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Transcriptome Analysis for Predictive Biomarkers of Muscle Viability During Ex Vivo Normothermic Limb Perfusion (EVNLP)
Abigail Meyers, Varun Kopparthy, Jesse Zhan, Payam Sadeghi, Antonio Rampazzo, Bahar Bassiri Gharb
Cleveland Clinic
2023-01-30
Presenter: Abigail Meyers
Affidavit:
Agree with above statement. This project was original work conducted by the medical student and research fellow team under the guidance of the primary investigator, Dr. Bahar Bassiri Gharb.
Director Name: Steven Bernard
Author Category: Medical Student
Presentation Category: Basic Science Research
Abstract Category: General Reconstruction
Purpose
Muscle viability remains the primary focus of limb preservation. We hypothesized that genes involved in the pathophysiology of tissue injury are differentially expressed during EVNLP compared to static cold storage (SCS).
Methods
Bilateral forelimbs were procured from Yorkshire pigs, allocated to EVNLP (n=4) or SCS (n=4). Skeletal muscle specimens were collected before limb procurement, at 0h, 6h, 12h, and perfusion end, multiplexed and sequenced on an Illumina NovaSeq 6000 system, then aligned to the porcine genome using Salmon 1.9.0. Transcripts were summarized as gene-level transcripts per million (TPM) abundances with tximport. Differential expression analysis was performed using DESEQ2. Pathway enrichment by significant genes (adj-p <.05) was performed using Panther Classification System and Gene Set Enrichment Analysis (GSEA).
Results
A total of 2,283 genes were differentially expressed in EVNLP. Compared to the baseline, perfused limbs exhibited enrichment of pathways involved in wound healing, inflammatory response, regulation of cellular stress, negative regulation of T-cell proliferation, negative regulation of apoptotic pathways and response to fluid shear stress (adj-p <0.05). SCS limbs exhibited a different transcriptome signature, primarily enriching for positive regulation of chemotaxis and anabolic metabolism pathways.
At 6h, both perfused and SCS limbs expressed 62 genes differentially as compared to 0h. However, when comparing samples from >16h vs. 0h, EVNLP showed 419 differentially expressed genes, while SCS had 37 (adj-p <0.05).
Conclusion
Increased genetic transcription during EVNLP is aligned with metabolic activity and inflammation. Identification of evoked pathways can allow development of a genetic test for real-time muscle viability assessment.
