Yunbo Ke

Intracellular Localization and Functional Effects of P21-Activated Kinase-1 (Pak1) in Cardiac Myocytes

Abstract— We investigated intracellular localization and substrate specificity of P21-activated kinase-1 (Pak1) in rat cardiac myocytes. Pak1 is a serine/threonine protein kinase that is activated by Rac1/Cdc42 and important in signaling of stress responses. Yet the localization and in vivo function of Pak1 in heart cells is poorly understood. Studies reported here indicate that Pak1 physically interacts with protein phosphatase 2a and localizes to the Z-disk, cell membrane, intercalated disc, and nuclear membrane of adult rat heart myocytes. We compared levels of phosphorylation of cardiac troponin I (cTnI) in control myocytes with phosphorylation of cTnI and myosin binding protein C (C-protein) in myocytes with increased Pak1 activity. The increase in activity was induced by infection of myocytes with a recombinant adenovirus (AdPak1) containing cDNA for a constitutively active Pak1. Control cells were infected with a virus (AdLacZ) containing LacZ. Basal levels of phosphorylation of cTnI and C-protein were relatively high in the myocytes infected with AdLacZ. However, phosphorylation of cTnI and C-protein in cells expressing constitutively active Pak1 was significantly reduced compared with those expressing LacZ. Measurement of Ca2+ tension relations in single myocytes demonstrated that this reduction in phosphorylation of cTnI and C-protein was associated with the predicted increase in sensitivity to Ca2+. Our data provide evidence for a novel pathway of phosphatase regulation in cardiac myocytes.

FTY720 prevents ischemia/reperfusion injury-associated arrhythmias in an ex vivo rat heart model via activation of Pak1/Akt signaling.

Recent studies demonstrated a role of sphingosine-1-phosphate (S1P) in the protection against the stress of ischemia/reperfusion (I/R) injury. In experiments reported here, we have investigated the signaling through the S1P cascade by FTY720, a sphingolipid drug candidate displaying structural similarity to S1P, underlying the S1P cardioprotective effect. In ex vivo rat heart and isolated sinoatrial node models, FTY720 significantly prevented arrhythmic events associated with I/R injury including premature ventricular beats, VT, and sinus bradycardia as well as A-V conduction block. Real-time PCR and Western blot analysis demonstrated the expression of the S1P receptor transcript pools and corresponding proteins including S1P1, S1P2, and S1P3 in tissues dissected from sinoatrial node, atrium and ventricle. FTY720 (25 nM) significantly blunted the depression of the levels of phospho-Pak1 and phospho-Akt with ischemia and with reperfusion. There was a significant increase in phospho-Pak1 levels by 35%, 199%, and 205% after 5, 10, and 15 min of treatment with 25 nM FTY720 compared with control nontreated myocytes. However, there was no significant difference in the levels of total Pak1 expression between nontreated and FTY720 treated. Phospho-Akt levels were increased by 44%, 63%, and 61% after 5, 10, and 15 min of treatment with 25 nM FTY720, respectively. Our data provide the first evidence that FTY720 prevents I/R injury-associated arrhythmias and indicate its potential significance as an important and new agent protecting against I/R injury. Our data also indicate, for the first time, that the cardioprotective effect of FTY720 is likely to involve activation of signaling through the Pak1.

Pak1 as a Novel Therapeutic Target for Antihypertrophic Treatment in the Heart

Background— Stress-induced hypertrophic remodeling is a critical pathogenetic process leading to heart failure. Although many signal transduction cascades are demonstrated as important regulators to facilitate the induction of cardiac hypertrophy, the signaling pathways for suppressing hypertrophic remodeling remain largely unexplored. In this study, we identified p21-activated kinase 1 (Pak1) as a novel signaling regulator that antagonizes cardiac hypertrophy. Methods and Results— Hypertrophic stress applied to primary neonatal rat cardiomyocytes (NRCMs) or murine hearts caused the activation of Pak1. Analysis of NRCMs expressing constitutively active Pak1 or in which Pak1 was silenced disclosed that Pak1 played an antihypertrophic role. To investigate the in vivo role of Pak1 in the heart, we generated mice with a cardiomyocyte-specific deletion of Pak1 (Pak1cko). When subjected to 2 weeks of pressure overload, Pak1cko mice developed greater cardiac hypertrophy with attendant blunting of JNK activation compared with controls, and these knockout mice underwent the transition into heart failure when prolonged stress was applied. Chronic angiotensin II infusion also caused increased cardiac hypertrophy in Pak1cko mice. Moreover, we discovered that the Pak1 activator FTY720, a sphingosine-like analog, was able to prevent pressure overload-induced hypertrophy in wild-type mice without compromising their cardiac functions. Meanwhile, FTY720 failed to exert such an effect on Pak1cko mice, suggesting that the antihypertrophic effect of FTY720 likely acts through Pak1 activation. Conclusions— These results, for the first time, establish Pak1 as a novel antihypertrophic regulator and suggest that it may be a potential therapeutic target for the treatment of cardiac hypertrophy and heart failure.

Activation of Pak1/Akt/eNOS signaling following sphingosine-1-phosphate release as part of a mechanism protecting cardiomyocytes against ischemic cell injury.

We investigated whether plasma long-chain sphingoid base (LCSB) concentrations are altered by transient cardiac ischemia during percutaneous coronary intervention (PCI) in humans and examined the signaling through the sphingosine-1-phosphate (S1P) cascade as a mechanism underlying the S1P cardioprotective effect in cardiac myocytes. Venous samples were collected from either the coronary sinus (n = 7) or femoral vein (n = 24) of 31 patients at 1 and 5 min and 12 h, following induction of transient myocardial ischemia during elective PCI. Coronary sinus levels of LCSB were increased by 1,072% at 1 min and 941% at 5 min (n = 7), while peripheral blood levels of LCSB were increased by 579% at 1 min, 617% at 5 min, and 436% at 12 h (n = 24). In cultured cardiac myocytes, S1P, sphingosine (SPH), and FTY720, a sphingolipid drug candidate, showed protective effects against CoCl induced hypoxia/ischemic cell injury by reducing lactate dehydrogenase activity. Twenty-five nanomolars of FTY720 significantly increased phospho-Pak1 and phospho-Akt levels by 56 and 65.6% in cells treated with this drug for 15 min. Further experiments demonstrated that FTY720 triggered nitric oxide release from cardiac myocytes is through pertussis toxin-sensitive phosphatidylinositol 3-kinase/Akt/endothelial nitric oxide synthase signaling. In ex vivo hearts, ischemic preconditioning was cardioprotective in wild-type control mice (Pak1f/f), but this protection appeared to be ineffective in cardiomyocyte-specific Pak1 knockout (Pak1cko) hearts. The present study provides the first direct evidence of the behavior of plasma sphingolipids following transient cardiac ischemia with dramatic and early increases in LCSB in humans. We also demonstrated that S1P, SPH, and FTY720 have protective effects against hypoxic/ischemic cell injury, likely a Pak1/Akt1 signaling cascade and nitric oxide release. Further study on a mouse model of cardiac specific deletion of Pak1 demonstrates a crucial role of Pak1 in cardiac protection against ischemia/reperfusion injury.

Regulation of L-Type Calcium Channel and Delayed Rectifier Potassium Channel Activity by p21-Activated Kinase-1 in Guinea Pig Sinoatrial Node Pacemaker Cells

Phosphorylation of ion channels plays an important role in the regulation of cardiac function, but signaling mechanisms controlling dephosphorylation are not well understood. We have tested the hypothesis that p21-activated kinase-1 (Pak1), a serine–threonine protein kinase regulated by Ras-related small G proteins, regulates sinoatrial node (SAN) ion channel activity through a mechanism involving protein phosphatase 2A. We report a novel role of Pak1-mediated signaling in attenuating isoproterenol-induced enhancement of L-type Ca2+ current (ICaL) and delayed rectifier potassium current (IK) in guinea pig SAN pacemaker cells. We demonstrate that in guinea pig SAN: (1) there is abundant expression of endogenous Pak1 in pacemaker cells; (2) expression of constitutively active Pak1 depresses isoproterenol-induced upregulation of ICaL and IK; (3) inhibition of protein phosphatase 2A increases the enhancement of IK and ICaL by isoproterenol in Ad-Pak1–infected cells; (4) protein phosphatase 2A coimmunoprecipitates with endogenous Pak1 in SAN tissue; and (5) expression of constitutively active Pak1 suppresses the chronotropic action of isoproterenol on pacemaker activity of intact SAN preparations. In conclusion, our data demonstrate that a Pak1 signaling pathway exists in cardiac pacemaker cells and that this novel pathway plays a role in the regulation of ion channel activity.

Ablation of p21-activated kinase-1 in mice promotes isoproterenol-induced cardiac hypertrophy in association with activation of Erk1/2 and inhibition of protein phosphatase 2A.

Earlier investigations in our lab indicated an anti-adrenergic effect induced by activation of p21-activated kinase (Pak-1) and protein phosphatase 2A (PP2A). Our objective was to test the hypothesis that Pak-1/PP2A is a signaling cascade controlling stress-induced cardiac growth. We determined the effects of ablation of the Pak-1 gene on the response of the myocardium to chronic stress of isoproterenol (ISO) administration. Wild-type (WT) and Pak-1-knockout (Pak-1-KO) mice were randomized into six groups to receive either ISO, saline (CTRL), or ISO and FR180204, a selective inhibitor of Erk1/2. Echocardiography revealed that hearts of the Pak-1-KO/ISO group had increased LV fractional shortening, reduced LV chamber volume in diastole and systole, increased cardiac hypertrophy, and enhanced transmitral early filling deceleration time, compared to all other groups. The changes were associated with an increase in relative Erk1/2 activation in Pak-1-KO/ISO mice versus all other groups. ISO-induced cardiac hypertrophy and Erk1/2 activation in Pak-1-KO/ISO were attenuated when the selective Erk1/2 inhibitor FR180204 was administered. Immunoprecipitation showed an association between Pak-1, PP2A, and Erk1/2. Cardiac myocytes infected with an adenoviral vector expressing constitutively active Pak-1 showed a repression of Erk1/2 activation. p38 MAPK phosphorylation was decreased in Pak-1-KO/ISO and Pak-1-KO/CTRL mice compared to WT. Levels of phosphorylated PP2A were increased in ISO-treated Pak-1-KO mice, indicating reduced phosphatase activity. Maximum Ca(2+)-activated tension in detergent-extracted bundles of papillary fibers from ISO-treated Pak-1-KO mice was higher than in all other groups. Analysis of cTnI phosphorylation indicated that compared to WT, ISO-induced phosphorylation of cTnI was blunted in Pak-1-KO mice. Active Pak-1 is a natural inhibitor of Erk1/2 and a novel anti-hypertrophic signaling molecule upstream of PP2A.

Regulation of cardiac excitation and contraction by p21 activated kinase-1.

Cardiac excitation and contraction are regulated by a variety of signaling molecules. Central to the regulatory scheme are protein kinases and phosphatases that carry out reversible phosphorylation of different effectors. The process of beta-adrenergic stimulation mediated by cAMP dependent protein kinase (PKA) forms a well-known pathway considered as the most significant control mechanism in excitation and contraction as well as many other regulatory mechanisms in cardiac function. However, although dephosphorylation pathways are critical to these regulatory processes, signaling to phosphatases is relatively poorly understood. Emerging evidence indicates that regulation of phosphatases, which dampen the effect of beta-adrenergic stimulation, is also important. We review here functional studies of p21 activated kinase-1 (Pak1) and its potential role as an upstream signal for protein phosphatase PP2A in the heart. Pak1 is a serine/threonine protein kinase directly activated by the small GTPases Cdc42 and Rac1. Pak1 is highly expressed in different regions of the heart and modulates the activities of ion channels, sarcomeric proteins, and other phosphoproteins through up-regulation of PP2A activity. Coordination of Pak1 and PP2A activities is not only potentially involved in regulation of normal cardiac function, but is likely to be important in patho-physiological conditions.

Expression of active p21-activated kinase-1 induces Ca2+ flux modification with altered regulatory protein phosphorylation in cardiac myocytes

p21-Activated kinase-1 (Pak1) is a serine-threonine kinase that associates with and activates protein phosphatase 2A in adult ventricular myocytes and, thereby, induces increased Ca2+ sensitivity of skinned-fiber tension development mediated by dephosphorylation of myofilament proteins (Ke Y, Wang L, Pyle WG, de Tombe PP, Solaro RJ. Circ Res 94: 194-200, 2004). We test the hypothesis that activation of Pak1 also moderates cardiac contractility through regulation of intracellular Ca2+ fluxes. We found no difference in field-stimulated intracellular Ca2+ concentration ([Ca2+]i) transient amplitude and extent of cell shortening between myocytes expressing constitutively active Pak1 (CA-Pak1) and controls expressing LacZ; however, time to peak shortening was significantly faster and rate of [Ca2+]i decay and time of relengthening were slower. Neither caffeine-releasable sarcoplasmic reticulum (SR) Ca2+ content nor fractional release was different in CA-Pak1 myocytes compared with controls. Isoproterenol application revealed a significantly blunted increase in [Ca2+]i transient amplitude, as well as a slowed rate of [Ca2+]i decay, increased SR Ca2+ content, and increased cell shortening, in CA-Pak1 myocytes. We found no significant change in phospholamban phosphorylation at Ser16 or Thr17 in CA-Pak1 myocytes. Analysis of cardiac troponin I revealed a significant reduction in phosphorylated species that are primarily attributable to Ser(23/24) in CA-Pak1 myocytes. Nonstimulated, spontaneous SR Ca2+ release sparks were significantly smaller in amplitude in CA-Pak1 than LacZ myocytes. Propagation of spontaneous Ca2+ waves resulting from SR Ca2+ overload was significantly slower in CA-Pak1 myocytes. Our data indicate that CA-Pak1 expression has significant effects on ventricular myocyte contractility through altered myofilament Ca2+ sensitivity and modification of the [Ca2+]i transient.

Splicing signals are required for S-phase regulation of the mouse thymidylate synthase gene.

The thymidylate synthase (TS) gene is expressed at a much higher level in cells undergoing DNA replication than in nondividing cells. In growth-stimulated mammalian cells, TS mRNA content increases 10 to 20-fold as cells progress from G1 through S phase. However, the rate of transcription of the TS gene does not increase during this interval, indicating that the gene is regulated at the posttranscriptional level. We have shown that both the promoter of the mouse TS gene and TS introns are necessary (although neither is sufficient) for S-phase-specific regulation of TS mRNA content. In the present study, we examined in more detail the role of introns in regulating TS mRNA levels in growth-stimulated cells. TS minigenes that contain normal or modified introns were stably transfected into mouse 3T6 fibroblasts, and the regulation of the minigenes was compared with that of the endogenous TS gene. TS minigenes that contain TS intron 1 or 2 maintain S-phase regulation. Deletion of most of the interior of the introns had only minor effects on regulation. However, when splicing of the intron was inhibited by alteration of the splice donor and acceptor sites, the minigene was expressed at a constant level following growth stimulation. Minigenes consisting of the TS promoter linked to either a luciferase or a human beta-globin indicator gene were growth regulated when spliceable introns were included in the minigenes. However, when the introns were eliminated, the minigenes were expressed at a constant level. These observations indicate that the splicing reaction itself, rather than a control sequence within the intron, is important for growth-regulated expression of the TS gene. Possible mechanisms to account for the dual requirement for the TS promoter and intron splicing for proper regulation of the TS gene are discussed.

A Novel Immunomodulator, FTY-720 Reverses Existing Cardiac Hypertrophy and Fibrosis From Pressure Overload by Targeting NFAT (Nuclear Factor of Activated T-cells) Signaling and Periostin

Background—Hypertension or aortic stenosis causes pressure overload, which evokes hypertrophic myocardial growth. Sustained cardiac hypertrophy eventually progresses to heart failure. Growing evidence indicates that restraining hypertrophy could be beneficial; here, we discovered that FTY-720, an immunomodulator for treating multiple sclerosis, can reverse existing cardiac hypertrophy/fibrosis. Methods and Results—Male C57/Bl6 mice underwent transverse aortic constriction (TAC) for 1 week followed by FTY-720 treatment for 2 weeks under continuing TAC. Compared with vehicle-treated TAC hearts, FTY-720 significantly reduced ventricular mass, ameliorated fibrosis, and improved cardiac performance. Mechanistic studies led us to discover that FTY-720 appreciably inhibited nuclear factor of activated T-cells (NFAT) activity. Moreover, we found that in primary cardiomyocytes (rat and human) pertussis toxin (Gi-coupled receptor inhibitor) substantially blocked the antihypertrophic effect of FTY-720. This observation was confirmed in a mouse model of pressure overload. Interestingly, gene array analysis of TAC hearts revealed that FTY-720 profoundly decreased gene expression of a group of matricellular proteins, of which periostin was prominent. Analysis of periostin protein expression in TAC-myocardium, as well as in rat and human cardiac fibroblasts, confirmed the array data. Moreover, we found that FTY-720 treatment or knockdown of periostin protein was able to inhibit transforming growth factor-&bgr; responsiveness and decrease collagen expression. Conclusions—FTY-720 alleviates existing cardiac hypertrophy/fibrosis through mechanisms involving negative regulation of NFAT activity in cardiomyocytes and reduction of periostin expression allowing for a more homeostatic extracellular compartment milieu. Together, FTY-720 or its analogues could be a promising new approach for treating hypertrophic/fibrotic heart disease.