Membrane-stabilizing copolymers confer marked protection to dystrophic skeletal muscle in vivo

Evelyne M Houang, Karen J Haman, Antonio Filareto, Rita C Perlingeiro, Frank S Bates, Dawn A Lowe, Joseph M Metzger, Evelyne M Houang, Karen J Haman, Antonio Filareto, Rita C Perlingeiro, Frank S Bates, Dawn A Lowe, Joseph M Metzger

Abstract

Duchenne muscular dystrophy (DMD) is a fatal disease of striated muscle deterioration. A unique therapeutic approach for DMD is the use of synthetic membrane stabilizers to protect the fragile dystrophic sarcolemma against contraction-induced mechanical stress. Block copolymer-based membrane stabilizer poloxamer 188 (P188) has been shown to protect the dystrophic myocardium. In comparison, the ability of synthetic membrane stabilizers to protect fragile DMD skeletal muscles has been less clear. Because cardiac and skeletal muscles have distinct structural and functional features, including differences in the mechanism of activation, variance in sarcolemma phospholipid composition, and differences in the magnitude and types of forces generated, we speculated that optimized membrane stabilization could be inherently different. Our objective here is to use principles of pharmacodynamics to evaluate membrane stabilization therapy for DMD skeletal muscles. Results show a dramatic differential effect of membrane stabilization by optimization of pharmacodynamic-guided route of poloxamer delivery. Data show that subcutaneous P188 delivery, but not intravascular or intraperitoneal routes, conferred significant protection to dystrophic limb skeletal muscles undergoing mechanical stress in vivo. In addition, structure-function examination of synthetic membrane stabilizers further underscores the importance of copolymer composition, molecular weight, and dosage in optimization of poloxamer pharmacodynamics in vivo.

Conflict of interest statement

J.M.M. is on the scientific advisory board of and holds shares in Phrixus Pharmaceuticals Inc., a company developing novel therapeutics for heart failure.

Figures

Figure 1
Figure 1
In vitro hypo-osmotic stress assay to screen block copolymers for membrane stabilization. (a) Schematic representation of the tested triblock copolymers and the exclusively polyethylene oxide (PEO)-based polymer PEG8000, showing relative polypropylene oxide (PPO) (red) and PEO composition (blue). (b) Satellite cell-derived myotubes from normal muscle were exposed to hypo-osmotic stress media and lactate dehydrogenase (LDH) enzyme release into the media was measured as a marker of membrane leak. The ability of P188 and other copolymers to decrease LDH leakage from stressed myotubes was assessed as a parameter of membrane stabilization. Data are presented as % LDH release normalized to total LDH release (*P < 0.05, via one-way analysis of variance compared to nontreated group, #P < 0.05 indicates 150 µmol/l P188 is significantly different from 1 µmol/l P188, &P < 0.05 indicates 1 µmol/l ext-P188 is significantly different from 0.1 µmol/l ext-P188). Mean values are derived from at least three independent experiments with three to four wells of densely cultured myotubes per experiment. Error bars shown as mean ± standard error of the mean.
Figure 2
Figure 2
Membrane stabilizers P188 and ext-P188 decrease hypo-osmotic stress-induced release of LDH in dystrophic myofibers and myotubes in vitro. (a) Isolated mdx flexor digitorum brevis (FDB) myofibers and (b) double knockout (mdx/utr–/–) myotubes were subjected to hypo-osmotic stress and lactate dehydrogenase (LDH) release was assessed in the absence (nontreated) or presence of increasing concentrations of P188 and ext-P188. Data presented as % LDH release normalized to total LDH release from nontreated. *P < 0.05 via one-way analysis of variance compared to nontreated group. Mean values are derived from at least three independent experiments with three to four wells of 20–30 isolated mdx FDB myofibers or densely cultured mdx/utr–/– myotubes per experiment. Error bars shown as mean ± standard error of the mean.
Figure 3
Figure 3
Intraperitoneal and intravenous delivery of P188 have no effect to protect against lengthening contraction-induced force loss in mdx mice in vivo. Force loss by the anterior crural muscles in adult mdx mice (n = 5–8 per group) treated with 460 mg/kg P188 or saline vehicle either (a) intraperitoneally (IP) or (b) intravenously (IV), at least 30 minutes before the injury protocol was assessed over the course of 50 lengthening contractions. Force loss is presented as a fraction of the initial maximal force ± standard error of the mean (SEM). Contractions #1–15 show the marked force deficit in mdx mice. Results from BL/10 control mice injected saline via each delivery route are also shown. Inset (a,b) shows all 50 contractions. Both IP and IV delivery of P188 had no significant effect to decrease force loss over the course of the protocol. Error bars shown as mean ± SEM (in some cases symbol size was larger than error bar).
Figure 4
Figure 4
Subcutaneous delivery of P188 markedly protects against lengthening contraction-induced force loss in mdx mice in vivo. (a) Representative individual force tracings during lengthening contractions #1 versus #10 for subQ saline and subQ P188 treated mdx mice. (b) SubQ P188 delivery confers significant protection against force loss from contractions #5–50 (*P < 0.05 via two-way analysis of variance compared to the mdx saline group. Contractions #1–15 are highlighted with the entire protocol shown in inset. Error bars shown as mean ± SEM (in some cases symbol size was larger than error bar).
Figure 5
Figure 5
P188 significantly decreases baseline and lengthening contraction-induced sarcolemmal instability. (a) Example images of Evans Blue dye uptake into TA myofibers 2 hours postinjury in mdx mice treated subQ with saline or P188. (b) Quantification of total Evans Blue Dye extracted from whole TA muscles. Evans Blue dye was extracted by incubating the whole TA muscle in 1 ml of formamide. “C” denotes the contralateral noninjured TA, “LC” denotes lengthening contraction. Data is shown as absorbance at 620 nm divided by mg of tissue. **P < 0.01, ***P < 0.001 via one-way analysis of variance. Error bars shown as mean ± standard error of the mean.
Figure 6
Figure 6
In vivo isometric force measurements in P188-treated mdx mice immediately postinjury. Intraperitoneal (a) and intravenous (b) P188 delivery had no significant effect on peak isometric force immediately after injury whereas subQ P188 treatment (c) significantly enhanced immediate postinjury isometric force recovery compared to mdx saline, and was not significantly different from BL/10 saline group. *P < 0.05 one-way analysis of variance compared to mdx saline, #P < 0.05 one-way analysis of variance compared to BL/10 saline. Error bars shown as mean ± standard error of the mean.
Figure 7
Figure 7
Dose and delivery route dependence of ext-P188 in protecting mdx mice skeletal muscle from lengthening contraction-induced force loss in vivo. Mdx mice were administered either low dose (60 mg/kg) (gray symbols) or high dose (160 mg/kg) (black symbols) ext-P188 via intraperitoneal (a) (IP), (b) intravenous (IV)), or (c) subcutaneous (subQ) injection at least 30 minutes prior to the protocol and the anterior crural muscle group tested for lengthening contraction-induced force loss over the course of 50 contractions. Force loss is presented as a fraction of the initial maximal force ± standard error of the mean (SEM). Contractions #1–15 are graphically emphasized. (a) High-dose ext-P188 (160 mg/kg) had no protective effect against lengthening contraction-induced force loss. However, low dose ext-P188 (60 mg/kg) significantly improved resistance to injury from contraction #8 through #50 (*P < 0.05 via two-way analysis of variance compared to mdx saline). (b,c) Neither low-dose nor high-dose ext-P188 administered IV or subQ had any significant effects on force deficit. (d) Low dosage 60 mg/kg ext-P188 was injected into the tibialis anterior (TA) muscle of mdx mice intramuscularly (IM) directly prior to the protocol. Ext-P188-treated mice had a highly significant and sustained resistance to lengthening contraction injury loss (contractions 6–50, *P < 0.05 two-way analysis of variance compared to mdx saline). Contractions #1–15 are graphically emphasized, entire protocol shown in inset. Error bars shown as mean ± SEM (in some cases symbol size was larger than error bar).
Figure 8
Figure 8
Isometric force measurements in ext-P188-treated mdx immediately postinjury. (a) Low-dose (60 mg/kg) ext-P188 delivered IP significantly prevented postlengthening protocol isometric force loss (*P < 0.05 one-way analysis of variance compared to mdx saline) although was significantly different from the BL/10 saline group loss (#P < 0.05 one-way analysis of variance compared to BL/10 saline). (b,c). No significant effect obtained for IV or subQ ext-P188-treated mice. (d) Postinjury isometric force of mdx mice treated with direct injection of 60 mg/kg ext-P188 into the TA muscle was not significantly different from BL/10 saline (#P = 0.74, one-way analysis of variance compared to BL/10 saline). Error bars shown as mean ± standard error of the mean.

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