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

Abstract

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.

Introduction

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 co-workers (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 myosin-binding protein C (cMyBP-C), as potential PKD substrates, and have shown that PKD induces cTnI dual phosphorylation at Ser²²/Ser²³, reduces myofilament Ca²⁺ 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 Ser²²/Ser²³, decreases myofilament Ca²⁺ sensitivity, and abolishes the positive inotropic response to this stimulus (6). Nevertheless, any causal link between PKD-mediated cTnI phosphorylation at Ser²²/Ser²³ and the regulation of myocardial contraction through reduced myofilament Ca²⁺ 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 Ser²²/Ser²³ in PKD-mediated regulation of myofilament Ca²⁺ sensitivity and cross-bridge cycle kinetics, using myocardial preparations from transgenic mice that express cTnI containing substitutions of Ser²² and Ser²³ by nonphosphorylatable Ala on a cTnI-null background. Our data reveal that PKD decreases myofilament Ca²⁺ sensitivity through cTnI phosphorylation at Ser²²/Ser²³ but accelerates cross-bridge cycle kinetics through a distinct mechanism. Our data also identify Ser³⁰² in cMyBP-C as a novel PKD phosphorylation 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 Ser²² and Ser²³ are replaced by two nonphosphorylatable Ala residues (cTnI-Ala₂) on a cTnI-null background have been described previously (7–9). Both cTnI-Ala₂ mice and wild-type (WT) control mice were given the β-adrenoreceptor antagonist propranolol (0.5 g/liter) in their drinking water for 3 days prior to sacrifice to minimize PKA-mediated protein phosphorylation during anesthesia and heart excision. Excised mouse hearts were frozen and stored in liquid N₂ 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 −20 °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 ∼2.2 μm in relaxing solution (18 °C). The skinned trabecula was then transferred into a Ca²⁺ activation solution, and Ca²⁺-activated force was measured as the steady-state force achieved during the contraction minus the passive force recorded in relaxing solution. At steady-state force, cross-bridge cycle kinetics were assessed by performing a release-restretch maneuver, using a modification of a previously described method (5, 13). The muscle was rapidly slackened to ∼80% length for 20 ms to forcibly detach cross-bridges during Ca²⁺ 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 Ca²⁺ activation solutions containing progressively greater [Ca²⁺] (pCa range 9.0–4.5) (Fig. 1C). Following measurements of Ca²⁺-activated force and k_tr in the basal state, the trabecula was 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, the trabecula was transferred back into relaxing solution, and the Ca²⁺-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.

FIGURE 1.

Ca²⁺ activated force and force redevelopment in mouse skinned trabeculae. A, a skinned trabecula, attached at either end to a force transducer and a high speed length controller. B, representative recording of force redevelopment after a release-restretch protocol at pCa 5.61. C, representative recording of Ca²⁺-activated force in bath solutions at pCa 5.81, 5.61, and 5.29.

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 His₆-tagged cMyBP-C C1C2 fragments (14), in WT form or containing a Ser/Ala mutation at Ser²⁷³, Ser²⁸², or Ser³⁰², 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-α-actinin; overnight at 4 °C) and then with secondary antibodies (goat Cy5-conjugated anti-rabbit and Cy3-conjugated anti-mouse; 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 63×/1.4NA oil immersion lens, using appropriate excitation and emission wavelengths.

Data Analysis

All of the data are given as the means ± 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 < 0.05 was considered significant.

RESULTS

Effects of PKD on cTnI Phosphorylation in WT and cTnI-Ala₂ Myocardium

We have shown previously that PKD phosphorylates cTnI at Ser²²/Ser²³ in both skinned and intact rat ventricular myocytes (5, 6). In the present study, we examined PKD-mediated phosphorylation of cTnI in skinned ventricular myocytes from WT and cTnI-Ala₂ 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 Ser²²/Ser²³ 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-Ala₂ mice even after incubation with PKD or PKA (Fig. 2B), confirming the utility of this mouse model in delineating the mechanistic roles of Ser²²/Ser²³ phosphorylation in the regulation of cardiac myofilament function.

FIGURE 2.

Effects of PKD and PKA on cTnI phosphorylation at Ser²²/Ser²³ in skinned ventricular myocytes from WT (A) and cTnI-Ala₂ (B) mice. The representative immunoblots (IB) show PKD- or PKA-mediated cTnI phosphorylation as detected by a phospho-specific Ser(P)^22/23 cTnI antibody, with protein loading illustrated by Coomassie staining of the membranes. The bar charts show quantitative data from such experiments using multiple skinned myocyte preparations (four hearts/group). cTnI phosphorylation is expressed relative to a common positive control sample included in each experiment. *, p < 0.05 versus no-kinase control (con).

Effects of PKD on the Ca²⁺ Sensitivity of Force Development in WT and cTnI-Ala₂ Myocardium

To determine the role of cTnI phosphorylation at Ser²²/Ser²³ in PKD-mediated regulation of myofilament Ca²⁺ sensitivity, Ca²⁺-activated force development was studied in skinned ventricular trabeculae from WT and cTnI-Ala₂ mice, before and after incubation with PKD. In WT trabeculae, PKD-mediated phosphorylation significantly decreased the Ca²⁺ sensitivity of force development, as indicated by a rightward shift of the force-pCa relationship and a significant reduction in pCa₅₀ after incubation with PKD (Fig. 3A). In contrast, in trabeculae from cTnI-Ala₂ mice, incubation with PKD did not significantly reduce the Ca²⁺ sensitivity of force development (Fig. 3B), indicating that PKD-mediated myofilament Ca²⁺ desensitization requires cTnI phosphorylation at Ser²²/Ser²³. Relative to PKD, incubation with PKA had a qualitatively similar impact on the Ca²⁺ sensitivity of force development in WT trabeculae (Fig. 3A). However, although the PKA-mediated decrease in myofilament Ca²⁺ sensitivity was also attenuated in cTnI-Ala₂ trabeculae, the reduction in pCa₅₀ remained statistically significant (Fig. 3B). Importantly, in time-matched control experiments, no significant reduction in the Ca²⁺ sensitivity of force development was observed in trabeculae from WT and cTnI-Ala₂ mice that were incubated for an equivalent time in the absence of either kinase (supplemental Table S1). Over the course of the experimental protocol (∼180 min), maximum force declined slightly (by ∼10% in all groups), and the steepness of the force-pCa relationship (Hill coefficient) did not change in trabeculae from WT and cTnI-Ala₂ mice that were incubated with PKD, PKA, or no kinase (supplemental Table S2).

FIGURE 3.

Effects of PKD and PKA on the Ca²⁺ sensitivity of force development in skinned ventricular trabeculae from WT (A) and cTnI-Ala₂ (B) mice. The mean force-pCa curves show data obtained before (pre, open circles) and after (post, filled circles) incubation of trabeculae with PKD or PKA, as indicated. Force values were normalized to the maximum force, measured at pCa 4.5. The bar charts show mean pCa₅₀ values obtained before (pre, open bars) and after (post, filled bars) incubation of trabeculae with PKD or PKA, as indicated. *, p < 0.05 versus pre-kinase (n = 7/group).

Effects of PKD on Cross-bridge Cycle Kinetics in WT and cTnI-Ala₂ 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 Ser²²/Ser²³ 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-Ala₂ hearts, before and after incubation with PKD. Because the level of active force is a major determinant of k_tr and changes in myofilament Ca²⁺ 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-Ala₂ mice (Fig. 4B). These findings indicate that PKD-mediated acceleration of cross-bridge cycle kinetics does not require cTnI phosphorylation at Ser²²/Ser²³ 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-Ala₂ 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-Ala₂ 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-Ala₂ 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).

FIGURE 4.

Effects of PKD and PKA on the rate of force redevelopment (k_tr) in skinned ventricular trabeculae from WT (A) and cTnI-Ala₂ (B) mice. The force-k_tr relationships show data obtained before (pre, open circles) and after (post, filled circles) incubation of trabeculae with PKD or PKA, as indicated. Force values were normalized to the maximum force, and the k_tr values were normalized to the maximum k_tr, both measured at pCa 4.5. The bar charts show mean relative k_tr at 50% maximum force obtained before (pre, open bars) and after (post, filled bars) incubation of trabeculae with PKD or PKA, as indicated. *, p < 0.05 versus pre-kinase (n = 7/group).

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 Ser²⁷³, Ser²⁸², and Ser³⁰², 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 Ser³⁰² by Ala in the substrate protein (Fig. 5B). These findings indicate that PKD selectively phosphorylates Ser³⁰² in the recombinant cMyBP-C C1C2 fragment.

FIGURE 5.

Phosphorylation by PKA (A) or PKD (B) of recombinant His₆-tagged C1C2 fragments of human cMyBP-C, in WT form, or with a Ser/Ala substitution at Ser²⁷³, Ser²⁸², or Ser³⁰². Phosphorylation was detected by immunoblot (IB) analysis using phospho-specific Ser(P)²⁷³, Ser(P)²⁸², or Ser(P)³⁰² cMyBP-C antibodies with protein loading illustrated by Coomassie staining of the membranes.

PKD-mediated Phosphorylation of Native cMyBP-C in WT and cTnI-Ala₂ Myocardium

We next determined the pattern of PKD-mediated phosphorylation of native full-length cMyBP-C in skinned ventricular myocytes from WT and cTnI-Ala₂ 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 Ser³⁰², but not at Ser²⁷³ or Ser²⁸², with no difference in the magnitude of the PKD-mediated Ser³⁰² phosphorylation between myocytes from WT mice (Fig. 6A) and those from cTnI-Ala₂ 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-Ala₂ mice (Fig. 6B). These findings confirm that Ser³⁰² in cMyBP-C is a novel target for PKD-mediated phosphorylation and additionally indicate that cMyBP-C phosphorylation by PKD (selectively at Ser³⁰²) or by PKA (at Ser²⁷³, Ser²⁸², and Ser³⁰²) is unaffected by Ala substitutions of Ser²²/Ser²³ in cTnI. Furthermore, the extent of cMyBP-C phosphorylation at Ser³⁰² in response to either kinase was not in excess of that observed in response to norepinephrine in intact ventricular myocytes (supplemental Fig. S1B).

FIGURE 6.

Effects of PKD and PKA on cMyBP-C phosphorylation at Ser²⁷³, Ser²⁸², and Ser³⁰² in skinned ventricular myocytes from WT (A) and cTnI-Ala₂ (B) mice. The representative immunoblots (IB) show PKD- or PKA-mediated cMyBP-C phosphorylation as detected by phospho-specific Ser(P)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 < 0.05 versus no-kinase control (con).

Finally, we determined the sarcomeric localization of Ser³⁰² phosphorylation in skinned myocytes from WT and cTnI-Ala₂ 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-Ala₂ mice (Fig. 7A, right panel), confirming that Ala substitutions of Ser²²/Ser²³ 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-Ala₂ mice (Fig. 7B). Taken together, these experiments indicate that PKD phosphorylates cMyBP-C at Ser³⁰², such phosphorylation occurs throughout the C-zones of sarcomeric M-bands, and Ala substitutions of Ser²²/Ser²³ 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-Ala₂ mice may arise from their common ability to induce cMyBP-C phosphorylation at Ser³⁰².

FIGURE 7.

Confocal microscope images showing the localization of total cMyBP-C (A) and cMyBP-C phosphorylated at Ser³⁰² (B) in skinned myocytes from WT and cTnI-Ala₂ (Ala2) mice following incubation with no kinase (CON), PKD, or PKA. The samples were immunolabeled additionally with an α-actinin antibody to demarcate the Z-discs, and the nuclei were stained with 4′,6′-diamino-2-phenylindole. The images were obtained from perinuclear regions of each sample. In the merged images, red indicates α-actinin labeling, green indicates cMyBP-C (total and Ser(P)³⁰²) labeling, and the nuclei are stained blue. Scale bar, 10 μm.

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 PKA-mediated phosphorylation, other kinases such as protein kinase C isoforms, protein kinase G, and Ca²⁺/calmodulin-dependent protein kinase have also been shown to play important regulatory roles (19). Previously, we have shown that PKD phosphorylates cTnI at Ser²²/Ser²³ (5, 6), an observation that was subsequently confirmed by others (20), and reported that such phosphorylation is associated with reduced myofilament Ca²⁺ 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 Ser²²/Ser²³ to explore the mechanistic importance of Ser²²/Ser²³ phosphorylation in PKD-mediated regulation of myofilament function. Our data provide the first direct evidence that cTnI phosphorylation at Ser²²/Ser²³ is responsible for PKD-mediated desensitization of myofilaments to Ca²⁺ but not for PKD-mediated acceleration of cross-bridge cycle kinetics. Furthermore, we identify Ser³⁰² in cMyBP-C as a novel PKD substrate and show that PKD phosphorylates Ser³⁰² without affecting the phosphorylation status of the other known PKA target sites in cMyBP-C, at Ser²⁷³ and Ser²⁸². These findings suggest that cMyBP-C phosphorylation at Ser³⁰² may be a critical 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 Ca²⁺ 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 (21–23) and mouse (16, 17, 24) skinned myocardial preparations. The PKD-mediated reduction in myofilament Ca²⁺ sensitivity was abolished in skinned trabeculae from cTnI-Ala₂ mice (Fig. 3B), indicating that cTnI phosphorylation at Ser²²/Ser²³ is the molecular mechanism underlying this response. The PKA-mediated reduction in myofilament Ca²⁺ sensitivity was also attenuated in skinned trabeculae from cTnI-Ala₂ 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 pCa₅₀ (from 5.83 ± 0.03 pre-PKA to 5.77 ± 0.02 post-PKA) in cTnI-Ala₂ myocardium was statistically significant (Fig. 3B), whereas the comparable effect reported by Stelzer et al. (9) was not (pCa₅₀ reduced from 5.77 ± 0.03 pre-PKA to 5.73 ± 0.03 post-PKA). This divergence may reflect a contribution from PKA-mediated phosphorylation of sarcomeric targets other than Ser²²/Ser²³ in cTnI under our experimental conditions or other differences between the two studies, such as a higher pre-PKA pCa₅₀ in our work (5.83 ± 0.03 versus 5.77 ± 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 Ca²⁺ sensitivity, Ala substitution of Ser²²/Ser²³ 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-Ala₂ 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 Ser²²/Ser²³.

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 (Ser²⁷³, Ser²⁸², and Ser³⁰²) 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 Ser²⁷³, Ser²⁸², and Ser³⁰² that were originally identified by Gautel et al. (14) in human cMyBP-C. However, our subsequent studies suggested that Ser²⁸² 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 Ser³⁰² and, unlike PKA, induces no detectable phosphorylation at Ser²⁷³ or Ser²⁸² (Fig. 5). Furthermore, PKD induces selective phosphorylation of Ser³⁰² also in native cMyBP-C that is incorporated within the sarcomeric lattice of mouse skinned myocytes (Fig. 6). These findings identify Ser³⁰² 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 Ser³⁰² motif (²⁹⁷LKKRDS³⁰²) conforms to the optimal PKD phosphorylation motif, which favors an aliphatic amino acid (Leu, Val, or Ile) at the −5 position and a basic amino acid (Arg or Lys) at the −3 position, relative to the targeted Ser residue (26). Our observation that PKD-mediated Ser³⁰² 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 Ser³⁰² in skinned myocytes from both WT and cTnI-Ala₂ mice, despite their markedly different effects on cMyBP-C phosphorylation at Ser²⁷³ and Ser²⁸² in these preparations, suggest that this may be the mechanism underlying their common ability to accelerate cross-bridge cycle kinetics in the presence and absence of cTnI phosphorylation at Ser²²/Ser²³. 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 Ser²⁷³, Ser²⁸², and Ser³⁰² 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 Ser²⁸² 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 Ser²⁷³, Ser²⁸², and Ser³⁰². 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 Ser²⁷³, Ser²⁸², and Ser³⁰² 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 Ser³⁰² 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 Ser³⁰², which is associated with familial hypertrophic 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 Ser³⁰²-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 Ser³⁰²) 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 phospho-mimetic amino acid substitutions at Ser²⁷³, Ser²⁸², and Ser³⁰² 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 Ser³⁰², 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-Ala₂ 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 Ca²⁺ sensitivity as a consequence of cTnI phosphorylation at Ser²²/Ser²³ but accelerates cross-bridge cycle kinetics through a distinct mechanism. It also identifies Ser³⁰² 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.