Abstract Body: The heart is a functional syncytium composed of millions of cells that must beat in unison to ensure effective blood circulation. This coordinated contraction requires the rapid transmission of electro-mechanical information from cardiomyocyte to cardiomyocyte. This transmission is facilitated by the Intercalated Disc (ICD) – a specialized microdomain of the cardiac sarcolemma that couples adjacent cardiomyocytes and is known to be dysregulated in disease. N-cadherin, the sole classical cadherin molecule at the ICD, serves as both a receptor and ligand for communicating mechanochemical signals between cells.
Obscurins are a family of cytoskeletal proteins with known regulatory roles in myofibrillogenesis and cellular adhesion. As signaling molecules, Obscurins act as linkers between the sarcomere and the various subcellular domains of muscle cells including the sarcoplasmic reticulum and the ICD. Obscurin-B (~870 kDa),the largest known isoform, has a unique dual kinase motif consisting of two enzymatically active kinases, Kin1 & Kin2. In particular, our group has demonstrated that Kin1 promotes cardiomyocyte adhesion and chemical coupling via phosphorylation of N-cadherin, at Serine 788.
Despite Kin1’s crucial role in cardiomyocyte communication, the regulatory mechanisms that modulate its function remain undefined. In vitro binding & kinase activity assays support Kin1’s regulation by calmodulin (CaM) as was predicted by sequence homology to the Myosin Light Chain Kinase (MLCK) family. Interestingly, in silico modeling suggests that mechanical force can induce unwinding of Kin1’s regulatory domain independent of CaM binding. This unwinding promotes Kin1’s conformational change to a new semi-stable intermediate state that we hypothesize is still capable of substrate binding and/or enzymatic activity. To address this hypothesis, the mechanical properties of Kin1 are being assessed using single molecule magnetic tweezer force spectroscopy. This approach facilitates measurement of key folding & unfolding parameters of Kin1 in the physiological force regime and will provide a mechanistic understanding of the effects that biomechanical stimuli elicit on Kin1’s activity.