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Review

The junctophilin family of proteins:

from bench to bedside

1 2 2,3

Andrew P. Landstrom , David L. Beavers , and Xander H.T. Wehrens

Department of Pediatrics, Cardiovascular Research Institute, Baylor College of Medicine, Houston, TX 77030, USA

Department of Molecular Physiology and Biophysics, Cardiovascular Research Institute, Baylor College of Medicine, Houston, TX

77030, USA

Department of Medicine (Cardiology), Cardiovascular Research Institute, Baylor College of Medicine, Houston, TX 77030, USA

Excitable tissues rely on junctional membrane com- cells from striated muscle to neurons. JPHs were originally

plexes to couple cell surface signals to intracellular discovered using an immunoproteomic monoclonal anti-

channels. The junctophilins have emerged as a family body library. This library was created from mice immu-

of proteins critical in coordinating the maturation and nized with rabbit skeletal muscle membranes taken from

maintenance of this cellular ultrastructure. Within skel- transverse tubules (T-tubules) and muscle triads [2]. JPHs

etal and cardiac muscle, junctophilin 1 and junctophilin contain speciﬁc protein domains which deﬁne them as a

2, respectively, couple sarcolemmal and intracellular family including eight amino-terminal membrane occupa-

calcium channels. In neuronal tissue, junctophilin 3 tion and recognition nexus (MORN) motifs separated by a

and junctophilin 4 may have an emerging role in cou- joining region, followed by an a-helix, divergent, and a

pling membrane neurotransmitter receptors and intra- C-terminal transmembrane motif that anchors the protein

cellular calcium channels. These important physiological into the endoplasmic reticulum (ER) or sarcoplasmic

roles are highlighted by the pathophysiology which

results when these proteins are perturbed, and a grow-

ing body of literature has associated junctophilins with

the pathogenesis of human disease. Glossary

Akinetic–rigid syndrome: a constellation of symptoms characterized by a

An emerging role in the pathophysiology of excitable paucity of voluntary movements which, when present, are slow and associated

cells with increased muscle tone/rigidity.

Alternative splicing: a molecular process during which the mRNA transcribed

Since the sentinel discovery of the junctophilin (JPH)

from a single gene can be processed differently to create various mature

family of proteins a decade ago [1], a rapidly progressing mRNA sequences which are then ultimately translated into different proteins.

Calcium-induced calcium release (CICR): the release of a relatively large

body of literature has linked these proteins to critical roles

amount of stored intracellular calcium triggered by a relatively small amount of

in all excitable cells with implications for human physiol-

calcium entering the cell.

ogy and pathophysiology. Through maintaining critical Chorea: a neurological disorder characterized by abnormal involuntary move-

ments which are generally abrupt, irregular, and nonstereotyped in nature. It is

subcellular architecture, and an emerging function as a

derived from the Greek word meaning ‘dance’.

direct regulator of calcium-handling proteins, impaired

Diastole: the active relaxation of the heart or cardiac myocyte.

JPHs have been implicated in a number of human dis- Dilated cardiomyopathy (DCM): enlargement and dilation of the heart

associated with impaired function and weak contraction.

eases. Investigations into the role of JPHs in cellular

Excitation–contraction coupling (EC coupling): mechanical cellular contrac-

physiology have elucidated novel mechanisms of skeletal

tion, such as in a striated muscle cell, is linked to cellular excitation such as a

muscle myopathy, cardiomyopathy, heart failure, ar- change in the sarcolemmal membrane potential.

Huntington’s disease (HD)-like syndrome: a clinical entity mimicking the clinical

rhythmia, behavioral and learning deﬁcits, motor control,

presentation of HD, including progressive involuntary and uncoordinated muscle

and Huntington’s disease (HD)-like pathology. Future

movements (chorea) and subcortical dementia in a patient without an identifiable

investigations hold the promise of novel therapeutic tar- trinucleotide expansion mutation in the HTT-encoding huntingtin protein.

Hypertrophic cardiomyopathy (HCM): asymmetric hypertrophy of predomi-

gets and molecular interventions for a number of these

nantly the left ventricle of the heart in the absence of a clinically identifiable

diseases. cause of hypertrophy such as comorbid hypertension.

Junctional membrane complex (JMC): a complex formed by a plasma

membrane and a juxtaposed intracellular membrane such as the ER or SR,

Protein phylogeny and structure

which hosts a number of proteins required for intracellular ion signaling.

JPHs are members of a family of junctional membrane Purkinje cells: a type of neuron located in the cerebellum of the brain.

Store-operated calcium entry (SOCE): a cellular process to replace a depleted

complex (JMC)-associated proteins found in all excitable

store of intracellular calcium, such as within the SR, characterized by a net

influx of calcium across the plasma membrane to replenish the store.

Corresponding author: Wehrens, X.H.T. ([email protected]). Transient: a rapid increase in cytosolic calcium resulting from release of

Keywords: calcium; excitation contraction coupling; JPH; junctophilin; mutation. intracellular store calcium.

Transverse tubule (T-tubule): a finger-like invagination of the sarcolemma of

1471-4914/$ – see front matter

striated muscle cells, which allows penetration of a membrane potential into the

myocyte.

Supraventricular tachycardia: an arrhythmia of the heart with rapid heart rate

with an arrhythmic origin above the ventricles.

Systole: contraction of the heart or cardiac myocyte.

Trends in Molecular Medicine, June 2014, Vol. 20, No. 6 353

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Review Trends in Molecular Medicine June 2014, Vol. 20, No. 6

(A) MORN I MORN II JPH1 Joining α-helical Divergent TM

1 LTCC 658

(B) S101R Y141H S165F E169K

JPH2 Joining α-helical Divergent TM 1 696

Exon 1 2a 2 3 4 5

JPH3 Joining α-helical Divergent TM 1 748 (D) JPH4 Joining α-helical Divergent TM 1 628

TRENDS in Molecular Medicine

Figure 1. Protein topology of the four junctophilin isoforms expressed in humans. The amino terminus has eight MORN motifs (light blue fill) distributed across two MORN

domains separated by a joining region (white fill). The carboxy terminus contains a transmembrane domain (white fill) which embeds this end of the protein into

intracellular membranes such as the sarcoplasmic reticulum in striated muscle. An a-helical domain (white fill) and a less evolutionarily conserved divergent region (dark

blue fill) join these two termini. The isoforms vary in length from 628 (JPH4) to 758 (JPH3) residues. (A) Protein topology of JPH1 found predominantly in skeletal muscle.

JPH1 binds to the LTCC via a binding domain within the joining region of the protein. (B) Protein topology of JPH2, the major cardiac isoform. Three JPH2 mutations have

been linked with the development of hypertrophic cardiomyopathy (blue text) and one to arrhythmogenesis (black text). (C) Top: Schematic of the two alternatively spliced

transcripts of JPH3 including the untranslated regions (white fill) and coding exons (blue fill). The full-length transcript consisting of five exons which encode JPH3 is

depicted as well as the two exon alternative transcript containing the CTG trinucleotide repeat expansion in the alternate exon 2 (2a). Below is the protein topology of JPH3.

(D) Protein topology of JPH4. Abbreviations: JPH, junctophilin; LTCC, L-type calcium channel binding domain; MORN, membrane occupation and recognition nexus; TM,

transmembrane domain.

reticulum (SR) [1,3]. The protein topology for each JPH region. Highly conserved across JPH isoforms, in silico

isoform is depicted in Figure 1. analysis predicts multiple a-helical stretches within the

MORN motifs have been found to be highly conserved primary sequence of this domain. Spanning approximately

across all JPH isoforms as well as across various species 100 amino acids, it is thought to bridge the gap between the

and demonstrate a consensus sequence of YxGxWxxGxRH- plasmalemma/sarcolemma and the ER/SR [1,3,7]. Given

GYG [4]. Initially identiﬁed by BLAST analysis, the eight the possible a-helical structure, this domain may provide

MORN motifs in JPHs are separated by a ‘joining region’, mechanical elasticity to regulate dyad distance [1,4]. Con-

which links MORN motifs I–VI to VII and VIII. As a whole, versely, the divergent region derives its name from the

the MORN motifs are thought to trafﬁc and bind JPHs to relatively low primary sequence conservation between the

the plasma membrane. This hypothesis was based on early various JPH isoforms, and its role is unclear at present [4].

experiments which truncated the carboxy terminus of the Overall, these presumed functional domains allow for

protein and resulted in localization of the MORN motif- JPHs to play critical roles in the maintenance of cellular

containing amino terminus strictly to the plasma mem- ultrastructure, particularly the interface between intracel-

brane [1]. Further, homologous MORN motifs in plants, lular membranes such as the ER/SR and the plasmalem-

such as Arabidopsis thaliana, have high afﬁnity for mem- ma/sarcolemma.

brane phospholipids such as phosphatidic acid and some- Initial studies in rodent models as well as in-depth

what lower afﬁnity for membrane phosphatidylinositol bioinformatic and phylogenic analysis have identiﬁed four

(PI)4P and PI(4,5)P2 [5]. In addition to JPHs, multiple major isoforms of JPH family members in higher chordate

MORN motifs are found in several other classes of proteins organisms, including humans [4]. Each isoform is prefer-

including histone–lysine N-methyltransferase and phos- entially expressed in various excitable tissues and include

phatidylinositol-4-phosphate 5-kinase proteins [1,6]. skeletal muscle JPH1-encoded junctophilin type 1 (JPH1),

The carboxy-terminal membrane-spanning domain of cardiac muscle JPH2-encoded JPH2, and neuronal tissue

JPH is believed to embed the protein into intracellular JPH3-encoded JPH3, and JPH4-encoded JPH4. A phylo-

membranes such as the ER or SR [1,3]. This domain is genic tree generated from multiple JPH isoforms across a

highly conserved across species and isoforms, and in silico diverse group of species is depicted in Figure 2.

secondary structure analysis as well as hydrophobicity

analysis predict the presence of this domain [1,4]. Linking Overview of calcium signaling in striated muscle

the MORN motifs and the membrane-spanning domain are A major role of Ca within muscle is the initiation and

two domains – the a-helical domain and the divergent coordination of myoﬁlament contraction [8–11]. In cardiac

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B.taurus_JPH4 coding exons localizing to 8q21 and encodes a 658 amino

R.norvegicus_JPH4

acid 90 kDa protein in humans [15]. JPH2-encoded JPH2

M.musculus_JPH4 JPH4

H.sapien_JPH4 is also expressed in skeletal muscle and is the predominant

C.familiaris_JPH4*

cardiac isoform [3]. JPH1 maintains the critical ultrastruc-

S.scrofa_JPH4*

D.rerio_JPH3* tural geometry of the skeletal muscle triad which com-

G.gallus_JPH3*

H.sapien_JPH3 prises three independent membranous structures

R.norvegicus_JPH3 including the T-tubular invagination of the sarcolemma

M.musculus_JPH3 JPH3

E.caballus_JPH3* ﬂanked on two sides by cytosolic SR [16]. Loss of JPH1

B.taurus_JPH3 expression has been shown to reduce the number of intact

C.familiaris_JPH3*

M.domesca_JPH3* triads and to deform intact triads [17]. A detailed explana-

D.rerio_JPH2

tion of skeletal muscle triads, cardiac dyads, and the

T.nigroviridis_JPH2*

G.gallus_JPH2* junctional membrane complex can be found in Box 1.

R.norvegicus_JPH2

In addition to a structural role, JPH1 directly binds and

M.musculus_JPH2 JPH2

O.cuniculus_JPH2 regulates a number of proteins within the skeletal muscle

H.sapien_JPH2

JMC. JPH1 directly interacts with the sarcolemmal LTCC

M.mulaa_JPH2*

M.domesca_JPH2* through a binding domain on JPH1 spanning from amino

D.rerio_JPH1

acids 232 to 369 within the joining region domain of the

T.nigroviridis_JPH1*

X.tropicalis_JPH1 protein. In addition, JPH1 directly interacts with sarco-

G.gallus_JPH1*

lemmal CAV3-encoded caveolin type 3 (Cav3) [18]. JPH1

C.familiaris_JPH1*

B.taurus_JPH1 JPH1 also directly interacts with the skeletal muscle-speciﬁc,

O.cuniculus_JPH1

H.sapien_JPH1 RYR1-encoded ryanodine receptor type 1 (RyR1) which

M.mulaa_JPH1* mediates release of Ca stored within the SR. This inter-

R.norvegicus_JPH1

M.musculus_JPH1 action is mediated through highly reactive thiol groups,

which are sensitive to oxidative insult, and changes in this

0.2

interaction can alter gating of RyR1. Overall, these inter-

TRENDS in Molecular Medicine

actions suggest a mechanism through which release of SR-

Figure 2. Junctophilin phylogenic tree. A phylogenic tree comprises the primary stored Ca via RyR1 is mediated by a direct interaction

sequences of 35 JPH isoforms derived from multiple species created using

with JPH1 in an oxidation-dependent manner [19]. A

MUSCLE alignment and PhyML for phylogeny and TreeDyn tree rendering [4,77–

cartoon depicting JPH1 within the skeletal muscle triad

79]. Clades containing each of the isoforms are indicated. *Indicates that the

protein sequence is a putative JPH isoform that has not been experimentally is illustrated in Figure 3.

validated. Abbreviation: JPH, junctophilin.

In vitro, colocalization of LTCC and RyR1 has been

demonstrated by immunoﬂuorescence into discrete areas

along C2C12 murine skeletal myoblasts. This localization

myocytes, Ca -mediated contraction signaling begins is disrupted following expression silencing of JPH1 and is

2+ 2+

with the opening of voltage-gated L-type Ca channels associated with impaired LTCC Ca inﬂux [18]. Further,

(LTCCs) at the sarcolemma in response to membrane JPH1 expression silencing in vitro decreases store-operat-

2+ 2+ 2+

excitation. LTCC Ca inﬂux down its electrochemical ed Ca entry (SOCE), which balances net Ca loss from

2+ 2+

gradient triggers a larger Ca release from the SR via intracellular Ca stores [17]. This results in reduced SR-

2+ 2+

the intracellular Ca release channel ryanodine receptor stored Ca release via RyR1 [17]. Follow-up studies sug-

2+ 2+

type 2 (RyR2) in a process known as Ca -induced Ca gest that impaired SOCE may occur through disruption

release (CICR; see Glossary). In this manner, Ca travel-

ing across the cardiac dyad triggers the opening of RyR2

2+ Box 1. Subcellular architecture and the junctional

and release of SR Ca without direct coupling between the

membrane complex

LTCC and RyR2. This process serves as the molecular

initiator for ATP-expending myoﬁlament-based mechani- Precise subcellular architecture of intracellular membrane and

associated proteins is required for effective transmission of

cal contraction of the heart. Contraction is terminated by

2+ membrane excitation to mechanical contraction of skeletal and

active removal of Ca from the cytosol into the SR via the

cardiac muscle. To facilitate this, the sarcolemma of striated muscle

action of the sarcoplasmic reticulum calcium ATPase

cells has deep, finger-like invaginations that penetrate the interior of

(SERCA2a) or removal from the cell by the sodium–calci- the myocyte. Running parallel along these T-tubules are portions of

the SR which are so-called junctional SR. In skeletal muscle, the SR

um exchanger (NCX1). Although skeletal muscle is analo-

2+ runs alongside a shared portion of the T-tubule on two opposing

gous in many ways, store Ca release is directly triggered

sides which create a triad of membranes observed on microscopy.

by alterations in sarcolemmal potential, a process known

In cardiac muscle, a single junctional SR runs alongside the T-tubule

as voltage-dependent calcium release [12]. During this over a given portion of the T-tubule appearing as a dyad. The

process, voltage changes directly trigger release of store distance of the triad or dyad is carefully controlled and is critical for

2+ 2+

efficient movement and signaling of ions, particularly Ca . In the

Ca through direct coupling of the LTCC and RyR1

[13,14]. cardiac cell, it is the movement of extracellular Ca across the

sarcolemma and the cardiac dyad which triggers CICR and allows

for efficient EC coupling. A host of proteins, including JPHs, are

Junctophilin type 1 and skeletal muscle crucial to the development and maintenance of this architecture.

JPH1-encoded JPH1 is the major JPH family member Further, the proteins which localize to the interface of these

membranes and are responsible for Ca crosstalk are referred to

expressed in skeletal muscle but also demonstrates a

as the JMC.

low level of expression in the heart. JPH1 contains ﬁve

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Skeletal muscle myopathy and damage

T-tubule Extracellular space

Cav3 Mice lacking JPH1 die shortly after birth due to defective

suckling. This early mortality cannot be rescued by place-

LTCC

ment of an orogastric tube for feeding, suggesting that milk

JPH1 RyR1

JMC aspiration or respiratory failure, probably due to pharyn-

geal muscle and diaphragmatic dysfunction, contributed to

S pup death [26]. Skeletal myocytes isolated from these mice

Skeletal myocyte demonstrate fewer apparent JMCs, reduced contractile

force, and reduced Ca -mediated twitch tension [26]. In Sarcolemma

Cytosol Ca addition, this perinatal mortality is associated with failure

SR of normal triad development as well as disruption and

vacuolization of normal SR structure [27]. These studies

Triad T-tubule suggest that JPH1 plays a key role in skeletal muscle EC

coupling and that disruption or impairment of JPH1 func-

tion is associated with poor skeletal muscle function

through a loss of cellular ultrastructure and triad develop-

TRENDS in Molecular Medicine ment.

JPH1 expression has been shown to decrease after

Figure 3. JPH1 and the skeletal muscle triad. Cartoon of JPH1 and the skeletal

myocyte triad. The N terminus of JPH1 localizes to the sarcolemmal membrane of skeletal muscle damage from eccentric contraction, a type

the skeletal myocyte T-tubule while simultaneously approximating the SR

of contraction in which the muscle elongates under tension

membrane via a C-terminal transmembrane domain, which allows it to

approximate the triad subcellular membrane architecture. The N-terminal aspect due to an opposing force, which is greater than the tradi-

of JPH1 (light blue) binds sarcolemmal-limited Cav3 (red) and the LTCC (dark blue), tional contractile force generated by the muscle [28]. This

whereas the C-terminal aspect directly binds RyR1 (green) within the SR. The

loss of expression coincides with loss of EC coupling, and

JPH1–RyR1 interaction is mediated by reactive thiols interactions (depicted by S)

which regulate RyR1 channel gating. Together, these proteins, along with other gradual return of JPH1 protein levels correlates with

neighboring proteins, comprise the JMC. Inset, a schematic depicting the

recovery of EC coupling and force generation. Further

ultrastructure of the triad within skeletal myocytes. Abbreviations: Ca , calcium;

investigation found that increased skeletal muscle contrac-

Cav3, caveolin 3; JMC, junctional membrane complex; JPH1, junctophilin 1; LTCC,

L-type calcium channel; RyR1, ryanodine receptor 1; SR, sarcoplasmic reticulum. tion, as well as directly increasing cytosolic Ca levels to

supraphysiological levels, result in calpain-mediated pro-

teolysis of JPH1 [29]. Speciﬁcally, JPH1 is cleaved proxi-

of stromal interaction molecule 1 (STIM1) and calcium mal to the C terminus creating a 75 kDa and a 17 kDa

release-activated calcium modulator 1 (ORAI1) [20]. In fragment with a loss of expression of full-length protein.

this model, depletion of SR store Ca allows for STIM1 Interestingly, aberrant JPH1 proteolysis was identiﬁed in

oligomerization within the SR and interacts with ORAI1, a murine mouse model of muscular dystrophy providing an

which allows for SOCE through the sarcolemma. This association with the development of primary muscle dis-

interaction is disrupted with loss of JPH1 expression ease [29]. These ﬁndings suggest that eccentric contrac-

impairing the ability of the myocyte to increase sarcolem- tion, prolonged muscle contraction, and other mechanical

2+ 2+

mal Ca inﬂux to balance SR Ca depletion. RyR1 func- stress may develop impaired contractile force and EC

tion is also regulated by the transient receptor potential coupling secondary to loss of JPH1 function.

channel canonical type 3 (TRPC3) [21]. Knockout of TRPC3

in primary skeletal myotubes demonstrates impaired Junctophilin type 2 and cardiac muscle

RyR1 excitation–contraction (EC) coupling gain and SR The JPH2 gene contains ﬁve coding exons localizing to

2+ 2+

Ca release with intact SR store Ca levels. Taken to- 20q13.12 and encodes a 696 amino acid 74 kDa protein

gether, these results support the role of JPH1 in regulating JPH2 in humans [15]. JPH2 is the major JPH family

the skeletal muscle triad ultrastructure and the JMC. This member expressed in cardiac muscle and shares signiﬁcant

role is critical to maintain efﬁcient EC coupling gain, SR primary sequence identity and functional parallels with

store Ca release, and SOCE. JPH1 in skeletal muscle. In contrast to skeletal muscle,

Although the precise mechanism of SOCE remains con- which demonstrates triadic ultrastructure, cardiac muscle

troversial, a body of evidence suggests that SOCE plays a characteristically has dyadic ultrastructure whereby a

minimal role, if any, during physiological muscle contrac- span of junctional SR runs parallel to one sarcolemmal

tion. As the majority of cytosolic Ca is removed from the T-tubular membrane at a given location [30,31]. JPH2

cytosol by SERCA into the SR, a relatively small amount of plays a critical role in maintaining this dyadic structure

2+ 2+

Ca is extruded from the myocyte [22]. Conversely, during and associated effective Ca signaling. A cartoon depict-

nonphysiological experimental conditions, or under states of ing JPH2 within the cardiac dyad is depicted in Figure 4.

muscle fatigue, SOCE plays a critical role in maintaining JPH2 null mice, although embryonic lethal, demon-

store Ca homeostasis [23,24]. Further, because there is strate a 90% increase in the distance of the cardiac dyad

minimal net extravasation of cytosolic Ca from intact as well as vacuolization of the SR [1]. This disruption in

skeletal myocytes during relaxation, SOCE is seen more cellular ultrastructure was associated with random cardi-

prominently with in vitro myoblasts, such as C2C12 cells omyocyte contraction and irregular Ca transients which

[25]. Thus, additional studies are needed to further eluci- occurred spontaneously without Ca inﬂux through the

date the role of JPH1 in regulating SOCE utilizing in vivo LTCC. Further, Ca transients in JPH2 null mice are

models of intact, adult skeletal myocytes. signiﬁcantly reduced in amplitude perhaps due to the

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These studies in developing cardiomyocytes correlate

LTCC Extracellular space

T-tubule Cav3 well with studies in differentiated cardiac cells. JPH2

expression silencing in cardiac cells in vitro has been

JPH2 2+

shown to impair CICR with decreased Ca transient

JMC RyR2 amplitude and blunting of spontaneous generation of

Ca transients [37]. These ﬁndings were seen with un-

changed expression of the LTCC, RyR2, NCX, SERCA, and

Cardiac myocyte phospholamban and suggest altered function of RyR2

Sarcolemma rather than altered protein expression. Induced JPH2

Cytosol Ca2+ knockdown in adult mice demonstrated RyR2 hyperactivi-

SR ty with increased spark frequency despite impaired CICR

Dyad SR

and EC coupling gain [7]. Co-immunoprecipitation showed

T-tubule

that RyR2 directly binds JPH2, suggesting a role in the

SR regulation of RyR2 gating. Further, single molecule super-

resolution microscopy in rat ventricular myocytes identi-

ﬁed RyR2 clusters in close proximity to similarly shaped

TRENDS in Molecular Medicine

clusters of JPH2 with approximately 80% colocalization

Figure 4. JPH2 and the cardiac muscle dyad. Analogous to JPH1, the N terminus of [38]. These studies suggest that direct binding of JPH2 to

JPH2 (light blue) localizes to the sarcolemma membrane of the cardiomyocyte. The

RyR2 is necessary for proper RyR2 gating and loss of JPH2

N terminus binds sarcolemmal-limited Cav3 (red) and directly interacts with RyR2

expression results in reduced EC coupling gain during

(green) via the MORN motif joining region and possibly the C-terminal aspect of

the protein. This interaction regulates RyR2 gating and is responsible for CICR and increased RyR2 Ca leakage during diastole.

pathological Ca (yellow) release in some JPH2 mutations. Together, these 2+

In addition to Ca signaling, JPH2 plays a key role in

proteins, along with other neighboring proteins, comprise the JMC. The white

the development and stability of myocyte ultrastructure.

circles on JPH2 indicate the position of the three mutations associated with HCM,

whereas the black circle denotes the location of the mutation associated with Mice with decreased levels of cardiac JPH2 showed defec-

arrhythmia. Inset, a schematic depicting the ultrastructure of the dyad within

2+ tive postnatal T-tubule maturation, whereas mice over-

cardiac myocytes. Abbreviations: Ca , calcium; Cav3, caveolin 3; JMC, junctional

membrane complex; JPH2, junctophilin 2; LTCC, L-type calcium channel; RyR2, expressing JPH2 had accelerated T-tubule maturation by

ryanodine receptor 2; SR, sarcoplasmic reticulum. postnatal day 8 [39]. Further, independent studies utiliz-

ing acute RNAi-mediated knockdown of JPH2 in cultured

disruption of the SR ultrastructure [1]. From these early cardiomyocytes have shown T-tubule disorganization [40].

studies, JPH2 was originally viewed as a structural protein Taken together, these studies suggest that JPH2 plays

serving as a molecular tether which maintained the cardi- both a structural as well as protein-regulatory role within

ac dyad ultrastructure required for effective CICR and the cardiomyocyte. Reﬂecting this critical role, perturba-

Ca signaling. Recent studies have added complexity to tions in JPH2 expression or mutations result in cardiac

the function of this protein. pathology.

Development of the junctional membrane complex and Cardiomyopathy and arrhythmogenesis

the T-tubule Primary myocardial diseases, such as cardiomyopathies,

JPH2 plays a key role in cardiomyocyte development and are a common cause of morbidity and mortality worldwide

differentiation. Although it is possible that the amino- [41]. JPH2 has been linked to the development of various

terminal MORN motifs of JPH2 might directly interact types of myocardial disease in both rodent models as well

with the inner leaﬂet of the sarcolemma, JPH2 has also as humans. Murine genetic models of dilated cardiomyop-

been shown to bind Cav3, a protein which localizes to the athy (DCM) and hypertrophic cardiomyopathy (HCM)

sarcolemma [32]. Cav3 is necessary for the formation of demonstrated reduced JPH2 protein expression [32]. In

‘little cave’-like invaginations of the cardiomyocyte sarco- addition, a rat model of aortic banding pressure-induced

lemma called caveolae, as well as regulation of several hypertrophy similarly demonstrated reduced JPH2 ex-

cardiac ion channels including the LTCC and various K pression and was associated with disruption of the cardiac

channels [33,34]. Immunoﬂuorescent localization of the dyad and junctional SR surface [42]. In silico modeling of

LTCC, RyR2, Cav3, and JPH2 in rat myocytes demonstrat- the effect of this disrupted junctional interaction between

ed a temporal correlation between development of inﬂux of LTCC and RyR on CICR predicts slow Ca transient

Ca from the LTCCa, CICR, and T-tubule formation [35]. generation and reduced transient amplitude [43]. These

Whereas expression of Cav3 and RyR2 was observed early studies suggest that loss of JPH2 expression, and the

in development, JPH2 expression was not seen until day 15 resulting disruption in Ca transient generation, are

and was associated with formation of the T-tubule and associated with early molecular remodeling that may pre-

maturation of CICR. Further, embryonic stem cell derived cede pathological cellular remodeling.

cardiomyocytes with reduced JPH2 expression demon- Based on these ﬁndings, a series of studies were con-

strate perturbed cardiac development and decreased ducted to determine whether loss of JPH2 expression could

Ca transients that are sporadic and irregular. These drive cardiomyopathic disease. Knockdown of JPH2 in

cells also demonstrated disordered myoﬁlament produc- immortalized cardiac cells induced cellular hypertrophy,

tion, perturbed energy-generating capacity, and Ca dys- upregulation of several hypertrophy markers, including

regulation, which culminated in fewer spontaneously skeletal actin, atrial natriuretic factor, brain natriuretic

contractile myocytes [36]. factor, myosin heavy chain, regulator of calcineurin 1–exon

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4 splice isoform, and was associated with reduced transient Ca release via RyR1 [50]. Immunoprecipitation assays

amplitude [37]. In vivo, conditional cardiac knockdown of suggest that the S165F mutation impairs protein kinase

JPH2 in mice rapidly leads to heart failure and early C-mediated phosphorylation resulting in impaired bind-

mortality [7]. These mice demonstrated increased cardiac ing to TRPC3 [50]. TRPC3 had been previously found to

mass with dilated ventricles and reduced fractional short- modulate the function of RyR1 through a direct interaction

ening as well as reduced CICR due to reduced EC coupling with JPH2 in skeletal muscle and binds JPH2 near the

gain [7]. These murine model ﬁndings were supported by joining region [51,52]. Thus, the S165F mutation is be-

complementary human clinical studies where myocardium lieved to lead to skeletal myocyte hypertrophy through

taken from surgical resection of HCM patients with left impaired regulation of RyR1 function. JPH2–Y141H has

ventricular outﬂow tract obstruction demonstrated re- also been linked to skeletal muscle hypertrophy and al-

duced, and in some cases undetectable, levels of JPH2 tered Ca signaling [53]. Overexpression of JPH2–Y141H

[37]. In addition, cardiomyocytes isolated from patients in primary skeletal myotubes demonstrated an increase in

with DCM and ischemic cardiomyopathy were observed to cellular diameter and reduced EC coupling; however,

have reduced and mislocalized T-tubule–SR junctions with unlike S165F, impairment of RyR1-mediated SR Ca

reduced JPH2 expression [43]. release was not observed. Rather, increased SOCE via

Based on the observation that altered JPH2 expression Orai1 and increased resting Ca levels was observed.

is associated with cardiomyopathic disease and heart These ﬁndings suggest that the JPH2–Y141H mutation

failure, several studies have sought to identify whether is associated with skeletal muscle hypertrophy through a

mutations in the gene encoding JPH2 might be similarly mechanism independent of RyR1 impairment. Interest-

associated with disease. The sentinel study in this regard ingly, the initial probands hosting the JPH2–S165F and

identiﬁed three mutations localizing to the amino termi- JPH2–Y141H mutations demonstrated no clinical evi-

nus ﬁrst MORN motif domain and the linker domain of dence of skeletal muscle myopathy such as muscular

JPH2 [44]. These mutations include JPH2–S101R, dystrophy. Further, these studied mutations have not

Y141H, and S165F. Each mutation was identiﬁed in a been replicated using in vivo models and the clinical

Caucasian individual who demonstrated clear clinical relevance of these mutations on skeletal muscle remains

disease yet did not have a mutation in a gene traditionally unclear at present.

associated with the development of HCM [44–46]. As with

reduced JPH2 expression, these mutations were found to Junctophilin type 3 and type 4 and neuronal tissue

reduce CICR amplitude and disrupt cellular ultrastruc- JPH3 and JPH4 are primarily expressed within the neu-

ture resulting in cellular hypertrophy when overex- rons of the brain. Although the precise role of these pro-

pressed [44]. Two additional genetic variants localizing teins remains enigmatic, an emerging body of evidence

to the divergent domain, JPH2–R436C and G505S, were suggests that neuronal JPHs may play roles in mediating

reported in a small cohort of Japanese patients with HCM balance and motor control through maintenance of efﬁcient

[47]. Further genetic analyses have identiﬁed each of these Ca signaling. JPH3 knockout mice demonstrate reduced

mutations in control cohorts of ostensibly healthy individ- balance and impaired motor coordination at 3 months of

uals without HCM, thus it is likely these variants are age without overt alterations in brain morphology or sig-

polymorphisms without clinical signiﬁcance [48]. Recent- niﬁcant defects in molecular signaling [54]. Whereas JPH1

ly, a JPH2 mutation has been identiﬁed in a small family and JPH2 knockout mice demonstrated disrupted ultra-

with HCM and prominent clinical arrhythmia [49]. The structure of the skeletal myocyte triad and cardiomyocyte

JPH2–E169K mutation was associated with development dyad, respectively, JPH3 knockout mice had no discernible

of supraventricular arrhythmias in a small multigenera- disruption in neuronal cellular architecture. In addition,

tional family. A murine model of this mutation exhibited a these mice demonstrated no apparent difference in Pur-

higher incidence of pacing-induced atrial ﬁbrillation sec- kinje cell action potentials and normal synaptic function

ondary to abnormal spontaneous Ca waves and in- [54]. A follow-up study utilizing JPH3 knockout and hemi-

creased spark frequency resulting from decreased zygous knockout mice, aged 6 and 9 months of age, identi-

RyR2–JPH2 interaction [49]. These genetic studies sug- ﬁed progressive defects in neuromuscular strength,

gest that JPH2 may function beyond JMC structural coordination, and balance which was greater in the knock-

integrity and may also have a regulatory role on RyR2 out mouse and progressive over time [55]. JPH4 localizes to

and perhaps other JMC-associated proteins. Finally, al- spatially discrete areas in the brain compared with JPH3;

though the frequency of JPH2 mutations among patients however, the knockout mouse has no discernible phenotype

with HCM is rare (<1%), loss of JPH2 expression may [56,57]. Detailed intracellular Ca signaling studies in

demonstrate a common feature of hypertrophic and ar- these mice have not been done to date, nor has a compre-

rhythmic remodeling [48]. hensive analysis of all major functional regions of the brain

been done to determine whether a more robust phenotype

Skeletal muscle myopathy linked to JPH2 mutations may be identiﬁable. Despite these shortcomings, these

Although JPH2 mutations have been associated with results suggest that JPH3 and JPH4 are not required

cardiomyopathy and arrhythmias, a small number of for cellular architecture stabilization and brain develop-

studies have linked these mutations to perturbed skeletal ment individually, which raises the possibility that the

muscle function in vitro. Overexpression of the JPH2– function of these isoforms may be redundant.

S165F mutation in primary murine skeletal myotubes Supporting this hypothesis, JPH3/4 double knockout

resulted in hypertrophy, reduced CICR, and reduced SR mice demonstrate disrupted intracellular Ca signaling

358

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Review Trends in Molecular Medicine June 2014, Vol. 20, No. 6

[57]. Indeed, disrupted communication between plasma- Extracellular space

lemmal Ca entry via the N-methyl-D-aspartate (NMDA) P/Q or NMDAR

glutamate receptors and intracellular neuronal RyRs, as SK

well as the small conductance Ca -activated potassium

(SK) channels, was noted in hippocampal neurons [57]. JPH3 JPH4

Under normal physiological conditions, these plasmalem- RyR

mal and intracellular channels are necessary for intracel-

lular Ca signaling and proper neuronal function [57]. In

addition to this molecular defect in hippocampal neurons,

JPH3/4 double knockout mice also demonstrate a similar

2+ Ca2+

impairment in Ca signaling of Purkinje cells [58]. Spe- ER

ciﬁcally, Purkinje cells obtained from double knockout

mice have impaired Ca crosstalk between plasmalemmal Cytosol

P/Q-type voltage-gated channels and intracellular RyR TRENDS in Molecular Medicine

channels impairing SK channel-mediated after-hyperpo-

Figure 5. JPH3/4 and neuronal ultrastructure. In Purkinje cells, the two isoforms,

larization [58]. These mice demonstrate reduced memory

JPH3 and JPH4 (light blue), may be responsible for maintaining appropriate

and exploratory activity, as well as irregular hind limb spacing between the neuronal plasma membrane and the ER. This is critical for

effective Ca signaling from the P/Q-type channel (red), RyR (green), and SK

reﬂexes, ultimately culminating in death of young mice

channels (yellow). In other neuronal subtypes, such as hippocampal neurons, the

within 5 weeks of birth [57]. Although the full mechanistic 2+

NMDA receptor has been shown to mediate influx of Ca . There is no clear

implications of this defective Ca signaling has yet to be evidence for direct binding of JPH3 or JPH4 with other members of the neuronal

JMC to date. Although it is hypothesized that particular RyR isoforms, including

clariﬁed, these early studies raise the possibility that 2+

2+ RyR1, RyR2, and RyR3, may have important roles in ER Ca release in a neuronal

defective store Ca release by RyR isoforms may be asso- type-specific manner, this has yet to be clearly demonstrated. Abbreviations: Ca ,

ciated with neuronal JPH loss. Although little is known calcium; ER, endoplasmic reticulum; JPH3, junctophilin 3; JPH4, junctophilin 4;

NMDAR, N-methyl-D-aspartate glutamate receptor; P/Q, P/Q-type voltage-gated

about the role of RyR within neuronal tissue, RyR3 has 2+

channel; RyR, ryanodine receptor; SK, small conductance Ca -activated

canonically been viewed as the major isoform of the brain potassium channel.

while RyR1 and RyR2 are also expressed [59,60]. Although

the speciﬁc RyR isoform responsible for ER Ca release

has not been clariﬁed for the numerous neuronal cell been designated HDL1 [66]. In 2001, a trinucleotide CAG/

subtypes in the brain, it is hypothesized that RyR isoforms CTG repeat expansion was identiﬁed in a family with HD-

may be neuronal subtype-speciﬁc [61,62]. In addition, like syndrome that was negative for mutations in HTT that

recent evidence has suggested that neuronal LTCCs localized to an alternatively spliced exon of JPH3 [67,68].

may couple with RyR1 in a manner analogous to volt- This has led to the labeling of JPH3 as the second HD-like

age-triggered RyR1 opening in skeletal muscle. Indeed, associated gene (HDL2). A more detailed explanation of

heterozygous knockin mice harboring a known RyR1– alternative splicing is provided in Box 2. Although large

I4895T mutation were noted to have signiﬁcantly impaired studies estimating the frequency of JPH3 mutations in HD

2+ 2+

voltage-induced Ca release of store Ca in addition to or HD-like syndromes have not been done, a study exam-

skeletal muscle myopathy [63]. These recent studies raise ining nine independent series of patients with HD-like

the possibility that molecular uncoupling of voltage-sensi- syndrome estimated that 0–15% of patients who are nega-

tive Ca channels from neuronal RyRs in JPH3/4-deﬁcient tive for mutations in HTT will host mutations in JPH3 [69].

mice may mirror the uncoupling of SOCE from RyR1 in A recent study of a cohort of Venezuelan patients with HD-

skeletal muscle with loss of JPH1. A cartoon depicting like syndrome found that approximately 25% (4 out of 16)

JPH3 and JPH4 within neurons is depicted in Figure 5. hosted JPH3 expansion mutations [70]. These numbers

represent early estimates derived from small studies; how-

Huntington’s disease-like syndrome ever, it is generally accepted that JPH3 mutations are a

JPH3 was the ﬁrst member of the junctophilin family to be relatively rare cause of HD-like syndrome.

implicated in the pathogenesis of human disease. An in-

sidiously progressive neurodegenerative disease, HD is

associated with development of involuntary and uncoordi-

Box 2. Alternative splicing and splice variants

nated muscle movements (chorea) and subcortical demen-

Alternative splicing is a process during which the messenger RNA

tia [64]. The etiology of this disease is traditionally believed

(mRNA) transcribed from a single gene can be processed differently

to be a CAG trinucleotide repeat expansion within the gene

resulting in multiple different proteins following translation [80]. In

HTT which encodes the huntingtin protein [65]. This tri-

this process, pre-mRNA is transcribed from the DNA of a gene

nucleotide repeat results in the insertion of a polygluta- including exons as well as intervening noncoding introns. This pre-

mine tract within the huntingtin protein. Although this mRNA is then processed, or ‘spliced’, by a group of proteins called a

spliceosome to remove specific portions of the pre-mRNA. Spliced

mutation represents the classic cause of HD, a subset of

material includes introns and may or may not include removal of

patients are negative for mutations in this gene and yet are

some exons and/or inclusion of other exons. These differentially

clinically indistinguishable from patients with mutations

spliced mature mRNAs are then translated into proteins and are

in HTT. These mutation-negative individuals are consid- referred to as splice variants. It is believed that nearly all multi-exon

genes are ultimately alternatively spliced prior to final protein

ered HD-like syndrome phenocopies. Some patients with

translation and that this is a mechanism for increasing the diversity

HD-like syndrome were found to host octapeptide repeat

of proteins with a relatively small pool of genes [81].

expansion in the PRNP-encoded prion protein and have

359

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Review Trends in Molecular Medicine June 2014, Vol. 20, No. 6

Although mutations in JPH3 underlie a relatively small Box 3. Outstanding questions

proportion of individuals with HD-like syndrome, multiple

Do JPH isoforms undergo post-translational modifications such

independent cohorts have identiﬁed this disease associa-

as phosphorylation and, if so, what is the effect of these

tion particularly in individuals of African ancestry [69,71– modifications on Ca signaling within the cell?

73]. Individuals with HDL2 demonstrate a triad of symp- What is the role of JPH1 mutations, if any, in the pathogenesis of

skeletal muscle myopathies?

toms marked by abnormal movements, most often chorea,

Are there additional binding partners of striated muscle-specific

dementia, and psychiatric disturbances, and the number of

JPH1 and JPH2 which may play roles in the development of

trinucleotide repeats is inversely proportional to the age of

myopathic disease?

symptom onset [69]. Recent familial studies have added Are there additional binding partners of cardiac JPH2 which may

play roles in arrhythmic disease?

additional complexity to the clinical phenotype associated

How do mutations in various functional domains of JPH1 and

with JPH3 mutations. Indeed, several large, multigenera-

JPH2 create differing disease phenotypes?

tional families hosting JPH3 trinucleotide repeat expan-

What are physiological and pathophysiological triggers for

sions demonstrated severe akinetic–rigid syndrome with a proteolytic degradation of JPH1 and JPH2, and can this form of

relatively minor degree of chorea [70,74]. Despite the post-translational modification be extended to JPH3 and JPH4?

Can overexpression of JPH2 in cardiac cells, or JPH3/4 in neuronal

possible emergence of distinct clinical presentations of

cells, be protective against cardiomyopathic and neurodegenera-

individuals with HDL2, there has been no reproducible

tive processes, respectively?

genotype–phenotype correlation which might explain why

What is the mechanism of disease pathogenesis triggered by the

a given family demonstrates hyperkinetic chorea symp- JPH3 trinucleotide expansion in patients with HD-like syndrome?

toms whereas another develops akinetic–rigid syndrome What is the role of mutations in JPH4, if any, in the pathogenesis

symptoms. of neurodegenerative disease?

Are there other brain or neuronal diseases that can be linked to

Although the precise mechanism of disease pathogene-

perturbed JPH expression or genetic mutations?

sis in HDL2 remains unknown, some progress has been in

Do JPH3 and/or JPH4 play a role in learning and memory?

made in clarifying the pathogenic mechanism of the JPH3

trinucleotide repeat expansion. Fluorescent in situ hybrid-

ization (FISH) analysis of neuronal specimens obtained

the possibility of a similar role in neuronal tissue such as

from HDL2 patients at autopsy demonstrated RNA foci

the brain, and mutations in neuronal-speciﬁc JPHs have

within the neurons which were absent in traditional HD

been linked to neurodegenerative disease. Although the

patients [75]. FISH for CAG/CTG repeat expansion of the

number of patients with clearly deﬁned mutations in JPH

mutated JPH3 colocalize to these areas of RNA foci. Fur-

genes remains small, understanding this class of proteins

ther, overexpression of the alternatively spliced exon 2A

may provide signiﬁcant understanding to the way in which

containing the repeat expansion resulted in cellular tox-

excitable cells function and how to intervene when they

icity in vitro [75]. These studies suggest that the trinucle-

fail. A list of outstanding questions and possible future

otide repeat expansion of JPH3 may result in aberrant

directions for investigation are presented in Box 3.

RNA transcription and formation of toxic RNA foci. This

possibility is supported by murine models overexpressing

Acknowledgments

the trinucleotide repeat that demonstrate accelerated de-

D.B. is supported by American Heart Association (AHA) Predoctoral

velopment of motor deﬁcits as well as neurodegeneration

Fellowships and by National Institutes of Health (NIH) grant

[76]. Recent in vitro studies have raised the possibility that T32HL007676-21A1. X.H.T.W. is an Established Investigator of the AHA

additional antisense transcription in an open reading (13EIA14560061), a W.M. Keck Foundation Distinguished Young Scholar,

and is supported by the Muscular Dystrophy Association, and NIH grants

frame within the JPH3 gene locus may also play a role

HL089598, HL091947, and HL117641, and the Juanita P. Quigley

in neuronal toxicity [55]. Although this possible antisense

Endowed Chair in Cardiology. This work was also supported in part by

transcript has yet to be identiﬁed in vivo, RNA toxicity

Fondation Leducq (‘Alliance for CaMKII Signaling in Heart’) to X.H.T.W.

remains an interesting possible mechanism of neuronal

dysfunction. Taken together, these studies demonstrate Disclaimer statement

that neuronal toxicity and death may be occurring through X.H.T.W. is a founding partner of Elex Biotech, a company that develops

new drugs targeting intracellular calcium leak.

multiple mechanisms including loss of JPH3 expression or

generation of toxic RNA.

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