Journal of Cardiovascular Development and Disease Review Functions of PDE3 Isoforms in Cardiac Muscle Matthew Movsesian 1,*, Faiyaz Ahmad 2,† and Emilio Hirsch 3 ID 1 Department of Internal Medicine/Division of Cardiovascular Medicine, University of Utah, Salt Lake City, UT 841132, USA 2 Vascular Biology and Hypertension Branch, Division of Cardiovascular Sciences, National Heart, Lung and Blood Institute, Bethesda, MD 20892, USA; [email protected] 3 Department of Molecular Biotechnology and Health Sciences, Center for Molecular Biotechnology, University of Turin, 10126 Turin, Italy; [email protected] * Correspondence: [email protected]; Tel.: +1-801-582-1565 † Beginning 18 February 2018: Sidra Medical Research Center, Doha, Qatar. Received: 9 January 2018; Accepted: 1 February 2018; Published: 6 February 2018 Abstract: Isoforms in the PDE3 family of cyclic nucleotide phosphodiesterases have important roles in cyclic nucleotide-mediated signalling in cardiac myocytes. These enzymes are targeted by inhibitors used to increase contractility in patients with heart failure, with a combination of beneficial and adverse effects on clinical outcomes. This review covers relevant aspects of the molecular biology of the isoforms that have been identified in cardiac myocytes; the roles of these enzymes in modulating cAMP-mediated signalling and the processes mediated thereby; and the potential for targeting these enzymes to improve the profile of clinical responses. Keywords: cyclic nucleotides; cAMP; cGMP; phosphodiesterase; PDE3; intracellular signalling; heart 1. Introduction Cyclic nucleotide phosphodiesterases regulate intracellular signalling by hydrolysing cAMP and/or cGMP. Enzymes in the PDE3 family of phosphodiesterases are dual-specificity enzymes with high affinities for both cAMP and cGMP but much higher turnover rates for cAMP [1–3]. In cardiac muscle, these enzymes have been studied principally in the context of their role in regulating cAMP-mediated signalling, and this is the focus of our review. Several isoforms of PDE3 are expressed in cardiac myocytes, and PDE3 inhibitors are used therapeutically to potentiate cAMP-mediated signalling in patients with heart failure. In the short term, these agents have the desired action of increasing myocardial contractility, but their long-term administration has been shown in several clinical trials to increase cardiovascular mortality [4–10]. This frustrating combination of beneficial and adverse effects of PDE3 inhibition presents a challenge that remains to be solved. Here we review the function of PDE3 isoforms in cardiac muscle and raise possibilities for targeting these isoforms so as to achieve more satisfying clinical outcomes. 2. The PDE3 Family of Cyclic Nucleotide Phosphodiesterases Cyclic nucleotide phosphodiesterases comprise a superfamily of enzymes. As of now, more than 50 mammalian isoforms have been described and classified into eleven gene families (PDE1 through PDE11) defined on the basis of sensitivity to pharmacologic inhibitors, kinetic activity, and regulatory mechanisms [11]. PDE1, 2, 3, 10, and 11 hydrolyse both cAMP and cGMP; PDE4, PDE7, and PDE8 selectively hydrolyse cAMP; and PDE5, PDE6, and PDE9 selectively hydrolyse cGMP [11–13]. The N-terminal regulatory regions of phosphodiesterases contain sequences involved in post-translational modifications and protein–protein interactions that target the enzymes to specific functional compartments. Transcription start sites and alternative splicing lead to the generation of multiple different isoforms of the same family. J. Cardiovasc. Dev. Dis. 2018, 5, 10; doi:10.3390/jcdd5010010 www.mdpi.com/journal/jcdd J. Cardiovasc. Dev. Dis. 2018, 5, x FOR PEER REVIEW 2 of 15 J. Cardiovasc. Dev. Dis. 2018, 5, 10 2 of 15 functional compartments. Transcription start sites and alternative splicing lead to the generation of multiple different isoforms of the same family. EnzymesEnzymes in in the the PDE3 PDE3 family family are are transcribed transcribed from from two two genes, genes, PDE3A PDE3A and and PDE3B PDE3B [14,15]. [14,15]. In In the the casecase of of PDE3A, PDE3A, three three isoforms isoforms (some (some prefer prefer the the te termrm ‘variants’) ‘variants’) are are generated generated by by transcription transcription from from alternativealternative starts starts sites sites in in the the gene, gene, yielding yielding two two mRNAs, mRNAs, as as well well as as translation translation from from alternative alternative start start sitessites in in the the smaller smaller mRNA mRNA (Figure (Figure 1)1)[ [16].16]. As As a a result result of of these these N-terminal N-terminal ‘deletions’, ‘deletions’, the the amino-acid amino-acid sequencessequences of thesethese three three isoforms isoforms differ differ only only with wi respectth respect to the lengthsto the lengths of their N-terminalof their N-terminal sequences. sequences.PDE3A1 (length: PDE3A1 996 (length: amino acids;996 amino MW: acids; 109,980), MW: which 109,980), is transcribed which is fromtranscribed an upstream from an start upstream site and starttranslated site and from translated the second from AUG the in second the PDE3A AUG open in the reading PDE3A frame—a open possiblyreading frame—a misleading possibly term in misleadingthis case, as term no isoform in this case, translated as no fromisoform the translated first AUG hasfrom been the described—hasfirst AUG has been an N-terminal described—has sequence an N-terminalcontaining sequence hydrophobic containing loops that hydrophobic insert into loops intracellular that insert membranes into intracellular [17,18], asmembranes well as three [17,18], sites asof well phosphorylation as three sites thatof phosphorylation regulate protein–protein that regulate interactions protein–protein [19–21]. interactions PDE3A2 (length: [19–21]. 842 PDE3A2 amino (length:acids; MW: 842 amino 93,600) acids; is transcribed MW: 93,600) from is transcribed a downstream from site a downstream in exon 1 and site translated in exon 1 from and translated the fourth fromAUG the in fourth the PDE3A AUG openin the readingPDE3A frame;open reading it lacks fram thee; most it lacks N-terminal the most phosphorylationN-terminal phosphorylation site and the sitetransmembrane and the transmembrane hydrophobic hydrophobic loops of PDE3A1. loops of PDE3A3PDE3A1. (length: PDE3A3 659 (length: amino 659 acids; amino MW: acids; 73,720) MW: is 73,720)translated is translated from the samefrom the mRNA same as mRNA PDE3A2 as andPDE3A2 lacks and all oflacks the all hydrophobic of the hydrophobic loops and loops the upstream and the upstreamphosphorylation phosphorylation sites. These sites. three These isoforms three isoforms are essentially are essentially indistinguishable indistinguishable with respect with respect to their tobasal their catalytic basal catalytic activity activity and their and sensitivity their sensitivity to catalytic to catalytic site inhibitors site inhibitors [22]. [22]. At this At this time, time, only only one oneisoform isoform of PDE3B of PDE3B (length: (length: 1112 1112 amino amino acids; acids; MW: MW 124,333): 124,333) has has been been described described [15 [15].]. Like Like PDE3A1, PDE3A1, its itsN-terminal N-terminal sequence sequence contains contains hydrophobic hydrophobic loops loops (six (six for for PDE3B, PDE3B, ascompared as compared to four to four for PDE3A1)for PDE3A1) and andphosphorylation phosphorylation sites, sites, and itsand C-terminal its C-terminal sequence sequence contains contains its catalytic its catalytic region [ 23region,24]. The[23,24]. sequence The sequenceof the catalytic of the regioncatalytic of region PDE3B of is PDE3B >80% identical is >80% toidentical that of PDE3Ato that of (both PDE3A contain (both a 44-amino-acidcontain a 44- amino-acidinsert absent insert from otherabsent phosphodiesterase from other phosphodiesterase families), and its families), catalytic activityand its andcatalytic inhibitor activity sensitivity and inhibitorare similar sensitivity to those are of PDE3A;similar to the those remainder of PDE3 ofA; the the PDE3B remainder sequence of the is PDE3B 20–30% sequence identical is to 20–30% that of identicalPDE3A [to3]. that of PDE3A [3]. FigureFigure 1. 1. StructureStructure and and subcellular subcellular localisation localisation of of the the PD PDE3E3 genes genes and and their their variants. variants. Length Length in in amino amino acidsacids (aa) (aa) is is provided provided at at the the top top of of the the two two PDE3 PDE3 isoforms. isoforms. PDE3A1 PDE3A1 is is translated translated from from the the second second AUGAUG codon ofof thethe open-reading open-reading frame frame found found in thein the PDE3 PDE3 mRNA. mRNA. While While the longest the longest variant variant of PDE3A, of PDE3A,PDE3A1, PDE3A1, is mainly is localisedmainly localised to the sarcoplasmic to the sarcop reticulum,lasmic reticulum, PDE3A2 PDE3A2 and PDE3A3 and PDE3A3 are found are bothfound in bothmembranes in membranes and cytoplasm. and cytoplasm. PDE3B isPDE3B mainly is localised mainly tolocalised plasma to membrane plasma membrane invaginations invaginations known as T knowntubules. as Coloured T tubules. diamonds Coloured indicate diamonds phosphorylation indicate phosphorylation sites. Selected sites. PDE3-interacting Selected PDE3-interacting proteins are proteinslisted where are listed the precise where bindingthe precise sequences binding are sequences known. Membrane-associatedare known. Membrane-associated N-terminal hydrophobic