A cardiac myosin light chain kinase regulates sarcomere assembly in the vertebrate heart
Osamu Seguchi, Seiji Takashima, Satoru Yamazaki, Masanori Asakura,, Yoshihiro Asano, Yasunori Shintani, Masakatsu Wakeno, Tetsuo Minamino, Hiroya Kondo, Hidehiko Furukawa, Kenji Nakamaru, Asuka Naito,, Tomoko Takahashi, Toshiaki Ohtsuka, Koichi Kawakami, Tadashi Isomura,, Soichiro Kitamura, Hitonobu Tomoike, Naoki Mochizuki, Masafumi Kitakaze
Osamu Seguchi, Seiji Takashima, Satoru Yamazaki, Masanori Asakura,, Yoshihiro Asano, Yasunori Shintani, Masakatsu Wakeno, Tetsuo Minamino, Hiroya Kondo, Hidehiko Furukawa, Kenji Nakamaru, Asuka Naito,, Tomoko Takahashi, Toshiaki Ohtsuka, Koichi Kawakami, Tadashi Isomura,, Soichiro Kitamura, Hitonobu Tomoike, Naoki Mochizuki, Masafumi Kitakaze
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Research Article Cardiology

A cardiac myosin light chain kinase regulates sarcomere assembly in the vertebrate heart

Abstract

Marked sarcomere disorganization is a well-documented characteristic of cardiomyocytes in the failing human myocardium. Myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v), which is involved in the development of human cardiomyopathy, is an important structural protein that affects physiologic cardiac sarcomere formation and heart development. Integrated cDNA expression analysis of failing human myocardia uncovered a novel protein kinase, cardiac-specific myosin light chain kinase (cardiac-MLCK), which acts on MLC2v. Expression levels of cardiac-MLCK were well correlated with the pulmonary arterial pressure of patients with heart failure. In cultured cardiomyocytes, knockdown of cardiac-MLCK by specific siRNAs decreased MLC2v phosphorylation and impaired epinephrine-induced activation of sarcomere reassembly. To further clarify the physiologic roles of cardiac-MLCK in vivo, we cloned the zebrafish ortholog z–cardiac-MLCK. Knockdown of z–cardiac-MLCK expression using morpholino antisense oligonucleotides resulted in dilated cardiac ventricles and immature sarcomere structures. These results suggest a significant role for cardiac-MLCK in cardiogenesis.

Authors

Osamu Seguchi, Seiji Takashima, Satoru Yamazaki, Masanori Asakura,, Yoshihiro Asano, Yasunori Shintani, Masakatsu Wakeno, Tetsuo Minamino, Hiroya Kondo, Hidehiko Furukawa, Kenji Nakamaru, Asuka Naito,, Tomoko Takahashi, Toshiaki Ohtsuka, Koichi Kawakami, Tadashi Isomura,, Soichiro Kitamura, Hitonobu Tomoike, Naoki Mochizuki, Masafumi Kitakaze

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Figure 1

Microarray analysis for candidate gene selection.

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Microarray analysis for candidate gene selection. (A) Flowchart for the ...
(A) Flowchart for the selection of candidate genes. (B) The relative expression levels of 75678_at correlated well with the relative PAP values in the respective patients. (C) Tissue localization of the candidate gene expression was analyzed using the GeneExpress database; 75678_at was specifically expressed in the heart. (D) Each MLCK family member possesses a highly conserved serine-threonine kinase domain in the C-terminal region of the protein. Amino acid residues on black backgrounds are the most commonly conserved residues at each position; residues on gray backgrounds are similar to the consensus amino acids. (E) Expression analysis of MLCK family members using multiple human tissue Northern blot membranes. The 2 transcripts transcribed from MYLK (encoding smMLCK) were ubiquitously expressed with the exception of skeletal muscle, thymus, and peripheral blood leukocytes. In contrast, MYLK2 (encoding skMLCK) and MYLK3 (encoding cardiac-MLCK) were only expressed in skeletal muscle and heart, respectively. (F) Fluorescence microscopy of cardiomyocytes cultured in serum-free conditions and infected with adenovirus encoding LacZ (Ad-LacZ) revealed predominantly round-shaped cells with disorganized sarcomere structures. Infection with adenovirus encoding cardiac-MLCK (Ad-cMK) at a MOI of 120 increased the number of the cells with organized sarcomere structures. Original magnification, ×1,000. (G) The percentage of cells with organized sarcomeres was significantly higher in cardiomyocytes infected with adenovirus encoding cardiac-MLCK than in those infected with adenovirus encoding LacZ. Values are mean ± SEM. *P < 0.001.