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.

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.

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.

Mesenchymal stromal cells are present in the heart and promote growth of adult stem cells in vitro.

BACKGROUND AIMS: For many years the human heart has been considered a terminally differentiated organ with no regenerative potential after injury. Recent studies, however, have cast doubt on this long-standing dogma. The objective of this study was to investigate the presence of and characterize mesenchymal stromal cells (MSC) in the adult mouse heart. The impact of MSC on growth and differentiation of adult cardiac stem cells (CSC) was also analyzed. METHODS: A combination of lineage-negative/c-kit-negative (Lin(-)/c-kit(-)) immunoselection with a plastic-adhesion technique was used to isolate cardiac-derived MSC. The differentiation capacity and expression of surface markers were analyzed. To investigate the impact of MSC on growth and differentiation of adult CSC, Green Fluorescent Protein (GFP(+)) adult CSC were co-cultured with GFP(-) cardiac-derived MSC. RESULTS: MSC were present in the adult mouse heart and they met the criteria established to define mouse MSC. They expressed surface markers and were able to differentiate, in a controlled manner, into multiple lineages. In addition, cardiac-derived MSC promoted the survival and expansion of adult CSC in vitro. CONCLUSIONS: MSC can be isolated from the mouse heart and they promote growth and differentiation of adult CSC. The findings from this study could have a significant beneficial impact on future heart failure treatment. Co-culture and co-implantation of cardiac-derived MSC with adult CSC could provide extensive cardiac regeneration and maintenance of the CSC population after implanted into the heart.

Role of arachidonic acid, lipoxygenase, and mitochondrial depolarization in reperfusion arrhythmias.

We have sought evidence that arachidonic acid (AA) induces mitochondrial depolarization in isolated myocytes by a lipoxygenase (LOX)-dependent mechanism and that such depolarization might contribute to arrhythmogenesis following ischemia-reperfusion injury. A method was developed for measuring mitochondrial depolarization in isolated adult rat myocytes in suspension, using tetramethylrhodamine ethyl ester. The addition of AA to myocytes resulted in mitochondrial depolarization that was inhibited by the LOX inhibitor baicalein, by the reactive oxygen species (ROS) scavenger mercaptoproprionylglycine, and by the anion channel inhibitor diisothiocyanatostilbene-disulfonic acid (DIDS). AA induced mitochondrial uncoupling and mitochondrial ATPase activity in myocytes, but both were insensitive to baicalein. We conclude that the metabolic effect of AA in myocytes puts mitochondria into an energetically compromised state where membrane potential is easily changed by the DIDS-sensitive LOX/ROS-mediated opening of an inner membrane anion channel. In an in vivo anesthetized rat model of coronary artery occlusion, baicalein was found to strongly inhibit arrhythmias induced by ischemia-reperfusion injury. Arrhythmias following ischemia-reperfusion injury have been previously associated with DIDS-sensitive ROS-mediated mitochondrial depolarization, and free fatty acids including AA were previously found to accumulate during such injury. We therefore conclude that arrhythmias following ischemia-reperfusion injury might originate from mitochondrial depolarization mediated by LOX and AA.

Protein kinase d in the cardiovascular system: emerging roles in health and disease.

The protein kinase D (PKD) family is a recent addition to the calcium/calmodulin-dependent protein kinase group of serine/threonine kinases, within the protein kinase complement of the mammalian genome. Relative to their alphabetically superior cousins in the AGC group of kinases, namely the various isoforms of protein kinase A, protein kinase B/Akt, and protein kinase C, PKD family members have to date received limited attention from cardiovascular investigators. Nevertheless, increasing evidence now points toward important roles for PKD-mediated signaling pathways in the cardiovascular system, particularly in the regulation of myocardial contraction, hypertrophy and remodeling. This review provides a primer on PKD signaling, using information gained from studies in multiple cell types, and discusses recent data that suggest novel functions for PKD-mediated pathways in the heart and the circulation.

Ca2+/calmodulin-dependent protein kinase IIdelta and protein kinase D overexpression reinforce the histone deacetylase 5 redistribution in heart failure.

Cardiac hypertrophy and heart failure (HF) are associated with reactivation of fetal cardiac genes, and class II histone deacetylases (HDACs) (eg, HDAC5) have been strongly implicated in this process. We have shown previously that inositol trisphosphate, Ca2+/calmodulin-dependent protein kinase II (CaMKII), and protein kinase (PK)D are involved in HDAC5 phosphorylation and nuclear export in normal adult ventricular myocytes and also that CaMKIIdelta and inositol trisphosphate receptors are upregulated in HF. Here we tested whether, in our rabbit HF model, nucleocytoplasmic shuttling of HDAC5 was altered either at baseline or in response to endothelin-1, which would indicate HDAC5 phosphorylation and transcription effects. The fusion protein HDAC5-green fluorescent protein (HDAC5-GFP) was more cytosolic in HF myocytes (F(nuc)/F(cyto) 3.3+/-0.3 vs 7.2+/-0.4 in control), and HDAC5 was more phosphorylated. Despite this baseline cytosolic HDAC5 shift, endothelin-1 produced more rapid HDAC5-GFP nuclear export in HF versus control myocytes. We also find that PKD and CaMKIIdelta(C) expression and activation state are increased in both rabbit and human HF. Inhibition of either CaMKII or PKD in HF myocytes partially restored the HDAC5-GFP F(nuc)/F(cyto) toward control, and simultaneous inhibition restored F(nuc)/F(cyto) to that in control myocytes. Moreover, adenovirus-mediated overexpression of PKD, CaMKIIdelta(B), or CaMKIIdelta(C) reduced baseline HDAC5 F(nuc)/F(cyto) in control myocytes (3.4+/-0.5, 3.8+/-0.5, and 5.2+/-0.5, respectively), approaching that seen in HF. We conclude that chronic upregulation and activation of inositol trisphosphate receptors, CaMKII, and PKD in HF shifts HDAC5 out of the nucleus, derepressing transcription of hypertrophic genes. This may directly contribute to the development and/or maintenance of HF.

Are Active Video Games Effective at Eliciting Moderate-Intensity Physical Activity in Children, and Do They Enjoy Playing Them?

BACKGROUND: Despite current physical activity (PA) guidelines, children spend an average of 1-3 hours/day playing video games. Some video games offer physically active components as part of gameplay. We sought to determine if these active video games (AVGs) can elicit at least moderate PA in children, identify game elements important for PA, and determine if they are fun to play. METHODS: Twenty children aged 8 to 16 years underwent cardiopulmonary exercise testing to determine their heart rate (HR) at ventilatory threshold. Participants played 2 different AVGs, and the gaming time that each participant's HR was above the HR thresholds for moderate and vigorous PA was determined. Gameplay elements that supported or inhibited active gameplay were also identified. Participants also completed questionnaires on physical activity, game engagement, and game experience. RESULTS: The Dance Central Spotlight and Kung-Fu for Kinect AVGs produced at least moderate PA, for a mean of 54.3% ± 29.5% and 87.8% ± 21.8% of gameplay time, respectively. Full-body movements, player autonomy, and self-efficacy were observed to be important elements of good AVG design. Although participants enjoyed these AVGs, they still preferred their favorite games (game engagement score of 1.82 ± 0.67 vs 0.95 ± 0.70 [Dance Central Spotlight] and 1.39 ± 0.37 [Kung Fu for Kinect]). CONCLUSIONS: AVGs can provide at least moderate PA and are enjoyable to play, but most popular video games do not incorporate active components. The implementation of government policies and a rating system concerning PA in video games may help address the widespread sedentary lifestyle of children.

CONTEXTE: Malgré les lignes directrices actuelles sur l'activité physique, les enfants passent en moyenne entre une et trois heures par jour à jouer à des jeux vidéo. Comme la jouabilité de certains jeux vidéo comporte des activités physiques, nous avons cherché à déterminer si les jeux vidéo dynamiques (JVD) pouvaient permettre aux enfants d'atteindre un degré d'activité physique au moins modéré, à cerner les éléments de jeu qui sont importants pour l'activité physique et à déterminer si ces jeux étaient amusants. MÉTHODOLOGIE: Nous avons soumis 20 enfants de 8 à 16 ans à des épreuves d'effort cardiopulmonaire pour déterminer leur fréquence cardiaque (FC) au seuil ventilatoire. Les participants ont joué à deux JVD différents et nous avons déterminé la durée pendant laquelle la FC de chaque participant était supérieure aux seuils de FC correspondant à une activité physique modérée et intense. Nous avons aussi cerné les éléments de la jouabilité qui favorisaient ou empêchaient la jouabilité dynamique. Les participants ont en outre rempli des questionnaires sur l'activité physique, l'intérêt des jeux et l'expérience de jeu. RÉSULTATS: Deux JVD, Dance Central Spotlight et Kung-Fu for Kinect, ont produit un degré d'activité physique au moins modéré pendant respectivement 54,3 % ± 29,5 % et 87,8 % ± 21,8 % de la durée de jouabilité. Nous avons constaté que les mouvements du corps entier, l'autonomie des joueurs et l'auto-efficacité étaient des éléments importants de la bonne conception d'un JVD. Les participants ont aimé ces JVD, mais ils préféraient toujours leurs jeux favoris (score d'intérêt à l'égard du jeu de 1,82 ± 0,67 vs 0,95 ± 0,70 [Dance Central Spotlight] et 1,39 ± 0,37 [Kung Fu for Kinect]). CONCLUSIONS: Les JVD semblent produire un degré d'activité physique au moins modéré et sont amusants, mais la plupart des jeux vidéo populaires ne font pas faire d'activité physique. La mise en œuvre de politiques gouvernementales et l'adoption d'un système de cotation concernant l'activité physique associée aux jeux vidéo pourraient permettre de s'attaquer au problème répandu que représente la sédentarité chez les enfants.

Protein kinase D selectively targets cardiac troponin I and regulates myofilament Ca2+ sensitivity in ventricular myocytes.

Protein kinase D (PKD) is a serine/threonine kinase with emerging myocardial functions; in skinned adult rat ventricular myocytes (ARVMs), recombinant PKD catalytic domain phosphorylates cardiac troponin I at Ser22/Ser23 and reduces myofilament Ca(2+) sensitivity. We used adenoviral gene transfer to determine the effects of full-length PKD on protein phosphorylation, sarcomere shortening and [Ca(2+)](i) transients in intact ARVMs. In myocytes transduced to express wild-type PKD, the heterologously expressed enzyme was activated by endothelin 1 (ET1) (5 nmol/L), as reflected by PKD phosphorylation at Ser744/Ser748 (PKC phosphorylation sites) and Ser916 (autophosphorylation site). The ET1-induced increase in cellular PKD activity was accompanied by increased cardiac troponin I phosphorylation at Ser22/Ser23; this measured approximately 60% of that induced by isoproterenol (10 nmol/L), which activates cAMP-dependent protein kinase (PKA) but not PKD. Phosphorylation of other PKA targets, such as phospholamban at Ser16, phospholemman at Ser68 and cardiac myosin-binding protein C at Ser282, was unaltered. Furthermore, heterologous PKD expression had no effect on isoproterenol-induced phosphorylation of these proteins, or on isoproterenol-induced increases in sarcomere shortening and relaxation rate and [Ca(2+)](i) transient amplitude. In contrast, heterologous PKD expression suppressed the positive inotropic effect of ET1 seen in control cells, without altering ET1-induced increases in relaxation rate and [Ca(2+)](i) transient amplitude. Complementary experiments in "skinned" myocytes confirmed reduced myofilament Ca(2+) sensitivity by ET1-induced activation of heterologously expressed PKD. We conclude that increased myocardial PKD activity induces cardiac troponin I phosphorylation at Ser22/Ser23 and reduces myofilament Ca(2+) sensitivity, suggesting that altered PKD activity in disease may impact on contractile function.

Structure-function relationships of Ca spark activity in normal and failing cardiac myocytes as revealed by flash photography.

We describe the two-dimensional imaging of excitation-induced Ca gradients in isolated myocytes under physiological conditions, using a novel method of flash photography of fluo-3 fluorescence. This method is useful for showing the spatial distribution and reproducibility of rapidly triggered Ca release events, and their relationship to underlying structures. In normal rat myocytes, Ca sparks were evident 6ms after stimulation emerging from around t-tubules, as judged by co-localization with di-8-ANEPPS staining. Gaps in the spark pattern coincided with gaps in di-8-ANEPPS staining. Vacuolar fluo-3 uptake, previously identified as lysosomal, was prominent in some of the gaps, suggesting possible areas of t-tubule turnover. In normal dog myocytes, the beat-to-beat variance of Ca sparks was very low, t-tubular voids were small, and Ca gradients resolved rapidly. In myocytes from dogs with failure induced by rapid pacing, a reduced Ca transient was observed associated with increased areas that were void of sparks and t-tubules, and a greater beat-to-beat spark variance. These abnormalities resulted in a non-uniform spatial distribution of sparks, leading to Ca gradients across the cell that persisted for longer times after stimulation. Such Ca gradients could cause heterogeneous contraction and contribute to contractile failure.

Crosstalk of beta-adrenergic receptor subtypes through Gi blunts beta-adrenergic stimulation of L-type Ca2+ channels in canine heart failure.

The mechanisms underlying the blunted contractile response to beta-adrenergic receptor (beta-AR) stimulation in heart failure (HF) are incompletely understood, especially with regard to beta-AR subtype-specific regulation of L-type Ca2+ channels. We evaluated the impact of HF induced by pacing tachycardia on beta-AR regulation of L-type Ca2+ channels in a canine model. To evaluate changes in the relative subcellular distribution of beta-AR subtypes, left ventricular membranes enriched in surface sarcolemma and T-tubular sarcolemma were prepared. Radioligand binding using [(125)I]cyanopindolol revealed that HF resulted in a comparable decrease in the density of beta1-ARs in both surface and T-tubule sarcolemma (55+/-4%, n=7, P<0.001; and 45+/-10%, n=7, P<0.01, respectively), but no significant change in beta2-AR density was observed. Whole-cell patch clamp studies demonstrated a markedly blunted increase in I(Ca,L) in response to saturating concentrations of the nonselective beta-AR agonist isoproterenol (0.1 micromol/L) in failing myocytes compared with control (129+/-20%, n=11, versus 332+/-35%, n=7; P<0.001). Experiments testing beta1-AR- and beta2-AR-selective stimulation showed that the major component of the blunted response to nonselective beta-AR stimulation in HF was caused by beta2-AR activation, resulting in a pertussis toxin-sensitive, Gi-mediated inhibition of the beta1-AR-induced increase in I(Ca,L). In conclusion, canine HF results in the following: (1) a uniform reduction in beta1-AR density in surface and T-tubule membrane fractions without a change in beta2-AR density; and (2) the emergence of distinct Gi-coupling to beta2-ARs resulting in accentuated antagonism of beta1-AR-mediated stimulation of I(Ca,L). These results have implications for optimizing the use of beta-AR drugs in HF.

Ras triggers acidosis-induced activation of the extracellular-signal-regulated kinase pathway in cardiac myocytes.

In cardiac myocytes, sustained (3 min) intracellular acidosis activates the ERK1/2 (extracellular-signal-regulated kinase 1/2) pathway and, through this pathway, increases sarcolemmal NHE (Na+/H+ exchanger) activity [Haworth, McCann, Snabaitis, Roberts and Avkiran (2003) J. Biol. Chem. 278, 31676-31684]. In the present study, we aimed to determine the time-dependence, pH-dependence and upstream signalling mechanisms of acidosis-induced ERK1/2 activation in ARVM (adult rat ventricular myocytes). Cultured ARVM were subjected to intracellular acidosis for up to 20 min by exposure to NH4Cl, followed by washout with a bicarbonate-free Tyrode solution containing the NHE1 inhibitor cariporide. After the desired duration of intracellular acidosis, the phosphorylation status of ERK1/2 and its downstream effector p90(RSK) (90 kDa ribosomal S6 kinase) were determined by Western blotting. This revealed a time-dependent transient phosphorylation of both ERK1/2 and p90(RSK) by intracellular acidosis (intracellular pH approximately 6.6), with maximum activation occurring at 3 min and a return to basal levels by 20 min. When the degree of intracellular acidosis was varied from approximately 6.8 to approximately 6.5, maximum ERK1/2 phosphorylation was observed at an intracellular pH of 6.64. Inhibition of MEK1/2 [MAPK (mitogen-activated protein kinase)/ERK kinase 1/2) by pre-treatment of ARVM with U0126 or adenoviral expression of dominant-negative D208A-MEK1 protein prevented the phosphorylation of ERK1/2 by sustained intracellular acidosis, as did inhibition of Raf-1 with GW 5074 or ZM 336372. Interference with Ras signalling by the adenoviral expression of dominant-negative N17-Ras protein or with FPT III (farnesyl protein transferase inhibitor III) also prevented acidosis-induced ERK1/2 phosphorylation, whereas inhibiting G-protein signalling [by adenoviral expression of RGS4 or Lsc, the RGS domain of p115 RhoGEF (guanine nucleotide-exchange factor)] or protein kinase C (with bisindolylmaleimide I) had no effect. Our data show that, in ARVM, sustained intracellular acidosis activates ERK1/2 through proximal activation of the classical Ras/Raf/MEK pathway.

Increased nitration of sarcoplasmic reticulum Ca2+-ATPase in human heart failure.

BACKGROUND: Reduced sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a isoform) activity is a major determinant of reduced contractility in heart failure. Ca2+-ATPase inactivation can occur through SERCA2a nitration. We therefore investigated the role of SERCA2a nitration in heart failure. METHODS AND RESULTS: We measured SERCA2a levels and nitrotyrosine levels in tissue from normal and failing human hearts using Western blots. We found that nitrotyrosine levels in idiopathic dilated cardiomyopathic (DCM) hearts were almost double those of control hearts in age-matched groups. Nitrotyrosine was dominantly present in a single protein with the molecular weight of SERCA2a, and immunoprecipitation confirmed that the protein recognized by the nitrotyrosine antibody was SERCA2a. There was a positive correlation between the time to half relaxation and the nitrotyrosine/SERCA2a content (P<0.01) in myocytes isolated from control and DCM hearts. In experiments with isolated SR vesicles from porcine hearts, we also showed that the Ca pump is inactivated by peroxynitrite exposure, and inactivation was prevented by protein kinase A pretreatment. CONCLUSIONS: We conclude that SERCA2a inactivation by nitration may contribute to Ca pump failure and hence heart failure in DCM.

Protein kinase D is a novel mediator of cardiac troponin I phosphorylation and regulates myofilament function.

Protein kinase D (PKD) is a serine kinase whose myocardial substrates are unknown. Yeast 2-hybrid screening of a human cardiac library, using the PKD catalytic domain as bait, identified cardiac troponin I (cTnI), myosin-binding protein C (cMyBP-C), and telethonin as PKD-interacting proteins. In vitro phosphorylation assays revealed PKD-mediated phosphorylation of cTnI, cMyBP-C, and telethonin, as well as myomesin. Peptide mass fingerprint analysis of cTnI by liquid chromatography-coupled mass spectrometry indicated PKD-mediated phosphorylation of a peptide containing Ser22 and Ser23, the protein kinase A (PKA) targets. Ser22 and Ser23 were replaced by Ala, either singly (Ser22Ala or Ser23Ala) or jointly (Ser22/23Ala), and the troponin complex reconstituted in vitro, using wild-type or mutated cTnI together with wild-type cardiac troponin C and troponin T. PKD-mediated cTnI phosphorylation was reduced in complexes containing Ser22Ala or Ser23Ala cTnI and completely abolished in the complex containing Ser22/23Ala cTnI, indicating that Ser22 and Ser23 are both targeted by PKD. Furthermore, troponin complex containing wild-type cTnI was phosphorylated with similar kinetics and stoichiometry (approximately 2 mol phosphate/mol cTnI) by both PKD and PKA. To determine the functional impact of PKD-mediated phosphorylation, Ca2+ sensitivity of tension development was studied in a rat skinned ventricular myocyte preparation. PKD-mediated phosphorylation did not affect maximal tension but produced a significant rightward shift of the tension-pCa relationship, indicating reduced myofilament Ca2+ sensitivity. At submaximal Ca2+ activation, PKD-mediated phosphorylation also accelerated isometric crossbridge cycling kinetics. Our data suggest that PKD is a novel mediator of cTnI phosphorylation at the PKA sites and may contribute to the regulation of myofilament function.

Abnormal Ca2+ release, but normal ryanodine receptors, in canine and human heart failure.

Sarcoplasmic reticulum (SR) Ca2+ transport proteins, especially ryanodine receptors (RyR) and their accessory protein FKBP12.6, have been implicated as major players in the pathogenesis of heart failure (HF), but their role remain controversial. We used the tachycardia-induced canine model of HF and human failing hearts to investigate the density and major functional properties of RyRs, SERCA2a, and phospholamban (PLB), the main proteins regulating SR Ca2+ transport. Intracellular Ca2+ is likely to play a role in the contractile dysfunction of HF because the amplitude and kinetics of the [Ca2+]i transient were reduced in HF. Ca2+ uptake assays showed 44+/-8% reduction of Vmax in canine HF, and Western blots demonstrated that this reduction was due to decreased SERCA2a and PLB levels. Human HF showed a 30+/-5% reduction in SERCA2a, but PLB was unchanged. RyRs from canine and human HF displayed no major structural or functional differences compared with control. The P(o) of RyRs was the same for control and HF over the range of pCa 7 to 4. Subconductance states, which predominate in FKBP12.6-stripped RyRs, were equally frequent in control and HF channels. An antibody that recognizes phosphorylated RyRs yields equal intensity for control and HF channels. Further, phosphorylation of RyRs by PKA did not appear to change the RyR/FKBP12.6 association, suggesting minor beta-adrenergic stimulation of Ca2+ release through this mechanism. These results support a role for SR in the pathogenesis of HF, with abnormal Ca2+ uptake, more than Ca2+ release, contributing to the depressed and slow Ca2+ transient characteristic of HF.

Effects of bisindolylmaleimide PKC inhibitors on p90RSK activity in vitro and in adult ventricular myocytes.

1 Bisindolylmaleimide inhibitors of protein kinase C (PKC), such as GF109203X and Ro31-8220, have been used to investigate the roles of PKC isoforms in many cellular processes in cardiac myocytes, but these agents may also inhibit p90RSK activity. 2 In in vitro kinase assays utilising 50 microM [ATP], GF109203X and Ro31-8220 inhibited p90RSK isoforms (IC50 values for inhibition of RSK1, RSK2 and RSK3, respectively, were 610, 310 and 120 nM for GF109203X, and 200, 36 and 5 nM for Ro31-8220) as well as classical and novel PKC isoforms (IC50 values for inhibition of PKCalpha and PKCepsilon, respectively, were 8 and 12 nM for GF109203X, and 4 and 8 nM for Ro31-8220). 3 At physiological [ATP] (5 mM), both GF109203X and Ro31-8220 exhibited reduced potency as inhibitors of RSK2, PKCalpha and PKCepsilon (IC50 values of 7400, 310 and 170 nM, respectively, for GF109203X, and 930, 150 and 140 nM, respectively, for Ro31-8220), with the latter agent retaining its relatively greater potency. 4 To determine the effects of GF109203X and Ro31-8220 on p90RSK activity in cultured adult rat ventricular myocytes (ARVM), phosphorylation of the eukaryotic elongation factor 2 kinase (eEF2K) at Ser366, a known p90RSK target, was used as the index of such activity. Adenoviral expression of a constitutively active form of mitogen-activated protein kinase (MAPK) or extracellular signal-regulated kinase (ERK) kinase 1 (MEK1) was used to induce PKC-independent p90RSK activation and downstream phosphorylation of eEF2K. 5 eEF2K phosphorylation was abolished by U0126 (1 microM), a selective inhibitor of MEK1, and was significantly reduced by GF109203X at > or =3 microM and by Ro31-8220 at > or =1 microM. At 1 microM, both agents inhibited PMA-induced PKC activity in ARVM. 6 These data show that GF109203X and Ro31-8220 inhibit various isoforms of PKC and p90RSK in vitro and in intact ARVM, with the former agent exhibiting relatively greater selectivity for PKC.

Regulatory effects of G protein-coupled receptors on cardiac sarcolemmal Na+/H+ exchanger activity: signalling and significance.

In cardiac myocytes, sarcolemmal Na+/H+ exchanger (NHE) activity is subject to regulation by a variety of G protein-coupled receptor (GPCR) systems. This regulation usually manifests as an increase in NHE activity (e.g. in response to the stimulation of alpha1-adrenergic, angiotensin AT1, endothelin and thrombin receptors), although some GPCR systems have been shown to inhibit sarcolemmal NHE activity (e.g. beta1-adrenergic receptors) or to attenuate its stimulation by other ligands (e.g. adenosine A1 and angiotensin AT2 receptors). The pertinent molecular signalling mechanisms are only now beginning to be unravelled, with the extracellular signal regulated kinase/ribosomal S6 kinase pathway and the protein kinase C pathway both appearing to play critical roles in the stimulation of sarcolemmal NHE activity. GPCR-mediated regulation of sarcolemmal NHE activity is likely to play significant roles in modulating myocardial function in both physiological and pathophysiological conditions. These roles include the regulation of (1) myocardial pH(i) and contractility, (2) myocardial susceptibility to injury and dysfunction during ischaemia and reperfusion, and (3) myocardial hypertrophy in response to neurohormonal and mechanical stimuli. Greater understanding of the pertinent molecular signalling mechanisms distal to GPCR stimulation may reveal novel targets for therapeutic manipulation.

Depletion of T-tubules and specific subcellular changes in sarcolemmal proteins in tachycardia-induced heart failure.

OBJECTIVE: The T-tubule membrane network is integrally involved in excitation-contraction coupling in ventricular myocytes. Ventricular myocytes from canine hearts with tachycardia-induced dilated cardiomyopathy exhibit a decrease in accessible T-tubules to the membrane-impermeant dye, di8-ANNEPs. The present study investigated the mechanism of loss of T-tubule staining and examined for changes in the subcellular distribution of membrane proteins essential for excitation-contraction coupling. METHODS: Isolated ventricular myocytes from canine hearts with and without tachycardia-induced heart failure were studied using fluorescence confocal microscopy and membrane fractionation techniques using a variety of markers specific for sarcolemmal and sarcoplasmic reticulum proteins. RESULTS: Probes for surface glycoproteins, Na/K ATPase, Na/Ca exchanger and Ca(v)1.2 demonstrated a prominent but heterogeneous reduction in T-tubule labeling in both intact and permeabilised failing myocytes, indicating a true depletion of T-tubules and associated membrane proteins. Membrane fractionation studies showed reductions in L-type Ca(2+) channels and beta-adrenergic receptors but increased levels of Na/Ca exchanger protein in both surface sarcolemma and T-tubular sarcolemma-enriched fractions; however, the membrane fraction enriched in junctional complexes of sarcolemma and junctional sarcoplasmic reticulum demonstrated no significant changes in the density of any sarcolemmal protein or sarcoplasmic reticulum protein assayed. CONCLUSION: Failing canine ventricular myocytes exhibit prominent depletion of T-tubules and changes in the density of a variety of proteins in both surface and T-tubular sarcolemma but with preservation of the protein composition of junctional complexes. This subcellular remodeling contributes to abnormal excitation-contraction coupling in heart failure.