Expression of Metabolic Transcripts with Deletion of Crk and CrkL Adaptor Proteins in Mice

Date of Award

5-2026

Degree Type

Thesis

Degree Name

Master of Science in Biomedical Sciences

First Advisor

Cathy J. Hatcher, PhD

Second Advisor

Marina D’Angelo, PhD

Third Advisor

Philip Reno, PhD

Abstract

Crk and CrkL are adaptor proteins that function in cell signaling pathways and share biochemical properties; however, functional roles of Crk and CrkL in the epicardium and EPDCs remain to be fully understood. Thus, Crk-CrkLepi+/- mice were generated to achieve tissue-specific deletion and were able to exercise on the treadmill under fatiguing conditions for a significantly longer duration than control mice. This study sought to determine the molecular mechanism that contributed to this enhanced exercise performance in our mutant mice.

RNA sequencing (RNAseq) of mRNA was performed to examine differences in transcriptome expression, and Metabolomics study was completed to shed light on possible metabolic processes occurring. We used RNAscope technique to examine expression and localization of PLAAT1 (Phospholipase A and acyltransferase 1), UCP1 (Uncoupling Protein 1), PCK1 (phosphoenolpyruvate carboxykinase), and PDK4 (Phosphoenolpyruvate Carboxykinase 4) transcripts and quantitative PCR (qPCR) to validate mRNA expression of these transcripts identified by RNAseq analysis.

Many of these altered transcripts in the RNA-seq analysis were involved in glucose and lipid metabolism which aligned with our cardiac metabolomics data where fatty acid metabolites were upregulated, and key glycolytic metabolites were downregulated in our Crk-CrkLepi+/- mouse hearts vs. controls. We detected slightly more punctate PLAAT1 transcript expression in the myocardium of mutant vs. control mouse hearts. More punctate and clustered PCK1 transcript expression was observed in the nuclei in the epicardium and myocardium of mutant mouse hearts by RNAscope. qPCR analysis revealed upregulation of PCK1 and PLAAT1 in the base region of the heart. Our complex breeding strategy inhibited ability to obtain enough 3aCKO mice for analysis of UCP1 and PDK4 transcripts in hearts.

Our data demonstrates that epicardial deletion of Crk and CrkL alters expression of cardiac transcripts that encode proteins involved in lipid and glucose metabolism in the heart. We speculate that both transcripts alter metabolic efficiency in the hearts of Crk-CrkLepi+/- mice to enhance their exercise performance.

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