MYLK3

MYLK3 is a cardiac-specific myosin light chain kinase that phosphorylates myosin regulatory light chain 2 (MLC2) and is essential for sarcomere organization and normal cardiac function (PMID:29235529, PMID:32213617). Loss-of-function mutations in MYLK3 in humans cause familial dilated cardiomyopathy with markedly reduced MLC2 phosphorylation (PMID:29235529), and a null mutation in mice recapitulates this phenotype with eccentric hypertrophy and sarcomere disorganization (PMID:32213617). MYLK3 expression in cardiomyocytes is post-transcriptionally repressed by miR-200c, which reduces p-MLC2 levels and contributes to cardiac hypertrophy (PMID:30680929).

1. 2017 High

   Establishing MYLK3 as a human disease gene: it was unknown whether MYLK3 loss of function causes cardiomyopathy in humans; whole-exome sequencing of familial dilated cardiomyopathy kindreds identified two distinct loss-of-function MYLK3 mutations that abolished protein expression and MLC2 phosphorylation, directly linking MYLK3 kinase activity to human cardiac disease.

   Evidence Whole exome sequencing with segregation analysis, in vitro expression of mutant constructs, immunohistochemistry for phospho-MLC2 in patient myocardium

   PMID:29235529

   Open questions at the time

   • Structural basis for how the read-through and frameshift mutations disrupt kinase function is not defined
   • Whether residual kinase-dead MYLK3 protein exerts dominant-negative effects versus simple haploinsufficiency is unresolved
   • No therapeutic rescue experiments were performed
2. 2019 Medium

   Defining post-transcriptional regulation of MYLK3: it was unclear how MYLK3 expression is modulated in cardiac stress; miR-200c was shown to directly target MYLK3 mRNA, and its manipulation bidirectionally controlled MYLK3 and p-MLC2 levels, establishing a regulatory axis linking a stress-responsive miRNA to sarcomere phosphorylation.

   Evidence miRNA mimic/inhibitor transfection in neonatal rat cardiomyocytes with Western blotting and qRT-PCR readouts; aortic banding rat hypertrophy model

   PMID:30680929

   Open questions at the time

   • Direct binding of miR-200c to the MYLK3 3′-UTR was not validated by luciferase reporter assay in this study
   • Whether miR-200c regulation of MYLK3 operates in adult human cardiomyocytes is untested
   • Contribution of miR-200c–MYLK3 axis relative to other hypertrophy pathways is not quantified
3. 2020 High

   Confirming MYLK3 essentiality in vivo: the question of whether complete MYLK3 loss is sufficient to cause cardiac failure was resolved by characterizing a spontaneous Mylk3-null mouse, which developed dilated cardiomyopathy with eccentric hypertrophy and sarcomere disorganization, validating the human genetic findings in a mammalian model.

   Evidence RNAseq variant calling, immunofluorescence of cardiomyocyte sarcomeres, echocardiography in C57BL/6N Mylk3-null mice

   PMID:32213617

   Open questions at the time

   • Whether cardiac-restricted re-expression of MYLK3 rescues the phenotype has not been tested
   • Specific MLC2 phosphorylation sites affected by MYLK3 loss were not mapped in this study
   • Temporal progression from MYLK3 loss to sarcomere disorganization is not characterized

• Key unresolved questions include the structural basis for MYLK3 substrate selectivity, whether MYLK3 phosphorylates additional cardiac substrates beyond MLC2, and whether pharmacological augmentation of MYLK3 activity or expression can reverse established dilated cardiomyopathy.
• No crystal structure or cryo-EM model of MYLK3 exists
• Full substrate repertoire of MYLK3 in the heart has not been determined by unbiased phosphoproteomics
• No gain-of-function rescue study has been reported in any cardiomyopathy model