Abstract Body: Introduction: Heart failure (HF) is the result of cardiac remodelling and impaired cardiac function due to cardiac insult. These mechanisms are strongly influenced by mechanical signalling such as those originating at extracellular matrix (ECM) adhesions. Talin 2 is a 270kDa protein that links cardiomyocyte actin filaments to integrins in the ECM. Its conformation-dependent interactions with accessory proteins regulate contractility in response to mechanical stress. Force-dependent post-translational modifications (PTMs) of Talin 2 alter binding partner competition, and may induce sustained, force-independent changes contributing to HF progression. This study aims at studying Talin 2 dynamics in health and disease using living myocardial slices (LMS).
Methods: The left ventricle of healthy human donor hearts was sectioned into 300 µm thick LMS using a high-precision vibratome. LMS were cultured on auxotonic load in a bioreactor, with electrical stimulation and media circulation. LMS were stretched at 23% or 34% of their slack length to recreate physiological load and volume overload, respectively. Cardiac function was monitored until day 7, when LMS were processed for further experiments. Immunostaining was performed to quantify cardiomyocyte size, sarcomere length and Talin 2. qPCR was performed to assess development of hypertrophy, fibrosis and inflammation. Protein lysates were used to perform immunoprecipitation of Talin 2 for mass spectrometry and phosphoproteomics.
Results: Active force production did not differ between groups. Volume overload increased sarcomere length as expected (2.43 µm vs. 2.77 µm) and cardiomyocyte length (P<0.05). ACTA1 expression was significantly upregulated in overload conditions, with trends of increased fibrotic and inflammatory markers FN1, MMP2, and IL6. Talin 2 gene expression was significantly reduced in overloaded LMS (P<0.05) (N=4). Mass spectrometry showed strong Talin 2 association (FDR<0.01) with proteins involved in cardiac contraction, cytoskeletal organization, and actin dynamics. Phosphoproteomics identified one phosphorylation site at Y1843.
Conclusion: Overload induces hypertrophic remodelling and downregulates Talin 2 gene expression in human LMS. Y1843 is a phosphorylation site located in the Talin 2 R10 rod domain, a mechanosensitive region that regulates recruitment of adhesion proteins such as vinculin and actin-associated complexes, which can be targeted to understand cardiac dysfunction.