Biochemical and myofilament responses of the right ventricle to severe pulmonary hypertension

Right ventricular (RV) failure is one of the strongest predictors of mortality both in the presence of left ventricular decompensation and in the context of pulmonary vascular disease. Despite this, there is a limited understanding of the biochemical and mechanical characteristics of the pressure-overloaded RV at the level of the cardiac myocyte. To better understand this, we studied ventricular muscle obtained from neonatal calves that were subjected to hypobaric atmospheric conditions, which result in profound pulmonary hypertension. We found that RV pressure overload resulted in significant changes in the phosphorylation of key contractile proteins. Total phosphorylation of troponin I was decreased with pressure overload, predominantly reflecting changes at the putative PKA site at Ser(22/23). Similarly, both troponin T and myosin light chain 2 showed a significant decline in phosphorylation. Desmin was unchanged, and myosin-binding protein C (MyBP-C) phosphorylation was apparently increased. However, the apparent increase in MyBP-C phosphorylation was not due to phosphorylation but rather to an increase in MyBP-C total protein. Importantly, these findings were seen in all regions of the RV and were paralleled by reduced Ca(2+) sensitivity with preserved maximal Ca(2+) saturated developed force normalized to cross-sectional area in isolated skinned right ventricular myocyte fragments. No changes in total force or cooperativity were seen. Taken together, these results suggest that RV failure is mechanistically unique from left ventricular failure.     

The significance of regulatory light chain phosphorylation in cardiac physiology

It has been over 35 years since the first identification of phosphorylation of myosin light chains in skeletal and cardiac muscle. Yet only in the past few years has the role of these phosphorylations in cardiac dynamics been more fully understood. Advances in this understanding have come about with further evidence on the control mechanisms regulating the level of phosphorylation by kinases and phosphatases. Moreover, studies clarifiying the role of light chain phosphorylation in short and long term control of cardiac contractility and as a factor in cardiac remodeling have improved our knowledge. Especially important in these advances has been the use of gain and loss of function approaches, which have not only testedthe role of kinases and phosphatases, but also the effects of loss of RLC phosphorylation sites. Major conclusions from these studies indicate that (i) two negatively-charged post-translational modifications occupy the ventricular RLC N-terminus, with mouse RLC being doubly phosphorylated (Ser 14/15), and human RLC being singly phosphorylated (Ser 15) and singly deamidated(Asn14/16 to Asp); (ii)a distinct cardiac myosin light kinase (cMLCK) and a unique myosin phosphatase targeting peptide (MYPT2) control phosphoryl group transfer;and (iii) ablation of RLC phosphorylationdecreases ventricular power, lengthens the duration of ventricular ejection, and may also modify other sarcomeric proteins (e.g., troponin I) as substrates for kinases and/or phosphatases. A long term effect of low levels of RLC phosphorylation in mouse models also involves remodeling of the heart with hypertrophy, depressed contractility, and sarcomeric disarray. Data demonstrating altered levels of RLC phosphorylation in comparisons of samples from normal and stressed human hearts indicate the significance of these findings in translational medicine.     

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.     

Mutations in beta-myosin S2 that cause familial hypertrophic cardiomyopathy (FHC) abolish the interaction with the regulatory domain of myosin-binding protein-C

The myosin filaments of striated muscle contain a family of enigmatic myosin-binding proteins (MyBP), MyBP-C and MyBP-H. These modular proteins of the intracellular immunoglobulin superfamily contain unique domains near their N termini. The N-terminal domain of cardiac MyBP-C, the MyBP-C motif, contains additional phosphorylation sites and may regulate contraction in a phosphorylation dependent way. In contrast to the C terminus, which binds to the light meromyosin portion of the myosin rod, the interactions of this domain are unknown. We demonstrate that fragments of MyBP-C containing the MyBP-C motif localise to the sarcomeric A-band in cardiomyocytes and isolated myofibrils, without affecting sarcomere structure. The binding site for the MyBP-C motif resides in the N-terminal 126 residues of the S2 segment of the myosin rod. In this region, several mutations in beta-myosin are associated with FHC; however, their molecular implications remained unclear. We show that two representative FHC mutations in beta-myosin S2, R870H and E924K, drastically reduce MyBP-C binding (Kd approximately 60 microM for R870H compared with a Kd of approximately 5 microM for the wild-type) down to undetectable levels (E924K). These mutations do not affect the coiled-coil structure of myosin. We suggest that the regulatory function of MyBP-C is mediated by the interaction with S2, and that mutations in beta-myosin S2 may act by altering the interactions with MyBP-C.     

Sarcoplasmic reticulum calcium defect in Ras-induced hypertrophic cardiomyopathy heart

The small G protein Ras-mediated signaling pathway has been implicated in the development of hypertrophy and diastolic dysfunction in the heart. Earlier cellular studies have suggested that the Ras pathway is responsible for reduced L-type calcium channel current and sarcoplasmic reticulum (SR) calcium uptake associated with sarcomere disorganization in neonatal cardiomyocytes. In the present study, we investigated the in vivo effects of Ras activation on cellular calcium handling and sarcomere organization in adult ventricular myocytes using a newly established transgenic mouse model with targeted expression of the H-Ras-v12 mutant. The transgenic hearts expressing activated Ras developed significant hypertrophy and postnatal lethal heart failure. In adult ventricular myocytes isolated from the transgenic hearts, the calcium transient was significantly depressed but membrane L-type calcium current was unchanged compared with control littermates. The expressions of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2a and phospholamban (PLB) were significantly reduced at mRNA levels. The amount of SERCA2a protein was also modestly reduced. However, the expression of PLB protein and gross sarcomere organization remained unchanged in the hypertrophic Ras hearts, whereas Ser(16) phosphorylation of PLB was dramatically inhibited in the Ras transgenic hearts compared with controls. Hypophosphorylation of PLB was also associated with a significant induction of protein phosphatase 1 expression. Therefore, our results from this in vivo model system suggest that Ras-induced contractile defects do not involve decreased L-type calcium channel activities or disruption of sarcomere structure. Rather, suppressed SR calcium uptake due to reduced SERCA2a expression and hypophosphorylation of PLB due to changes in protein phosphatase expression may play important roles in the diastolic dysfunction of Ras-mediated hypertrophic cardiomyopathy.     

PKCepsilon increases phosphorylation of the cardiac myosin binding protein C at serine 302 both in vitro and in vivo

Cardiac myosin binding protein C (cMyBPC) phosphorylation is essential for normal cardiac function. Although PKC was reported to phosphorylate cMyBPC in vitro, the relevant PKC isoforms and functions of PKC-mediated cMyBPC phosphorylation are unknown. We recently reported that a transgenic mouse model with cardiac-specific overexpression of PKCepsilon (PKCepsilon TG) displayed enhanced sarcomeric protein phosphorylation and dilated cardiomyopathy. In the present study, we have investigated cMyBPC phosphorylation in PKCepsilon TG mice. Western blotting and two-dimensional gel electrophoresis demonstrated a significant increase in cMyBPC serine (Ser) phosphorylation in 12-month-old TG mice compared to wild type (WT). In vitro PKCepsilon treatment of myofibrils increased the level of cMyBPC Ser phosphorylation in WT mice to that in TG mice, whereas treatment of TG myofibrils with PKCepsilon showed only a minimal increase in cMyBPC Ser phosphorylation. Three peptide motifs of cMyBPC were identified as the potential PKCepsilon consensus sites including a 100% matched motif at Ser302 and two nearly matched motifs at Ser811 and Ser1203. We treated synthetic peptides corresponding to the sequences of these three motifs with PKCepsilon and determined phosphorylation by mass spectrometry and ELISA assay. PKCepsilon induced phosphorylation at the Ser302 site but not at the Ser811 or Ser1203 sites. A S302A point mutation in the Ser302 peptide abolished the PKCepsilon-dependent phosphorylation. Taken together, our data show that the Ser302 on mouse cMyBPC is a likely PKCepsilon phosphorylation site both in vivo and in vitro and may contribute to the dilated cardiomyopathy associated with increased PKCepsilon activity.     

Phosphorylation of human inhibitor-1 at Ser67 and/or Thr75 attenuates stimulatory effects of protein kinase A signaling in cardiac myocytes

The depressed function of failing hearts has been partially attributed to increased protein phosphatase-1 through its impaired regulation by inhibitor-1. Phosphorylation of inhibitor-1 at Thr35 by PKA results in potent inhibition of protein phosphatase-1 activity, while phosphorylation at Ser67 or Thr75 by PKC attenuates the inhibitory activity. To examine the functional role of dual-site (Ser67, Thr75) phosphorylation of inhibitor-1 by PKC, the constitutively phosphorylated Ser67 (S67D) and/or Thr75 (T75D) human inhibitor-1 forms were expressed in adult cardiomyocytes. Expression of either single or double phosphorylated inhibitor-1 was associated with similar decreases in cardiac contractility, indicating that maximal inhibition can be elicited by each of these sites alone and that their inhibitory effects are not additive. Notably, activation of the cAMP pathway could only partially reverse the depressed contractile parameters. Accordingly, protein phosphatase-1 activity remained elevated, phosphorylation of phospholamban at Ser16 was decreased, and the EC(50) values of the sarcoplasmic reticulum calcium transport system were higher compared with controls. Thus phosphorylation of Ser67 and/or Thr75 in inhibitor-1 may mitigate the stimulatory effects of the cAMP pathway, resulting in compromised cardiac function.     

COOH-terminal truncated human cardiac MyBP-C alters myosin filament organization

Myosin-binding protein C (MyBP-C) is thought to play structural and/or regulatory role in striated muscles. The cardiac isoform of MyBP-C is one of the disease genes associated with familial hypertrophic cardiomyopathy and most of the mutations produce COOH truncated proteins. In order to determine the consequences of these mutations on myosin filament organization, we have characterized the effect of a 52-kDa NH2-terminal peptide of human cardiac MyBP-C on the alpha-myosin heavy chain (alpha-MyHC) filament organization. This peptide lacks the COOH-terminal MyHC-binding site and retains the two MyHC-binding domains located in the N-terminal part of MyBP-C. For this characterization, cDNA constructs (rat alpha-MyHC, full-length and truncated human cardiac MyBP-C) were transiently expressed singly or in pairwise combination in COS cells. In conformity with previous works performed on the skeletal isoform of MyBP-C, we observed that full-length cardiac MyBP-C organizes the MyHC into dense structures of uniform width. While the truncated protein is stable and can interact with MyHC in COS cells, it does not result in the same organization of sarcomeric MyHC that is seen with the full-length MyBP-C. These results suggest that the presence of truncated cardiac MyBP-C could, at least partly, disorganize the sarcomeric structure in patients with familial hypertrophic cardiomyopathy.     

Phosphorylation switches specific for the cardiac isoform of myosin binding protein-C: a modulator of cardiac contraction?

Cardiac myosin binding protein-C (cardiac MyBP-C, cardiac C protein) belongs to a family of proteins implicated in both regulatory and structural functions of striated muscle. For the cardiac isoform, regulatory phosphorylation in vivo by cAMP-dependent protein kinase (PKA) upon adrenergic stimulation is linked to modulation of cardiac contraction. The sequence of human cardiac MyBP-C now reveals regulatory motifs specific for this isoform. Site-directed mutagenesis identifies a LAGGGRRIS loop in the N-terminal region of cardiac MyBP-C as the key substrate site for phosphorylation by both PKA and a calmodulin-dependent protein kinase associated with the native protein. Phosphorylation of two further sites by PKA is induced by phosphorylation of this isoform-specific site. This phosphorylation switch can be mimicked by aspartic acid instead of phosphoserine. Cardiac MyBP-C is therefore specifically equipped with sensors for adrenergic regulation of cardiac contraction, possibly implicating cardiac MyBP-C in cardiac disease. The gene coding for cardiac MyBP-C has been assigned to the chromosomal location 11p11.2 in humans, and is therefore in a region of physical linkage to subsets of familial hypertrophic cardiomyopathy (FHC). This makes cardiac MyBP-C a candidate gene for chromosome 11-associated FHC.     

Changes in cardiac contractility related to calcium-mediated changes in phosphorylation of myosin-binding protein C.

Ca ions can influence the contraction of cardiac muscle by activating kinases that specifically phosphorylate the myofibrillar proteins myosin-binding protein C (MyBP-C) and the regulatory light chain of myosin (RLC). To investigate the possible role of Ca-regulated phosphorylation of MyBP-C on contraction, isolated quiescent and rhythmically contracting cardiac trabeculae were exposed to different concentrations of extracellular Ca and then chemically skinned to clamp the contractile system. Maximum Ca-activated force (F(max)) was measured in quiescent cells soaking in 1) 2.5 mM Ca for 120 min, 2) 1.25 mM for 120 min, or 3) 1.25 mM for 120 min followed by 10 min in 7.5 mM, and 4) cells rhythmically contracting in 2.5 mM for 20 min. F(max) was, respectively, 21.5, 10.5, 24.7, and 32.6 mN/mm(2). Changes in F(max) were closely associated with changes in the degree of phosphorylation of MyBP-C and occurred at intracellular concentrations of Ca below levels associated with phosphorylation of RLC. Monophosphorylation of MyBP-C by a Ca-regulated kinase is necessary before beta-adrenergic stimulation can produce additional phosphorylation. These results suggest that Ca-dependent phosphorylation of MyBP-C modulates contractility by changing thick filament structure.     

Inhibition of PKC phosphorylation of cTnI improves cardiac performance in vivo

Protein kinase C (PKC) modulates cardiomyocyte function by phosphorylation of intracellular targets including myofilament proteins. Data generated from studies on in vitro heart preparations indicate that PKC phosphorylation of troponin I (TnI), primarily via PKC-epsilon, may slow the rates of cardiac contraction and relaxation (+dP/dt and -dP/dt). To explore this issue in vivo, we employed transgenic mice [mutant TnI (mTnI) mice] in which the major PKC phosphorylation sites on cardiac TnI were mutated by alanine substitutions for Ser(43) and Ser(45) and studied in situ hemodynamics at baseline and increased inotropy. Hearts from mTnI mice exhibited increased contractility, as shown by a 30% greater +dP/dt and 18% greater -dP/dt than FVB hearts, and had a negligible response to isoproterenol compared with FVB mice, in which +dP/dt increased by 33% and -dP/dt increased by 26%. Treatment with phenylephrine and propranolol gave a similar result; FVB mouse hearts demonstrated a 20% increase in developed pressure, whereas mTnI mice showed no response. Back phosphorylation of TnI from mTnI hearts demonstrated that the mutation of the PKC sites was associated with an enhanced PKA-dependent phosphorylation independent of a change in basal cAMP levels. Our results demonstrate the important role that PKC-dependent phosphorylation of TnI has on the modulation of cardiac function under basal as well as augmented states and indicate interdependence of the phosphorylation sites of TnI in hearts beating in situ.     

Ablation of Ventricular Myosin Regulatory Light Chain Phosphorylation in Mice Causes Cardiac Dysfunction in Situ and Affects Neighboring Myofilament Protein Phosphorylation

There is little direct evidence on the role of myosin regulatory light chain phosphorylation in ejecting hearts. In studies reported here we determined the effects of regulatory light chain (RLC) phosphorylation on in situ cardiac systolic mechanics and in vitro myofibrillar mechanics. We compared data obtained from control nontransgenic mice (NTG) with a transgenic mouse model expressing a cardiac specific nonphosphorylatable RLC (TG-RLC(P-). We also determined whether the depression in RLC phosphorylation affected phosphorylation of other sarcomeric proteins. TG-RLC(P-) demonstrated decreases in base-line load-independent measures of contractility and power and an increase in ejection duration together with a depression in phosphorylation of myosin-binding protein-C (MyBP-C) and troponin I (TnI). Although TG-RLC(P-) displayed a significantly reduced response to β₁-adrenergic stimulation, MyBP-C and TnI were phosphorylated to a similar level in TG-RLC(P-) and NTG, suggesting cAMP-dependent protein kinase signaling to these proteins was not disrupted. A major finding was that NTG controls were significantly phosphorylated at RLC serine 15 following β₁-adrenergic stimulation, a mechanism prevented in TG-RLC(P-), thus providing a biochemical difference in β₁-adrenergic responsiveness at the level of the sarcomere. Our measurements of Ca²⁺ tension and Ca²⁺-ATPase rate relations in detergent-extracted fiber bundles from LV trabeculae demonstrated a relative decrease in maximum Ca²⁺-activated tension and tension cost in TG-RLC(P-) fibers, with no change in Ca²⁺ sensitivity. Our data indicate that RLC phosphorylation is critical for normal ejection and response to β₁-adrenergic stimulation. Our data also indicate that the lack of RLC phosphorylation promotes compensatory changes in MyBP-C and TnI phosphorylation, which when normalized do not restore function.Phosphorylation of sarcomeric proteins tunes the intensity and dynamics of cardiac contraction and relaxation independent of membrane Ca²⁺ fluxes to meet physiologic demands (1, 2). We focus here on ventricular myosin regulatory light chain, which is phosphorylated in vivo (3–5) but whose functional role in control of cardiac dynamics has remained unclear. The identification of RLC² mutations linked to familial hypertrophic cardiomyopathy (6) underscores the importance of understanding its action as a regulator of contraction. Functionally, in vitro cardiac RLC phosphorylation by MLCK produces a sensitizing shift in the force-Ca²⁺ relation in skinned fibers (7–11). Moreover, studies show that RLC phosphorylation manifests as a gradient across the wall of the heart, which may be important for both normalizing wall stress and for generation of torsion about the long axis of the ejecting heart (12–14). Yet there remains a lack of understanding of the in situ functional effects of RLC phosphorylation and whether phosphorylation of RLC influences other sarcomeric sites as substrates for kinases and phosphatases.Understanding the precise mechanisms by which phosphorylation of RLC affects function of ejecting ventricles is particularly important, because mechanisms downstream of Ca²⁺ fluxes at the level of the sarcomere appear to dominate ejection and to sustain ventricular elastance (15). Myosin motors are important in this, and RLC is well positioned at the S1-S2 junction to modulate myosin heavy chain directly by fine-tuning lever arm motion and indirectly by interacting with the essential light chain, the thick filament backbone, and MyBP-C (16, 17). Accordingly, the hypothesis underlying this study was that ablation of N-terminal RLC phosphorylation would elicit a depression in ventricular ejection and compensatory changes in phosphorylation of sarcomeric proteins neighboring RLC.To understand the role of RLC phosphorylation in the ejection phase of the cardiac cycle, we determined in situ pressure-volume functions in ejecting, auxotonically loaded ventricles expressing either wild type RLC (NTG) or a nonphosphorylatable RLC (TG-RLC(P-)) (10). Our experiments provide novel data demonstrating the importance of RLC phosphorylation in systolic pump function and provide new insights into how a lack of phosphorylation of RLC induces a redistribution of charge among myofilament proteins. Furthermore, our data demonstrate an enigmatic blunting of TG-RLC(P-) functional response to β₁-adrenergic simulation despite a normal TnI and MyBP-C phosphorylation profile. RLC serine 15 phosphorylation increased significantly in NTG controls but was not permitted in TG-RLC(P-) (RLC S14/15/19/A), suggesting that a change in RLC phosphorylation following β₁-adrenergic simulation may be critical for eliciting a normal response.     

Cypher/ZASP Is a Novel A-kinase Anchoring Protein

PKA signaling is important for the post-translational modification of proteins, especially those in cardiomyocytes involved in cardiac excitation-contraction coupling. PKA activity is spatially and temporally regulated through compartmentalization by protein kinase A anchoring proteins. Cypher/ZASP, a member of PDZ-LIM domain protein family, is a cytoskeletal protein that forms multiprotein complexes at sarcomeric Z-lines. It has been demonstrated that Cypher/ZASP plays a pivotal structural role in the structural integrity of sarcomeres, and several of its mutations are associated with myopathies including dilated cardiomyopathy. Here we show that Cypher/ZASP, interacting specifically with the type II regulatory subunit RIIα of PKA, acted as a typical protein kinase A anchoring protein in cardiomyocytes. In addition, we show that Cypher/ZASP itself was phosphorylated at Ser²⁶⁵ and Ser²⁹⁶ by PKA. Furthermore, the PDZ domain of Cypher/ZASP interacted with the L-type calcium channel through its C-terminal PDZ binding motif. Expression of Cypher/ZASP facilitated PKA-mediated phosphorylation of the L-type calcium channel in vitro. Additionally, the phosphorylation of the L-type calcium channel at Ser¹⁹²⁸ induced by isoproterenol was impaired in neonatal Cypher/ZASP-null cardiomyocytes. Moreover, Cypher/ZASP interacted with the Ser/Thr phosphatase calcineurin, which is a phosphatase for the L-type calcium channel. Taken together, our data strongly suggest that Cypher/ZASP not only plays a structural role for the sarcomeric integrity, but is also an important sarcomeric signaling scaffold in regulating the phosphorylation of channels or contractile proteins.     

Hypercontractile properties of cardiac muscle fibers in a knock-in mouse model of cardiac myosin-binding protein-C

Myosin-binding protein-C (MyBP-C) is a component of all striated-muscle sarcomeres, with a well established structural role and a possible function for force regulation. Multiple mutations within the gene for cardiac MyBP-C, one of three known isoforms, have been linked to familial hypertrophic cardiomyopathy. Here we generated a knock-in mouse model that carries N-terminal-shortened cardiac MyBP-C. The mutant protein was designed to have a similar size as the skeletal MyBP-C isoforms, whereas known myosin and titin binding sites as well as the phosphorylatable MyBP-C motif were not altered. We have shown that mutant cardiac MyBP-C is readily incorporated into the sarcomeres of both heterozygous and homozygous animals and can still be phosphorylated by cAMP-dependent protein kinase. Although histological characterization of wild-type and mutant hearts did not reveal obvious differences in phenotype, left ventricular fibers from homozygous mutant mice exhibited an increased Ca(2+) sensitivity of force development, particularly at lower Ca(2+) concentrations, whereas maximal active force levels remained unchanged. The results allow us to propose a model of how cMyBP-C may affect myosin-head mobility and to rationalize why N-terminal mutations of the protein in some cases of familial hypertrophic cardiomyopathy could lead to a hypercontractile state.     

Ontogeny of phosphoinositide 3-kinase signaling in developing heart: effect of acute beta-adrenergic stimulation

Signaling pathways underlying transition of cardiomyocyte growth from hyperplasia in fetal/newborn to hypertrophy in postnatal/adult hearts are not well understood. We have shown that beta-adrenergic receptor (beta-AR)-mediated regulation of neonatal cardiomyocyte proliferation involves p70 ribosomal protein S6 kinase (p70S6K). Here we examined the ontogeny of phosphoinositide 3-kinase (PI3K)/p70S6K signaling pathway in rat hearts and investigated the influence of beta-AR on this pathway during development. Cardiac PI3K and p70S6K1 activities were high in the embryonic day 20 fetus, decreased gradually postnatally, and were low in the adult. In contrast, p70S6K2 was barely detectable. Phosphorylation of p70S6K1, Akt, and phosphoinositide-dependent protein kinase 1 were markedly increased in late gestation and early postnatal life but not in adult hearts. Phosphatase and tensin homolog on chromosome 10 (PTEN), a negative regulator of PI3K, was highly expressed in adult hearts but only at low levels and mostly in the phosphorylated (inactivated) form in the fetus. Beta-AR stimulation resulted in increased cardiac p70S6K1 activity only in animals > or = 2 wk old, whereas Akt level was increased in all developmental stages tested. These increases were accompanied by increased Bcl-2 associated death promoter (Ser136) phosphorylation without changes in PTEN level. Thus there is globally high input of cardiac PI3K signaling during the fetal-neonatal transition period. Inactivation of PTEN may in part contribute to the high activity of PI3K signaling, which coincides with the period of high cardiomyocyte proliferation. Beta-AR stimulation activates cardiac p70S6K1 and Akt in postnatal animals and may activate cardiac survival signals. These data provide further evidence for the importance of beta-AR and PI3K signaling in the regulation of cardiac growth during development.     

Specific enhancement of sarcomeric response to Ca2+ protects murine myocardium against ischemia-reperfusion dysfunction

Alteration in myofilament response to Ca2+ is a major mechanism for depressed cardiac function after ischemia-reperfusion (I/R) dysfunction. We tested the hypothesis that hearts with increased myofilament response to Ca2+ are less susceptible to I/R. In one approach, we studied transgenic (TG) mice with a constitutive increase in myofilament Ca2+ sensitivity in which the adult form of cardiac troponin I (cTnI) is stoichiometrically replaced with the embryonic/neonatal isoform, slow skeletal TnI (ssTnI). We also studied mouse hearts with EMD-57033, which acts specifically to enhance myofilament response to Ca2+. We subjected isolated, perfused hearts to an I/R protocol consisting of 25 min of no-flow ischemia followed by 30 min of reperfusion. After I/R, developed pressure and rates of pressure change were significantly depressed and end-diastolic pressure was significantly elevated in nontransgenic (NTG) control hearts. These changes were significantly blunted in TG hearts and in NTG hearts perfused with EMD-57033 during reperfusion, with function returning to nearly baseline levels. Ca2+- and cross bridge-dependent activation, protein breakdown, and phosphorylation in detergent-extracted fiber bundles were also investigated. After I/R NTG fiber bundles exhibited a significant depression of cross bridge-dependent activation and Ca2+-activated tension and length dependence of activation that were not evident in TG preparations. Only NTG hearts demonstrated a significant increase in cTnI phosphorylation. Our results support the hypothesis that specific increases in myofilament Ca2+ sensitivity are able to diminish the effect of I/R on cardiac function.     

Modulation of Tau Phosphorylation Within Its Microtubule-Binding Domain by Cellular Thiols

Tau is a microtubule-stabilizing protein that is functionally modulated by alterations in its phosphorylation state. Because phosphorylation regulates both normal and pathological tau functioning, it is of importance to identify the signaling pathways that regulate tau phosphorylation in vivo. The present study examined changes in tau phosphorylation and function in response to modulation of cellular thiol content. Treatment of cells with phenylarsine oxide, which reacts with vicinal thiols, selectively increased tau phosphorylation within its microtubule-binding domain, at the non-Ser/Thr-Pro sites Ser262/356, while decreasing tau phosphorylation at Ser/ Thr-Pro sites outside this region. This increase in tau phosphorylation correlated with a decrease in the amount of tau associated with the cytoskeleton and decreased microtubule stability. Phenylarsine oxide-induced tau phosphorylation was inhibited by oxidants and by the protein kinase inhibitor staurosporine. Although staurosporine completely eliminated the increase in tau phosphorylation at Ser262/356, as detected by immunostaining with 12E8, it had a comparatively minor effect on the changes in tau localization induced by phenylarsine oxide. The results suggest that regulation of cellular thiols is important for modulating tau phosphorylation and function in situ. Additionally, although phosphorylation of Ser262/356 decreases tau's interaction with the cytoskeleton, phosphorylation of these residues alone is not sufficient for the phenylarsine oxide-induced changes in tau localization.     

Comparative phosphoproteomic analysis of neonatal and adult murine brain

Developmental processes are governed by diverse regulatory mechanisms including a suite of signaling pathways employing reversible phosphorylation. With the advent of large-scale phosphoproteomics, it is now possible to identify thousands of phosphorylation sites from tissues at distinct developmental stages. We describe here the identification of over 6000 nonredundant phosphorylation sites from neonatal murine brain. When compared to nearly three times the number of phosphorylation sites identified from 3-week-old murine brain, remarkably one-third of the neonatal sites were unique. This fraction only dropped to one-quarter when allowing the site to stray plus or minus 15 residues. This provides evidence for considerable change in the profiles of developmentally regulated phosphoproteomes. Using quantitative MS we characterized a novel phosphorylation site (Ser265) identified uniquely in the neonatal brain on doublecortin (Dcx), a protein essential for proper mammalian brain development. While the relative levels of Dcx and phospho-Ser265 Dcx between embryonic and neonatal brain were similar, their levels fell precipitously by postnatal day 21, as did phospho-Ser297, a site required for proper neuronal migration. Both sites lie near the microtubule-binding domain and may provide functionally similar regulation via different kinases.     

Role of dual-site phospholamban phosphorylation in intermittent hypoxia-induced cardioprotection against ischemia-reperfusion injury

Cardioprotection by intermittent high-altitude (IHA) hypoxia against ischemia-reperfusion (I/R) injury is associated with Ca(2+) overload reduction. Phospholamban (PLB) phosphorylation relieves cardiac sarcoplasmic reticulum (SR) Ca(2+)-pump ATPase, a critical regulator in intracellular Ca(2+) cycling, from inhibition. To test the hypothesis that IHA hypoxia increases PLB phosphorylation and that such an effect plays a role in cardioprotection, we compared the time-dependent changes in the PLB phosphorylation at Ser(16) (PKA site) and Thr(17) (CaMKII site) in perfused normoxic rat hearts with those in IHA hypoxic rat hearts submitted to 30-min ischemia (I30) followed by 30-min reperfusion (R30). IHA hypoxia improved postischemic contractile recovery, reduced the maximum extent of ischemic contracture, and attenuated I/R-induced depression in Ca(2+)-pump ATPase activity. Although the PLB protein levels remained constant during I/R in both groups, Ser(16) phosphorylation increased at I30 and 1 min of reperfusion (R1) but decreased at R30 in normoxic hearts. IHA hypoxia upregulated the increase further at I30 and R1. Thr(17) phosphorylation decreased at I30, R1, and R30 in normoxic hearts, but IHA hypoxia attenuated the depression at R1 and R30. Moreover, PKA inhibitor H89 abolished IHA hypoxia-induced increase in Ser(16) phosphorylation, Ca(2+)-pump ATPase activity, and the recovery of cardiac performance after ischemia. CaMKII inhibitor KN-93 also abolished the beneficial effects of IHA hypoxia on Thr(17) phosphorylation, Ca(2+)-pump ATPase activity, and the postischemic contractile recovery. These findings indicate that IHA hypoxia mitigates I/R-induced depression in SR Ca(2+)-pump ATPase activity by upregulating dual-site PLB phosphorylation, which may consequently contribute to IHA hypoxia-induced cardioprotection against I/R injury.     

Protein kinase-mediated regulation of the Na(+)/H(+) exchanger in the rat myocardium by mitogen-activated protein kinase-dependent pathways.

We examined regulation of the Na(+)/H(+) exchanger isoform 1 by phosphorylation in the rat myocardium. We utilized cell extracts from adult rat hearts, adult rat extracts fractionated by fast performance liquid chromatography, and extracts from cultured neonatal cardiac myocytes. The carboxyl-terminal 178 amino acids of the Na(+)/H(+) exchanger were expressed in Escherichia coli fused with glutathione S-transferase. The purified protein was used as a substrate for in vitro phosphorylation and in-gel kinase assays. Unfractionated extracts from neonatal myocytes or adult hearts phosphorylated the COOH-terminal domain of the antiporter. Western blot analysis revealed that mitogen-activated protein (MAP) kinase (44 and 42 kDa) and p90(rsk) (90 kDa) were present in specific fractions of cardiac extracts that phosphorylated the COOH-terminal protein. In-gel kinase assays confirmed that protein kinases of approximately 44 and 90 kDa could phosphorylate this domain. MAP kinase and p90(rsk)-dependent phosphorylation of the antiporter could be demonstrated by immunoprecipitation of these kinases from extracts of neonatal cardiac myocytes. PD98059, a mitogen-activated protein kinase kinase inhibitor, decreased MAP kinase and p90(rsk) phosphorylation of the antiporter and abolished serum and endothelin 1-stimulated increases in steady-state pH(i). These results confirm the presence of MAP kinase-dependent phosphorylation in the regulation of the Na(+)/H(+) exchanger in the rat myocardium and suggest an important role for p90(rsk) phosphorylation in regulation of the protein by endothelin-mediated stimulation of the antiporter.