Phosphatase regulatory subunit MYPT2 knockout partially compensates for the cardiac dysfunction in mice caused by lack of myosin light chain kinase 3

Figures

• 

  Figure 1. MYPT2 null C57BL/6N mice are viable and have reduced PP1Cδ in the heart.A, agarose gel electrophoresis of MYPT2 genotyping PCR products in WT (+/+), heterozygous (+/−), and homozygous (−/−) mice. The WT allele produces a 775 base pair product, and the mutant allele produces a 195 base pair product. B, Western blots of heart lysates from 12-week-old WT (+/+), heterozygous (+/−), and homozygous (−/−) MYPT2 KO mice using pan-MYPT, PP1Cδ, PP1Cγ, and β-actin antibodies. C–F, quantification of MYPT2 (panel C), MYPT1 (panel D), PP1Cδ (panel E), and PP1Cγ (panel F) expression from Western blots performed as in panel B. Graphs represent means ± SD; ∗p > 0.05%, ∗∗∗p > 0.001 using multiple comparisons (panel C) or two-tailed Students t test (panel E). C, non-template control; L, DNA ladder; MYPT2, Myosin Phosphatase Targeting Subunit 2.

• 

  Figure 2. Phosphorylation of MLC-2v is increased in MYPT2 null C57BL/6N mice.A, heart lysates were separated by glycerol/urea gel electrophoresis, in which phosphorylated MLC-2v runs faster than unphosphorylated MLC-2v. Protein were transferred to nitrocellulose membranes and blotted with an anti-MLC-2v antibody. Blots were developed using chemiluminescent substrate and proteins were quantified using a Li-COR Odyssey imaging system. B, quantification of four experiments performed as described in panel A. C and D, in vitro phosphorylation of purified murine MLC-2v by MLCK3. Phosphorylation was detected by autoradiography (panel C) or immunoblotting using an antibody specific for Ser15-phosphorylated MLC-2v (panel D). Anti-Flag blots show the relative amount of Flag-tagged MLCK3 used in each reaction (lower blots). E and F, heart lysates were separated by SDS-PAGE and analyzed by immunoblotting using the Ser15-phosphorylated MLC-2v antibody (panel E) or a Ser23/Ser24-phosphorylated troponin I antibody (panel F). G, heart lysates were separated by Phos-tag gel electrophoresis and analyzed by immunoblotting using anti-MYBPC3 antibodies. H, quantification of three mice per genotype as shown in…

• 

  Figure 3. MYPT2 KO C57BL/6N mice have reduced heart weights.A–D, twelve-week-old mice were euthanized using ketamine/xylazine, and organ weights were measured. E, quantitative PCRs were performed to measure the expression levels of mRNA for Nppa, Nppb, Myh7, and Tnnt1 in twelve-week-old mice. Graphs represent means ± SD; ∗p > 0.05%, ∗∗p > 0.01 using two-tailed Students t test; ns, nonsignificant. MYPT2, Myosin Phosphatase Targeting Subunit 2.

• 

  Figure 4. MYPT2 knock-out leads to improved cardiac performance in C57Bl/6N mice. Twenty-four-week-old male mice analyzed by cardiac echo. A, representative M-mode images. B, left ventricular mass (LVM) over body weight (BW). C, fractional shortening (FS). D, left ventricular inner diameter at diastole (LVIDd) over BW. E, left ventricular inner diameter at systole (LVIDs) over BW. Graphs represent means ± SD; ∗p > 0.05%, ∗∗p > 0.01, ∗∗∗p > 0.001, ∗∗∗∗p > 0.0001 using multiple comparisons; ns, nonsignificant. MYPT2, Myosin Phosphatase Targeting Subunit 2.

• 

  Figure 5. Schema showing the proposed model for MYPT2 function. In C57BL/6J mice with MLCK3 and MYPT2, the balanced activity of MLCK3-induced phosphorylation and MYPT2-mediated dephosphorylation leads to a basal MLC-2v phosphorylation of 40% and normal cardiac function. The lack of MLCK3 in C57BL/6N mice disrupts this balance, and the basal level of MLC-2v phosphorylation drops to ∼10%, causing reduced cardiac function. Knocking out MYPT2 in C57BL/6N mice increases MLC-2v phosphorylation to ∼28% and improves cardiac function. P, Ser15 phosphorylation. MYPT2, Myosin Phosphatase Targeting Subunit 2; MLCK, myosin light chain kinase.