The enzymatic activity of the type 2A protein phosphatase (PP2A) holoenzyme, a major serine/threonine phosphatase in the heart, is conferred by its catalytic subunit (PP2AC). PP2AC activity and subcellular localisation can be regulated by reversible carboxylmethylation of its C-terminal leucine309 (leu309) residue. Previous studies have shown that the stimulation of adenosine type 1 receptors (A1.Rs) induces PP2AC carboxylmethylation and altered subcellular distribution in adult rat ventricular myocytes (ARVM). In the current study, we show that the enzymatic components that regulate the carboxylmethylation status of PP2AC, leucine carboxylmethyltransferase-1 (LCMT-1) and phosphatase methylesterase-1 (PME-1) are abundantly expressed in, and almost entirely localised in the cytoplasm of ARVM. The stimulation of Gi-coupled A1.Rs with N6-cyclopentyladenosine (CPA), and of other Gi-coupled receptors such as muscarinic M2 receptors (stimulated with carbachol) and angiotensin II AT2 receptors (stimulated with CGP42112) in ARVM, induced PP2AC carboxylmethylation at leu309 in a concentration-dependent manner. Exposure of ARVM to 10 µM CPA increased the cellular association between PP2AC and its methyltransferase LCMT-1, but not its esterase PME-1. Stimulation of A1.Rs with 10 µM CPA increased the phosphorylation of protein kinase B at ser473, which was abolished by the PI3K inhibitor LY294002 (20 µM), thereby confirming that PI3K activity is upregulated in response to A1.R stimulation by CPA in ARVM. A1.R-induced PP2AC translocation to the particulate fraction was abrogated by adenoviral expression of the alpha subunit (Gαt1) coupled to the transducin G-protein coupled receptor. A similar inhibitory effect on A1.R-induced PP2AC translocation was also seen with LY294002 (20 µM). These data suggest that in ARVM, A1.R-induced PP2AC translocation to the particulate fraction occurs through a GiPCR-Gβγ-PI3K mediated intracellular signalling pathway, which may involve elevated PP2AC carboxylmethylation at leu309.
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.
Protein kinase D has multiple roles in cardiac myocytes, where its regulatory mechanisms remain incompletely defined. In the present study we identify four-and-a-half LIM domains proteins 1 and 2 as novel binding partners and regulators of protein kinase D in this cell type.
cardiac myocyte; four-and-a-half LIM (FHL); histone deacetylase; neurohormonal stimulation; protein kinase; signal transduction; ARVM, adult rat ventricular myocyte; BPKDi, bipyridyl PKD inhibitor; CaMK, Ca2+/calmodulin-dependent protein kinase; caPKD, constitutively active catalytic domain of PKD; cMyBP-C, cardiac myosin-binding protein C; CRM1, chromosome region maintenance 1; cTnI, inhibitory subunit of cardiac troponin; ERK, extracellular-signal-regulated kinase; ET1, endothelin 1; FHL, four-and-a-half LIM domains; HDAC, histone deacetylase; IVK, in vitro kinase; MEF2, myocyte enhancer factor 2; MOI, multiplicity of infection; MuRF, muscle RING finger; NRVM, neonatal rat ventricular myocyte; PE, phenylephrine; pfu, plaque-forming unit; PKC, protein kinase C; PKD, protein kinase D; TAC, transverse aortic constriction
Background: Telethonin mutations are associated with cardiomyopathy through unknown mechanisms.
Results: Telethonin is a substrate for CaMK family kinases and exists in a bis-phosphorylated state in cardiomyocytes, in which non-phosphorylated telethonin disrupts transverse tubule organization and intracellular calcium transients.
Conclusion: Telethonin phosphorylation is critical for the maintenance of normal cardiomyocyte morphology and calcium handling.
Significance: Disruption of phospho-telethonin functions may contribute to pathogenesis in cardiomyopathy.
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 Ca2+/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 Ca2+ 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 Ca2+ transients.
CaMKII; Cardiac Muscle; Cardiomyopathy; Excitation-Contraction Coupling; Protein Kinase D (PKD); Protein Phosphorylation
Multiple stimuli of physiological and pathophysiological significance, including α1-adrenoceptor agonists, stimulate the cardiac sarcolemmal Na+/H+ exchanger isoform 1 (NHE1) through activation of the mitogen-activated or extracellular signal-regulated kinase (ERK) kinase (MEK) ERK-90-kDa ribosomal S6 kinase (RSK) signaling cascade. However, the individual contributions of ERK and RSK, which can each phosphorylate the NHE1 regulatory domain, to such stimulation are unknown. In the present study, we have used the novel RSK inhibitor fmk to determine the role of RSK as a direct regulator of NHE1 phosphorylation and activity in response to α1-adrenergic stimulation, in ventricular myocytes isolated from the adult rat heart. Initial experiments confirmed that pretreatment of myocytes with fmk before exposure to the α1-adrenoceptor agonist phenylephrine inhibited RSK C-terminal kinase activity and thereby RSK N-terminal kinase activation, without affecting MEK or ERK activation. Pretreatment of myocytes with fmk also inhibited phenylephrine-induced increases in NHE1 phosphorylation and the rate of NHE1-mediated H+ efflux under conditions of intracellular acidosis. These findings reveal, for the first time to our knowledge, that RSK is the principal regulator of NHE1 phosphorylation and activity after α1-adrenergic stimulation in adult myocardium.
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.
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 β-adrenergic (β-AR) 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 Cε can also phosphorylate Ser-273 and Ser-302. In contrast, Ca2+-calmodulin-activated kinase II (CaMKII) targets Ser-302 but can also target Ser-282 at non-physiological calcium concentrations. Strikingly, Ser-302 phosphorylation by CaMKII was abolished by ablating Ser-282's ability to be phosphorylated via alanine substitution. To determine the sites’ functional roles in vivo, three transgenic lines, which expressed cMyBP-C containing either Ser-273-Ala-282-Ser-302 (cMyBP-CSAS), Ala-273-Asp-282-Ala-302 (cMyBP-CADA) or Asp-273-Ala-282-Asp-302 (cMyBP-CDAD), 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 residues’ abilities to be phosphorylated while serine to aspartate substitutions were used to mimic the charged state conferred by phosphorylation. Compared to control non-transgenic mice, as well as transgenic mice expressing wild-type cMyBP-C, the transgenic cMyBP-CSAS(t/t), cMyBP-CADA(t/t) and cMyBP-CDAD(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 β-adrenergic response. In vivo hemodynamic studies revealed that contractility was unaffected but that cMyBP-CSAS(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 β-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 β-adrenergic responsiveness.
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 β-agonist stimulation.
Myofilament Phosphorylation; Contractile Function; Myosin Binding Protein-C
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 Ca2+ 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 Ca2+ sensitivity in skinned ventricular trabeculae, ii) lower diastolic sarcomere length, and faster Ca2+ 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 Ca2+ sensitivity, faster Ca2+ transient decay, and diastolic dysfunction. These data demonstrate that myofilament Ca2+ sensitization and diastolic dysfunction are early phenotypic consequences of Mybpc3 mutations independent of LVH. The accelerated Ca2+ 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.
► Absence of left ventricular hypertrophy in heterozygous Mybpc3-targeted knock-in mice. ► Myofilament Ca2+ sensitization in heterozygous Mybpc3-targeted knock-in mice. ► Diastolic dysfunction independent of left ventricular hypertrophy. ► Hypertrophic cardiomyopathy as a model of diastolic heart failure.
cMyBP-C, cardiac myosin-binding protein C; cTnI, cardiac troponin I; CSQ, calsequestrin; HCM, hypertrophic cardiomyopathy; Het, heterozygous Mybpc3-targeted knock-in mice; KI, homozygous Mybpc3-targeted knock-in mice; KO, homozygous Mybpc3-targeted knock-out mice; LVH, left ventricular hypertrophy; max F, maximal Ca2+-activated force; MYBPC3, human cardiac myosin-binding protein C gene; Mybpc3, mouse cardiac myosin-binding protein C gene; NCX, Na+/Ca2+ exchanger; nH, Hill coefficient; pCa50, log of [Ca2+] required for 50% of maximal activation; PKA, cAMP-dependent protein kinase A; PLB, phospholamban; SERCA2, SR-Ca2+ ATPase; SL, sarcomere length; SR, sarcoplasmic reticulum; Ca2+ sensitivity; Ca2+ transient; Diastolic dysfunction; Hypertrophy; Mouse model
The Na+/H+ exchanger (NHE-1) plays a key role in pHi recovery from acidosis and is regulated by pHi and the ERK1/2-dependent phosphorylation pathway. Since acidosis increases the activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in cardiac muscle, we examined whether CaMKII activates the exchanger by using pharmacological tools and highly specific genetic approaches. Adult rat cardiomyocytes, loaded with the pHi indicator SNARF-1/AM were subjected to different protocols of intracellular acidosis. The rate of pHi recovery from the acid load (dpHi/dt), -an index of NHE-1 activity in HEPES buffer or in NaHCO3 buffer in the presence of inhibition of anion transporters-, was significantly decreased by the CaMKII-inhibitors KN-93 or AIP. pHi recovery from acidosis was faster in CaMKII-overexpressing myocytes than in overexpressing β-galactosidase myocytes, (dpHi/dt: 0.195±0.04 vs. 0.045±0.010 min-1 respectively, n=8), and slower in myocytes from transgenic mice with chronic cardiac CaMKII inhibition (AC3-I) than in controls (AC3-C). Inhibition of CaMKII and/or ERK1/2 indicated that stimulation of NHE-1 by CaMKII was independent of and additive to the ERK1/2 cascade. In vitro studies with fusion proteins containing wild-type or mutated (Ser/Ala) versions of the C-terminal domain of NHE-1, indicate that CaMKII phosphorylates NHE-1 at residues other than the canonical phosphorylation sites for the kinase (Ser648, Ser703 and Ser796). These results provide new mechanistic insights and unequivocally demonstrate a role of the already multifunctional CaMKII on the regulation of the NHE-1 activity. They also prove clinically important in multiple disorders which, like ischemia/reperfusion injury or hypertrophy, are associated with increased NHE-1 and CaMKII.
Hypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy, increased ventricular stiffness and impaired diastolic filling. We investigated to what extent myocardial functional defects can be explained by alterations in the passive and active properties of human cardiac myofibrils. Skinned ventricular myocytes were prepared from patients with obstructive HCM (two patients with MYBPC3 mutations, one with a MYH7 mutation, and three with no mutation in either gene) and from four donors. Passive stiffness, viscous properties, and titin isoform expression were similar in HCM myocytes and donor myocytes. Maximal Ca2+-activated force was much lower in HCM myocytes (14 ± 1 kN/m2) than in donor myocytes (23 ± 3 kN/m2; P < 0.01), though cross-bridge kinetics (ktr) during maximal Ca2+ activation were 10% faster in HCM myocytes. Myofibrillar Ca2+ sensitivity in HCM myocytes (pCa50 = 6.40 ± 0.05) was higher than for donor myocytes (pCa50 = 6.09 ± 0.02; P < 0.001) and was associated with reduced phosphorylation of troponin-I (ser-23/24) and MyBP-C (ser-282) in HCM myocytes. These characteristics were common to all six HCM patients and may therefore represent a secondary consequence of the known and unknown underlying genetic variants. Some HCM patients did however exhibit an altered relationship between force and cross-bridge kinetics at submaximal Ca2+ concentrations, which may reflect the primary mutation. We conclude that the passive viscoelastic properties of the myocytes are unlikely to account for the increased stiffness of the HCM ventricle. However, the low maximum Ca2+-activated force and high Ca2+ sensitivity of the myofilaments are likely to contribute substantially to any systolic and diastolic dysfunction, respectively, in hearts of HCM patients.
► The passive stiffness of skinned HCM cardiac myocytes was similar to that of normal (donor) myocytes. ► Maximum Ca-activated force production was reduced by 40% in HCM vs donor myocytes. ► This loss of force could contribute to systolic dysfunction in HCM hearts. ► Myofibrillar Ca sensitivity was higher in HCM than in donor myocytes. ► The enhanced Ca sensitivity could compensate for the smaller maximum force but would tend to cause diastolic dysfunction. ► These characteristics were common to all HCM patients studied, suggesting the changes were secondary consequence of the underlying genetic variants.
Hypertrophic cardiomyopathy; Skinned cardiac myocytes; Viscoelasticity; Ca2+ sensitivity; Cross-bridge kinetics
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.
Cardiac myocyte; PKD; Phosphodiesterase; PKA; Histone deacetylase
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 B56α. Immunoblot analysis of myocyte fractions confirmed that β-adrenergic stimulation by isoproterenol decreased the B56α 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 B56α from Z-disc and M-band regions but increased association of MYPT1/2 with A-band regions of the sarcomere following β-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.