Presentation of Early Heart Failure Phenotypes in βII-Spectrin Conditional Knockout Mice

Abstract

Background βII spectrin is an actin associated protein which regulates the localization of cytoskeletal and plasma membrane protein complexes necessary for normal cellular function. In cardiac myocytes βII spectrin recruits ankyrin-B to the cardiac dyad and modulates the functions of Na+/Ca2+ exchanger, ryanodine receptor 2, ankyrin-B, actin, and αII spectrin. Disruption of the βII spectrin/ankyrin-B complex due to genetic mutation results in the onset of fatal arrhythmias. This was associated to the dysregulation of Ca2+ channels via ryanodine receptor 2 which forms the cardiac dyad. We hypothesize that loss of spectrin leads to acceleration of heart failure (HF), and plays a critical role in the SMAD pathway in the heart, and spectrin based arrhythmias are caused by abnormalities with the cardiac dyad. Methods Transgenic βII conditional knockout (cKO) mice were regenerated with a cleaner genetic profile (C57BL/6J) via Cre/LoxP system. Cardiac function of flox and cKO confirmed mice is assessed every 4 weeks via transthoracic echocardiogram (TTE) and electrocardiogram. We used the following antibodies: βII-spectrin (lab created), Ankryin B (lab created), βI (Novos), Nav (Abcam), Cav1.2 (Abcam), RyR receptor 2 (Abcam), Girk1 (alomone), Girk4 (alomone). A 2-tailed Student's t-test was performed on all data obtained from immunoblots. A p value of Results βII spectrin cKO mice display HF phenotypes as early as 4 weeks, verified by TTE. The observed ejection fraction is notably lower in the βII spectrin cKO mice compared to control (Flox) mice (LVEF 46.2% vs 57.8%, p=0.046). Expression of ankyrin-B was lower in the conditional knockout mice cardio myocytes relative to flox control (55.6 % vs 76.5%, n=3, p=0.38). Consistent with our previous work, expression of Ryanodine receptor 2 is significantly decreased in cKO relative to flox control (0.78 % vs 2.02, n=3, p=0.036). Conclusions The regeneration of cardiac specific βII-spectrin knockout mice model using the genetically homogenous C57BL/6J mice has been successful and confirmed to have decreased βII spectrin expression. Of note, these cKO mice do not require an external stressor for the development of heart failure phenotypes and exhibit this phenotype quite early on. Future studies we will explore other spectrin based regulatory pathways (SMADs) and assess the role of spectrins in organizing the cardiac dyad.