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Research ArticleArticle

Effects of Sarcomere Activators and Inhibitors Targeting Myosin Cross-Bridges on Ca2+-Activation of Mature and Immature Mouse Cardiac Myofilaments

Monika Halas, Paulina Langa, Chad M. Warren, Paul H. Goldspink, Beata M. Wolska and R. John Solaro
Molecular Pharmacology May 2022, 101 (5) 286-299; DOI: https://doi.org/10.1124/molpharm.121.000420

Article Figures & Data

Figures

  • Figure
  • Fig. 1.
    Fig. 1.

    Chemical structure depiction of (A) omecamtive mecarbil (source: https://pubchem.ncbi.nlm.nih.gov/compound/Omecamtiv-mecarbil) and (B) mavacamten (source: https://pubchem.ncbi.nlm.nih.gov/compound/Mavacamten).

  • Fig. 2.
    Fig. 2.

    Western blot and SDS-PAGE analysis demonstrating myofilament isoform abundance differences in adult and immature NTG and TG-cTnT-R92Q mouse hearts. (A) Western blot probing for troponin I isoforms in adult samples, cardiac troponin I, and slow skeletal troponin I, with an antibody recognizing both isoforms simultaneously. (B) Histograms quantifying the percent of TG-ssTnI in the total TnI population of adult samples from panel (A). (C) Histograms quantifying the percent of TG-ssTnI in the total TnI population of 7- and 14-day-old samples from panel (A). (D) Western blot probing for TnT isoforms with an antibody recognizing all isoforms simultaneously. (E) Histograms quantifying the percent of TG-cTnT-R92Q in the total TnT population of adult samples from panel (D). (F) Histograms quantifying the percent of TG-cTnT-R92Q in the total TnT population of 7- and 14-day-old samples from panel (D). (G) Representative SDS-PAGE stained with Coomassie blue showing the myosin heavy chain (MHC) α and β isoforms. (H) Histograms quantifying the percent of β-MHC in the total MHC in adult samples from panel (G). Statistical significance was determined by an unpaired, two-tailed T-test. No significance was found. (I) Histograms quantifying the percent of β-MHC in the total MHC in 7- and 14-days old samples from panel (G). Data presented as mean ± S.D., n = 4, **P < 0.0042, ****P < 0.0001. Statistical significance was determined by two-way ANOVA test, followed by a Tukey’s multiple comparison post hoc test for panels C, F, and I. No statistical test was performed for panels (B) or (E). Day, age of animals in days; WT, wild-type.

  • Fig. 3.
    Fig. 3.

    Effect of OM on pCa-tension relations of skinned fiber bundles from hearts of adult control NTG mice and TG-ssTnI mice. (A) pCa-tension relations in NTG and TG adult mouse skinned fiber bundles ± OM treatment. (B) Scatter plot of maximum tension (mN/mm2). (C) Scatter plot of pCa50 values. (D) Scatter plot of Hill slopes (nH). (E) Activation represented as OM-induced percent increase in control tension over a range of submaximal pCa values. Data presented as mean ± S.D., n = 5, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Exact P values are found in Table 1. Statistical significance was determined by two-way ANOVA test, followed by a Tukey’s multiple comparison post hoc test (A–D); and by unpaired two-tailed Student’s t test for (E).

  • Fig. 4.
    Fig. 4.

    Effect of OM on pCa-tension relations of skinned fiber bundles from hearts of 7- and 14-day-old NTG mice. (A) pCa-tension relations of NTG 7- and 14-day-old mouse skinned fiber bundles ± OM treatment. (B) Scatter plot of maximum tension (mN/mm2). (C) Scatter plot of pCa50 values. (D) Scatter plot of Hill slopes (nH). (E) Activation represented as percent increase in control tension over a range of submaximal pCa values. Data presented as mean ± S.D., n = 6–7, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Exact P values are found in Table 2. Statistical significance was determined by two-way ANOVA test followed by a Tukey’s multiple comparison post hoc test (A–D); and by unpaired two-tailed Student’s t test for (E).

  • Fig. 5.
    Fig. 5.

    Effect of Mava on pCa-tension relations of skinned fiber bundles from hearts of adult control NTG mice and TG-ssTnI mice. (A) pCa-tension relations in NTG and TG-ssTnI adult skinned fiber bundles ± Mava treatment. (B) Scatter plot of maximum tension (mN/mm2). (C) Scatter plot of pCa50 values. (D) Scatter plot of Hill slopes (nH). (E) Inhibition represented as percent of control tension over a range of submaximal pCa values. Data represented as mean ± S.D., n = 5–6, *P < 0.05, ***P < 0.001, ****P < 0.0001. Exact P values are found in Table 3. Statistical significance was determined by two-way ANOVA test followed by a Tukey’s multiple comparison post hoc test (A–D); and by unpaired two-tailed Student’s t test for (E).

  • Fig. 6.
    Fig. 6.

    Effect of Mava on pCa-tension relations of skinned fiber bundles from hearts of 7-day-old NTG and 7-day-old TG-cTnT-R92Q mice. (A) pCa-tension relations of NTG 7-day-old mouse skinned fiber bundles ± Mava treatment. (B) Scatter plot of maximum tension (mN/mm2). (C) Scatter plot of pCa50 values. (D) Scatter plot of Hill slopes (nH). (E) Inhibition represented as percent of control tension over a range of submaximal pCa values. Data presented as mean ± S.D., n = 3–7, *P < 0.05, ***P < 0.001. Exact P values are found in Table 4. Statistical significance was determined by two-way ANOVA test followed with a Tukey’s multiple comparison post hoc test (A, C–D); and by unpaired two-tailed Student’s t test for (E).

  • Fig. 7.
    Fig. 7.

    Effect of Mava on pCa-tension relations of skinned fiber bundles from hearts of 14-day-old NTG mice and 14-day-old TG-cTnT-R92Q mice. (A) pCa-tension relations of NTG 14-day-old skinned fiber bundles ± Mava treatment. (B) Scatter plot of max tension (mN/mm2). (C) Scatter plot of pCa50 values. (D) Scatter plot of Hill slope (nH). (E) Inhibition represented as percent of control tension over a range of submaximal pCa values. Data represented as mean ± S.D., n = 4–5, *P < 0.05, **P < 0.01, ****P < 0.0001. Exact P values are found in Table 5. Statistical significance was determined by two-way ANOVA test followed by a Tukey’s multiple comparison post-hoc test (A–D); and by unpaired two-tailed Student’s t test for (E). Data presented as mean ± S.D.

  • Fig. 8.
    Fig. 8.

    Mechanisms of regulation of cardiac myofilament activation modified by myosin activators and inhibitors. As shown by the right side of the figure, the reaction of cross-bridges with thin filaments promotes ATP hydrolysis, powering shortening and tension generation. At the level of the thin filaments, Ca-binding to cTnC triggers an interaction of a Sw of the inhibitory protein (TnI) to switch on the thin filament and release the inhibition imposed by a TnI-inhibitory peptide (Ip), the mobile domain of TnI, and TnT interactions with actin-Tm interface. The result is a movement of Tm permitting the actin-cross-bridge reaction. The extent of myofilament activation is governed by the number of cross-bridges reacting with the thin filament, which is determined by the proportion of cTnC sites with bound Ca2+ related to the amounts of Ca2+ released and to the population of cross-bridges existing in an SRX and a DRX. As shown by the green arrow extending along the myofilaments, the extent of activation is also determined by the cooperative spread of activation from an active regulatory unit to near neighbors among the regulatory units. Cross-bridge interactions with thin filament promote this spread, as do modifications in energies of interaction among neighboring thin filament proteins, especially interactions at the ends of contiguous Tm strands. Critical regions of TnI differ between cTnI and ssTnI. The N-terminal extension is specific to cTnI. In homologous regions the cTnI Ip T143 is P111 in ssTnI, in the cTnI Sw Q155, A162 and E164 are R125, H132, V134 in ssTnI, and in the cTnI mobile domain cTnI H172 and K207 are N141 and M175 in ssTnI. Data from our studies indicate that myosin activators and inhibitors are affected by this difference, which affect activation and inhibition of tension. See text for further discussion.

Tables

  • Table 1

    Ca2+-activated tension parameters for skinned fibers of adult TG-ssTnI mice and NTG controls treated with 0.3 µM OM

    Data are presented as mean ± S.D. Two-way ANOVA test followed by a Tukey’s multiple comparison post hoc test.

    Skinned FibersTreatmentMax Tension (mN/mm2)pCa50Hill Slope
    NTG adult (n = 5)Control33.50 ± 3.075.73 ± 0.035.65 ± 0.30
    NTG adult (n = 5)OM 0.3 µM31.12 ± 3.225.89 ± 0.08a2.49 ± 0.69b
    TG-ssTnI adult (n = 5)Control26.62 ± 2.75c5.94 ± 0.05d3.12 ± 0.36e
    TG-ssTnI adult (n = 5)OM 0.3 µM26.66 ± 4.71f6.07 ± 0.07g,h,i1.96 ± 0.21j,k

    • aP = 0.0035, 95% confidence interval (CI) of diff. [-0.27 to -0.05] when compared with NTG adult control

    • bP < 0.0001, 95% CI of diff. [−3.94 to −2.39] when compared with NTG adult control

    • cP = 0.0319, 95% CI of diff. [0.51 to 13.24] when compared with NTG adult control

    • dP = 0.0002, 95% CI of diff. [−0.32 to −0.10] when compared with NTG adult control

    • eP < 0.0001, 95% CI of diff. [−3.30 to −1.76] when compared with NTG adult control

    • fP = 0.0329, 95% CI of diff. [0.48 to 13.21] when compared with NTG adult control

    • gP = 0.0164, 95% CI of diff. [−0.24 to −0.02] when compared with TG-ssTnI adult control

    • hP = 0.001, 95% CI of diff. [−0.29 to −0.07] when compared with NTG adult 0.3 µM OM

    • iP < 0.0001, 95% CI of diff. [−0.45 to −0.24] when compared with NTG adult control

    • jP = 0.0027, 95% CI of diff. [−1.94 to −0.39] when compared with TG-ssTnI adult control

    • kP < 0.0001, 95% CI of diff. [−4.47 to −2.92] when compared with NTG adult control

  • Table 2

    Ca2+-activated tension parameters for skinned fiber bundles of 7- and 14-day-old NTG mice treated with 0.3 µM OM

    Data are presented as mean ± S.D. Two-way ANOVA test followed by a Tukey’s multiple comparison post hoc test.

    Skinned FibersTreatmentMax Tension (mN/mm2)pCa50Hill Slope
    NTG 7d (n = 7)Control16.80 ± 2.315.88 ± 0.053.72 ± 0.57
    NTG 7d (n = 7)OM 0.3 µM14.25 ± 2.06a6.07 ± 0.05b,c,d3.28 ± 0.42e
    NTG 14d (n = 6)Control22.65 ± 3.27f5.80 ± 0.03g5.29 ± 1.08h
    NTG 14d (n = 6)OM 0.3 µM21.83 ± 3.01i,j5.86 ± 0.063.21 ± 0.72k

    • aP < 0.0001, 95% CI of diff. [−12.52 to −4.29] when compared with NTG 14-day control

    • bP < 0.0001, 95% CI of diff. [−0.26 to −0.12] when compared with NTG 7-day control

    • cP < 0.0001, 95% CI of diff. [0.20 to 0.34] when compared with NTG 14-day control

    • dP < 0.0001, 95% CI of diff. [0.14 to 0.28] when compared with NTG 14-day 0.3 µM OM

    • eP = 0.0003, 95% CI of diff. [0.89 to 3.13] when compared with NTG 14-day control

    • fP = 0.0035, 95% CI of diff. [−9.97 to −1.74] when compared with NTG 7-day control

    • gP = 0.0202, 95% CI of diff. [0.01 to 0.15] when compared with NTG 7-day control

    • hP = 0.0037, 95% CI of diff. [0.46 to 2.69] when compared with NTG 7-day control

    • iP = 0.013, 95% CI of diff. [−9.14 to −0.91] when compared with NTG 7-day control

    • jP = 0.0002, 95% CI of diff. [−11.69 to −3.47] when compared with NTG 7-day 0.3 µM OM

    • kP = 0.0003, 95% CI of diff. [−3.24 to −0.92] when compared with NTG 14-day control

  • Table 3

    Ca2+-activated tension parameters for skinned fiber bundles of adult TG-ssTnI mice and NTG controls treated with 0.3 µM Mava

    Data are presented as mean ± S.D. Two-way ANOVA test followed by a Tukey’s multiple comparison post hoc test.

    Skinned FibersTreatmentMax Tension(mN/mm2)pCa50Hill Slope
    NTG adult (n = 5)Control35.38 ± 4.935.68 ± 0.084.05 ± 0.86
    NTG adult (n = 5)Mava 0.3 µM19.89 ± 3.92a5.55 ± 0.074.96 ± 0.95
    TG-ssTnI adult (n = 6)Control29.05.05 ± 4.27b5.88 ± 0.13c,d3.24 ± 0.68e
    TG-ssTnI adult (n = 6)Mava 0.3 µM14.19 ± 4.41f,g5.73 ± 0.123.46 ± 0.96h

    • aP = 0.0001, 95% CI of diff. [7.65 to 23.34] when compared with NTG adult control

    • bP = 0.0139, 95% CI of diff. [−16.68 to −1.65] when compared with NTG 0.3 µM Mava

    • eP = 0.029, 95% CI of diff. [−0.38 to −0.02] when compared with NTG adult control

    • fP = 0.0005, 95% CI of diff. [−0.51 to −0.14] when compared with NTG 0.3 µM Mava

    • gP = 0.0201, 95% CI of diff. [−3.19 to −0.23] when compared with NTG adult 0.3µM Mava

    • cP < 0.0001, 95% CI of diff. [13.68 to 28.71] when compared with NTG adult control

    • dP < 0.0001, 95% CI of dif. [7.70 to 22.03] when compared with TG-ssTnI adult control

    • hP = 0.0465, 95% CI of diff. [−2.98 to −0.02] when compared with NTG adult 0.3 µM Mava

  • Table 4

    Ca2+-activated tension parameters for skinned fiber bundles of 7-day-old TG-cTnT-R92Q mice and NTG controls treated with 0.3 µM Mava

    Data are presented as mean ± S.D. Two-way ANOVA test followed with a Tukey’s multiple comparison post hoc test.

    Skinned FibersTreatmentMax Tension(mN/mm2)pCa50Hill Slope
    NTG 7d (n = 3)Control13.93 ± 2.386.21 ± 0.342.82 ± 0.29
    NTG 7d (n = 3)Mava 0.3 µM6.87 ± 1.31a6.07 ± 0.293.54 ± 0.31
    TG-cTnT-R92Q 7d (n = 7)Control15.04 ± 3.52b6.08 ± 0.233.33 ± 0.45
    TG-cTnT-R92Q 7d (n = 7)Mava 0.3 µM7.88 ± 1.66c,d5.97 ± 0.253.26 ± 0.49

    • aP = 0.0186, 95% CI of diff. [1.05 to 13.07] when compared with NTG 7-day control

    • bP = 0.0015, 95% CI of diff. [−13.24 to −3.09] when compared with NTG 7-day 0.3 µM Mava

    • cP = 0.0004, 95% CI of diff. [3.23 to 11.09] when compared with TG-cTnT-R92Q 7-day control

    • dP = 0.0169, 95% CI of diff. [0.98 to 11.13] when compared with NTG 7-day control

  • Table 5

    Ca2+-activated tension parameters for skinned fiber bundles of 14-day-old TG-cTnT-R92Q mice and NTG controls treated with 0.3 µM Mava

    Data are presented as mean ± S.D. Two-way ANOVA test followed with a Tukey’s multiple comparison post hoc test.

    Skinned FibersTreatmentMax Tension (mN/mm2)pCa50Hill Slope
    NTG 14d (n = 4)Control26.49 ± 4.205.73 ± 0.025.04 ± 0.69
    NTG 14d (n = 4)Mava 0.3 µM13.30 ± 2.90a5.59 ± 0.045.62 ± 0.37b,c
    TG-cTnT-R92Q 14d (n = 5)Control24.93 ± 4.83d5.94 ± 0.06e,f2.71 ± 0.34g
    TG-cTnT-R92Q 14d (n = 5)Mava 0.3 µM13.97 ± 2.97h,i5.75 ± 0.11j,k3.44 ± 0.57l

    • aP = 0.0013, 95% CI of diff. [5.29 to 21.09] when compared with NTG 14-day control

    • bP < 0.0001, 95% CI of diff. [−3.91 to −1.92] when compared with TG-cTnT-R92Q 14-day control

    • cP < 0.0001, 95% CI of diff. [−3.17 to −1.18] when compared with TG-cTnT-R92Q 14-day 0.3 µM Mava

    • dP = 0.0024, 95% CI of diff. [−19.13 to −4.14] when compared with NTG 14-day 0.3 µM Mava

    • eP = 0.0022, 95% CI of diff. [−0.34 to −0.07] when compared with NTG 14-day control

    • fP < 0.0001, 95% CI of diff. [−0.48 to −0.22] when compared with NTG 14-day 0.3 µM Mava

    • gP < 0.0001, 95% CI of diff. [−3.33 to −1.34] when compared with NTG 14-day control

    • hP = 0.0025, 95% CI of diff. [3.89 to 18.03] when compared with TG-cTnT-R92Q 14-day control

    • iP = 0.0013, 95% CI of diff. [5.02 to 20.01] when compared with NTG 14-day control

    • jP = 0.0162, 95% CI of diff. [−0.29 to −0.03] when compared with NTG 14-day 0.3 µM Mava

    • kP = 0.0034, 95% CI of diff. [0.06 to 0.31] when compared with TG-cTnT-R92Q 14-day control

    • lP = 0.0018, 95% CI of diff. [−2.59 to −0.60] when compared with NTG 14-day control

Additional Files

  • Data Supplement

    • Supplemental Data -

      Supplemental Figure S1. Western blot images probed with β-actin.

      Supplemental Table S1. Ca2+-activated percent control change of tension for TG and NTG mouse fibers treated with 0.3 µM OM or Mava.
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Research ArticleArticle

Myosin Activators/Inhibitors in Immature Cardiac Sarcomeres

Monika Halas, Paulina Langa, Chad M. Warren, Paul H. Goldspink, Beata M. Wolska and R. John Solaro
Molecular Pharmacology May 1, 2022, 101 (5) 286-299; DOI: https://doi.org/10.1124/molpharm.121.000420
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