Titin truncating variants impair myocardial thick-filament activation during stretch and blunt length-dependent activation mediating Frank-Starling

Abstract Background Genetic truncating variants in titin’s A-band (TTNtv) are commonly associated with dilated cardiomyopathy (DCM). In the clinic, patients with TTNtv are known to have impaired Frank-Starling responses, limiting the ability of the heart to adequately adapt to changing loading conditions and/or myocardial stretch. The mechanism(s) mediating the impaired stretch-response in TTNtv myocardium remains to be elucidated, however, altered thick-filament mechano-sensing during stretch, resulting in blunted recruitment of contractile myosin-heads, has been hypothesized to underlie such dysfunction. These studies leveraged a novel large-animal model and 3D engineered heart tissues (EHT) of human induced pluripotent stem cells (iPSCs) derived myocytes, both carrying TTNtv mutations to evaluate thick-filament structure and mechano-sensing. Methods Biomechanical relationships and sarcomere structure (via small-angle x-ray diffraction) were evaluated (at varying sarcomere lengths, SL) in permeabilized LV fibers from heterozygous TTNtv Yucatan minipigs and age-matched wild-type animals. Thick-filament reflections indicative of myosin-head recruitment in response to stretch (SL increase from 2.1 to 2.3 µm) at rest were studied. Intact twitch-force parameters at rest, and in response to ±6 % changes in resting length were studied in iPSC-EHTs with either a TTNtv or isogenic controls. Results At rest (at 2.1µm), thick-filament organization (OFF/ON states) appeared vastly indistinguishable between WT and TTNtv fibers, showing similar myosin head mass distribution between the thick and thin filaments (I1,1/I1,0: 0.28 ± 0.01 vs. 0.29 ± 0.02, NS). TTNtv fibers, however, expressed wider spacing between thick filaments (d1,0: 36.83 ± 0.17 vs. 37.39 ± 0.16, nm, P < 0.05), suggesting impaired sarcomere structure and a lower probability of cross-bridge formation. Upon stretch (to 2.3 µm), TTNtv fibers also had blunted responses in the M6 (ΔSM6: 0.25 ± 0.03 vs. 0.15 ± 0.03, %, P < 0.05) and M3 (ΔIM3: -29.09 ± 2.27 vs. -20.91 ± 2.45, %, P < 0.05) reflections, indicating improper transmission of stress to the thick-filament and limited myosin-head OFF-ON transition. In line with these observations, TTNtv EHTs had comparable twitch force time areas to controls (at resting length, nFTI: 0.20 ± 0.01 vs. 0.19 ± 0.01 mN*s, NS), but markedly blunted force/length relationships (LDA slope: 9.9 ± 0.5 vs. 13.3 ± 0.6, P< 0.05). Conclusion Taken together, these multi-system observations demonstrate that titin truncating variants (TTNtv) impair thick-filament mechano-sensing, limiting the recruitment of working myosin-heads during stretch, and blunting length-dependent activation (LDA) mediating the Frank-Starling law of the heart. If confirmed in DCM, these data could suggest the pharmacological enhancement of myosin-recruitment and LDA as a potential mechanism-based targeted therapy for TTNtv patients.