Abstract 14037: A Shorter Myosin Light Chain Kinase 3 Isoform Maintains Myosin Light Chain 2 Phosphorylation but Does Not Attenuate the Dilated Cardiomyopathy Seen in C57BL/6N Mice

Introduction: The C57BL/6J and C57BL/6N are two of the most commonly used mouse strains in biological research. We recently discovered C57BL/6N males develop a modest dilated cardiomyopathy (DCM) by 12 months, which is absent from the closely related C57BL/6J. We identified a variant (Chr8:85365179A>T, c.-5T>A) in myosin light chain kinase 3 ( Mylk3 ) in the 5’ UTR of the primary isoform resident to C57BL/6N substrains, which abolishes translation of this isoform. Myosin light chain 2 (MYL2) is the primary target of MYLK3, and its phosphorylation is vital for survival. Hypothesis: Loss of MYLK3 expression alters phosphorylation of key cardiac proteins. Results: Surprisingly, we found no difference in MYL2 phosphorylation by western blot between C57BL/6N and C57BL/6J hearts, at any of the reported target residues. Phosphoproteomics corroborated this, but highlighted significantly different phosphorylation at a number of phosphosites in other proteins linked to cardiomyopathies and cardiac development, particularly a reduction of a number of residues in ACTN2. We also detected peptide sequences that mapped to the C-terminus and middle of MYLK3 in the C57BL/6N. Western blotting with a C-terminal specific antibody revealed expression of a shorter isoform, present at lower levels in both strains, that is unaffected by the variant due to a distinct N-terminus. Using this antibody in IHC showed similar spatial expression of MYLK3 in hearts from both strains, localizing to the Z-disc, although the expression was much lower in the C57BL/6N presumed to be due to lack of the more abundant full-length isoform. Using immunoprecipitation, we found both isoforms bound to MYL2 in vivo and in vitro , and both were capable of phosphorylating MYL2 in vitro. However, the shorter isoform was not as readily bound to alpha-actinin-2 (ACTN2), a key sarcomeric protein that is reported to bind full-length MYLK3 isoform by its N-terminus. Conclusions: This data indicates loss of full-length MYLK3 causes DCM via a MYL2-independent mechanism, possibly due to lack of phosphorylation of alternative target(s), such as ACTN2. The expression of the shorter MYLK3 isoform in C57BL/6N mice may explain the persistence of MYL2 phosphorylation in these mice and enable their survival.