Abstract 10240: Key Pathomechanisms of Cardiomyopathy Due to TTN Truncation

Abstract

Introduction: Heterozygous truncating variants in TTN (TTNtv) coding for titin cause dilated cardiomyopathy (DCM) but the underlying pathomechanisms are unclear and disease management remains uncertain. Here, we aim to elucidate the key pathomechanisms of TTNtv-DCM. Hypothesis: Stable expression of truncated titin proteins and titin haploinsufficiency characterize TTNtv-DCM and represent potential targets for therapy. Methods and Results: We studied left ventricular tissues from 14 nonfailing donor hearts and 113 endstage failing DCM patients and identified a TTNtv in 22 DCM patients (19.5%) by next generation sequencing. Using titin protein gel electrophoresis and western blot we demonstrate, for the first time, titin haploinsufficiency in TTNtv-DCM hearts. Strikingly, all 21 adult TTNtv-DCM hearts of our cohort showed stable expression of truncated titin proteins. Expression was variable, up to one-half of the total titin protein pool, and negatively correlated to patient age at heart transplantation. Truncated titin proteins were not detected in sarcomeres but were present in intracellular aggregates. Deregulated ubiquitin-dependent protein quality control was apparent. Next, we produced human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), comparing wildtype controls to cells with a patient-derived, prototypical A-band TTNtv or a CRISPR/Cas9-edited M-band TTNtv. TTNtv hiPSC-CMs showed reduced wildtype titin expression and contained truncated titin proteins, whose proportion increased upon inhibition of proteasomal activity. In engineered heart muscle generated from hiPSC-CMs, depressed contractility caused by TTNtv could be reversed by correction of the mutation using CRISPR/Cas9, thereby eliminating truncated titin proteins and raising wildtype titin content. Functional improvement also occurred when titin protein content was increased by proteasome inhibition. Conclusions: Our study reveals the major pathomechanisms of TTNtv-DCM, which include titin haploinsufficiency as a life-long condition and truncated titin protein enrichment with aggregate formation, along with aberrant protein quality control. Results can be exploited for new therapies of TTNtv cardiomyopathies.