The p21‐activated kinase 1 (Pak1) signalling pathway in cardiac disease: from mechanistic study to therapeutic exploration

Abstract

p21‐activated kinase 1 (Pak1) is a member of the highly conserved family of serine/threonine protein kinases regulated by Ras‐related small G‐proteins, Cdc42/Rac1. It has been previously demonstrated to be involved in cardiac protection. Based on recent studies, this review provides an overview of the role of Pak1 in cardiac diseases including disrupted Ca²⁺ homoeostasis‐related cardiac arrhythmias, adrenergic stress‐ and pressure overload‐induced hypertrophy, and ischaemia/reperfusion injury. These findings demonstrate the important role of Pak1 mediated through the phosphorylation and transcriptional modification of hypertrophy and/or arrhythmia‐related genes. This review also discusses the anti‐arrhythmic and anti‐hypertrophic, protective function of Pak1 and the beneficial effects of fingolimod (an FDA‐approved sphingolipid drug), a Pak1 activator, and its ability to prevent arrhythmias and cardiac hypertrophy. These findings also highlight the therapeutic potential of Pak1 signalling in the treatment and prevention of cardiac diseases.

Linked Articles

This article is part of a themed section on Spotlight on Small Molecules in Cardiovascular Diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.8/issuetoc

Abbreviations

Ad‐CaPak1

constitutively active Pak1

AID

autoinhibitory domain

Ang II

angiotensin II

BAD

Bcl‐2‐associated death promoter

Bcl‐2

beta cell lymphoma 2

Cdc42

cell division control protein 42

cTnI/T

cardiac troponin I/T

DI

dimerization domain

ECM

extracellular matrix

Fbxo32

F‐box protein 32

Foxo

Forkhead box

FS

fractional shortening

FTY720

fingolimod

HW/TL

heart weight/tibia length

I/R

ischaemia/reperfusion

MLCK

myosin light chain kinase

MyBP‐C

myosin‐binding protein C

NFAT

nuclear factor of activated T‐cells

NRCM

neonatal rat cardiomyocyte

Pak

p21‐activated kinase

Pak1^cko

cardiac‐specific Pak1 knockout

Pak1^cTG

cardiac‐specific overexpression of Ad‐CaPak1 mice

PP1

protein phosphatase 1

PP2A

protein phosphatase 2A

Rac1

Ras‐related C3 botulinum toxin substrate

S1P

sphingosine‐1‐phosphate

SERCA2a

SR Ca²⁺‐ATPase 2a

Smad3

small mother against decapentaplegic 3

SR

sarcolemma reticulum

SRF

serum response factor

TAC

transverse aortic constriction

Introduction

The p21‐activated kinases (Paks) are a family of serine/threonine kinases activated by Cdc42 (cell division control protein 42) and Rac1 (Ras‐related C3 botulinum toxin substrate). They were discovered as binding proteins of small GTPase in rodent brain tissue (Kumar et al., 2017). In mammals, the Pak family consists of six distinct isoforms, Pak1–6. Discrete expression patterns are found among the Pak isoforms, which suggests they have distinct functions (Kelly et al., 2013). In particular, Pak1 is highly expressed in the brain, blood vessels and the heart (Kichina et al., 2010). Pak1 has been demonstrated to be abundantly expressed in all regions of the heart, including the sino‐atrial node (SAN), the atria and the ventricles (Ke et al., 2004; Ke et al., 2007). Extensive research on Pak1 has been conducted on its structural characterization and activation cascade, providing a basis for subsequent findings. Pak1 has been shown to possess important roles in many cellular functions, including cell cycle, cell motility, apoptosis and cell survival. The ability of Pak1 to modulate the motility of cancer cells, neurodevelopment, neuroplasticity and maturation of the nervous system has been well documented (Koth et al., 2014; Kumar and Li, 2016). However, investigations into the functions of Pak1 in the heart were only begun two decades ago (Clerk and Sugden, 1997).

In ventricular cells, Pak1 is localized to the cellular and nuclear membrane, Z‐discs and intercalated disc, and activated Pak1 has also been shown to be present within the cytoplasm. In sino‐atrial node cells of guinea pigs, endogenous Pak1 was shown to be distributed evenly in distinct subcellular patterns (Ke et al., 2004; 2007). Further studies showed that in the heart, Pak1 is a regulator of ion channels and contractile proteins through its effects on protein phosphatases and thus its potential ability to prevent arrhythmias by modifying Ca²⁺ homoeostasis in myocytes was established (Ke et al., 2007; 2008). In vitro and in vivo studies demonstrated that various hypertrophic stresses activate Pak1 phosphorylation, which protects the heart from hypertrophy through the JNK/NFAT (nuclear factor of activated T‐cells) signalling pathway. A lack of Pak1 exacerbates pressure overload‐induced hypertrophy, which indicates its potential as an anti‐hypertrophic agent (Liu et al., 2011b). A more recent study demonstrated that Pak1 is involved in ischaemia/reperfusion (I/R) injury in vivo; furthermore, Pak1 was suggested as a novel therapeutic target for the treatment of I/R. A deficiency in Pak1 results in changes in the phosphorylation of myofilament proteins and, consequently, impedes the recovery of cardiac function after I/R (Monasky et al., 2012). By using fingolimod (FTY720) to elevate the activity of Pak1/Akt signalling, Pak1 was revealed to have a protective effect in the rat heart (Egom et al., 2010a). Pak1 has been shown to be involved in a variety of cellular events in the heart through distinct signalling pathways. In this review, we have evaluated the recent studies of Pak1 in the heart and focused on its regulatory roles in cardiac arrhythmias, in particular cardiac contractility dysfunction, hypertrophy and I/R. Pak1 has been identified as having anti‐arrhythmic and anti‐hypertrophic effects. Elucidation of the downstream components involved in Pak1 signalling pathways has also revealed novel potential therapeutic targets for the treatment of cardiovascular diseases.

Structure of Pak1 and its functions in the cell cycle, cytoskeletal dynamics and cell survival

Structure and activation of Pak1

Pak1 contains a kinase domain at the carboxyl terminus, a p21 binding domain and an autoinhibitory domain (AID) at the amino terminus (Jaffer and Chernoff, 2002). Pak1 exists as homodimers adopting a trans conformation for the preservation of its inactive state through autoinhibition (Figure 1). The trans conformation permits interaction between the kinase domain of one monomer towards the inhibitory domain of the opposing monomer. The dimer core consists of salt bridges linking glutamic acid at site 82 of one strand to histidine at site 86 of the opposing strand, hydrophobic interactions within residues of the dimerization domain (DI) and inhibitory switch domain, as well as seven hydrogen bonds within the DI domain in the form of an antiparallel β ribbon (Lei et al., 2000; Parrini et al., 2002). GTP‐bound Rac1or Cdc42 (GTP‐Rac1 or GTP‐Cdc42) binds to the Cdc42/Rac1 interactive binding domain of Pak1, which causes a conformational change in its structure that dissociates the AID domain from the kinase domain and removes the trans inhibitory switch, therefore allowing autophosphorylation of Pak1 (Lei et al., 2005; Pirruccello et al., 2006; Strochlic et al., 2010) (Figure 1).

Figure 1.

Structure of Pak1. Upper panel shows the detailed schematic diagram of the trans structure of Pak1 existing as a homodimer. Each Pak1 monomer consists of a proline rich region, Pix binding motif, potential integrin binding domains, a Cdc42/Rac1 interactive binding (CRIB) region and a DI region. The Pak1 monomers interact with each other between the kinase domains towards the inhibitory domain of the opposing monomer. The lower panel shows the Pak1 activation process. Pak1 monomer forms a trans inhibitory homodimer. The binding of GTP–Cdc42/Rac1 to the CRIB domain relieves the binding of the two opposing monomers and allows the autophosphorylation within the kinase domain, consequently activating Pak1. PBD, p21 binding domain.

Pak1 and the cell cycle

Pak1 has been reported to be involved in the cell cycle through its effects on the proliferation in HeLa cells. An overexpression of Pak1 causes degradation of the inhibitor of κB, thus activating NFκB; this increases the expression of cyclin D1 protein and results in cell proliferation (Dadke et al., 2003; Balasenthil et al., 2004). Pak1 has also been shown to interact with and phosphorylate the centrosomal kinase aurora‐A, consequently promoting centrosome duplication and separation (Zhao et al., 2005).

Pak1 and cytoskeletal dynamics

Pak1 is involved in the regulation of microtubule and actin–myosin dynamics and reorganization of actin filaments. Pak1 has been reported to affect microtubule dynamics through tubulin cofactor B (TCoB), as phosphorylation of TCoB on centrosomes causes multiple spindle formation (Vadlamudi et al., 2005). In addition, Pak1 phosphorylation of Op18/stathmin inhibits its ability to destabilize microtubules (Wittmann et al., 2004). Pak1 induces the phosphorylation of myosin light chain kinase (MLCK), which inhibits the activity of MLCK and prevents the phosphorylation of its target, MLC, thus resulting in decreased actin and myosin assembly (Sanders et al., 1999). Furthermore, activation of Pak1 inhibits the actin binding protein cofilin through phosphorylation of Lim kinase. This inhibits cell motility, as activated cofilin stimulates F‐actin cycling and its inhibition leads to actin stabilization (Delorme et al., 2007).

Pak1 and cell survival

Pak1 prevents cell apoptosis through the regulation of several signalling pathways, including the Raf1 pathway, Akt pathway and Forkhead box (Foxo) pathway. Raf1 protects cells from apoptosis by stimulating complexes between Raf1 and B‐cell lymphoma 2 (Bcl‐2), as well as inhibiting Bcl‐2‐associated death promoter (BAD) (Mao et al., 2008). Additionally, Pak1 stimulates Raf1 translocation through phosphorylation; subsequently, Raf1 phosphorylates BAD and thus prevents apoptosis (Jin et al., 2005). Akt is an intracellular kinase that mediates cell survival via phosphorylation of BAD. Arg‐binding protein 2 was reported to regulate Akt/Pak1 phosphorylation of BAD through its interaction with Pak1 in a phosphorylation‐dependent manner, thus promoting cell survival (Yuan et al., 2005). Moreover, in oestrogen‐induced breast cancer cells, Pak1 was demonstrated to phosphorylate the pro‐apoptotic transcription factor, Foxo, resulting in Foxo nuclear exclusion, which again promoted cell survival (Mazumdar and Kumar, 2003).

Pak1 and the heart

The role of Pak1 in arrhythmia

A disrupted intracellular Ca²⁺ homoeostasis causes abnormal biochemical and electrophysiological activities in the heart, which lead to cardiac arrhythmias through contractile dysfunction (Bers, 2002). Electrophysiological studies in animals and isolated cardiomyocytes independently implicate Pak1 in the maintenance of cardiac excitation and contraction dynamics through its ability to modify the ion currents. This includes changes in the Ca²⁺sensitivity evoked by affecting the phosphorylation status and, thus, the activity of downstream proteins, such as L‐type Ca²⁺ channels, sarcolemma reticulum (SR) Ca²⁺‐ATPase 2a (SERCA2a), cardiac troponin I (cTnI), cardiac troponin T (cTnT) and myosin‐binding protein C (MyBP‐C).

Pak1 regulation of calcium handling

In Pak1 cardiac‐specific knockout (Pak1^cko) mice, an increased heart rate was detected but the electrocardiographic parameter measurements were unaffected. Meanwhile, ectopic beats were occasionally exhibited in the unconscious ECG recordings (Wang et al., 2014b). Increased ventricular tachyarrhythmia susceptibility occurred in both Pak1^cko hearts and isolated Pak1^cko ventricular myocytes. The occurrence of arrhythmic events due to irregular Ca²⁺ transients and/or Ca²⁺ waves increased with amplified stimulation frequencies compared with control myocytes and were particularly more severe in the presence of isoprenaline (Wang et al., 2014b). Studies in isolated cardiomyocytes demonstrated that the Pak1 deficiency induced abnormal Ca²⁺ homoeostasis, particularly during chronic β‐adrenoceptor‐mediated stress induced by isoprenaline. Decreased Ca²⁺ transient amplitude and delayed action potential repolarization were observed in global Pak1‐deficient (Pak1KO) mice (Desantiago et al., 2013). Consistently, increased diastolic Ca²⁺ and decreased systolic Ca²⁺ were demonstrated in Pak1^cko myocytes, along with a prolonged refill rate of the sarcolemma reticulum, especially after treatment with isoprenaline, indicating the impaired function of SERCA during Pak1‐deficient conditions (Wang et al., 2014b). In Pak1KO mice, exercise failed to increase the sensitivity of myofilament responsiveness to Ca²⁺, and Ca²⁺‐dependent sarcomere tension was unaffected (Davis et al., 2015). In another study, Pak1KO mice also exhibited decreased t‐tubular density and altered Ca²⁺ transient kinetics due to t‐tubular remodelling (Desantiago et al., 2013). Furthermore, Pak1KO mice exhibited reduced peak shortening of the sarcomere and a prolonged rate of relaxation during chronic isoprenaline treatment (Wang et al., 2014b).

The protective role of Pak1 was further explored through the use of in vitro studies, using cardiomyocytes transfected with adenovirus expressing constitutively active Pak1 (Ad‐CaPak1). When stimulated by isoprenaline, Ad‐CaPak1 transfected myocytes showed an attenuated elevation of the decay constant and amplitude of Ca²⁺ transient; demonstrating a reduction in Ca²⁺ spark amplitude and reduced spatial spread. These results indicate that the kinetics of Ca²⁺ release from the sarcolemma reticulum (SR) were improved with reductions in Ca²⁺ upstroke, which reduced the time constant of Ca²⁺ decay and reduced the velocity of propagation. Thus, an overexpression of Ad‐CaPak1 abolished isoprenaline‐induced elevation of fractional SR Ca²⁺ release (Sheehan et al., 2009). Moreover, the Ad‐CaPak1‐infected myocytes exhibited faster shortening time and prolonged relengthening time when compared to control myocytes (Solaro et al., 2002),

Interplay between Pak1 and PKA

Recent studies within the heart have provided molecular evidence for the regulatory role of Pak1 signalling in Ca²⁺ handling and Ca²⁺ homeostasis under both physiological and adrenergic stress conditions. Activation of the adrenergic pathway leads to increased levels of cAMP, subsequently leading to activation of cAMP‐dependent PKA (Sheehan et al., 2007). In cardiac myocytes, ion channels that are targets for PKA phosphorylation are frequently also targets for protein phosphatase 2A (PP2A) and protein phosphatase 1 (PP1) (Ke et al., 2008; Weber et al., 2015). It has been demonstrated that PKA can be regulated positively or negatively through the nuclear membrane‐tethered protein, PKA‐anchoring protein (mAKAP), which serves as a scaffold for PKA, cAMP‐dependent PDE4D3 and PP2A (Dodge‐Kafka et al., 2010). The negative feedback loop occurs when PKA phosphorylates PDE4D3 within the mAKAP complex, resulting in decreased activation of PKA through reduced cAMP; the positive feedback loop requires the binding of PP2A to the mAKAP complex followed by phosphorylation by PKA (Dodge‐Kafka et al., 2010). PKA has also been shown to regulate PP1 through phosphorylation of protein phosphatase inhibitor‐1 (I‐1), at threonine 35, resulting in potent inhibition of PP1 (Endo et al., 1996). Earlier studies demonstrated that activated Pak1 forms a complex with PP2A, resulting in the activation of PP2A, proposed to be through auto‐dephosphorylation at position Y307 (Ke et al., 2004). Due to the common targets that PP2A and PKA share, it is therefore vital to discuss the implications of these actions.

Increasing studies have indicated that PKA agonists affect the activity of L‐type Ca²⁺ channels through PKA‐dependent phosphorylation (Osterrieder et al., 1982). H‐89, a PKA inhibitor, was reported to reduce I _CaL density via inhibiting the α_1c‐subunit phosphorylation of L‐type Ca²⁺ channels in ventricular myocytes (Bryant et al., 2014). In addition, the dephosphorylation of L‐type Ca²⁺ channels is shown to decrease the currents mediated by these channels in rat ventricular myocytes (Dubell et al., 1996). A selective PP2A inhibitor, fostriecin, normalized the t‐tubule I _CaL density in isoprenaline‐treated ventricular myocytes, indicating that PP2A suppresses the activity of L‐type Ca²⁺ channels during isoprenaline stimulation (Chen et al., 2002; Kashihara et al., 2012).

Sphingosine ‐1‐phosphate (S1P, a circulating bioactive sphingolipid) was found to regulate intracellular Ca²⁺ handling by L‐type Ca²⁺ channels through the Pak1–PP2A‐mediated signalling pathway (Egom et al., 2016). S1P alleviated the increase in I _CaL amplitude and spontaneous Ca²⁺ waves induced by isoprenaline in rat ventricular myocytes. This study also showed increased co‐localization of Pak1 and PP2A, suggesting that S1P blunted the effects evoked by stimulation of the β‐adrenoceptors via the Pak1–PP2A interaction (Egom et al., 2016). Numerous studies have suggested that S1P can modulate intracellular Ca²⁺ levels in myocytes (Guo et al., 1999), without affecting the basal activity of L‐type Ca²⁺ channels (Nakajima et al., 2000). Furthermore, the non‐selective PP1 and PP2A inhibitor, okadaic acid, partially reversed the isoprenaline‐induced increase in I _CaL in cultured Ad‐CaPak1 infected sino‐atrial node cells. This confirms that Pak1 regulates the activity of L‐type Ca²⁺ channels by elevating the activity of PP2A (Ke et al., 2007).

Calcium is recycled back into the SR through SERCA2a, which is inhibited by dephosphorylated phospholamban. Phospholamban can be phosphorylated by PKA at serine site 16, thus preventing its inhibition of SERCA2a and promoting the reuptake of Ca²⁺ into the SR (Kranias, 1985; Kuschel et al., 1999). PP1 and PP2A can both dephosphorylate phospholamban; PP1 is responsible for 60–70% of phospholamban dephosphorylation (Macdougall et al., 1991). Recent studies have shown that bradykinin can increase the autophosphorylation of Pak1 and alter its translocation. This was accompanied by the dephosphorylation of phospholamban through PP2A, consequently inhibiting Ca²⁺ reuptake by the SR, hence slowing the Ca²⁺ transient (slow relaxation) (Ke et al., 2010). However, this effect was not seen in studies using phenylephrine or isoprenaline as a stimulus. Instead, under these stress conditions the disruption in Ca²⁺ homoeostasis caused by Pak1 deficiency was demonstrated to be a result of reduced SERCA2a expression. In this study, Pak1 was demonstrated to regulate SERCA2a expression through the transcription factor, serum response factor (SRF); expression levels of SERCA2a were attenuated in neonatal rat cardiomyocytes (NRCMs) pretreated with siRNA to knockdown SRF, prior to infection with Ad‐CaPak1 (Wang et al., 2014b). Furthermore, phosphorylated and total phospholamban protein levels were unaltered in Ad‐CaPak1‐infected myocytes, indicating that either the absence of Pak1 or participation of Pak1 in the dephosphorylation activity at this site was not sufficient to oppose the PKA‐mediated phospholamban phosphorylation and thus SR Ca²⁺ uptake (Sheehan et al., 2009).

Pak1 regulation of myofilaments

In addition, Pak1 has also been identified as an important regulator of myocyte contractility dynamics. In Pak1 deficient conditions the exercise‐induced cardiac remodelling was attenuated; an effect mediated through altered calcineurin signalling (Davis et al., 2015). Post‐exercise training, the Pak1KO mice displayed elevated levels of cTnT phosphorylation, a reduction in tropomyosin phosphorylation, reduced total phospholamban levels and a reduction in phosphorylated phospholamban, as well as decreased SERCA2a levels. These data indicate that exercise‐induced increase in myofilament responsiveness to Ca²⁺ and phosphorylation of MyBP‐C is mediated by Pak1 (Davis et al., 2015). Conversely, increasing the activity of Pak1 decreased the phosphorylation levels of cTnI and MyBP‐C and this was associated with enhanced Ca²⁺ sensitivity in myofilaments (Ke et al., 2004). The overexpression of Pak1 induced a functional reduction in the Ser^23/24 phosphorylation of cTnI, thus improving the kinetics of myocyte contractility (Sheehan et al., 2009).

The role of Pak1 in cardiac hypertrophy

Cardiac hypertrophy is a compensatory condition which commonly leads to heart failure. It is characterized by alterations in several signalling pathways. Pak1 has been identified as a novel anti‐hypertrophic compound through its regulation of the JNK/NFAT pathway (Liu et al., 2011b). In vitro studies have demonstrated that hypertrophic stimuli including angiotensin II (Ang II), stimuli of α‐ and β‐adrenoceptors, phenylephrine and isoprenaline, respectively, are all capable of activating Pak1 through phosphorylation.

Pak1 phosphorylation is increased in ventricular tissues of control mice subjected to transverse aortic constriction (TAC) (Liu et al., 2011b). Pak1^cko mice exhibited increased heart weight/tibia length (HW/TL) ratio, a larger cardiomyocyte cross‐sectional area and reduced fractional shortening (FS) level compared with wild‐type mice subjected to TAC (Liu et al., 2011b). This indicates that Pak1 acts as an anti‐hypertrophic agent in the heart. Furthermore, a lack of Pak1 also resulted in increased interstitial fibrosis and cardiomyocyte apoptosis in Pak1^cko/TAC mice, whilst Ad‐CaPak1‐infected NRCMs displayed higher survival rates despite being subjected to the cardiotoxic anticancer drug, doxorubicin (Yuan et al., 2005; Mao et al., 2008; Liu et al., 2011b).

Pak1 antagonizes cardiac hypertrophy not only by mechanical stress‐induced membrane receptor activation but also by neuroendocrine agonist stimulation. This was demonstrated through studies using Ang II stress or isoprenaline stress; when subjected to stress Pak1‐deficient mice showed increased susceptibility to LV (left ventricle) myocardial hypertrophy associated with enhanced LV systolic function with impaired LV diastolic relaxation (Taglieri et al., 2011; Liu et al., 2011b; Wang et al., 2014a). Furthermore, hypertrophic effects induced by phenylephrine were inhibited in NRCMs infected with Ad‐CaPak1, whereas hypertrophy progressed as predicted in phenylephrine‐treated control NRCMs (Liu et al., 2011b). Consistently, the cardioprotective effects of Pak1 were further confirmed by subjecting mice with cardiac‐specific overexpression of Ad‐CaPak1 (Pak1^cTG) to TAC stress. The detrimental effects of TAC stress were significantly reduced in the Pak1^cTG mice; the TAC‐induced increases in fibrosis, LV mass, LV end‐diastolic diameter, LV end‐systolic diameter and reduced fraction shortening were diminished in these mice (Tsui et al., 2015).

Pak1 in MAPK signalling pathways

Post‐activation of Pak1 by upstream Cdc42/Rac1 results in the activation of downstream MAPK kinases (MAPKK), MKK4 and MKK7, and post‐activation of either kinase phosphorylates the JNK1/2 cascade. The loss of Cdc42 in cardiomyocytes rendered mice more capable of cardiac hypertrophic growth, as Cdc42 is required for JNK activation in response to hypertrophic stress (Maillet et al., 2009). Cdc42 can also initiate the JNK cascade through MEKK1 (Yujiri et al., 1998; Witowsky and Johnson, 2003). In vitro and in vivo studies showed unaltered phosphorylation levels of MKK4, MKK7 and JNK1/2 in Pak1^cko mice subjected to TAC, whereas elevations in phosphorylation levels of these kinases were observed in TAC‐stressed control mice. Thus, Pak1 protects the heart from hypertrophic stress through phosphorylation of the MKK4/MKK7–JNK pathway, which subsequently phosphorylates NFAT (Liu et al., 2011a). Conversely, activation of ERK1/2 was higher in isoprenaline‐treated Pak1KO mice, indicating that Pak1 potentially has an inhibitory effect on ERK1/2 activation in vivo following isoprenaline treatment (Taglieri et al., 2011). An ERK1/2 inhibitor attenuated the development of myocardial and myocyte hypertrophy in isoprenaline‐treated wild‐type and Pak1KO mice, indicating ERK1/2 is the major kinase driving isoprenaline‐induced myocardial hypertrophy in the absence of Pak1 (Taglieri et al., 2011). Previous studies have demonstrated that the Pak1 signalling cascade activates PP2A and suppresses the effects of β‐adrenoceptor stimulation (Ke et al., 2004). Studies have shown that β‐adrenoceptor stimulation enhances the phosphorylation of ERK1/2 due to a suppression of PP2A activation in the absence of Pak1, thereby promoting ERK‐induced LV cardiac hypertrophy (Lorenz et al., 2009).

A novel anti‐hypertrophic pathway of Pak1 was identified through small mother against decapentaplegic 3 (Smad3) transcriptional regulation of F‐box protein 32 (Fbxo32) (Tsui et al., 2015). In the absence of Pak1 in the heart TAC stress led to exacerbated increased protein levels of calcineurin as the up‐regulation of Fbxo32 was abolished. In control NRCMs PE stress induces an up‐regulation of Fbxo32 at the mRNA and protein levels, which was eliminated in Pak1 knockdown NRCMs, resulting in the accumulation of calcineurin. Ad‐CaPak1‐infected NRCMs showed elevated protein expression levels of Fbxo32 and subsequent down‐regulation of calcineurin (Tsui et al., 2015). Thus, Pak1 negatively regulates calcineurin, through Fbxo32, in response to hypertrophic stress. It has been reported that the hearts of Smad3‐null mice have an increased susceptibility to TAC‐induced hypertrophy (Divakaran et al., 2009). Previous studies have demonstrated that Smad3 can be phosphorylated by JNK in lung epithelial cells, p38 in breast carcinoma cells or Pak1 in mesangial cells (Chen et al., 2013; Velden et al., 2011; Kamaraju and Roberts, 2005). This is interesting as a lack of Smad3 phosphorylation was also demonstrated in Pak1‐deficient hearts under hypertrophic conditions (Tsui et al., 2015). This study further confirmed that knockdown of MKK7 or JNK abolished the phenylephrine‐induced phosphorylation of Smad3 despite the presence of Pak1, indicating that Pak1 activation of Smad3 is likely to occur through the MKK7/JNK pathway (Tsui et al., 2015). These results highlight the therapeutic potential of Fbxo32 itself and a pharmacological activator of Fbxo32, berberine, has been reported to improve cardiac function (Huang et al., 2015; Lau et al., 2001; Zhang et al., 2014). Berberine prevented hypertrophic remodelling by improving cardiac functions in both Pak1^cko and wild‐type mice subjected to TAC stress. These data suggest that in mice subjected to TAC stress, the induction of Fbxo32 antagonizes the pathological hypertrophic remodelling resulting from a deficiency in Pak1 (Tsui et al., 2015).

In other studies, the Pak‐activating peptide, which directly enhances Pak1 function, was found to abolish Ang II‐induced cardiac hypertrophy in vitro and in vivo, as well as associated ventricular arrhythmias (Wang et al., 2014a). Alternatively, miR‐30c could potentially target Cdc42 and Pak1 mRNAs; the overexpression of miR‐30c attenuated cardiomyocyte hypertrophy by facilitating the increased expression of Cdc42 and Pak1 induced by glucose (Raut et al., 2015).

Overall, these results suggest that activation of Pak1 exerts beneficial effects in the heart, preventing cardiac hypertrophy by modifying the activities and phosphorylation status of downstream proteins. Hence, Pak1 and its associated components are promising potential therapeutic targets for the future treatment of hypertrophy.

The role of Pak1 in I/R injury

Myocardial I/R injury is caused by the sudden re‐establishment of the coronary blood supply; this results in reduced myocardial mechanical function associated with cell death in the myocardium. The no‐ or low‐reflow phenomenon after I/R injury, caused by endothelial oedema, neutrophil and platelet plugging, may lead to inadequate coronary perfusion that further compromises cardiac function (Yang et al., 2016). There is emerging evidence indicating that Pak1 has a role in I/R, as Pak1/Akt and Pak1/PP2A have been suggested as downstream signalling molecules for S1P and bradykinin in cardiac tissue, which induce cardioprotection during I/R (Egom et al., 2010b). PI3K has been demonstrated to activate Pak1, resulting in activation of the PI3K/Akt signalling pathway (Menard and Mattingly, 2003), which protects the heart during I/R by inhibiting cardiac myocyte apoptosis. In Pak1KO mice the recovery of cardiac function during I/R was attenuated; the percentage recovery of LV‐developed pressure was reduced and increase in end‐diastolic pressure was exacerbated. This indicates that Pak1 is involved in the regulation of developed pressure during I/R in mice heart (Monasky et al., 2012). Moreover, hearts with an increased myofilament response to Ca²⁺ have been reported to be less susceptible to I/R injury (Arteaga et al., 2005), which indicates that Pak1‐induced elevation of myofilament Ca²⁺ sensitivity could possibly diminish the effects of I/R on cardiac function.

Pak1 in Akt signalling pathway

Akt has been well documented for its cardioprotective role during I/R, as well as its regulation of myocardial growth and myocardial survival (Fujio et al., 2000; Shiojima and Walsh, 2006). Pak1 directly activates Akt, and Akt itself can phosphorylate Pak1. Using both gain‐ and loss‐of‐function approaches in vitro and in vivo, Pak1 has been demonstrated to be sufficient and essential for growth factor‐induced Akt activity in myocytes (Mao et al., 2008). The functional significance of Pak1/Akt signalling is underscored by the observation that the pro‐survival effects of Pak1 are diminished by Akt inhibition (Shiojima and Walsh, 2006). S1P, an activator of Pak1, has been reported to mediate cardiac ischaemic pre‐ and postconditioning in knockout animal studies; S1P has been demonstrated to protect isolated mice hearts from global I/R damage (Jin et al., 2002, 2007). S1P receptor agonists were also demonstrated to activate downstream Akt; thus, it is likely that S1P protects the heart against I/R through the Pak1/Akt signalling pathway (Hofmann et al., 2009; Egom et al., 2010b). Further studies have also shown that Akt increases the phosphorylation of eNOS, resulting in the activation of eNOS and consequently NO‐mediated cytoprotection against ischaemic damage (Michell et al., 1999).

Pak1 and phosphatases

Pak1 and PP2A activation have been demonstrated to improve cardiac contractile function during I/R by affecting the phosphorylation levels of cTnT and MLC2 (Monasky et al., 2012). Bradykinin has been shown to play a protective role during pre‐conditioning and I/R (Baxter and Ebrahim, 2002). Recent studies have shown that bradykinin can increase the autophosphorylation of Pak1 and alter its translocation. This was accompanied by dephosphorylation of phospholamban through PP2A, consequently inhibiting Ca²⁺ reuptake by the SR, which resulted in a reduction in the Ca²⁺ transient (slow relaxation) (Ke et al., 2010). Furthermore, studies have shown that ablation of MLC2 phosphorylation resulted in decreased ventricular contractility, thus lengthening the duration of ventricular ejection, and other sarcomeric proteins (Scruggs et al., 2009). In co‐immunoprecipitation studies, Pak1 and cTnT are demonstrated to directly associate with each other in a protein–protein interaction; this suggests that Pak1 may play a regulatory role in the phosphorylation of cTnT (Monasky et al., 2012). The finding that Pak1 is involved in the prevention of arrhythmogenesis associated with I/R is vital to the ongoing development of current therapies for the treatment of I/R injury‐induced ventricular arrhythmias.

Pak1 as a therapeutic target

The involvement of Pak1 in current activities and Ca²⁺ homoeostasis suggests its potential role in preventing cardiac arrhythmias. Sphingosine and sphingosine‐derived lipids have been shown to directly activate Pak1 (Bokoch, 2003), and sphingosine has been suggested to play an important role in cardiac protection against I/R including studies utilizing fingolimod (FTY720; FDA‐approved sphingolipid drug exhibiting similar structure to S1P), derived from the component myriocin found in the Chinese herb Iscaria sinclarii (Adachi et al., 1995).

Our previous study has shown that fingolimod prevents arrhythmias through activation of the Pak1/Akt signalling pathway in studies of ex vivo rat heart subjected to I/R (Egom et al., 2010a). Treatment with fingolimod prevented I/R injury‐induced arrhythmic events in langendorff ex vivo heart models, including the occurrence of premature ventricular beats, ventricular tachycardia, sinus brady arrhythmias and atrioventricular conduction block (Egom et al., 2010a). Our results indicated that fingolimod triggers the autophosphorylation of Pak1 and subsequent Akt phosphorylation through G_i‐mediated signalling pathways in cardiomyocytes (Egom et al., 2010a). Moreover, fingolimod also stimulates NO production via the PI3K/Akt/eNOS signalling pathway (Egom et al., 2011a,b).

In more recent in vitro and in vivo studies, we have demonstrated that fingolimod exerts cardioprotective effects by inhibiting the development of hypertrophy as well as reversing existing hypertrophy. This protection is mediated through the activation of Pak1 and subsequent activation of the MKK7–JNK1/2 pathway (Liu et al., 2011b). TAC‐stressed mice treated with fingolimod exhibited less pathological hypertrophic remodelling in the heart, as evidenced by their reduced HW/TL ratio, reduced cardiomyocyte cross‐sectional area and reduced interstitial fibrosis (Liu et al., 2013). Fingolimod also ameliorated cardiac functional performance in mice subjected to TAC, as shown by increased FS, increased ejection fraction and decreased diastolic velocity (Liu et al., 2013). Fingolimod was demonstrated to activate the Pak1/NFAT signalling pathway by interacting with G_i‐coupled S1P receptors, as pertussis toxin was able to inhibit these effects of fingolimod (Liu et al., 2013). Fingolimod was shown to reverse hypertrophy by activating Pak1 and thereby negatively affecting NFAT activity through MKK7 and JNK1/2 (Liu et al., 2013). The reduction in fibrosis following fingolimod treatment was demonstrated through decreased expression levels of periostin in the extracellular matrix (ECM); this was accompanied by a reduction in TGF‐β signalling (Liu et al., 2013). Studies using periostin‐deficient mice also exhibited reduced fibrosis and hypertrophy following pressure overload, whereas the overexpression of periostin resulted in age‐related spontaneous hypertrophy (Oka et al., 2007; Lorts et al., 2009). Furthermore, fingolimod treatment reduced the expression of periostin induced by AngII (Sidhu et al., 2010; Snider et al., 2008). Studies have shown that periostin itself can activate TGF‐β signalling, whilst TGF‐β signalling can increase periostin expression levels. Hence, the reduced periostin expression induced by fingolimod is probably due to its inhibitory effect on TGF‐β activation, which in turn further down‐regulates periostin expression levels. Collectively, fingolimod reverses hypertrophy, reduces the fibrotic response, restores ECM integrity and improves cardiac performance (Liu et al., 2013).

These cardioprotective effects of fingolimod demonstrated through the protection of cardiac function during I/R, as well as the prevention and reversal of hypertrophy, have revealed that it has potential as a very promising pharmacological treatment of heart disease.

Conclusion

Over the past decades, significant progress has been made in exploring the role of Pak1 in the heart. This ranges from discovering its regulatory effects in Ca²⁺ homoeostasis‐ and Ca²⁺ handling‐related arrhythmias, its cardioprotective effects against pressure overload‐induced hypertrophy and protective effects against myocardial I/R injury (Figure 2). The cardiac phenotypes of gain‐ and loss‐of‐function model of Pak1, Cdc42/Rac1, PKA, PP2A, PP1, MKK4/7, JNK, Smad3 and F‐Box32 were clearly described in Table 1. As demonstrated by fingolimod and berberine, components of the Pak1 signalling pathway, as well as Pak1 itself, are promising potential targets for future therapeutic treatment of cardiac diseases.

Figure 2.

The signalling pathway of Pak1 in the heart. Pak1 can be activated by Cdc42/Rac1, and it regulates the Ca²⁺ handling proteins through activation of PP2A, which allows dephosphorylation of phospholamban, cTnI, RyR and LTCC and phosphorylation of cTnT and MLC2. Pak1 protects the heart from arrhythmias by activating SRF and transcriptionally regulating SERCA2a through the MKK4/7/JNK pathway. Pak1 also prevents hypertrophy through negative regulation of calcineurin via the Smad3/F‐Bxo32 cascade. Black arrows indicate stimulation/phosphorylation/activation; red lines indicate inhibition/dephosphorylation.

Table 1.

Cardiac phenotypes of molecules’ gain ‐or loss‐of‐function models

Molecules	Cardiac phenotype
	Gain‐of‐function	Loss‐of‐function
Pak1	Anti‐hypertrophy (Liu et al., 2011b), anti‐arrhythmias in I/R model (Egom et al., 2011a,b)	Cardiac hypertrophy (Liu et al., 2011b), ventricular arrhythmias and abnormal Ca2+ homeostasis (Wang et al., 2014a,b)
Cdc42/Rac1	Anti‐hypertrophy (Maillet et al., 2009), reduced cardiomyocyte apoptosis (Wang et al., 2015), hypercontractile, dilated cardiomyopathy (Sussman et al., 2000)	Enhanced apoptosis and necrosis, hypertrophy, susceptibility to heart failure (Maillet et al., 2009), abnormal cardiomyocyte adherens/desmosome junction formation (Li et al., 2017), decreased cardiomyocyte proliferation and regeneration (Peng et al., 2016)
PKA	Dilated cardiomyopathy, cardiac sudden death (Antos et al., 2001), cardiac hypertrophy (Houser and Molkentin, 2008; Mishra et al., 2010; Yang et al., 2014)	Reduced heart beating rate magnitude and kinetics (Yaniv et al., 2015)
PP2A	Dilated cardiomyopathy (Brewis et al., 2000), impaired cardiac contractility (Gergs et al., 2004), reduced heart rate, increased heart rate variability, conduction defects (Little et al., 2015), reduced L‐type Ca2+ current (Christ et al., 2004)	Cardiac hypertrophy and fibrosis (Li et al., 2016), increased phosphorylation of Cav1.2 and L‐type Ca2+ current (Shi et al., 2012)
PP1	Reduced L‐type Ca2+ current (Christ et al., 2004), increased RyR2 activity and atrial fibrillation susceptibility (Chiang et al., 2014), impaired cardiac contractility, dilated cardiomyopathy (Carr et al., 2002), mild β‐adrenergic desensitization, protective effect from isoprenaline‐induced cardiac hypertrophy and arrhythmias (El‐Armouche et al., 2008)	Enhanced LV contractility (Miyazaki et al., 2012), hypertrophy (El‐Armouche et al., 2008), enhanced cardiac contractile responses to β‐agonists (Carr et al., 2002)
MKK4/7	Protection against cardiomyocyte apoptosis, prevention of cardiac hypertrophy and ventricular remodelling (Choukroun et al., 1999; Liu et al., 2009; Liu et al., 2011a), diastolic dysfunction and premature death with signs of congestive heart failure (Petrich et al., 2004)	Increased atrial fibrosis and atrial arrhythmias susceptibility (Davies et al., 2014), cardiac hypertrophy and apoptosis (Liu et al., 2009), reduced I/R‐induced apoptosis and protection from myocardial cell injury during post‐ischaemic recovery (Liao et al., 2007)
JNK	Hypertrophy (Choukroun et al., 1999), apoptosis during I/R (Xie et al., 2009), abnormal gap junction structure and slowed conduction velocity (Petrich et al., 2002), perivascular remodelling (Jesmin et al., 2006), conduction disruption in aged atrium (Jones and Lancaster, 2015)	Aggravated cardiac and mitochondrial dysfunction from IR injury (Jang and Javadov, 2014), attenuation of Gq‐induced hypertrophy (Minamino et al., 2002), increased fibrosis‐induced by pressure overload (Tachibana et al., 2006), apoptosis (Kyoi et al., 2006)
SMAD3	Increased cardiac fibrosis (Chen et al., 2017), anti‐hypertrophy (Tsui et al., 2015)	Reduced collagen deposition and development of cardiac fibrosis (Bujak et al., 2007), protection against pressure overload‐induced cardiac fibrosis (Zhang et al., 2016; Zhou et al., 2017)
Fbxo32	Anti‐hypertrophy (Tsui et al., 2015)	Dilated cardiomyopathy (Al‐Hassnan et al., 2016; Al‐Yacoub et al., 2016)

Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Southan et al., 2016), and are permanently archived in the Concise Guide to PHARMACOLOGY 2015/16 (Alexander et al., 2015a,b).

Conflict of interest

The authors declare no conflicts of interest.

Wang, Y. , Wang, S. , Lei, M. , Boyett, M. , Tsui, H. , Liu, W. , and Wang, X. (2018) The p21‐activated kinase 1 (Pak1) signalling pathway in cardiac disease: from mechanistic study to therapeutic exploration. British Journal of Pharmacology, 175: 1362–1374. doi: 10.1111/bph.13872.