A Critical Function for Ser-282 in Cardiac Myosin Binding
Protein-C Phosphorylation and Cardiac Function
Sakthivel Sadayappan, James Gulick, Hanna Osinska, David Barefield, Friederike Cuello,
Metin Avkiran, Valerie M. Lasko, John N. Lorenz, Marjorie Maillet, Jody L. Martin,
Joan Heller Brown, Donald M. Bers, Jeffery D. Molkentin, Jeanne James, Jeffrey Robbins
Rationale: Cardiac myosin-binding protein-C (cMyBP-C) phosphorylation at Ser-273, Ser-282, and Ser-302 regulates
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myocardial contractility. In vitro and in vivo experiments suggest the nonequivalence of these sites and the potential
importance of Ser-282 phosphorylation in modulating the protein’s overall phosphorylation and myocardial function.
Objective: To determine whether complete cMyBP-C phosphorylation is dependent on Ser-282 phosphorylation
and to define its role in myocardial function. We hypothesized that Ser-282 regulates Ser-302 phosphorylation
and cardiac function during ␤-adrenergic stimulation.
Methods and Results: Using recombinant human C1-M-C2 peptides in vitro, we determined that protein kinase
A can phosphorylate Ser-273, Ser-282, and Ser-302. Protein kinase C can also phosphorylate Ser-273 and
Ser-302. In contrast, Ca2ⴙ-calmodulin-activated kinase II targets Ser-302 but can also target Ser-282 at
nonphysiological calcium concentrations. Strikingly, Ser-302 phosphorylation by Ca2ⴙ-calmodulin-activated
kinase II was abolished by ablating the ability of Ser-282 to be phosphorylated via alanine substitution. To
determine the functional roles of the sites in vivo, three transgenic lines, which expressed cMyBP-C containing
either Ser-273-Ala-282-Ser-302 (cMyBP-CSAS), Ala-273-Asp-282-Ala-302 (cMyBP-CADA), or Asp-273-Ala-282Asp-302 (cMyBP-CDAD), were generated. Mutant protein was completely substituted for endogenous cMyBP-C
by breeding each mouse line into a cMyBP-C null (t/t) background. Serine-to-alanine substitutions were used to
ablate the abilities of the residues to be phosphorylated, whereas serine-to-aspartate substitutions were used to
mimic the charged state conferred by phosphorylation. Compared to control nontransgenic mice, as well as
transgenic mice expressing wild-type cMyBP-C, the transgenic cMyBP-CSAS(t/t), cMyBP-CADA(t/t), and cMyBPCDAD(t/t) mice showed no increases in morbidity and mortality and partially rescued the cMyBP-C(t/t) phenotype.
The loss of cMyBP-C phosphorylation at Ser-282 led to an altered ␤-adrenergic response. In vivo hemodynamic
studies revealed that contractility was unaffected but that cMyBP-CSAS(t/t) hearts showed decreased diastolic
function at baseline. However, the normal increases in cardiac function (increased contractility/relaxation) as a
result of infusion of ␤-agonist was significantly decreased in all of the mutants, suggesting that competency for
phosphorylation at multiple sites in cMyBP-C is a prerequisite for normal ␤-adrenergic responsiveness.
Conclusions: Ser-282 has a unique regulatory role in that its phosphorylation is critical for the subsequent
phosphorylation of Ser-302. However, each residue plays a role in regulating the contractile response to
␤-agonist stimulation. (Circ Res. 2011;109:141-150.)
Key Words: contractile function 䡲 myofilament 䡲 myosin-binding protein-C 䡲 phosphorylation
C
ardiovascular disease, particularly ischemia, myocardial
infarction, and heart failure, constitutes a growing health
and economic problem, afflicting approximately five million
people in the United States each year at an estimated cost of
$29.6 billion.1 Heart failure is associated with diminished
␤-adrenergic (␤-AR) receptor responsiveness, loss of cardiac
contractility, abnormalities in Ca2⫹ handling,2,3 and altered
contractile protein phosphorylation.4 Altered phosphorylation
Original received February 11, 2011; revision received May 5, 2011; accepted May 9, 2011. In April 2011, the average time from submission to first
decision for all original research papers submitted to Circulation Research was 15 days.
From the Department of Cell and Molecular Physiology (S.S., D.B., J.L.M.), Stritch School of Medicine, Loyola University Chicago, IL; Division of
Molecular Cardiovascular Biology (J.G., H.O., M.M., J.D.M., J.J., J.R.), Department of Pediatrics, The Cincinnati Children’s Hospital Medical Center,
Cincinnati, OH; Cardiovascular Division (F.C., M.A.), King’s College London, The Rayne Institute, St. Thomas’ Hospital, London, United Kingdom;
Department of Molecular and Cellular Physiology (V.M.L., J.N.L.), University of Cincinnati College of Medicine, University of Cincinnati, Cincinnati,
OH; Department of Pharmacologyy, University of California, La Jolla, CA; Department of Pharmacology (D.M.B.), University of California Davis,
Genomics and Biological Sciences Facility, Davis, CA.
This manuscript was sent to Ali J. Marian, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Correspondence to Jeffrey Robbins, PhD, Division of Molecular Cardiovascular Biology, Cincinnati Children’s Hospital Medical Center, MLC 7020,
240 Albert Sabin Way, Cincinnati, OH 45229-3039. E-mail [email protected]
© 2011 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.111.242560
141
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July 8, 2011
Non-standard Abbreviations and Acronyms
AllPⴚ
cMyBP-C
TG
NTG
AllPⴙ
CaMKII
a cMyBP-C in which 3 phosphorylatable serines have
been mutated to alanine
cardiac myosin binding protein-C
transgenic
nontransgenic
cMyBP-C in which 3 phosphorylatable serines have been
mutated to aspartate
Ca2⫹-calmodulin-activated kinase II
structure and function using three TG mouse models in which
mutated cMyBP-C replaced the endogenous cardiac protein.
cMyBP-C carrying the mutations Ser273-Ala282-Ser302
(SAS), Ala273-Asp282-Ala302 (ADA), or Asp273-Ala282Asp302 (DAD) were generated and expressed specifically in
cardiomyocytes. Our data demonstrate that Ser-282 phosphorylation is a critical determinant of Ser-302 phosphorylation in vitro and in vivo and that phosphorylation at each of
the residues participates in the contractile response to ␤-AR
stimulation in vivo.
Methods
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of the contractile proteins may partially underlie cardiac
dysfunction in heart failure.4,5 Cardiac myosin-binding
protein-C (cMyBP-C) phosphorylation at multiple sites can
impact myocardial function,6,7 metabolism,8 and cardioprotection,9 but the precise functional consequences of phosphorylation are not characterized fully. cMyBP-C is a 140-kDa
structural protein that is localized at the inner two-thirds of
the A-band in the cardiac sarcomere.10,11 cMyBP-C binds
myosin at the S2 region12 and light meromyosin,13,14 where it
modulates myosin assembly10 and actin–myosin interaction.15,16 It also binds to titin via domains C8 to C1017 and
presumably to actin as well; however, this interaction is less
well-defined,18 suggesting that these interactions may be
necessary for the stability or function of cMyBP-C in the
sarcomeres.
The cardiac isoform differs from the skeletal isoform in
that it contains an extra domain at the N-terminus (C0) and a
phosphorylation motif (M-domain) localized between the
immunoglobulin I-like C1 and C2 domains,19,8 in which
Ser-273, Ser-282, and Ser-302 each can be phosphorylated.20
The three sites are differentially phosphorylated by the
enzymes protein kinase (PK) A,21 PKC,21 Ca2⫹-calmodulinactivated kinase II (CaMKII),19 PKD,22 and the 90-kDa
ribosomal s6 kinase.23 To begin to explore the physiological
roles of cMyBP-C phosphorylation, we previously established two transgenic (TG) mouse models in which the three
phosphorylation sites were mutated to either alanine
(phospho-ablation, AllP⫺) or aspartate (phospho-mimetic,
AllP⫹).7,9,24,25 Our findings suggested that cMyBP-C phosphorylation was essential for normal cardiac function and that
charged residues at 273, 282, and 302 sites conferred cardioprotection against ischemia-reperfusion injury in either the
␣-myosin heavy chain or the ␤-myosin heavy chain backgrounds. However, these data did not resolve the functional
roles of the individual sites. Previously, hierarchical phosphorylation patterns for cMyBP-C have been defined in
vitro,19 where Ca2⫹-dependent CaMKII phosphorylation at
Ser-282 appeared to be necessary for phosphorylation of
Ser-273 and Ser-302.19,21,26 Furthermore, monophosphorylation of cMyBP-C by CaMKII is associated with myocardial
stunning20 and enhanced contractility27 under certain conditions.26 However, the critical role of Ser-282 phosphorylation
in vivo remains to be systematically investigated.
In the present study, we explored the necessity and sufficiency of Ser-282 phosphorylation in mediating cardiac
TG Constructs
cMyBP-C wild-type (WT) cDNA was subjected to site-directed
mutagenesis to generate cMyBP-CSAS, cMyBP-CDAD, and cMyBPCADA constructs. Cardiomyocyte-specific TG mice were generated
using the mouse cardiac-specific ␣-myosin heavy chain promoter.7,9
An N-terminal myc-tag, which has no effect on cMyBP-C function
and stability,7,9,28 was introduced to differentiate TG protein from
endogenous cMyBP-C. All biochemical and functional assays used
hearts from 10- to 12-week-old mice. Animals were handled in
accordance with the principles and procedures of the Guide for the
Care and Use of Laboratory Animals. The Institutional Animal Care
and Use Committee at Cincinnati Children’s Hospital approved all
experimental procedures.
Characterization of TG Mice
TG mice were identified by polymerase chain reaction.7 Transgene copy
number was determined by southern blotting (Figure I, online data
supplement available online at http://circres.ahajournals.org) and transgene expression in the cardiomyocytes determined by real-time polymerase chain reaction and confirmed in some cases by northern blot
analysis with a gene-specific ␥32P-labeled probe7 using actin and
GAPDH as loading controls (Online Figure I). To ensure that the
observed phenotype was not attributable to insertional mutagenesis, at
least two different TG lines were used from each group when assessing
the functional consequences of complete cMyBP-C mutant protein
replacement in the cMyBP-C(t/t) background.7,9 The presence of necrosis, fibrosis, and disarray, as well as localization and integration of
cMyBP-C mutant proteins into the sarcomere, were evaluated by
immunofluorescence microscopy as described.7 To determine sarcomeric A-band, I-band, and M-line organization and thick filament
distances, structural analyses at the light and electron microscopy levels
were performed.7,9
Evaluation of cMyBP-C Phosphorylation
To define the phosphorylation status of Ser-273, Ser-282, and
Ser-302, myofibrils from nontransgenic (NTG) and TG mouse hearts
were treated with either PKA, PKC␧, CaMKII␦, or phosphatase
(Sigma), electrophoresed on SDS-PAGE, and Western blots were
performed using an anti-cMyBP-C2–14 antibody that was raised
against the first 14 residues of cMyBP-C (ProSci) and phosphospecific cMyBP-C antibodies as described previously.22,24 In addition, the importance of each phosphorylation site was confirmed
using recombinant human mutant C1-M-C2 peptides and sitespecific cMyBP-C phospho-antibodies.22,24
Cardiac Function
Cardiac function was determined in the intact closed chest model as
described.7 Increasing doses of dobutamine were administered during 3-minute constant infusions (0.1 ␮L/min/g body weight) to
determine ␤-AR responsiveness. To observe changes in left ventricular chamber size and fractional shortening, noninvasive M-mode
echocardiography was used.9
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Figure 1. Differential substrate specificities and regulation of Ser-273, Ser-282,
and Ser-302 phosphorylation.
A, His6-tagged C1-M-C2
recombinant human cardiac
myosin-binding protein-C
(cMyBP-C) peptide that
includes the C1-M-C2
domains with the three phosphorylation sites. In the normal
endogenous protein, each of
these three serines is phosphorylatable. B, The ability of
each residue to be phosphorylated by protein kinase (PK) A,
Ca2⫹-calmodulin-activated
kinase II (CaMKII␦), or PKC␧ is
shown in (B) and (C), respectively. A dash indicates that
the endogenous fragment with
all three serines intact is present, and immunoblotting is
indicated by “IB” (Western
blotting). The mutation of serine (S) to alanine (A) at each
residue is indicated as Ser to
Ala. Phosphorylation of each
residue with each enzyme was
assayed using our phosphoresidue–specific antibodies via
Western blot analyses.
Ponceau-S staining of the
membranes confirmed equal
amounts of protein were
loaded. D and E, Western blot
analyses, using site-specific phospho cMyBP-C antibodies, of neonatal rat cardiomyocytes infected with constitutively active (CA) and
dominant negative (DN) CaMKII␦ and (F and G) PKC␧ adenoviruses (Ad) at a multiplicity of infection of 100 (n⫽5). C, Negative control
using an empty vector for infection, isoproterenol-treated (ISO); 50 nmol/L ISO neonatal rat cardiomyocytes. H and I, At different time
points after transverse aortic constriction (TAC) in mice, Western blot analysis of total heart proteins using site-specific phospho
cMyBP-C antibodies demonstrated that phosphorylation increased significantly after 30 minutes, but that Ser-282 phosphorylation was
significantly reduced after 24 hours compared to Ser-273 and Ser-302 sites (n⫽6). *P⬍0.01 vs control or sham (n⫽5).
Statistical Analysis
All biochemical and functional assays were performed in mice
obtained at 10 to 12 weeks of age along with mixed-gender controls.
One-way ANOVA was used to test for overall significance, followed
by the Student–Newman–Keuls test for multiple comparison testing
between the various groups (Sigma Plot 11). Data are presented as
means⫾SEM.
Results
Phosphorylation of Ser-282 Is Critical for
Ser-302 Phosphorylation
We focused on three sites in the cardiac-specific insertion
region of cMyBP-C to determine their roles in modulating
cardiac function. Initially, to determine the substrate specificity of these sites by PKA,21 PKC,21 and CaMKII,19 recombinant human His-tagged C1-M-C2 fragments of cMyBP-C
were generated in which the three phosphorylatable sites were
individually mutated to alanine, as well as a wild-type control
(Figure 1A).22 Peptides were treated with PKA, PKC␧, or
CaMKII␦, and their phosphorylation status was determined by
Western blot analysis using site-specific cMyBP-C phosphoantibodies (Figure 1B, 1C). Experiments with PKA-mediated
and PKC-mediated phosphorylation confirmed that PKA phosphorylates all three sites, whereas PKC phosphorylates only
Ser-273 and Ser-302 (Figure 1C). At low Ca2⫹ concentrations,
CaMKII␦ targets Ser-302, whereas at high Ca2⫹ concentrations
Ser-282 also can be a substrate (Online Figure I, available online
at http://circres.ahajournals.org), confirming that Ca2⫹dependent CaMKII differentially phosphorylates Ser-282 and
Ser-302. Phosphorylation at Ser-282 appears to be required for
Ser-302 phosphorylation in vitro (Figure 1B, panel 4, antip302).
To explore further the site-specificity of Ser-273, Ser-282,
and Ser-302 for PKA, PKC, and CaMKII, we infected
neonatal rat cardiomyocytes with adenovirus expressing constitutively active and dominant-negative forms of rat
CaMKII␦C29 and rabbit PKC␧.30 Western blot analysis
showed 10⫾2-fold overexpression of CaMKII␦ and 4⫾1.2fold overexpression of PKC␧ in neonatal rat cardiomyocytes
(Figure 1D, 1F). Cardiomyocytes were treated with isoproterenol as a positive control for the PKA activation. We then
performed Western blot analysis using our site-specific
cMyBP-C phospho-antibodies24 and compared those results
with isoproterenol-treated cardiomyocytes as a positive control.24 Results show that overexpression of constitutively
active CaMKII␦ specifically increased Ser-302 phosphorylation compared to controls, confirming that Ser-302 is a
substrate for CaMKII␦ ex vivo, whereas the PKC␧ transfections confirmed that Ser-273 and Ser-302 are both PKC
substrates (Figure 1D–1G). To demonstrate active regulation
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Figure 2. Transgenic expression of cardiac myosin-binding protein-C (cMyBP-C) mutants in the cMyBP-C(t/t) null background.
A, Schematic diagram of cMyBP-C illustrates domain structure and regions that interact with the other filament systems in the sarcomere. B, Mutations in the cMyBP-C transgenes. C, SDS-PAGE analysis of myofibrillar proteins from nontransgenic (NTG), cMyBP-C
null (t/t), transgenic (TG) cMyBP-CWT (wild-type [WT]), cMyBP-CSAS (Ser273-Ala282-Ser302 [SAS]), cMyBP-CADA (Ala273-Asp282Ala302 [ADA]), and cMyBP-CDAD (Asp273-Ala282-Asp302 [DAD]) hearts. D, Expression levels of total cMyBP-C and the presence of
myc-tagged (TG) cMyBP-C in the same mice were confirmed by Western blot analysis. E, Western blot analyses using phosphospecific antibodies on the in vitro dephosphorylated and phosphorylated NTG, cMyBP-CWT, and cMyBP-CSAS myofibrils with phosphatase (control), protein kinase (PK) C␧, PKA, and Ca2⫹-calmodulin-activated kinase II (CaMKII␦) treatment. PKA phosphorylates all three
sites, whereas PKC phosphorylates Ser-273 and Ser-302. CaMKII␦ was used at two different Ca2⫹ concentrations. F, Quantitative analysis of Western blot data (E). No significant differences were found between NTG and WT(t/t) groups. Values represent means⫾standard
error for each group (n⫽3), and designations denote significant differences (P⬍0.001) from *WT(t/t) and #controls.
of these sites during cardiac stress, a transverse aortic
constriction-induced pressure-overload mouse model was
used. Western blot analyses, using myofilament proteins from
the transverse aortic constriction-induced pressure-overload
model, showed that Ser-273 and Ser-302 phosphorylation
was significantly increased after 5 minutes but decreased
after 4 weeks of transverse aortic constriction (Figure 1H, 1I).
In contrast, Ser-282 phosphorylation was significantly increased after 5 minutes but decreased after 24 hours, showing
the sites were differentially regulated in terms of their
steady-state phosphorylation levels. These in vivo experiments are consistent with the hypothesis that Ser-282 phosphorylation is an early event in the stressed heart but does not
resolve the question of whether its posttranslational modification is necessary for the subsequent phosphorylation of the
other sites.19,21 To resolve this question, we turned to the
intact animal and replaced endogenous cMyBP-C with protein that could not be phosphorylated at Ser-282.
Three Transgenic Mouse Models
Three separate constructs were used to generate multiple lines
of TG mice to test the hypothesis that Ser-282 phosphorylation influenced the phosphorylation levels of residue 302
(Figure 2A, 2B). In the cMyBP-CSAS mouse model, Ser-282
of cMyBP-C was mutated to a nonphosphorylatable alanine,
preventing Ser-282 phosphorylation. In the cMyBP-CADA
mouse model, the PKC sites (Ser-273 and Ser-302) were
mutated to nonphosphorylatable alanines and Ser-282 was
replaced with aspartate, resulting in a Ser-282 phosphomimetic. In contrast, the third mouse model, cMyBP-CDAD,
contains charged residues (aspartates) at amino acids 273 and
302, mimicking constitutive PKC-mediated phosphorylation
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Figure 3. Phenotypic analyses of sarcomere architecture. A, Incorporation of
Myc-tagged mutant cardiac myosin-binding
protein-C (cMyBP-C) was confirmed by
immunofluorescent staining of cMyBP-C
with either an anti-myc (A) or an anticMyBP-C antibody (B). C, Longitudinal sections of the heart stained with hematoxylin
and eosin (20⫻) or (D) Masson-trichrome
(20⫻) to assess fibrosis. Note the areas of
disrupted myocyte organization and fibrosis
in the (t/t) and Asp273-Ala282-Asp302
(DAD(t/t)) hearts. E, Transmission electron
micrographs of sarcomeres show loss of
the normal M-line structures in both
cMyBP-C(t/t) and cMyBP-CDAD(t/t) hearts.
For each line, between 240 and 539 individual sarcomeres were scored. For the control hearts, wild-type (WT) (t/t), 544 sarcomeres were counted with 9% blindly
scored as “abnormal.” For the Ser273Ala282-Ser302 (SAS(t/t)) or Ala273-Asp282Ala302 (ADA(t/t)) animals, 15% to 18% had
abnormal M-lines, whereas for the DAD(t/t)
animals 244 sarcomeres were counted and
83% had scores indicating abnormal
M-lines.
in the absence of phosphorylation at Ser-282, resulting in
Ser-282 phospho-ablation. All TG mice were characterized
before crossing into the cMyBP-C(t/t) background to assess
any effects of TG expression. In each TG mouse model, we
obtained three lines with variable transgene expression,
resulting in 25% to 45% replacement of the endogenous
cMyBP-C with TG protein (Online Figure II). None of the
lines showed any obvious phenotype or increased morbidity
and mortality over the course a 10-month period, suggesting
that the SAS, ADA, and DAD mutations are benign in the
unstressed animals (data not shown).
To obtain complete replacement of endogenous cMyBP-C
with the TG mutant proteins, the individual cMyBP-CSAS,
cMyBP-CADA, and cMyBP-CDAD lines were bred into the
cMyBP-C null background (t/t).31 SDS-PAGE confirmed
normal levels of cMyBP-C in this background, driven by
Table 1.
transgenic expression of the mutant cMyBP-C and levels of
other sarcomeric proteins, such as actin and myosin, were
unaltered (Figure 2C). Using antibodies against the myctag as well as to the first 14 N-terminal residues of
cMyBP-C, the incorporation of the mutant cMyBP-C into
the sarcomere was confirmed as well as the ability of the
protein to maintain the proper stoichiometry by Western
blotting (Figure 2D).
To determine if elimination of Ser-282 phosphorylation
affected Ser-302 phosphorylation, we treated myofilaments
derived from cMyBP-CSAS(t/t) hearts with PKA, PKC, and
CaMKII and determined the phosphorylation status of each
residue by Western analyses using the residue-specific
phospho-antibodies (Figure 2E). As expected, the Ser-282
site-specific phospho-antibody showed no reactivity under
any conditions with cMyBP-CSAS(t/t)-derived protein. Strik-
In Vivo Cardiac Function Assayed by M-Mode Echocardiography
NTG (n⫽7)
(t/t) (n⫽15)
WT(t/t) (n⫽7)
SAS(t/t) (n⫽12)
ADA(t/t) (n⫽8)
DAD(t/t) (n⫽8)
IVSd, mm
0.73⫾0.12
1.15⫾0.15‡
0.74⫾0.10㛳
1.02⫾0.13‡
1.14⫾0.15‡
1.21⫾0.10‡
LVPW, mm
0.80⫾0.06
1.06⫾0.20*
0.79⫾0.12§
0.87⫾0.13
0.96⫾0.10
1.04⫾0.18*
LVIDd, mm
3.86⫾0.17
4.73⫾0.49‡
3.93⫾0.32㛳
4.29⫾0.34§
4.33⫾0.38
4.53⫾0.38*
LVIDs, mm
2.63⫾0.23
3.86⫾0.53‡
2.85⫾0.38¶
3.27⫾0.38
3.40⫾0.32*
3.46⫾0.51*
FS, %
32⫾4
18⫾5‡
28⫾5§
24⫾5
22⫾4*
24⫾7
LV VOLd, mm
64⫾7
105⫾25‡
68⫾12㛳
83⫾15
85⫾18
95⫾18†
LV VOLs, mm
26⫾6
66⫾21‡
31⫾9㛳
44⫾13
48⫾11*
51⫾17*
LV EF, %
60⫾5
38⫾9‡
54⫾8§
48⫾8
44⫾7*
47⫾11
LV Mass
84⫾17
193⫾44‡
87⫾18¶
134⫾31§
153⫾29
184⫾21‡
25.9⫾0.36
25.7⫾0.42
26.3⫾0.39
24.95⫾0.51
26.17⫾0.37
25.6⫾0.46
Body weight, g
ADA indicates Ala273-Asp282-Ala302; DAD, Asp273-Ala282-Asp302; EF, ejection fraction; FS, fractional shortening; IVSd, interventricular septum; LV, left
ventricular; LVIDd, left ventricular inner diameter in diastole; LVIDs, left ventricular inner diameter in systole; NTG, nontransgenic; PW, posterior wall; SAS,
Ser273-Ala282-Ser302; VOLd, volume in diastole; VOLs, volume in systole; WT, wild-type.
Measurement values in mm were averaged from at least three separate cardiac cycles. All values given as mean⫾standard deviation.
*P⬍0.05 vs NTG; †P⬍0.01 vs NTG; ‡P⬍0.001 vs NTG; §P⬍0.05 vs (t/t); ¶P⬍0.01 vs (t/t); 㛳P⬍0.001 vs (t/t).
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Figure 4. Cardiac myosin-binding protein-C (cMyBP-C) phosphorylation and cardiac function. A and B, Phosphorylation status of Ser273, Ser-282, and Ser-302 sites before and after dobutamine treatment
was determined by Western blots using phospho-specific antibodies
(n⫽5). C, Contractile function was measured at baseline and dobutamine (32 ng/mL min). Maximal rate of ventricular contraction (dP/dtmax)
and relaxation (dP/dtmin). Data are presented as mean⫾standard error
(n⫽6). *P⬍0.05, **P⬍0.005 compared with nontransgenic (NTG);
¶P⬍0.05, ¶¶P⬍0.005, ¶¶¶P⬍0.0005 compared with (t/t); §P⬍0.05
compared with basal (absence of dobutamine stimulation)
measurements.
ingly, the loss of Ser-282 phosphorylation impacted significantly on the ability of PKA, PKC, and CaMKII to phosphorylate Ser-302 (Figure 2E). Specifically, CaMKII-mediated
phosphorylation was completely abolished in the SAS myofilament preparations (Figure 2E and Figure 4). To determine
whether increased Ca2⫹ concentrations could overcome this
block in the phosphor-ablated cardiac sample, 25 mmol/L
Ca2⫹ was used with CaMKII to treat the cMyBP-CSAS(t/t)
myofilaments. Western blot analyses showed that increased
Ca2⫹ concentrations can stimulate Ser-282 phosphorylation
in the NTG and cMyBP-CWT(t/t) myofilaments, whereas
Ser-302 phosphorylation in the absence of Ser-282 phosphorylation was reduced, with the site being hypophosphorylated
relative to normal cMyBP-C (Figure 2F).
Sarcomere Architecture in the SAS, ADA, and
DAD Mutant Hearts
We reported previously that expression of cMyBP-CWT(t/t)
and cMyBP-CAllP⫹(t/t) effectively rescued the cMyBP-C(t/t)
phenotype, but that expression of the nonphosphorylatable
cMyBP-CAllP⫺ failed to rescue the null, suggesting the
necessity of cMyBP-C phosphorylation for normal sarcomere and cardiac function.7,9 To explore this observation at the
individual residue level, we first determined whether the
phospho-specific mutants incorporated normally into the sarcomere. Immunohistochemical analyses were performed with
both anti-myc and anti-cMyBP-C antibodies (Figure 3A, 3B).
Results show that SAS, ADA, and DAD proteins were
incorporated normally into the sarcomere in the cMyBP-C(t/t)
mouse background with striations indistinguishable from
those seen in the NTG and cMyBP-CWT(t/t) sarcomeres
(Figure 3A, 3B). Histological analyses showed that, as
expected, the cMyBP-C(t/t) hearts displayed cardiac hypertrophy with fibrosis and disarray. However, there were no
obvious abnormalities, fibrosis, calcification, or disarray
observed in the cMyBP-CSAS(t/t) and cMyBP-CADA(t/t) samples compared to NTG and cMyBP-CWT(t/t) controls (Figure
3C and 3D). In contrast with these data, the cMyBP-CDAD(t/t)
mouse hearts showed fibrosis and disarray, mimicking the
cMyBP-Ct/t phenotype. Considering that this mouse contains
the cMyBP-C phospho-mimetic in which residues 273 and
302 are constitutively charged, the data emphasize the importance of Ser-282 phosphorylation for cMyBP-C function.
Transmission electron microscopy confirmed the previous
observations of subtle organizational changes at the center of
the sarcomere, at the M-line, and in the cMyBP-C–null mice,
with the characteristic electron-dense M-line largely absent or
irregular (Figure 3E).7,9,31 Consistent with the lack of disarray
and fibrosis observed in the cMyBP-CWT(t/t), cMyBP-CSAS(t/t), and
cMyBP-CADA(t/t) hearts, the center of those sarcomeres contained a well-defined M line that was indistinguishable from
those observed in normal sarcomeres in 81% to 91% of the
sarcomeres. In contrast, 83% of the cMyBP-CDAD(t/t) sarcomeres displayed significant M-line abnormalities, suggesting
that Ser-282 phosphorylation is necessary for the structural
functions of cMyBP-C.
Sadayappan et al
Table 2.
Cardiac Myosin Binding Protein-C Phosphorylation
147
In Vivo Cardiac Hemodynamic Measurements
Heart rate, bpm
Basal
Dobutamine (32 ng/g/min)
MAP, mm Hg/s
Systolic pressure,
mm Hg/s
LVP, mm Hg/s
dP/dtmax, mm Hg/s
LVEDP, mm Hg/s
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dP/dt40, mm Hg/s
(t/t) (n⫽8)
WT(t/t) (n⫽6)
SAS(t/t) (n⫽6)
ADA(t/t) (n⫽5)
DAD(t/t) (n⫽5)
411⫾8
383⫾7*
400⫾5
363⫾7†
353⫾12†
361⫾20
589⫾10
533⫾16*
587⫾10‡
555⫾20
514⫾10†
518⫾33
Basal
72.8⫾3
66⫾2
74⫾4
68⫾4
68⫾5
59⫾6
Dobutamine (32 ng/g/min)
67.9⫾10
58⫾6
70⫾3
60⫾5
60⫾5
61⫾4
Basal
86.8⫾7
76⫾5
88⫾5
78⫾5
88⫾8
70⫾5
Dobutamine (32 ng/g/min)
85⫾8
72⫾7
85⫾3
70⫾6
82⫾9
80⫾6
Basal
94.7⫾5
88⫾2
98⫾6
90⫾4
90⫾5
81⫾4*
Dobutamine (32 ng/g/min)
106.9⫾4
91⫾4
105⫾4‡
93⫾3
93⫾4
91⫾4
7853⫾385
7842⫾464
7472⫾582
7058⫾805
6592⫾964
Basal
Dobutamine (32 ng/g/min)
dP/dtmin, mm Hg/s
NTG (n⫽5)
8611⫾703
14 858⫾1362*
19 016⫾664‡
15 283⫾1426*
14 242⫾1629*
15 268⫾1328*
Basal
⫺10 008⫾914
⫺4486⫾224†
⫺7720⫾361¶
⫺5868⫾483†‡
⫺6112⫾724*
⫺6477⫾824*
Dobutamine (32 ng/g/min)
⫺14 548⫾2091
⫺5225⫾409*
⫺10 138⫾952§
⫺8077⫾495*§
⫺7143⫾727*‡
⫺7264⫾505*‡
10⫾1
15⫾1†
6⫾2
Basal
19 480⫾1227
7.2⫾0.3
13⫾2
5⫾1‡
Dobutamine (32 ng/g/min)
2.6⫾0.8
3⫾0.7
4⫾2
3⫾1
Basal
8053⫾508
7358⫾350
7106⫾294
3⫾1
7082⫾618
3⫾0.8
6270⫾1005
5945⫾901
Dobutamine (32 ng/g/min)
16367⫾499
13 678⫾995*
16 037⫾284‡
13 536⫾1140*
12 831⫾1364*
13 776⫾902*
ADA indicates Ala273-Asp282-Ala302; DAD, Asp273-Ala282-Asp302; dP/dtmax, maximum rate of heart contraction and relaxtion; dP/dtmin, minimum rate of heart
contraction and relaxtion; EDP, end-diastolic pressure; LV, left ventricular; LVP, left ventricular pressure; NTG, nontransgenic; MAP, mean arterial pressure; SAS,
Ser273-Ala282-Ser302; WT, wild-type.
Ventricular contraction and relaxation rates were measured at baseline and during ␤-agonist stimulation in the intact closed chest model, including telemetric
measurements of heart rate, MAP, LV systolic pressure and LVEDP, LVP, ⫹dP/dtmax, ⫺dP/dtmin, and the rate of LVP increase determined at an LVP of 40 mm Hg
(dP/dt40). Cardiovascular stress response to increasing doses of dobutamine was determined during 3-minute constant infusions (0.1 ␮L/min/g body weight), and data
were analyzed using a PowerLab system (AD Instruments). Data are presented as mean⫾SE.
*P⬍0.05; †P⬍0.005 compared with NTG; ‡P⬍0.05; §P⬍0.005; ¶P⬍0.0005 compared with (t/t).
The heart-to-body weight ratios were normal in cMyBPCSAS(t/t) (0.57⫾0.02) and cMyBP-CADA(t/t) (0.57⫾0.02) mice
compared to cMyBP-C WT(t/t) (0.56⫾0.02) and NTG
(0.50⫾0.013) controls at 3 months. Consistent with the
histology, cMyBP-CDAD(t/t) mouse hearts showed significantly increased heart-to-body weight ratios (0.70⫾0.015,
P⬍0.0001 vs NTG), as did the cMyBP-C(t/t) hearts
(0.81⫾0.02, P⬍0.0001 vs NTG),7 but with normal systolic
function (Table 1). In contrast, cMyBP-CADA(t/t) mice showed
normal heart-to-body weight ratios but reduced systolic
function, suggesting that phospho-specific mutations have
differential effects on cardiac structure and function. M-mode
echocardiographic measurements showed that expression of
normal cMyBP-C (cMyBP-CWT(t/t)) rescued the cMyBP-C(t/t)
phenotype as previously shown (Table 1).7,9 In contrast to
those data, cMyBP-CSAS(t/t), cMyBP-CADA(t/t), and cMyBPCDAD(t/t) mice showed only partial rescue of the null phenotype. Each model had significantly increased intraventricular
septal thickness and left ventricular end-diastolic and endsystolic dimensions (Table 1), indicating that none was as
effective at restoring normal cardiac hemodynamics as the
WT protein (Table 1). The cMyBP-CSAS(t/t), cMyBP-CDAD(t/t)
and cMyBP-CADA(t/t) hearts, all showed reduced fractional
shortening, although only the cMyBP-CADA(t/t) hearts reached
statistical significance. These data suggest that phosphorylation at Ser-273, Ser-282, and Ser-302, individually or in
combination, is required for normal cardiac function to be
maintained long-term.
␤-AR Stimulation and cMyBP-C Phosphorylation
Phospho-ablation at Ser-282 attenuates Ser-302 phosphorylation, and we hypothesized that this would critically impact
the ability of the heart to respond to ␤-AR stimulation. The
cMyBP-CSAS(t/t) hearts contain only cMyBP-CSAS cMyBP-C,
allowing us to determine the functional consequence of the
inability of Ser-282 to be phosphorylated in vivo during
␤-AR stimulation. We noted that Ser-273 appeared to be
hyperphosphorylated in the cMyBP-CSAS hearts at baseline,
in contrast to the hypophosphorylation observed at Ser-302
(Figure 4A, 4B). At baseline, all groups showed normal
systolic function, suggesting that mutating the three selected
phosphorylation sites does not affect systolic function (Table
2). On ␤-AR stimulation (dobutamine infusion), a reduced
response was observed in all of the TG hearts, with the
exception of those animals expressing the normal cMyBP-C
protein (cMyBP-CWT/(t/t)). The cMyBP-CSAS(t/t) hearts further
showed significantly decreased phosphorylation at Ser-302
during ␤-agonist stimulation compared to NTG and cMyBPCWT:(t/t) controls, confirming the importance of Ser-282
phosphorylation in mediating Ser-302 phosphorylation as
well (Figure 4C). Interestingly, the mice containing
phosphor-mimetics (cMyBP-CDAD:(t/t)) and phospho-ablation
(cMyBP-CADA:(t/t)) at residues 273 and 302 could not respond
normally to dobutamine, emphasizing the importance of
normal and, potentially, reversible phosphorylation at each of
the three sites in mediating normal function and
␤-agonist⫺induced stimulation.
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Figure 5. Cardiac myosin-binding protein-C (cMyBP-C) domains and kinases that target the three phosphorylation motifs.
cMyBP-C is the 140-kDa-thick filament sarcomeric proteins that consists of 11 domains; 8 immunoglobulin (oval), and 3 fibronectin
type III (square) domains. The C0 domain, phosphorylation motif (m), and a cardiac-unique insertion in the C5 domain are unique (light
green) to cMyBP-C when compared to skeletal MyBP-C. The proline-rich/alanine-rich region (Pro-Ala) and hypothesized actin and myosin binding sites are marked. The three phosphorylation motifs, Ser-282, Ser-273, and Ser-302, are shown (reference to the mouse
identification number), which are targeted by protein kinase (PK) A,21 PKC,21 Ca2⫹-calmodulin-activated kinase II (CaMKII),19 PKD,22
and the 90-kDa ribosomal s6 kinase (RSK).23 Amino acid sequences are numbered using Uniprot and National Center for Biotechnology Information reference numbers. Note that Ser-282 in the mouse corresponds to Ser-284 in the human sequence.
Discussion
Increasing data suggest that cMyBP-C phosphorylation can
regulate myocardial function,7 sarcomere integrity,9 and, in
some cases, can be cardioprotective.9,25 It is currently the only
thick filament protein that is differentially phosphorylated by
five important kinases (Figure 5) that can impact on cardiac
contraction: PKA,21 PKC,21 CaMKII,19 PKD,22 and ribosomal
s6 kinase.23 Our present study focused on deciphering the
phosphorylation pattern of cMyBP-C and determining which
phosphorylatable sites are necessary or perhaps sufficient for
normal cardiac function. Although cMyBP-C is extensively
phosphorylated under basal conditions, the level of cMyBP-C
phosphorylation decreases in animal models during development of heart failure, myocardial injury, pathological hypertrophy, and myocardial stunning.5,20,24,32,33 Previously, to
better-understand the mechanisms and significance of
cMyBP-C phosphorylation, we established two TG mouse
models to determine the necessity and sufficiency of
cMyBP-C for normal cardiac function.7,9 Our findings
showed that cMyBP-C phosphorylation is essential for normal cardiac function7 and that a phospho-mimetic at the
Ser-273, Ser-282, and Ser-302 phosphorylation sites conferred cardioprotection against myocardial ischemia reperfusion injury in either the ␣-myosin heavy chain9 or ␤-myosin
heavy chain backgrounds.24
Phosphorylation of Ser-282 was a focus of the present
study because phosphorylation levels of this residue are
decreased in patients with heart failure32,34 and atrial fibrilla-
tion.4 Strikingly, Ser-282 is also selectively phosphorylated
by ribosomal s6 kinase, which regulates myofilament function.23 In vitro studies demonstrated that phosphorylation at
Ser-282 is a prerequisite for phosphorylation of the Ser-273
and Ser-302 sites.19,21 The data obtained from those studies
also showed that the ability to phosphorylate Ser-273 and
Ser-302 is markedly reduced when Ser-282 is mutated to
alanine, suggesting that hierarchical phosphorylation may be
involved in cMyBP-C–mediated regulation of contraction in
cardiac muscle. In the present study, we removed the ability
of Ser-282 to be phosphorylated to investigate the importance
of Ser-282 phosphorylation in maintaining normal cardiac
function at the whole organ function. Our data demonstrate
that Ser-282 regulates the subsequent phosphorylation of
Ser-302, but not Ser-273.19,21,22 We hypothesize that Ser-282
is both a regulatory and functional site for phosphorylation,
with the cMyBP-CDAD:(t/t) mice showing significant pathology at the light and ultrastructural levels (Figure 3D, 3E).
cMyBP-C is a substrate for PKC, which can phosphorylate
Ser-273 and Ser-302 in cardiomyocytes.21 PKC␧ can also
phosphorylate cMyBP-C,35 and this series of enzymes, some
more selective than others, makes cMyBP-C phosphorylation
exquisitely responsive to the changing environment.
cMyBP-C phosphorylation, which is partially mediated by
PKC, appears to play a critical role in cardiac function.9
However, the roles of these sites in cardiac physiology have
not been characterized. PKC phosphorylation mediates a
major mechanism by which the myofilament modulates
Sadayappan et al
Cardiac Myosin Binding Protein-C Phosphorylation
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changes in myocardial function.35 Gautel et al19 obtained in
vitro evidence that phosphorylation of Ser-282 may potentiate the PKC phosphorylation sites (Ser-273 and Ser-302), and
thus phosphorylation could function as a switch that might
also be triggered to mediate accelerated cardiac function
during ␤-AR activation. Evidence from other studies suggests
that cMyBP-C phosphorylation influences actomyosin Mg2⫹ATPase activity, the kinetics of cross-bridge cycling, and the
rate of relaxation.19,36 –38 Therefore, the second objective of
the study was to assess the necessity and sufficiency of
PKC-mediated cMyBP-C phosphorylation for sarcomeric
integrity and for normal cardiac function. cMyBP-CADA(t/t)
and cMyBP-CDAD(t/t) hearts showed decreased hemodynamics and contractility relative to cMyBP-CWT(t/t) controls
during ␤-AR stimulation because of the constitutive phosphoablation or phospho-mimetic “phosphorylation” (charged residues) of the PKC phosphorylation sites in cMyBP-C.
Importantly, cMyBP-C ADA(t/t) mice unambiguously
showed the effect of Ser-282 phosphorylation in the absence
of PKC site phosphorylation on whole organ anatomy and
function and partially rescued the cMyBP-C(t/t) phenotype.
Conversely, despite the charged residues at Ser-273 and
Ser-302, the cMyBP-CDAD(t/t) hearts displayed extensive
damage compared to cMyBP-CADA(t/t) and cMyBP-CSAS(t/t)
mice and damage to cellular components (Figure 3) and had
hypertrophy over time, mimicking the t/t null phenotype.
Why do the cMyBP-CDAD(t/t) hearts display more overt
abnormal pathology than the cMyBP-CSAS(t/t) mice when
residue 282 is mutated in both? Overexpression of PKC
isoforms in the heart causes hypertrophy in adult mice.39 We
hypothesize that mimicking constitutive activation of these
two PKC sites in cMyBP-C by replacing the two serines with
aspartates in the cMyBP-CDAD(t/t) cardiomyocytes is detrimental to the hearts. In contrast, serines 273 and 302 in the
cMyBP-CSAS(t/t) will not behave as if they were chronically
phosphorylated; in fact, serine 302 will be hypophosphorylated in these hearts and thus not exhibit the effects of chronic
phosphorylation, as manifested both by a hypertrophic response and a decreased M-line definition.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
Conclusion
In conclusion, our data confirm the critical importance of
Ser-282 in maintaining normal cardiac (Figure 3C, 3D) and
sarcomere (Figure 3E) architecture regardless of the phosphorylation status of the PKC sites. These data provide strong
evidence that cMyBP-C phosphorylation directly affects the
heart’s contractile properties and sarcomere organization.
17.
18.
19.
Sources of Funding
This research was supported by National Institutes of Health grants
P01HL69799, P50HL074728, P50HL077101, P01HL059408,
R01HL087862 (J.R.), and R01HL105826 (S.S.), and by an American
Heart Association Scientist Development grant (0830311N to S.S.).
20.
21.
Disclosure
None.
22.
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35.
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37.
38.
39.
Novelty and Significance
What Is Known?
●
●
●
Cardiac myosin binding protein-C (cMyBP-C) can be phosphorylated
at multiple sites, with phosphorylation levels decreasing during
cardiac stress and the development of heart failure.
Three of the serines in cMyBP-C (Ser-273, Ser-282, and Ser-302) are
substrates for protein kinase A, protein kinase C, protein kinase D,
90-kDa ribosomal s6 kinase, and Ca2⫹-calmodulin-activated
kinase II.
It is unclear whether phosphorylation of these sites is functionally
equivalent.
What New Information Does This Article Contribute?
●
●
During cardiac stress, Ser-282 phosphorylation is differentially regulated, compared to the other two sites, suggesting that Ser-282
phosphorylation has a unique role in myocardial function.
A phosphorylated serine at position 282 is required for Ser-302
phosphorylation, suggesting that Ser-282 phosphorylation is a
critical determinant of cMyBP-C phosphorylation at Ser-302.
●
●
Ser-302 is differentially phosphorylated by calmodulin-dependent
protein kinase II.
The data suggest that Ser-282 phosphorylation may act in a
regulatory manner. In addition, Ser-302 is also a functional site,
partially mediating the contractile response to ␤-adrenergic
stimulation.
We previously determined that cMyBP-C phosphorylation is vital
for normal cardiac function and that phosphorylation of the
Ser-273, Ser-282, and Ser-302 sites conferred cardioprotection
against ischemic reperfusion injury in either the ␣-myosin heavy
chain or the ␤-myosin heavy chain backgrounds. In the present
study, we discovered that Ser-282 phosphorylation is a critical
determinant of Ser-302 phosphorylation and that Ser-282
partially regulates the contractile response to ␤-adrenergic
stimulation in vivo. Future studies will define the functional and
mechanistic effects of cMyBP-C phosphorylation at individual
sites in mediating the interactions of cMyBP-C with other
sarcomeric proteins, as well as posttranslational effects on
sarcomeric integrity and myocardial function.
Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017
A Critical Function for Ser-282 in Cardiac Myosin Binding Protein-C Phosphorylation
and Cardiac Function
Sakthivel Sadayappan, James Gulick, Hanna Osinska, David Barefield, Friederike Cuello, Metin
Avkiran, Valerie M. Lasko, John N. Lorenz, Marjorie Maillet, Jody L. Martin, Joan Heller
Brown, Donald M. Bers, Jeffery D. Molkentin, Jeanne James and Jeffrey Robbins
Circ Res. 2011;109:141-150; originally published online May 19, 2011;
doi: 10.1161/CIRCRESAHA.111.242560
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Online Supplement Figures
A Critical Function for Ser-282 in Cardiac Myosin Binding Protein-C
Phosphorylation and Cardiac Function
Sakthivel Sadayappan, James Gulick, Lisa A. Martin, Hanna Osinska, David Barefield,
Friederike Cuello, Metin Avkiran, Valerie M. Lasko, John N. Lorenz, Marjorie Maillet,
Jody L. Martin, Joan Heller Brown, Donald M. Bers, Jeffery D. Molkentin, Jeanne James
and Jeffrey Robbins
From the Department of Cell and Molecular Physiology, Stritch School of Medicine,
Loyola University Chicago, 2160 South First Avenue, Maywood, IL 60153, USA (S.S.,
D.B., J.L.M.); Division of Molecular Cardiovascular Biology, Department of Pediatrics,
The Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229, USA (J.G.,
L.A.M., H.O., M.M., J.D.M., J.J., J.R.); Cardiovascular Division, King’s College
London, The Rayne Institute, St Thomas’ Hospital, London SE1 7EH, United Kingdom
(F.C., M.A.); Department of Molecular and Cellular Physiology, University of Cincinnati
College of Medicine, University of Cincinnati, Cincinnati, OH 45279, USA (V.M.L,
J.N.L.); Department of Pharmacology, University of California, La Jolla, CA 92093-0636
(J.H.B); Department of Pharmacology, University of California Davis, Genomics and
Biological Sciences Facility, 451 Health Sciences Drive, Davis, CA 95616, USA
(D.M.B.).
Running title: Phosphorylation of Cardiac Myosin Binding Protein-C
Correspondence to Jeffrey Robbins PhD, Division of Molecular Cardiovascular Biology,
Cincinnati Children’s Hospital Medical Center, MLC 7020, 240 Albert Sabin Way,
Cincinnati, OH 45229-3039. Tel: (513) 636-8098; Fax: (513) 636-5958. E-mail:
[email protected]
Online Supplement Figure I. CaMKII requires high levels of Ca2+ to phosphorylate Ser282 in cMyBP-C. Recombinant His6-tagged C1-M-C2 peptide of human cMyBP-C (100
pmol) was incubated with pre-activated CaMKII at either 2mM or 25mM CaCl2, 1.2µM
CaM, 200 µM ATP for 10 min at 30°C, with recombinant Calmodulin-dependent Protein
Kinase II (CaMKII); New England Biolabs Cat. No. P6060S or the PKA catalytic subunit
(Millipore) for 20 min at 30°C in a final volume of 50 µl (30mM Tris pH 7.4; 15mM
MgCl2; 1mM DTT; 100µM ATP). Proteins were subsequently resolved on 12%
acrylamide gels, transferred nitrocellulose membranes and immunoblotted with antipSer273 (1:3000), anti-pSer282 (1:3000) or anti-pSer302 (1:10000) phospho-specific
antibodies or anti-cMyBP-C2-14 antibody. Results show that Ser-302 is preferentially
phosphorylated by the enzyme at low Ca2+ concentrations while at higher Ca2+
concentrations Ser-282 is phosphorylatable by CaMKII.
Online Supplement Figure II. Transgenic (TG) expression of cMyBP-C mutant lines.
A, Southern blot analysis of genomic DNA from non-transgenic (NTG), wild-type (WT)
TG line 21, three lines from cMyBP-CSAS (lines 142, 153 and 155), three lines of cMyBPCADA (lines 14, 22 and 25) and three lines of cMyBP-CDAD (lines 212, 214 and 151). Ten
µg of total genomic DNA was digested with EcoRI, separated on agarose, transferred to
nitrocellulose and probed with an α-myosin heavy chain promoter specific probe (1000
bp), which recognize both endogenous and TG sequences. Quantitation of the signal
indicated that the cMyBP-CSAS, cMyBP-CADA and cMyBP-CDAD lines used for the
subsequent studies inherited 5, 4 and 2 transgene copies, respectively, compared to 6
copies in the WT TG line 21. Expression of the cMyBP-C mutant constructs did not
result in any detectable embryonic lethality. B-C, Northern blot analyses of total mRNA
from left ventricles derived from 3-month old NTG, WT-Tg line 21 and 3 lines from the
cMyBP-C mutant mice. To confirm expression of the intact and correctly sized transcript,
Northern blot analysis was performed with cMyBP-C and GAPDH probes. Results show
that compared to the NTG, significantly increased cMyBP-C transcript levels were
observed in the selected TG lines (*P<0.01, n=3). D, Representative SDS-PAGE analysis
shows the conservation of the sarcomeric protein levels in these mice compared with a
NTG sample. Five µg of total myofilament proteins were loaded on a 4-15% gradient gel
(Bio-Rad, Cat. No. 456-1083) and stained with SYPRO-RUBY (Bio-Rad, Cat. No. 1703138). E, Western blot analysis shows the absence of TG cMyBP-C in the NTG hearts
and the presence of myc-tagged Tg cMyBP-C in TG mice. cTnI was used as a loading
control (Cell Signaling Technology, Cat. No. 4002S). F-H, Total cMyBP-C levels as well
as the presence of myc-tagged cMyBP-C protein was confirmed by Western blot
analysis. 5 µg of total myofilament proteins were loaded from the respective lines on the
same SDS-PAGE for Western blot analysis using monoclonal anti-Myc tag antibody
(Roche, Cat. No. 11667149001). NTG hearts do not express the Myc-tagged cMyBP-C.
The purpose of loading total myofilament proteins from both the single TG lines and the
TG line in the (t/t) background mice on the same gel, side by side, was to normalize the
loading and allow us to calculate the cMyBP-C Myc-tag replacement. In the homozygous
(t/t) background, TG cMyBP-C levels are equal to 100% as there was no trace of cMyBPC determined in the (t/t) background (Figure 2).1-3 TG Myc-tagged cMyBP-C was first
normalized with α-actin (TG Myc/actin). TG Myc-tagged cMyBP-C in the (t/t)
background was then normalized with α- actin (TG Myc(t/t)/actin). Then to obtain the
amount of replacement in the transgenic lines, the value of [TG Myc/actin]/[ TG
Myc(t/t)/actin]*100 was determined. The data obtained from these analyses were used to
determine the level of cMyBP-C mutant replacement in the TG lines, compared to the
expression in the cMyBP-C null (t/t) background. α- actin was used as a loading control
(Sigma, Cat. No. A2172).
References:
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2.
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Sadayappan S, Gulick J, Klevitsky R, Lorenz JN, Sargent M, Molkentin JD,
Robbins J. Cardiac myosin binding protein-c phosphorylation in a {beta}-myosin
heavy chain background. Circulation. 2009;119:1253-1262
Sadayappan S, Gulick J, Osinska H, Martin LA, Hahn HS, Dorn GW, 2nd,
Klevitsky R, Seidman CE, Seidman JG, Robbins J. Cardiac myosin binding
protein-c phosphorylation and cardiac function. Circ Res. 2005;97:1156-1163
Sadayappan S, Osinska H, Klevitsky R, Lorenz JN, Sargent M, Molkentin JD,
Seidman CE, Seidman JG, Robbins J. Cardiac myosin binding protein-c
phosphorylation is cardioprotective. Proc Natl Acad Sci U S A. 2006;103:1691816923