Quantitative Analysis of the Cardiac Phosphoproteome in Response to Acute ß-Adrenergic Receptor Stimulation In Vivo.

ß-adrenergic receptor (ß-AR) stimulation represents a major mechanism of modulating cardiac output. In spite of its fundamental importance, its molecular basis on the level of cell signalling has not been characterised in detail yet. We employed mass spectrometry-based proteome and phosphoproteome analysis using SuperSILAC (spike-in stable isotope labelling by amino acids in cell culture) standardization to generate a comprehensive map of acute phosphoproteome changes in mice upon administration of isoprenaline (ISO), a synthetic ß-AR agonist that targets both ß1-AR and ß2-AR subtypes. Our data describe 8597 quantitated phosphopeptides corresponding to 10,164 known and novel phospho-events from 2975 proteins. In total, 197 of these phospho-events showed significantly altered phosphorylation, indicating an intricate signalling network activated in response to ß-AR stimulation. In addition, we unexpectedly detected significant cardiac expression and ISO-induced fragmentation of junctophilin-1, a junctophilin isoform hitherto only thought to be expressed in skeletal muscle. Data are available via ProteomeXchange with identifier PXD025569.

Cyclic AMP represses pathological MEF2 activation by myocyte-specific hypo-phosphorylation of HDAC5.

Class IIa histone deacetylases (HDACs) critically regulate cardiac function through the repression of the activity of myocyte enhancer factor 2 (MEF2)-dependent gene programs. Protein kinase D (PKD) and Ca2+/Calmodulin-dependent kinase II (CaMKII) activate MEF2 by phosphorylating distinct HDAC isoforms and thereby creating 14-3-3 binding sites for nucleo-cytoplasmic shuttling. Recently, it has been shown that this process is counteracted by cyclic AMP (cAMP)-dependent signaling. Here, we investigated the specific mechanisms of how cAMP-dependent signaling regulates distinct HDAC isoforms and determined their relative contributions to the protection from pathological MEF2 activation. We found that cAMP is sufficient to induce nuclear retention and to blunt phosphorylation of the 14-3-3 binding sites of HDAC5 (Ser259/498) and HDAC9 (Ser218/448) but not HDAC4 (Ser246/467/632). These regulatory events could be observed only in cardiomyocytes and myocyte-like cells but not in non-myocytes, pointing to an indirect myocyte-specific mode of action. Consistent with one previous report, we found that blunted phosphorylation of HDAC5 and HDAC9 was mediated by protein kinase A (PKA)-dependent inhibition of PKD. However, we show by the use of neonatal cardiomyocytes derived from genetic HDAC mouse models that endogenous HDAC5 but not HDAC9 contributes specifically to the repression of endogenous MEF2 activity. HDAC4 contributed significantly to the repression of MEF2 activity but based on the mechanistic findings of this study combined with previous results we attribute this to PKA-dependent proteolysis of HDAC4. Consistently, cAMP-induced repression of agonist-driven cellular hypertrophy was blunted in cardiomyocytes deficient for both HDAC5 and HDAC4. In conclusion, cAMP inhibits MEF2 through both nuclear accumulation of hypo-phosphorylated HDAC5 and through a distinct HDAC4-dependent mechanism.

Monophosphorylation of cardiac troponin-I at Ser-23/24 is sufficient to regulate cardiac myofibrillar Ca2+ sensitivity and calpain-induced proteolysis.

The acceleration of myocardial relaxation produced by ß-adrenoreceptor stimulation is mediated in part by protein kinase A (PKA)-mediated phosphorylation of cardiac troponin-I (cTnI), which decreases myofibrillar Ca2+ sensitivity. Previous evidence suggests that phosphorylation of both Ser-23 and Ser-24 in cTnI is required for this Ca2+ desensitization. PKA-mediated phosphorylation also partially protects cTnI from proteolysis by calpain. Here we report that protein kinase D (PKD) phosphorylates only one serine of cTnI Ser-23/24. To explore the functional consequences of this monophosphorylation, we examined the Ca2+ sensitivity of force production and susceptibility of cTnI to calpain-mediated proteolysis when Ser-23/24 of cTnI in mouse cardiac myofibrils was nonphosphorylated, mono-phosphorylated, or bisphosphorylated (using sequential incubations in λ-phosphatase, PKD, and PKA, respectively). Phos-tag gels, Western blotting, and high-resolution MS revealed that PKD produced >90% monophosphorylation of cTnI, primarily at Ser-24, whereas PKA led to cTnI bisphosphorylation exclusively. PKD markedly decreased the Ca2+ sensitivity of force production in detergent-permeabilized ventricular trabeculae, whereas subsequent incubation with PKA produced only a small further fall of Ca2+ sensitivity. Unlike PKD, PKA also substantially phosphorylated myosin-binding protein-C and significantly accelerated cross-bridge kinetics (ktr). After phosphorylation by PKD or PKA, cTnI in isolated myofibrils was partially protected from calpain-mediated degradation. We conclude that cTnI monophosphorylation at Ser-23/24 decreases myofibrillar Ca2+ sensitivity and partially protects cTnI from calpain-induced proteolysis. In healthy cardiomyocytes, the basal monophosphorylation of cTnI may help tonically regulate myofibrillar Ca2+ sensitivity.

ß-Adrenergic regulation of cardiac type 2A protein phosphatase through phosphorylation of regulatory subunit B56δ at S573.

BACKGROUND: Type 2A protein phosphatase (PP2A) enzymes are serine/threonine phosphatases which comprise a scaffold A subunit, a regulatory B subunit and a catalytic C subunit, and have been implicated in the dephosphorylation of multiple cardiac phosphoproteins. B subunits determine subcellular targeting, substrate specificity and catalytic activity, and can themselves be regulated by post-translational modifications. We explored potential ß-adrenergic regulation of PP2A in cardiomyocytes through phosphorylation of the regulatory B subunit isoform B56δ. METHODS AND RESULTS: Phosphate affinity SDS-PAGE and immunoblot analysis revealed increased phosphorylation of B56δ in adult rat ventricular myocytes (ARVM) exposed to the ß-adrenergic receptor (ßAR) agonist isoprenaline (ISO). Phosphorylation of B56δ occurred at S573, primarily through stimulation of the ß1AR subtype, and was dependent on PKA activity. The functional role of the phosphorylation was explored in ARVM transduced with adenoviruses expressing wild type (WT) or non-phosphorylatable (S573A) B56δ, fused to GFP at the N-terminus. C subunit expression was increased in ARVM expressing GFP-B56δ-WT or GFP-B56δ-S573A, both of which co-immunoprecipitated with endogenous C and A subunits. PP2A activity in cell lysates was increased in response to ISO in ARVM expressing GFP-B56δ-WT but not GFP-B56δ-S573A. Immunoblot analysis of the phosphoproteome in ARVM expressing GFP-B56δ-WT or GFP-B56δ-S573A with antibodies detecting (i) phospho-serine/threonine residues in distinct kinase substrate motifs or (ii) specific phosphorylated residues of functional importance in selected proteins revealed a comparable phosphorylation profile in the absence or presence of ISO stimulation. CONCLUSIONS: In cardiomyocytes, ßAR stimulation induces PKA-mediated phosphorylation of the PP2A regulatory subunit isoform B56δ at S573, which increases associated PP2A catalytic activity. This is likely to regulate the phosphorylation status of specific B56δ-PP2A substrates, which remain to be identified.

Roles and post-translational regulation of cardiac class IIa histone deacetylase isoforms.

Cardiomyocyte hypertrophy is an integral component of pathological cardiac remodelling in response to mechanical and chemical stresses in settings such as chronic hypertension or myocardial infarction. For hypertrophy to ensue, the pertinent mechanical and chemical signals need to be transmitted from membrane sensors (such as receptors for neurohormonal mediators) to the cardiomyocyte nucleus, leading to altered transcription of the genes that regulate cell growth. In recent years, nuclear histone deacetylases (HDACs) have attracted considerable attention as signal-responsive, distal regulators of the transcriptional reprogramming that in turn precipitates cardiomyocyte hypertrophy, with particular focus on the role of members of the class IIa family, such as HDAC4 and HDAC5. These histone deacetylase isoforms appear to repress cardiomyocyte hypertrophy through mechanisms that involve protein interactions in the cardiomyocyte nucleus, particularly with pro-hypertrophic transcription factors, rather than via histone deacetylation. In contrast, evidence indicates that class I HDACs promote cardiomyocyte hypertrophy through mechanisms that are dependent on their enzymatic activity and thus sensitive to pharmacological HDAC inhibitors. Although considerable progress has been made in understanding the roles of post-translational modifications (PTMs) such as phosphorylation, oxidation and proteolytic cleavage in regulating class IIa HDAC localisation and function, more work is required to explore the contributions of other PTMs, such as ubiquitination and sumoylation, as well as potential cross-regulatory interactions between distinct PTMs and between class IIa and class I HDAC isoforms.

Phosphoregulation of the titin-cap protein telethonin in cardiac myocytes.

Telethonin (also known as titin-cap or t-cap) is a muscle-specific protein whose mutation is associated with cardiac and skeletal myopathies through unknown mechanisms. Our previous work identified cardiac telethonin as an interaction partner for the protein kinase D catalytic domain. In this study, kinase assays used in conjunction with MS and site-directed mutagenesis confirmed telethonin as a substrate for protein kinase D and Ca(2+)/calmodulin-dependent kinase II in vitro and identified Ser-157 and Ser-161 as the phosphorylation sites. Phosphate affinity electrophoresis and MS revealed endogenous telethonin to exist in a constitutively bis-phosphorylated form in isolated adult rat ventricular myocytes and in mouse and rat ventricular myocardium. Following heterologous expression in myocytes by adenoviral gene transfer, wild-type telethonin became bis-phosphorylated, whereas S157A/S161A telethonin remained non-phosphorylated. Nevertheless, both proteins localized predominantly to the sarcomeric Z-disc, where they partially replaced endogenous telethonin. Such partial replacement with S157A/S161A telethonin disrupted transverse tubule organization and prolonged the time to peak of the intracellular Ca(2+) transient and increased its variance. These data reveal, for the first time, that cardiac telethonin is constitutively bis-phosphorylated and suggest that such phosphorylation is critical for normal telethonin function, which may include maintenance of transverse tubule organization and intracellular Ca(2+) transients.

Four-and-a-half LIM domains proteins are novel regulators of the protein kinase D pathway in cardiac myocytes.

PKD (protein kinase D) is a serine/threonine kinase implicated in multiple cardiac roles, including the phosphorylation of the class II HDAC5 (histone deacetylase isoform 5) and thereby de-repression of MEF2 (myocyte enhancer factor 2) transcription factor activity. In the present study we identify FHL1 (four-and-a-half LIM domains protein 1) and FHL2 as novel binding partners for PKD in cardiac myocytes. This was confirmed by pull-down assays using recombinant GST-fused proteins and heterologously or endogenously expressed PKD in adult rat ventricular myocytes or NRVMs (neonatal rat ventricular myocytes) respectively, and by co-immunoprecipitation of FHL1 and FHL2 with GFP-PKD1 fusion protein expressed in NRVMs. In vitro kinase assays showed that neither FHL1 nor FHL2 is a PKD1 substrate. Selective knockdown of FHL1 expression in NRVMs significantly inhibited PKD activation and HDAC5 phosphorylation in response to endothelin 1, but not to the α1-adrenoceptor agonist phenylephrine. In contrast, selective knockdown of FHL2 expression caused a significant reduction in PKD activation and HDAC5 phosphorylation in response to both stimuli. Interestingly, neither intervention affected MEF2 activation by endothelin 1 or phenylephrine. We conclude that FHL1 and FHL2 are novel cardiac PKD partners, which differentially facilitate PKD activation and HDAC5 phosphorylation by distinct neurohormonal stimuli, but are unlikely to regulate MEF2-driven transcriptional reprogramming.

Neurohormonal regulation of cardiac histone deacetylase 5 nuclear localization by phosphorylation-dependent and phosphorylation-independent mechanisms.

RATIONALE: Myocyte enhancer factor 2 (MEF2) transcription factors drive the genetic reprogramming that precipitates pathological cardiac hypertrophy and remodeling. Class II histone deacetylase (HDAC) isoforms, such as HDAC5, act as signal-responsive repressors of MEF2 activity in cardiac myocytes and their nuclear export provides a key mechanism for the neurohormonal induction of such activity. OBJECTIVE: To delineate the mechanism(s) through which 2 clinically relevant neurohormonal stimuli, endothelin-1 (ET1) and the ß-adrenergic receptor (ß-AR) agonist isoproterenol (ISO), may regulate HDAC5 nuclear localization in adult cardiac myocytes. METHODS AND RESULTS: ET1 induced HDAC5 phosphorylation and nuclear export in ventricular myocytes from the adult rat heart. Use of a novel, highly selective protein kinase D (PKD) inhibitor and a nonphosphorylatable HDAC5 mutant revealed that PKD-mediated phosphorylation was necessary for ET1-induced HDAC5 nuclear export. In contrast, ISO reduced HDAC5 phosphorylation in the presence or absence of ET1 but still induced HDAC5 nuclear export. ISO-induced HDAC5 nuclear export occurred through a ß(1)-AR-mediated oxidative process that was independent of PKD, protein kinase A, and Ca(2+)/calmodulin-dependent kinase II activities. Although ET1 and ISO shared a similar ability to induce HDAC5 nuclear export, albeit through distinct phosphorylation-dependent versus phosphorylation-independent mechanisms, ISO induced a significantly greater increase in MEF2 activity. CONCLUSIONS: PKD-mediated HDAC5 phosphorylation and nuclear export are unlikely to be of major importance in regulating MEF2-driven cardiac remodeling in the presence of sympathetic activity with intact ß(1)-AR signaling, which would not only counteract HDAC5 phosphorylation but also induce HDAC5 nuclear export through a novel phosphorylation-independent, oxidation-mediated mechanism. Inhibition of this mechanism may contribute to the therapeutic efficacy of ß(1)-AR antagonists in heart failure.

A critical function for Ser-282 in cardiac Myosin binding protein-C phosphorylation and cardiac function.

RATIONALE: Cardiac myosin-binding protein-C (cMyBP-C) phosphorylation at Ser-273, Ser-282, and Ser-302 regulates myocardial contractility. In vitro and in vivo experiments suggest the nonequivalence of these sites and the potential importance of Ser-282 phosphorylation in modulating the protein's overall phosphorylation and myocardial function. OBJECTIVE: To determine whether complete cMyBP-C phosphorylation is dependent on Ser-282 phosphorylation and to define its role in myocardial function. We hypothesized that Ser-282 regulates Ser-302 phosphorylation and cardiac function during ß-adrenergic stimulation. METHODS AND RESULTS: Using recombinant human C1-M-C2 peptides in vitro, we determined that protein kinase A can phosphorylate Ser-273, Ser-282, and Ser-302. Protein kinase C can also phosphorylate Ser-273 and Ser-302. In contrast, Ca(2+)-calmodulin-activated kinase II targets Ser-302 but can also target Ser-282 at nonphysiological calcium concentrations. Strikingly, Ser-302 phosphorylation by Ca(2+)-calmodulin-activated kinase II was abolished by ablating the ability of Ser-282 to be phosphorylated via alanine substitution. To determine the functional roles of the sites in vivo, three transgenic lines, which expressed cMyBP-C containing either Ser-273-Ala-282-Ser-302 (cMyBP-C(SAS)), Ala-273-Asp-282-Ala-302 (cMyBP-C(ADA)), or Asp-273-Ala-282-Asp-302 (cMyBP-C(DAD)), were generated. Mutant protein was completely substituted for endogenous cMyBP-C by breeding each mouse line into a cMyBP-C null (t/t) background. Serine-to-alanine substitutions were used to ablate the abilities of the residues to be phosphorylated, whereas serine-to-aspartate substitutions were used to mimic the charged state conferred by phosphorylation. Compared to control nontransgenic mice, as well as transgenic mice expressing wild-type cMyBP-C, the transgenic cMyBP-C(SAS(t/t)), cMyBP-C(ADA(t/t)), and cMyBP-C(DAD(t/t)) mice showed no increases in morbidity and mortality and partially rescued the cMyBP-C((t/t)) phenotype. The loss of cMyBP-C phosphorylation at Ser-282 led to an altered ß-adrenergic response. In vivo hemodynamic studies revealed that contractility was unaffected but that cMyBP-C(SAS(t/t)) hearts showed decreased diastolic function at baseline. However, the normal increases in cardiac function (increased contractility/relaxation) as a result of infusion of ß-agonist was significantly decreased in all of the mutants, suggesting that competency for phosphorylation at multiple sites in cMyBP-C is a prerequisite for normal ß-adrenergic responsiveness. CONCLUSIONS: Ser-282 has a unique regulatory role in that its phosphorylation is critical for the subsequent phosphorylation of Ser-302. However, each residue plays a role in regulating the contractile response to ß-agonist stimulation.

Inhibition of miR-199a-3p in a murine hypertrophic cardiomyopathy (HCM) model attenuates fibrotic remodeling.

Background: Hypertrophic cardiomyopathy (HCM) is an autosomal dominant genetic disorder, characterized by cardiomyocyte hypertrophy, cardiomyocyte disarray and fibrosis, which has a prevalence of ∼1: 200-500 and predisposes individuals to heart failure and sudden death. The mechanisms through which diverse HCM-causing mutations cause cardiac dysfunction remain mostly unknown and their identification may reveal new therapeutic avenues. MicroRNAs (miRNAs) have emerged as critical regulators of gene expression and disease phenotype in various pathologies. We explored whether miRNAs could play a role in HCM pathogenesis and offer potential therapeutic targets. Methods and results: Using high-throughput miRNA expression profiling and qPCR analysis in two distinct mouse models of HCM, we found that miR-199a-3p expression levels are upregulated in mutant mice compared to age- and treatment-matched wild-type mice. We also found that miR-199a-3p expression is enriched in cardiac non-myocytes compared to cardiomyocytes. When we expressed miR-199a-3p mimic in cultured murine primary cardiac fibroblasts and analyzed the conditioned media by proteomics, we found that several extracellular matrix (ECM) proteins (e.g., TSP2, FBLN3, COL11A1, LYOX) were differentially secreted (data are available via ProteomeXchange with identifier PXD042904). We confirmed our proteomics findings by qPCR analysis of selected mRNAs and demonstrated that miR-199a-3p mimic expression in cardiac fibroblasts drives upregulation of ECM gene expression, including Tsp2, Fbln3, Pcoc1, Col1a1 and Col3a1. To examine the role of miR-199a-3p in vivo, we inhibited its function using lock-nucleic acid (LNA)-based inhibitors (antimiR-199a-3p) in an HCM mouse model. Our results revealed that progression of cardiac fibrosis is attenuated when miR-199a-3p function is inhibited in mild-to-moderate HCM. Finally, guided by computational target prediction algorithms, we identified mRNAs Cd151 and Itga3 as direct targets of miR-199a-3p and have shown that miR-199a-3p mimic expression negatively regulates AKT activation in cardiac fibroblasts. Conclusions: Altogether, our results suggest that miR-199a-3p may contribute to cardiac fibrosis in HCM through its actions in cardiac fibroblasts. Thus, inhibition of miR-199a-3p in mild-to-moderate HCM may offer therapeutic benefit in combination with complementary approaches that target the primary defect in cardiac myocytes.

Increased myofilament Ca2+ sensitivity and diastolic dysfunction as early consequences of Mybpc3 mutation in heterozygous knock-in mice.

Hypertrophic cardiomyopathy (HCM) is frequently caused by mutations in MYBPC3 encoding cardiac myosin-binding protein C (cMyBP-C). The mechanisms leading from gene mutations to the HCM phenotype remain incompletely understood, partially because current mouse models of HCM do not faithfully reflect the human situation and early hypertrophy confounds the interpretation of functional alterations. The goal of this study was to evaluate whether myofilament Ca(2+) sensitization and diastolic dysfunction are associated or precede the development of left ventricular hypertrophy (LVH) in HCM. We evaluated the function of skinned and intact cardiac myocytes, as well as the intact heart in a recently developed Mybpc3-targeted knock-in mouse model carrying a point mutation frequently associated with HCM. Compared to wild-type, 10-week old homozygous knock-in mice exhibited i) higher myofilament Ca(2+) sensitivity in skinned ventricular trabeculae, ii) lower diastolic sarcomere length, and faster Ca(2+) transient decay in intact myocytes, and iii) LVH, reduced fractional shortening, lower E/A and E'/A', and higher E/E' ratios by echocardiography and Doppler analysis, suggesting systolic and diastolic dysfunction. In contrast, heterozygous knock-in mice, which mimic the human HCM situation, did not exhibit LVH or systolic dysfunction, but exhibited higher myofilament Ca(2+) sensitivity, faster Ca(2+) transient decay, and diastolic dysfunction. These data demonstrate that myofilament Ca(2+) sensitization and diastolic dysfunction are early phenotypic consequences of Mybpc3 mutations independent of LVH. The accelerated Ca(2+) transients point to compensatory mechanisms directed towards normalization of relaxation. We propose that HCM is a model for diastolic heart failure and this mouse model could be valuable in studying mechanisms and treatment modalities.

Novel role for p90 ribosomal S6 kinase in the regulation of cardiac myofilament phosphorylation.

In myocardium, the 90-kDa ribosomal S6 kinase (RSK) is activated by diverse stimuli and regulates the sarcolemmal Na(+)/H(+) exchanger through direct phosphorylation. Only limited information is available on other cardiac RSK substrates and functions. We evaluated cardiac myosin-binding protein C (cMyBP-C), a sarcomeric regulatory phosphoprotein, as a potential RSK substrate. In rat ventricular myocytes, RSK activation by endothelin 1 (ET1) increased cMyBP-C phosphorylation at Ser(282), which was inhibited by the selective RSK inhibitor D1870. Neither ET1 nor D1870 affected the phosphorylation status of Ser(273) or Ser(302), cMyBP-C residues additionally targeted by cAMP-dependent protein kinase (PKA). Complementary genetic gain- and loss-of-function experiments, through the adenoviral expression of wild-type or kinase-inactive RSK isoforms, confirmed RSK-mediated phosphorylation of cMyBP-C at Ser(282). Kinase assays utilizing as substrate wild-type or mutated (S273A, S282A, S302A) recombinant cMyBP-C fragments revealed direct and selective Ser(282) phosphorylation by RSK. Immunolabeling with a Ser(P)(282) antibody and confocal fluorescence microscopy showed RSK-mediated phosphorylation of cMyBP-C across the C-zones of sarcomeric A-bands. In chemically permeabilized mouse ventricular muscles, active RSK again induced selective Ser(282) phosphorylation in cMyBP-C, accompanied by significant reduction in Ca(2+) sensitivity of force development and significant acceleration of cross-bridge cycle kinetics, independently of troponin I phosphorylation at Ser(22)/Ser(23). The magnitudes of these RSK-induced changes were comparable with those induced by PKA, which phosphorylated cMyBP-C additionally at Ser(273) and Ser(302). We conclude that Ser(282) in cMyBP-C is a novel cardiac RSK substrate and its selective phosphorylation appears to regulate cardiac myofilament function.

cMyBP-C as a promiscuous substrate: phosphorylation by non-PKA kinases and its potential significance.

It is now generally accepted that phosphorylation of cMyBP-C is critically important in maintaining normal cardiac function. Although much of the work to date on phospho-regulation of cMyBP-C has focused on the role of protein kinase A (PKA, also known as cAMP-dependent protein kinase), recent evidence suggests that a number of non-PKA serine/threonine kinases, such as Ca(2+)/calmodulin-dependent protein kinase II, protein kinase C, protein kinase D and the 90-kDa ribosomal S6 kinase are also capable of targeting this key regulatory sarcomeric protein. This article reviews such evidence and proposes a hypothetical role for some of the pertinent signalling pathways in phospho-regulation of cMyBP-C in the setting of heart failure.

Proteomics analysis of the cardiac myofilament subproteome reveals dynamic alterations in phosphatase subunit distribution.

Myofilament proteins are responsible for cardiac contraction. The myofilament subproteome, however, has not been comprehensively analyzed thus far. In the present study, cardiomyocytes were isolated from rodent hearts and stimulated with endothelin-1 and isoproterenol, potent inducers of myofilament protein phosphorylation. Subsequently, cardiomyocytes were "skinned," and the myofilament subproteome was analyzed using a high mass accuracy ion trap tandem mass spectrometer (LTQ Orbitrap XL) equipped with electron transfer dissociation. As expected, a small number of myofilament proteins constituted the majority of the total protein mass with several known phosphorylation sites confirmed by electron transfer dissociation. More than 600 additional proteins were identified in the cardiac myofilament subproteome, including kinases and phosphatase subunits. The proteomic comparison of myofilaments from control and treated cardiomyocytes suggested that isoproterenol treatment altered the subcellular localization of protein phosphatase 2A regulatory subunit B56alpha. Immunoblot analysis of myocyte fractions confirmed that beta-adrenergic stimulation by isoproterenol decreased the B56alpha content of the myofilament fraction in the absence of significant changes for the myosin phosphatase target subunit isoforms 1 and 2 (MYPT1 and MYPT2). Furthermore, immunolabeling and confocal microscopy revealed the spatial redistribution of these proteins with a loss of B56alpha from Z-disc and M-band regions but increased association of MYPT1/2 with A-band regions of the sarcomere following beta-adrenergic stimulation. In summary, we present the first comprehensive proteomics data set of skinned cardiomyocytes and demonstrate the potential of proteomics to unravel dynamic changes in protein composition that may contribute to the neurohormonal regulation of myofilament contraction.

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 Ser(22)/Ser(23), reduces myofilament Ca(2+) sensitivity, and accelerates cross-bridge cycle kinetics. Whether PKD regulates cardiac myofilament function entirely through cTnI phosphorylation at Ser(22)/Ser(23) remains to be established. To determine the role of cTnI phosphorylation at Ser(22)/Ser(23) in PKD-mediated regulation of cardiac myofilament function, we used transgenic mice that express cTnI in which Ser(22)/Ser(23) are substituted by nonphosphorylatable Ala (cTnI-Ala(2)). In skinned myocardium from wild-type (WT) mice, PKD increased cTnI phosphorylation at Ser(22)/Ser(23) and decreased the Ca(2+) sensitivity of force. In contrast, PKD had no effect on the Ca(2+) sensitivity of force in myocardium from cTnI-Ala(2) mice, in which Ser(22)/Ser(23) were unavailable for phosphorylation. Surprisingly, PKD accelerated cross-bridge cycle kinetics similarly in myocardium from WT and cTnI-Ala(2) 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 Ser(273), Ser(282), and Ser(302), and revealed that PKD phosphorylates only Ser(302). Furthermore, PKD phosphorylated Ser(302) selectively and to a similar extent in native cMyBP-C of skinned myocardium from WT and cTnI-Ala(2) mice, and this phosphorylation occurred throughout the C-zones of sarcomeric A-bands. In conclusion, PKD reduces myofilament Ca(2+) sensitivity through cTnI phosphorylation at Ser(22)/Ser(23) but accelerates cross-bridge cycle kinetics by a distinct mechanism. PKD phosphorylates cMyBP-C at Ser(302), which may mediate the latter effect.

A chemical genetic approach reveals that p38alpha MAPK activation by diphosphorylation aggravates myocardial infarction and is prevented by the direct binding of SB203580.

The use of nonselective pharmacological inhibitors has resulted in controversy regarding the mechanism and consequences of p38 activation during myocardial infarction. Classic p38 inhibitors such as SB203580 rely on a critical "gatekeeper" threonine residue for binding. We addressed these controversies by using mice in which the p38alpha alleles were targeted to cause substitution of the gatekeeper residue and resistance to inhibition. In homozygous drug-resistant compared with wild-type hearts, SB203580 failed to inhibit the activating phosphorylation of p38 or to reduce the infarction caused by myocardial ischemia. However, BIRB796, a p38 inhibitor not reliant on the gatekeeper for binding, similarly reduced p38-activating phosphorylation and infarction in both wild-type and knock-in mice, thereby excluding a nonspecific inhibitor-dependent phenotype resulting from the targeting strategy. Furthermore, the activation during myocardial ischemia involved phosphorylation of both the threonine and tyrosine residues in the activation loop of p38 despite the phosphorylation of the threonine alone being sufficient to create the epitope for dual phosphospecific antibody binding. Finally, SB203580 failed to reduce infarction in heterozygous drug-resistant hearts, suggesting that near complete inhibition of p38alpha kinase activity is necessary to elicit protection. These results indicate that, during myocardial ischemia, p38alpha (i) is the dominant-active p38 isoform, (ii) contributes to infarction, (iii) is responsible for the cardioprotective effect of SB203580, and (iv) is activated by a mechanism consistent with autodiphosphorylation despite this necessitating the phosphorylation of a tyrosine residue by an archetypal serine/threonine kinase.

Regulation by phosphodiesterase isoforms of protein kinase A-mediated attenuation of myocardial protein kinase D activation.

Protein kinase D (PKD) targets several proteins in the heart, including cardiac troponin I (cTnI) and class II histone deacetylases, and regulates cardiac contraction and hypertrophy. In adult rat ventricular myocytes (ARVM), PKD activation by endothelin-1 (ET1) occurs via protein kinase Cε and is attenuated by cAMP-dependent protein kinase (PKA). Intracellular compartmentalisation of cAMP, arising from localised activity of distinct cyclic nucleotide phosphodiesterase (PDE) isoforms, may result in spatially constrained regulation of the PKA activity that inhibits PKD activation. We have investigated the roles of the predominant cardiac PDE isoforms, PDE2, PDE3 and PDE4, in PKA-mediated inhibition of PKD activation. Pretreatment of ARVM with the non-selective PDE inhibitor isobutylmethylxanthine (IBMX) attenuated subsequent PKD activation by ET1. However, selective inhibition of PDE2 [by erythro-9-(2-hydroxy-3-nonyl) adenine, EHNA], PDE3 (by cilostamide) or PDE4 (by rolipram) individually had no effect on ET1-induced PKD activation. Selective inhibition of individual PDE isoforms also had no effect on the phosphorylation status of the established cardiac PKA substrates phospholamban (PLB; at Ser16) and cTnI (at Ser22/23), which increased markedly with IBMX. Combined administration of cilostamide and rolipram, like IBMX alone, attenuated ET1-induced PKD activation and increased PLB and cTnI phosphorylation, while combined administration of EHNA and cilostamide or EHNA and rolipram was ineffective. Thus, cAMP pools controlled by PDE3 and PDE4, but not PDE2, regulate the PKA activity that inhibits ET1-induced PKD activation. Furthermore, PDE3 and PDE4 play redundant roles in this process, such that inhibition of both isoforms is required to achieve PKA-mediated attenuation of PKD activation.