Cardiac MLC2 kinase is localized to the Z-disc and interacts with α-actinin2

Figures

• 

  Figure 1. Expression of cMLCK proteins in mouse and human hearts. Representative co-immunostaining images of cMLCK, MLC2v and α-actinin2 using hearts from wild-type (A,B) or conditional Mylk3 knockout mice (C). Co-immunostaining of cMLCK and α-actinin2 in wild-type mouse (D) and human hearts without apparent disease (E). (F) Co-immunostaining of cMLCK and α-actinin2 (left panel) and cMLCK and M-line protein myomesin (right panel) in isolated mouse adult cardiomyocytes. Bars = 10 µm in A, B, D, E; 20 µm in C; 100 or 20 µm in F.

• 

  Figure 2. Interaction between cMLCK and α-actinin2. (A) A schematic of screening methodology to identify proteins interacting with cMLCK. (B) Representative mass spectra for cMLCK and α-actinin2 showing relative intensity (y-axis) and mass-to-charge ratio (m/z, x-axis). Co-immunoprecipitation of cMLCK and α-actinin2 using mouse heart lysates (C) and 293 cell lysates expressing exogenous HA-cMLCK and/or Myc-α-actinin2 (D). (E) A schematic of experiment for analysis of binding kinetics of cMLCK and α-actinin2 using biolayer interferometry (BLI). (F) Representative data showing association and dissociation kinetics of cMLCK to α-actinin2 using four different cMLCK concentrations. Mean values ± S.E.M (n = 2).

• 

  Figure 3. Identification of four amino acids located in the domain unique to the cardiac isoform required for interacting with α-actinin2. (A) Schematics of cMLCK protein including four amino acids 171–174 at the N-terminus region specific for cardiac isoform. (B) Two N-terminus deletion mutants, HA-tagged cMLCK(171–795) and cMLCK(175–795), were mixed with Myc-tagged full-length α-actinin2. Presence ( + ) and absence (−) of interaction between two proteins were indicated. (C) Internal deletion mutant of cMLCK (Δ171–174), and (D) four amino acid substituted mutants from 171DVKP174 to 171AAAA174 were mixed with Myc-tagged full-length α-actinin2. (E) Schematics of α-actinin2 protein including the second actin binding site. (F) Two Myc-tagged N-terminus deletion mutants of α-actinin2 with or without ABS2 domain were mixed with HA-full-length cMLCK. The additional results were available in Supplemental Fig. 1. ABS, F-actin binding site; SR, spectrin repeat; EF, EF hand motif.

• 

  Figure 4. 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 Mylk3flox/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.

• 

  Figure 5. Effect of cMLCK mutations in adult cardiomyocytes. (A) Representative beta gal-stained heart 1 week after a single injection of adenovirus-lacZ (1 × 1011 viral particle in 30 µl) into the anterior LV wall. (B) Time schedule of experiments. (C) Representative Western blotting with HA and GAPDH antibodies using whole heart lysates at 2, 4 and 7 days after injection of adenovirus. (D) Representative adult cardiomyocytes isolated from Mylk3flox/flox/Cre mice infected with three viral constructs co-immunostained with HA (green) and α-actinin2 (red). Enlarged images were shown at the bottom. See Supplemental Fig. 3 for additional immunostaining images. (E) Summary of cellular area (µm2), long-axis (µm), and short-axis (µm). Number of cardiomyocytes measured was indicated on graph. Mean ± S.E.M. Results were compared using one-way ANOVA followed by post-hoc test (SPSS ver. 25). *P < 0.05.