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. 2015 Jul 28;112(30):E4138-46.
doi: 10.1073/pnas.1505819112. Epub 2015 Jun 29.

Constitutive phosphorylation of cardiac myosin regulatory light chain prevents development of hypertrophic cardiomyopathy in mice

Chen-Ching Yuan  1 , Priya Muthu  1 , Katarzyna Kazmierczak  1 , Jingsheng Liang  1 , Wenrui Huang  1 , Thomas C Irving  2 , Rosemeire M Kanashiro-Takeuchi  1 , Joshua M Hare  1 , Danuta Szczesna-Cordary  3
Affiliations

Constitutive phosphorylation of cardiac myosin regulatory light chain prevents development of hypertrophic cardiomyopathy in mice

Chen-Ching Yuan et al. Proc Natl Acad Sci U S A. .

Abstract

Myosin light chain kinase (MLCK)-dependent phosphorylation of the regulatory light chain (RLC) of cardiac myosin is known to play a beneficial role in heart disease, but the idea of a phosphorylation-mediated reversal of a hypertrophic cardiomyopathy (HCM) phenotype is novel. Our previous studies on transgenic (Tg) HCM-RLC mice revealed that the D166V (Aspartate166 → Valine) mutation-induced changes in heart morphology and function coincided with largely reduced RLC phosphorylation in situ. We hypothesized that the introduction of a constitutively phosphorylated Serine15 (S15D) into the hearts of D166V mice would prevent the development of a deleterious HCM phenotype. In support of this notion, MLCK-induced phosphorylation of D166V-mutated hearts was found to rescue some of their abnormal contractile properties. Tg-S15D-D166V mice were generated with the human cardiac RLC-S15D-D166V construct substituted for mouse cardiac RLC and were subjected to functional, structural, and morphological assessments. The results were compared with Tg-WT and Tg-D166V mice expressing the human ventricular RLC-WT or its D166V mutant, respectively. Echocardiography and invasive hemodynamic studies demonstrated significant improvements of intact heart function in S15D-D166V mice compared with D166V, with the systolic and diastolic indices reaching those monitored in WT mice. A largely reduced maximal tension and abnormally high myofilament Ca(2+) sensitivity observed in D166V-mutated hearts were reversed in S15D-D166V mice. Low-angle X-ray diffraction study revealed that altered myofilament structures present in HCM-D166V mice were mitigated in S15D-D166V rescue mice. Our collective results suggest that expression of pseudophosphorylated RLC in the hearts of HCM mice is sufficient to prevent the development of the pathological HCM phenotype.

Keywords: X-ray structure; cardiomyopathy; hemodynamics; myocardial contraction; myosin RLC.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Characterization of Tg-S15D-D166V transgenic mice. All assessments were made using three to four hearts of ∼5-mo-old female and male mice of NTg, WT, D166V, and Homo- and Hetero-rescue mice (see SI Methods for details). (A) Transgenic protein expression. Two lines of S15D-D166V mice (L1 = 95 ± 3%, n = 9 and L3 = 97 ± 2%, n = 5) with nearly complete replacement of the endogenous mouse RLC with S15D-D166V are called “Homo” (homozygote); whereas in one line (L5 = 56 ± 3%, n = 11) RLC was replaced ∼50% and is referred to as “Hetero” (heterozygote)-rescue mice. Transgenic S15D-D166V lines were compared with previously generated D166V (93 ± 3%) (5) and WT (L2 = 99 ± 1%) (18). The assessment of transgene expression was achieved in mouse atrium due to different molecular weights of RLC-atrial vs. RLC-ventricular. In ventricles, both mouse endogenous and Tg-human RLC migrate with the same speed due to their similar MW. (B) RLC phosphorylation. Phosphorylation of mouse purified myosin RLC was tested in all groups of mice. Note largely reduced phosphorylation in D166V vs. WT mice, no phosphorylation in Homo-rescue, and ∼50% in Hetero-rescue mice. +P-RLC WT std., positive phosphorylation control; –P RLC WT std., negative phosphorylation control. (C) cMLCK expression by qPCR. The changes in mRNA cMLCK expression in LV heart lysates from WT, D166V, and S15D-D166V Homo- and Hetero-rescue mice are presented as fold change vs. NTg mice (FC = 1). (D) Expression of cMLCK protein by Western blotting. LV tissue from the hearts of all mice was homogenized in CHAPS solution, and 5-µg samples, dissolved in Laemmli buffer, were loaded into 12% (wt/vol) SDS/PAGE. Membranes were probed with a cMLCK-specific antibody (16). GAPDH was probed with ab9485 (Abcam), which was also used as the loading control. No significant differences were observed in cMLCK expression in the hearts of mice from all groups. (E) Phosphorylation of myosin binding protein C (MyBP-C). Phosphorylation of MyBP-C at Ser282 and Ser302 was tested in LV heart lysates from all groups of mice. Approximately 20-µg samples were loaded into 10% SDS/PAGE, and the membranes were probed with phospho-specific MyBP-C antibodies (29). A rabbit polyclonal antibody was used to detect total MyBP-C. GAPDH was used as a loading control. Note no changes in MyBP-C phosphorylation or total MyBP-C protein expression between the groups. (F) Phosphorylation of troponin I (TnI). Phosphorylation of Troponin I at Ser23/24 was tested using 15% SDS/PAGE (∼20 µg protein per lane) and phospho-Ser23/24 and total-TnI antibodies. Total RLC served as a loading control. Note no changes in P-TnI between WT, D166V, and Homo/Hetero-rescue mice.
Fig. 2.
Fig. 2.
Gross morphology (A), histopathology (B), and EM (C) in WT, D166V, and Homo- and Hetero-rescue mice. (A) The sizes of ∼5- and ∼9-mo-old female (F) rescue mice were compared with age- and sex-matched Tg-WT and Tg-D166V mice. Note that the hearts of ∼5-mo-old mice are the same in size whereas those of ∼9-mo-old Tg-D166V mice are much larger than control Tg-WT mice. No changes in heart size of rescue mice were noticed in both age groups of mice. (B) H&E- and Masson’s trichrome-stained heart sections from ∼5-mo-old Tg-WT, Tg-D166V, and Tg-S15D-D166V female mice. Note that the myocardium of D166V-HCM mice shows morphological abnormalities and fibrotic lesions that are not observed in the myocardium of Tg-S15D-D166V mice. (C) H&E- and Masson’s Trichrome-stained heart sections from ∼9-mo-old Tg-WT, Tg-D166V, and Tg-S15D-D166V mice. Similar to histopathology images of the hearts from young D166V-HCM mice, ∼9-mo-old animals also show distinct abnormalities and severe fibrotic lesions. In contrast, the hearts of rescue mice display no histopathological changes, and the myocardium of Tg-S15D-D166V mice looks similar to age-matched Tg-WT mice. (D) Rescue of sarcomeric ultrastructure in the hearts of Tg-S15D-D166V mice. Note severe myofilament disarray in the myocardium from D166V mice compared with WT mice. Sarcomeric abnormalities seen in HCM-D166V mice were lessened or not present in Homo- and/or Hetero-rescue mice.
Fig. 3.
Fig. 3.
Hemodynamic assessment of ∼5-mo-old WT, D166V, and S15D-D166V rescue-mice by pressure-volume loops. (A) dP/dtmax-EDV (peak rate of rise in LV pressure–end diastolic volume relationship). (B) PRSW (preload recruited stroke work). (C) ESPVR (slope = contractility of end-systolic pressure-volume relationship). (D) Relaxation time, Tau in ms. Note significantly altered heart function in HCM-D166V mice and substantial improvement of systolic and diastolic indices in Homo- and Hetero-rescue mice.
Fig. 4.
Fig. 4.
Improvement of contractile function in skinned cardiac papillary muscle strips from LV of Homo- and Hetero-rescue mice compared with HCM-D166V mice. (A) Maximal tension per cross-section of muscle strip in kN/m2. Note significantly lower tension in D166V compared with WT mice. The ability to produce force was significantly increased in Homo- and Hetero-rescue mice compared with D166V. (B) Force-pCa relationship in transgenic mice. Note significantly increased Ca2+ sensitivity of force in D166V mice (black circles, pCa50 = 5.94 ± 0.01, nH = 2.09 ± 0.07, n = 37) compared with Tg-WT (open circles, pCa50 = 5.68 ± 0.01, nH = 2.86 ± 0.09, n = 30). The mutation-induced sensitizing effect was significantly reversed in Homo-rescue (gray triangles, pCa50 = 5.77 ± 0.01, nH = 1.89 ± 0.05, n = 32) and Hetero-rescue (open squares, pCa50 (midpoint) = 5.76 ± 0.01, nH (Hill coefficient) = 2.02 ± 0.05, n = 43) mice. P < 0.001 indicates significance between curves of D166V vs. WT and between D166V vs. Homo- and hetero-rescue mice. (C) Resistance to stretch monitored in skinned muscle fibers from Tg mice. Note significantly elevated passive tension in skinned papillary muscle strips from in D166V mice compared with WT. Resistance to stretch was significantly recovered in Homo- and Hetero-rescue mice (P < 0.01, as determined by ANOVA for repeated measurements). All data are average of n fibers ± SEM isolated from ∼5-mo-old female and male mice (about 10 mice per group).
Fig. 5.
Fig. 5.
Equatorial reflections intensity ratio I1,1/I1,0 in skinned papillary muscle fibers from WT, D166V, and Homo- and Hetero-rescue mice in relaxation. The I1,1/I1,0 was established for sarcomere length (SL) = 2.1 and 2.3 µm. Note the increased I1,1/I1,0 in D166V vs. WT mice depicting mutation-induced changes in cross-bridge mass distribution, positioning them closer to the thin filaments. Pseudophosphorylation significantly reversed an abnormal cross-bridge arrangement in Homo- and Hetero-rescue mouse fibers. The number of X-rayed fibers (n) from WT, D166V, and Homo- and Hetero-rescue hearts were, respectively: for SL = 2.1 μm, n = 18, 18, 16 and 14; and for SL = 2.3 μm, n = 17, 17, 16, and 12. Errors are SEM. **P < 0.001 for D166V vs. WT and #P < 0.05 and ##P < 0.001 for S15D-D166V vs. D166V.
Fig. 6.
Fig. 6.
Modeled structure of the human ventricular RLC-WT, D166V, and S15D-D166V mutant proteins. Note the mutation-rendered changes in the C-α distance between the site of HCM mutation and the myosin RLC phosphorylation site at Serine15 (S15). The predicted structures of RLCs were based on PDB structures 3jvtB, 1prwA, 4ik1A, 2mysA, 4i2yA, and 2w4aB (SI Methods).
See this image and copyright information in PMC

Comment in

  • Myosin light chain phosphorylation to the rescue.
    Granzier HL, de Tombe PP. Granzier HL, et al. Proc Natl Acad Sci U S A. 2015 Jul 28;112(30):9148-9. doi: 10.1073/pnas.1511455112. Epub 2015 Jul 8. Proc Natl Acad Sci U S A. 2015. PMID: 26157138 Free PMC article. No abstract available.

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