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. 2009 Mar 10;119(9):1253-62.
doi: 10.1161/CIRCULATIONAHA.108.798983. Epub 2009 Feb 23.

Cardiac myosin binding protein-C phosphorylation in a {beta}-myosin heavy chain background

Sakthivel Sadayappan  1 , James Gulick, Raisa Klevitsky, John N Lorenz, Michelle Sargent, Jeffery D Molkentin, Jeffrey Robbins
Affiliations

Cardiac myosin binding protein-C phosphorylation in a {beta}-myosin heavy chain background

Sakthivel Sadayappan et al. Circulation. .

Abstract

Background: Cardiac myosin binding protein-C (cMyBP-C) phosphorylation modulates cardiac contractility. When expressed in cMyBP-C-null (cMyBP-C((t/t))) hearts, a cMyBP-C phosphomimetic (cMyBP-C(AllP+)) rescued cardiac dysfunction and protected the hearts from ischemia/reperfusion injury. However, cMyBP-C function may be dependent on the myosin isoform type. Because these replacements were performed in the mouse heart, which contains predominantly alpha-myosin heavy chain (alpha-MyHC), the applicability of the data to humans, whose cardiomyocytes contain predominantly beta-MyHC, is unclear. We determined the effect(s) of cMyBP-C phosphorylation in a beta-MyHC transgenic mouse heart in which >80% of the alpha-MyHC was replaced by beta-MyHC, which is the predominant myosin isoform in human cardiac muscle.

Methods and results: To determine the effects of cMyBP-C phosphorylation in a beta-MyHC background, transgenic mice expressing normal cMyBP-C (cMyBP-C(WT)), nonphosphorylatable cMyBP-C (cMyBP-C(AllP)(-)), or cMyBP-C(AllP+) were bred into the beta-MyHC background (beta). These mice were then crossed into the cMyBP-C((t/t)) background to ensure the absence of endogenous cMyBP-C. cMyBP-C((t/t)/beta) and cMyBP-C(AllP)(-)(:(t/t)/beta) mice died prematurely because of heart failure, confirming that cMyBP-C phosphorylation is essential in the beta-MyHC background. cMyBP-C(AllP+:(t/t)/beta) and cMyBP-C(WT:(t/t)/beta) hearts showed no morbidity and mortality, and cMyBP-C(AllP+:(t/t)/beta) hearts were significantly cardioprotected from ischemia/reperfusion injury.

Conclusions: cMyBP-C phosphorylation is necessary for basal myocardial function in the beta-MyHC background and can preserve function after ischemia/reperfusion injury. Our studies justify exploration of cMyBP-C phosphorylation as a therapeutic target in the human heart.

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Figures

Figure 1
Figure 1
cMyBP-C interacts with both β-MyHC and α-MyHC in a phosphorylation-dependent fashion. A, The myosin and cMyBP-C domains are depicted with the region of interaction shown. The essential light chain (ELC), regulatory light chain (RLC), subfragment-2 (S2) and light meromyosin (LMM) regions are indicated. Phospho-ablation of cMyBP-C (AllP-) promotes the protein’s interaction with myosin S2, whereas the phosphomimetic (AllP+) abolishes the interaction. B, A pull-down assay demonstrates the phosphorylation-dependent interaction of C1-C2 with β-MyHC. Two hundred μg of total β-TG heart ventricular lysate was mixed with either 20 μg of His-tagged C1-C2WT peptide, PKA-treated WT peptides without (WT/P) and with PKA inhibitors (WT/P/I), C1-C2AllP- and C1C2AllP+ peptides and the interacting proteins collected with Ni-NTA resin as described. The proteins were separated in 4-15% SDS-PAGE and analyzed by western blots using anti-β-MyHC, anti-α-MyHC (BA-G5) and anti-His antibodies.
Figure 2
Figure 2
Phospho-ablation (AllP-) of cMyBP-C is deleterious to the heart in a β-MyHC background. Representative hematoxylin-eosin stained longitudinal sections of mouse heart at 4X (A) and 20X (B). Masson’s trichrome-stained myocardial sections, 20X (C). D, Representative SYPRO Ruby stained glycerol gel shows MyHC isoform content of the samples shown in A. E, Survival curves show that cMyBP-C(t/t)/β and cMyBPCAllP-:(t/t)/β mice die within 7 weeks post-birth due to severe HF. NTG, cMyBP-C(t/t) β-TG and cMyBP-CWT:(t/t)/β mice showed normal survival. One mouse in the cMyBP-CAllP+:(t/t)/β group died from other causes (n=10 per group).
Figure 3
Figure 3
cMyBP-CAllP+ in a β-MyHC background. A, SDS-PAGE analyses of myofibrillar proteins from NTG, β-TG, cMyBP-CWT:(t/t)/β and cMyBP-CAllP+:(t/t)/β hearts. B, Representative western blot analyses using anti-cMyBP-C antibodies show that total cMyBP-C content is unchanged. α-sarcomeric actin was used as a loading control. C, RNA dot-blot analyses. As expected, β-MyHC is up-regulated in the β-MyHC background. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control (n=3). D, Heart weight/body weight (HW/BW) ratios and E, percentages of fractional shortening (%FS) are unchanged among the four groups (n=6) at 12 weeks.
Figure 4
Figure 4
cMyBP-CAllP+ in a β-MyHC background improves myocardial function in vivo. cMyBP-CAllP+:(t/t)/β mouse hearts show enhanced in vivo contraction (dP/dtmax, A) and relaxation (dP/dtmin, B), compared to β-MyHC TG and cMyBP-CWT:(t/t)/β mouse hearts, which show decreased function compared to the NTG controls (n=6, Table 2). Measurements were made at basal levels and during β-agonist stimulation. C, Effects on maximal Ca2+-activated Mg2+-ATPase activities of the cMyBP-C phosphomimetic (AllP+) in a β-MyHC background (n=5) at pCa 4.0 (Table 3). *P<0.05 versus NTG and #P<0.05 versus β-TG.
Figure 5
Figure 5
β-MyHC kinetics. A, E, Isotonic quick release data were utilized to determine the force-velocity relationships and (I) maximum shortening velocities at pCa 5.0 and sarcomere length of 2.1 μm. m.l./s; muscle lengths per second. B, F, Normalized power output-force relationship and (J) maximum power output at pCa 5.0. C, G, Rate of force redevelopment and (K) maximum unloaded shortening velocities were determined using the slack test. The changes in sarcomere length (Δlength) amplitude versus duration (Δtime) of unloaded shortening are shown (C and G) to determine cross-bridge turnover. The isometric force at different calcium (pCa) concentrations (D and H) and maximum force relationships (L) are shown before and after treatment with PKA at 22°C. PKA treatment accelerates the kinetics of cross-bridge rate and Ca2+ sensitivity. The values of fiber force, shortening velocity and power output are summarized in Table 4. Data are mean±SE (n=5). *P<0.05 versus NTG and #P<0.05 versus β-TG and cMyBP-CWT:(t/t)/β.
Figure 6
Figure 6
I-R injury. A, Representative I-R injured mouse hearts stained with Evans blue and triphenyltetrazolium chloride. The white areas represent the infarcted region and the red shows the area at risk region. B, Area at risk (AAR) and infarcted area (IA) normalized to total LV area and AAR, respectively (n=6; *P<0.05). C, Myosin isoform shift in sham (S) and I-R injured hearts. D, Immunohistochemistry shows TUNEL-positive nuclei (red), cardiac TnI (green) and nuclei (blue). E, Quantitation of TUNEL-positive cardiomyocytes expressed as a percentage of total cardiomyocytes in hearts after sham and I-R injury. Values are expressed as mean±SE (*P<0.01 versus NTG, β-TG and cMyBP-CWT:(t/t)/β, n=3 hearts/group). F, DNA fragmentation assays by ligation-mediated PCR as an indication of apoptosis after I-R. G, Western blot of cMyBP-C shows decreased cMyBP-C degradation after I-R in cMyBP-CAllP+:(t/t)/β hearts compared to NTG, β-TG and cMyBP-CWT:(t/t)/β hearts (small arrow= cMyBP-C degradation products). H, Percentage of fractional shortening (%FS) and I, fractional area change (%FAC) assessed by echocardiography (*P<0.001, **P<0.05 versus sham; #P<0.05 versus NTG, β-TG and cMyBP-CWT:(t/t)/β after I-R (n=6)).
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