4. Discussion
In this manuscript we describe the identification of PDE4DIP as
the genetic modifier of DES . Desmin is a type III intermediate
filament protein that plays a pivotal role in the electromechanical
functioning of cardiomyocytes (Mado et al., 2019). It connects the Z
discs, which are at the center of the contractile unit, to the plasma
membrane, nucleus and mitochondria and plays a pivotal role in the
contractile function of cardiomyocytes. Mutations in DES are
associated with autosomal dominant dilated cardiomyopathy with
incomplete penetrance. Over 40 pathogenic DES mutations have been
causally linked to myofibrillar myopathies involving skeletal
myopathies, non-ischemic cardiomyopathies and cardiac conduction disease
(van Tintelen et al., 2009). The p.S13F substitution occurs within a
highly conserved motif in the non-helical head domain of the protein
that is shared by type III and IV intermediate filaments (Bar, Strelkov,
Sjoberg, Aebi, & Herrmann, 2004; Pica et al., 2008). This substitution
eliminates serine at a known phosphorylation site for protein kinase C,
thus interfering with the protein organization and likely leading to
deleterious Desmin aggregation and disruption of the cytoskeletal
network (Kitamura et al., 1989). Although Desmin is widely known as a
skeletal muscle protein it is highly expressed within heart and within
the conduction system of the heart and the pulmonary vein myocardial
sleeves that are foci of origin of AF (Kugler et al., 2018).
Accordingly, the mutations have pleotropic effects with cardiomyopathy
being the most prominent trait, followed by cardiac conduction disease
and much less commonly AF (van Spaendonck-Zwarts et al., 2011). The
genetic modifiers of DES have not been identified. Interestingly,
six members of the Lebanese kindred who were carriers of the
pathological Desmin p.S13F substitution had slightly higher incidence of
NICM compared to the literature (50% vs 38% in published data), but
considerably higher incidence of early onset AF (83% vs 11% in
published data) and heart block requiring a pacemaker (67% vs 38% in
published data) (supplementary table 1) (Bergman et al., 2007; Pica et
al., 2008; van Tintelen et al., 2009). The high incidence of AF and
heart block suggested epistatic interactions between the DESmutation and other genetic mutations and provided an exceptional
opportunity to search for genetic modifiers.
Our study identified the PDE4DIPpA123T mutation
as a genetic modifier of DES that increases the penetrance of
heart block and early onset AF in DES mutation carriers. Also
known as myomegalin, PDE4DIP is highly expressed in cardiac
atrial and ventricular tissue (supplementary figure 1). It is a large
gene located on the long arm of chromosome 1 and contains 64 exons
coding for a 2,362 amino acid protein that was first isolated in 2001 in
a study of cardiac genes (Soejima et al., 2001) with several splicing
variants . The protein is composed of alpha-helical and coiled-coil
structures and is also heavily expressed in skeletal muscles
(supplementary figure 1). Within myocytes, PDE4DIP is predominantly
localized in the Golgi compartment, in proximity to the cytoskeletal
apparatus and in the nucleus (Bouguenina et al., 2017; Soejima et al.,
2001; Wang, Zhang, & Qi, 2014; Wu et al., 2016). It is an
A-kinase-anchoring protein that is involved in the assembly of the cAMP
dependent protein kinase A (PKA)/phosphodiesterase 4D (PDE4D) cAMP
signaling module in a multiprotein complex (Dodge et al., 2001).
Co-compartmentalization of both PKA and PDE4D is critical for sustained
specificity of adrenergic signaling to subcellular locations,
contractility of cardiomyocytes and timely termination of the second
messenger response (Fink et al., 2001).
PDE4D hydrolyzes cAMP and regulates its levels within cardiac myocytes
where it also complexes with proteins mediating sympathetic signals to
the heart, including β-adrenergic receptors (Mongillo et al., 2004;
Perry et al., 2002; Xiang et al., 2005) . The activity of PDE4D has been
also localized to the transverse (T) tubule/sarcoplasmic reticulum (SR)
junctional space thus mediating cAMP/calcium homeostasis that is
involved in excitation-contraction coupling (Mika, Richter, & Conti,
2015; Mongillo et al., 2004; Zaccolo & Pozzan, 2002). Of note, loss of
function of PDE4D is associated with cardiomyopathy and AF (Jorgensen,
Yasmeen, Iversen, & Kruuse, 2015).
We further established an association between multiple rare
non-conservative PDE4DIP mutations with large effects in four out
of seven unrelated families referred for early onset familial AF and
slow conduction without structural heart disease. The functional
significance of these mutations was established by the combined
statistical power, high conservation of the mutated bases, and in vitro
functional studies.
The in vitro characterization of PDE4DIPpA123Tsuggested a gain of function mutation leading to increased PKA activity.
This effect is likely due to the disruption of the spatial-temporal
activity of PDE4D, which is normally anchored to PDE4DIP and showed
reduced colocalization with PDE4DIPpA123T.
Consequently, there was increased cAMP activation by
PDE4DIPpA123T vs. wildtype PDE4DIP in response to
isoproterenol as assayed by FRET imaging. Under normal circumstances,
the rise of cAMP in response to isoproterenol triggers PDE4D activation
as well as desensitization of the G-protein coupled β2AR receptor via
phosphorylation by PKA and G protein-coupled receptor serine/threonine
kinases (GRKs) at distinct phosphorylation sites (Xin, Tran, Richter,
Clark, & Rich, 2008). PKA mediated phosphorylation of the β2AR at
Ser345/346 by PKA causes a switch from stimulatory Gs to inhibitory Gi
protein (Zamah, Delahunty, Luttrell, & Lefkowitz, 2002). On the other
hand, prolonged β2AR stimulation results in its GRK mediated
phosphorylation at Ser 355/356, its binding to β-arrestin, termination
of G protein-mediated signaling, and facilitated receptor endocytosis
resulting in receptor desensitization (Nobles et al., 2011; Shenoy &
Lefkowitz, 2005). In addition, PDE4D binds to β-arrestin and is
subsequently recruited to the plasma membrane to regulate PKA mediated
phosphorylation of β2AR.(Houslay & Baillie, 2005; Perry et al., 2002;
Willoughby et al., 2007). Interestingly, there was more pronounced
phosphorylation of the β2AR at the PKA residue (Ser 346) in the mutant
versus control cells while the phosphorylation at the GRK residues
(Ser355-356) was unchanged. The former may underlie the mechanism of
slow heart rate in patients with AF.
Like all A-kinase-anchoring proteins, PDE4DIP binds to the regulatory
subunit of PKA and anchors it to the N-terminal region of cMyBPC,
whereby it mediates cMyBPC phosphorylation and plays an important role
in regulation of cardiac contractility. The PDE4DIP colocalization with
cMyBPC and cTNI has been shown to increase upon β adrenergic activation
(Uys et al., 2011). Desmin is also known to be a phosphorylation target
for cAMP-dependent kinases (Gard & Lazarides, 1982; O’Connor, Gard, &
Lazarides, 1981). Specifically, PKA phosphorylation of Desmin regulates
its function and assembly (Inagaki et al., 1988; Tao & Ip, 1991).
Interestingly, our studies in C2C12 cells showed that the β adrenergic
activation reduces colocalization between mutant PDE4DIP and
phospho-Desmin compared to wildtype PDE4DIP. This suggests that PDE4DIP
directs PKA to Desmin, but PDE4DIPpA123T induced loss
of compartmentalization of both PDE4D and PKA leads to PKA
phosphorylation of β2AR but reduced phosphorylation of the Desmin, which
has also lost a PKC phosphorylation site by the p.S13F mutation.
Phosphorylation changes in Desmin are shown to alter its assembly and
trigger accumulation of toxic preamyloid oligomers in acquired heart
failure (Bouvet et al., 2016). Thus, the perturbed compartmentalization
of PDE4DIPpA123T and further reduction in Desmin
phosphorylation is a mechanistic link to the modifier effect of the
PDE4DIP variant on the mutant Desmin, leading to increased penetrance of
heart block and AF in Desmin p.S13F carriers.
It is noteworthy that a common nonsynonymous variant (rs1778155) of
PDE4DIP gene, resulting in arginine for histidine substitution at codon
1761 has been associated with ischemic stroke in NHLBI Exome Sequence
Project ( odds ratio: 2.15; p-value: 2.63 × 10−8)
(Auer et al., 2015). This disorder is believed to be associated with AF.
Thus, our findings may provide a molecular link between ischemic stroke
and slow AF, which is often subclinical due to the absence of
tachycardia and palpitations.
Our success in identifying the disease gene and its modifier was largely
due to our systematic study of outlier subjects with well-characterized
disease and the segregation analysis of the rare variants. The
investigated kindreds in our study all had early onset AF with slow
conduction and demonstrated an autosomal dominant pattern of
inheritance. This approach is particularly superior to case–control
association studies for identification of disease-causing rare variants
of large genes with high mutation burden such as PDE4DIP . It is,
however, noteworthy that not all identified segregating PDE4DIPmutations may have sufficient power to be independently disease causing;
this is particularly true for variants with higher allele frequencies.
One limitation of our study is that it does not examine the effect of
the PDE4DIP mutation on K channel and ryanodine receptor RyR2. PDE4D has
been shown to regulate the slow delayed rectifier K current (Terrenoire,
Houslay, Baillie, & Kass, 2009) and RyR2 (Lehnart et al., 2005; Mika,
Richter, Westenbroek, Catterall, & Conti, 2014). Its inhibition leads
to increased PKA activation of the slow delayed rectifier K current
(Terrenoire et al., 2009), a mechanism that may underlie the AF (Sampson
et al., 2008). It remains to be determined if PDE4DIP mutation generate
a functional substrate for AF in the atrium by altering K current. In
addition, a Mendelian randomization study examining link between
rs1778155 variant, AF, and stroke could support the potential link
between PDE4DIP common variant and the risk for atrial fibrillation and
should be a topic for future investigations.