Experimentally Altering the Compliance of Titin's Spring Region

Abstract

Chapter 1 of this work focuses on alternative splicing of titin as a proof of concept therapy for treating diastolic dysfunction and restrictive filling in a genetic murine model (Ttn^(ΔIAjxn)). The Ttn^(ΔIAjxn) mouse has increased strain on the spring region of titin and acts as a mechanical analogue of the titin-based increase in passive myocardial stiffness found in patients with heart failure and preserved ejection fraction (HFpEF). HFpEF is a complex disease characterized by diastolic dysfunction, exercise intolerance, and concentric hypertrophic remodeling. Approximately half all of heart failure patients suffer from diastolic dysfunction, however, no effective therapy exists for treating this pervasive syndrome. Titin, the largest known protein and molecular spring in the heart, has emerged as a prime candidate for therapeutic targets aimed at restoring compliance to the sarcomere in order to improve diastolic function. Titin has two main cardiac isoforms that are regulated by alternative splicing; the smaller N2B isoform (~3.0 MDa) and the larger more compliant N2BA isoform (~3.3 MDa). Diastolic stiffness of the left ventricle is dependent upon the N2BA:N2B isoform ratio. In the first half of this work, we modified these two primary isoforms by inhibiting the known titin splicing factor Rbm20. We demonstrate that Rbm20 reduction restores diastolic function, improves exercise tolerance and attenuates afterload induced pathologic remodeling of the left ventricle in Ttn^(ΔIAjxn) mice.The work in chapter 2 is focused on studies using the previously published N2B knock out (KO) murine model. The N2B spring element found in cardiac titin's I-band region has been proposed as a sensor and signaling "hot spot" in the sarcomere. This study investigates the role of titin's cardiac specific N2B element as a mechano-sensor for stress and strain induced remodeling of the heart. The N2B KO mouse was subjected to a variety of stressors including transverse aortic constriction (TAC), aortocaval fistula (ACF), chronic swimming, voluntary running and isoproterenol stimulation. Our data revealed that the N2B element is essential in preload stimulated cardiac hypertrophy as well as remodeling due to beta-adrenergic stress. Cardiac hypertrophy is a common maladaptive feature of heart failure patients and the mechanical triggers that determine pathologic growth are not well understood. My work in the N2B KO mouse reveal titin's important role in cardiac remodeling.