Repression of phosphatidylinositol transfer protein α ameliorates the pathology of Duchenne muscular dystrophy

Abstract

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease caused by X-linked inherited mutations in the DYSTROPHIN (DMD) gene. Absence of dystrophin protein from the sarcolemma causes severe muscle degeneration, fibrosis, and inflammation, ultimately leading to cardiorespiratory failure and premature death. Although there are several promising strategies under investigation to restore dystrophin protein expression, there is currently no cure for DMD, and identification of genetic modifiers as potential targets represents an alternative therapeutic strategy. In a Brazilian golden retriever muscular dystrophy (GRMD) dog colony, two related dogs demonstrated strikingly mild dystrophic phenotypes compared with those typically observed in severely affected GRMD dogs despite lacking dystrophin. Microarray analysis of these "escaper" dogs revealed reduced expression of phosphatidylinositol transfer protein-α (PITPNA) in escaper versus severely affected GRMD dogs. Based on these findings, we decided to pursue investigation of modulation of PITPNA expression on dystrophic pathology in GRMD dogs, dystrophin-deficient sapje zebrafish, and human DMD myogenic cells. In GRMD dogs, decreased expression of Pitpna was associated with increased phosphorylated Akt (pAkt) expression and decreased PTEN levels. PITPNA knockdown by injection of morpholino oligonucleotides in sapje zebrafish also increased pAkt, rescued the abnormal muscle phenotype, and improved long-term sapje mutant survival. In DMD myotubes, PITPNA knockdown by lentiviral shRNA increased pAkt and increased myoblast fusion index. Overall, our findings suggest PIPTNA as a disease modifier that accords benefits to the abnormal signaling, morphology, and function of dystrophic skeletal muscle, and may be a target for DMD and related neuromuscular diseases.

Keywords: Duchenne muscular dystrophy; genetic modifier; phosphatidylinositol transfer protein-α; skeletal muscle.

Conflict of interest statement

Conflict of interest statement: L.M.K. is a consultant for Pfizer, Inc., Summit Corporation PLC, and Sarepta Therapeutics for muscle disease drug therapies.

Figures

Fig. 1.

Mildly affected escaper GRMD dogs exhibit decreased Pitpna expression and increased pAKT. (A) Venn diagram showing the number of genes differentially expressed in normal, escaper, and severely affected GRMD dogs. Pitpna was identified as the one gene differentially expressed in the mildly affected escaper dogs versus the severely affected dogs in the microarray. FDR was 5%. (B) Quantification of Pitpna mRNA expression in normal, escaper, and severely affected GRMD dogs. Data are represented as means ± SDM. *P < 0.005; **P < 0.05 by Student’s t test. (C) Western blot of protein isolated from normal, escaper, and severely affected GRMD dogs showing down-regulation of PITPNA and PTEN and up-regulation of pAkt in escaper dogs. n = 3, normal; n = 2, escaper; and n = 4, severely affected.

Fig. 2.

Morpholino-mediated pitpnaa knockdown in zebrafish elicits dose-dependent changes in morphology and signaling. (A) Phase contrast images of sapje larvae injected with pitpnaa MO or control MO at 1 and 4 dpf. Embryos injected with 3 ng of pitpnaa MO at the one-cell stage showed abnormal development and were not viable, whereas 1 ng of pitpnaa MO did not negatively impact development. (B) Western blot showing decreased Pitpna expression with increasing doses of pitpnaa MO injected into sapje embryos. Protein lysates were harvested from sapje larvae at 4 dpf. Experiment performed in triplicate, n = 300 per replicate.

Fig. 3.

Pitpnaa knockdown rescues sapje zebrafish muscle phenotype, improves swim velocity, and increases long-term survival. (A) Birefringence assay (Upper) and immunofluorescent staining for myosin heavy chain (Lower) showing the typical abnormal muscle phenotype of affected homozygous-null sapje fish at 4 dpf. Injection of pitpnaa MO at the one-cell stage prevented manifestation of the muscle phenotype in some homozygous-null fish, termed escaper fish, which showed healthy muscle morphology comparable to normal fish. [Scale bars: 1 mm (Upper), 100 μm (Lower).] (B) Percent of affected sapje fish as determined by birefringence assay at 4 dpf. Mating of heterozygous sapje fish normally yields ∼25% of affected embryos. Cohorts injected with 1 ng of pitpnaa MO at the one-cell stage showed significantly lower percentages of affected fish compared with control MO. Ten replicate experiments were performed, n = 200–300 per replicate. Bars represent means ± SDM. *P < 0.0001 by Student’s t test. (C) Swim velocity of sapje fish tracked on the DanioVision system. Injection with 1 ng of pitpnaa MO increased swim velocity of affected fish during a 15-min tracking period performed at 4 dpf. Bars represent means ± SEM; *P < 0.05 by two-way ANOVA and Bonferroni post hoc test. (D) Long-term survival assay showing increased survival of affected fish injected with 1 ng of pitpnaa MO at the one-cell stage. Affected fish were identified by birefringence assay at 4 dpf and followed until 30 dpf. Data represent means ± SDM. *P < 0.05, **P < 0.002 versus noninjected by Student’s t test.

Fig. S1.

Genotyping of sapje zebrafish injected with pitpnaa morpholino oligonucleotides confirms homozygous-null escaper sapje population. The genotypes of noninjected, control MO-injected, and pitpnaa MO-injected fish were determined, and it was confirmed that a subset of phenotypically normal fish were in fact homozygous null fish. Injection of either control MO or pitpnaa MO did not increase fish death or expected genotype ratios.

Fig. S2.

Pitpnaa knockdown by morpholino injection improves swim distance and time of homozygous-null sapje zebrafish. (A) Swim distance and (B) swim time of sapje fish were tracked on the DanioVision system. Injection with 1 ng of pitpnaa MO increased swim velocity of affected fish during a 15-min tracking period performed at 4 dpf. Bars represent means ± SEM; *P < 0.05 by two-way ANOVA and Bonferroni post hoc test.

Fig. 4.

PITPNA knockdown by lentiviral shRNA in DMD human myotubes increases pAkt and fusion index. Normal and DMD patient-derived myoblasts were transduced with doxycycline-inducible lentivirus containing shRNA targeting PITPNA mRNA. Myoblasts were induced to differentiate into myotubes in differentiation medium with (+) and without (−) 0.25 μg/mL Dox for 5 d. (A) Western blot showing decreased PITPNA and PTEN expression and increased active pAkt (Ser473) expression with Dox treatment in transduced DMD cells. tRFP expression confirmed expression of the lentiviral construct. (B–D) Western blot quantification showing decreased PITPNA and PTEN, and increased pAKT in DMD myotubes expressing the lentiviral PITPNA shRNA. (E) Quantification of fusion index showing significant increase in DMD cells (+) Dox expressing the PITPNA shRNA compared with (−) Dox. Nuclei were counted from 10 random fields per sample, and fusion index was calculated as the percentage of nuclei within fused myotubes per total nuclei. Representative phase contrast images are shown from which fusion index was calculated. (F) Representative phase contrast images of myotubes taken at 20× magnification. Bars represent means ± SEM; *P < 0.05 by two-way ANOVA and Bonferroni post hoc test, n = 3 normal, three DMD biopsy cell lines (Scale bar: 20 μm.)