https://doi.org/10.1007/s00392-024-02526-y
1Universitätsklinikum Münster Institut für Physiologie II Münster, Deutschland; 2Universitätsklinikum Münster Klinik für Radiologie Münster, Deutschland; 3Universitätsklinikum Schleswig-Holstein Klinik für Innere Medizin V mit dem Schwerpunkt Angiologie Kiel, Deutschland
Question:
Titin abnormalities play a crucial role in various heart disorders. However, the impact of titin stiffness loss on cardiac activity is incompletely understood. Using the titin cleavage (TC) mouse model, we aimed to investigate how cleavage of elastic titin in living hearts may affect cell growth, tissue remodeling, and cardiac function.
Titin abnormalities play a crucial role in various heart disorders. However, the impact of titin stiffness loss on cardiac activity is incompletely understood. Using the titin cleavage (TC) mouse model, we aimed to investigate how cleavage of elastic titin in living hearts may affect cell growth, tissue remodeling, and cardiac function.
Methods:
The TC mouse contains a tobacco etch virus protease (TEVp) recognition cassette in the titin springs. Titin was cleaved in vivo by cardiac specific overexpression of AAV9-TEVp plasmid, with AAV9-eGFP-injection as a control. Immunostaining of Ki67 and cardiomyocyte (CM) nuclei marker pericentriolar material 1 (PCM1), along with DAPI staining, allowed counting of cycling cells. Wheat germ agglutinin (WGA) was marked for quantification of CM number and area; picrosirius red staining helped evaluate fibrosis. Sarcomere integrity was assessed by transmission electron microscopy (TEM), ventricular morphology and function by cardiac MRI.
The TC mouse contains a tobacco etch virus protease (TEVp) recognition cassette in the titin springs. Titin was cleaved in vivo by cardiac specific overexpression of AAV9-TEVp plasmid, with AAV9-eGFP-injection as a control. Immunostaining of Ki67 and cardiomyocyte (CM) nuclei marker pericentriolar material 1 (PCM1), along with DAPI staining, allowed counting of cycling cells. Wheat germ agglutinin (WGA) was marked for quantification of CM number and area; picrosirius red staining helped evaluate fibrosis. Sarcomere integrity was assessed by transmission electron microscopy (TEM), ventricular morphology and function by cardiac MRI.
Results:
Two weeks after AAV9-TEVp injection, 48 ± 3.4% (mean ± SEM) of titin were cleaved in homozygous TC mice (N=5). Cardiac MRI analyses on days 6 and 13 post-injection revealed a significant decrease in left ventricular diameter and volume during both systole and diastole, in TEVp-injected mice (N=12) compared to eGFP-controls (N=6). The systolic thickness of the interventricular septum increased, while the outer heart diameter remained unchanged. Cardiac output was significantly reduced but not ejection fraction. Microscopic analysis of CM count per area and CM size showed no evidence of hypertrophy or hyperplasia in titin-cleaved (N=6) vs. control mice (N=5). Time-resolved Ki67-immunostaining revealed a mitotic index of 7.64 ± 0.55% (N=3) on day 6 post-TEVp-injection, declining to 2.22 ± 0.38% (N=4) on day 13 post-injection, whereas in eGFP-injected mice (N=9), less than 1% of cells were Ki67-positive. Actively cycling cells exclusively were PCM-1-negative non-CMs, probably fibroblasts. Fibrotic cardiac area was 1.44 ± 0.52% in AAV9-GFP-injected hearts (N=6), increasing to 18.12 ± 2.19% two weeks post-TEVp-injection (N=12). At that time point, ultrastructural changes to CMs included the formation of degenerated areas and granulofilamentous aggregates, high abundance of lysosomes, and sarcomere damage, including Z-disc streaming and loss of myofibril orientation.
Conclusions:
Specific in-vivo titin cleavage causes concentric cardiac growth, likely explained by fibrotic remodeling, and severely compromises living heart function.