Chronic Hindlimb Ischemia Induces Alterations of Muscle Transcriptomes Based on Muscle Type

Abstract

Introduction: Peripheral artery disease (PAD) is a progressive atherothrombotic disorder of the arteries supplying the lower limb and is the most prevalent cardiovascular disease, impacting over 230 million people worldwide. PAD is associated with lower extremity myopathy characterized by myofiber degeneration and fibrosis. Increased proportions of type I muscle fibers are associated with improved patient outcomes, but most studies, human and animal, only analyze the gastrocnemius (gastroc), which contains mixed amounts of type I and type II fibers. To elucidate the impacts of ischemia on muscles comprising mainly type I fibers, we evaluated the differential effect of hindlimb ischemia (HLI) on the soleus and gastroc, hypothesizing that chronic HLI would more severely impact the soleus. Methods: Chronic HLI was induced in Sprague-Dawley male rats with an endovascular iliofemoral artery occlusion approach. Time-dependent laser Doppler perfusion imaging assessed hindlimb perfusion before and after HLI up to 42 days. Myopathy was evaluated 28 days post-HLI by staining muscle cross-sections with Hematoxylin and Eosin (H&E) and Masson trichrome. Quantification of muscle fibrosis, average fiber cross-sectional area, and presence of central nuclei were used to determine muscle pathology. RNAsequencing was used to determine the differences in transcriptomes of the gastroc and soleus in both sham and HLI conditions. Results: Histological analysis revealed that there were significantly increased levels of fibrosis after HLI, but only in the soleus (18.24±7.23 vs. 7.71±2.54 %, n = 8-16, p-value <0.0001). The average fiber cross-sectional area was significantly reduced in the HLI soleus compared to the sham (1556.00±459.60 vs. 3072.00±831.50 μm, n = 8-16, p-value <0.0001), but not between HLI and sham gastroc. Conversely, HLI increased the proportions of centralized nuclei in both muscles (soleus — 22.72±12.46 vs. 1.12±1.49 %, n = 8-16, p-value = 0.0002, gastroc — 12.75±10.17 vs. 0.00±0.00 %, n = 8-16, p-value = 0.0002), indicating prior muscle damage. The HLI soleus exhibited worse outcomes in all parameters compared to the HLI gastroc. These data were substantiated by RNAseq, which showed that the HLI soleus transcriptome contained more differentially expressed genes (DEGs, ~1000 altered genes) than its sham counterpart compared to HLI gastroc, which possessed <100 DEGs (n = 5-6). Moreover, the HLI soleus transcriptome was highly fibrotic, pro-angiogenic, and promoted myoblast fusion, a combination that could explain the histological myopathy findings. Conclusions: Chronic HLI induces alterations of muscle ultrastructure and transcription. These effects occur predominantly in the soleus rather than the gastroc. The results of the present study implicate that the pathophysiology is different for distinct muscles and may result from varying fiber composition. This work can help identify the mechanisms responsible for the myopathy of PAD and may lead to the design of targeted interventions for the care of PAD patients. R01HL169205-01. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.