Myomegalin has been characterized as a protein with the properties of a scaffold or structural protein that is expressed at high levels in skeletal and cardiac tissue, suggesting an important function in muscle, and which interacts with a cAMP-specific phosphodiesterase . However, the precise function and interactions of this protein, and its five isoforms, have been largely unknown. We here describe how the smallest MMGL isoform, isoform 4, binds to known and predicted PKA targets in the cardiac myocyte, including some sarcomeric proteins, viz. cMyBPC, cTNI, ENO1, ENO3, CARP and COMMD4 (Tables 1 and 2). Moreover, we show that MMGL isoform 4 interacts with two regulatory subunits of PKA (Figure 3). Together these results describe MMGL isoform 4 as a novel sarcomeric AKAP, which, like mAKAP , is involved in assembling a PKA/PDE cAMP signalling module.
In addition to interacting with both types of regulatory subunits, viz. RI and RII, which qualifies MMGL isoform 4 as a dual-specific AKAP [11, 19]. MMGL also meets the three other criteria for classification as an AKAP: By verifying its interaction with its proposed binding partners, we demonstrate that the targeting domains contained within the proposed AKAP participates in protein-protein interactions . Further, it has previously been shown that MMGL is a phosphodiesterase 4D-interacting protein , thus, it meets the criterion for being able to coordinate multiple signalling pathways by anchoring additional signalling enzymes [11, 20]. Lastly, we have shown in other Y2H screens that cMyBPC also binds to COMMD4 (unpublished results), here shown to be a MMGL interactor, while COMMD4 itself also binds to ENO1 and SNX3 (unpublished results). This strongly suggests that MMGL is part of a larger, multiprotein unit [11, 20], and that MMGL isoform 4 may function as a vital link in signaling between upstream activators and numerous downstream targets .
Co-compartmentalization of both PKA and PDE4D is crucial for maintaining specificity of adrenergic signaling, and for maintaining contractility in cardiac cells . We here established an important and novel link between PKA and PDE4D co-compartmentalization at the sarcomere level, and cMyBPC phosphorylation, and hence, regulation of cardiac contraction. The mechanism of docking of PKA to cMyBPC for phosphorylation of the MyBPC motif has previously not been elucidated; this study strongly suggests that MMGL isoform 4 anchors PKA to the N-terminal region, viz. C1-C2, of cMyBPC. The interaction between MMGL isoform 4, PKA, PDE4D and the N-terminal region of cMyBPC therefore sheds light on how second messenger responses are regulated in this particular part of the sarcomere.
We also found that β-adrenergic stimulation led to greater co-localization of myomegalin with both cMyBPC and cTNI in live cardiomyocytes, as evidenced by the increase in yellow staining during fluorescence microscopy in Figures 1 & 5, respectively. Thus, although MMGL is apparently present within the sarcomere under normal conditions, this implies that under adrenergic stimulation, and consequential increased intracellular levels of cAMP, PKA is dynamically recruited by MMGL isoform 4 to distinct sarcomeric locations. This translocation of MMGL to the sarcomeric region is therefore compatible with a mechanism that would lead to increased phosphorylation of cMyBPC and cTNI, which is known to form part of the cardiac cellular stress response that leads to increased cardiac contraction . Furthermore, given MMGL's known interaction with PDE4D , the mechanism for termination of the second messenger response, by degrading cAMP, would also be on site; lower levels of cAMP may then cause this multiprotein complex to dissociate again.
The knockdown studies of MMGL further suggests that MMGL not only acts as an AKAP at the MyBPC motif, but by implication plays a role in cardioprotection during adrenergic signaling. Although, in the presence of MMGL, all phosphorylation isoforms of cMyBPC are expressed well in H9C2 cells, and the level of the trisphosphorylated cMyBPC is increased in such cells under conditions of β-adrenergic stimulation as is to be expected, knockdown of MMGL under adrenergic conditions dramatically reduced cMyBPC expression (Figure 7). The latter finding suggests that when MMGL expression is reduced, cMyBPC phosphorylation is hindered, rendering the protein vulnerable to cleavage by proteases and reducing cMyBPC protein levels in the cell, as described by others [17, 18, 23]. Typically, annulment of the effect of an AKAP is commonly more noticeable on the target protein only after adrenergic stimulation: for instance, the studies of McConnell et al. (2009)  and Fink et al. (2001)  showed no significant difference in the level of phosphorylation of key cardiac proteins between wild-type cardiomyocytes and those in which AKAP function had been impaired with the Ht31 peptide. However, upon isoproterenol stimulation, dramatically decreased levels of PKA phosphorylation of all these proteins was observed in Ht31-transfected cells compared to isoproterenol-stimulated wild-type cells. Fink et al. (2001)  also demonstrated that AKAPs regulate phosphorylation of these PKA targets, including cTNI and cMyBPC, in response to β-adrenergic stimulation, although it has been unknown which AKAP/s are responsible for the phosphorylation of these two proteins up until our study. Thus, it appears that MMGL is an essential part in the β-adrenergic pathway leading to trisphosphorylation of cMyBPC and protection of the protein against degradation and normal sarcomeric integrity, which in turn is essential for normal physiological cardiac function, as well as cardioprotection during ischemia-reperfusion injury .
The subcellular localization and functions of some of the putative PKA targets identified via the Y2H library screen suggest that MMGL may act as AKAP in regions outside the sarcomere as well. Although some of the identified interactions, such as those with cMyBPC and cTNI, certainly occur within the sarcomere, associations with the other identified interactors (Table 2) do not necessarily occur within the sarcomere, nor do they necessarily all occur concurrently. In fact, a multiprotein subunit in the sarcomere consisting simultaneously of all identified MMGL-ligands would most likely sterically hinder crossbridge formation, and is therefore improbable. However, interaction of MMGL with proteins such as COMMD4, CARP, ENO1 and ENO3 may facilitate control of efficient PKA phosphorylation of these proteins; the protein-protein interactions and/or activation of these proteins may thus be regulated.
Although this study prioritized investigation of the interaction between the N-terminal of cMyBPC and MMGL, the other putative interactions of this region of cMyBPC should be further explored. The absence of cMyBPC as a prey in the MMGL Y2H library screen is most likely explained by the known absence of cDNAs representing the N-terminals of large proteins in oligo dT-primed libraries, while the absence of PRKAR1A and PRKAR2A may relate to the stringency of selection during heterologous bait-matings during this Y2H screen .
Interaction between myomegalin, PKA and cMyBPC or cTNI is also relevant to understanding of HCM patho-aetiology, as both of the latter proteins cause HCM when defective. It is known that point mutations in the C1-C2 region and in the MyBPC motif lead to HCM [3, 27]; a potential mechanism for this may be involve disruption of binding between MMGL and cMyBPC. Such disruption would have consequences for PKA-phosphorylation of the cMyBPC motif (with implications for regulation of cardiac contractility), implying a poison-peptide mechanism underlying disease, as well as maintenance of sufficient cMyBPC levels within the sarcomere, particularly under conditions of adrenergic stress, implying a haplo-insufficiency mechanism. Point mutations in cTNI may have a similar patho-aetiology. It could be further speculated that, mutations in MMGL could similarly lead to inadequate binding of PKA and/or its sarcomeric partners, which might affect cMyBPC or cTNI phosphorylation and hence affect adaptation of cardiac contractility to adrenergic stimulation. Future studies could therefore consider MMGL as either a candidate causal gene or a potential modifier gene for HCM.