In vivo cleavage of cardiac titin springs disrupts cardiomyocyte connectivity and integrity, rapidly triggers fibrosis, and leads to concentric remodeling with impaired diastolic and systolic function

The giant protein titin serves as a molecular spring and a template for sarcomere assembly in cardiomyocytes (CM). Cardiac titin governs CM passive stiffness and is a crucial contributor to the elasticity of the whole heart. Titin-based forces finely regulate diastolic strain, bear passive load, and help determine the Frank-Starling mechanism. Changes in titin stiffness are thought to disrupt the mechanical properties and compromise the function of failing hearts. However, the impact of titin stiffness loss on beating heart function remains largely unknown.

To study the effects of in vivo elastic titin cleavage on cardiac remodeling and living heart function using the titin cleavage (TC) mouse model.

In the TC mouse, a tobacco etch virus protease (TEVp) recognition site is cloned into the titin springs. Titin was cleaved in vivo through AAV9-mediated, cardiac-specific overexpression of TEVp plasmid, with AAV9-eGFP plasmid injection as a control. On heart tissue sections, wheat germ agglutinin (WGA) staining marked cell borders; anti-Myotilin and anti-TTN-M were used to assess sarcomere integrity, and Ki67, PCM1, and periostin staining to identify proliferating cells. Living heart structure and function were evaluated by transthoracic echocardiography (TTE) and cardiac magnetic resonance imaging (cMRI).

In homozygous TC mice, ~10% of cardiac titin were cleaved 3 days post-TEVp injection, increasing to ~40% on day 6 and ~55% on day 13. CM hypertrophy and hyperplasia were absent in TEVp-injected mice vs. eGFP controls. However, proliferating cardiac cells were identified using marker Ki67: on day 6 post-TEVp injection, 6.05±0.56% (mean±SEM) of cells were Ki67-positive, declining to 1.70±0.34% by day 13, whereas in eGFP-injected controls, less than 1% of cells were Ki67-positive. Actively cycling cells were identified as PCM1-negative non-CM and periostin-positive proliferating fibroblasts. On day 13 post-TEVp injection, but not yet on day 6, massive fibrosis was present. Anti-Myotilin/WGA/TTN-M co-staining of cardiac tissue revealed increasingly expanded intercalated discs, progressive loss of sarcomeres, and widening of the inter-CM space, in titin-cleaved hearts between days 6 and 13 post-TEVp injection. TTE and cMRI showed significant reductions in left ventricular (LV) diameter and volume at both end-systole and end-diastole, in TEVp vs. eGFP controls, beginning on day 6 post-injection. Thickening of the interventricular septum and LV posterior wall was also evident, but LV mass remained unchanged. LV stroke volume and cardiac output significantly declined, while LV ejection fraction was preserved. Pulsed-wave Doppler imaging showed a decrease in both E wave (early filling) and A wave (late filling) velocities, along with a lower E’/A’ ratio calculated from tissue Doppler. The pressure gradient across the pulmonary valve (PV) and PV annular velocity were reduced, and right ventricular output was significantly decreased, in titin-cleaved hearts.

Cleavage of cardiac titin springs in living hearts disrupts CM connectivity and substructure, induces fibroblast activation followed by fibrosis, and thus impairs both left and right ventricular performance. Loss of titin stiffness does not trigger eccentric remodeling but instead, causes diastolic dysfunction characterized by slowed elastic recoil, restricted ventricular filling, and concentric remodeling, accompanied by systolic dysfunction.