Browsing by Subject "Rheumatoid arthritis"

Rheumatoid Arthritis (RA) is a chronic inflammatory auto-immune disease typically involving the joints, mainly the diarthrodial joint and generally starts between the age of 30 and 60 in women and somewhat later in life in men. It is the most common inflammatory arthritis and about one percent of the population is affected by RA. A complex interaction between various genetic and environmental factors lead to the development of the disease, though the specific cause of RA is not known. The goal of this study is to characterize the maturation of skeletal muscle bundles made with myoblasts isolated from RA patients and compare it with maturation of age-matched controls. Moreover, the engineered myobundles were treated with pro-inflammatory cytokines to assess their effect on the bundle maturation and to replicate the pro-inflammatory phenotype of RA.

Myobundles were prepared with human skeletal muscle (HSkM) samples obtained from young controls, age-matched controls and RA patients through biopsy of vastus lateralis muscle (biopsy of hamstring muscle was taken for young controls). We measured nuclei count, cross-sectional area, Myogenin count, Sarcomeric alpha-actinin (SAA) positive area and the myofiber diameter for each time course studies and cytokine treated bundles.

Contrary to our expectations, the time course study did not indicate significant reduction in fiber formation. This may be due to the effect of medications taken by the RA patient which might be helping the muscle function. Another possible reason might be that the cells could have regained their normal function once they were taken out from the inflammatory environment induced by the pro-inflammatory cytokines. Yet another possible reason may be that the time course considered may not be enough to access changes in the maturation and a longer time period may be required.

We then moved forward to replicate the disease pro-inflammatory phenotype by carrying out cytokine treatments on the engineered myobundles. IFNγ, IFNγ+GMCSF, TNFα+GMCSF and IFNγ+TNFα+GMCSF were chosen for the cytokine treatments. According to our results, the cross-sectional area, nuclei count/CSA, MyoG count/CSA, MyoG/Nuclei count, SAA+ area and the myofiber diameter each decreased with cytokine treatments indicating that the cytokines may indeed affect the regeneration ability of skeletal muscle cells.

The results from cytokines treatment studies indicate that cytokines do play a role in disease development and progression. A longer time course study say for up to 10 days or more post differentiation, more patient data regarding the disease severity and medications might also be helpful in further investigation.

Tissue-engineered skeletal muscle can be designed and optimized to serve as a platform for disease modeling and drug testing. In vitro models such as these can be used to explore basic research questions that may be difficult to study in vivo. To achieve this, it is important that the engineered skeletal muscle mimic its in vivo equivalent both phenotypically and functionally. Our engineered human skeletal muscle constructs (myobundles) generate quantitative contractile forces in response to electrical stimulation. The 3D myobundles provide a more realistic in vitro environment than that experienced by cells cultured in 2D monolayers.

The overall goal of this work was to develop an in vitro myobundle model of rheumatoid arthritis (RA), a chronic inflammatory disorder, to (1) characterize muscle function of RA patients, (2) further our understanding of the underlying disease mechanisms, and (3) test potential therapeutics that can reduce muscle damage and loss in RA. We first characterized myobundles made with cells from the vastus lateralis muscle of RA patients and aged healthy donors, as well as from the hamstring muscle of young healthy donors as a benchmark. Next, we investigated RA myobundle sensitivity to pro-inflammatory cytokine exposure, compared to aged healthy controls. Finally, we evaluated the effect of pharmacologic agents on functional recovery of RA myobundles.

Surprisingly, in 3D culture, contractile force production by RA myobundles was greater compared to aged controls. In support of this finding, assessment of RA myofiber maturation showed increased area of sarcomeric α-actinin expression over time compared to aged controls. Furthermore, a linear regression test indicated a positive correlation between sarcomeric α-actinin protein levels and tetanus force production in RA and controls. Our findings suggest that medications prescribed to RA patients may maintain—or even enhance—muscle function, and this effect is retained and observed in in vitro culture.

We demonstrated that RA myobundles were more sensitive to IFN-γ treatment leading to reduced contractile force and reduced contractile protein levels compared to aged healthy controls. RNA sequencing (RNA-seq) and gene set enrichment analysis (GSEA) was performed to identify pathways associated with altered gene expression. Gene sets that were enriched in IFN-γ-treated RA myobundles, but not IFN-γ-treated controls, were genes upregulated in response to hypoxia and genes upregulated during unfolded protein response. From the hypoxia gene set, Pim1 and MT-1 were identified as potential therapeutic targets for treating RA-associated muscle dysfunction. Furthermore, we showed that treatment with tofacitinib fully restores contractile force and contractile protein levels in IFN-γ-treated RA myobundles.

To our knowledge, this represents the first study to use tissue-engineered human muscle to characterize muscle function of RA patients. Our in vitro RA myobundle model enables us to (1) model key variables affecting the progression of RA and (2) serve as a platform for pharmaceutical testing allowing for ineffective drugs to be quickly identified. Since chronic inflammation and muscle loss play a role in other diseases such as osteoarthritis, sarcopenia, and cachexia in heart failure and cancer, this work serves as a proof-of-principle for modeling and treating inflammation and fibrosis of muscle.