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. 2019 Aug 29;9:12580. doi: 10.1038/s41598-019-48884-w

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Generation of a kinase-deficient cMLCK mutant and effects of cMLCK mutations in neonatal cardiomyocytes. (A) Schematics of cMLCK protein including two amino acids ⁵²⁰Lys and ⁵²³Lys in the catalytic domain. (B) In vitro kinase assay using wild-type and kinase-deficient mutant cMLCK(K520/523R) using GST-fused MLC2v as substrates with different concentrations (0.012–3 µM). (C) Representative neonatal cardiomyocytes isolated from Mylk3^flox/flox/Cre mice infected with five adenoviral constructs co-immunostained with α-actinin2 (red) and HA (green). (D) Summary of cellular area (µm²). Number of cardiomyocytes measured indicated on graph. (E) Representative Western blotting with HA, cMLCK and GAPDH antibodies using cardiomyocyte lysates. Mean ± S.E.M. Results were compared using one-way ANOVA followed by post-hoc test (SPSS ver. 25). *P < 0.05.