Increased Stiffness in Aged Skeletal Muscle Impairs Muscle Progenitor Cell Proliferative Activity

<div><p>Background</p><p>Skeletal muscle aging is associated with a decreased regenerative potential due to the loss of function of endogenous stem cells or myogenic progenitor cells (MPCs). Aged skeletal muscle is characterized by the deposition of extracellular matrix (ECM), which in turn influences the biomechanical properties of myofibers by increasing their stiffness. Since the stiffness of the MPC microenvironment directly impacts MPC function, we hypothesized that the increase in muscle stiffness that occurs with aging impairs the behavior of MPCs, ultimately leading to a decrease in regenerative potential.</p><p>Results</p><p>We showed that freshly isolated individual myofibers from aged mouse muscles contain fewer MPCs overall than myofibers from adult muscles, with fewer quiescent MPCs and more proliferative and differentiating MPCs. We observed alterations in cultured MPC behavior in aged animals, where the proliferation and differentiation of MPCs were lower and higher, respectively. These alterations were not linked to the intrinsic properties of aged myofibers, as shown by the similar values for the cumulative population-doubling values and fusion indexes. However, atomic force microscopy (AFM) indentation experiments revealed a nearly 4-fold increase in the stiffness of the MPC microenvironment. We further showed that the increase in stiffness is associated with alterations to muscle ECM, including the accumulation of collagen, which was correlated with higher hydroxyproline and advanced glycation end-product content. Lastly, we recapitulated the impaired MPC behavior observed in aging using a hydrogel substrate that mimics the stiffness of myofibers.</p><p>Conclusions</p><p>These findings provide novel evidence that the low regenerative potential of aged skeletal muscle is independent of intrinsic MPC properties but is related to the increase in the stiffness of the MPC microenvironment.</p></div

MPCs in aged myofibers display decreased myogenic activity.

<p><b>(A</b>) Histograms showing the total number of MPCs per myofiber on 5–109 freshly isolated and cultured (6 days) myofibers from adult and aged mice (<i>n</i> = 5 mice per group). <b>(B, C)</b> Histograms showing the percentage of quiescent SCs (Pax7⁺MyoD⁻) and proliferating (Pax7⁺MyoD⁺) and differentiating MPCs (Pax7⁻MyoD⁺) per myofiber on intact <b>(B)</b> and damaged <b>(C)</b> myofibers. The Pax7 and MyoD proteins were immunostained as shown in the photomicrograph on the right. The count includes all parts and depths of the myofibers. The representative image on the right shows that Pax7 and MyoD proteins are expressed at lower levels in aged myofibers. **<i>p</i><0.01 and ***<i>p</i><0.0001 versus adult. ^†††<i>p</i><0.001 versus intact counterparts. All data are expressed as means ± SEM.</p

Damaged myofibers from aged mice display robust stiffness that impairs MPC activity.

<p>Graphs showing Young’s modulus values (kPa) and stiffness measurements of freshly isolated (<b>A</b>) and cultured intact and damaged myofibers (<b>B</b>) from adult (<i>n</i> = 3) and aged (<i>n</i> = 5) muscles. One measurement (three force-indentation curves collected for each measurement) per myofiber was performed on 6–50 myofibers. **<i>p</i><0.01; ***<i>p</i><0.0001 versus adult counterpart. ^†††<i>p</i><0.001 versus intact counterpart. All data are expressed as means ± SEM.</p

Hydrogels mimicking myofiber stiffness recapitulate MPC behavior.

<p>Histograms showing the frequency of quiescent, proliferative, and differentiating MPCs (Pax7⁺MyoD^-, Pax7⁺MyoD⁺ and Pax7⁻MyoD⁺, respectively), as a function of hydrogel stiffness (0.5, 2.0, and 18 kPa). Primary myoblasts from adult (<i>n</i> = 5) mice were used for this experiment. Cells were plated and grown for 48 h. The cells were then fixed and stained with DAPI and immunostained with Pax7 and MyoD antibodies. We counted the number of cells in five random fields in four plates for each stiffness preparation. All data are expressed as means ± SEM (*<i>p</i><0.05; **<i>p</i><0.001).</p

Aging is associated with increased skeletal muscle stiffness.

<p><b>(A)</b> Representative images of Masson trichrome-stained TA sections from adult and aged male C57Bl/6 mice. <b>(B)</b> Graph showing the quantification of whole muscle stiffness from 100-μm-thick slices of TA and Quad muscles from adult (<i>n</i> = 4) and aged (<i>n</i> = 7) mice. Ten measurements per slice with three force-indentation curves collected per measurement were performed on 3–5 sections for each mouse. (<b>C</b>) Graph showing the biochemical quantification of total hydroxyproline content of Quad muscles from adult (<i>n</i> = 5) and aged (<i>n</i> = 5) mice. <b>(D)</b> Graph showing hydroxyproline values converted to collagen content. (<b>E</b>) Measurement of total advanced glycation end-product (AGE) content of Quad muscles from adult (<i>n</i> = 5) and aged (<i>n</i> = 5) mice. *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.0001 versus adult group. All data are expressed as means ± SEM.</p

Diabetic GK/Par β-cells are resistant to oxidative stress <i>in vitro</i>.

<p>Wistar or GK/Par islets were perifused with medium containing (in mmol/l) 2.8 glucose (G2.8), 16.7 glucose+1 acetylcholine (G16.7+ACh1), G2.8, or 50 KCl (KCl50). Vehicle or oxidative agent (H₂O₂ 50 µmol/l, or alloxan 1 mmol/l, or streptozotocin (STZ) 1 mmol/l) was added as indicated (<i>A,B</i>). ΔIns values (insulin secretion AUC) derived from <i>panels</i> (<i>A</i>) and (<i>B</i>) indicated that GK/Par insulin secretion at G16.7 and KCl50 was strongly resistant to the toxic effects of all oxidative agents used, unlike Wistar. In another set of experiments insulin secretion at G16.7+ACh1 was measured (static incubation, 30 min) in the presence of the GSH-oxidizing agent <i>tert</i>-butylhydroperoxide (<i>t</i>-BH) at different concentration as indicated (<i>C</i>). Data are the means±SEM of 3–6 experiments in each group. *<i>p</i><0.05 <i>vs.</i> age-matched Wistar group; ⁺<i>p</i><0.05 <i>vs.</i> vehicle in the same group.</p

Unlike Wistar β-cells, diabetic neonatally streptozotocin-treated (n-STZ) are resistant to oxidative stress <i>in vitro</i>, as GK/Par β-cells.

<p>The perifusion experiments are the same that those described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006500#pone-0006500-g002" target="_blank">Fig. 2</a>. Data are the means±SEM of 4–6 experiments in each group.</p

Antioxidant defense status tended to be lower in prediabetic GK/Par rat islets and was associated with enhanced reactive oxygen (ROS) production.

<p>Protocols (see the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006500#pone-0006500-g005" target="_blank">Fig. 5</a>) were repeated with 7-day-old (D7) Wistar or D7 GK/Par rats. The mRNA levels for genes encoding antioxidant proteins (<i>A</i>). The mRNA levels for genes encoding proteins instrumental in the production of the antioxidant cofactor NADPH (<i>B</i>). Intra-insular reduced glutathione (Eq GSH) content tended to be decreased in D7 GK/Par rats (<i>C</i>). ROS generation was assessed as fluorescence intensity (AU) normalized by total islet proteins in D7 Wistar or D7 GK/Par islets incubated for 30 min at 2.8 (G2.8) or 16.7 mmol/l glucose (G16.7), in the presence or the absence of blockers of the electron transfer chain complexes I (rotenone, 10 µmol/l) or III (antimycin A, 20 µmol/l), or the H₂O₂ scavenger (trolox, 1 mmol/l) (<i>D</i>). Data are the means±SEM of 10–28 experiments (<i>n</i> = 15–51 animals) in each group. *<i>p</i><0.05 <i>vs.</i> age-matched Wistar group; ⁺<i>p</i><0.05 <i>vs.</i> G2.8 or G16.7 in the same group; ^†<i>p</i><0.05 <i>vs.</i> G16.7 in the same experimental condition.</p

Antioxidant defense status was higher in diabetic GK/Par islets.

<p>The mRNA levels for genes encoding antioxidant proteins (<i>A</i>). The mRNA levels for genes encoding proteins instrumental in the production of the antioxidant cofactor NADPH (<i>B</i>). Intra-insular reduced glutathione GSH (Eq GSH) content was higher in GK/Par than Wistar rats (<i>C</i>). Data are the means±SEM of 8–16 experiments in each group. *<i>p</i><0.05 <i>vs.</i> age-matched Wistar group.</p

In 2.5-month-old diabetic GK/Par pancreases, nitrotyrosine, 4-hydroxy-2-nonenal (HNE)-modified proteins and 8-hydroxy-2′-deoxyguanosine (8-OHdG) accumulate in the peri-islet vascular and inflammatory compartments.

<p>Immunolabelling of nitrotyrosine, HNE-adducts or 8-OHdG (arrows) in pancreatic tissues of GK/Par and Wistar rats (<i>A</i>). An islet is encircled in each quadrant. Nitrotyrosine-positive (arrows) material was closely associated with macrophages (CD68) and granulocytes (MCA967) at the islet periphery (<i>B</i>), and in the vicinity of pancreatic ducts (<i>C</i>). Original magnification×250.</p