Research Paper Volume 8, Issue 9 pp 2062—2080

Age-associated repression of type 1 inositol 1, 4, 5-triphosphate receptor impairs muscle regeneration

Figure 5. Intramuscular adenovirus-mediated ITPR1 silencing delays muscle regeneration. (A) Scheme of the in vivo experimental procedure. TA muscles of C57BL/6 mice were injected with 50 μl of 20 μM CTX using an insulin syringe at day 0. The next day, injured TA muscle tissues were injected with adenovirus encoding shITPR1-RFP or scrambled shRNA-RFP (1.4x109 particles) using an insulin syringe. At the indicated days, muscle tissues were harvested for biochemical and histological analyses. (B) Immunoblot analysis of ITPR1 knockdown and control mouse TA muscle lysates with anti-ITPR1, anti-MyHC, anti-phospho ERK1/2 and anti-ERK1/2 antibodies, with actinin as the loading control. (C) qRT-PCR analysis of the time-courses of Myod1 and Myog mRNA expression in ITPR1-silenced and control TA muscle samples subjected to CTX injury, relative to 36b4. n = 3 for each group. (D) Representative immunohistochemistry images of Laminin (green) and DAPI (blue) staining of ITPR1-silenced and control TA muscle fibers on day 14 after CTX injury. Cross-sectional areas of muscle fiber were measured using NIS-Elements Microscope Imaging software (Nikon) and fiber size distributions are presented as means ± S.D. (n = 3).