Single Muscle Fiber Gene Expression of tPA, uPA, and PAI-1

Abstract

PURPOSE: We have previously shown that the fibrinolytic proteins tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) are expressed in human muscle samples (unpublished observations). However, muscle biopsy samples contain both muscle fibers and endothelial cells, a known site of tPA and PAI-1 synthesis. To demonstrate that skeletal muscle fibers express tPA and PAI-1, we extracted 22 single fibers from a muscle sample and assessed gene expression of tPA, PAI-1, and urokinase plasminogen activator (uPA, another activator of the fibrinolytic system). METHODS: A muscle biopsy sample was obtained from the vastus lateralis of a young male using the percutaneous needle muscle biopsy technique. The sample was stored in RNAlater at −80ËšC until analysis. Twenty-two single fibers were extracted from the muscle sample using an inverted microscope while samples were in RNAlater (Ambion). Single fibers were homogenized in Tri-Reagent (MRC, Inc), and total RNA was extracted and purified. RNA was DNase treated using DNA-free (Ambion) prior to the reverse-transcription polymerase chain reaction (RT-PCR). RT was performed using the Superscript III RT kit (Invitrogen) with oligo-dT primers. PCR was performed using a multiplex PCR kit (Qiagen) and gene specific primers for tPA, uPA, PAI-1, and actin. Actin was assessed to verify the PCR reaction. PCR products were electrophoresed using a 3% agarose gel with 1 mg/ml ethidium bromide, and then PCR bands were determined by ultraviolet light and an imager. Additionally, a portion of the whole muscle sample was subjected to identical procedures as the single fibers to determine gene expression of uPA, tPA, and PAI-1. RESULTS: Whole muscle sample analysis showed expression of the uPA, tPA, and PAI-1 genes. Single fiber analysis showed that all 22 fibers expressed the tPA, PAI-1, and actin genes, but none of the 22 fibers expressed the uPA gene. CONCLUSIONS: Our data shows that muscle biopsy samples that contain both muscle fibers and endothelial cells express uPA, tPA and PAI-1, while single muscle ibers express tPA andPAI-1, but not uPA. This suggests that the source of uPA in a whole muscle sample is the endothelial cells and not skeletal muscle fibers. Furthermore, these data suggest that human skeletal muscle fibers express both the tPA and PAI-1 genes.