Cardiac myosin binding protein-C phosphorylationregulates the super-relaxed state of myosinJames W. McNamaraa,1, Rohit R. Singha, and Sakthivel Sadayappana,1

aHeart, Lung and Vascular Institute, Department of Internal Medicine, Division of Cardiovascular Health and Disease, University of Cincinnati,Cincinnati, OH 45267

Edited by Jonathan Seidman, Harvard Medical School, Boston, MA, and approved May 7, 2019 (received for review December 19, 2018)

Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) ac-celerates cardiac contractility. However, the mechanisms by whichcMyBP-C phosphorylation increases contractile kinetics have notbeen fully elucidated. In this study, we tested the hypothesis thatphosphorylation of cMyBP-C releases myosin heads from the inhibitedsuper-relaxed state (SRX), thereby determining the fraction ofmyosin available for contraction. Mice with various alanine (A) oraspartic acid (D) substitutions of the three main phosphorylatableserines of cMyBP-C (serines 273, 282, and 302) were used to addressthe association between cMyBP-C phosphorylation and SRX. Single-nucleotide turnover in skinned ventricular preparations demonstratedthat phosphomimetic cMyBP-C destabilized SRX, whereas phospho-ablated cMyBP-C had a stabilizing effect on SRX. Strikingly, phos-phorylation at serine 282 sitewas found toplay a critical role in regulatingthe SRX. Treatment of WT preparations with protein kinase A(PKA) reduced the SRX, whereas, in nonphosphorylatable cMyBP-Cpreparations, PKA had no detectable effect. Mice with stable SRXexhibited reduced force production. Phosphomimetic cMyBP-Cwith reduced SRX exhibited increased rates of tension redevelop-ment and reduced binding to myosin. We also used recombinantmyosin subfragment-2 to disrupt the endogenous interaction be-tween cMyBP-C and myosin and observed a significant reduction inthe population of SRX myosin. This peptide also increased forcegeneration and rate of tension redevelopment in skinned fibers.Taken together, this study demonstrates that the phosphorylation-dependent interaction between cMyBP-C andmyosin is a determinantof the fraction of myosin available for contraction. Furthermore, thebinding between cMyBP-C and myosin may be targeted to improvecontractile function.

MYBPC3 | myosin S2 | myofilament | sarcomere | SRX

Cardiac myosin binding protein-C (cMyBP-C) is a thickfilament-associated sarcomeric protein present in vertebrate

cardiomyocytes (1). It consists of 11 Ig-like and fibronectin-likedomains, termed C0–C10, and localizes as seven to nine stripesin the inner two thirds of each half A-band, termed the C-zone(2). Cardiac contractility is modulated by cMyBP-C through itsability to transiently interact with thick and thin filaments to regu-late actomyosin interactions (3–5). The importance of cMyBP-C ishighlighted by the fact that mutations in the gene encoding it,MYBPC3, are the most common cause of hypertrophic cardio-myopathy (HCM) (6). cMyBP-C primarily mediates contractilitythrough its phosphorylation status. cMyBP-C is highly phosphory-lated, with three phosphorylatable serines within the M-domain,namely serines 273, 282, and 302, all of which have been inten-sively characterized (7–9). These sites are differentially phosphory-lated by a range of kinases, including protein kinase A (PKA),protein kinase C, and Ca2+/calmodulin-dependent protein kinaseII (8). The importance of cMyBP-C phosphorylation is moreevident by the pathological consequences of its dephosphorylatedstate in many cardiac diseases (10, 11). Transgenic overexpressionof phosphomimetic cMyBP-C was shown to prevent disease de-velopment in cMyBP-C–null mice, whereas dephosphorylatedcMyBP-C could not (12, 13). Despite its clear role in cardiac functionin health and disease, the precise mechanism(s) by which cMyBP-C

phosphorylation increases cardiac inotropy (contraction of myo-cardium) and lusitropy (myocardial relaxation) has/have not beenfully elucidated.It is possible that cMyBP-C phosphorylation regulates cardiac

function by modulating the myosin super-relaxed state (SRX), asubpopulation of myosin heads characterized by their highly inhibitedATP turnover (reviewed in ref. 14). Myosin heads in the SRX adoptan evolutionarily conserved conformation of myosin II, termedthe interacting-heads motif (IHM), which is suggested to be afundamental regulator of muscle contractility (15). Through multipleinter- and intramolecular interactions, myosin heads in the SRXadopt an ordered, quasihelical arrangement around the thick filamentbackbone, where they cannot bind actin (16). These interactions re-duce ATP turnover of SRX myosin to a rate approximately 10 timesslower than that of myosin in the disordered-relaxed state (DRX),commonly known as the detached state, in which relaxed myosinheads protrude into the interfilament space (17). Thus, the ratio ofSRX to DRXmyosin heads determines the energy utilization of themyofilaments and the number of myosin heads that can contributeto contraction.Previously, we have demonstrated that cMyBP-C stabilized the

SRX in that mice lacking cMyBP-C, or humans with cMyBP-Cmutations, exhibited a significant shift of myosin heads from theSRX to the DRX (18, 19). Here, we tested the hypothesis thatphosphorylation of cMyBP-C may promote a loss of SRX myosinheads, thereby regulating the number of force-producing myosinheads. To test this hypothesis, single-nucleotide turnover was

Significance

Cardiac myosin binding protein-C (cMyBP-C) is a sarcomericprotein closely linked to cardiac contractility. Importantly, althoughthe phosphorylation status of cMyBP-C regulates the force and rateof cardiac contraction, the exact molecular mechanism(s) remain(s)unclear. Previously, loss of, or mutations in, cMyBP-C resulted in aloss of the inhibited super-relaxed state (SRX) of myosin, whichexplains the hypercontractile phenotype. Thus, we asked ifcMyBP-C phosphorylation would increase contractile functionby the release of myosin heads from SRX to a state more conduciveto their interaction with actin. We found that a site-specific,phosphorylation-dependent interaction between cMyBP-C andmyosin does modulate the fraction of SRX myosin that controlscontractility.

Author contributions: J.W.M. and S.S. conceived the study; J.W.M. and S.S. designedexperiments; J.W.M. and R.R.S performed research; J.W.M., R.R.S., and S.S. analyzed data;and J.W.M. and S.S. wrote the paper.

Conflict of interest statement: S.S. provided consulting and collaborative services toAstraZeneca, Merck, and Amgen unrelated to the content of this manuscript. No otherdisclosures are reported.

This article is a PNAS Direct Submission.

Published under the PNAS license.1To whom correspondence may be addressed. Email: mcnamajw@ucmail.uc.edu orsadayasl@ucmail.uc.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1821660116/-/DCSupplemental.

Published online May 29, 2019.

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measured in four transgenic cMyBP-C phosphomimetic mousemodels compared with wild-type (WT) controls. Based on theseresults, we measured force production, rate of tension re-development, and binding between myosin and cMyBP-C. Re-sults tended to show that a stable SRX reduced maximal forceproduction, which correlated with the interaction between myosinand cMyBP-C. Therefore, we reasoned that molecules that inhibitthe interaction between cMyBP-C and myosin may, at the sametime, increase contractility. It was found that competitive inhibi-tion of myosin–cMyBP-C interaction does indeed reduce thenumber of myosin heads in the SRX and, strikingly, also resultedin increased force production and rate of force generation. Thus,regulatory mechanisms in the biology of cMyBP-C and its role inthe SRX are revealed. Furthermore, we demonstrate that theinteraction between cMyBP-C and myosin may be targeted toincrease cardiac contractility.

ResultsTransgenesis Reveals cMyBP-C Phospho-Regulation of the SRX. Totest the hypothesis that phosphorylation of cMyBP-C can mediatethe SRX, the fluorescent ATP analog 2′-3′-O-(N′-methyanthraniloyl)-ATP (mant-ATP) was used in a pulse-chase to measure the single-nucleotide turnover in detergent-skinned multicellular ventricularpreparations from WT, cMyBP-C phosphomimetic (DDD; as-partate mutations in 273, 282, and 302 sites), and phospho-ablated(AAA; alanine mutations in 273, 282, and 302 sites) mice. An in-depth description of this assay is provided in SI Appendix, Mea-surement of the SRX. By Western blot, it was determined thatWT controls were ∼50% phosphorylated at each of these serines(SI Appendix, Fig. S2). Representative traces from each model areshown in Fig. 1A. Consistent with previously published data (19),WTmice had a P2 of 17 ± 2% and a lifetime of ATP turnover (T2)of 130 ± 11 s (Fig. 1C). The same experiment in AAA mice yieldeda P2 of 15.2 ± 0.7% and a T2 of 112 ± 12 s, indicating that nochanges in the SRX occurred in these mice. However, when

DDD mice were assayed, a significant shift of myosin from theSRX to the DRX was observed compared with WT mice (P1,79 ± 1% vs. 88 ± 1%; P < 0.0001; P2, 17 ± 2% vs. 8 ± 1%; P =0.0001). No difference in T2 was observed between these groups.Having determined that phosphomimetic cMyBP-C destabi-

lizes the SRX in transgenic mice, we explored the possibility ofsite-specific phospho-regulation of the SRX, and, to accomplishthis, two additional models were used. In the first mouse model,DAD, the PKC-phosphorylatable sites ser-273 and ser-302 weremutated to aspartic acid, and the remaining site, ser-282, wasmutated to alanine. The second model, ADA, reversed the con-dition of the first in that ser-282 was mutated to aspartic acid andser-273 and ser-302 were mutated to alanine. Multicellular prepara-tions were tested to measure the SRX in these models, as describedearlier (traces in Fig. 1B). Interestingly, the SRX in DAD mice wassimilar to that in WT and AAA mice (P1, 76 ± 3%; P2, 18 ± 2%;Fig. 1 C and D). Strikingly, however, the slow phase of single-nucleotide turnover in ADA mice was significantly reduced com-pared with WT experiments, indicating a shift of myosin heads fromthe SRX to the DRX (P1, 85 ± 1%; P2, 8.8 ± 0.5%; both P < 0.01vs. WT). As ser-282 phosphorylation was sufficient to shift myosinheads from the SRX to the DRX, these data implicate phosphor-ylation of ser-282 as the primary cMyBP-C phosphorylation site thatregulates the SRX. No changes were found in the lifetime of ATPturnover in the fast or slow phases (SI Appendix, Fig. S3).

cMyBP-C Is the Main PKA-Mediated Regulator of the SRX. It wasapparent that the amino acid substitutions used to mimic phos-phorylation of cMyBP-C might be completely representative ofthe structural changes accompanying endogenous serine phos-phorylation. Indeed, a recent report suggests that this may be thecase (20). Thus, to test whether destabilization of the SRX, asobserved in phosphomimetic hearts, was representative of endog-enously phosphorylated cMyBP-C, WT preparations were pre-treated with PKA to phosphorylate myofilament targets beforeperforming single-nucleotide turnover assays. PKA treatment ofWT myofilaments elicited close to a 50% increase in phosphory-lation for each serine site (SI Appendix, Fig. S2). Following PKAphosphorylation, mant-nucleotide turnover was faster than that ofuntreated samples (Fig. 2A). This corresponded to a 10% increasein the P1 (79 ± 1% vs. 89 ± 1%; P < 0.0001, Fig. 2D). Concurrently,the P2 of PKA-treated samples was significantly decreased com-pared with nontreated samples (18 ± 1% vs. 8 ± 1%; P < 0.0001;Fig. 2F) in a manner that closely resembled results fromDDDmice.Whereas the lifetime of the fast phase was unaffected (Fig. 2C), aslight, near-significant increase was observed in the lifetime of SRXATP turnover (120 ± 8 s vs. 160 ± 12 s; P = 0.0523; Fig. 2E).However, this could be attributed to the instability of fitting a sec-ond exponential to such a fast-decaying curve.It is also possible that other PKA targets within the myofila-

ment may have contributed to the decrease in SRX myosin ob-served upon PKA phosphorylation. To address this concern, wealso compared the SRX in AAA mice with or without PKAtreatment. As seen in Fig. 2B, the traces of AAA and AAA+PKAare nearly indistinguishable, and subsequent fittings were un-changed (Fig. 1 C andD). In sum, these data confirm that cMyBP-Cphosphorylation does indeed modulate the SRX in cardiac muscle,thus determining the fraction of myosin heads available for con-traction. Furthermore, as PKA treatment of AAA fibers containingnonphosphorylatable cMyBP-C had no effect on the SRX, it can beinferred that cMyBP-C is the primary target for PKA-mediatedregulation of the SRX.

Modulation of the SRX by cMyBP-C Phosphorylation in Turn RegulatesForce and Rate Development.To determine whether changes in thefraction of SRX myosin affects contractile function, we measuredforce generation and tension redevelopment in skinned LVmyocardial preparations from each group (Fig. 3 A and B). The

Fig. 1. Comparison of SRX in skinned fibers from transgenic cMyBP-Cphosphomimetics. (A and B) Mant-ATP single-nucleotide turnover experi-ments comparing WT with AAA and DDD (A) andWT with DAD and ADA (B).Between-group comparisons of amplitudes of fast phase (C) and slow phase(D) representing the fraction of SRX myosin, as determined from curve fit-ting with double exponential fits. Lifetimes of exponential fits are reportedin SI Appendix, Fig. S3. n = 8 (24) for WT, n = 5 (16) for AAA, n = 5 (17) forDDD, n = 3 (13) for ADA, and n = 4 (13) for DAD (values inside parenthesesrepresent number of fibers). Error bars indicate ± SEM (*P < 0.05, **P < 0.01,***P < 0.001, and ****P < 0.0001).

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maximal force generation fromWT preparations was 26 ± 2 mN/mm2

(Fig. 3C). The force production by DDD mice was unchangedcompared with WT (23 ± 2 mN/mm2). Strikingly, the force pro-duced by AAA muscle fibers was significantly reduced comparedwith WT (14 ± 1 mM/mm2; P < 0.001). When only serine 282 wasmutated to mimic phosphorylation (i.e., ADA), force productionwas similar to WT and DDD preparations (30 ± 2 mN/mm2; P =0.8023). However, phosphorylation of only the PKC sites ofcMyBP-C (i.e., DAD) resulted in a significant reduction in maximaltension (14 ± 1 mN/mm2), consistent with previously publishedvalues of maximal force using the DAD model (21). The averageforce–pCa curve for each group is shown in SI Appendix, Fig. S4 Aand B. No major changes were observed in calcium sensitivity (WTEC50 = 1.54 ± 0.06 μM) or Hill coefficient (WT nH = 3.5 ± 0.8)between groups (SI Appendix, Fig. S4 C and D).Next, to investigate the effect(s) of SRX stability on regulating

the kinetics of muscle contraction, we performed a slack-restretchmaneuver on myocardial preparations at submaximal calciumconcentrations (pCa 5.7). This technique involves a 20-ms 20%slack in fiber length to allow a period of unloaded shorteningbefore a rapid restretch to the starting length to measure the rateof tension redevelopment (ktr). At this submaximal calciumconcentration, the ktr in WT mice was 3.6 ± 0.4 s−1, which is con-sistent with previously reported values of ∼3 s−1 (22). We noted atrend toward slower ktr in AAA mice compared with WT (2.6 ±0.1 s−1); however, although this did not reach significance byone-way ANOVA with multiple comparisons, it did reach sig-nificance with the unpaired t test. Conversely, ktr was acceleratedin DDD myocardial preparations compared with WT (6.1 ±0.7 s−1 vs. 3.6 ± 0.4 s−1; P < 0.05). We also noted a trend toward

accelerated ktr in ADA samples compared with WT (5.5 ± 0.4 s−1 vs.3.6 ± 0.4 s−1; P = 0.1). Interestingly, a less apparent trend towardaccelerated ktr was observed in DAD samples compared with WT(5.1 ± 0.5 s−1 vs. 3.6 ± 0.4 s−1; P = 0.25).

N-Terminal cMyBP-C Binding to Myosin Is Phosphorylation-Dependentin a Site-Specific Manner. We hypothesized that phosphoregulationof SRX most likely resulted from changes in the interaction be-tween myosin and cMyBP-C, thereby releasing an inhibitory tetheron the myosin. To test this notion, we performed cosedimentationassays by using varying phosphomimetic recombinant C0C2 (rC0C2)peptides and purified murine cardiac myosin. Recombinant C0C2is soluble at low KCl concentrations (100 mM), and, when sub-jected to high-speed ultracentrifugation alone, it remains in thesupernatant, whereas myosin will form synthetic thick filamentsthat form sediment under centrifugation. Fig. 4A displays repre-sentative SDS/PAGE gels for pellet, supernatants, and standardsused to determine the molar ratio of binding (Fig. 4B). Using WTrC0C2, the interaction with myosin displayed a binding affinity of2.8 ± 0.6 μM and a Bmax of 0.72 ± 0.13. The binding associationwas largely unchanged in AAA and DAD rC0C2 compared withWT (Fig. 4 C and D). Strikingly, when DDD and ADA rC0C2peptides were studied with cosedimentation, very little bindingcould be observed, and reliable fittings were unobtainable (Fig. 4C and D). Because DDD and ADA cMyBP-C both exhibiteddestabilized SRX and reduced binding to myosin, these findingssupport the hypothesis that interaction between cMyBP-C andmyosin is dynamically regulated by phosphorylation, particularlyby serine 282 phosphorylation, and is likely important for regu-lation of the SRX. Importantly, these results were validated bysolid-phase protein-binding assays (SI Appendix, Fig. S5).

Proximal Myosin Subfragment-2 Destabilizes the SRX and ImprovesForce Generation and Kinetics. Having demonstrated that thebinding of cMyBP-C to myosin likely regulates the ratio of SRXto DRX myosin, we next asked if a peptide that inhibited theinteraction between cMyBP-C and myosin would also destabilizethe SRX, thereby increasing the number of DRX myosin heads.To examine this question, we expressed a recombinant peptide

Fig. 2. SRX comparison of PKA-treated WT and AAA skinned fibers. (A andB) Mant-ATP traces comparing WT (A) and AAA (B) fibers with or withoutPKA pretreatment. Lifetime of nucleotide turnover associated with fast (C)and slow (E) phases of fluorescence decays. Amplitudes of fast (D) and slow(F) phases, indicating the fraction of DRX and SRX myosin, respectively. n = 4(21 and 13 for −PKA and +PKA, respectively); n = 3 (12 and 13 for −PKAand +PKA, respectively). Error bars indicate ± SEM (***P < 0.001 and ****P <0.0001).

Fig. 3. Comparison of mechanical properties of cMyBP-C phosphomimeticmice. (A and B) Normalized force–pCa traces comparing WT with AAA andDDD (A) or with DAD and ADA (B). Maximal force production for each groupmeasured at pCa 4.5 (C). Submaximal ktr comparison measured at pCa 5.7(D). Total force–pCa traces are shown in SI Appendix, Fig. S4 (n = 3; n = 5/6fibers). Error bars indicate ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, and****P < 0.0001).

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encoding the proximal region of myosin subfragment-2 (rS2).This proximal S2 contains a putative binding region for the N-terminal domains of cMyBP-C. We theorized that the presenceof excess exogenous S2 would compete with endogenous myosinfor cMyBP-C, thereby reducing the stabilizing effect of cMyBP-Con myosin SRX. Single-nucleotide turnover was measured in thepresence or absence of 45 μM of rS2. Strikingly, the presence ofmyosin rS2 significantly reduced the number of myosin heads inthe SRX in WT mice (17.3 ± 1.2% vs. 8.1 ± 1.5%; P < 0.0001;Fig. 5 A and B). This effect was also observed in AAA mice (16 ±1% vs. 5.8 ± 1.9%; P < 0.0001). These data demonstrate thatexogenous rS2 can destabilize cardiac SRX, most likely throughdisruption of cMyBP-C/myosin interaction. Next, we measuredsteady-state mechanics and tension redevelopment on skinnedWT muscle fibers to determine whether this destabilizationresulted in any changes in contractile function. Strikingly, theaddition of 45 μM rS2 into muscle fibers significantly increasedtension generation in WT muscle fibers (23 ± 1 mN/mm2 vs. 32 ±2 mN/mm2; P < 0.01). Furthermore, submaximal ktr was morethan twice as fast upon addition of rS2 (4.0 ± 0.5 s−1 vs. 11 ± 1 s−1).From these results, it can be concluded that cMyBP-C interactionwith myosin can be targeted for disruption and that this may impartfavorable contractile effects in skinned muscle.

DiscussionThe SRX of cardiac myosin has been intensively studied in re-cent years. Myosin heads in super-relaxed state are thought to bestructurally analogous to the “OFF” state of myosin in which theactin binding domain of one myosin head (“blocked head”) inter-acts with the converter domain of the other (“free head”), thusinhibiting myosin ATPase (23). Multiple intra- and intermolecularinteractions between blocked and free myosin heads, myosin lightchains, myosin S2, as well as between myosin molecules of adja-cent crowns, act to stabilize SRX myosin to the thick filamentsurface in a quasihelical arrangement (16). On the contrary, in theDRX state, myosin heads may sway freely in the interfilamentspace with the ability to readily contribute to contraction. Thus,the ratio of SRX to DRX myosin determines the efficiency ofmyofibrillar energetics and the fraction of myosin heads availablefor contraction. Recently published work has demonstrated thatmany cardiomyopathy-linked myosin mutations may disrupt IHMby changes in electrostatic charge or by disruption of the putativebinding regions within the myosin mesa, a relatively flat region ofthe myosin S1 with the characteristics of a protein binding domain(24, 25). Although a number of HCM-associated myosin mutantsactually exhibit reduced intrinsic force capacity, disruption ofthe IHM would explain why they commonly associate with a

hypercontractile phenotype by increasing the fraction of myosinavailable to interact with actin (26–28). Recently, it was demon-strated that the mode of action of the HCM drug mavacamten, alsoknown as SAR-439152 and MYK-461, occurs at least partiallythrough the stabilization of SRX myosin (29–31). Despite theclear importance of understanding SRX biology, much remains tobe explored.We previously reported that cMyBP-C plays a role in the stabi-

lization of the SRX. Mice lacking cMyBP-C exhibited significantreductions in SRX myosin, consistent with structural data dem-onstrating myosin head disordering (19, 32). Similarly, humanHCM samples containing MYBPC3 mutations had greater de-stabilization of SRX myosin than both nonfailing and HCM sampleswithout sarcomeric mutations (18). Of note, these findings have sincebeen independently verified (31). Despite this, the mechanism(s) bywhich cMyBP-C contributes to SRX regulation remain(s) poorlyunderstood. cMyBP-C interacts with the myosin light meromyosinand titin at its C terminus, stabilizing the thick filament structure(33). At its N terminus, cMyBP-C may bind myosin S2 and reg-ulatory light chain (RLC) and may also directly interact with themyosin subfragment 1 (S1) (34–36). Furthermore, the interactionswith S1 and S2 are phospho-dependent (13, 34). Thus, it is pos-sible that C- and/or N-terminal interactions contribute to SRXregulation. Here, we hypothesized that the phosphorylation ofcMyBP-C reduces its interaction with myosin, allowing myosinheads to readily transition out of the SRX. To test this hypothesis,single-nucleotide turnover was measured in a range of transgenicphosphomimetic mice. These experiments were performed underrelaxing conditions whereby myosin RLC phosphorylation wasexpected to be uniformly low (37). As myosin RLC phosphorylation

Fig. 4. Cosedimentation analysis of N-terminal C0C2 domains (lower band)of cMyBP-C binding to full-length myosin (upper band). (A) Representativegels from pellet and supernatant fractions, as well as standard. (B) Standardcurves were generated for each assay. Comparison of binding among WT,AAA and DDD C0C2 (C), and WT, ADA, and DAD C0C2 (n = 5 for WT; n = 4for AAA, DDD, and ADA; n = 3 for DAD). Error bars indicate ± SEM.

Fig. 5. Effect of myosin S2 on the SRX and mechanical properties of skinnedfibers. (A) Mant-ATP traces of WT with and without myosin S2. (B) Com-parison of P2 values (as percentages) from WT and AAA fibers with andwithout myosin S2. (C) Normalized force–pCa traces of WT fibers with orwithout myosin S2 and total force production (D). Representative sub-maximal ktr (E) and comparison of values (F). Error bars represent SEM (**P <0.01, ***P < 0.001, and ****P < 0.0001).

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also destabilizes the SRX, we could specifically probe the effectof cMyBP-C phosphorylation (22). We first found that phos-phorylation of cMyBP-C releases myosin heads from the SRX, asdetermined by single-nucleotide turnover from DDD and PKA-treated WT mice. Our analysis of chronically (phosphomimetic)and acutely (WT PKA-treated fibers) phosphorylated cMyBP-Cwas critical to confirm that cMyBP-C phosphorylation regulatesthe SRX. Indeed, although the use of phosphomimetic cMyBP-Callowed for the specific isolation of cMyBP-C–mediated effects,the use of PKA-treated fibers allowed us to circumnavigate anypotential structural changes as a result of the aspartic acid mutationof cMyBP-C. Second, the phosphorylation state of serine 282 ap-pears to be critical in regulating this effect on the SRX. Finally, thisregulation is likely mechanistically driven by the phosphorylation-dependent interaction between cMyBP-C and myosin, providing apotential target to modulate contractile function.cMyBP-C phosphorylation positively regulates cardiac inotropy

and lusitropy; however, the mechanism(s) that underlie(s) suchregulation is/are not fully understood. Specifically, phosphoryla-tion of cMyBP-C increases the rate of force redevelopment andaccelerates the rate of relaxation and force development duringstretch activation in submaximally activated myocardial prepara-tions (9, 38, 39). Furthermore, PKA treatment of skinned cardiacmuscle significantly increases power output and speed of unloadedshortening (40–42), whereas AAAmice expressing phospho-ablatedcMyBP-C develop diastolic dysfunction (9, 40). Our finding thatcMyBP-C phosphorylation disrupts SRX myosin agrees withstructural data (39, 43), and by increasing the fraction of possiblecross-bridges, readily explains how cMyBP-C phosphorylationmodulates many of the aforementioned phenomena such as in-creased ktr and power output. It is less clear how cMyBP-Cphosphorylation accelerates myocardial relaxation, but it likelyresults from structural changes within the catalytic domain ofmyosin that accelerate ADP release.Strikingly, we also report a strong dependence on the phos-

phorylation status of serine 282 in the cMyBP-C regulation of theSRX. Specifically, when just this site was mutated to mimic phos-phorylation (i.e., ADA), we found that the change in SRX:DRX ratiowas similar to that observed in DDD mice, whereas phosphomimeticsubstitution of PKC-phosphorylatable sites (i.e., DAD) exhibitedstable SRX. Although structural studies have not been performedon these models, these data predict reduced IHM and disorderedthick filaments in ADA mice, but stabilized thick filaments inDAD models. Supporting this, we found a reduced interactionbetween ADA C0C2 and myosin. Furthermore, force productionin DAD skinned myocardial preparations was significantly reduced,with no changes in ADA mice, as previously reported (21). Al-though cMyBP-C phosphorylation is known to be high underphysiological conditions, dobutamine treatment significantly in-creased phosphorylation of all three serine sites in vivo and exvivo (8, 44). We found similar increases in the phosphorylationlevel of each site upon PKA treatment (SI Appendix, Fig. S2).This demonstrates that cMyBP-C regulation of the SRX is pos-sible at physiological level. In general, we observed that greaterstability of the SRX correlated with reduced maximal force pro-duction. An exception to this was the WT model, which exhibiteda stable SRX while producing force similar to that of DDD andADA models. The reason for this is uncertain. However, it ispossible that the basal phosphorylation levels of WT cMyBP-C,although sufficient to maintain the SRX in relaxation, may allowfor the recruitment of these myosin heads in response to calcium.Interestingly, it was recently demonstrated that phosphorylationof serine 302 reduces cardiomyocyte fractional shortening inresponse to PKC, which agrees with our results (45). Furthermore,PKC depressed myofilament function and could not be rescued byPKA treatment (46). Although these effects can be attributed tocontributions of cTnI and cMyBP-C phosphorylation, they agreewith our findings that residue-specific phosphorylation of cMyBP-C

regulates contractile function. Conversely, phospho-ablation ofonly serine 302, leaving serines 273 and 282 unmutated (i.e.,SSA), blunted the β-adrenergic response (7). The origin of thesecontrasts remains unknown, and because we did not quantify theSRX in the SSA model, it is possible that cMyBP-C regulation ofthe SRX is even more intricate.Current structural models for the SRX suggest exceptional

importance for myosin S2 in the formation of the IHM whereby themesa of blocked and free heads cradles the proximal region ofmyosin S2 (24, 34). Furthermore, exciting new work has demon-strated the biochemical presence of an inhibited SRX-like state insoluble myosin that is dependent on the presence of a proximalmyosin S2 (29, 30). Given the intrinsic capacity of myosin to forman IHM, it is interesting that cMyBP-C exhibits such dramatic ef-fects on the SRX in muscle fibers (18, 19, 31). It seems reasonableto argue that cMyBP-C acts to stabilize one IHM, allowing theformation of neighboring IHMs in a cooperative manner through-out the C-zone. Proximal myosin S2 also binds to N-terminal do-mains of cMyBP-C, and in the model proposed by Nag andcolleagues, it would likely act to stabilize the IHM (34). Our resultsdemonstrate that exogenous myosin S2 can destabilize the SRX,which agrees with the aforementioned model (34). Excess myosinS2 has previously been demonstrated to block the interaction be-tween full-length myosin and cMyBP-C, suggesting that this is in-deed the mode of action in our assays (35). Increased contractilityof intact cardiomyocytes upon myosin S2 permeabilization has alsobeen described, supporting our findings that this peptide increasedforce production and rate of tension redevelopment (47).We and others have determined that ∼40–50% of myosin

occupies the biochemical SRX state in skinned myocardial fibers.However, X-ray diffraction of intact trabeculae suggests a fargreater number of myosin heads occupying the structural OFF-state during diastole (48). Furthermore, under resting conditions,treatment of intact trabeculae with isoprenaline, a β1-agonist, didnot destabilize the OFF-state of myosin motors, whereas directPKA phosphorylation of skinned trabeculae could (39, 49). How-ever, consistent with the model proposed in this paper, isoprenalinetreatment resulted in a reduced intensity of the M1 cluster relatedto cMyBP-C (49). It is possible that the loss of osmotic compressionassociated with the skinning process promotes propensity towardloss of thick filament stability and subsequent myosin head disorder.An excellent editorial piece has recently addressed these disparatefindings, suggesting that, within the intensely crowded milieu of theintact lattice, small movements of the myosin heads may not bedetectable (50). Alternatively, multiple biochemical states of myosinmay exist within the structural OFF-state, such as between blockedand free myosin heads. Clearly, extensive investigations are stillrequired to characterize the relationship between the biochemical andstructural properties of these states in physiologically relevant settings.In conclusion, we have defined a regulatory mechanism of the

SRX by phosphorylation of cMyBP-C, significantly advancingour understanding of the mechanisms controlling SRX biology.Additionally, we demonstrated that the phospho-dependent in-teraction between cMyBP-C and myosin could be targeted toimprove contractile function. Future work will further characterizethe specific interaction between cMyBP-C and myosin and definein depth the effects of exogenous myosin S2 on cardiac function.

Materials and MethodsTransgenic Mouse Models.An expanded description of the study methods andmaterials is provided in SI Appendix. Transgenic mice overexpressing phos-phomimetic cMyBP-C have been previously characterized (8, 12, 13). Micewere deeply anesthetized by isoflurane inhalation, followed by cervicaldislocation and bilateral thoracotomy. Hearts were excised, snap-frozen inliquid nitrogen, and stored at −80 °C until use. All animal experiments wereapproved by the institutional animal care and use committees at LoyolaUniversity Chicago and University of Cincinnati and followed the policiesdescribed in the Guide for the Use and Care of Laboratory Animals publishedby the National Institutes of Health (51).

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In Vitro Assays. To measure the SRX, single-nucleotide turnover assays wereperformed by using skinned LV from the free wall as described in SI Appendix(19). Cardiac muscle mechanics were performed by using the Aurora Scien-tific 1400A permeabilized fiber system (18, 19, 22). Recombinant peptideswere produced in Escherichia coli by using the pET28a+ expression systemand purified by His-tag affinity purification, and cosedimentation assay wasperformed as described previously (4). Statistical analysis was performed byusing GraphPad Prism 7 with significance accepted at P < 0.05.

ACKNOWLEDGMENTS. The authors thank Dr. Matt Kofron and the confocalimaging core at Cincinnati Children’s Hospital for use of their microscopyfacilities. This work was supported by American Heart Association PostdoctoralFellowship 17POST33630095 (to J.W.M.); NIH Grants R01 HL130356, R56 HL139680,R01 AR067279, and R01 HL105826 (to S.S.); American Heart Association (S.S.); In-stitute for Precision Cardiovascular Medicine Cardiovascular Genome–PhenomeStudy Grant 15CVGPSD27020012 and Catalyst Award 17CCRG33671128(to S.S.); AstraZeneca (S.S.); Merck (S.S.); and Amgen (S.S.).

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