Cardiac myosin light chain 1 dependent regulatory mechanisms in dilated cardiomyopathy
Final Report Abstract
Genes associated with sarcomeric proteins are implicated in Dilated Cardiomyopathy (DCM), marked by an enlarged heart chamber. The essential myosin light chain (ELC) plays a pivotal role in force transmission and heart contractility. In transgenic zebrafish, the absence of ELC phosphorylation at serine 195 hampers cardiac function. The regulatory landscape of ELC phosphorylation in DCM remains unclear, and the responsible kinases are yet to be identified. Using the APEX assay, we validated the interaction between ELC and NEK9. Employing CRISPR Cas9, we created two NEK9 transgenic zebrafish lines (78del and 500del), resulting in embryos exhibiting a heart failure phenotype reminiscent of ELC deficiency. Specific immunoprecipitation and immunoblotting revealed reduced interaction between ELC and truncated NEK978del, pinpointing the interaction site to the protein kinase domain. This observation was further confirmed with human deletion mutants of NEK9, indicating a decline in interaction with protein kinase domain deletion. Utilizing the Cre-Lox system, we developed an inducible NEK9 heart-specific knockout line, exhibiting dilated cardiomyopathy at five days post-induction and signs of hypertrophy after two weeks. Analysis of ELC phosphorylation through 2D electrophoresis showed significantly reduced phosphorylation at 5 days post-induction, while mouse heart samples after 2 weeks displayed a compensatory increase in ELC posphorylation, indicating alternative kinase activation in the absence of NEK9. In summary, our study affirms the role of NEK9 in regulating ELC phosphorylation, mapping the interaction to the protein kinase domain. We elucidated the diverse roles of NEK9 in murine cardiac function at different time points and highlighted alterations in ELC phosphorylation patterns following NEK9 deficiency. Gene profiling analysis identified potential candidates responsible for ELC phosphorylation in the absence of NEK9, introducing a novel sarcomeric regulatory system and suggesting promising therapeutic targets for future heart disease interventions.
Publications
-
NIMA-related kinase 9 regulates the phosphorylation of the essential myosin light chain in the heart. Nature Communications, 13(1).
Müller, Marion; Eghbalian, Rose; Boeckel, Jes-Niels; Frese, Karen S.; Haas, Jan; Kayvanpour, Elham; Sedaghat-Hamedani, Farbod; Lackner, Maximilian K.; Tugrul, Oguz F.; Ruppert, Thomas; Tappu, Rewati; Martins, Bordalo Diana; Kneuer, Jasmin M.; Piekarek, Annika; Herch, Sabine; Schudy, Sarah; Keller, Andreas; Grammes, Nadja; Bischof, Cornelius ... & Meder, Benjamin
