Advance Publication by-J-STAGE Circulation Journal REVIEW Official Journal of the Japanese Circulation Society http://www.j-circ.or.jp Molecular Variants of Soluble Guanylyl Cyclase Affecting Cardiovascular Risk Jana Wobst; Philipp Moritz Rumpf, MD; Tan An Dang; Maria Segura-Puimedon, PhD; Jeanette Erdmann, PhD; Heribert Schunkert, MD Soluble guanylyl cyclase (sGC) is the physiological receptor for nitric oxide (NO) and NO-releasing drugs, and is a key enzyme in several cardiovascular signaling pathways. Its activation induces the synthesis of the second mes- senger cGMP. cGMP regulates the activity of various downstream proteins, including cGMP-dependent protein kinase G, cGMP-dependent phosphodiesterases and cyclic nucleotide gated ion channels leading to vascular relaxation, inhibition of platelet aggregation, and modified neurotransmission. Diminished sGC function contributes to a number of disorders, including cardiovascular diseases. Knowledge of its regulation is a prerequisite for under- standing the pathophysiology of deficient sGC signaling. In this review we consolidate the available information on sGC signaling, including the molecular biology and genetics of sGC transcription, translation and function, including the effect of rare variants, and present possible new targets for the development of personalized medicine in vascu- lar diseases. Key Words: Cardiovascular disease; Cyclic guanosine-3’,5’-monophosphate (cGMP); Molecular variants; Nitric oxide (NO); Soluble guanylyl cyclase (sGC) he major components of the nitric oxide (NO)/cyclic varied and include vascular smooth muscle cell (VSMC) relax- guanosine-3’,5’-monophosphate (cGMP) pathway ation,11 inhibited platelet aggregation12 and modified neuro- T were identified in the late 1980s.1,2 NO is biosynthe- transmission,13 for example. sized endogenously through sequential oxidation of the amino acid L-arginine by an enzyme family called the NO synthases (NOS).3 Three isoforms of NOS with different tissue distribu- sGC Subunits tions are known: neuronal NOS (nNOS/NOS1), inducible NOS sGC is composed of 2 subunits: α and β.14 In 1981 Gerzer et in macrophages (iNOS/NOS2) and endothelial NOS (eNOS/ al showed that sGC contains a heme in form of ferroprotopor- NOS3).4 In the cardiovascular system, eNOS is the main source phyrin IX.15 However, it was unknown for a long time whether of NO production.5,6 The formation of NO by eNOS is increased this prosthetic heme group is sandwiched between the α and β by various stimuli such as platelet-derived factors, shear stress, subunits or whether it exclusively binds to the β subunit. In acetylcholine, and cytokines.7 1997, Zhao and Marletta demonstrated the ferrous heme to be NO mediates its functions through its primary receptor: sol- ligated to the N-terminal part of the β subunit at His105.16 uble guanylyl cyclase (sGC). Together with the adenylate In humans, 2 types of each subunit exist: α1 and α2 for the cyclases, sGC belongs to the class III purine nucleotidyl cyclase α subunit and β1 and β2 for the β subunit. These 4 proteins are family.8 Binding of NO to the heme moiety of sGC induces encoded by 4 distinct genes: GUCY1A3 (α1), GUCY1A2 (α2); the transition from basal to activated sGC. Activated sGC GUCY1B3 (β1), and GUCY1B2 (β2). The genes for human α1 converts guanosine-5’-triphosphate (GTP) to cGMP and pyro- and β1 have been mapped to chromosome 4,17 and those encod- phosphate (PPi).9 PPi is emerging as a major factor in prevent- ing α2 and β2 to chromosomes 1118 and 13,19 respectively ing vascular calcification.10 cGMP acts as a ubiquitous second (Table 1). Dimerization of the enzyme is a prerequisite for its messenger in intracellular signaling cascades, which serves to catalytic activity.20 Both α subunits give rise to a functional regulate the activity of a number of downstream proteins, enzyme when coexpressed with β1 (ie, both α1/β1 and α2/β1 including cGMP-dependent protein kinase G (PKG), cGMP- heterodimers are activated by NO).21 Although heterodimers dependent phosphodiesterases (PDE) and cyclic nucleotide seem to be the preferred form in cells, the features that deter- gated ion channels. The signals propagated through cGMP are mine this preference, and any possible role for homodimers, Received January 13, 2015; accepted January 14, 2015; released online February 6, 2015 Department of Cardiovascular Diseases, German Heart Center Munich, Technical University Munich, Munich (J.W., P.M.R., T.A.D., H.S.); Institute for Integrative and Experimental Genomics, University of Lübeck, Lübeck (M.S.-P., J.E.); German Center for Cardiovascular Research (DZHK), Partner Site Hamburg/Kiel/Lübeck, Lübeck (J.E.); and German Center for Cardiovascular Research (DZHK), Partner Site Munich Heart Alliance, Munich (H.S.), Germany Mailing address: Heribert Schunkert, Professor Dr, MD, Klinik für Herz- und Kreislauferkrankungen, Deutsches Herzzentrum München, Technische Universität München, Lazarettstr. 36, 80636 Munich, Germany. E-mail: [email protected] ISSN-1346-9843 doi: 10.1253/circj.CJ-15-0025 All rights are reserved to the Japanese Circulation Society. For permissions, please e-mail: [email protected] Advance Publication by-J-STAGE WOBST J et al. Table 1. Overview of Human Soluble Guanylyl Cyclase Subunit Isoforms Chromosomal Protein size Gene Transcript variant Exons Isoform location (aa) GUCY1A3 4q31.1-q31.2 GUCY1A3-Tr1 11 α1-IsoA 690 GUCY1A3-Tr2 10 GUCY1A3-Tr3 10 GUCY1A3-Tr4 8 GUCY1A3-Tr5 10 α1-IsoB 455 GUCY1A3-Tr7 9 α1-IsoD 624 GUCY1A3-Tr8 10 α1-IsoA 690 GUCY1A2 11q21-q22 GUCY1A3-Tr1 9 α2i 763 GUCY1A3-Tr2 8 α2 732 GUCY1B3 4q31.3-q33 GUCY1B3-Tr1 15 β1-Iso1 641 GUCY1B3-Tr2 14 β1-Iso2 619 GUCY1B3-Tr3 15 β1-Iso3 599 GUCY1B3-Tr4 16 β1-Iso4 594 GUCY1B3-Tr5 14 β1-Iso5 586 GUCY1B3-Tr6 13 β1-Iso6 551 GUCY1B2 13q14.3 – 17 – 617 are not fully defined. Koglin et al could show thatβ 2 does not which likely affects mRNA stability. exhibit cyclase activity when expressed with either α1 or α2 but In mammals, a splice variant of α2 (α2i) generates a dominant that β2 is active in the absence of an α subunit.22 This implies negative variant when forming a dimer with β1 because α2i con- that the β2 protein can function as a homodimer ex vivo. How- tains an in-frame insertion of 31 amino acids within the cata- ever, the physiological role of the β2 subunit in cGMP signal- lytic domain.37 Concerning β2, the NCBI nucleotide database ing remains elusive. By contrast, Zabel et al were able to just provides information on a single human β2 transcript overexpress α1/α1 and β1/β1 homodimers in Sf9 cells, both (Table 1). Because at least 3 different β2 transcripts have been being catalytically inactive.23 observed in humans38–40 and different β2 isoforms have been cloned from rats,41 there is evidence that β2 also exists as dif- ferent isoforms in humans. Alternative Splicing Recently, Martin et al36 conducted a study of the role of alter- Diminished expression and function of sGC contributes to the native splicing of GUCY1A3 and GUCY1B3 sGC in healthy pathogenesis of several cardiovascular disorders such as coro- and diseased human vascular tissue. As splicing regulation of nary artery disease (CAD), atherosclerosis and hypertension.24 sGC and its biological role in vascular tissue has not been Most recently, the chromosomal locus harboring GUCY1A3 previously examined in vivo, they were the first to show splic- (α1) and GUCY1B3 (β1) was shown to contain genetic variants ing diminishing sGC function. Using a semiquantitative with genome-wide significant association to CAD and hyper- reverse transcriptase PCR approach, they uncovered various tension.25–27 Thus, the GUCY1A3/GUCY1B3 locus adds to the GUCY1A3 and GUCY1B3 splice variants in human aorta. growing list of those contributing to CAD and myocardial Quantifying the total levels of GUCY1A3 and GUCY1B3 sGC infarction (MI) risk.28,29 transcripts using quantitative PCR revealed a 3.2- and 2.3-fold The expression of sGC subunits is modulated at different increase in, respectively, GUCY1A3 and GUCY1B3 mRNA in levels, including inhibition of transcription,30,31 destabilization aortas with aneurysms compared with healthy control aortas. of mRNA,32,33 and protein degradation.34 The role of alterna- Interestingly, the aortas with aneurysms demonstrated decreased tive splicing in this process still needs to be uncovered. Precise sGC activity that correlated with increased expression of dys- understanding of sGC splicing regulation could serve as a tar- functional sGC splice variants, because the composition of the get for new therapeutic interventions and help to personalize splice forms in the aortas with aneurysms differed from that in sGC-targeting therapies in the treatment of vascular disease. control aortas (Figure 1). As demonstrated by several studies, human α1, α2 and β1 exist GUCY1A3-Tr1 coding for α1-IsoA and GUCY1A3-Tr7 cod- as different isoforms because of alternatively spliced transcript ing for α1-IsoD were higher in diseased aortas. α1-IsoD lacks variants35–37 (Table 1). Besides several predicted sequences, 66 C-terminal amino acids and has impaired enzymatic activ- NCBI nucleotide database research reveals a total of 7 alter- ity. In contrast, the level GUCY1A3-Tr5 coding for α1-IsoB natively spliced human transcript variants for GUCY1A3, 2 for was lower in the aneurysm samples. α1-IsoB is 235 amino GUCY1A2 and 6 for GUCY1B3. In the case of GUCY1A2 (α2 acids shorter at the N-terminus and oxidation resistant.42 The and α2i) and GUCY1B3 (β1-Iso1 to Iso6), each transcript vari- immunoprecipitation studies and activity evaluation presented ant codes for 1 unique isoform. However, the 7 alternatively by Martin et al clearly demonstrated that α1-IsoB forms a func- spliced variants of GUCY1A3 only code for 3 different iso- tional heterodimer with β1 subunit in aorta in vivo.