Abstract Tu097: Titin cleavage induces loss of myocardial tensional homeostasis driving a rapid TGF-beta-independent fibrotic response

Abstract

Background: Cardiomyocytes (CMs) are subjected to different types of mechanical loads in the myocardium. Depending on its origin these loads can be classified as extracellular (extracellular matrix-ECM towards CMs), intracellular (exerted by the CM contraction machinery, i.e. the sarcomeres) or intercellular (exerted between CMs). In the myocardium, all these loads are actively maintained in tensional homeostasis (TH). In this context, the study of the sarcomere protein titin is essential, because its I-band region is considered as a hub for mechanotransduction, mostly of intracellular mechanical cues. Research Questions: It has been shown that mechanical disruption of the ECM affects the myocardial TH, leading to morphological changes inside CMs. However, potential alteration of TH resulting from defective mechanics of CM proteins remains unknown due to the lack of specific tools for in vivo studies. Aims: Here we explore the in vivo effects on myocardial TH of an abrupt tensional unloading of titin. Methods: We have experimentally challenged CMs to a tensional unloading by means of titin cleavage in vivo using TEVs-TTN mice. In this model, a cassette with a Tobacco Etch Virus protease (TEVp) recognition sequence has been included in the I-band of titin. Transducing TEVp with adeno-associated virus (AAV), we were able to cleave titin in vivo , ceasing only the mechanical properties of the protein. Samples were analyzed using an array of techniques including histology, immunofluorescence, and bulk and single nuclei RNA sequencing. Results: Our results show that a mosaic expression of TEVp, which does not affect cell viability, cleaving no more than 30% of total titin. However, this leads to a fast fibrotic response (less than six days), characterized by an increase in interstitial collagen and a response from cardiac fibroblasts population. Transcriptional and phospo-SMAD2/3 analysis results suggest that this ECM response occurs without noticeable activation of the TGFβ canonical pathway. Conclusion: Taken together, our results show experimentally for the first time that titin is not only a mechanotransducer of intracellular cues. In fact, mechanical abrogation of the I-band leads to a fast ECM response mediated by cardiac fibroblasts.