Thyroid-Stimulating Hormone Receptor Expression on Primary Cultured Human Extraocular Muscle Myoblasts

ABSTRACT

Purpose

To isolate and culture human extraocular muscle (EOM) myoblasts and facilitate their differentiation to myotubes in vitro, and to determine whether these myoblasts express thyroid-stimulating hormone receptor (TSHR).

Materials and methods

Human EOM myoblasts were isolated from EOM samples, and identified by immunostaining for PAX7 and MYOD1 (markers of human skeletal myoblasts), and western blot for desmin (muscle marker). In addition, we investigated the expressions of SHOX2 (a genetic marker of EOM myoblasts) and HOXC10 (an exclusive marker of hind-limb muscle-derived myoblasts) by RT-PCR. Fusion index and myotube area were measured to quantify myotube differentiation. TSHR immunostaining and western blot were used to determine the presence of TSHR on human EOM myoblasts and investigate its expression during myogenesis.

Results

Human EOM myoblasts were immunopositive for PAX7 and MYOD1 staining, and had desmin expression during myogenesis. The EOM-specific gene SHOX2 was detected by RT-PCR, but HOXC10 was not detected. The significant change in both fusion index and myotubes were shown at 8 days after induction of differentiation myotubes. Immunostaining revealed TSHR was expressed on human EOM myoblasts and western blot demonstrated the presence of TSHR protein and highest TSHR protein expression was shown at 10 days after myogenic differentiation.

Conclusions

Human EOM myoblasts were cultured and underwent myogenic differentiation in vitro. TSHR protein was detected on human EOM myoblasts and increasing TSHR expression during myogenic differentiation.

KEYWORDS:

• Human extraocular muscle
• human extraocular muscle myoblasts
• thyroid-stimulating hormone receptor
• thyroid-associated ophthalmopathy
• myogenesis

Supplementary material

Supplemental data for this article can be accessed here.

Declaration of interest

The authors have no conflict of interest to declare and are wholly responsible for the contents of this paper.

Additional information

Funding

This study was supported by a Biomedical Research Institute Grant (2014-07), Pusan National University Hospital. The funding organization played no role in the design or conduct of this study.