PAK1 is a novel cardiac protective signaling molecule

Abstract

We review here the novel cardiac protective effects of the multifunctional enzyme, p21-activated kinase 1 (PAK1), a member of a serine/threonine protein kinase family. Despite the large body of evidence from studies in noncardiac tissue indicating that PAK1 activity is key in the regulation of a number of cellular functions, the role of PAK1 in the heart has only been revealed over the past few years. In this review, we assemble an overview of the recent findings on PAK1 signaling in the heart, particularly its cardiac protective effects. We present a model for PAK1 signaling that provides a mechanism for specifically affecting cardiac cellular processes in which regulation of protein phosphorylation states by protein phosphatase 2A (PP2A) predominates. We discuss the anti-adrenergic and antihypertrophic cardiac protective effects of PAK1, as well as its role in maintaining ventricular Ca²⁺ homeostasis and electrophysiological stability under physiological, β-adrenergic and hypertrophic stress conditions.

Keywords

p21-activated kinase 1 (PAK1) / heart

Introduction

Protein kinases are versatile signaling molecules that are involved in the regulation of most physiological responses in biological systems. The p21-activated kinases (PAKs) are a group of serine/threonine protein kinases that are activated by Cdc42 and Rac1, and were first discovered in rat brain tissue two decades ago [ 1]. The structure, substrate specificity and functional role of PAKs are evolutionarily conserved from protozoa to mammals. Vertebrate PAKs are particularly important for cytoskeletal remodeling and focal adhesion assembly, thereby contributing to dynamic processes such as cell migration and synaptic plasticity [ 2– 4]. Our work, along with the work of others over the past few years, has led to the identification of new roles of PAK1 in cardiac physiology, such as the regulation of cardiac excitability and contractility. More recent studies have revealed that PAK1-deficient mice were vulnerable to cardiac hypertrophy, ischemia/reperfusion injury and readily progress to failure under sustained pressure overload. The PAK1 activator FTY720 was able to prevent this pressure overload-induced hypertrophy and ischemia/reperfusion injury in wild-type mice without compromising their cardiac functions. Taken together, these studies suggest that PAK1 is more important in the heart than previously thought, particularly in terms of cardioprotection and therapeutic potential.

Anti-adrenergic effects

In our previous study [ 5], we demonstrated the functional effects of PAK1 in sinoatrial node (SAN) pacemaker cells. In SAN expressing the constitutively active (CA) PAK1, responses to adrenergic stimulation by isoproterenol (ISO), assessed by the frequency of firing electrical impulses, were significantly lower compared with those of controls, indicating that PAK1 antagonises β-adrenergic stimulation. Whole cell patch clamping demonstrated that both L-type Ca²⁺ current (I_Ca,L) and the delayed rectifier K⁺ current had a repressed response to ISO in cells expressing the CA PAK1. In the SAN cells, CA PAK1 redirects intracellular localization of protein phosphatase 2A (PP2A) [ 5]. Our further biochemical study indicated that the regulatory role of PAK1 on I_Ca,L and delayed rectifier K⁺ channel currents is attributable to increased activity of PP2A. Based on these findings, we suggested that signaling through PAK1 provides a mechanism for specifically affecting cardiac processes. The dynamic balance of phosphorylation is brought about through dedicated regulation by cAMP-PKA signaling (a well-established pathway) and PAK1-PP2A signaling (an under-studied pathway).

Recent studies have also provided evidence for the regulatory role of PAK1 on Ca²⁺ handling and Ca²⁺ homeostasis [ 6– 8]. Overexpression of CA-PAK1 altered Ca²⁺ transient decay constants (τ_Ca) [ 6], promoted anti-adrenergic signaling by attenuating the ISO-induced increase in activities of I_Ca,L and other proteins regulating Ca²⁺ handling, likely through PP2A activation. In ventricular myocytes from mice with cardiomyocyte-specific knockout of PAK1 (PAK1^cko), abnormal Ca²⁺ homeostasis including increased diastolic [Ca²⁺]_i, altered sarcoplasmic reticulum Ca²⁺ content and sarcoplasmic reticulum Ca²⁺ ATPase (SERCA) function was observed, particularly under β-adrenergic stress conditions [ 8]. Such altered Ca²⁺ homeostasis is associated with high incidences of ventricular arrhythmias and electrophysiological instability during either acute or chronic β-adrenergic challenge induced by ISO in PAK1^cko hearts [ 8]. These results indicate that modulation of PAK1 activity can have a significant impact on Ca²⁺ handling under both physiological and β-adrenergic challenge conditions. PAK1 is likely to regulate Ca²⁺ handling in cardiac myocytes through either PP2A-mediated or PP2A- independent regulation of the cellular proteins that regulate Ca²⁺ fluxes, including L-type Ca²⁺ channels, ryanodine receptors and phospholamban. However, the data from Sheehan et al. and DeSantiago et al. [ 6, 7] do not show changes in phospholamban or ryanodine receptor open probability, respectively with either overexpression or deletion of Pak1. Interestingly, the study by DeSantiago et al. [ 7] also provides evidence for a prominent role of PAK1 activity, not only in the functional regulation of ventricular myocyte excitation-contraction coupling, but also for the structural maintenance of the T-tubular system, whose remodeling is an integral feature of hypertrophic remodeling. Another recent study by DeSantiago et al. [ 9] also provided evidence that changes in the Ca²⁺ handling properties of Pak1 deficient ventricular myocytes depended on the increased levels of NOX2 dependent ROS production in these cells. Their results suggest that Pak1 is a critical negative regulator of NOX2 dependent ROS production and that a latent ROS dependent stimulation of NCX activity can predispose ventricular myocytes to Ca²⁺ overload under conditions where no significant changes in excitation-contraction coupling are yet evident [ 9].

Antihypertrophic effects

In response to acute or chronic insults the heart initially undergoes hypertrophic growth as an adaptive response to normalize ventricular wall stress. However, the capacity of this compensation is limited. Prolonged application of deleterious stimuli eventually causes decompensated remodeling, leading to chamber dilation, reduced contractile function, interstitial fibrosis and myocyte loss. These maladaptive effects subsequently lead to heart failure, a devastating condition affecting millions of people worldwide. Since hypertrophy is generally regarded as a determinant of heart failure, studies to discover the molecular and cellular mechanisms underlying hypertrophic remodeling, and to identify potential therapeutic approaches for treating heart failure, are of paramount importance.

Using both primary cardiomyocytes and PAK1^cko mice, we discovered that PAK1 acts as a novel signaling hub relaying antihypertrophic and survival signals from small GTPases to the JNK cascade in the heart [ 10]. The Pak^cko mice showed a greater degree of cardiac hypertrophy controls. They also showed clearcut apoptosis following 2 weeks of pressure overload, and a rapid progression to heart failure after 5 weeks of load [ 10]. The PAK1^cko mice also demonstrated enhanced hypertrophy in response to angiotensin II infusion. Furthermore, we observed that application of FTY720, a synthetic analog of sphingosine with an ability to activate PAK1 [ 10, 11], prevented the development of cardiac hypertrophy in load-stressed wild-type mice. This antihypertrophic effect was likely due to the activation of PAK1 by FTY720. Mechanistically, the loss of PAK1 led to a relative inability to activate the JNK signaling cascade and to an enhancement in the transcriptional activity of NFAT (nuclear factor of activated T cell). Furthermore, application of FTY720 induced PAK1 activation and prevented the development of cardiac hypertrophy in pressure overload stressed-wild type mice, but not in PAK1^cko mice, suggesting the antihypertrophic effect of FTY720 was likely due to its function to activate PAK1 [ 11]. Furthermore, a recent examination of PAK1 global knockout mice [ 12] revealed a similar phenotype as that in PAK1^cko mice. Absence of PAK1 in the whole heart rendered mice more capable of hypertrophic growth under conditions of ISO stimulation. Overall, these data demonstrate for the first time that PAK1 activation exerts a beneficial effect by preventing stress-induced hypertrophic remodeling. PAK1 may thus be a potential therapeutic target for antihypertrophic treatment.

Anti-ischemia/reperfusion injury

Acute myocardial ischemia/reperfusion injury is the major pathophysiological manifestation of ischemic heart disease (i.e., coronary heart disease). Interestingly, the heart has the ability to render itself resistant to ischemia/reperfusion injury. This phenomenon was first discovered by Murry et al. and was described as ischemic preconditioning (IPC) [ 13]. “Conditioning” the heart to tolerate the effects of acute ischemia/reperfusion injury can be initiated through inducing brief non-lethal episodes of ischemia and reperfusion to the heart either prior to, during, or even after an episode of sustained lethal myocardial ischemia—a phenomenon termed ischemic preconditioning (IPC), preconditioning or postconditioning, respectively. To date, at least three different cardioprotective protein kinase programs have been proposed to be initiated by different G-protein coupled receptors including the adenosine, bradykinin, opioid, protease activated receptor 2 (PAR2), and sphingosine-1-phosphate (S1P) receptors, etc. [ 14].

S1P has been shown to be an important mediator (through S1P receptor signaling) of cardiac ischemic pre- and postconditioning in both pharmacological and knockout animal studies [ 15, 16]. FTY720 protected against organ ischemia/reperfusion (I/R) injury in animal models [ 17, 18]. In our recent study, we demonstrated that prevention of arrhythmias by FTY720 in an I/R model was attributed to the activation of PAK1/AKT. FTY720 is able to stimulate the autophosphorylation of both PAK1 and AKT and their kinase activities in cardiomyocytes. Further experiments demonstrated that FTY720 triggered NO release from cardiac myocytes through pertussis toxin (PTX)-sensitive PI3K/Akt/eNOS signaling [ 19]. AKT is a well-established regulator of myocardial growth and survival, contractile function, and coronary angiogenesis [ 20]. Using both gain- and loss-of-function approaches in vitro and in vivo, Mao et al. demonstrated that PAK1 alone is sufficient to activate AKT [ 21]. The functional significance of this PAK1-AKT link is underscored by the observation that the pro-survival effect of PAK1 is diminished by AKT inhibition [ 21]. The PAK1-conferred protection was blocked by the AKT inhibitor X (2-​chloro-​N,​N-​diethyl-​10H-​phenoxazine-​10-​butanamine), suggesting that the protective effect of PAK1 is mediated, at least in part, by AKT signaling. Our more recent study also demonstrated that PAK1 improves cardiac contractile function in the I/R model of PAK1 global KO mice through regulation of troponin-T and myosin light chain 2 phosphorylation [ 22].

Clinical perspectives

Heart failure (HF) is a devastating disease with a lifetime risk of 1 in 5 among both men and women. Its incidence is still rising rapidly due to an increase in three major risk factors: population aging, obesity and diabetes. Despite advances in diagnostic and therapeutic technology during past decades, HF mortality rates still remain significantly high. Innovative, cost effective therapeutic approaches are urgently needed to provide the best quality of care when prevention fails. While a considerable amount of knowledge has been generated by biomedical research over the past few decades, the development of new and effective therapies for HF is stagnating. Modulating specific intracellular signaling pathways such as PAK1 signaling to offset/regulate myocardial remodeling, offers a cost effective therapeutic strategy for the management of HF, in particular, by preventing the progression of maladaptive hypertrophy and HF.

In summary, as illustrated in Fig. 1, these novel PAK1 effects complement its previously established actions upon PP2A and the resulting balance between kinase and phosphatase activity controlling cardiac ion channel activity and rhythmic Ca²⁺ cycling. PAK1-PP2A signaling modulation exerts anti-adrenergic and antihypertrophic effects through MKK4/MKK7/JNK signaling in ventricular cells [ 23]. PAK1 offers a potential new target for developing novel therapy for cardiac disease conditions.

References

[1] Manser E, Leung T, Salihuddin H, Zhao ZS, Lim L. A brain serine/threonine protein kinase activated by Cdc42 and Rac1. Nature1994; 367(6458): 40-46

[2] Manser E, Lim L. Roles of PAK family kinases. Prog Mol Subcell Biol1999; 22: 115-133

[3] Hofmann C, Shepelev M, Chernoff J. The genetics of Pak. J Cell Sci2004; 117(19): 4343-4354

[4] Zhao ZS, Manser E. PAK and other Rho-associated kinases—effectors with surprisingly diverse mechanisms of regulation. Biochem J2005; 386(2): 201-214

[5] Ke Y, Lei M, Collins TP, Rakovic S, Mattick PAD, Yamasaki M, Brodie MS, Terrar DA, Solaro RJ. Regulation of L-type calcium channel and delayed rectifier potassium channel activity by p21-activated kinase-1 in guinea pig sinoatrial node pacemaker cells. Circ Res2007; 100(9): 1317-1327

[6] Sheehan KA, Ke Y, Wolska BM, Solaro RJ. Expression of active p21-activated kinase-1 induces Ca2+ flux modification with altered regulatory protein phosphorylation in cardiac myocytes. Am J Physiol Cell Physiol2009; 296(1): C47-C58

[7] DeSantiago J, Bare DJ, Ke Y, Sheehan KA, Solaro RJ, Banach K. Functional integrity of the T-tubular system in cardiomyocytes depends on p21-activated kinase 1. J Mol Cell Cardiol2013; 60: 121-128

[8] Wang Y, Tsui H, Ke Y, Shi Y, Li Y, Davies L, Cartwright EJ, Venetucci L, Zhang H, Terrar DA, Huang CLH, Solaro RJ, Wang X, Lei M. Pak1 is required to maintain ventricular ca2+ homeostasis and electrophysiological stability through serca2a regulation in mice. Circ Arrhythm Electrophysiol2014<month>Sep</month><day>12</day>[Epub ahead of print]

[9] DeSantiago J, Bare DJ, Xiao L, Ke Y, Solaro RJ, Banach K. p21-Activated kinase1 (Pak1) is a negative regulator of NADPH-oxidase 2 in ventricular myocytes. J Mol Cell Cardiol2014; 67: 77-85

[10] Liu W, Zi M, Naumann R, Ulm S, Jin J, Taglieri DM, Prehar S, Gui J, Tsui H, Xiao RP, Neyses L, Solaro RJ, Ke Y, Cartwright EJ, Lei M, Wang X. Pak1 as a novel therapeutic target for antihypertrophic treatment in the heart. Circulation2011; 124(24): 2702-2715

[11] Liu W, Zi M, Tsui H, Chowdhury SK, Zeef L, Meng QJ, Travis M, Prehar S, Berry A, Hanley NA, Neyses L, Xiao RP, Oceandy D, Ke Y, Solaro RJ, Cartwright EJ, Lei M, Wang X. 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. Circ Heart Fail2013; 6(4): 833-844

[12] Taglieri DM, Monasky MM, Knezevic I, Sheehan KA, Lei M, Wang X, Chernoff J, Wolska BM, Ke Y, Solaro RJ. 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. J Mol Cell Cardiol2011; 51(6): 988-996

[13] Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation1986; 74(5): 1124-1136

[14] Heusch G, Boengler K, Schulz R. Cardioprotection: nitric oxide, protein kinases, and mitochondria. Circulation2008; 118(19): 1915-1919

[15] Jin ZQ, Zhang J, Huang Y, Hoover HE, Vessey DA, Karliner JS. A sphingosine kinase 1 mutation sensitizes the myocardium to ischemia/reperfusion injury. Cardiovasc Res2007; 76(1): 41-50

[16] Lecour S, Smith RM, Woodward B, Opie LH, Rochette L, Sack MN. Identification of a novel role for sphingolipid signaling in TNF alpha and ischemic preconditioning mediated cardioprotection. J Mol Cell Cardiol2002; 34(5): 509-518

[17] Egom EEA, Ke Y, Musa H, Mohamed TMA, Wang T, Cartwright E, Solaro RJ, Lei M. FTY720 prevents ischemia/reperfusion injury-associated arrhythmias in an ex vivo rat heart model via activation of Pak1/Akt signaling. J Mol Cell Cardiol2010; 48(2): 406-414

[18] Hofmann U, Burkard N, Vogt C, Thoma A, Frantz S, Ertl G, Ritter O, Bonz A. Protective effects of sphingosine-1-phosphate receptor agonist treatment after myocardial ischaemia-reperfusion. Cardiovasc Res2009; 83(2): 285-293

[19] Egom EEA, Mohamed TMA, Mamas MA, Shi Y, Liu W, Chirico D, Stringer SE, Ke Y, Shaheen M, Wang T, Chacko S, Wang X, Solaro RJ, Fath-Ordoubadi F, Cartwright EJ, Lei M. Activation of Pak1/Akt/eNOS signaling following sphingosine-1-phosphate release as part of a mechanism protecting cardiomyocytes against ischemic cell injury. Am J Physiol Heart Circ Physiol2011; 301(4): H1487-H1495

[20] Shiojima I, Walsh K. Regulation of cardiac growth and coronary angiogenesis by the Akt/PKB signaling pathway. Genes Dev2006; 20(24): 3347-3365

[21] Mao K, Kobayashi S, Jaffer ZM, Huang Y, Volden P, Chernoff J, Liang Q. Regulation of Akt/PKB activity by P21-activated kinase in cardiomyocytes. J Mol Cell Cardiol2008; 44(2): 429-434

[22] Monasky MM, Taglieri DM, Patel BG, Chernoff J, Wolska BM, Ke Y, Solaro RJ. p21-activated kinase improves cardiac contractility during ischemia-reperfusion concomitant with changes in troponin-T and myosin light chain 2 phosphorylation. Am J Physiol Heart Circ Physiol2012; 302(1): H224-H230

[23] Nelson TJ, Balza R Jr, Xiao Q, Misra RP. SRF-dependent gene expression in isolated cardiomyocytes: regulation of genes involved in cardiac hypertrophy. J Mol Cell Cardiol2005; 39(3): 479-489

RIGHTS & PERMISSIONS

Higher Education Press and Springer-Verlag Berlin Heidelberg