Research ArticleCARDIAC FUNCTION

Cardiac myosin binding protein-C Ser302 phosphorylation regulates cardiac β-adrenergic reserve

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Science Advances  10 Mar 2017:
Vol. 3, no. 3, e1602445
DOI: 10.1126/sciadv.1602445

Figures

  • Fig. 1 Determination of expression, PKA-mediated phosphorylation of MyBP-C, and other sarcomeric proteins.

    (A) MyBP-C is composed of eight immunoglobulin (ovals) and three fibronectin III (rectangles) domains labeled C0 to C10 (N to C terminus). The conserved M-domain in the linker between domains C1 and C2 contains three serines (S273, S282, S302; mouse numbering) in the NTG sequence that are targets for PKA phosphorylation. The substitution used to prevent S302 phosphorylation in TGS302A is shown in red. (B) Representative Western blot showing S273, S282, and S302 phosphorylation, before and after PKA incubation. No Ser302 expression was detected in TGS302A samples, whereas fully phosphorylatable Ser273 and Ser282 were observed. (C) Representative 5% tris-HCl gel, stained with silver stain, showing MHC isoform expression in the myocardial samples. (D) Representative gels shown are stained by Pro-Q (left) for protein phosphorylation, and the same gel is shown for total protein (right) stained with Coomassie Blue. cMyBP-C, cardiac myosin binding protein-C; pMyBP-C, phosphorylated cardiac myosin binding protein-C. (E) Relative protein phosphorylation (phosphorylated signal/total protein signal) was calculated for each protein and is expressed as % of PKA-treated NTG values for that protein. Values are expressed as means ± SEM, from three to six hearts in each group. *P < 0.05, different from non–PKA-treated samples from the same line; P < 0.05, different from the corresponding NTG group.

  • Fig. 2 Analysis of cardiac morphology.

    (A) Representative NTG, TG3SA, and TGS302A formalin-fixed hearts. TGS302A hearts showed a similar overall morphology compared to NTG hearts, in contrast to the enlarged heart size observed in TG3SA hearts. Scale bar, 1 mm. (B) Representative cross sections of NTG, TG3SA, and TGS302A hearts from the mid-LV were stained with Masson’s trichrome and imaged at ×100 magnification, demonstrating no observable increase in fibrosis in TGS302A hearts. Scale bars, 25 μm.

  • Fig. 3 Analysis of the β-adrenergic acceleration in pressure development.

    (A) Developed pressure, where end diastolic pressure was subtracted from instantaneous ventricular pressure, was plotted during early systolic contraction, to yield developed pressure. Dobutamine (DOB) increased the developed pressure in NTG mice starting at 8 ms after the start of contraction compared to baseline (P = 0.015) but did not increase developed pressure in TG3SA or TGS302A mice. Following dobutamine treatment, developed pressure in TGS302A mice was not higher than that in NTG mice before dobutamine treatment at any point examined (that is, NTG at baseline). (B) The rate of pressure development (dP/dt) was measured during early systole. Dobutamine accelerated dP/dt starting at 6 ms after the start of contraction in NTG (P < 0.005). In TGS302A mice, the acceleration in dP/dt was blunted compared to that in NTG mice and was only faster than baseline after 9 ms (P < 0.05). dP/dt in TGS302A mice was not faster after dobutamine than NTG dP/dt before dobutamine treatment (that is, NTG at baseline) at any time point examined. *P < 0.05, NTG + dobutamine versus NTG without dobutamine treatment; P < 0.05, TGS302A + dobutamine versus TGS302A without dobutamine treatment; P < 0.05, NTG + dobutamine versus TGS302A + dobutamine.

  • Fig. 4 Effect of PKA treatment on the stretch activation responses in NTG, TG3SA, and TGS302A skinned myocardium.

    Representative force responses elicited by a sudden 2% stretch in muscle length (ML) in isometrically contracting (A) NTG, (B) TG3SA, and (C) TGS302A myocardial preparations before (black) and following (red) PKA incubation. (D) Representative stretch activation traces following PKA treatment for NTG (red), TG3SA (green), and TGS302A (blue) myocardial preparations. Following PKA treatment, NTG preparations exhibited faster XB contractile behavior, whereas TG3SA exhibited slower XB contractile behavior and TGS302A exhibited intermediate XB contractile behavior. In (A), highlighted are the important phases of the force transients and the various stretch activation parameters that are derived from the elicited force response (see Materials and Methods for explanation of each phase and parameters). PKA incubation accelerated both krel (P < 0.005) and kdf (P < 0.005) in NTG skinned myocardium. However, in the TGS302A skinned myocardium, the PKA-mediated acceleration in krel is less pronounced (P = 0.02) than that in the NTG coupled with no PKA-mediated accelerations in kdf. There were no PKA-mediated accelerations in krel and kdf in TG3SA skinned myocardium. AU, arbitrary units.

  • Fig. 5 Effect of PKA on stretch activation parameters.

    Measurements in the skinned ventricular preparations were first made under basal conditions (no PKA). Measurements were again made on the same preparations following PKA incubation. The net changes in stretch activation parameters after PKA incubation were calculated and are expressed as % increases from baseline for (A) the magnitude of new steady-state force attained in response to stretch in ML, P3; (B) the magnitude of overall XB recruitment in response to stretch in ML, Pdf; and (C) the rate of XB detachment, krel in the NTG, TG3SA, and TGS302A groups. PKA treatment enhanced the Pdf in all the groups, but the PKA-mediated enhancements in Pdf were lower in the TG3SA and TGS302A groups when compared to the NTG group, indicating that the magnitude of XB recruitment occurs to a lesser extent in the TG3SA and TGS302A groups. NTG preparations displayed accelerations in krel following PKA incubation; however, this acceleration in krel was less pronounced in the TGS302A group and is completely absent in the TG3SA group. Similar effects were observed for P3. Values are expressed as means ± SEM. *P < 0.05; 12 preparations isolated from four hearts were used for all the groups.

Tables

  • Table 1 LV morphology and in vivo cardiac performance measured by echocardiography.

    BW, body weight; LV mass/BW, ratio of LV and body weight; HR, heart rate; PWd, posterior wall thickness in diastole; PWs, posterior wall thickness in systole; IVRT, isovolumic relaxation time; EF, ejection fraction. Values are expressed as means ± SEM from 10 mice per group.

    NTGTG3SATGS302A
    BW (g)27.5 ± 0.526.2 ± 1.326.9 ± 0.6
    LV mass/BW3.8 ± 0.25.8 ± 0.4*4.0 ± 0.2
    HR (beats/min)418 ± 8431 ± 13425 ± 10
    PWd (mm)0.88 ± 0.011.13 ± 0.03*0.91 ± 0.02
    PWs (mm)1.18 ± 0.031.39 ± 0.1*1.23 ± 0.02
    IVRT (ms)18.1 ± 1.528.5 ± 2.1*19.4 ± 1.4
    EF (%)75.1 ± 2.662.0 ± 2.2*73.1 ± 2.2

    *P < 0.05, different compared to NTG.

    • Table 2 Left ventricular hemodynamic function measured by P-V loop analysis.

      HR, heart rate; Pmax, maximal systolic pressure; EDP, end diastolic pressure; dP/dtmax, maximum rate of pressure development; τ, time constant of pressure relaxation; DOB, dobutamine. Values are expressed as means ± SEM. n = 9 for NTG, 8 for TG3SA, and 10 for TGS302A.

      GroupHR
      (beats/min)
      Pmax
      (mmHg)
      EDP
      (mmHg)
      dP/dtmax
      (mmHg/s)
      τ
      (ms)
      DOB
      NTG456 ± 1195.3 ± 3.76.1 ± 0.97,487 ± 5128.1 ± 0.3
      TG3SA451 ± 1187.7 ± 3.16.2 ± 1.17,216 ± 2909.2 ± 0.6
      TGS302A470 ± 592.9 ± 3.46.4 ± 0.66,690 ± 4018.5 ± 0.3
      + DOB
      NTG537 ± 7*96.9 ± 3.44.8 ± 0.6*13,962 ± 919*6.7 ± 0.4*
      TG3SA531 ± 9*85.0 ± 2.64.4 ± 0.48,141 ± 6528.8 ± 0.6
      TGS302A553 ± 6*87.0 ± 1.93.7 ± 0.2*8,725 ± 337*†6.9 ± 0.5*

      *P < 0.05, different versus the corresponding baseline group (without dobutamine).

      P < 0.05, different versus the corresponding NTG group.

      • Table 3 Dynamic stretch-activation parameters measured in NTG, TG3SA, and TGS302A skinned myocardium.

        Twelve preparations isolated from four hearts were used for all the groups. P1, XB stiffness; P2, magnitude of XB detachment; P3, the new steady-state force attained in response to the imposed stretch in ML; Pdf, magnitude of XB recruitment; P0, prestretch isometric force; krel, rate of XB detachment; kdf, rate of XB recruitment. Traces in the top row highlight the portion of the stretch activation trace that the particular parameter represents. Values are expressed as means ± SEM.


        Embedded Image

        *Different versus the corresponding NTG group.

        †Different versus the corresponding (−PKA) group, P < 0.05.

        ‡Different versus the corresponding TG3SA group.

        • Table 4 Steady-state contractile parameters measured in NTG, TG3SA, and TGS302A skinned myocardium.

          Fmin, Ca2+-independent force measured at pCa 9.0; Fmax, Ca2+-activated maximal force measured at pCa 4.5; nH, Hill coefficient of the force-pCa relationship; pCa50, pCa required for the generation of half-maximal force. Values are expressed as means ± SEM.

          GroupFmin
          (mN/mm2)
          Fmax
          (mN/mm2)
          nHpCa50
          − PKA
          NTG1.21 ± 0.1317.21 ± 2.052.50 ± 0.155.86 ± 0.02
          TG3SA1.48 ± 0.1820.44 ± 2.472.66 ± 0.275.88 ± 0.01
          TGS302A1.18 ± 0.2017.46 ± 2.482.37 ± 0.155.89 ± 0.01
          + PKA
          NTG1.10 ± 0.1617.86 ± 1.782.87 ± 0.165.75 ± 0.01*
          TG3SA1.17 ± 0.2120.77 ± 3.542.80 ± 0.205.77 ± 0.02*
          TGS302A1.07 ± 0.2316.25 ± 2.392.42 ± 0.175.78 ± 0.02*

          *Different versus the corresponding (− PKA) group; 12 preparations isolated from four hearts were used for all the groups.

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