High mobility group box 1 (HMGB1) is a potential disease biomarker in cell and mouse models of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder affecting 1:3500 male births and is associated with myofiber degeneration, regeneration, and inflammation. Glucocorticoid treatments have been the standard of care due to immunomodulatory/immunosuppressive properties but novel genetic approaches, including exon skipping and gene replacement therapy, are currently being developed. The identification of additional biomarkers to assess DMD-related inflammatory responses and the potential efficacy of these therapeutic approaches are thus of critical importance. The current study uses RNA sequencing of skeletal muscle from two mdx mouse models to identify high mobility group box 1 (HMGB1) as a candidate biomarker potentially contributing to DMD-related inflammation. HMGB1 protein content was increased in a human iPSC-derived skeletal myocyte model of DMD and microdystrophin treatment decreased HMGB1 back to control levels. In vivo, HMGB1 protein levels were increased in vehicle treated B10-mdx skeletal muscle compared to B10-WT and significantly decreased in B10-mdx animals treated with adeno-associated virus (AAV)-microdystrophin. However, HMGB1 protein levels were not increased in D2-mdx skeletal muscle compared to D2-WT, demonstrating a strain-specific difference in DMD-related immunopathology.

Aberrations in Energetic Metabolism and Stress-Related Pathways Contribute to Pathophysiology in the Neb Conditional Knockout Mouse Model of Nemaline Myopathy.

Nemaline myopathy (NM) is a genetically and clinically heterogeneous disease that is diagnosed on the basis of the presence of nemaline rods on skeletal muscle biopsy. Although NM has typically been classified by causative genes, disease severity or prognosis cannot be predicted. The common pathologic end point of nemaline rods (despite diverse genetic causes) and an unexplained range of muscle weakness suggest that shared secondary processes contribute to the pathogenesis of NM. We speculated that these processes could be identified through a proteome-wide interrogation using a mouse model of severe NM in combination with pathway validation and structural/functional analyses. A proteomic analysis was performed using skeletal muscle tissue from the Neb conditional knockout mouse model compared with its wild-type counterpart to identify pathophysiologically relevant biological processes that might impact disease severity or provide new treatment targets. A differential expression analysis and Ingenuity Pathway Core Analysis predicted perturbations in several cellular processes, including mitochondrial dysfunction and changes in energetic metabolism and stress-related pathways. Subsequent structural and functional studies demonstrated abnormal mitochondrial distribution, decreased mitochondrial respiratory function, an increase in mitochondrial transmembrane potential, and extremely low ATP content in Neb conditional knockout muscles relative to wild type. Overall, the findings of these studies support a role for severe mitochondrial dysfunction as a novel contributor to muscle weakness in NM.

ACTA1 H40Y mutant iPSC-derived skeletal myocytes display mitochondrial defects in an in vitro model of nemaline myopathy.

Nemaline myopathies (NM) are a group of congenital myopathies that lead to muscle weakness and dysfunction. While 13 genes have been identified to cause NM, over 50% of these genetic defects are due to mutations in nebulin (NEB) and skeletal muscle actin (ACTA1), which are genes required for normal assembly and function of the thin filament. NM can be distinguished on muscle biopsies due to the presence of nemaline rods, which are thought to be aggregates of the dysfunctional protein. Mutations in ACTA1 have been associated with more severe clinical disease and muscle weakness. However, the cellular pathogenesis linking ACTA1 gene mutations to muscle weakness are unclear To evaluate cellular disease phenotypes, iPSC-derived skeletal myocytes (iSkM) harboring an ACTA1 H40Y point mutation were used to model NM in skeletal muscle. These were generated by Crispr-Cas9, and include one non-affected healthy control (C) and 2 NM iPSC clone lines, therefore representing isogenic controls. Fully differentiated iSkM were characterized to confirm myogenic status and subject to assays to evaluate nemaline rod formation, mitochondrial membrane potential, mitochondrial permeability transition pore (mPTP) formation, superoxide production, ATP/ADP/phosphate levels and lactate dehydrogenase release. C- and NM-iSkM demonstrated myogenic commitment as evidenced by mRNA expression of Pax3, Pax7, MyoD, Myf5 and Myogenin; and protein expression of Pax4, Pax7, MyoD and MF20. No nemaline rods were observed with immunofluorescent staining of NM-iSkM for ACTA1 or ACTN2, and these mRNA transcript and protein levels were comparable to C-iSkM. Mitochondrial function was altered in NM, as evidenced by decreased cellular ATP levels and altered mitochondrial membrane potential. Oxidative stress induction revealed the mitochondrial phenotype, as evidenced by collapsed mitochondrial membrane potential, early formation of the mPTP and increased superoxide production. Early mPTP formation was rescued with the addition of ATP to media. Together, these findings suggest that mitochondrial dysfunction and oxidative stress are disease phenotypes in the in vitro model of ACTA1 nemaline myopathy, and that modulation of ATP levels was sufficient to protect NM-iSkM mitochondria from stress-induced injury. Importantly, the nemaline rod phenotype was absent in our in vitro model of NM. We conclude that this in vitro model has the potential to recapitulate human NM disease phenotypes, and warrants further study.

Drug-impregnated, pressurized gas expanded liquid-processed alginate hydrogel scaffolds for accelerated burn wound healing.

While the benefits of both hydrogels and drug delivery to enhance wound healing have been demonstrated, the highly hydrophilic nature of hydrogels creates challenges with respect to the effective loading and delivery of hydrophobic drugs beneficial to wound healing. Herein, we utilize pressurized gas expanded liquid (PGX) technology to produce very high surface area (~200 m2/g) alginate scaffolds and describe a method for loading the scaffolds with ibuprofen (via adsorptive precipitation) and crosslinking them (via calcium chelation) to create a hydrogel suitable for wound treatment and hydrophobic drug delivery. The high surface area of the PGX-processed alginate scaffold facilitates >8 wt% loading of ibuprofen into the scaffold and controlled in vitro ibuprofen release over 12-24 h. In vivo burn wound healing assays demonstrate significantly accelerated healing with ibuprofen-loaded PGX-alginate/calcium scaffolds relative to both hydrogel-only and untreated controls, demonstrating the combined benefits of ibuprofen delivery to suppress inflammation as well as the capacity of the PGX-alginate/calcium hydrogel to maintain wound hydration and facilitate continuous calcium release to the wound. The use of PGX technology to produce highly porous scaffolds with increased surface areas, followed by adsorptive precipitation of a hydrophobic drug onto the scaffolds, offers a highly scalable method of creating medicated wound dressings with high drug loadings. STATEMENT OF SIGNIFICANCE: While medicated hydrogel-based wound dressings offer clear advantages in accelerating wound healing, the inherent incompatibility between conventional hydrogels and many poorly water-soluble drugs of relevance in wound healing remains a challenge. Herein, we leveraged supercritical fluids-based strategies to both process and subsequently impregnate alginate, followed by post-crosslinking to form a hydrogel, to create a very high surface area alginate hydrogel scaffold loaded with high hydrophobic drug contents (here, >8 wt% ibuprofen) without the need for any pore-forming additives. The impregnated scaffolds significantly accelerated burn wound healing while also promoting regeneration of the native skin morphology. We anticipate this approach can be leveraged to load clinically-relevant and highly bioavailable dosages of hydrophobic drugs in hydrogels for a broad range of potential applications.