Biomechanical characterization of a desminopathy in primary human myoblasts

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Abstract

Heterozygous mutations of the human desmin gene on chromosome 2q35 cause hereditary and sporadic myopathies and cardiomyopathies. The expression of mutant desmin brings about partial disruption of the extra sarcomeric desmin cytoskeleton and abnormal protein aggregation in the sarcoplasm of striated muscle cells. The precise molecular pathways and sequential steps that lead from a desmin gene defect to progressive muscle damage are still unclear. We tested whether mutant desmin changes the biomechanical properties and the intrinsic mechanical stress response of primary cultured myoblasts derived from a patient carrying a heterozygous R350P desmin mutation. Compared to wildtype controls, undifferentiated mutant desmin myoblasts revealed increased cell death and substrate detachment in response to cyclic stretch on flexible membranes. Moreover, magnetic tweezer microrheometry of myoblasts using fibronectin-coated beads showed increased stiffness of diseased cells. Our findings provide the first evidence that altered mechanical properties may contribute to the progressive striated muscle pathology in desminopathies. We postulate that the expression of mutant desmin leads to increased mechanical stiffness, which results in excessive mechanical stress in response to strain and consecutively to increased mechanical vulnerability and damage of muscle cells.

Highlights

► Mutations of the human desmin gene cause hereditary and sporadic myopathies and cardiomyopathies. ► Desminopathic myoblasts show higher stiffness compared to control cells. ► Higher cell stiffness leads mechanical stress contributes to higher vulnerability to excessive mechanical strain which may contribute to the progressive disease process.

Introduction

Mutations of the human desmin gene cause myopathies affecting skeletal and cardiac muscle. Desminopathies classically exhibit an autosomal dominant inheritance, but rare autosomal recessive cases and an increasing number of sporadic forms have been reported [1], [2]. Desminopathies are usually manifest in the second to the fourth decade of life. Cardiac involvement including arrhythmias or truly dilated, hypertrophic or restrictive cardiomyopathy may precede, coincide with, or succeed skeletal muscle weakness. To date, no specific or ameliorating therapy is available [1], [2]. Moreover, it is a matter of clinical debate if physical exercise has a beneficial or deteriorating effect in patients.

Desminopathies are the best-studied disease entity within the clinically and genetically heterogeneous group of myofibrillar myopathies, which are morphologically characterized by desmin-positive protein aggregates and myofibrillar changes [2]. The complex molecular pathophysiology of desminopathies seems primarily related to toxic effects of mutant desmin proteins on the formation and maintenance of the extra sarcomeric intermediate filament network. Further, mutant desmin affects essential protein–protein interactions, cell signaling cascades, mitochondrial function, and protein quality control mechanisms [3].

We previously characterized the pathological consequences of a heterozygous R350P desmin missense mutation at the clinical, myopathological, biochemical, and molecular level [4], [5]. Using primary myoblasts derived from diagnostic muscle biopsies from a patient carrying a heterozygous R350P desmin mutation, we investigated to what extent the expression of mutant desmin contributes to mechanical changes and causes abnormal cellular response to mechanical perturbation. For this purpose, we adapted a biomechanical protocol that imposes cyclic mechanical strain to adherent myoblasts on flexible membranes and measured the cell viability in response to cyclic strain [6]. In addition, we performed magnetic tweezer experiments with fibronectin-coated beads to measure cell mechanical properties. Our data provide the first evidence that mutant desmin myoblasts show altered mechanical properties and higher vulnerability to mechanical stretch, which may contribute to the progressive disease process.

Section snippets

Cells and cell culture

Primary human myoblasts derived from diagnostic skeletal muscle biopsies of one healthy female control and one female patient with a genetically proven heterozygous R350P desmin mutation were supplied from the Muscle Tissue Culture Collection (MTCC) at the Friedrich Baur Institute, Munich, Germany. Cells were cultured in skeletal muscle cell growth medium (PromoCell) supplemented with 5% PromoCell supplement Mix (PromoCell), 1.5% Glutamax (Gibco), 0.3% Gentamycin (Gibco), and 10% FCS.

R350P desmin myoblasts show increased mechanical vulnerability to cyclic stretch

To investigate the response of primary human myoblasts to cyclic mechanical stretch, we cultured the cells on flexible PDMS substrates and exposed them to uniaxial cyclic stretch with a peak-to-peak amplitude of 30% at 0.25 Hz for 60 min. In time-matched control experiments (no stretch), the percentage of dead cells was around 2% in both control and diseased cells (Fig. 3A). The percentage of dead and detached cells after 1 h of cyclic 30% stretch was 10.9% in control cells and 16.6% in mutant

Discussion

Higher mechanical vulnerability of skeletal muscle fibers in response to shear and pulling force during muscle contraction, especially during eccentric muscle contraction, has been previously suggested as a central mechanism for muscle fiber degeneration in a number of hereditary myopathies [15]. Since direct biomechanical assessment of human skeletal muscle fibers and tissue from diagnostic biopsies has serious technical and ethical limitations, we developed a novel approach to characterize

Acknowledgments

We thank Werner Schneider and Wolfgang Rubner for technical help. This work was supported by grants from the Deutsche Gesellschaft für Muskelkranke (DGM), the Johannes und Frieda Marohn-Stiftung, and the Deutsche Forschungsgesellschaft (DFG).

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