Requirement for Crk and CrkL in the Epicardial Cell Lineage for Cardiovascular Development and Function

Introduction: Crk and CrkL are intracellular cytoplasmic adaptor proteins that contribute to congenital heart disease. They mediate multiple signaling pathways involved in biological processes including cell migration, proliferation, apoptosis, adhesion, survival and differentiation. Both Crk and CrkL have shared functions in the cardiac neural crest cells for cardiac outflow tract septation as well as shared functions in the endocardial lineage for atrioventricular valve development. Recently, we observed Crk and CrkL expression in the epicardium and epicardium-derived cells (EPDCs) including the coronary vessels, in the embryonic mouse heart. The roles of these proteins in epicardium and EPDC development have not been established. Study Objective: This study aims to explore the role of Crk and CrkL in the epicardium and its derivatives and their impact on cardiovascular development, structure and function. Methods and Results: Ubiquitous heterozygous deletion of Crk and CrkL in mice with the CAG-Cre promoter caused lethality by embryonic day (E) 18.5. These Crk/CrkL del/+ embryos had cardiovascular defects affecting ventricular septation and outflow tract development while maturation of epicardial cells into its derivatives was also impaired. We circumvented the embryonic lethality caused by global deletion through tissue-specific deletion of Crk and CrkL. Crk lox/lox ; CrkL lox/lox mice were mated with Wilms’ tumor 1 (Wt1) Cre mice to achieve heterozygous deletion in the epicardium and EPDCs. Mice with epicardial lineage-specific heterozygous deletion of Crk and CrkL (Crk/CrkL-epi del/+ ) survived to adulthood. We examined cardiovascular structure and function in these mice through echocardiography and treadmill tests of maximum exercise tolerance under fatiguing conditions. Crk/CrkL-epi del/+ mice exhibited a longer duration of exercise and greater total running distance on the treadmill than control mice at 13-14 weeks of age. Echocardiographic analysis of 4-month old mice revealed slight increases in cardiovascular function of Crk/CrkL-epi del/+ versus control hearts as determined with the contractility parameters of stroke volume, cardiac output, and fractional shortening. Also, we observed a mild increase in the coronary velocity time integral of Crk/CrkL-epi del/+ mouse hearts compared to control mice indicating the distance blood travels within the coronary arteries. Conclusion: Our data reveal that maintenance of Crk and CrkL expression are important for development of the epicardium and its derivatives. Epicardial-specific Crk/CrkL deletion appears to impact contractility of the heart to support the increasing demands of the systemic circulation during maximal exercise. PCOM Center for Chronic Disorders of Aging; PCOM Division of Research This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.