REVIEW URRENT C OPINION
New pharmacologic interventions to increase cardiac contractility: challenges and opportunities Matthew Movsesian
Purpose of review The most extensively studied inotropic agents in patients with heart failure are phosphodiesterase (PDE) 3 inhibitors, which increase contractility by raising intracellular cyclic adenosine monophosphate content. In clinical trials, the inotropic benefits of these agents have been outweighed by an increase in sudden cardiac death. Here, I review recent findings that help explain what are likely to be distinct mechanisms involved in the beneficial and adverse effects of PDE3 inhibition. Recent findings The proapoptotic consequences of PDE3 inhibition are becoming more apparent. Moreover, it has also become clear that individual PDE3 isoforms in cardiac myocytes are selectively regulated to interact with different proteins in different intracellular compartments. The beneficial and adverse effects of PDE3 inhibition may thus be attributable to the inhibition of different isoforms in different intracellular domains. In particular, PDE3A1 has been shown to interact directly with sarcoplasmic/endoplasmic reticulum Ca2þ ATPase (SERCA2) in the sarcoplasmic reticulum through a phosphorylation of a site in its unique N-terminal domain, making it possible that this isoform can be selectively targeted to increase intracellular Ca2þ cycling. Summary Conventional PDE3 inhibitors target several functionally distinct isoforms of these enzymes. Isoform-selective and/or compartment-selective targeting of PDE3, through its protein–protein interactions, may produce the inotropic benefits of PDE3 inhibition without the adverse consequences. Keywords contractility, cyclic adenosine monophosphate, heart failure, phosphodiesterase inhibitors
INTRODUCTION Five million seven hundred thousand Americans suffer from heart failure, a syndrome characterized by the inability of the heart to meet the body’s circulatory demands. Each year, more than 550 000 new patients are diagnosed in the United States, of which approximately 50% involve dilated cardiomyopathy and impaired myocardial contractility [1,2]. Restoring normal contractility is an obvious therapeutic goal. A number of agents are effective in increasing myocardial contractility in these patients in the short term. The most extensively studied have been drugs that inhibit the cyclic adenosine monophosphate (cAMP)-hydrolytic activity of phosphodiesterase (PDE) 3, the principal cAMP PDE regulating contractile responses in cardiomyocytes [3 ]. Although PDE3 inhibitors augment contractility in patients with dilated cardiomyopathy, with longterm administration this benefit is offset by an increase in sudden cardiac death (Fig. 1) [4]. In fact, &&
no inotropic agent has yet been shown convincingly to increase survival with sustained use. The development of an agent that could maintain the inotropic benefits of PDE3 inhibition without increasing mortality would address one of the most glaring unmet needs in cardiovascular medicine. In this review, I examine the mechanisms responsible for the inotropic response to PDE3 inhibitors, about which much is known, and those responsible for the long-term increase in sudden cardiac death, which are less well understood but are likely to be distinct from those to which Cardiology Section, VA Salt Lake City Health Care System, Salt Lake City, Utah, USA Correspondence to Matthew Movsesian, MD, Cardiology Section, VA Salt Lake City Health Care System, 500 Foothill Blvd, Salt Lake City, UT 84148, USA. Tel: +1 801 582 1565x4156; fax: +1 801 584 2532; e-mail: [email protected] Curr Opin Cardiol 2015, 30:285–291 DOI:10.1097/HCO.0000000000000165
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KEY POINTS
PDE3 inhibition
PDE3 inhibitors, which raise intracellular cAMP content, increase contractility in patients with heart failure, but they also increase sudden cardiac death through what are likely to be separate proapoptotic and prohypertrophic mechanisms. It has recently become clear that PDE3 inhibitors are targeting several functionally distinct isoforms that are localized to different intracellular compartments of cardiac myocytes and regulated through separate mechanisms. New therapies that target sarcoplasmic reticulumassociated PDE3 – possibly through its unique protein– protein interactions – may yield inotropic actions without the adverse consequences of conventional PDE3 inhibition.
inotropic responses are attributable. I consider the challenges and opportunities involved in the development of new inotropic agents in this context.
PHOSPHODIESTERASE 3 INHIBITION IN THE TREATMENT OF DILATED CARDIOMYOPATHY Cyclic nucleotide PDEs regulate intracellular signaling by hydrolyzing cAMP and/or cyclic guanosine monophosphate (cGMP). By blocking the hydrolysis of these second messengers, PDE inhibitors potentiate cyclic nucleotide-mediated signaling, increasing the phosphorylation of intracellular proteins by cAMP-dependent and cGMP-dependent protein kinases A (PKA) and G (PKG). Eleven families of PDEs have been described, comprising a total of over 50 isoforms that are selectively expressed in different cells (a cell may express more than one PDE) [5].
cAMP-mediated signaling
Phosphorylation of ryanodine-sensitive Ca2+channels and phospholamban
ICER ?
Ca2+cycling
Pro-apoptotic signaling ?
Contractility
Mortality
Short term
Long term
FIGURE 2. Mechanisms contributing to the inotropic effects of PDE3 inhibition and to the increase in cardiac death. cAMP, cyclic adenosine monophosphate; ICER, inducible cAMP early repressor; PDE, phosphodiesterase. Reproduced from Movsesian, Wever-Pinzon and Vandeput, Current Opinion in Pharmacology 2011; 11:707–713.
Enzymes in the PDE3 family have a particularly prominent role in regulating cAMP-mediated signaling in cardiac muscle [6]. Two subfamilies, PDE3A and PDE3B, have been identified [7,8]. Experiments in mice indicate that myocardial contractility is regulated specifically by the PDE3A subfamily, and Pde3a ablation increases the phosphorylation of two proteins in the sarcoplasmic reticulum of cardiac myocytes that are involved in intracellular Ca2þ cycling [3 ]. Phosphorylation of phospholamban stimulates the activity of sarcoplasmic/endoplasmic reticulum Ca2þ ATPase (SERCA2), the Ca2þ-transporting ATPase of the sarcoplasmic reticulum, increasing Ca2þ uptake during diastole, whereas phosphorylation of ryanodine-sensitive Ca2þ channels increases Ca2þ release from the sarcoplasmic reticulum during systole [9,10]. These changes in protein phosphorylation augment myocardial contractility by increasing the amplitude of intracellular Ca2þ transients (Fig. 2) [3 ]. The role of PDE3A in regulating Ca2þ uptake by the sarcoplasmic reticulum is likely to be linked to its integration into a sarcoplasmic-reticulum signaling complex that includes SERCA2, A-kinase (PKA)-anchoring protein (AKAP18), phospholamban, and PKA [3 ]. In dilated cardiomyopathy, decreases in myocardial b-adrenergic receptor density, together with increases in the activity of Gai and b-adrenergic receptor kinase, attenuate the stimulation of cAMP formation by catecholamines, leading to decreases in myocardial cAMP content and, consequently, in the amplitude of intracellular Ca2þ transients [11–20]. PDE3 inhibitors are used to ‘overcome’ this reduction in intracellular cAMP content. In the &&
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Annual incidence (%)
25
Placebo
PDE3 inhibition
20
15
&&
10
5
0 Cardiac death
Sudden death
FIGURE 1. Effects of PDE3 inhibition on cardiac death and sudden death. PDE, phosphodiesterase. Reproduced from [4]. 286
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short term, PDE3 inhibitors have the desirable hemodynamic actions of raising cardiac output and lowering left ventricular filling pressures [21–27]. With long-term administration, however, these improvements in hemodynamics are outweighed by an increase in mortality attributable to an increase in sudden cardiac death [4]. It is possible that the increase in sudden cardiac death in these clinical trials might have been averted by the use of implantable cardiac defibrillators, which were not ‘standard-of-care’ for dilated cardiomyopathy at the time most of these trials were carried out. There is also the possibility that the concomitant use of antiarrhythmic agents would have been beneficial. In one retrospective study, patients treated with PDE inhibitors for extended periods while awaiting cardiac transplant were treated concurrently with amiodarone, and an increase in sudden cardiac death was not observed [28]. At this point, however, given the costs that would be incurred and the fact that most of the agents used starting in the 1990s are now off-patent, it is unlikely that a prospective randomized trial will be conducted.
MECHANISMS UNDERLYING THE INCREASE IN SUDDEN CARDIAC DEATH Understanding the mechanisms underlying the increase in sudden cardiac death in patients treated with PDE3 inhibitors has proven challenging. In my opinion, they are likely, for two reasons, to be separate from those that augment contractility. First, the inotropic response to PDE3 inhibitors is immediate, and, as demonstrated in some of the first clinical trials, was often not sustained with longterm use, whereas the increase in sudden cardiac death is seen over a period of months to a year [21–27]. This suggests that direct proarrhythmic effects of PDE3 inhibition may not be responsible. Moreover, an increase in SERCA2 activity, the immediate consequence of the increase in phospholamban phosphorylation brought about by PDE3 inhibition, has antiarrhythmic effects in animal models of ischemia/reperfusion and chronic heart failure [29,30]. It seems probable, therefore, that the increase in sudden cardiac death reflects a gradual response to proapoptotic or other maladaptive changes in cAMP-mediated signaling rather than an acute proarrhythmic effect concomitant with the increase in contractility. PDE3 inhibition in rats and Pde3a ablation in mice lead to increases in the phosphorylation of cAMP response element-binding protein and consequent increases in the expression of inducible cAMP early repressors, promoting apoptosis (Fig. 2) [3 ,31,32]. Conversely, PDE3A &&
overexpression in mice reduces inducible cAMP early repressor expression, increases Bcl-2 expression and reduces apoptosis following ischemia/ reperfusion injury (but reduces myocardial contractility) [33 ]. Such proapoptotic consequences of PDE3 inhibition are likely to contribute to pathologic remodeling in dilated cardiomyopathy [34]. Part of the problem in trying to identify a mechanism to explain the increased mortality in patients treated with PDE3 inhibitors is the complexity of cAMP-mediated signaling in cardiac myocytes. Canonically, cAMP activates PKA, leading to the phosphorylation of its protein substrates. A role for cAMP directly modulating the open probability of ion channels has also been noted [35]. More recently, it has become clear that effects are also mediated by guanine-nucleotide-exchange proteins activated by cAMP (Epacs), which influence protein phosphorylation through diverse mechanisms [36,37]. In cardiac myocytes, Epac activation augments contractility through signaling pathways that increase the phosphorylation of ryanodine-sensitive Ca2þ channels of the sarcoplasmic reticulum by Ca2þ/calmodulin-activated protein kinase II (CamKII) – which was seen in Pde3a/ mice1 – and of sarcomeric proteins such as cardiac myosin-binding protein C and troponin I by CamKII and protein kinase C [38–41]. Epac activation also has prohypertrophic actions that result, at least in part, from the activation of CamKII, as well as the protein phosphatase calcineurin [42,43]. These observations suggest that changes in intracellular cAMP content in cardiac myocytes are likely to lead to the activation of multiple protein kinases and phosphatases and to affect the phosphorylation of a large number of proteins. It may be that the aggregate of these actions yields a pathologically altered myocardium with an increased susceptibility to sudden cardiac death, and identifying a single pathway to which this can be fully ascribed may be impossible. &
PHOSPHODIESTERASE 3 INHIBITION HAS DIFFERENT EFFECTS IN CHILDREN AND ADULTS A recent study comparing the effects of long-term PDE3 inhibition on cAMP-mediated signaling in children and adults with heart failure may offer new insight [44 ]. Phospholamban phosphorylation, which is diminished in failing myocardium in both age groups, was restored to normal levels in children treated with PDE3 inhibitors, who do not appear to have the adverse responses to PDE3 inhibitors seen in adults. In the latter group, in contrast, long-term administration of PDE3 inhibitors led to a
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reduction in phospholamban phosphorylation relative to adults with heart failure who were not treated with PDE3 inhibitors. This may explain the tachyphylactic inotropic response to PDE3 inhibition noted above [21–27]. In view of the beneficial long-term actions of b-adrenergic receptor antagonists in patients with dilated cardiomyopathy, an intervention that inhibits cAMP hydrolysis in cardiac myocytes and increases the phosphorylation of the ‘undesirable’ PKA substrates without increasing the phosphorylation of proteins responsible for inotropic responses – such as phospholamban – may have effects that are, on balance, harmful. Elucidating the reasons for these fundamentally different consequences of PDE3 inhibition in adults and children with heart failure may therefore be very instructive as to the mechanisms involved.
COMPARTMENTATION OF CYCLIC ADENOSINE MONOPHOSPHATEMEDIATED SIGNALING IN CARDIAC MYOCYTES Would it be possible to target PDE3 so as to increase the phosphorylation of proteins involved in inotropic responses without increasing the phosphorylation of proteins involved in the increase in sudden cardiac death, and, in so doing, to avoid the adverse consequences of PDE3 inhibition seen in clinical trials? Here, it is useful to consider the ‘compartmentation’ of cAMP-mediated signaling in cardiac myocytes, a term that refers to the fact that cAMP content is regulated differentially in spatially and functionally distinct compartments of these cells. It has long been known that exposure to b-adrenergic receptor agonists increases cAMP content in cytosolic and microsomal fractions of cardiac muscle and augments contractility, whereas exposure to prostaglandin E1 increases cAMP content only in cytosolic fractions, without inotropic effects [45,46]. This observation suggested that inotropic responses are likely to result specifically from increases in the phosphorylation of membrane-associated proteins. The discovery that Pde3a ablation in mice increases the phosphorylation of sarcoplasmic reticulum proteins involved in intracellular Ca2þ handling is consistent with this hypothesis [3 ]. This compartmentation of cAMP-mediated signaling is a feature of the pathophysiology of dilated cardiomyopathy in humans (both ischemic and nonischemic), where the reduction in cAMP content is much more pronounced in microsomes than in cytosolic fractions [19]. Over the past decade, the prominent involvement of PDEs in this compartmentation has become &&
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apparent. In rat cardiac myocytes, b-adrenergic receptor agonists induce increases in intracellular cAMP content that are highly localized in the absence of PDE inhibitors, but are diffuse in their presence [47]. Individual PDEs, which are targeted by protein– protein interactions to specific intracellular domains, have distinct roles. In rat heart, PDE4 has a greater role than PDE3 in regulating glucagon and catecholamine-mediated increases in intracellular cAMP content, whereas PDE3 has a greater role in regulating forskolin-induced increases [48,49]. PDE2 has a major role in regulating b-adrenergic receptor-mediated increases in intracellular cAMP content but only a small role in regulating forskolin-induced increases, and PDE2 and PDE3 regulate ‘opposing’ effects of cGMP on cAMP-mediated signaling in rat heart in functionally separate compartments [50,51]. These studies focused on PDE families, but individual isoforms within a family have precise and distinct roles. This has been examined most extensively in the PDE4 family. PDE4D3 is present in multiprotein complexes regulating KCNQ1/KCNE1 Kþ channels and ryanodine-sensitive Ca2þ channels [52,53]. The latter are hyperphosphorylated in Pde4d/ mice, leading to abnormalities of sarcoplasmic reticulum Ca2þ release associated with arrhythmias and the development of dilated cardiomyopathy [53]. In contrast, experiments in Pde4d/ and Pde4b/ mice showed that the stimulation of L-type Ca2þ currents by b-adrenergic receptor agonists is controlled specifically by PDE4B [54]. These unique roles for individual PDE4 variants derive principally from the differences in their intracellular targeting, which reflect the distinct protein–protein interactions through which they are recruited to intracellular signaling complexes [55,56]. Although PDE4, which has a prominent role in rodent hearts, contributes less to the regulation of contractility in human heart, the delineation of unique roles for individual PDE4 isoforms is a relevant paradigm for PDE3A. In human cardiac myocytes, the PDE3A gene gives rise to several isoforms, the amino acid sequences of which are identical save for the presence of different lengths of N-terminal sequence. These N-terminal sequences are involved in intracellular localization, protein– protein interactions and allosteric regulation of catalytic activity, which resides in the C-terminus [57]. The most important isoforms appear to be PDE3A1 and PDE3A2. PDE3A1, a 136-kDa protein, has a unique N-terminal extension containing hydrophobic loops that insert into intracellular membranes [58,59], and three well-characterized phosphorylation sites, S293, S312 and S428 [60–62]. PDE3A1 is found solely in the sarcoplasmic Volume 30 Number 3 May 2015
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reticulum of cardiac myocytes. PDE3A2, a 118-kDa protein transcribed from a downstream site in exon 1, lacks the N-terminal extension and S293, and is recovered in cytosolic as well as membrane-enriched fractions of human myocardium.
COMPARTMENT-SELECTIVE PHOSPHODIESTERASE 3A INHIBITION? With PDE3A1 being restricted to the sarcoplasmic reticulum of cardiac myocytes, it would seem that an agent that could target this isoform without targeting PDE3A2 would have the potential to selectively increase the phosphorylation of proteins involved in intracellular Ca2þ signaling. Unsurprisingly, in view of their C-terminal sequence identity, PDE3A1 and PDE3A2 are functionally identical with respect to their basal catalytic activity and sensitivity to existing PDE3 inhibitors [63], making it extremely unlikely that an agent could target the catalytic site of PDE3A1 without also targeting PDE3A2. Moreover, recent studies have shown important distinctions between these isoforms. One is that the two isoforms are selectively phosphorylated at distinct protein-interacting sites in their common N-terminal sequence in response to different extracellular stimuli, and that these differences in N-terminal phosphorylation lead to different allosteric effects on the catalytic activity of PDE3A1 and PDE3A2 [64 ]. Perhaps more importantly, with respect to therapeutic potential, phosphorylation of PDE3A1 and PDE3A2 regulates their interactions with different proteins [64 ]. The characterization of the interactomes of each isoform is at a very early stage, but it was recently shown that PDE3A1 – but not PDE3A2 – interacts with the 5-hydroxytryptamine receptor [65 ]. Taken together, these observations indicate that existing PDE3 inhibitors, which have no significant selectivity for individual PDE3 isoforms, are actually targeting several functionally distinct enzymes in cardiac myocytes likely to have discrete roles in cAMP-mediated signaling. Although nonselective PDE3 inhibition can be expected to affect the phosphorylation of a diverse set of proteins (phosphoproteome), with beneficial and adverse consequences, isoform-selective inhibition could separate these effects by regulating a smaller number of proteins with greater precision. Although isoform-selective catalytic-site inhibition is unfeasible, the differences in protein–protein interactions attributable to the N-terminal sequence differences between PDE3A1 and PDE3A2 suggest that PDE3A1 could be selectively targeted in this region. An agent that could bind selectively to the N-terminus of PDE3A1 and block its incorporation into a SERCA2 complex in the &&
&&
&
sarcoplasmic reticulum has the potential to increase the phosphorylation of a relatively limited subset of proteins involved in intracellular Ca2þ cycling, thereby increasing contractility without proapoptotic or other sequelae that increase sudden cardiac death with long-term administration. It has recently been shown, in fact, that PDE3A1 and PDE3A2 interact with SERCA2 through separate molecular mechanisms, making it likely that PDE3A1’s interaction with SERCA2 can be selectively targeted in this manner [66]. Of course, the notion of using small molecules to disrupt protein–protein interactions brings technological challenges of its own. Protein–protein interfaces may be large, hydrophobic, discontinuous or flat, lacking the deep pockets into which small molecules typically insert. Nonetheless, advances in high-throughput screening for protein–protein interaction inhibitors have been promising, with several examples in cancer therapeutics now in clinical trials [67]. In principle, there is no reason to think this could not work as well in the treatment of cardiovascular diseases.
OTHER POTENTIAL TARGETS TO IMPROVE CONTRACTILITY Given that PDE3 and PDE5 inhibitors have made it into clinical practice as agents that target intracellular proteins successfully, it seems natural to ask whether agents that target PDEs other than PDE3 might be useful as inotropic agents. One of the beststudied families is PDE4. Inhibitors of enzymes in this family have inotropic properties in animal hearts. To my knowledge, however, inotropic responses to PDE4 inhibition in human myocardium have not been demonstrated. To some degree this is surprising, given that PDE4, like PDE3, forms complexes with phospholamban [68]; thus, PDE4 inhibition might be expected to have inotropic actions. On the contrary, the amount of PDE4 as a fraction of total PDE activity is much lower in human heart than in rodent models [68]. In sarcoplasmic reticulum-enriched microsomes from failing human hearts, PDE3 inhibition potentiated phospholamban phosphorylation and SERCA2 activity, and the effect was not seen with PDE4 inhibition [66]. In the past decade, the fact that isoforms in the PDE1 family represent a large fraction of the cAMP(and cGMP-)hydrolytic activity in human myocardium has been recognized [69,70]. Inhibition of PDE1 has been shown to have antihypertrophic actions in rodent cardiomyocytes [71], raising the possibility that this could be a mechanism for increasing contractility with fewer adverse consequences.
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Moreover, inotropic actions attributable to PDE1 inhibition have not, to my knowledge, been demonstrated [72], and research has been hampered by the lack of access to inhibitors with high specificity for the PDE1 family, let alone for specific isoforms. Finally, levosimendan, a compound said to have both PDE3 inhibitory and Ca2þ-sensitizing actions, has been used as an alternative to PDE3 inhibitors for raising contractility in patients with heart failure. The underlying notion has been that its Ca2þ-sensitizing action, which would increase myocardial contraction as a function of intracellular [Ca2þ] independently of effects mediated through intracellular [cAMP], might avoid the adverse longterm consequences of PDE3 inhibition. Benefits of repetitive treatment with levosimendan for patients with advanced heart failure have been demonstrated with respect to hemodynamics and hospital readmissions [73], but no clinical trial has to my knowledge shown a convincing benefit versus PDE3 inhibitors with respect to long-term survival. This past year, a study provided evidence that inotropic responses to levosimendan are due principally to PDE3 inhibition rather than Ca2þ-sensitization, which may explain the difficulty in demonstrating superior safety with long-term use [74].
CONCLUSION The challenge in the field of inotropic therapy has been that of developing an agent that can yield a sustained increase in cardiac contractility without reducing survival. In the case of PDE3 inhibitors, which potentiate cAMP-mediated signaling and therefore affect the phosphorylation of a wide range of proteins, it seems likely that the beneficial and adverse effects occur through distinct and potentially separable mechanisms. New information suggests that it may be possible to target individual isoforms of PDE3 with greater precision, raising the possibility of selectively increasing the phosphorylation of a more limited set of proteins involved specifically in the inotropic responses. Whether this will prove feasible remains to be seen. Acknowledgements None. Financial support and sponsorship This work is supported by medical research funds from the US Department of Veterans Affairs and the National Institutes of Health (NIH 2R01 HD014938-30). Conflicts of interest The author is the inventor of two patents issued to the University of Utah and the US Department of Veterans Affairs for isoform-selective PDE3 inhibition: 290
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‘N-terminally truncated isoforms of PDE3A cyclic phosphodiesterases’. European Patent Office, EP1430140. ‘Isoform-selective inhibitors and activators of PDE3 cyclic nucleotide phosphodiesterases’. United States Patent & Trademark Office, 8722866.
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Proc Natl Acad Sci U S A 2009; 106:14144– 14149. 42. Morel E, Marcantoni A, Gastineau M, et al. cAMP-binding protein epac induces cardiomyocyte hypertrophy. Circ Res 2005; 97:1296–1304. 43. Pereira L, Ruiz-Hurtado G, Morel E, et al. Epac enhances excitation-transcription coupling in cardiac myocytes. J Mol Cell Cardiol 2012; 52:283–291. 44. Nakano SJ, Miyamoto SD, Movsesian M, et al. Age-related differences in && phosphodiesterase activity and effects of chronic phosphodiesterase inhibition in idiopathic dilated cardiomyopathy. Circ Heart Fail 2015; 8:57–63. This article shows that phospholamban phosphorylation is increased in children with heart failure treated with PDE3 inhibitors but not in adults. This may help explain the lack of sustained inotropic benefits with long-term administration of PDE3 inhibitors in adults. 45. Hayes JS, Brunton LL, Mayer SE. Selective activation of particulate cAMPdependent protein kinase by isoproterenol and prostaglandin E1. J Biol Chem 1980; 255:5113–5119. 46. Buxton IL, Brunton LL. Compartments of cyclic AMP and protein kinase in mammalian cardiomyocytes. J Biol Chem 1983; 258:10233–10239. 47. Zaccolo M, Pozzan T. Discrete microdomains with high concentration of cAMP in stimulated rat neonatal cardiac myocytes. Science 2002; 295:1711–1715. 48. Mongillo M, McSorley T, Evellin S, et al. Fluorescence resonance energy transfer-based analysis of cAMP dynamics in live neonatal rat cardiac myocytes reveals distinct functions of compartmentalized phosphodiesterases. Circ Res 2004; 95:67–75.
49. Rochais F, Abi-Gerges A, Horner K, et al. A specific pattern of phosphodiesterases controls the cAMP signals generated by different Gs-coupled receptors in adult rat ventricular myocytes. Circ Res 2006; 98:1081–1088. 50. Mongillo M, Tocchetti CG, Terrin A, et al. Compartmentalized phosphodiesterase-2 activity blunts beta-adrenergic cardiac inotropy via an NO/cGMPdependent pathway. Circ Res 2006; 98:226–234. 51. Stangherlin A, Gesellchen F, Zoccarato A, et al. cGMP signals modulate cAMP levels in a compartment-specific manner to regulate catecholaminedependent signaling in cardiac myocytes. Circ Res 2011; 108:929–939. 52. Terrenoire C, Houslay MD, Baillie GS, Kass RS. The cardiac IKs potassium channel macromolecular complex includes the phosphodiesterase PDE4d3. J Biol Chem 2009; 284:9140–9146. 53. Lehnart SE, Wehrens XH, Reiken S, et al. Phosphodiesterase 4d deficiency in the ryanodine-receptor complex promotes heart failure and arrhythmias. Cell 2005; 123:25–35. 54. Leroy J, Richter W, Mika D, et al. Phosphodiesterase 4b in the cardiac L-type Ca(2)(þ) channel complex regulates Ca(2)(þ) current and protects against ventricular arrhythmias in mice. J Clin Invest 2011; 121:2651–2661. 55. Houslay MD, Baillie GS, Maurice DH. cAMP-specific phosphodiesterase-4 enzymes in the cardiovascular system: a molecular toolbox for generating compartmentalized cAMP signaling. Circ Res 2007; 100:950–966. 56. Houslay MD. Underpinning compartmentalised cAMP signalling through targeted cAMP breakdown. Trends Biochem Sci 2010; 35:91–100. 57. Wechsler J, Choi YH, Krall J, et al. Isoforms of cyclic nucleotide phosphodiesterase PDE3a in cardiac myocytes. J Biol Chem 2002; 277:38072– 38078. 58. Kenan Y, Murata T, Shakur Y, et al. Functions of the N-terminal region of cyclic nucleotide phosphodiesterase 3 (PDE 3) isoforms. J Biol Chem 2000; 275:12331–12338. 59. Shakur Y, Takeda K, Kenan Y, et al. Membrane localization of cyclic nucleotide phosphodiesterase 3 (PDE3). Two N-terminal domains are required for the efficient targeting to, and association of, PDE3 with endoplasmic reticulum. J Biol Chem 2000; 275:38749–38761. 60. Han SJ, Vaccari S, Nedachi T, et al. Protein kinase B/Akt phosphorylation of PDE3a and its role in mammalian oocyte maturation. EMBO J 2006; 25:5716–5725. 61. Pozuelo Rubio M, Campbell DG, Morrice NA, Mackintosh C. Phosphodiesterase 3a binds to 14-3-3 proteins in response to PMA-induced phosphorylation of Ser428. Biochem J 2005; 392:163–172. 62. Hunter RW, Mackintosh C, Hers I. Protein kinase c-mediated phosphorylation and activation of PDE3a regulate cAMP levels in human platelets. J Biol Chem 2009; 284:12339–12348. 63. Hambleton R, Krall J, Tikishvili E, et al. Isoforms of cyclic nucleotide phosphodiesterase PDE3 and their contribution to cAMP hydrolytic activity in subcellular fractions of human myocardium. J Biol Chem 2005; 280:39168– 39174. 64. Vandeput F, Szabo-Fresnais N, Ahmad F, et al. Selective regulation of cyclic && nucleotide phosphodiesterase PDE3a isoforms. Proc Natl Acad Sci U S A 2013; 110:19778–19783. This work demonstrates that PDE3A1 and PDE3A2 are selectively regulated and interact with different sets of proteins, making it likely they are functionally distinct and selectively targetable. 65. Weninger S, Van Craenenbroeck K, Cameron RT, et al. Phosphodiesterase 4 & interacts with the 5-HT4(b) receptor to regulate cAMP signaling. Cell Signal 2014; 26:2573–2582. This article gives a specific example of a protein–protein interaction specific to PDE3A1, providing further evidence of the unique function and targetability of this isoform. 66. Ahmad F, Shen W, Vandeput F, et al. Regulation of SERCA2 activity by PDE3a in human myocardium: phosphorylation-dependent interaction of PDE3a1 with SERCA2. J Biol Chem 2015. [Epub ahead of print] 67. Ivanov AA, Khuri FR, Fu H. Targeting protein–protein interactions as an anticancer strategy. Trends Pharmacol Sci 2013; 34:393–400. 68. Richter W, Xie M, Scheitrum C, et al. Conserved expression and functions of PDE4 in rodent and human heart. Basic Res Cardiol 2011; 106:249–262. 69. Vandeput F, Wolda SL, Krall J, et al. Cyclic nucleotide phosphodiesterase PDE1C1 in human cardiac myocytes. J Biol Chem 2007; 282:32749– 32757. 70. Vandeput F, Krall J, Ockaili R, et al. cGMP-hydrolytic activity and its inhibition by sildenafil in normal and failing human and mouse myocardium. J Pharmacol Exp Ther 2009; 330:884–891. 71. Miller CL, Oikawa M, Cai Y, et al. Role of Ca2þ/calmodulin-stimulated cyclic nucleotide phosphodiesterase 1 in mediating cardiomyocyte hypertrophy. Circ Res 2009; 105:956–964. 72. Johnson WB, Katugampola S, Able S, et al. Profiling of cAMP and cGMP phosphodiesterases in isolated ventricular cardiomyocytes from human hearts: comparison with rat and guinea pig. Life Sci 2012; 90:328–336. 73. Nieminen MS, Altenberger J, Ben-Gal T, et al. Repetitive use of levosimendan for treatment of chronic advanced heart failure: clinical evidence, practical considerations, and perspectives: an expert panel consensus. Int J Cardiol 2014; 174:360–367. 74. Orstavik O, Ata SH, Riise J, et al. Inhibition of phosphodiesterase-3 by levosimendan is sufficient to account for its inotropic effect in failing human heart. Br J Pharmacol 2014; 171:5169–5181.
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New pharmacologic interventions to increase cardiac contractility: challenges and opportunities Matthew Movsesian
Purpose of review The most extensively studied inotropic agents in patients with heart failure are phosphodiesterase (PDE) 3 inhibitors, which increase contractility by raising intracellular cyclic adenosine monophosphate content. In clinical trials, the inotropic benefits of these agents have been outweighed by an increase in sudden cardiac death. Here, I review recent findings that help explain what are likely to be distinct mechanisms involved in the beneficial and adverse effects of PDE3 inhibition. Recent findings The proapoptotic consequences of PDE3 inhibition are becoming more apparent. Moreover, it has also become clear that individual PDE3 isoforms in cardiac myocytes are selectively regulated to interact with different proteins in different intracellular compartments. The beneficial and adverse effects of PDE3 inhibition may thus be attributable to the inhibition of different isoforms in different intracellular domains. In particular, PDE3A1 has been shown to interact directly with sarcoplasmic/endoplasmic reticulum Ca2þ ATPase (SERCA2) in the sarcoplasmic reticulum through a phosphorylation of a site in its unique N-terminal domain, making it possible that this isoform can be selectively targeted to increase intracellular Ca2þ cycling. Summary Conventional PDE3 inhibitors target several functionally distinct isoforms of these enzymes. Isoform-selective and/or compartment-selective targeting of PDE3, through its protein–protein interactions, may produce the inotropic benefits of PDE3 inhibition without the adverse consequences. Keywords contractility, cyclic adenosine monophosphate, heart failure, phosphodiesterase inhibitors
INTRODUCTION Five million seven hundred thousand Americans suffer from heart failure, a syndrome characterized by the inability of the heart to meet the body’s circulatory demands. Each year, more than 550 000 new patients are diagnosed in the United States, of which approximately 50% involve dilated cardiomyopathy and impaired myocardial contractility [1,2]. Restoring normal contractility is an obvious therapeutic goal. A number of agents are effective in increasing myocardial contractility in these patients in the short term. The most extensively studied have been drugs that inhibit the cyclic adenosine monophosphate (cAMP)-hydrolytic activity of phosphodiesterase (PDE) 3, the principal cAMP PDE regulating contractile responses in cardiomyocytes [3 ]. Although PDE3 inhibitors augment contractility in patients with dilated cardiomyopathy, with longterm administration this benefit is offset by an increase in sudden cardiac death (Fig. 1) [4]. In fact, &&
no inotropic agent has yet been shown convincingly to increase survival with sustained use. The development of an agent that could maintain the inotropic benefits of PDE3 inhibition without increasing mortality would address one of the most glaring unmet needs in cardiovascular medicine. In this review, I examine the mechanisms responsible for the inotropic response to PDE3 inhibitors, about which much is known, and those responsible for the long-term increase in sudden cardiac death, which are less well understood but are likely to be distinct from those to which Cardiology Section, VA Salt Lake City Health Care System, Salt Lake City, Utah, USA Correspondence to Matthew Movsesian, MD, Cardiology Section, VA Salt Lake City Health Care System, 500 Foothill Blvd, Salt Lake City, UT 84148, USA. Tel: +1 801 582 1565x4156; fax: +1 801 584 2532; e-mail: [email protected] Curr Opin Cardiol 2015, 30:285–291 DOI:10.1097/HCO.0000000000000165
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KEY POINTS
PDE3 inhibition
PDE3 inhibitors, which raise intracellular cAMP content, increase contractility in patients with heart failure, but they also increase sudden cardiac death through what are likely to be separate proapoptotic and prohypertrophic mechanisms. It has recently become clear that PDE3 inhibitors are targeting several functionally distinct isoforms that are localized to different intracellular compartments of cardiac myocytes and regulated through separate mechanisms. New therapies that target sarcoplasmic reticulumassociated PDE3 – possibly through its unique protein– protein interactions – may yield inotropic actions without the adverse consequences of conventional PDE3 inhibition.
inotropic responses are attributable. I consider the challenges and opportunities involved in the development of new inotropic agents in this context.
PHOSPHODIESTERASE 3 INHIBITION IN THE TREATMENT OF DILATED CARDIOMYOPATHY Cyclic nucleotide PDEs regulate intracellular signaling by hydrolyzing cAMP and/or cyclic guanosine monophosphate (cGMP). By blocking the hydrolysis of these second messengers, PDE inhibitors potentiate cyclic nucleotide-mediated signaling, increasing the phosphorylation of intracellular proteins by cAMP-dependent and cGMP-dependent protein kinases A (PKA) and G (PKG). Eleven families of PDEs have been described, comprising a total of over 50 isoforms that are selectively expressed in different cells (a cell may express more than one PDE) [5].
cAMP-mediated signaling
Phosphorylation of ryanodine-sensitive Ca2+channels and phospholamban
ICER ?
Ca2+cycling
Pro-apoptotic signaling ?
Contractility
Mortality
Short term
Long term
FIGURE 2. Mechanisms contributing to the inotropic effects of PDE3 inhibition and to the increase in cardiac death. cAMP, cyclic adenosine monophosphate; ICER, inducible cAMP early repressor; PDE, phosphodiesterase. Reproduced from Movsesian, Wever-Pinzon and Vandeput, Current Opinion in Pharmacology 2011; 11:707–713.
Enzymes in the PDE3 family have a particularly prominent role in regulating cAMP-mediated signaling in cardiac muscle [6]. Two subfamilies, PDE3A and PDE3B, have been identified [7,8]. Experiments in mice indicate that myocardial contractility is regulated specifically by the PDE3A subfamily, and Pde3a ablation increases the phosphorylation of two proteins in the sarcoplasmic reticulum of cardiac myocytes that are involved in intracellular Ca2þ cycling [3 ]. Phosphorylation of phospholamban stimulates the activity of sarcoplasmic/endoplasmic reticulum Ca2þ ATPase (SERCA2), the Ca2þ-transporting ATPase of the sarcoplasmic reticulum, increasing Ca2þ uptake during diastole, whereas phosphorylation of ryanodine-sensitive Ca2þ channels increases Ca2þ release from the sarcoplasmic reticulum during systole [9,10]. These changes in protein phosphorylation augment myocardial contractility by increasing the amplitude of intracellular Ca2þ transients (Fig. 2) [3 ]. The role of PDE3A in regulating Ca2þ uptake by the sarcoplasmic reticulum is likely to be linked to its integration into a sarcoplasmic-reticulum signaling complex that includes SERCA2, A-kinase (PKA)-anchoring protein (AKAP18), phospholamban, and PKA [3 ]. In dilated cardiomyopathy, decreases in myocardial b-adrenergic receptor density, together with increases in the activity of Gai and b-adrenergic receptor kinase, attenuate the stimulation of cAMP formation by catecholamines, leading to decreases in myocardial cAMP content and, consequently, in the amplitude of intracellular Ca2þ transients [11–20]. PDE3 inhibitors are used to ‘overcome’ this reduction in intracellular cAMP content. In the &&
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Annual incidence (%)
25
Placebo
PDE3 inhibition
20
15
&&
10
5
0 Cardiac death
Sudden death
FIGURE 1. Effects of PDE3 inhibition on cardiac death and sudden death. PDE, phosphodiesterase. Reproduced from [4]. 286
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short term, PDE3 inhibitors have the desirable hemodynamic actions of raising cardiac output and lowering left ventricular filling pressures [21–27]. With long-term administration, however, these improvements in hemodynamics are outweighed by an increase in mortality attributable to an increase in sudden cardiac death [4]. It is possible that the increase in sudden cardiac death in these clinical trials might have been averted by the use of implantable cardiac defibrillators, which were not ‘standard-of-care’ for dilated cardiomyopathy at the time most of these trials were carried out. There is also the possibility that the concomitant use of antiarrhythmic agents would have been beneficial. In one retrospective study, patients treated with PDE inhibitors for extended periods while awaiting cardiac transplant were treated concurrently with amiodarone, and an increase in sudden cardiac death was not observed [28]. At this point, however, given the costs that would be incurred and the fact that most of the agents used starting in the 1990s are now off-patent, it is unlikely that a prospective randomized trial will be conducted.
MECHANISMS UNDERLYING THE INCREASE IN SUDDEN CARDIAC DEATH Understanding the mechanisms underlying the increase in sudden cardiac death in patients treated with PDE3 inhibitors has proven challenging. In my opinion, they are likely, for two reasons, to be separate from those that augment contractility. First, the inotropic response to PDE3 inhibitors is immediate, and, as demonstrated in some of the first clinical trials, was often not sustained with longterm use, whereas the increase in sudden cardiac death is seen over a period of months to a year [21–27]. This suggests that direct proarrhythmic effects of PDE3 inhibition may not be responsible. Moreover, an increase in SERCA2 activity, the immediate consequence of the increase in phospholamban phosphorylation brought about by PDE3 inhibition, has antiarrhythmic effects in animal models of ischemia/reperfusion and chronic heart failure [29,30]. It seems probable, therefore, that the increase in sudden cardiac death reflects a gradual response to proapoptotic or other maladaptive changes in cAMP-mediated signaling rather than an acute proarrhythmic effect concomitant with the increase in contractility. PDE3 inhibition in rats and Pde3a ablation in mice lead to increases in the phosphorylation of cAMP response element-binding protein and consequent increases in the expression of inducible cAMP early repressors, promoting apoptosis (Fig. 2) [3 ,31,32]. Conversely, PDE3A &&
overexpression in mice reduces inducible cAMP early repressor expression, increases Bcl-2 expression and reduces apoptosis following ischemia/ reperfusion injury (but reduces myocardial contractility) [33 ]. Such proapoptotic consequences of PDE3 inhibition are likely to contribute to pathologic remodeling in dilated cardiomyopathy [34]. Part of the problem in trying to identify a mechanism to explain the increased mortality in patients treated with PDE3 inhibitors is the complexity of cAMP-mediated signaling in cardiac myocytes. Canonically, cAMP activates PKA, leading to the phosphorylation of its protein substrates. A role for cAMP directly modulating the open probability of ion channels has also been noted [35]. More recently, it has become clear that effects are also mediated by guanine-nucleotide-exchange proteins activated by cAMP (Epacs), which influence protein phosphorylation through diverse mechanisms [36,37]. In cardiac myocytes, Epac activation augments contractility through signaling pathways that increase the phosphorylation of ryanodine-sensitive Ca2þ channels of the sarcoplasmic reticulum by Ca2þ/calmodulin-activated protein kinase II (CamKII) – which was seen in Pde3a/ mice1 – and of sarcomeric proteins such as cardiac myosin-binding protein C and troponin I by CamKII and protein kinase C [38–41]. Epac activation also has prohypertrophic actions that result, at least in part, from the activation of CamKII, as well as the protein phosphatase calcineurin [42,43]. These observations suggest that changes in intracellular cAMP content in cardiac myocytes are likely to lead to the activation of multiple protein kinases and phosphatases and to affect the phosphorylation of a large number of proteins. It may be that the aggregate of these actions yields a pathologically altered myocardium with an increased susceptibility to sudden cardiac death, and identifying a single pathway to which this can be fully ascribed may be impossible. &
PHOSPHODIESTERASE 3 INHIBITION HAS DIFFERENT EFFECTS IN CHILDREN AND ADULTS A recent study comparing the effects of long-term PDE3 inhibition on cAMP-mediated signaling in children and adults with heart failure may offer new insight [44 ]. Phospholamban phosphorylation, which is diminished in failing myocardium in both age groups, was restored to normal levels in children treated with PDE3 inhibitors, who do not appear to have the adverse responses to PDE3 inhibitors seen in adults. In the latter group, in contrast, long-term administration of PDE3 inhibitors led to a
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reduction in phospholamban phosphorylation relative to adults with heart failure who were not treated with PDE3 inhibitors. This may explain the tachyphylactic inotropic response to PDE3 inhibition noted above [21–27]. In view of the beneficial long-term actions of b-adrenergic receptor antagonists in patients with dilated cardiomyopathy, an intervention that inhibits cAMP hydrolysis in cardiac myocytes and increases the phosphorylation of the ‘undesirable’ PKA substrates without increasing the phosphorylation of proteins responsible for inotropic responses – such as phospholamban – may have effects that are, on balance, harmful. Elucidating the reasons for these fundamentally different consequences of PDE3 inhibition in adults and children with heart failure may therefore be very instructive as to the mechanisms involved.
COMPARTMENTATION OF CYCLIC ADENOSINE MONOPHOSPHATEMEDIATED SIGNALING IN CARDIAC MYOCYTES Would it be possible to target PDE3 so as to increase the phosphorylation of proteins involved in inotropic responses without increasing the phosphorylation of proteins involved in the increase in sudden cardiac death, and, in so doing, to avoid the adverse consequences of PDE3 inhibition seen in clinical trials? Here, it is useful to consider the ‘compartmentation’ of cAMP-mediated signaling in cardiac myocytes, a term that refers to the fact that cAMP content is regulated differentially in spatially and functionally distinct compartments of these cells. It has long been known that exposure to b-adrenergic receptor agonists increases cAMP content in cytosolic and microsomal fractions of cardiac muscle and augments contractility, whereas exposure to prostaglandin E1 increases cAMP content only in cytosolic fractions, without inotropic effects [45,46]. This observation suggested that inotropic responses are likely to result specifically from increases in the phosphorylation of membrane-associated proteins. The discovery that Pde3a ablation in mice increases the phosphorylation of sarcoplasmic reticulum proteins involved in intracellular Ca2þ handling is consistent with this hypothesis [3 ]. This compartmentation of cAMP-mediated signaling is a feature of the pathophysiology of dilated cardiomyopathy in humans (both ischemic and nonischemic), where the reduction in cAMP content is much more pronounced in microsomes than in cytosolic fractions [19]. Over the past decade, the prominent involvement of PDEs in this compartmentation has become &&
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apparent. In rat cardiac myocytes, b-adrenergic receptor agonists induce increases in intracellular cAMP content that are highly localized in the absence of PDE inhibitors, but are diffuse in their presence [47]. Individual PDEs, which are targeted by protein– protein interactions to specific intracellular domains, have distinct roles. In rat heart, PDE4 has a greater role than PDE3 in regulating glucagon and catecholamine-mediated increases in intracellular cAMP content, whereas PDE3 has a greater role in regulating forskolin-induced increases [48,49]. PDE2 has a major role in regulating b-adrenergic receptor-mediated increases in intracellular cAMP content but only a small role in regulating forskolin-induced increases, and PDE2 and PDE3 regulate ‘opposing’ effects of cGMP on cAMP-mediated signaling in rat heart in functionally separate compartments [50,51]. These studies focused on PDE families, but individual isoforms within a family have precise and distinct roles. This has been examined most extensively in the PDE4 family. PDE4D3 is present in multiprotein complexes regulating KCNQ1/KCNE1 Kþ channels and ryanodine-sensitive Ca2þ channels [52,53]. The latter are hyperphosphorylated in Pde4d/ mice, leading to abnormalities of sarcoplasmic reticulum Ca2þ release associated with arrhythmias and the development of dilated cardiomyopathy [53]. In contrast, experiments in Pde4d/ and Pde4b/ mice showed that the stimulation of L-type Ca2þ currents by b-adrenergic receptor agonists is controlled specifically by PDE4B [54]. These unique roles for individual PDE4 variants derive principally from the differences in their intracellular targeting, which reflect the distinct protein–protein interactions through which they are recruited to intracellular signaling complexes [55,56]. Although PDE4, which has a prominent role in rodent hearts, contributes less to the regulation of contractility in human heart, the delineation of unique roles for individual PDE4 isoforms is a relevant paradigm for PDE3A. In human cardiac myocytes, the PDE3A gene gives rise to several isoforms, the amino acid sequences of which are identical save for the presence of different lengths of N-terminal sequence. These N-terminal sequences are involved in intracellular localization, protein– protein interactions and allosteric regulation of catalytic activity, which resides in the C-terminus [57]. The most important isoforms appear to be PDE3A1 and PDE3A2. PDE3A1, a 136-kDa protein, has a unique N-terminal extension containing hydrophobic loops that insert into intracellular membranes [58,59], and three well-characterized phosphorylation sites, S293, S312 and S428 [60–62]. PDE3A1 is found solely in the sarcoplasmic Volume 30 Number 3 May 2015
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reticulum of cardiac myocytes. PDE3A2, a 118-kDa protein transcribed from a downstream site in exon 1, lacks the N-terminal extension and S293, and is recovered in cytosolic as well as membrane-enriched fractions of human myocardium.
COMPARTMENT-SELECTIVE PHOSPHODIESTERASE 3A INHIBITION? With PDE3A1 being restricted to the sarcoplasmic reticulum of cardiac myocytes, it would seem that an agent that could target this isoform without targeting PDE3A2 would have the potential to selectively increase the phosphorylation of proteins involved in intracellular Ca2þ signaling. Unsurprisingly, in view of their C-terminal sequence identity, PDE3A1 and PDE3A2 are functionally identical with respect to their basal catalytic activity and sensitivity to existing PDE3 inhibitors [63], making it extremely unlikely that an agent could target the catalytic site of PDE3A1 without also targeting PDE3A2. Moreover, recent studies have shown important distinctions between these isoforms. One is that the two isoforms are selectively phosphorylated at distinct protein-interacting sites in their common N-terminal sequence in response to different extracellular stimuli, and that these differences in N-terminal phosphorylation lead to different allosteric effects on the catalytic activity of PDE3A1 and PDE3A2 [64 ]. Perhaps more importantly, with respect to therapeutic potential, phosphorylation of PDE3A1 and PDE3A2 regulates their interactions with different proteins [64 ]. The characterization of the interactomes of each isoform is at a very early stage, but it was recently shown that PDE3A1 – but not PDE3A2 – interacts with the 5-hydroxytryptamine receptor [65 ]. Taken together, these observations indicate that existing PDE3 inhibitors, which have no significant selectivity for individual PDE3 isoforms, are actually targeting several functionally distinct enzymes in cardiac myocytes likely to have discrete roles in cAMP-mediated signaling. Although nonselective PDE3 inhibition can be expected to affect the phosphorylation of a diverse set of proteins (phosphoproteome), with beneficial and adverse consequences, isoform-selective inhibition could separate these effects by regulating a smaller number of proteins with greater precision. Although isoform-selective catalytic-site inhibition is unfeasible, the differences in protein–protein interactions attributable to the N-terminal sequence differences between PDE3A1 and PDE3A2 suggest that PDE3A1 could be selectively targeted in this region. An agent that could bind selectively to the N-terminus of PDE3A1 and block its incorporation into a SERCA2 complex in the &&
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&
sarcoplasmic reticulum has the potential to increase the phosphorylation of a relatively limited subset of proteins involved in intracellular Ca2þ cycling, thereby increasing contractility without proapoptotic or other sequelae that increase sudden cardiac death with long-term administration. It has recently been shown, in fact, that PDE3A1 and PDE3A2 interact with SERCA2 through separate molecular mechanisms, making it likely that PDE3A1’s interaction with SERCA2 can be selectively targeted in this manner [66]. Of course, the notion of using small molecules to disrupt protein–protein interactions brings technological challenges of its own. Protein–protein interfaces may be large, hydrophobic, discontinuous or flat, lacking the deep pockets into which small molecules typically insert. Nonetheless, advances in high-throughput screening for protein–protein interaction inhibitors have been promising, with several examples in cancer therapeutics now in clinical trials [67]. In principle, there is no reason to think this could not work as well in the treatment of cardiovascular diseases.
OTHER POTENTIAL TARGETS TO IMPROVE CONTRACTILITY Given that PDE3 and PDE5 inhibitors have made it into clinical practice as agents that target intracellular proteins successfully, it seems natural to ask whether agents that target PDEs other than PDE3 might be useful as inotropic agents. One of the beststudied families is PDE4. Inhibitors of enzymes in this family have inotropic properties in animal hearts. To my knowledge, however, inotropic responses to PDE4 inhibition in human myocardium have not been demonstrated. To some degree this is surprising, given that PDE4, like PDE3, forms complexes with phospholamban [68]; thus, PDE4 inhibition might be expected to have inotropic actions. On the contrary, the amount of PDE4 as a fraction of total PDE activity is much lower in human heart than in rodent models [68]. In sarcoplasmic reticulum-enriched microsomes from failing human hearts, PDE3 inhibition potentiated phospholamban phosphorylation and SERCA2 activity, and the effect was not seen with PDE4 inhibition [66]. In the past decade, the fact that isoforms in the PDE1 family represent a large fraction of the cAMP(and cGMP-)hydrolytic activity in human myocardium has been recognized [69,70]. Inhibition of PDE1 has been shown to have antihypertrophic actions in rodent cardiomyocytes [71], raising the possibility that this could be a mechanism for increasing contractility with fewer adverse consequences.
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Moreover, inotropic actions attributable to PDE1 inhibition have not, to my knowledge, been demonstrated [72], and research has been hampered by the lack of access to inhibitors with high specificity for the PDE1 family, let alone for specific isoforms. Finally, levosimendan, a compound said to have both PDE3 inhibitory and Ca2þ-sensitizing actions, has been used as an alternative to PDE3 inhibitors for raising contractility in patients with heart failure. The underlying notion has been that its Ca2þ-sensitizing action, which would increase myocardial contraction as a function of intracellular [Ca2þ] independently of effects mediated through intracellular [cAMP], might avoid the adverse longterm consequences of PDE3 inhibition. Benefits of repetitive treatment with levosimendan for patients with advanced heart failure have been demonstrated with respect to hemodynamics and hospital readmissions [73], but no clinical trial has to my knowledge shown a convincing benefit versus PDE3 inhibitors with respect to long-term survival. This past year, a study provided evidence that inotropic responses to levosimendan are due principally to PDE3 inhibition rather than Ca2þ-sensitization, which may explain the difficulty in demonstrating superior safety with long-term use [74].
CONCLUSION The challenge in the field of inotropic therapy has been that of developing an agent that can yield a sustained increase in cardiac contractility without reducing survival. In the case of PDE3 inhibitors, which potentiate cAMP-mediated signaling and therefore affect the phosphorylation of a wide range of proteins, it seems likely that the beneficial and adverse effects occur through distinct and potentially separable mechanisms. New information suggests that it may be possible to target individual isoforms of PDE3 with greater precision, raising the possibility of selectively increasing the phosphorylation of a more limited set of proteins involved specifically in the inotropic responses. Whether this will prove feasible remains to be seen. Acknowledgements None. Financial support and sponsorship This work is supported by medical research funds from the US Department of Veterans Affairs and the National Institutes of Health (NIH 2R01 HD014938-30). Conflicts of interest The author is the inventor of two patents issued to the University of Utah and the US Department of Veterans Affairs for isoform-selective PDE3 inhibition: 290
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‘N-terminally truncated isoforms of PDE3A cyclic phosphodiesterases’. European Patent Office, EP1430140. ‘Isoform-selective inhibitors and activators of PDE3 cyclic nucleotide phosphodiesterases’. United States Patent & Trademark Office, 8722866.
REFERENCES AND RECOMMENDED READING Papers of particular interest, published within the annual period of review, have been highlighted as: & of special interest && of outstanding interest 1. Roger VL, Go AS, Lloyd-Jones DM, et al. Heart disease and stroke statistics– 2011 update: a report from the American Heart Association. Circulation 2011; 123:e18–e209. 2. Roger VL. Epidemiology of heart failure. Circ Res 2013; 113:646–659. 3. Beca S, Ahmad F, Shen W, et al. Phosphodiesterase type 3a regulates basal && myocardial contractility through interacting with sarcoplasmic reticulum calcium ATPase type 2a signaling complexes in mouse heart. Circ Res 2013; 112:289– 297. This article identifies PDE3A as the specific subclass of PDE3A responsible for inotropic responses to PDE3 inhibition and identifies a SERCA2 complex with which PDE3A1 is associated. It provides a rationale for targeting the association of PDE3A1 with other proteins in this complex as a new strategy. 4. Amsallem E, Kasparian C, Haddour G, et al. Phosphodiesterase III inhibitors for heart failure. Cochrane Database Syst Rev (Online) 2005; CD002230. 5. Ahmad F, Murata T, Shimizu K, et al. Cyclic nucleotide phosphodiesterases: important signaling modulators and therapeutic targets. Oral Dis 2015; 21:e25–e50. 6. Francis SH, Blount MA, Corbin JD. Mammalian cyclic nucleotide phosphodiesterases: molecular mechanisms and physiological functions. Physiol Rev 2011; 91:651–690. 7. Taira M, Hockman SC, Calvo JC, et al. Molecular cloning of the rat adipocyte hormone-sensitive cyclic GMP-inhibited cyclic nucleotide phosphodiesterase. J Biol Chem 1993; 268:18573–18579. 8. Meacci E, Taira M, Moos M Jr, et al. Molecular cloning and expression of human myocardial cGMP-inhibited cAMP phosphodiesterase. Proc Natl Acad Sci U S A 1992; 89:3721–3725. 9. Simmerman HK, Jones LR. Phospholamban: protein structure, mechanism of action, and role in cardiac function. Physiol Rev 1998; 78:921–947. 10. Takasago T, Imagawa T, Shigekawa M. Phosphorylation of the cardiac ryanodine receptor by cAMP-dependent protein kinase. J Biochem (Tokyo) 1989; 106:872–877. 11. Bristow MR, Ginsburg R, Minobe W, et al. Decreased catecholamine sensitivity and beta-adrenergic-receptor density in failing human hearts. N Engl J Med 1982; 307:205–211. 12. Bristow MR, Ginsburg R, Umans V, et al. Beta 1- and beta 2-adrenergicreceptor subpopulations in nonfailing and failing human ventricular myocardium: coupling of both receptor subtypes to muscle contraction and selective beta 1-receptor down-regulation in heart failure. Circ Res 1986; 59:297–309. 13. Ungerer M, Parruti G, Bohm M, et al. Expression of beta-arrestins and betaadrenergic receptor kinases in the failing human heart. Circ Res 1994; 74:206–213. 14. Ungerer M, Bohm M, Elce JS, et al. Altered expression of beta-adrenergic receptor kinase and beta 1-adrenergic receptors in the failing human heart. Circulation 1993; 87:454–463. 15. Neumann J, Schmitz W, Scholz H, et al. Increase in myocardial G-proteins in heart failure. Lancet 1988; 2:936–937. 16. Feldman AM, Cates AE, Veazey WB, et al. Increase of the 40,000-mol wt pertussis toxin substrate (G protein) in the failing human heart. J Clin Invest 1988; 82:189–197. 17. Feldman MD, Copelas L, Gwathmey JK, et al. Deficient production of cyclic AMP: pharmacologic evidence of an important cause of contractile dysfunction in patients with end-stage heart failure. Circulation 1987; 75:331–339. 18. Danielsen W, v der Leyen H, Meyer W, et al. Basal and isoprenalinestimulated cAMP content in failing versus nonfailing human cardiac preparations. J Cardiovasc Pharmacol 1989; 14:171–173. 19. Bohm M, Reiger B, Schwinger RH, Erdmann E. cAMP concentrations, cAMP dependent protein kinase activity, and phospholamban in nonfailing and failing myocardium. Cardiovasc Res 1994; 28:1713–1719. 20. Beuckelmann DJ, Nabauer M, Erdmann E. Intracellular calcium handling in isolated ventricular myocytes from patients with terminal heart failure. Circulation 1992; 85:1046–1055. 21. Baim DS, McDowell AV, Cherniles J, et al. Evaluation of a new bipyridine inotropic agent–milrinone–in patients with severe congestive heart failure. N Engl J Med 1983; 309:748–756. 22. Benotti JR, Grossman W, Braunwald E, et al. Hemodynamic assessment of amrinone. A new inotropic agent. N Engl J Med 1978; 299:1373–1377.
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