Expression of Metabolic Transcripts is Altered with Crk-CrkL Deletion in Mouse Hearts
Location
Philadelphia, PA
Start Date
17-4-2026 1:30 PM
End Date
17-4-2026 2:30 PM
Description
Introduction:
Crk and CrkL are adaptor proteins that function in cell signaling pathways and share biochemical properties. They appear in embryonic and adult mouse hearts and contribute to congenital heart disease. Crk and CrkL contribute to outflow tract separation, maturation of arteries and vascular remodeling during cardiovascular development. Crk and CrkL are expressed in the embryonic mouse heart within the epicardium and derivatives of the epicardium (EPDCs) that include coronary vascular cells. The functional roles of Crk and CrkL in the epicardium and EPDCs remain to be fully understood. Thus, we generated mice with epicardial-specific deletion of Crk and CrkL (Crk-CrkL-epidel/+) using a Cre-Lox recombination strategy to achieve tissue-specific deletion. Adult Crk-CrkL-epidel/+ mice were able to exercise on the treadmill under fatiguing conditions for a significantly longer duration with a greater total running distance than control mice. We sought to determine the molecular mechanism that contributed to this enhanced exercise performance in our mutant mice.
Methods:
We isolated hearts from adult control and mutant Crk-CrkL-epidel/+ mice. Total RNA was extracted from these hearts. RNA sequencing (RNAseq) of mRNA was performed to examine differences in transcriptome expression. Bioinformatic analysis was conducted on the resultant datasets to identify transcripts and explore gene ontology data. We used the RNAscope technique to examine expression of 2 transcripts through hybridization of specific probes to sections of control and mutant hearts. The number of transcripts expressed were quantified using Image J software in three regions throughout the left ventricle at the base, mid-ventricle, and apex of the hearts. Also, we performed quantitative PCR (qPCR) with probes to validate mRNA expression of these transcripts identified by RNAseq analysis. In addition, we analyzed metabolites in flash-frozen hearts collected from these mice.
Results:
With RNAseq analysis, we observed 1544 transcripts to be differentially expressed between our control and mutant mice. Many of these altered transcripts 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-CrkL-epidel/+ mouse hearts vs. controls. Two notable transcripts were PLAAT1 (Phospholipase A and acyltransferase 1) and PCK1 (phosphoenolpyruvate carboxykinase). We observed a 3.4-fold increase in PLAAT1 expression in mutant vs. control hearts while PCK1 expression was increased by 2.9-fold in mutant hearts via RNAseq analysis. 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 epicardium and myocardium of mutant mouse hearts by RNAscope. qPCR analysis is being conducted to validate mRNA expression of these transcripts.
Conclusion:
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. PLAAT1 functions in lipid metabolism while PCK1 is a key enzyme in gluconeogenesis. We speculate that both transcripts alter metabolic efficiency in hearts of Crk-CrkL-epidel/+ mice to enhance their exercise performance.
Embargo Period
5-20-2026
Expression of Metabolic Transcripts is Altered with Crk-CrkL Deletion in Mouse Hearts
Philadelphia, PA
Introduction:
Crk and CrkL are adaptor proteins that function in cell signaling pathways and share biochemical properties. They appear in embryonic and adult mouse hearts and contribute to congenital heart disease. Crk and CrkL contribute to outflow tract separation, maturation of arteries and vascular remodeling during cardiovascular development. Crk and CrkL are expressed in the embryonic mouse heart within the epicardium and derivatives of the epicardium (EPDCs) that include coronary vascular cells. The functional roles of Crk and CrkL in the epicardium and EPDCs remain to be fully understood. Thus, we generated mice with epicardial-specific deletion of Crk and CrkL (Crk-CrkL-epidel/+) using a Cre-Lox recombination strategy to achieve tissue-specific deletion. Adult Crk-CrkL-epidel/+ mice were able to exercise on the treadmill under fatiguing conditions for a significantly longer duration with a greater total running distance than control mice. We sought to determine the molecular mechanism that contributed to this enhanced exercise performance in our mutant mice.
Methods:
We isolated hearts from adult control and mutant Crk-CrkL-epidel/+ mice. Total RNA was extracted from these hearts. RNA sequencing (RNAseq) of mRNA was performed to examine differences in transcriptome expression. Bioinformatic analysis was conducted on the resultant datasets to identify transcripts and explore gene ontology data. We used the RNAscope technique to examine expression of 2 transcripts through hybridization of specific probes to sections of control and mutant hearts. The number of transcripts expressed were quantified using Image J software in three regions throughout the left ventricle at the base, mid-ventricle, and apex of the hearts. Also, we performed quantitative PCR (qPCR) with probes to validate mRNA expression of these transcripts identified by RNAseq analysis. In addition, we analyzed metabolites in flash-frozen hearts collected from these mice.
Results:
With RNAseq analysis, we observed 1544 transcripts to be differentially expressed between our control and mutant mice. Many of these altered transcripts 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-CrkL-epidel/+ mouse hearts vs. controls. Two notable transcripts were PLAAT1 (Phospholipase A and acyltransferase 1) and PCK1 (phosphoenolpyruvate carboxykinase). We observed a 3.4-fold increase in PLAAT1 expression in mutant vs. control hearts while PCK1 expression was increased by 2.9-fold in mutant hearts via RNAseq analysis. 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 epicardium and myocardium of mutant mouse hearts by RNAscope. qPCR analysis is being conducted to validate mRNA expression of these transcripts.
Conclusion:
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. PLAAT1 functions in lipid metabolism while PCK1 is a key enzyme in gluconeogenesis. We speculate that both transcripts alter metabolic efficiency in hearts of Crk-CrkL-epidel/+ mice to enhance their exercise performance.