Biology subjects

Agbulut, O.

Publications and source records attributed to Agbulut, O..

6 recordsLinked to original sources

bioRxiv2024

Fibrotic-like collagen matrices as innovative 3D in vitro models for investigating the impact of pathological ECM on muscle regeneration in muscular dystrophies

Muscular dystrophies are characterized by impaired skeletal muscle contraction due to genetic mutations. Beyond the disruption of muscle function, the extracellular matrix (ECM) surrounding muscle fibers becomes fibrotic, further impeding tissue repair and function. In this study, we designed both healthy and fibrotic matrices to examine the impact of persistent fibrosis on muscle cell behavior. Fibrotic matrices were synthesized by 3D printing of dense collagen solutions in air, followed by slow gelation to yield non-porous, isotropic hydrogels. These matrices were subsequently crosslinked using EDC/NHS chemistry, resulting in a Youngs modulus of approximately 50 kPa. The behavior of C2C12 myoblasts cultured within the fibrotic matrices was compared to cells grown in healthy matrices. Our results showed that the fibrotic matrix had a detrimental effect on cell behavior. Myoblasts were unable to differentiate into mature myotubes, exhibited poor alignment, and suffered from hypoxia. Furthermore, these cells failed to proliferate, secreted inflammatory cytokines, and were unable to remodel their ECM. Using matrices possessing a single characteristic of the fibrotic matrix (i.e stiff or non porous), the predominant factor for each feature of the cell phenotype was determined. These findings underscore the detrimental effects of a fibrotic persistent ECM on muscle homeostasis. The development of these two distinct 3D muscle models, one representing healthy muscle and the other fibrotic, offers a valuable tool for investigating the pathophysiology of muscular dystrophy. HighlightsO_LIStiff and non-porous dense collagen matrices synthesized by 3D printing recapitulate the key characteristics of fibrosis observed in muscular dystrophies C_LIO_LIFibrotic matrices negatively impact myoblast proliferation and differentiation into mature myocytes C_LIO_LIFibrotic matrices impede the 3D organotypic organization of myocytes C_LIO_LIThis 3D pathological model of skeletal muscle offers a valuable tool for investigating how persistent fibrosis influences the progression of muscular dystrophy C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/630059v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@c2403corg.highwire.dtl.DTLVardef@1074147org.highwire.dtl.DTLVardef@14eb055org.highwire.dtl.DTLVardef@11ac419_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

Critical contribution of mitochondria in the development of cardiomyopathy linked to desmin mutation

Beyond the observed alterations in cellular structure and mitochondria, the cellular mechanisms linking genetic mutations to the development of heart failure in patients affected by desmin defects remain unclear due, in part, to the lack of relevant human cardiomyocyte models. We investigated the role of mitochondria using cardiomyocytes derived from human induced pluripotent stem cells carrying the heterozygous DESE439K desmin mutation, that were either isolated from a patient or generated by gene editing. To increase physiological relevance, cells were either cultured on an anisotropic surface to obtain elongated and aligned cardiomyocytes, or as spheroids to create a micro- tissue. When applicable, results were confirmed with heart biopsies from the family harboring DESE439K mutation. We show that mutant cardiomyocytes reproduce critical defects in mitochondrial architecture, respiratory capacity and metabolic activity as observed in patients heart tissue. To challenge the pathological mechanism, normal mitochondria were transferred inside the mutant cardiomyocytes. This treatment restored mitochondrial and contractile functions. This work demonstrates the crucial role of mitochondrial abnormalities in the pathophysiology of desmin-related cardiomyopathy, and opens-up new potential therapeutic perspectives.

pathology↗

bioRxiv2022

Anisotropic dense collagen hydrogels possessing two ranges of porosity to create the adequate microenvironment for muscle bundles: a step towards skeletal muscle modeling

Despite the crucial role of the extracellular matrix (ECM) in the organotypic organization and function of skeletal muscles, most 3D models do not mimic its specific characteristics, namely its biochemical composition, stiffness, anisotropy, and porosity. Here, a novel 3D in vitro model of muscle extracellular matrix was developed to differentiate myogenic cells (C2C12 line) into myotubes and reproduce their natural cell/cell and cell/matrix interactions. An anisotropic hydrogel mimicking the perimysium was obtained thanks to unidirectional 3D printing of dense collagen with aligned collagen fibrils. The space between the different layers was tuned to generate an intrinsic porosity (100 {micro}m) suitable for nutrient and oxygen diffusion. By modulating the gelling conditions, the mechanical properties of the construct reached those measured in the physiological muscle ECM. The addition of large channels (600 {micro}m) by molding permitted to create a second range of porosity suitable for cell colonization without altering the physical properties of the hydrogel. C2C12 cells embedded in Matrigel(R), seeded within the channels, organized in 3D, and differentiated into multinucleated mature myotubes. This organization reproduced the global muscular bundles, i.e., the endomysium encompassing myotubes. These results show that porous and anisotropic dense collagen hydrogels colonized with myoblasts are promising biomaterials to model skeletal muscle. Table of contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=39 SRC="FIGDIR/small/496716v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@bae7e8org.highwire.dtl.DTLVardef@cf453corg.highwire.dtl.DTLVardef@1ca69a2org.highwire.dtl.DTLVardef@1dd13cd_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIA novel extracellular matrix-like hydrogel increases the physiological relevance of the skeletal muscle model. C_LIO_LIPorous and anisotropic dense collagen hydrogels mimic the muscle ECM physical properties. C_LIO_LIUnidirectional printing of dense collagen creates a porous and anisotropic scaffold in a single step. C_LIO_LIAnisotropic dense collagen hydrogels promote C2C12 differentiation into myotubes and their 3D organization. C_LI

bioengineering↗

bioRxiv2022

Receptor interacting protein kinase-3 promotes both myopathy and cardiomyopathy in dystrophin-deficient mice

BackgroundDuchenne muscular dystrophy (DMD) is a progressive muscle degenerative disorder, culminating in a complete loss of ambulation, hypertrophic cardiomyopathy and a fatal cardiorespiratory failure. Necroptosis is the form of necrosis that is dependent upon the receptor-interacting protein kinase (RIPK) 3; it is involved in several inflammatory and neurodegenerative conditions. We previously identified RIPK3 as a key player in the acute myonecrosis affecting the hindlimb muscles of the dystrophic mouse model, mdx. Whether necroptosis also mediates respiratory and heart disorders in DMD is currently unknown. MethodsEvidence of activation of the necroptotic axis was examined in dystrophic tissues from Golden retriever muscular dystrophy (GRMD) dogs and R-DMDdel52 rats. A functional assessment of the involvement of necroptosis in dystrophic animals was performed on mdx mice that were genetically depleted for RIPK3. Dystrophic mice aged from 12 to 18 months were analyzed by histology and molecular biology to compare the phenotype of muscles from mdxRipk3+/+ and mdxRipk3-/- mice. Heart function was also examined by echocardiography in 40-week-old mice. ResultsQuantification of RIPK3 transcripts in sartorius and biceps femoris muscles from GRMD dogs positively correlated to myonecrosis levels (r=0.81; p=0.0076). RIPK3 was also found elevated in the diaphragm (p=0<0.05). In the slow progressing heart phenotype of GRMD dogs, the phosphorylated form of RIPK1 at the Serine 161 site was dramatically increased in cardiomyocytes. A similar p-RIPK1 upregulation characterized the cardiomyocytes of R-DMDdel52 rats, associated with a marked overexpression of Ripk1 (p=0.007) and Ripk3 (p=0.008), indicating primed activation of the necroptotic pathway in the dystrophic heart. MdxRipk3-/- mice displayed decreased compensatory hypertrophy of the heart (p=0.014), and echocardiography showed a 19% increase in the relative wall thickness (p<0.05) and 29% reduction in the left ventricle mass (p=0.0144). Besides, mdxRipk3-/- mice presented no evidence of a regenerative default or sarcopenia in skeletal muscles, moreover around 50% less affected by fibrosis (p<0.05). ConclusionsOur data provide evidence of the activation of the necroptotic pathway in degenerative tissues from dystrophic animal models, including the diaphragm and the heart. The genetic inhibition of necroptosis in dystrophic mice improves both cardiac function and histological features of muscles, suggesting that prevention of necroptosis is susceptible to providing multiorgan beneficial effects for DMD.

cell biology↗

bioRxiv2021

The beneficial effect of chronic muscular exercise on muscle fragility is increased by Prox1 gene transfer in dystrophic mdx muscle

PurposeGreater muscle fragility is thought to cause the exhaustion of the muscle stem cells during successive degeneration/repair cycles, leading to muscle wasting and weakness in Duchenne muscular dystrophy. Chronic voluntary exercise can partially reduce the susceptibility to contraction induced-muscle injury, i.e., muscle fragility, as shown by a reduced immediate maximal force drop following lengthening contractions, in the dystrophic mdx mice. Here, we studied the effect of Prospero-related homeobox factor 1 gene (Prox1) transfer (overexpression) on fragility in chronically exercised mdx mice, because Prox1 promotes slower type fibres in healthy mice and slower fibres are less fragile in mdx muscle. Methodsmdx mice received or not Prox1 transfer into the tibialis anterior muscle and performed voluntary running into a wheel during 1 month. We also performed Prox1 transfer in sedentary mdx mice. In situ maximal force production of the muscle in response to nerve stimulation was assessed before, during and after 10 lengthening contractions. Molecular and cellular parameters were also evaluated. ResultsInterestingly, Prox1 transfer reduced the force drop following lengthening contractions in exercised mdx mice (p < 0.05 to 0.01), but not in sedentary mdx mice. It also increased the muscle expression of Myh7 (p < 0.001), MHC-2x (p < 0.01) and Trpc1 (p < 0.01), whereas it reduced that one of Myh4 (p < 0.001) and MHC-2b (p < 0.01) in exercised mdx mice. Moreover, Prox1 transfer decreased the maximal force (p < 0.01) before lengthening contraction in exercised mdx mice (p < 0.01), and reduced muscle weight (p < 0.0001) despite increased Mstn expression (p < 0.001). ConclusionOur results indicate that the beneficial effect of Prox1 transfer on muscle fragility is only observed in chronically exercised mdx mice. Thus, Prox1 transfer combined to chronic exercise have the potential to substantially slow the progression of the dystrophic disease in the long term.

physiology↗

bioRxiv2019

Desmin is a modifier of dystrophic muscle features in Mdx mice

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disease, caused by dystrophin deficiency. Desmin is like dystrophin associated to costameric structures bridging sarcomeres to extracellular matrix that are involved in force transmission and skeletal muscle integrity. In the present study, we wanted to gain further insight into the roles of desmin which expression is increased in the muscle from the mouse Mdx DMD model. We show that a deletion of the desmin gene (Des) in Mdx mice (DKO, Mdx:desmin-/-) induces a marked worsening of the weakness (reduced maximal force production) as compared to Mdx mice. Fragility (higher susceptibility to contraction-induced injury) was also aggravated and fatigue resistance was reduced in DKO mice. Moreover, in contrast to Mdx mice, the DKO mice did not undergo a muscle hypertrophy because of smaller and less numerous fibers, with reduced percentage of centronucleated fibres. Interestingly, Desmin cDNA transfer with adeno-associated virus in 1-month-old DKO mice and newborn Mdx mice improved muscle weakness. Overall, desmin plays important and beneficial roles on muscle performance, fragility and remodelling in dystrophic Mdx mice.

pathology↗