Abstract Body:
State of the art
Myocardial remodeling including reactive fibrosis and disconnection of cardiomyocytes is at the basis of arrhythmias and heart failure associated to many conditions of the heart. Although many fibrotic mechanisms have been uncovered in recent years, there is still limited understanding of the full landscape of fibrotic pathways in the heart, limiting therapeutic opportunities.

Hypothesis
Based on the observation that cleavage of titin, a fundamental protein of the sarcomere, is found in several conditions that result in myocardial remodeling, including ischemia/reperfusion damage, chemotherapy-induced cardiotoxicity and the metabolic syndrome, here we hypothesized that titin cleavage itself could be a trigger of fibrotic remodeling.

Methods
To test our hypothesis, we have engineered a method that allows, for the first time to the best of our knowledge, specific cleavage of proteins systemically. Our approach relies on the specific insertion of a tobacco etch virus protease (TEVp) recognition site in titin (TEVs-TTN). Upon tail vein injection of adeno associated viruses expressing TEVp, titin in TEVs-TTN mice is cleaved specifically. Wild-type animals are not affected by the treatment. Tissues are studied using immunofluorescence, fibrosis markers and transcriptomics including single-nuclei RNAseq. We have also implemented the titin cleavage system in primary cardiomyocytes to study cell-autonomous mechanisms in relation to cardiomyocyte adhesion.

Results
We show that 30% mosaic cardiac titin cleavage leads to fast reactive fibrosis (less than 6 days after viral delivery) affecting the whole myocardium and in the absence of cardiomyocyte death. This remodeling response results from activation of cardiac fibroblasts involving ERK1/2 signaling. In addition, titin cleavage results in loss of connexin 43 and reduced cardiomyocyte-cardiomyocyte adhesion strength. Single-nuclei RNAseq captures specific activation of integrin alpha5-beta1 in the approximately 15% cardiomyocytes that contain cleaved titin and mostly disrupted sarcomeres. By analyzing earlier time points after TEVp delivery, we find that cardiomyocyte disengagement and fibrosis are concurrent.

Conclusion
Our work uncovers a previously unappreciated mechanistic role of titin cleavage in the development of myocardial reactive fibrosis.