The Pak1 signalling pathway in cardiac disease: from

mechanistic study to therapeutic exploration

Correspondence: Xin Wang, Wei Liu and Hoyee Tsui, Faculty of Biology, Medicine and

Health, The University of Manchester, M13 9NT, Manchester, UK, E-mail:

xin.wang@manchester.ac.uk; wei.liu@manchester.ac.uk; Hoyee_Jenny@hotmail.com

Yanwen Wang1, Shunyao Wang1, Ming Lei2, Mark Boyett1, Hoyee Tsui1, Wei Liu1,

Xin Wang1

1Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, UK, 2Department

of Pharmacology, The University of Oxford, Oxford, UK

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 assembles an overview of the role of Pak1 in

cardiac diseases including disrupted Ca2+ homoeostasis related cardiac arrhythmias, β-

adrenergic stress and pressure overload-induced hypertrophy, and

ischemia/reperfusion injury. These findings demonstrate the importance of Pak1

regulation through phosphorylation and transcriptional modification of hypertrophy

and/or arrhythmia-related genes. This review also discusses the anti-arrhythmic and

anti-hypertrophic protective role of Pak1 and beneficial effects of FTY-720

(Fingolimod, FDA approved sphingolipid drug), a Pak1 activator in the prevention of

arrhythmias and cardiac hypertrophy. These findings shed light on the therapeutic

1

potential of Pak1 signalling in the treatment and prevention of cardiac diseases.

Abbreviations

Ad-CaPak1

AID

Ang II

BAD

Bcl-2

Cdc42

CRIB

cTnI/T

DI

ECM

Fbxo32

Foxo

FS

FTY720

HW/TL

ISO

I/R

constitutively active Pak1

auto inhibitory domain

Angiotensin II

Bcl-2-associated death promoter

B-cell lymphoma 2

cell division control protein 42

Cdc42/Rac interactive-binding

cardiac troponin I/T

dimerization domain

extracellular matrix

F-box protein 32

Forkhead box

fractional shortening

fingolimod

heart weight/tibia length

isoproterenol

ischemia/reperfusion

2

LTCC

LW/TL

MLC

MLCK

MyBP-C

NFAT

NRCM

OA

Pak

Pak1cko

Pak1cTG

L-type Ca2+ channel

lung weight/tibia length

myosin light chain

myosin light chain kinase

myosin-binding protein C

nuclear factor of activated T-cells

neonatal rat cardiomyocytes

okadaic acid

p21-activated kinase

cardiac-specific Pak1 knockout

cardiac-specific overexpression of constitutively active Pak1 mice

PAP

PBD

PE

PI3K

PP1, PP2A

PLN

PTX

Rac1

Pak1 activating peptide

p21 binding domain

phenylephrine

phosphoinositide 3-kinase

protein phosphatase 1, protein phosphatase 2A

phospholamban

pertussis toxin

ras-related C3 botulinum toxin substrate

3

RCAN1.4

SAN

SERCA2a

Smad3

S1P

SR

SRF

SPH

TAC

regulator of calcineurin 1 variant 4

sino-atrial node

sarcolemma reticulum Ca2+-ATPase 2a

small mother against decapentaplegic 3

Sphingosine-1-phosphate

sarcolemma reticulum

serum response factor

Sphingosine

transverse aortic constriction

4

Introduction

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. Restricted expression patterns are found among Pak isoforms

suggesting 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

characterisation 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 cellular functions of Pak1 in

modulating motility of cancer cells, neurodevelopment, neuroplasticity and

maturation of nervous system have been well documented (Kumar and Li, 2016; Koth

et al., 2014). However, the uncovering of Pak1functions in the heart only begun two

decades ago (Clerk and Sugden, 1997).

In ventricular cells, Pak1 is shown to be localised to the cellular and nuclear

membrane, Z-discs and intercalated disc, and activated Pak1 was shown to be also

present within the cytoplasm. In SAN cells of guinea pigs, endogenous Pak1 was

shown to be distributed evenly in distinct subcellular patterns (Ke et al., 2004; Ke et

al., 2007). Further studies show that in the heart, Pak1 was as a regulator of ion

channels and contractile proteins through its regulation of protein phosphatases, and

thus establishing its potential function in the prevention of arrhythmias through

modifying Ca2+ homoeostasis in myocytes (Ke et al., 2008; Ke et al., 2007). In vitro

and in vivo studies demonstrated that various hypertrophic stress activated Pak1

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phosphorylation, which protected the heart from hypertrophy through the JNK/NFAT

(cJun N-terminal kinase/nuclear factor of activated T-cells) signalling pathway. As a

potential anti-hypertrophic regulator, loss of Pak1 exacerbates pressure overload-

induced hypertrophy (Liu et al., 2011b). A more recent study demonstrated Pak1 is

involved in ischemia/reperfusion injury (I/R) in vivo, furthermore, Pak1 was

suggested as a novel therapeutic target for the treatment of I/R. Pak1 deficiency

induces altered phosphorylation of myofilament proteins and consequently leads to

depressed recovery of cardiac function after I/R (Monasky et al., 2012). Elevated

activity of Pak1/Akt signalling through activation by fingolimod (FTY720) revealed a

cardiac protection mechanism in the rat heart (Egom et al., 2010a). Pak1 has been

involved in a range of cellular events in the heart through distinct signalling pathways.

In this review, we have combined the recent studies of Pak1 in the heart, and focus on

its regulatory roles in cardiac arrhythmias in particular cardiac contractility

dysfunction, hypertrophy and I/R. Pak1 has been identified as an anti-arrhythmic and

anti-hypertrophic regulator. Elucidation of the downstream components involved in

Pak1 signalling pathways also uncovers novel potential therapeutic targets for the

treatment of cardiovascular diseases.

Structure and Functions in Cell Cycle, Cytoskeletal

Dynamics and Cell Survival

Structure and Activation of Pak1

Pak1contains a kinase domain at the carboxyl terminus, a p21 binding domain

(PBD) 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

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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 7 hydrogen bonds within the DI domain

in the form of an antiparallel β ribbon (Parrini et al., 2002; Lei et al., 2000). GTP-

bound Rac1or Cdc42 (GTP-Rac1 or GTP-Cdc42) binds to the CRIB (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).

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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 CRIB region and a DI region. The Pak1 monomers interact with each other between the kinase domain 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 auto-phosphorylation within the kinase domain, consequently activating Pak1.

Pak1 in Cell Cycle

Pak1 has been reported to be involved in the cell cycle through its role in cell

proliferation in HeLa cells. Overexpression of Pak1 caused degradation of the

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inhibitor of kappa B, thus activating nuclear factor kappa B; this increases cyclin D1

protein expression resulting in cell proliferation (Dadke et al., 2003; Balasenthil et al.,

2004). Pak1 has also been shown to interact and phosphorylate the centrosomal kinase

Aurora-A, consequently promoting centrosome duplication and separation (Zhao et

al., 2005).

Pak1 in Cytoskeletal Dynamics

Pak1 is involved in microtubule regulation, actin-myosin regulation and

reorganisation of actin filaments to mediate actin dynamics. Pak1 has been reported to

be involved in microtubule regulation 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 function to destabilise microtubules (Wittmann et al., 2004). Phosphorylation of

myosin light chain kinase (MLCK) by Pak1 inhibits MLCK activity; this inhibition

prevents phosphorylation of the MLCK target, myosin light chain (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, its inhibition leads to actin stabilisation (Delorme et al., 2007).

Pak1 in Cell Survival

Pak1 prevents cell apoptosis through 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

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(Jin et al., 2005). Akt is an intracellular kinase for 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 has demonstrated to phosphorylation the pro-apoptotic transcription

factor, Foxo, resulting in Foxo nuclear exclusion, which again promoted cell survival

(Mazumdar and Kumar, 2003).

Pak1 in the heart

The role of Pak1 in Arrhythmia

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 modified current activities. This includes

altered Ca2+sensitivity by regulating the phosphorylation status thus the activity of

downstream proteins, such as L-type Ca2+ channel (LTCC), sarcolemma reticulum

Ca2+-ATPase 2a (SERCA2a), cardiac troponin I (cTnI), cardiac troponin T (cTnT) and

myosin-binding protein C (MyBP-C).

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Pak1 regulation of Calcium handling

In Pak1 cardiac-specific knockout (Pak1cko) mice, an increased heart rate was

detected with unaltered electrocardiographic parameter measurements. Meanwhile,

ectopic beats were occasionally exhibited with unconscious ECG (Wang et al.,

2014b). Increased ventricular tachyarrhythmia susceptibility occurred in both Pak1cko

heart and isolated Pak1cko ventricular myocytes. The occurrence of frequent

arrhythmic events due to irregular Ca2+ transient and/or Ca2+ waves increased with

amplified stimulation frequencies than control myocytes and were particularly more

severe under isoproterenol (ISO) conditions (Wang et al., 2014b). Studies in isolated

cardiomyocytes demonstrated Pak1 deficiency induced abnormal Ca2+ homoeostasis,

particular during chronic β-adrenergic stress induced by ISO. Decreased Ca2+ transient

amplitude and delayed AP repolarization were observed in Pak1 global deficiency

(Pak1KO) mice (DeSantiago et al., 2013). Consistently, increased diastolic Ca2+,

decreased systolic Ca2+ were demonstrated in Pak1cko myocytes, along with prolonged

SR refill rate, especially under ISO conditions indicating the impaired function of

SERCA with Pak1 deficiency (Wang et al., 2014b). In Pak1KO mice, exercise failed

to increase the sensitivity of myofilament responsiveness to Ca2+, and Ca2+-dependent

sarcomere tension was unaltered (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 under

chronic ISO 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 induced by ISO, 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 the kinetics of Ca2+ release from the sarcolemma reticulum (SR) were

11

improved with reductions in Ca2+ upstroke, which reduced the time constant of Ca2+

decay and reduced velocity of propagation. Thus, overexpression of constitutively

active Pak1 abolished ISO-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 on Ca2+ handling and Ca2+ homoestasis under both

physiological and β-adrenergic stressed conditions. Activation of the β-adrenergic

pathway leads to an increase in the levels of cyclic-adenosine monophosphate

(cAMP), subsequently leading to activation of cAMP-dependent protein kinase A

(PKA) (Sheehan et al., 2007). In cardiac myocytes, ion channels which 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, protein kinase A-anchoring protein (mAKAP), which

serves as a scaffold for PKA, cAMP-dependent phosphodiesterase 4D3 (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

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

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the implications.

Increasing studies have indicated that PKA agonists are responsible for the

regulation of LTCC activity through PKA-dependent phosphorylation (Osterrieder et

al., 1982). H-89, a PKA inhibitor, was reported to reduce ICaL density via inhibiting

LTCC α1c-subunit phosphorylation in ventricular myocytes (Bryant et al., 2014). In

addition, dephosphorylation of LTCC is shown to decrease LTCC currents in rat

ventricular myocytes (duBell et al., 1996). A selective PP2A inhibitor, fostriecin,

normalised the t-tubule ICaL density in ISO-treated ventricular myocytes; indicating

PP2A suppresses LTCC activity under ISO stimulation (Chen et al., 2002; Kashihara

et al., 2012).

Sphingosine-1-phosphate (S1P, a circulating bioactive sphingolipid), was

found to regulate intracellular Ca2+ handling through LTCC through the Pak1-PP2A-

mediated signalling pathway (Egom et al., 2016). S1P alleviated the increase of ICaL

amplitude and spontaneous Ca2+ waves induced by ISO in rat ventricular myocytes.

This study also showed increased co-localisation of Pak1 and PP2A, suggesting that

S1P blunted the β-adrenergic stimulation via Pak1-PP2A interaction (Egom et al.,

2016). Numerous studies suggested S1P can modulate intracellular Ca2+ levels in

myocytes (Guo et al., 1999), without affecting the basal activity of LTCC (Nakajima

et al., 2000). Furthermore, the non-selective PP1 and PP2A inhibitor, okadaic acid

(OA), partially reversed the ISO-induced responsiveness on ICaL in cultured Ad-

CaPak1 infected SAN cells. This confirms that Pak1 regulates the activity of LTCC

via elevation of PP2A activity (Ke et al., 2007).

Calcium is recycled back into the sarcolemma reticulum (SR) through

SERCA2a, which is inhibited by dephosphorylated phospholamban (PLN). PLN can

be phosphorylated by PKA at serine site 16, thus relieving its inhibition on SERCA2a

and promoting the re-uptake of Ca2+ into the SR (Kranias, 1985; Kuschel et al., 1999).

PP1 and PP2A can both dephosphorylate PLN; PP1 responsible for 60%-70% of PLN

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dephosphorylation (Macdougall et al., 1991). Recent studies have shown that

bradykinin can increase autophosphorylation of Pak1 and alter its translocation. This

was accompanied by dephosphorylation of PLN through PP2A, and 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

ISO stimulation. Instead, under such stress 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 was

attenuated in neonatal rat cardiomyocytes (NRCM) pre-treated with siRNA to

knockdown SRF, prior to infection with Ad-CaPak1 (Wang et al., 2014b).

Furthermore, phosphorylated and total PLN protein levels were unaltered in Ad-

CaPak1 infected myocytes, indicating either the absence of Pak1 participation or

participation of Pak1 in dephosphorylation activity at this site was not sufficient to

oppose the PKA-mediated PLN 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. Pak1 deficiency attenuated exercise-induced cardiac

remodelling through altered calcineurin signalling (Davis et al., 2015). Post-exercise

training, the Pak1KO mice displayed elevated levels in cTnT phosphorylation, a

reduction in tropomyosin phosphorylation, reduced total PLN levels and a reduction

in PLN phosphorylated, as well as decreased SERCA2a levels. This data implicates

that exercise-induced increase in myofilament responsiveness to Ca2+ and

phosphorylation of MyBP-C is Pak1-mediated (Davis et al., 2015). Conversely,

increased activity of Pak1 decreased the phosphorylation levels of cTnI and MyBP-C,

this was associated with enhanced Ca2+ sensitivity in myofilaments (Ke et al., 2004).

Over-expression of Pak1 induced a functional reduction in the Ser23/24

14

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 characterised by alterations of several signalling pathways. Pak1 has been

identified as a novel anti-hypertrophic regulator 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), α-adrenergic stimuli

phenylephrine (PE), or β-adrenergic stimuli ISO, are all capable of activating Pak1

through phosphorylation.

Pak1 phosphorylation is increased in ventricular tissues of control mice subjected

to transverse aortic constriction (TAC) stress (Liu et al., 2011b). Pak1cko mice

exhibited increased heart weight/tibia length (HW/TL) ratio, enlarger cardiomyocyte

cross-sectional area and reduced fractional shortening (FS) level compared to wild

type mice under TAC stress (Liu et al., 2011b). This indicates that Pak1 acts as an

anti-hypertrophic regulator in the heart. Furthermore, loss of Pak1 also resulted in

increased interstitial fibrosis and cardiomyocyte apoptosis in Pak1cko/TAC mice,

whilst Ad-CaPak1 infected NRCMs displayed higher survival rates despite being

subjected to the cardiotoxic anticancer drug, doxorubicin (Liu et al., 2011b; Mao et

al., 2008; Yuan et al., 2005).

Pak1 antagonises 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 ISO stress where Pak1

deficient mice showed increased susceptibility to LV (left ventricle) myocardial

hypertrophy associated with enhanced LV systole function with impaired LV diastole

relaxation (Taglieri et al., 2011; Liu et al., 2011b; Wang et al., 2014a). Hypertrophic

15

effects caused by PE stress was inhibited in NRCMs infected by Ad-CaPak1, whereas

hypertrophy progressed as predicted in PE treated control NRCMs (Liu et al., 2011b).

Consistently, the cardioprotective effects of Pak1 was further confirmed by subjecting

cardiac-specific overexpression of constitutively active Pak1 (Pak1cTG) mice to TAC

stress. The detrimental effects of TAC stress were significantly reduced in Pak1cTG

mice, this was evident as TAC-induced increases in fibrosis, LV mass, LV end-

diastolic diameter, LV end-systolic diameter, and reduced fraction shortening were

diminished (Tsui et al., 2015).

Pak1 in MAPK Signalling Pathways

Post activation by upstream Cdc42/Rac1, Pak1 activates downstream mitogen

activated protein kinase kinases (MAPKK), MKK4 and MKK7, whereby post

activation of either kinase phosphorylate the JNK1/2 cascade (Coso et al., 1995;

Liang et al., 2003). Loss of Cdc42 in cardiomyocytes rendered mice more capable of

cardiac hypertrophic growth and 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 Pak1cko/TAC mice 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 maximal in ISO-treated Pak1KO mice, indicating Pak1 potentially

plays an inhibitory role on ERK1/2 activation in vivo under ISO treatment (Taglieri et

al., 2011). ERK1/2 inhibitor attenuated the development of myocardial and myocyte

hypertrophy in ISO-treated wild type and Pak1KO mice, indicating ERK1/2 as a

major kinase driving ISO-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 from β-adrenergic stimulation (Ke

16

et al., 2004). Studies have shown that β-adrenergic stimulation triggers enhanced

ERK1/2 phosphorylation due to a suppression of PP2A activation in the absence of

Pak1, therefore 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). Under TAC-stress, the absence of Pak1 in the heart led to

exacerbated increased protein levels of calcineurin due to abolished up-regulation of

Fbxo32. PE stress induced an up-regulation of Fbxo32 at mRNA and protein level,

which was eliminated in Pak1 knockdown NRCMs, resulted in calcineurin

accumulation. 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 Smad3-null mice expressed increased

susceptibility to TAC-induced hypertrophy in the heart (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 PE-induced Smad3 phosphorylation despite

the presence of Pak1; indicating Pak1 activation of Smad3 is likely through the

MKK7/JNK pathway (Tsui et al., 2015). These results highlight the therapeutic

potential of Fbxo32 itself through its pharmacological activator Berberine, which is

reported to improve cardiac function (Huang et al., 2015; Lau et al., 2001; Zhang et

al., 2014). Berberine prevented TAC-induced hypertrophic remodelling by improving

cardiac functions in both Pak1cko and wild type mice under TAC stress. These data

suggest that Fbxo32 induction antagonises pathological hypertrophic remodelling

caused by Pak1 deficiency under TAC (Tsui et al., 2015).

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Other studies directly enhanced Pak1 function through utilising the Pak activating

peptide, PAP, which abolished Ang II-induced hypertrophy in vitro and in vivo, as

well as associated ventricular arrhythmias (Wang et al., 2014a). Alternatively, miR-

30c was suggested to potentially target Cdc42 and Pak1 mRNAs. Over-expression of

miR-30c attenuated cardiomyocyte hypertrophy through mediating the high glucose-

induced elevation of Cdc42 and Pak1 expression (Raut et al., 2015).

Overall, these results suggest that activation of Pak1 exerts beneficial effects in the

prevention of cardiac hypertrophy through modifying the activities and

phosphorylation status of downstream proteins, hence, Pak1 and its associated

components act as promising potential therapeutic targets for the future treatment of

hypertrophy.

The role of Pak1 in Ischemia/Reperfusion (I/R) Injury

Myocardial I/R injury is caused by severe re-established coronary blood supply,

which 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).

Emerging evidence indicates a role for Pak1 in I/R as Pak1/Akt and Pak1/PP2A

signalling pathways have been suggested as downstream signalling molecules for S1P

and bradykinin in cardiac tissue; resulting in cardioprotection during I/R (Egom et al.,

2010b). Phosphoinositide 3-kinase (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 through inhibition of cardiac myocyte

apoptosis. Pak1KO mice demonstrated depressed recovery of cardiac function during

I/R, including reduced percentage recovery of LV developed pressure and an

exacerbated increase in end diastolic pressure. This indicates the involvement of Pak1

in the regulation of developed pressure during I/R in mice heart (Monasky et al.,

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2012). Moreover, increased myofilament response to Ca2+ within the heart has been

reported to be less susceptible to I/R (Arteaga et al., 2005), this implicates that Pak1-

induced elevation of myofilament Ca2+ sensitivity could possibly diminish I/R

originated effects 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 ischemic pre- and postconditioning in

knockout animal studies; S1P has been demonstrated to protect isolated mice hearts

against global I/R damage (Jin et al., 2007; Jin et al., 2002). S1P receptor agonists

were demonstrated to also 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 eNOS

phosphorylation, resulting in the activation of eNOS and consequently NO-mediated

cytoprotection against ischemic damage (Michell et al., 1999).

Pak1 and Phosphatases

Pak1 and PP2A activation have been demonstrated to improve cardiac contractile

function during I/R through regulating the phosphorylation levels of cTnT and MLC2

(myosin light chain 2) (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 autophosphorylation of Pak1 and

19

alter its translocation. This was accompanied by dephosphorylation of PLN through

PP2A, and consequently inhibiting Ca2+ reuptake by the SR, hence slowing the Ca2+

transient (slow relaxation) (Ke et al., 2010). Furthermore, studies have shown that

ablation of MLC2 phosphorylation caused decreased ventricular contractility, thus

lengthening the duration of ventricular ejection and other sarcomeric proteins

(Scruggs et al., 2009). Through 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 uncovering of Pak1 in the prevention of

arrhythmogenesis associated with I/R is vital to the on-going development of current

therapies for the treatment of I/R injury-induced ventricular arrhythmias.

Pak1 as a Therapeutic Target

The involvement of Pak1 on current activities and Ca2+ homoeostasis suggests its

potential role in preventing cardiac arrhythmias. Sphingosine (SPH) and SPH-derived

lipids have been shown to directly activate Pak1 (Bokoch, 2003), and SPH has been

suggested to play an important role in cardiac protection against I/R including studies

utilising FTY720 (Fingolimod, 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 FTY720 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 FTY720 prevented I/R injury-induced

arrhythmic events in langendorff ex vivo heart models, including the occurrence of

premature ventricular beats, VT (ventricular tachycardia), sinus brady arrhythmias

and atrioventricular conduction block (Egom et al., 2010a). Our study indicates that

FTY720 triggers auto-phosphorylation of Pak1 and subsequent Akt phosphorylation

through Gi-mediated signalling pathways in cardiomyocytes (Egom et al., 2010a).

20

Moreover, FTY720 also stimulates NO production via the PI3K/Akt/eNOS signalling

pathway (Egom et al., 2011).

Our more recent in vitro and in vivo studies have demonstrated FTY720 has

cardioprotective potential through inhibition of hypertrophy as well as the reversal of

hypertrophy. This protection is through activation of Pak1 and subsequent activation

of the MKK7-JNK1/2 pathway (Liu et al., 2011b). FTY720 treated TAC stressed mice

exhibited a reduction in pathological hypertrophic remodelling in the heart, evidenced

by reduced HW/TL ratio, reduced cardiomyocyte cross-sectional area, and reduced

interstitial fibrosis (Liu et al., 2013). FTY720 ameliorated cardiac functional

performance as shown by increased FS, increased ejection fraction and decreased

diastolic velocity; when under TAC stress (Liu et al., 2013). FTY720 was

demonstrated to activate the Pak1/NFAT signalling pathway through interacting with

Gi-coupled sphingosine 1-phosphate receptors; as PTX was able to inhibit the effects

of FTY720 (Liu et al., 2013). FTY720 treatment was demonstrated to activate Pak1,

and thereby negatively regulate NFAT activity through MKK7 and JNK1/2, resulting

in the reversal of hypertrophy (Liu et al., 2013). The reduction in fibrotic response

following FTY720 treatment is demonstrated through decreased expression levels of

periostin in the extracellular matrix (ECM); this was accompanied with reduced TGF-

β signalling (Liu et al., 2013). Studies using periostin deficient mice also exhibited

reduced fibrosis and hypertrophy following pressure overload, whereas

overexpression of periostin caused age-related spontaneous hypertrophy (Oka et al.,

2007, Lorts et al., 2009). Furthermore, FTY720 treatment reduced Ang II-induced

periostin expression (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, reduced periostin expression by FTY720 treatment

is likely due to the blunting of TGF-β activation, which in turn further down-regulates

periostin expression levels. Collectively, FTY720 reverses hypertrophy, reduces the

fibrotic response, restores ECM integrity and improves cardiac performance(Liu et

al., 2013).

21

The cardioprotective effects of FTY720 demonstrated through the protection of

cardiac function during I/R, as well as the prevention and reversal of hypertrophy, has

revealed FTY720 as a very promising pharmacological component for the treatment

of heart disease.

Conclusion

Over 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 FTY720 and berberine,

components of the Pak1 signalling pathway, as well as Pak1 itself, are promising

potential targets for future therapeutic treatment of cardiac diseases.

22

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 PLN, 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.

23

Molecules Cardiac phenotypeGain-of-function Loss-of-function

Pak1 Anti-hypertrophy (Liu et al., 2011b), anti-arrhythmias in I/R model (Egom et al., 2011)

Cardiac hypertrophy (Liu et al., 2011b), ventricular arrhythmias and abnormal

Ca2+ homeostasis (Wang et

al., 2014)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 et al., 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)

PP1Reduced 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.,

Enhanced LV contractility (Miyazaki et al., 2012), hypertrophy (El-Armouche et al., 2008), enhanced cardiac contractile responses to β-agonists (Carr et al., 2002)

24

2002), mild β-adrenergic desensitisation, protective effect from isoprenaline-induced cardiac hypertrophy and arrhythmias (El-Armouche et al., 2008)

MKK4/7 Protection against cardiomyocyte apoptosis, prevention of cardiac hypertrophy and ventricular remodelling (Choukroun et al., 1999a; 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-ischemic recovery (Liao et al., 2007)

JNK Hypertrophy (Choukroun et al., 1999b), 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 et al., 2015)

Aggravated cardiac and mitochondrial dysfunction from IR injury (Jang et al.,

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)

F-Box32  Anti-hypertrophy (Tsui et al., 2015)

 Dilated cardiomyopathy (Al-Hassnan et al., 2016; Al-Yacoub et al., 2016)

Table 1. Cardiac phenotypes of molecule’s gain -or loss-of-function models.

25

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., 2015).

Acknowledgements

This study was supported by the British Heart Foundation

(PG/14/71/31063, PG/14/70/31039 and FS/15/16/31477).

Competing Interests’ Statement

None

26

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