Distinct Sarcomeric Substrates Are Responsible for Protein Kinase D-mediated Regulation of Cardiac Myofilament Ca2+ Sensitivity and Cross-bridge Cycling*

Protein kinase D (PKD), a serine/threonine kinase with emerging cardiovascular functions, phosphorylates cardiac troponin I (cTnI) at Ser22/Ser23, reduces myofilament Ca2+ sensitivity, and accelerates cross-bridge cycle kinetics. Whether PKD regulates cardiac myofilament function entirely through cTnI phosphorylation at Ser22/Ser23 remains to be established. To determine the role of cTnI phosphorylation at Ser22/Ser23 in PKD-mediated regulation of cardiac myofilament function, we used transgenic mice that express cTnI in which Ser22/Ser23 are substituted by nonphosphorylatable Ala (cTnI-Ala2). In skinned myocardium from wild-type (WT) mice, PKD increased cTnI phosphorylation at Ser22/Ser23 and decreased the Ca2+ sensitivity of force. In contrast, PKD had no effect on the Ca2+ sensitivity of force in myocardium from cTnI-Ala2 mice, in which Ser22/Ser23 were unavailable for phosphorylation. Surprisingly, PKD accelerated cross-bridge cycle kinetics similarly in myocardium from WT and cTnI-Ala2 mice. Because cardiac myosin-binding protein C (cMyBP-C) phosphorylation underlies cAMP-dependent protein kinase (PKA)-mediated acceleration of cross-bridge cycle kinetics, we explored whether PKD phosphorylates cMyBP-C at its PKA sites, using recombinant C1C2 fragments with or without site-specific Ser/Ala substitutions. Kinase assays confirmed that PKA phosphorylates Ser273, Ser282, and Ser302, and revealed that PKD phosphorylates only Ser302. Furthermore, PKD phosphorylated Ser302 selectively and to a similar extent in native cMyBP-C of skinned myocardium from WT and cTnI-Ala2 mice, and this phosphorylation occurred throughout the C-zones of sarcomeric A-bands. In conclusion, PKD reduces myofilament Ca2+ sensitivity through cTnI phosphorylation at Ser22/Ser23 but accelerates cross-bridge cycle kinetics by a distinct mechanism. PKD phosphorylates cMyBP-C at Ser302, which may mediate the latter effect.

Protein kinase D (PKD) 2 is a serine/threonine kinase whose cardiovascular functions are becoming increasingly recognized (1). With regard to cardiac biology, Olson and coworkers (2) were the first to show that neurohormonal activation of PKD in rat cardiomyocytes leads to the phosphorylation and nuclear export of class II histone deacetylase isoforms, such as histone deacetylase 5, thereby initiating transcriptional reprogramming toward hypertrophy (2,3). Recently, the same group has shown that PKD is necessary for the full manifestation of pathologic cardiac remodeling following chronic pressure overload or neurohormonal stimulation in mice (4). Concurrently, work in our laboratory has provided evidence that PKD regulates cardiac myofilament function. We have identified several sarcomeric proteins, including the inhibitory subunit of cardiac troponin (cTnI) and cardiac myosinbinding protein C (cMyBP-C), as potential PKD substrates, and have shown that PKD induces cTnI dual phosphorylation at Ser 22 /Ser 23 , reduces myofilament Ca 2ϩ sensitivity, and accelerates isometric cross-bridge cycle kinetics in rat permeabilized ("skinned") ventricular myocytes (5). Subsequently, we reported that increased expression of PKD in intact rat ventricular myocytes by adenoviral gene transfer potentiates endothelin-1-induced cTnI phosphorylation at Ser 22 /Ser 23 , decreases myofilament Ca 2ϩ sensitivity, and abolishes the positive inotropic response to this stimulus (6). Nevertheless, any causal link between PKD-mediated cTnI phosphorylation at Ser 22 / Ser 23 and the regulation of myocardial contraction through reduced myofilament Ca 2ϩ sensitivity and/or accelerated cross-bridge cycle kinetics remained to be established.
In the present study, we have investigated the causal role of cTnI phosphorylation at Ser 22 /Ser 23 in PKD-mediated regulation of myofilament Ca 2ϩ sensitivity and cross-bridge cycle kinetics, using myocardial preparations from transgenic mice that express cTnI containing substitutions of Ser 22 and Ser 23 by nonphosphorylatable Ala on a cTnI-null background. Our data reveal that PKD decreases myofilament Ca 2ϩ sensitivity through cTnI phosphorylation at Ser 22 /Ser 23 but accelerates cross-bridge cycle kinetics through a distinct mechanism. Our data also identify Ser 302 in cMyBP-C as a novel PKD phos-phorylation site that is likely to participate in PKD-mediated regulation of cross-bridge cycle kinetics.

EXPERIMENTAL PROCEDURES
Expanded methodology is provided in the supplemental materials.
Preparation of Mouse Cardiac Trabeculae-Transgenic mice that express cTnI in which Ser 22 and Ser 23 are replaced by two nonphosphorylatable Ala residues (cTnI-Ala 2 ) on a cTnI-null background have been described previously (7)(8)(9). Both cTnI-Ala 2 mice and wild-type (WT) control mice were given the ␤-adrenoreceptor antagonist propranolol (0.5 g/liter) in their drinking water for 3 days prior to sacrifice to minimize PKAmediated protein phosphorylation during anesthesia and heart excision. Excised mouse hearts were frozen and stored in liquid N 2 until they were thawed in relaxing solution. Trabeculae were then dissected from the right ventricle, skinned with Triton X-100, and stored in relaxing solution containing 50% glycerol at Ϫ20°C for up to 3 days (10, 11), until they were used for the assessment of myofilament function.
Assessment of Myofilament Function-Mechanical function of skinned mouse cardiac trabeculae was assessed using equipment and protocols similar to those described previously for skinned myocyte preparations (5,12). Individual trabeculae were attached to a force transducer and a high speed length controller at each end (Fig. 1A), and the sarcomere length was set to ϳ2.2 m in relaxing solution (18°C). The skinned trabecula was then transferred into a Ca 2ϩ activation solution, and Ca 2ϩ -activated force was measured as the steadystate force achieved during the contraction minus the passive force recorded in relaxing solution. At steady-state force, crossbridge cycle kinetics were assessed by performing a releaserestretch maneuver, using a modification of a previously described method (5,13). The muscle was rapidly slackened to ϳ80% length for 20 ms to forcibly detach cross-bridges during Ca 2ϩ activation and then restretched to its initial length (Fig.  1B). The data obtained during force redevelopment following the release-restretch protocol were fitted to a single exponential curve to determine the rate of force redevelopment (k tr ) as the index of cross-bridge cycle kinetics. After the release-restretch protocol, the trabecula was returned to relaxing solution, and the sequence was then repeated in Ca 2ϩ activation solutions containing progressively greater [Ca 2ϩ ] (pCa range 9.0 -4.5) (Fig. 1C). Following measurements of Ca 2ϩ -activated force and k tr in the basal state, the trabecula was incubated in relaxing solution containing PKD, PKA, or no kinase (timematched control) and the phosphatase inhibitor calyculin A for 30 min at room temperature. Subsequently, the trabecula was transferred back into relaxing solution, and the Ca 2ϩ -activated force and k tr measurements were repeated. Therefore, each trabecula served as its own control, allowing paired comparison of pre-and post-kinase data.
Assessment of Myofilament Protein Phosphorylation-Following the dissection of trabeculae, the myocytes were mechanically dissociated from the remaining ventricular tissue by homogenization in a Waring blender and skinned for 15 min at 4°C (5). The skinned myocytes were then incubated in relaxing solution containing PKD, PKA, or no kinase (time-matched control) and the phosphatase inhibitor calyculin A for 30 min at room temperature. Subsequently, Laemmli sample buffer was added, and myofilament phosphorylation status was determined by SDS/ PAGE and immunoblot analysis using phospho-specific antibodies for cTnI and cMyBP-C (6). In complementary experiments, recombinant N-terminally His 6 -tagged cMyBP-C C1C2 fragments (14), in WT form or containing a Ser/Ala mutation at Ser 273 , Ser 282 , or Ser 302 , were incubated in kinase solution containing PKD or PKA for 20 min at 30°C, with phosphorylation status again assessed by SDS/PAGE and immunoblot analysis with phospho-specific antibodies.
Immunolabeling and Confocal Microscopy-Skinned myocytes were centrifuged onto polylysine-coated glass slides and fixed with 4% paraformaldehyde. Unspecific binding sites were then blocked by incubation in 5% nonspecific goat serum, followed by incubation first with primary antibodies (rabbit anti-cMyBP-C (Ser(P) 302 or total) and mouse anti-␣actinin; overnight at 4°C) and then with secondary antibodies (goat Cy5-conjugated anti-rabbit and Cy3-conjugated antimouse; 4 h at room temperature). After final washing, the slides were mounted with coverslips in the presence of n-propyl gallate as an anti-fading reagent (15) and imaged on a Leica TCS SP5 confocal microscope equipped with a 63ϫ/1.4NA oil immersion lens, using appropriate excitation and emission wavelengths.
Data Analysis-All of the data are given as the means Ϯ S.E. Statistical comparisons were by paired or unpaired Student's t test, as appropriate, when comparing data between two groups or by analysis of variance followed by the Bonferroni test, when comparing data between multiple groups. p Ͻ 0.05 was considered significant.

Effects of PKD on cTnI Phosphorylation in WT and cTnI-Ala 2
Myocardium-We have shown previously that PKD phosphorylates cTnI at Ser 22 /Ser 23 in both skinned and intact rat ventricular myocytes (5,6). In the present study, we examined PKDmediated phosphorylation of cTnI in skinned ventricular myocytes from WT and cTnI-Ala 2 mice by immunoblot analysis with a previously characterized phospho-specific antibody that recognizes dually phosphorylated (Ser(P) 22/23 ) cTnI (5). PKD induced a significant increase in cTnI phosphorylation at Ser 22 /Ser 23 in WT myocytes, and the magnitude of such phosphorylation was comparable with that achieved by PKA, an established cTnI kinase that targets the same sites ( Fig. 2A). Furthermore, the extent of cTnI phosphorylation observed in response to either kinase was not in excess of that observed in response to the endogenous sympathetic neurotransmitter norepinephrine in intact ventricular myocytes (supplemental Fig. S1A). As expected, the Ser(P) 22/23 phospho-specific cTnI antibody failed to detect any phosphorylation in myocytes from cTnI-Ala 2 mice even after incubation with PKD or PKA (Fig.  2B), confirming the utility of this mouse model in delineating the mechanistic roles of Ser 22 /Ser 23 phosphorylation in the regulation of cardiac myofilament function.
Effects of PKD on the Ca 2ϩ Sensitivity of Force Development in WT and cTnI-Ala 2 Myocardium-To determine the role of cTnI phosphorylation at Ser 22 /Ser 23 in PKD-mediated regulation of myofilament Ca 2ϩ sensitivity, Ca 2ϩ -activated force development was studied in skinned ventricular trabeculae from WT and cTnI-Ala 2 mice, before and after incubation with PKD. In WT trabeculae, PKD-mediated phosphorylation significantly decreased the Ca 2ϩ sensitivity of force development, as indicated by a rightward shift of the force-pCa relationship and a significant reduction in pCa 50 after incubation with PKD (Fig. 3A). In contrast, in trabeculae from cTnI-Ala 2 mice, incubation with PKD did not significantly reduce the Ca 2ϩ sensitivity of force development (Fig. 3B), indicating that PKD-mediated myofilament Ca 2ϩ desensitization requires cTnI phosphorylation at Ser 22 /Ser 23 . Relative to PKD, incubation with PKA had a qualitatively similar impact on the Ca 2ϩ sensitivity of force development in WT trabeculae (Fig. 3A). However, although the PKA-mediated decrease in myofilament Ca 2ϩ sensitivity was also attenuated in cTnI-Ala 2 trabeculae, the reduction in pCa 50 remained statistically significant (Fig.  3B). Importantly, in time-matched control experiments, no significant reduction in the Ca 2ϩ sensitivity of force development was observed in trabeculae from WT and cTnI-Ala 2 mice that were incubated for an equivalent time in the absence of either kinase (supplemental Table S1). Over the course of the experimental protocol (ϳ180 min), maximum force declined slightly (by ϳ10% in all groups), and the steepness of the force-pCa relationship (Hill coefficient) did not change in trabeculae from WT and cTnI-Ala 2 mice that were incubated with PKD, PKA, or no kinase (supplemental Table S2).
Effects of PKD on Cross-bridge Cycle Kinetics in WT and cTnI-Ala 2 Myocardium-We have shown previously that PKD-mediated phosphorylation accelerates cross-bridge cycle kinetics in rat skinned ventricular myocytes (5). To determine the role of cTnI phosphorylation at Ser 22 /Ser 23 in PKD-mediated acceleration of cross-bridge cycle kinetics, the rate of force redevelopment (k tr ) after a release-restretch maneuver (Fig. 1B) was determined at each pCa in skinned trabeculae from WT and cTnI-Ala 2 hearts, before and after incubation with PKD. Because the level of active force is a major determinant of k tr and changes in myofilament Ca 2ϩ sensitivity alter active force at each pCa, we plotted the relationship between force and k tr to examine phosphorylation-induced changes in k tr (16). Following incubation of trabeculae with PKD, k tr was increased at submaximal forces, such that k tr at 50% maximal force was significantly greater post-PKD than pre-PKD, with little difference between the responses of trabeculae from WT mice (Fig. 4A) and those from cTnI-Ala 2 mice (Fig. 4B). These findings indicate that PKD-mediated acceleration of cross-bridge cycle kinetics does not require cTnI phosphorylation at Ser 22 /Ser 23 but occurs through a distinct mechanism. Qualitatively and quantitatively similar results were obtained in muscle preparations incubated with PKA, which increased k tr at 50% maximal force significantly in trabeculae from both WT and cTnI-Ala 2 mice (Fig. 4). In time-matched control experiments, there was no significant change in k tr at 50% maximal force in trabeculae from WT and cTnI-Ala 2 mice that were incubated for an equivalent time in the absence of either kinase (supplemental Table  S1). As seen with maximum force, there was a small decline in maximum k tr (k tr at pCa 4.5) in trabeculae from WT and cTnI-Ala 2 mice over the course of the experiment, but the extent of this did not differ between groups incubated with PKD, PKA, or no kinase (supplemental Table S2).
PKD-mediated Phosphorylation of Recombinant cMyBP-C C1C2 Domain-Recent evidence indicates that cMyBP-C phosphorylation is predominantly responsible for PKA-mediated acceleration of cross-bridge cycle kinetics in mouse myocardium (9,17). PKA is known to phosphorylate cMyBP-C at three sites, namely Ser 273 , Ser 282 , and Ser 302 , within the conserved linker region between its immunoglobulin I-like C1 and C2 domains (14). Although we have shown previously that PKD phosphorylates a recombinant cMyBP-C fragment that contains this linker region (5), the specific PKD-targeted residue(s) have not been identified. To determine whether PKD phosphorylates cMyBP-C at any of its established PKA phosphorylation sites, we performed in vitro kinase assays using recombinant cMyBP-C C1C2 fragments, in WT form or containing a S273A, S282A, or S302A mutation, with phosphorylation detected using recently developed phospho-specific cMyBP-C antibodies (18). Experiments with PKA-mediated phosphorylation confirmed the specificity of the Ser(P) 273 , Ser(P) 282 , and Ser(P) 302 phospho-specific cMyBP-C antibodies, with a robust signal detected with all three antibodies when WT C1C2 fragment was used as substrate and a loss of signal with each antibody only upon substitution of the pertinent Ser residue by Ala (Fig. 5A). Interestingly, following PKD-mediated phosphorylation, WT C1C2 showed no increased signal with the Ser(P) 273 and Ser(P) 282 phospho-specific cMyBP-C antibodies but a strongly increased signal with the Ser(P) 302 phospho-specific cMyBP-C antibody (Fig. 5B). Furthermore, this signal was lost only upon targeted substitution of Ser 302 by Ala in the substrate protein (Fig. 5B). These findings indicate that PKD selectively phosphorylates Ser 302 in the recombinant cMyBP-C C1C2 fragment.
PKD-mediated Phosphorylation of Native cMyBP-C in WT and cTnI-Ala 2 Myocardium-We next determined the pattern of PKD-mediated phosphorylation of native full-length cMyBP-C in skinned ventricular myocytes from WT and cTnI-Ala 2 mice, using the Ser(P) 273 , Ser(P) 282 , and Ser(P) 302 phospho-specific cMyBP-C antibodies. In these experiments, PKD catalyzed significant phosphorylation of native cMyBP-C at Ser 302 , but not at Ser 273 or Ser 282 , with no difference in the magnitude of the PKD-mediated Ser 302 phosphorylation between myocytes from WT mice (Fig. 6A) and those from cTnI-Ala 2 mice (Fig. 6B). In contrast, PKA led to significant phosphorylation of native cMyBP-C at all three sites, once again with comparable responses seen in myocytes from WT mice (Fig. 6A) and cTnI-Ala 2 mice (Fig. 6B). These findings confirm that Ser 302 in cMyBP-C is a novel target for PKD-mediated phosphorylation and additionally indicate that cMyBP-C phosphorylation by PKD (selectively at Ser 302 ) or by PKA (at Ser 273 , Ser 282 , and Ser 302 ) is unaffected by Ala substitutions of Ser 22 / Ser 23 in cTnI. Furthermore, the extent of cMyBP-C phosphorylation at Ser 302 in response to either kinase was not in excess of that observed in response to norepinephrine in intact ventricular myocytes (supplemental Fig. S1B).
Finally, we determined the sarcomeric localization of Ser 302 phosphorylation in skinned myocytes from WT and cTnI-Ala 2 mice by immunolabeling and confocal microscopy. As expected, total cMyBP-C was detected as a doublet located either side of the M-line in the C-zones of the sarcomeric A-band, in myocytes from WT mice (Fig. 7A, left panel). An identical pattern was observed in myocytes from cTnI-Ala 2 mice (Fig. 7A,  right panel), confirming that Ala substitutions of Ser 22 /Ser 23 in cTnI do not disrupt sarcomeric structure. Importantly, the Ser(P) 302 phospho-specific cMyBP-C antibody showed only weak labeling in control myocytes but revealed a pattern that was identical to that seen with the total cMyBP-C antibody upon PKD-or PKA-mediated phosphorylation, with comparable images observed in myocytes from WT and cTnI-Ala 2 mice (Fig. 7B). Taken together, these experiments indicate that PKD phosphorylates cMyBP-C at Ser 302 , such phosphorylation occurs throughout the C-zones of sarcomeric M-bands, and Ala substitutions of Ser 22 /Ser 23 in cTnI do not interfere with cMyBP-C phosphorylation by PKD or PKA. These findings raise the possibility that the common ability of PKD and PKA to accelerate the cross-bridge cycle rate in skinned trabeculae from both WT and cTnI-Ala 2 mice may arise from their common ability to induce cMyBP-C phosphorylation at Ser 302 .

DISCUSSION
Multiple kinase pathways regulate cardiac myofilament function through the phosphorylation of sarcomeric proteins (19). Although much of the focus to date has been on PKAmediated phosphorylation, other kinases such as protein kinase C isoforms, protein kinase G, and Ca 2ϩ /calmodulin-dependent protein kinase have also been shown to play important regulatory roles (19). Previously, we have shown that PKD phosphorylates cTnI at Ser 22 /Ser 23 (5,6), an observation that was subsequently confirmed by others (20), and reported that such phosphorylation is associated with reduced myofilament Ca 2ϩ sensitivity (5,6) and accelerated cross-bridge cycle kinetics (5). In the present study, we have used myocardial preparations from transgenic mice that express cTnI-containing Ala substitutions of Ser 22 /Ser 23 to explore the mechanistic importance of Ser 22 /Ser 23 phosphorylation in PKD-mediated regulation of myofilament function. Our data provide the first direct evidence that cTnI phosphorylation at Ser 22 /Ser 23 is responsible for PKD-mediated desensitization of myofilaments to Ca 2ϩ but not for PKD-mediated acceleration of cross-bridge cycle kinetics. Furthermore, we identify Ser 302 in cMyBP-C as a novel PKD substrate and show that PKD phosphorylates Ser 302 without affecting the phosphorylation status of the other known PKA target sites in cMyBP-C, at Ser 273 and Ser 282 . These findings suggest that cMyBP-C phosphorylation at Ser 302 may be a crit-ical mechanism through which the cross-bridge cycle rate is regulated.
Role of cTnI Phosphorylation in PKD-mediated Regulation of Myofilament Function-In the present study, we found that both PKD and PKA decreased myofilament Ca 2ϩ sensitivity and accelerated cross-bridge cycle kinetics in skinned trabeculae from WT mice (Figs. 3A and 4A). These findings are consistent with our earlier work with PKD in rat skinned ventricular myocytes (5) and comparable studies with PKA by multiple laboratories in rat (21)(22)(23) and mouse (16,17,24) skinned myocardial preparations. The PKD-mediated reduction in myofilament Ca 2ϩ sensitivity was abolished in skinned trabeculae from cTnI-Ala 2 mice (Fig. 3B), indicating that cTnI phosphorylation at Ser 22 /Ser 23 is the molecular mechanism underlying this response. The PKA-mediated reduction in myofilament Ca 2ϩ sensitivity was also attenuated in skinned trabeculae from cTnI-Ala 2 mice, which is consistent with recent evidence from the Moss laboratory, who used skinned myocardium from the same mouse model (9). Notably, the small PKA-mediated reduction of pCa 50 (from 5.83 Ϯ 0.03 pre-PKA to 5.77 Ϯ 0.02 post-PKA) in cTnI-Ala 2 myocardium was statistically significant (Fig. 3B), whereas the comparable effect reported by Stelzer et al. (9) was not (pCa 50 reduced from 5.77 Ϯ 0.03 pre-PKA to 5.73 Ϯ 0.03 post-PKA). This divergence may reflect a contribution from PKA-mediated phosphorylation of sarcomeric targets other than Ser 22 /Ser 23 in cTnI under our experimental conditions or other differences between the two studies, such as a higher pre-PKA pCa 50 in our work (5.83 Ϯ 0.03 versus 5.77 Ϯ 0.03 in Stelzer et al. (9)) or the use of distinct myocardial preparations (trabeculae versus mechanically isolated multicellular preparations). In contrast to its marked effect on the PKD-mediated reduction in myofilament Ca 2ϩ sensitivity, Ala substitution of Ser 22 /Ser 23 in cTnI had no impact on PKD-mediated acceleration of cross-bridge cycle kinetics (Fig. 4), indicating that phosphorylation of these sites is not the mechanism through which the latter effect is achieved. PKA-mediated acceleration of cross-bridge cycle kinetics was also of similar magnitude in skinned trabeculae from WT and cTnI-Ala 2 mice (Fig. 4), which is consistent with the findings of Stelzer et al. (9) and again points to an underlying mechanism that is distinct from cTnI phosphorylation at Ser 22 /Ser 23 .
Role of cMyBP-C Phosphorylation in PKD-mediated Regulation of Myofilament Function-cMyBP-C is localized to the cross-bridge-containing C-zones of cardiac sarcomeric A-bands, where it interacts with multiple other proteins, including myosin subfragment 2 (S2) at its N terminus and titin at its C terminus, and has been ascribed roles in both physiology and disease (25). Recent studies by the Moss laboratory in two different genetically modified mouse models, one with targeted deletion of the cMyBP-C gene Mybpc3 (9) and the other expressing cMyBP-C in which three Ser residues (Ser 273 , Ser 282 , and Ser 302 ) were replaced by nonphosphorylatable Ala (17), have provided robust evidence that cMyBP-C phosphorylation plays an essential role in PKA-mediated acceleration of cross-bridge cycle kinetics. In this context, our previous work has shown that PKD can phosphorylate a recombinant cMyBP-C fragment (5), which included the PKA phosphorylation sites at Ser 273 , Ser 282 , and Ser 302 that were originally identified by Gautel et al. (14) in human cMyBP-C. However, our subsequent studies suggested that Ser 282 in native cMyBP-C of rat myocytes was unlikely to be a PKD target (6). Our present findings provide novel evidence that PKD phosphorylates the recombinant cMyBP-C C1C2 fragment at Ser 302 and, unlike PKA, induces no detectable phosphorylation at Ser 273 or Ser 282 (Fig. 5). Furthermore, PKD induces selective phosphorylation of Ser 302 also in native cMyBP-C that is incorporated within the sarcomeric lattice of mouse skinned myocytes (Fig. 6). These findings identify Ser 302 in cMyBP-C as a novel PKD substrate. In the light of this finding, it is pertinent to note that, of the three PKA sites in cMyBP-C, only the Ser 302 motif ( 297 LKKRDS 302 ) conforms to the optimal PKD phosphorylation motif, which favors an aliphatic amino acid (Leu, Val, or Ile) at the Ϫ5 position and a basic amino acid (Arg or Lys) at the Ϫ3 position, relative to the targeted Ser residue (26). Our observation that PKD-mediated Ser 302 phosphorylation appears to occur throughout the C-zones of sarcomeric A-bands (Fig. 7) suggests that it may have functional consequences. Indeed, the quantitatively and spatially similar effects of PKD and PKA on cMyBP-C phosphorylation at Ser 302 in skinned myocytes from both WT and cTnI-Ala 2 mice, despite their markedly different effects on cMyBP-C phosphorylation at Ser 273 and Ser 282 in these preparations, suggest that this may be the mechanism underlying their common ability to accelerate crossbridge cycle kinetics in the presence and absence of cTnI phosphorylation at Ser 22 /Ser 23 . It also suggests that the two proteins are phosphorylated independently of each other.
PKA-mediated phosphorylation of cMyBP-C is believed to accelerate cross-bridge cycle kinetics by relieving a tether-like constraint imposed on myosin heads by cMyBP-C binding to myosin, which then increases their proximity to and probability of interaction with actin (27). An alternative mechanism, the switch of an actin binding site, was also recently proposed (28); however, this interaction would need to be significantly weaker to not impede actomyosin filament sliding, which is in agreement with the strict myosin filament localization of N-terminal cMyBP-C fragments in cardiomyocytes (29). The relative importance of the individual PKA phosphorylation sites at Ser 273 , Ser 282 , and Ser 302 in regulating cMyBP-C binding to myosin (or actin) is unknown, although there is evidence that suggests an ordered phosphorylation of these sites by PKA, with Ser 282 phosphorylation occurring before phosphorylation of the other sites (14,30). The N-terminal myosin-binding region of cMyBP-C, which interacts with myosin at its S2 segment in a phosphorylation-regulated manner, comprises the immunoglobulin I-like domains C1 and C2 connected by a presumably flexible linker that contains Ser 273 , Ser 282 , and Ser 302 . Based on their structural analyses of the myosin S2 interactions of cMyBP-C domains C1 and C2 (31, 32), Ababou et al. (32) have recently proposed a model in which the C1-C2 linker extends toward the C terminus of S2 to reach the area around residue 930. In this model, the phosphorylation sites at Ser 273 , Ser 282 , and Ser 302 appose negatively charged patches on the surface of S2, such that their phosphorylation would lead to charge repulsion and destabilization of the interaction (32). In the context of our present findings regarding the potential importance of Ser 302 phosphorylation in regulating cross-bridge cycle kinetics, it is intriguing to note that a E924K mutation in the S2 region that was proposed to appose Ser 302 , which is associated with familial hypertrophic  273 , Ser(P) 282 , and Ser(P) 302 cMyBP-C antibodies, with protein loading illustrated by Coomassie staining of the membranes. The bar charts show quantitative data from such experiments using multiple skinned myocyte preparations (4 hearts/group). cMyBP-C phosphorylation is expressed relative to a common positive control sample included in each experiment. *, p Ͻ 0.05 versus no-kinase control (con).
cardiomyopathy, abolishes C1C2 binding to S2 (33) without affecting the binding of either the C1 or the C2 domain alone (32). It appears therefore that the interaction between this region of S2 and the Ser 302 -containing region of the C1-C2 linker in cMyBP-C may be critical in maintaining the binding of the cMyBP-C N terminus to myosin, such that interference with this interaction (for example, as a result of S2 mutation or cMyBP-C phosphorylation at Ser 302 ) may be sufficient to have structural and functional consequences, providing a likely mechanism for PKD-mediated acceleration of cross-bridge cycle kinetics. Further investigation is clearly required to test this hypothesis. It will also ultimately be necessary to determine the atomic structure of the phosphorylated linker, which was recently proposed to adopt a compact conformation (34). Of broader potential physiological significance, a series of studies by Sadayappan et al. (18,35,36) in genetically modified mice that express cMyBP-C with nonphosphorylatable or phosphomimetic amino acid substitutions at Ser 273 , Ser 282 , and Ser 302 suggest that the functional importance of cMyBP-C phosphorylation extends beyond the regulation of cross-bridge cycle kinetics, for example to determining post-ischemic cardiac function. Once again, further investigation is required to determine the contributions of individual phosphorylation sites, including Ser 302 , to such functional consequences of cMyBP-C phosphorylation.
Relative Roles of PKD versus PKA in Myofilament Phosphorylation-In the present study, we treated WT and cTnI-Ala 2 mice with propranolol to minimize PKA-mediated protein phosphorylation arising from sympathetic activation during anesthesia and surgical excision of the heart. Such an approach has been used previously by us (5) and by other investigators (23,37) in exploring the functional consequences of kinase-mediated myofilament protein phosphorylation in skinned preparations. Performing experiments using hearts from untreated mice would not provide useful information on the functional consequences of PKD-mediated phosphorylation of sites that are targeted also by PKA, because those sites would be already occupied. Thus, the use of propranolol prevents the very high levels of PKA-induced phosphorylation that are an artifact of the anesthesia and dissection procedure and that cannot be present in the resting animal (otherwise sympathetic stimulation would have no effect on the heart).
As we have suggested recently (1), PKD-mediated phosphorylation of substrate proteins that are targeted also by PKA at the same site(s) is likely to be of greater functional significance in settings where the PKA pathway is down-regulated, such as in heart failure. Other observations that suggest a greater role for PKD in the heart failure setting include our recent finding that PKA inhibits PKD activation (38) and reports that PKD expression is increased in failing myocardium, as recently reviewed (1).
In conclusion, our study provides direct evidence that PKD decreases myofilament Ca 2ϩ sensitivity as a consequence of cTnI phosphorylation at Ser 22 /Ser 23 but accelerates crossbridge cycle kinetics through a distinct mechanism. It also identifies Ser 302 in cMyBP-C as a novel PKD phosphorylation site, which provides a potential mechanism through which PKD may regulate cross-bridge cycle kinetics. These observations add to the growing evidence that PKD-mediated pathways regulate cardiac myofilament function through coordinated phosphorylation of multiple sarcomeric proteins.