Telmisartan Modulates Exercise Responses in Peripheral Artery Disease: Analyses of Skeletal Muscle from the TELEX Trial
Background: In people with peripheral artery disease (PAD), the TELEX randomized clinical trial tested whether telmisartan (TEL), with or without exercise, significantly improved 6-minute walk distance at 6-month follow-up, compared to placebo (PLA). Objectives: The present study analyzed the effects of TEL on exploratory outcomes of muscle cellular and molecular characteristics. Methods: Baseline and 6-month follow-up muscle biopsies were obtained from 13 participants with PAD in the TELEX trial randomized to exercise + TEL or exercise + PLA. Immunohistochemistry was used to assess measures including myofiber cross-sectional area and satellite cell content, and the GeoMx digital spatial profiling system was used for transcriptomic analyses of alpha-smooth muscle actin (a-SMA)-positive and a-SMA-negative cells (primarily myofibers). Results: Myofiber cross-sectional area (CSA) and the number of satellite cells associated with Type II myofibers were significantly increased in the exercise + TEL group relative to exercise + PLA (p = 0.038 and p = 0.031, respectively). Among participants undergoing supervised exercise, those randomized to TEL had up-regulated N-linked glycosylation and down-regulated Ras signaling pathways in a-SMA-positive cells, compared to PLA. In a-SMA-negative cells, participants randomized to TEL showed up-regulated PPAR? activation-related pathways, including NO-cGMP-PKG signaling, and fatty acid oxidation. TEL also significantly reduced myostatin expression (adj. p = 0.0097) relative to PLA in a-SMA-negative cells. Conclusions: Overall, these findings suggest that TEL may regulate the effects of exercise on muscle in individuals with PAD by reducing myostatin expression, increasing myofiber size, and increasing activation of PPAR?. Overall design: Muscle tissue samples were stained with a fluorescent-conjugated antibody against a-SMA (1:400, Abcam, Cambridge, United Kingdom, #ab202368, RRID: AB_2924381) and a nucleic acid stain (SYTO 83, Thermo Fisher Scientific, #S11364), as well as GeoMX DSP oligo-conjugated RNA detection probes (GeoMx Human Whole Transcriptome Atlas [WTA] Human RNA for Illumina Systems, NanoString Technologies, Seattle, WA, #GMX-RNA-NGSHuWTA). Following region on interest (ROI) selection (660 µm x 785 µm), DSP barcodes were UV-cleaved, collected, and oligos were dispensed into 96-well plates prior to library preparation. Sequencing was performed using a NovaSeq 6000 S4 flow cell (Illumina, San Diego, CA). FASTQ files were processed via the GeoMX NGS Pipeline on DRAGEN via BaseSpace Sequence Hub (Illumina), and counts data were uploaded to the GeoMx DSP Analysis Suite (NanoString Technologies) for analyses. For quality control (QC) and filtering, probes with low counts (<5 reads in more than 2 ROIs) and probes failing global outlier (ratio of geometric mean over all ROIs to geometric mean of individual target over all ROIs <0.1) and Grubbs outlier (outlier probes in >20% of ROIs) tests were excluded. A total of 45 ROIs were analyzed for a-SMA+ cells, and 44 ROIs were analyzed for a-SMA- cells