RL MyoD− cells were significantly smaller compared with the other groups (Figure 4G). Taken together, these data demonstrate that cultured I-RL SCs are composed of cells with morphology characteristic of death and genomic damage.

Delayed and Incomplete Regenerative Capacity in Prepubertal Irradiated Skeletal Muscle

Although we observed severe deficiencies in the I-RL SCs, we postulated that some regenerative potential may exist. Thus, we sought to determine the regenerative capacity of I-RL muscle. To test this, CTL and I-RL tibialis anterior (TA)/EDL muscles were intramuscularly injected with a 1.2% BaCl2 solution to induce degenerative injury immediately post-radiation treatment (Figure 5A). Regenerating muscles were examined at 5, 15, and 30 days post-injury (dpi). At 5 dpi, centrally nucleated regenerated myofibers were readily apparent in CTL muscles (Figure 5B). Consistent with regenerative decline, few, if any, centrally nucleated regenerated myofibers were observed in injured areas of 5-dpi I-RL muscles (Figure 5B). However, by 15 dpi, such fibers were observed in I-RL regenerating muscles, albeit of reduced size (Figure 5C). This trend continued at 30 dpi, with I-RL muscle having significantly smaller centrally nucleated fibers (Figure 5C). Additionally, the number of SCs associated with regenerating centrally nucleated fibers was reduced in 30-dpi I-RL muscles (Figure 5D). Therefore, after a degenerative myofiber injury, prepubertal I-RL muscles are capable of mounting a delayed regenerative response, albeit with reduced integrity.

Figure 5.

Figure 5

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Delayed Regenerative Potential of I-RL Skeletal Muscle Following Injury

(A) Scheme representing BaCl2 injury experiments performed immediately following irradiation. Animals were sacrificed at 5, 15, or 30 days post injury (dpi).

(B) Representative H&Es of 5-, 15-, and 30-dpi CTL and I-RL.

(C) Quantification of 15- and 30-dpi centrally nucleated fiber (CNF) cross-sectional area. Unpaired t test.

(D) Quantification of SCs associated with CNF at 30 dpi in CTL and I-RL sections. Unpaired t test.

(E) Scheme representing injury experiments of P7nTnG. Mice were injected at ∼3.5 weeks of age with tamoxifen (Tmx) to label SCs and irradiated at 4 weeks of age. BaCl2 injuries were performed immediately following irradiation. Animals were sacrificed at 5 or 15 dpi. (F) Representative cross sections of 5- and 15-dpi P7nTnG CTL and I-RL sections. N = 3–4 per condition.

To examine SC contribution to I-RL muscle regeneration, P7nTnG were injected with tamoxifen at ∼3.5 weeks of age to indelibly label SCs with nGFP and irradiated at 4 weeks (Figure 5E). Immediately (∼2–4 hrs) after the final radiation dose, the TA/EDL muscles were subjected to muscle degenerative injury. At 5 dpi the vast majority (∼90%) of CTL regenerating muscles were SCderived based on the abundance nGFP+ central nucleated regenerated myofibers (Figure 5F). Consistent with regenerative delays, we only observed clusters of SC-derived nGFP+ cells in 5-dpi I-RL muscles (Figure 5F). However, at 15 dpi, similar to CTL, the majority of I-RL central nucleated regenerated myofibers were nGFP+ (Figure 5F). Our data indicate that after prepubertal irradiation, remaining SCs are able to contribute to muscle regeneration, albeit in a delayed manner.

As we observed a delay in muscle regeneration immediately after irradiation, we reasoned that an extended period of recovery may allow for the repair of radiation-induced effects to occur, thereby accelerating muscle regeneration. To examine this possibility, TA/EDL muscles were injected with BaCl2 to induce muscle degenerative injury, 3 weeks after the final radiation fraction (Figure S6A). As expected, myofiber regeneration was readily apparent in CTL 5-dpi muscles (Figure S6B). In contrast, yet similar to I-RL, 5-dpi RL injured areas were devoid of centrally nucleated regenerated myofibers; however, mononuclear infiltrate was readily apparent (Figure S6B). Additionally, we repeated these injures in our P7nTnG model and observed similar delays in terms of SC nGFP+ contribution to skeletal muscle regeneration (Figure S6C). Thus, extended recovery after prepubertal irradiation did not accelerate or improve the skeletal muscle regenerative response.

Prepubertal Irradiation Results in Lifelong Deficits in SC Number and Regenerative Capacity

To understand the long-term consequences of prepubertal irradiation, we aged mice to 14 months post-radiation (∼16 months of age) (Figure 6A). The aged irradiated cohort displayed graying of the hair only at the targeted region on the irradiated limb, evidence of the precision of the SARRP and successful treatment ∼14 months prior (Figure 6B). We first assessed raw muscle weight for lower limb irradiated muscles. RL TA, EDL, and SOL muscles all weighed significantly less than CL counterparts (Figures 6C–6E). We also assessed SC number in our aged cohort, finding TA RL muscles to have ∼68% less SCs than CL (Figure S7A). This reduction in SC number was not accompanied by any changes in fiber number in EDL and SOL muscles, or any change in tibial length (Figure S7B–S7D). We next examined the regenerative potential of muscles 14 months post-irradiation. TA and EDL muscles were injured with BaCl2 and harvested at 5 and 15 dpi (Figure 6F). Consistent with what we observed at the immediate and 3-week post-irradiation time points, 5-dpi RL muscles were nearly devoid of centrally nucleated regenerating myofibers (Figure 6G). Surprisingly, this deficit carried over to 15 dpi, as we still observed vast swaths of non-regenerated tissue (Figure 6G). The RL fibers that were actively regenerating at 15 dpi were significantly smaller than CL counterparts (Figure 6H). Thus, impairment of muscle regenerative capacity is a lifelong consequence of prepubertal irradiation.

Figure 6.

Figure 6

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Prepubertal Irradiation Results in Lifelong Deficits in Muscle Mass and Regenerative Capacity

(A) Scheme representing aged irradiation paradigm. Mice were irradiated at 4 weeks with the fractionated radiation regimen and aged to 16 months.

(B) Representative image of a mouse ∼14 months post-radiation. The field of radiation treatment can be observed based on the graying of hair in the treated limb. (C–E) Raw muscle weights for TA (C), EDL (D), and SOL (E) muscles from the aged radiation cohort. Unpaired t test.

(F) Scheme representing BaCl2 injury experiments performed on aged CL and RL. Animals were sacrificed at 5 or 15 dpi.

(G) Representative H&Es of 5- and 15-dpi CL and RL sections.

(H) Quantification of 15-dpi centrally nucleated fiber (CNF) cross-sectional area. Unpaired t test.

Prepubertal Irradiation Results in Lifelong Deficits in Myofiber CSA and Nuclear Number

As SCs are the principal source of muscle nuclei (myonuclei) required for skeletal muscle growth and regeneration, we next assessed myofiber CSA and myonuclear number of individual EDL and SOL myofibers (Bachman et al., 2018). In both aged RL EDL and SOL myofibers, we observed significant reductions in CSA compared with CL myofibers (Figures 7A–7E, S8B, and S8E). RL EDL myofibers were ∼78% the size of CL myofibers, and RL SOL myofibers were ∼72% the size of those from CL SOL. Assessing the number of myonuclei per millimeter of myofiber length (MN/mm), RL EDL, and SOL myofibers had less MN/mm when compared with CL counterparts (Figures 7A–7E, S8C, and S8F). An ∼ 20% loss of MN/mm was observed in both RL EDL and SOL myofibers when compared with their respective CL muscles. Last, we quantified myonuclear domain (MND) in our aged RL myofibers. MND is defined as the ratio of cytoplasm to myonuclear number, which is used as a readout of myonuclear function (Allen et al., 1999). Surprisingly, there was no difference in aged RL MND in EDL or SOL myofibers when compared with CL MND (Figures S8A and S8D). Thus, prepubertal irradiation resulted in lifelong deficits in myofiber CSA and myonuclear number, without affecting MND.

Figure 7.

Figure 7

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Prepubertal Irradiation and SC Depletion Result in Lifelong Deficits in Myofiber CSA and Myonuclear Number

(A) Scheme representing aged irradiation paradigm. Mice were irradiated at 4 weeks with the fractionated radiation regimen and aged to 16 months.

(B and C) Representative myofibers from CL and RL EDL (B) and SOL (C) muscles.

(D and E) Quantification of myofiber CSA and myonuclear number (MN/mm) for EDL (D) and SOL (E) muscles. Unpaired t test.

(F) Scheme representing P7DTA paradigm: tamoxifen was administered at 4 weeks and tissue harvested between 16 and 18 months of age.

(G and H) Representative myofibers from Cre- and Cre + EDL (G) and SOL (H) muscles.

(I and J) Quantification of myofiber CSA and myonuclear number (MN/mm) for EDL (I) and SOL (J) muscles. Unpaired t test.

Prepubertal SC Depletion Mimics Long-Term Radiation-Induced Deficits in Myofiber CSA and Myonuclear Number

Last, we wanted to ascertain if solely ablating SCs during prepuberty could mimic our radiation-induced long-term deficits in myofiber CSA and myonuclear number. For this purpose, we utilized a previously described mouse model of inducible SC depletion: Pax7CreERT2/+; Rosa26DTA/+ (P7DTA) (Bachman et al., 2018; Klose et al., 2018; Liu et al., 2015, 2017). Both Cre- (control) and Cre+ (experimental) littermates were injected with tamoxifen three times (every other day) beginning at 4 weeks and allowed to age to 16–18 months (Figure 7F). Utilizing this SC depletion model, Cre+ TA muscles had ∼65% less SCs when compared with Cre− littermates (Figure S7E). This was not accompanied by any changes in fiber number in EDL and SOL muscles (Figures S7F and S7G).

Subsequently, we assessed myofiber CSA and MN/mm in both EDL and SOL myofibers. Similar to aged irradiated myofibers, Cre+ myofibers had a significantly smaller CSA when compared with Cre− (Figures 7G–7J, S8H, and S8K). On average, EDL and SOL Cre+ myofibers were reduced in size to 83% and 81% of the CSA of Cre− myofibers, respectively. We next counted MN/mm and again found similar results to our aged radiation cohort. Cre+ EDL and SOL myofibers had significantly less MN/mm when compared with Cre-myofibers (Figures 7G–7J, S8I, and S8L). On average EDL and SOL Cre+ myofibers had 84% and 80% the MN/mm of Cre− myofibers respectively. Last, we assessed MND in our aged P7DTA cohort. Mimicking aged irradiated myofibers, there was no difference when comparing Cre+ and Cre− myofiber MND (Figures S8G and S8J). Taken together, these data demonstrate that insults that interfere with residual SC activity and contribution during prepubertal growth result in lifelong deficits in myofiber size proportional to a reduction in myonuclear number. Additionally, these data support our conclusion that radiation-induced prepubertal SC loss is the principle mechanism for lifelong deficits in myofiber size and myonuclear number.

Discussion

Skeletal muscle development and regeneration is dependent on SC myonuclear contributions (Relaix and Zammit, 2012). We have previously characterized residual SC activity during prepuberty, a period of dynamic juvenile growth after weaning up to adolescence (Bachman et al., 2018). Consistent with this, prepubertal SCs are susceptible to radiation injury. Through the examination of Pax7 expression, SC content, and SC fate analysis we identified a reduction in SC number and function both immediately and 3 weeks after prepubertal irradiation. Despite this loss, some SCs persisted and were able to activate and commit to the myogenic lineage. This raises the question as to how and why some prepubertal SCs are resistant to radiation exposure? The answer could lie within the intrinsic heterogeneity of the SC pool. Label-retaining experiments have been used in stem cell biology to identify subpopulations that divide less frequently, and thus, are less susceptible to stressors such as radiation (Fuchs, 2009). Subpopulations of label-retaining SCs have been identified that contribute less vigorously to postnatal myofiber growth (Chakkalakal et al., 2014; Schultz, 1996). Recently, Scaramozza et al. employed Mx1 reporter labeling strategies and an impressive array of mouse genetics to identify a subset of H2B-GFP reporter label-retaining SCs that could contribute to muscle regeneration following the stress of 9-Gy adult whole-body radiation (Scaramozza et al., 2019). However, whether our prepubertal fractionated radiation paradigm reveals similar levels of SC heterogeneity is unknown.

Cytotoxic insults to developing tissue can have prolonged, sometimes unforeseen, consequences into adulthood. Juvenile cancer survivors suffer from numerous physical limitations, including frailty, exercise intolerance, muscle atrophy, and muscle weakness (Armstrong et al., 2010; Ness et al., 2005, 2015; Paulino, 2004). To understand the long-term consequences of prepubertal irradiation, we aged mice to 14 months post-radiation (∼16 months of age). The regenerative capacity of this aged cohort was still delayed in injured RL muscles. Additionally, we observed a significant loss of myofiber CSA and myonuclei in RL EDL, and SOL muscles when compared with CL. Notably, we observed no difference in MND in either muscle. Thus, based on the similar percent decline in CSA and myonuclear number, we conclude that the lifelong reduction in myofiber CSA is due to the loss of SC-derived myonuclei during prepubertal growth. To formally test if the long-term deficits in prepubertal irradiated myofiber size and nuclear content were a consequence of SC loss, we utilized the P7DTA mouse model. These mice have been utilized to demonstrate the indispensable function of SCs in skeletal muscle late embryonic growth and muscle regeneration, and additional roles for SCs in skeletal muscle maturation, adaptation, and repair (Bachman et al., 2018; Fry et al., 2016; Hutcheson et al., 2009; Klose et al., 2018; Liu et al., 2015; Murach et al., 2017; Murphy et al., 2011). Following SC depletion during prepuberty (∼4 weeks of age), we aged P7DTA mice to 16–18 months. Remarkably, prepubertal SC depletion alone was able to mimic radiation-induced deficits in myofiber size and CSA, without affecting MND. These results emphasize that SC loss and consequent reduction of myonuclear contribution is the principal mechanism whereby prepubertal radiation leads to long-term deficits in skeletal muscle.

Skeletal muscle is an organ system composed of many different resident progenitors with unique roles (Judson et al., 2013). In this study we focused upon the SCs; however, it stands to reason that other populations of cells are likely affected by irradiation during prepuberty. Endothelial cells, in primary cardiovascular contexts, have been described as radiosensitive and prone to senescence under moderate to high radiation dose exposure (Venkatesulu et al., 2018; Wang et al., 2016). Within skeletal muscle, endothelial cells have been shown to partner with SCs to promote an angio-myogenic response (Christov et al., 2007). Fibro-adipogenic progenitors (FAPS) have been shown to play a key role in assisting with myogenic differentiation by providing a favorable environment for muscle regeneration (Biferali et al., 2019; Joe et al., 2010; Wosczyna et al., 2019). However, in states of chronic stress FAPS can be a source of infiltrating fibroblasts potentially resulting in aberrant extracellular matrix accumulation (Lemos et al., 2015; Madaro et al., 2018). Thus, it will be of interest to better understand how other resident skeletal muscle progenitor populations are affected by irradiation of prepubertal skeletal muscle.

To better understand radiation-induced mechanisms of skeletal muscle dysfunction, we can look to mediators of radiation damage in other organ systems. Due to its high rate of self-renewal, the intestinal epithelium has been heavily studied in the context of ionizing radiation (Chaves-Perez et al., 2019; Metcalfe et al., 2014). Recently, Chaves-Perez et al. demonstrated that overexpression of the molecular chaperone unconventional prefoldin RPB5 interactor (URI) was protective in the context of radiation-induced gastrointestinal syndrome (Chaves-Perez et al., 2019). By reducing URI expression, they found that label-retaining cells became more proliferative and thus more radiosensitive. This then leaves a potential therapeutic avenue where URI would be overexpressed before the radiation treatment, causing cells to become more dormant and less sensitive to ionizing radiation. It would be interesting to utilize a similar paradigm in our context of prepubertal skeletal muscle development. Utilizing a molecular target to make prepubertal SCs more “quiescent” before the radiation treatment could have favorable outcomes. However, the developmental role of these cells is to give rise to myonuclei required for the maturation of myofibers. Interfering with their ability to divide may lead to similar problems induced by irradiation, a sustained and lifelong loss of myonuclei. Thus, while considering therapeutic modalities and interventions it will be imperative to choose ones that mitigate radiation-induced damage but do not interfere with normal skeletal muscle developmental processes. In addition, care would be needed to ensure that similar protections are not imparted to target tumor cells.

Another potential therapeutic approach is to attempt to galvanize the remaining SCs following prepubertal irradiation to improve regenerative and homeostatic outcomes. Exercise, through the use of voluntary wheel running (VWR) has been shown to stimulate SC function and improve aged skeletal muscle integrity (Brett et al., 2020; Fujimaki et al., 2014; Kurosaka et al., 2009; Valdez et al., 2010). In juvenile rat models, VWR has been shown to increase SC number and enhance muscle growth into adulthood (Smith and Merry, 2012). In terms of radiotherapy, a recent study saw improvements in total SC number in adult mice exercised following 3-Gy whole-body radiation (D'Souza et al., 2019). Whether such benefits of exercise are observed after prepubertal irradiation is unknown, however should be tested since exercise is beneficial in reducing radiation and cancer-induced maladies (Segal et al., 2009; Sprod et al., 2010).

Ionizing radiation possesses the raw untamed power capable of inducing long-term organismal deficits. Our study highlights the radiosensitive nature of SCs during prepuberty, a critical time of organismal development (Bachman et al., 2018). We also highlight the importance of prepubertal SC-derived myonuclear contribution to the long-term maintenance of myofiber integrity. By better understanding the consequences of irradiation during developmental stages, this study could provide insight toward limiting lifelong normal tissue damage after pediatric radiation treatment.

Limitations of Study

In this study, we demonstrated the sensitivity of SCs to irradiation during prepubertal growth. The loss of prepubertal SC-derived myonuclear contribution after fractionated irradiation was associated with lifelong deficits in myofiber size with a corresponding reduction in nuclear number, phenotypes that were mimicked by prepubertal genetic ablation of SCs. Although we did not observe a change in MND ∼1 year after prepubertal radiation, a limitation of this study is that earlier examination could reveal myofiber adaptations consistent with dysregulation of hypertrophy and atrophy programs (Schiaffino et al., 2013). Another limitation of this study is the lack of DNA damage response regulator, apoptotic marker, and cell cycle inhibitor activity analysis in SCs and myofibers. As a fractionated irradiation strategy was employed, it will be important to characterize the kinetics of expression, activity, and persistence of these effectors and readouts of DNA damage, cell loss, and senescence. Finally, our study is limited by the absence of a relevant pediatric tumor model. Additionally, patients with pediatric cancer typically receive a chemotherapeutic as well, which this study lacks. Thus, whether the SC and myofiber deficits observed here are exacerbated in a model of pediatric tumor treatment with radiation and/or chemotherapeutic is unknown.

Resource Availability

Lead Contact

Further information and requests for resources and reagents should be directed to the Lead Contact, Joe V. Chakkalakal (joe_chakkalakal@urmc.rochester.edu).

Materials Availability

This study did not generate new unique reagents.

Data and Code Availability

All RNA-seq data in this study are available at National Center for Biotechnology Information's Gene Expression Omnibus database with the accession number GSE160064.

Methods

All methods can be found in the accompanying Transparent Methods supplemental file.

Acknowledgments

This work was supported by URMC Wilmot Cancer Institute pilot funding and NIH grants R01AG051456 and R01CA220467 to J.V.C and F31 training grant F31AR076175 to J.F.B. The SARRP was acquired through NIH grant 1S10OD021548-01 (J.P.W.).

Author Contributions

J.F.B., J.P.W., and J.V.C. designed the experiments. J.F.B. and J.V.C. wrote the manuscript, with contributing comments from J.P.W. J.F.B. performed the presented experiments. J.G.K. assisted in RNA-seq interpretation and analysis. R.S.B. assisted with cell culture experiments. J.F.B., C.J.J., E.H., and N.D.P., carried out mouse radiotherapy, and J.F.B. and N.D.P. harvested tissues.

Declaration of Interests

The authors declare no competing interests.

Published: November 20, 2020

Footnotes

Supplemental Information can be found online at https://doi.org/10.1016/j.isci.2020.101760.

Supplemental Information

Document S1. Transparent Methods and Figures S1–S8
mmc1.pdf (22.1MB, pdf)
Table S1. Radiation-Induced Gene Expression Alterations
mmc2.xlsx (181.8KB, xlsx)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Transparent Methods and Figures S1–S8
mmc1.pdf (22.1MB, pdf)
Table S1. Radiation-Induced Gene Expression Alterations
mmc2.xlsx (181.8KB, xlsx)

Data Availability Statement

All RNA-seq data in this study are available at National Center for Biotechnology Information's Gene Expression Omnibus database with the accession number GSE160064.

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