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.