Reply to Cristóbal et al.

Letter to the EditorReply to Cristóbal et al.Florentina Pluteanu and Uwe KirchheferFlorentina PluteanuDepartment of Anatomy, Animal Physiology and Biophysics, University of Bucharest, Bucharest, Romania and Uwe KirchheferInstitute of Pharmacology and Toxicology, University of Muenster, Muenster, GermanyPublished Online:01 Jun 2022https://doi.org/10.1152/ajpheart.00236.2022MoreSectionsPDF (208 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat REPLY: We thank the authors Cristóbal et al. (1) for highlighting our study in their Letter to the Editor, and we are thankful for the feedback on our article, “Activation of PKC results in improved contractile effects and Ca2+ cycling by inhibition of PP2A-B56α” (2), published in a recent issue of the American Journal of Physiology-Heart and Circulatory Physiology.Protein phosphatase 2A (PP2A) is a Ser/Thr phosphatase that controls the phosphorylation level of the target molecules of Ser/Thr kinases, such as PKA or PKC, kinases highly active in cellular events such as proliferation, signal transduction, and apoptosis. PP2A has the versatility to localize in different compartments and to associate with many kinases or substrates; therefore, endogenous or experimental modulation will trigger cell-specific responses. Thus, the investigations on PP2A generate both interesting and controversial results, because of the involvement of PP2A in multiple intracellular cascades. PP2A is considered the “master regulator of cell cycle” (3). There are studies showing the role of PP2A in reducing the current through Na+ channels (4) and Ca2+ channels (5) or increasing the flow through some K+ channels (6), suggesting that PP2A is an important player in excitability regulation. Although this topic emerges more from studies investigating PP2A implication in cardiomyocytes or neurons, PP2A’s role in ion channel modulation remains understudied. More research in this direction will add more knowledge to the already-established roles in development, cell mobility and cytoskeletal dynamics, cell proliferation, the cell cycle, and cell signaling (7).PP2A regulatory B subunits, such as B56α, emerged as molecules important for fine tuning the activity of PP2A. Through biochemical and cell physiological work, to our knowledge, our group demonstrated for the first time that activation of PKC leads to an increased phosphorylation of the regulatory subunit B56α at serine-41 and thus to inhibition of the PP2A holoenzyme (8). This inhibition was accompanied by an increase in the intracellular Ca2+ concentration. With our recent article (2), we were able to demonstrate the functional relevance of this postulated signaling pathway in the heart, where the increase in contractility in wild-type mice after stimulation of PKC was attenuated in a transgenic mouse model with mutation of this phosphorylation site. Activation of PKC in the latter study was achieved by phorbol-12-myristat-13-acetate, a phorbol ester, whereas the original work used phenylephrine, an α1-adrenoceptor agonist. Overall, the expression of α1-adrenoceptors in the heart is very low (9), suggesting that an activation of other sarcolemmal receptors, such as the AT1 receptor, might better result in a functionally relevant activation of PKC with downstream inhibition of PP2A.Our studies (2, 8, 10) contributed to understanding the role of B56α in the cross talk between PKC and PP2A and its effects on Ca2+ homeostasis in ventricular myocytes. In contrast to the myocardium, vascular smooth muscle exhibits a high receptor density of α1-adrenoceptors. Thus, it is quite possible that a PKC-dependent inhibition of PP2A, which is also highly expressed in smooth muscle (11), might contribute to the contractile effects in this tissue. By the mid-1980s, numerous papers had already demonstrated that okadaic acid, a potent inhibitor of protein phosphatases 2 A and 1, induced contraction of vascular smooth muscle preparations (12). The increase in contraction was explained by an inhibition of total phosphatase activity and a concomitant increase in the phosphorylation of regulatory proteins (13, 14). However, all these studies could not clarify which type of protein phosphatase is responsible for these contractile and biochemical effects. This may also be due to the fact that the effects caused by the individual inhibitors of protein phosphatases are concentration-dependent. We now know that low concentrations (<1 µM) of okadaic acid relax precontracted smooth muscle preparations, whereas muscle contraction is achieved by higher concentrations (15). However, regarding the more potent inhibition of PP2A compared with PP1 by okadaic acid, the relaxing effects are due to PP2A and the contractile effects are due to PP1. This suggests that PP2A might play an important role in regulating vascular muscle relaxation via unknown mechanisms. Consistently, treatment with 5 nM okadaic acid or ectopic expression of siRNA of the catalytic subunit of PP2A resulted in an enhanced phosphorylation of eNOS at serine-1179 and a higher NO production (16). B56α was identified as the mediator of the stimulatory effects on eNOS phosphorylation. Thus, inhibition of PP2A-B56α after PKC activation would contribute to an enhanced vascular relaxation also through processes in the endothelium. It can be assumed that under physiological and pathophysiological conditions, mechanisms of an endogenous inhibition of PP2A carry out the function of naturally occurring inhibitors.Taken together, these findings raise the question of whether the PKC-phosphoB56α-PP2A signaling pathway is transferrable to other tissues, such as vascular smooth muscle cells or atrial myocytes. The high protein expression of PP2A-B56α and the biochemical and physiological experiments performed using naturally occurring inhibitors of PP2A certainly suggest this. The need for a deeper understanding of the role of PP2A in the vasculature, its activation and inhibition, and interaction with its target proteins is highlighted by recent data on the mechanisms of aortic aneurysm development and progression (17). In that study, activation of PP2A by so-called small molecule activators (SMAPs) was identified as a novel therapeutic strategy. It will therefore also be necessary for future studies to better understand to what extent and in what manner target substrates are selectively dephosphorylated specifically by the PP2A in association with individual regulatory subunits, such as B56α. It is likely that PP2A regulatory subunits not only act as targeting subunits but also assume additional functions at the site-specific dephosphorylation recognizing certain motifs (18). The identification of these amino acid sequences will have significant implications for our understanding of how PP2A interferes with intracellular signaling in the heart and blood vessels. Therefore, there is an open field of PP2A research that may benefit from genetically modified mice, the discovery of new endogenous modulatory proteins, and the development of new specific inhibitors for PP2A.DISCLOSURESNo conflicts of interest, financial or otherwise, are declared by the authors.AUTHOR CONTRIBUTIONSF.P. and U.K. drafted manuscript; edited and revised manuscript; and approved final version of manuscript.REFERENCES1. Cristóbal I, Santos A, Rojo F, Garcia-Foncillas J. The PP2A pathway plays a crucial role controlling cardiac physiology. Am J Physiol Heart Circ Physiol 323: TBA, 2022. doi:10.1152/ajpheart.00203.2022.Link | ISI | Google Scholar2. Pluteanu F, Boknik P, Heinick A, König C, Müller FU, Weidlich A, Kirchhefer U. Activation of PKC results in improved contractile effects and Ca2+ cycling by inhibition of PP2A-B56α. Am J Physiol Heart Circ Physiol 322: H427–H441, 2022. doi:10.1152/ajpheart.00539.2021. Link | ISI | Google Scholar3. Wlodarchak N, Xing Y. 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Crossref | PubMed | ISI | Google ScholarAUTHOR NOTESCorrespondence: U. Kirchhefer ([email protected]de). Download PDF Previous Back to Top FiguresReferencesRelatedInformation Related ArticlesThe PP2A pathway plays a crucial role in controlling cardiac physiology 01 Jun 2022American Journal of Physiology-Heart and Circulatory Physiology More from this issue > Volume 323Issue 1July 2022Pages H67-H68 Crossmark Copyright & PermissionsCopyright © 2022 the American Physiological Society.https://doi.org/10.1152/ajpheart.00236.2022PubMed35648098History Received 13 May 2022 Accepted 16 May 2022 Published online 1 June 2022 Published in print 1 July 2022 Metrics