Abstract P2005: Loss Of Full-length Mylk3 Causes Dilated Cardiomyopathy Via A MYL2-independent Mechanism

Introduction: We recently discovered C57BL/6N mice develop dilated cardiomyopathy (DCM), while the closely related C57BL/6J do not. We identified a variant (Chr8:85365179A>T, c.-5T>A) in myosin light chain kinase 3 ( Mylk3 ) in the C57BL/6N substrain that abolishes translation of the predominant transcript (ENSMUST00000034133). MYLK3 phosphorylates many sarcomere proteins, in particular myosin light chain 2 (MYL2), vital for cardiac development and function. Hypothesis: The MYLK3 variant is sufficient to drive the development of DCM. Results: We generated a novel isogenic mouse line carrying the Mylk3 c.-5T>A variant on the C57BL/6J background (C57BL/6J Mylk3.6N ). We found symptoms of DCM in male mutant mice at just 4 weeks old, with increased left ventricular diameter and volume, thinning of the posterior wall and a reduced ejection fraction. These data suggest the Mylk3 variant alone is sufficient to cause DCM. Surprisingly, we found no difference in phosphorylation of myosin light chain 2 (MYL2) by Phos-Tag® blotting at any age. This was unexpected, as previous reports in Mylk3 -/- mice found complete loss of MYL2 phosphorylation with a similar profile of DCM. However, in our mouse model, loss of MYLK3 is caused by a single point mutation in exon 1 which ablates the primary isoform (ENSMUST00000034133; Q3UIZ8) but western blotting with a C-terminal antibody revealed expression of a shorter isoform that is unaffected by the variant due to a distinct N-terminus (ENSMUST00000122452; D3Z630). There was no difference between the isoforms in their ability to phosphorylate MYL2 suggesting the shorter isoform can compensate. MYLK3 may also interact with ACTN2 and phosphorylate TNNI3. We found no difference in the expression or localisation of ACTN2 in our tissues, although co-IP in cells showed the primary isoform binds more readily to ACTN2, however, there was a significant reduction in TNNI3 S23/24 phosphorylation at all ages in C57BL/6J Mylk3.6N mice. This could affect cross-bridge cycling kinetics by altering the function of TNNI3 within the tropomyosin/troponin complex. Conclusions: Loss of full-length MYLK3 causes DCM via a MYL2-independent mechanism, possibly due to lack of phosphorylation of alternative target(s), such as TNNI3.