Published online before print October 13, 2005, doi: 10.1161/01.RES.0000190605.79013.4d
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© 2005 American Heart Association, Inc.
Cellular Biology |
Cardiac Myosin-Binding Protein-C Phosphorylation and Cardiac Function
From the Department of Pediatrics (S.S., J.G., H.O., R.K., L.A.M., J.R), Division of Molecular Cardiovascular Biology, Cincinnati Children’s Hospital Medical Center, OH; Department of Medicine (H.S.H., G.W.D), University of Cincinnati, OH; Department of Genetics (C.E.S., J.G.S), Howard Hughes Medical Institute and Harvard Medical School, Boston, Mass; and Cardiovascular Division (C.E.S.), Brigham and Women’s Hospital, Boston, Mass.
Correspondence to Jeffrey Robbins, MLC 7020, 3333 Burnet Ave, Cincinnati, OH 45229-3039. E-mail jeff.robbins{at}cchmc.org
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Abstract |
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The role of cardiac myosin binding protein-C (cMyBP-C) phosphorylation in cardiac physiology or pathophysiology is unclear. To investigate the status of cMyBP-C phosphorylation in vivo, we determined its phosphorylation state in stressed and unstressed mouse hearts. cMyBP-C phosphorylation is significantly decreased during the development of heart failure or pathologic hypertrophy. We then generated transgenic (TG) mice in which the phosphorylation sites of cMyBP-C were changed to nonphosphorylatable alanines (MyBP-CAllP–). A TG line showing &40% replacement with MyBP-CAllP– showed no changes in morbidity or mortality but displayed depressed cardiac contractility, altered sarcomeric structure and upregulation of transcripts associated with a hypertrophic response. To explore the effect of complete replacement of endogenous cMyBP-C with MyBP-CAllP–, the mice were bred into the MyBP-C(t/t) background, in which less than 10% of normal levels of a truncated MyBP-C are present. Although MyBP-CAllP– was incorporated into the sarcomere and expressed at normal levels, the mutant protein could not rescue the MyBP-C(t/t) phenotype. The mice developed significant cardiac hypertrophy with myofibrillar disarray and fibrosis, similar to what was observed in the MyBP-C(t/t) animals. In contrast, when the MyBP-C(t/t) mice were bred to a TG line expressing normal MyBP-C (MyBP-CWT), the MyBP-C(t/t) phenotype was rescued. These data suggest that cMyBP-C phosphorylation is essential for normal cardiac function.
Key Words: mouse • mouse mutants • muscle • muscle contraction • myocardial contractility
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Introduction |
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Understanding the structure/function relations for cardiac myosin binding protein-C (cMyBP-C) is clinically relevant, as cMyBP-C mutations are a widely recognized cause of familial hypertrophic cardiomyopathy.1 Various cMyBP-C transgenic (TG) and gene-targeted mouse models have demonstrated the importance of the protein for long-term integrity of sarcomeric structure and for maintaining normal cardiac contractility.2,3 Functional insight can be gained from appreciating the crucial structural differences between cMyBP-C and the skeletal isoform. Only the cardiac isoform contains an extra immunoglobulin domain at the N terminus (C0), an insertion of 28 residues within the C5 domain, and three phosphorylation sites (Ser273, -282, -302) that are substrates for cAMP-dependent protein kinase A (PKA), Ca2+-calmodulin-activated kinase and protein kinase C.
In vivo, PKA-mediated phosphorylation of cMyBP-C is linked to modulation of cardiac contraction.4 On adrenergic stimulation, PKA phosphorylates Ser273, -282, and -302, whereas protein kinase C phosphorylates only Ser273 and -302.5 These residues, located near the N terminus of the protein, are of particular interest, as this region binds to the S2 segment of the myosin heavy chain (MHC),6,7 which is close to the lever arm domain of myosin. It has been hypothesized that cMyBP-C/MHC interactions are dynamically regulated by the phosphorylation/dephosphorylation of cMyBP-C.8 In vitro experiments showed that after phosphorylation of cMyBP-C by PKA, the thick filaments exhibited a relative loose structure,9 preventing binding to myosin and, thus, changing the maximum Ca2+-activated force (Fmax). Electron microscopy of isolated thick filaments confirmed that phosphorylation of cMyBP-C initiates crossbridge movement away from the thick-filament backbone, although this interaction may also be determined by the MHC isoform that is present.10 In theory, cMyBP-C phosphorylation could change both filament orientation and contractile mechanics,7,11 although cMyBP-C phosphorylation did not alter the Ca2+ sensitivity of Mg2+-ATPase activity in reconstituted contractile protein systems.12,13 Additionally, phosphorylation of cMyBP-C did not affect the Ca2+ sensitivity of Mg2+-ATPase activity, force-Ca2+ relationship, or sarcomere length dependency of contraction in intact skinned fiber experiments,14,15 suggesting that MyBP-C phosphorylation plays a relatively minor role in regulating contraction. Given the above body of data, the role that cMyBP-C phosphorylation plays in either normal or pathological cardiac function remains unclear.
We wished to determine whether cMyBP-C phosphorylation played an essential role in the maintenance of cardiac function. We first defined the phosphorylation status of cMyBP-C in the normal and diseased heart and showed that significant changes occurred during development of cardiac pathology. We then generated a TG mouse model in which the known phosphorylation sites in cMyBP-C were converted to nonphosphorylatable alanines (MyBP-CAllP–) and compared this model to a model with comparable levels of TG expression of normal cMyBP-C (MyBP-CWT). Both TG models were subsequently bred into the homozygous cMyBP-C(t/t) background.3 The data establish that cMyBP-C phosphorylation is essential for normal cardiac function.
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Materials and Methods |
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An expanded Materials and Methods section can be found in the online data supplement available at http://circres.ahajournals.org.
Transgenic and Targeted Lines
The cDNA for mouse cMyBP-C (MyBP-CWT) was obtained by RT-PCR using RNA isolated from the mouse cardiac ventricle. The cMyBP-C phosphorylation sites (Ser273, Ser282, and Ser302), along with 2 adjacent sites that could be potentially phosphorylated (Thr272, Thr281), were converted to alanine and a sequence encoding the myc epitope incorporated as described previously.2 The cDNA was subcloned into the mouse 
-MHC promoter and used to generate multiple lines of TG mice for each construct. To ensure the absence of endogenous phosphorylatable cMyBP-C, mice showing germline transmission of the transgene, termed MyBP-CAllP–, were bred with mice that expressed less than 10% of normal cMyBP-C levels (MyBP-C(t/t)). The small amount of protein that is present in these animals is truncated and unstable.3 An animal that expressed the normal cardiac isoform at equivalent levels to MyBP-CAllP–, MyBP-CWT,2 was also bred to the nulls to serve as a control. All protocols complied with the Guide for the Use and Care of Laboratory Animals published by the National Institutes of Health.
Molecular and Protein Analyses
For assessment of cMyBP-C transcript size, expression levels, and expression of hypertrophic marker genes, RNA transcript and blot analyses were performed as described.2,16 Enriched myofibrillar proteins were isolated using F60 buffer and solubilized in urea buffer.17 MyBP-CAllP– was identified via SDS-PAGE followed by Western blots using anti-myc monoclonal antibodies and anti–cMyBP-C rabbit polyclonal antibodies raised against the C0–C1 domains.18
Phosphorylation and Isoelectric Focusing
Myofibrillar proteins were treated with phosphatase and PKA as described.17 One-dimensional isoelectric focusing (IEF) was performed to identify the phosphorylated forms.18
Cardiac Function
In vivo cardiac function was assessed both by noninvasive echocardiography in nonsedated or sedated animals and by invasive catheterization.19
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Results |
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Phosphorylation States of cMyBP-C in Normal and Heart Failure Mouse Models
As is the case for a number of other contractile proteins, cMyBP-C can be reversibly phosphorylated at multiple sites in response to altered physiological conditions.20 However, the in vivo phosphorylation levels of the protein under basal and stressed conditions are not well defined. To understand the level of phosphorylation in the nontransgenic (NTG) animals before we perturbed the cMyBP-C protein complement, cMyBP-C phosphorylation under normal and stressed conditions was determined using IEF followed by Westerns using a cMyBP-C C0–C1 motif–specific antibody.18 Total enriched myofibrillar proteins were extracted from unstressed, NTG adults, animals that had undergone transverse aortic constriction (TAC) to produce pressure overload hypertrophy, animals in failure as a result of cardiac specific expression of calcineurin,21 mlp-deficient mice that develop dilated heart failure,22 and animals that had been subjected to ischemia reperfusion. Because the degree to which cMyBP-C and its phosphorylated forms regulate cardiac function may depend, in part, on the MHC isoform that is present,23 we also determined cMyBP-C phosphorylation levels in a mouse model in which we had replaced approximately 70% of the normal
-MHC with ß-MHC.24 Under basal conditions, the mono-, bi-, and triphosphorylated species of cMyBP-C made up >90% of the cMyBP-C population (Figure 1A, lane 5). A ß-MHC shift, in of itself, had no effect on the normal phosphorylation pattern (Figure 1B, lane 8). In all of the models in which cardiac function was significantly compromised by either surgical or genetic manipulation, total cMyBP-C phosphorylation, particularly the triphosphorylated species, was decreased (Figure 1A and 1C; supplemental Table I). Invariably, in the animals that displayed overt cardiac failure as determined by labored breathing, anasarca, and failure to groom, the triphosphorylated state was reduced or absent (Figure 1, lanes 4, 6, 7, and 12). These data show that increased dephosphorylation of cMyBP-C is associated with contractile dysfunction and heart failure.
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Genetic Modulation of cMyBP-C
The phosphorylation motif of cMyBP-C is conserved among the human, mouse, chicken, and frog (Figure 2A). To investigate the role of cMyBP-C phosphorylation in relation to cardiac function, we generated a construct (MyBP-CAllP–) in which the 3 phosphorylation sites (Ser273, -282, and -302) and 2 neighboring potential alternative phosphorylation sites (Thr272 and -281) were altered to alanine (Figure 2A). Previously, we prepared a TG mouse that expressed myc-tagged, normal cMyBP-C (line 21, MyBP-CWT),2 and mice that expressed MyBP-CAllP– at approximately the same level (line 262) were used along with NTG littermates for comparison of transcript and protein levels for the MyBP-CAllP– mice (Figure 2B, 2C, and 2E). Despite the increase in transcript levels in both the MyBP-CAllP– and MyBP-CWT hearts (Figure 2B and 2C), no absolute increase in the level of cMyBP-C was observed, nor were the levels of the other contractile proteins overtly affected (Figure 2D and 2E).
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To evaluate the level of MyBP-CAllP– replacement and define the phosphorylation status of cMyBP-C in the TG mice, cMyBP-C derived from NTG, MyBP-CWT, and MyBP-CAllP– hearts were treated with either protein phosphatase or PKA and resolved in IEF gels for Western blot analysis. Cardiac MyBP-C treated with protein phosphatase migrated at a pI of 6.06, as expected for the completely dephosphorylated species (Figure 2F). The introduction of the myc tag did not result in a detectable difference in migration from the endogenous protein, and the dephosphorylated, endogenous form comigrated with MyBP-CAllP–. PKA treatment resulted in essentially complete conversion of endogenous cMyBP-C to the di- and triphosphorylated species, and expression of MyBP-CWT had no effect on the overall levels of phosphorylation (Figure 2F, lane 2). As expected, cMyBP-C isolated from the MyBP-CAllP– hearts showed 2 species when treated with PKA, the nonphosphorylated MyBP-CAllP– (P0), and the phosphorylated endogenous cMyBP-C (P2, P3), allowing us to estimate the degree of replacement of the endogenous cMyBP-C with the transgenically-encoded species. Approximately 40% replacement with the MyBP-CAllP– occurred in line 262, which is equivalent to the level of replacement previously measured in the MyBP-CWT-expressing hearts derived from line 21.2
Histological and Ultrastructural Consequences of MyBP-CAllP– Expression
The gross histology of 3-month MyBP-CAllP– hearts was unremarkable, with no obvious abnormalities, fibrosis, calcification, or disarray compared with NTG controls (Figure 3A). However, transmission electron microscopy revealed subtle ultrastructural changes. Whereas NTG hearts showed typical, well-organized and aligned sarcomeres with regularly distributed mitochondria, sarcoplasmic reticulum, and T-tubules (Figure 3B and 3C), MyBP-CAllP– hearts occasionally displayed regions that lacked the regular sarcomere–mitochondria distribution, and some sarcomeres showed altered H-zones and M-lines that were relatively ill defined (Figure 3B, right panel; Figure 3C, lower panels).
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The lack of definition at the central part of the sarcomere is reminiscent of the altered sarcomeres that were observed in mice in which cMyBP-C expression was ablated.3 To confirm that MyBP-CAllP– was being incorporated normally into the sarcomere, we performed immunohistochemical analyses with both anti-myc and polyclonal anti–cMyBP-C antibodies. Both MyBP-CAllP– and MyBP-CWT proteins were incorporated normally into the sarcomere (Figure 3D).
Despite the subtle ultrastructural changes, no cardiac hypertrophy and/or dilation in the MyBP-CAllP– could be detected. The heart/body weight ratios did not significantly differ between NTG (0.53±0.05, n=8) and MyBP-CAllP– (0.60±0.05, n=8; P<0.08) littermates at 3 months. However, we reasoned that the ultrastructural changes reflected a structural deficit that might result in subtle alterations in the transcriptional patterns and, indeed, found that ß-MHC and atrial natriuretic factor (ANF) transcript levels, which can serve as sensitive molecular markers for cardiac stress, were significantly increased in MyBP-CAllP– hearts when compared with either NTG or MyBP-CWT samples (Figure 3E and 3F).
Depressed Cardiac Function in MyBP-CAllP– Mice
On the basis of the altered ultrastructure, we considered that partial replacement of cMyBP-C with a nonphosphorylatable form might affect whole-organ function. Cardiac function was evaluated both noninvasively and by catheterization in the intact animals at 3 months. M-mode echocardiography in nonsedated animals showed that the MyBP-CAllP– animals had normal cardiac dimensions and function under baseline conditions (supplemental Table II). As cMyBP-C phosphorylation occurs in response to alterations in intracellular calcium levels (activating calcium/calmodulin-dependent kinase) and ß-adrenergic stress (activating PKA), we reasoned that adrenergic stimulation might reveal a deficit in the ability of the MyBP-CAllP– animals to respond to dobutamine. Hemodynamic load can be significantly altered via ß-adrenergic stimulation, activation of PKA, and the subsequent phosphorylation of phospholamban, cTnI, and MyBP-C. Phosphorylation of cTnI and phospholamban leads to increases in crossbridge cycling and enhanced relaxation, but the role that cMyBP-C plays in these processes, if any, is unclear. Cardiac function was assessed by in vivo catheterization and dobutamine stimulation. The complete data are shown in supplemental Table III. At baseline in this model, we were able to detect significant differences in the basal left ventricular (LV) end-systolic pressure (83.6±1.0), dP/dtmax (6051.1±241.5), and dP/dtmin (–5541.1±151.2) in the MyBP-CAllP– hearts, compared with the NTG and MyBP-CWT groups. The MyBP-CAllP– hearts had decreased contractile performance (peak dP/dtmax) at maximum dobutamine stimulation, suggesting that a lack of cMyBP-C phosphorylation inhibits maximum contractility. To confirm that these changes were not attributable to compensatory phosphorylation of the other contractile proteins, such as the myosin light chains, the phosphorylation status of these proteins was examined using 2D electrophoresis and found to be unchanged in the MyBP-CAllP– mouse hearts (supplemental Figure I).
MyBP-CAllP– Fails to Rescue the MyBP-C(t/t) Phenotype
The data indicated that partial replacement with a nonphosphorylatable cMyBP-C led to both structural and functional deficits. If phosphorylatable cMyBP-C serves an essential function in the heart, complete replacement of endogenous cMyBP-C with MyBP-CAllP– should lead to significant functional deficits or even death. To test this hypothesis, line 262 was bred with MyBP-C(t/t),3 which should result in a homogenous complement of MyBP-CAllP–, as MyBP-C(t/t) produces only a small amount of protein that is truncated and nonfunctional. The MyBP-CWT line was also bred to MyBP-C(t/t) to confirm that a transgenic strategy could, in fact, rescue the MyBP-C(t/t) phenotype. Northern and dot-blot analysis confirmed robust expression of both MyBP-CWT and MyBP-CAllP– in the MyBP-C(t/t) background (Figure 4A). SDS-PAGE (Figure 4B) and Western blot analysis (Figure 4C) using both anti-myc and anti–MyBP-C antibodies demonstrated the absence of cMyBP-C in MyBP-C(t/t) hearts and the presence of MyBP-CWT and MyBP-CAllP– in the MyBP-C(t/t) background, at approximately the levels observed for the endogenous protein in NTG hearts. IEF was subsequently performed on untreated, PKA-treated, and phosphatase-treated samples to define the phosphorylation states of the total cMyBP-C complement in the mice and confirmed that MyBP-CAllP– "replacement" in the MyBP-C(t/t) background was complete (Figure 4D).
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The MyBP-C(t/t) mice have been characterized previously.3 The homozygous animals are viable but soon after birth display a progressive dilated cardiomyopathy. Myocyte hypertrophy, disarray, fibrosis, and calcification are observed, and these progress as the animals mature. The effectiveness of MyBP-CWT expression in preventing reactivation of a fetal transcription program was underscored by the lack of ß-MHC expression, which serves as a sensitive marker for a nascent hypertrophic response (Figure 4E).
TG expression of MyBP-CWT also effectively rescued the overt hypertrophy displayed by the MyBP-C(t/t) hearts. In contrast, equal levels of MyBP-CAllP– expression did not rescue the MyBP-C(t/t)-induced hypertrophy (Figure 5A). The MyBP-CAllP–:(t/t) and MyBP-C(t/t) mouse hearts showed markedly enlarged chambers, with significant cardiac hypertrophy and myocyte disarray, as compared with the NTG and MyBP-CWT:(t/t) mice (Figure 5B). Light microscopic analyses showed pathology typical of cardiac hypertrophy in both the MyBP-C(t/t) and MyBP-CAllP–:(t/t) ventricles, whereas the MyBP-CWT:(t/t)-derived sections appeared normal (Figure 5C and 5D). Heart/body weights in the MyBP-C(t/t) and MyBP-CAllP–:(t/t) mice were significantly elevated, whereas, in contrast, the values derived from the MyBP-CWT:(t/t) mice were essentially normal, confirming the absence of physiological hypertrophy (Figure 5E).
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Ultrastructural analysis showed the expected lack of M-band definition in the MyBP-C(t/t) sarcomeres, as described previously in this model.3 In contrast, regular A- and I-bands and M-lines in both the MyBP-CAllP–:(t/t) and MyBP-CWT:(t/t) compared with MyBP-C(t/t) sarcomeres were apparent (Figure 6A). To confirm correct incorporation of MyBP-CAllP– and MyBP-CWT in the MyBP-C(t/t) background, we performed immunohistochemistry with both cMyBP-C and myc antibody. As expected, cMyBP-C was absent in the MyBP-C(t/t) hearts, but each transgenically encoded species showed the expected pattern of incorporation at approximately equal levels (Figure 6B).
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The inability of MyBP-CAllP– to rescue the MyBP-C(t/t) phenotype was confirmed at the functional level. M-mode echocardiography showed the MyBP-CAllP–:(t/t) and MyBP-C(t/t) mice had increased LV end-diastolic and end-systolic dimensions, as well as reduced fractional shortening, whereas normal shortening fractions were observed in the MyBP-CWT:(t/t) hearts (Figure 7A and the Table). Although both MyBP-CAllP– and MyBP-CWT incorporate normally into the sarcomere, MyBP-CWT expression appears to be able to rescue the MyBP-C(t/t) phenotype, whereas MyBP-CAllP– cannot. To define this more completely, we looked for activation of the fetal gene program in these mice, as upregulation of ANF, brain natriuretic peptide (BNP), ß-MHC, and skeletal -actin and downregulation of -MHC, phospholamban (PLN), and the sarcoplasmic reticulum Ca2+ pump (SERCA) often serve as sensitive markers for hypertrophy or cardiac stress.25 The data illustrate the completeness of the rescue, as no differences, even at the molecular level, were detected between the NTG and MyBP-CWT:(t/t) groups (Figure 7B and 7C). In contrast, ANF, ß-MHC, BNP, and skeletal -actin were upregulated, whereas -MHC, PLN, and SERCA were significantly downregulated in both the MyBP-C(t/t) and MyBP-CAllP–:(t/t) groups (Figure 7B and 7C), a pattern consistent with compromised cardiac function.
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-32P–labeled probes. Hypertrophic molecular markers such as ANF, BNP, skeletal actin, and ß-MHC were significantly up regulated in the MyBP-Ct/t and MyBP-CAllP–:(t/t) groups, whereas the NTG pattern of expression is conserved in the MyBP-CWT:(t/t) hearts. The quantitative data are summarized in C.
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Discussion |
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Heart failure is associated with diminished ß-adrenergic responsiveness, loss of cardiac contractility, abnormalities in Ca2+ handling,26,27 and altered contractile protein phosphorylation.18,28 Although cMyBP-C is extensively phosphorylated under basal conditions, it becomes dephosphorylated during the development of heart failure or pathological hypertrophy, with the triphosphorylated form largely or completely absent in the advanced stages of heart failure (Figure 1). This phenomenon appears to be relatively independent of the type of cardiac stress, as pressure overload, ischemic-reperfusion injury, and various genetic alterations in the cardiac machinery all resulted in significantly decreased phosphorylation. Basal levels of cMyBP-C phosphorylation may be necessary for maintaining thick-filament orientation, dynamic regulation, and contractile mechanics,4,7,11 and compromised phosphorylation patterns almost certainly reflect alterations in these cardiac parameters. Protein phosphatase activities appear to be increased in heart failure,29 and this increased activity, along with decreased ß-adrenergic responsiveness during heart failure, may be responsible for the decrease in cMyBP-C phosphorylation.
We next addressed whether altered cMyBP-C phosphorylation patterns could actually cause cardiac disease. Mice in which approximately 40% of the endogenous cMyBP-C was replaced with MyBP-CAllP– appeared overtly normal. However, ultrastructural analysis did show subtle alterations at multiple foci, consistent with the hypothesis that normal cMyBP-C phosphorylation levels are needed to maintain normal myofibril structure.18 These mice also showed upregulation of transcripts usually associated with a nascent hypertrophic response, and invasive catheterization showed that contraction and relaxation were significantly decreased. Rapid and reversible changes in thick-filament structure and ordering of myosin heads can be produced in cardiac muscle by changes in the degree of cMyBP-C phosphorylation,10,30 and these changes in structure are accompanied by changes in force production.31 Cardiac MyBP-C and its phosphorylation state may play an important role in determining thick/thin-filament interaction, with the force of contraction and time to half-relaxation dependent on the phosphorylated state of the C1–C2 domains.4,10,32 Although it is not clear that dephosphorylated cMyBP-C directly causes cardiac disease, our data suggest that cMyBP-C phosphorylation patterns can have a significant effect on whole-heart function and compromise cardiac hemodynamics.
The inability of MyBP-CAllP– to rescue the MyBP-C(t/t) phenotype is consistent with the hypothesis that cMyBP-C phosphorylation is essential for normal cardiac function. Strikingly, equivalent TG expression of MyBP-CWT effectively rescued the MyBP-C(t/t) mice, resulting in restoration of normal morphology, preventing activation of the fetal gene program, and resulting in normal cardiac hemodynamics (Table). Both MyBP-CAllP– and MyBP-CWT appear to incorporate normally into the sarcomere, yet only the phosphorylatable form is effective in suppressing the MyBP-C(t/t) phenotype. In addition to the structural roles of cMyBP-C,32 and ability to bind to titin,33 phosphorylation of cMyBP-C extends the crossbridges from the backbone of the thick filament, changes their orientation, increases the degree of order of the crossbridges, and decreases crossbridge flexibility.23,31,34,35 Although our data do not address crossbridge mechanics directly, it is clear that the mutant cMyBP-C is present in the sarcomere in a pattern that is indistinguishable from normal protein but appears to lack some critical function necessary for maintaining the overall sarcomere architecture, as manifested by the alterations observed in the sarcomere–mitochondrial spatial relationships. Further studies using these mouse models should provide valuable insight into the mechanical consequences of cMyBP-C phosphorylation.
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Acknowledgments |
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This work was supported by NIH grants HL69799, HL60546, HL52318, HL60546, and HL56370 (to J.R.) and by the American Heart Association, Ohio Valley Affiliate (to S.S.). We thank Dr Robert S. Decker for sharing technical expertise in isolating and analyzing the phosphorylated forms of cMyBP-C.
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Footnotes |
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Original received August 22, 2005; revision received September 20, 2005; accepted October 4, 2005.
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References |
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