Author + information
- Received October 23, 2018
- Revision received November 20, 2018
- Accepted December 10, 2018
- Published online March 11, 2019.
- Carmen C. Sucharov, PhDa,∗ (, )@CUAnschutz,
- Stephanie J. Nakano, MDb,
- Dobromir Slavov, PhDa,
- Jessica A. Schwisow, BSa,
- Erin Rodriguez, BSa,
- Karin Nunley, MSa,
- Allen Medway, MDa,
- Natalie Stafford, MPHa,
- Penny Nelson, MSa,
- Timothy A. McKinsey, PhDa,c,
- Matthew Movsesian, MDd,e,f,
- Wayne Minobe, BSa,
- Ian A. Carroll, PhDg,
- Matthew R.G. Taylor, MD, PhDa and
- Michael R. Bristow, MD, PhDa,g
- aDivision of Cardiology and Cardiovascular Institute, University of Colorado Denver, Aurora, Colorado
- bDepartment of Pediatrics, University of Colorado Denver, Children's Hospital Colorado, Aurora, Colorado
- cUniversity of Colorado Anschutz Medical Campus Consortium for Fibrosis Research & Translation, Aurora, Colorado
- dCardiology Section, George E. Wahlen Department of Veterans Affairs Medical Center, Salt Lake City, Utah
- eDepartment of Internal Medicine (Cardiovascular Medicine), University of Utah School of Medicine, Salt Lake City, Utah
- fDepartment of Pharmacology & Toxicology, University of Utah School of Medicine, Salt Lake City, Utah
- gARCA Biopharma, Westminster, Colorado
- ↵∗Address for correspondence:
Dr. Carmen C. Sucharov, University of Colorado Anschutz Medical Campus, 12700 East 19th Avenue P-15, Room 8003, Campus Box B-139, PO Box 6511, Aurora, Colorado 80045.
Background The phosphodiesterase 3A (PDE3A) gene encodes a PDE that regulates cardiac myocyte cyclic adenosine monophosphate (cAMP) levels and myocardial contractile function. PDE3 inhibitors (PDE3i) are used for short-term treatment of refractory heart failure (HF), but do not produce uniform long-term benefit.
Objectives The authors tested the hypothesis that drug target genetic variation could explain clinical response heterogeneity to PDE3i in HF.
Methods PDE3A promoter studies were performed in a cloned luciferase construct. In human left ventricular (LV) preparations, mRNA expression was measured by reverse transcription polymerase chain reaction, and PDE3 enzyme activity by cAMP-hydrolysis.
Results The authors identified a 29-nucleotide (nt) insertion (INS)/deletion (DEL) polymorphism in the human PDE3A gene promoter beginning 2,214 nt upstream from the PDE3A1 translation start site. Transcription factor ATF3 binds to the INS and represses cAMP-dependent promoter activity. In explanted failing LVs that were homozygous for PDE3A DEL and had been treated with PDE3i pre-cardiac transplantation, PDE3A1 mRNA abundance and microsomal PDE3 enzyme activity were increased by 1.7-fold to 1.8-fold (p < 0.05) compared with DEL homozygotes not receiving PDE3i. The basis for the selective up-regulation in PDE3A gene expression in DEL homozygotes treated with PDE3i was a cAMP response element enhancer 61 nt downstream from the INS, which was repressed by INS. The DEL homozygous genotype frequency was also enriched in patients with HF.
Conclusions A 29-nt INS/DEL polymorphism in the PDE3A promoter regulates cAMP-induced PDE3A gene expression in patients treated with PDE3i. This molecular mechanism may explain response heterogeneity to this drug class, and may inform a pharmacogenetic strategy for a more effective use of PDE3i in HF.
Activation of β-adrenergic receptors (β-ARs) increases adenylate cyclase (AC) activity, resulting in cyclic adenosine monophosphate (cAMP) synthesis, activation of protein kinase A (PKA), and phosphorylation of various downstream effectors. The cardiac β-AR/AC/cAMP/PKA axis has an important role in regulating heart rate and myocardial contraction and relaxation. In the failing human heart, β-AR signal transduction is desensitized, and cAMP levels are decreased despite increased adrenergic activity (1). These changes may compromise cardiac function as well as redirect signaling to more biologically adverse “PKA-independent” pathways, such as Ca2+/calmodulin-dependent protein kinase II (1).
Cyclic nucleotide phosphodiesterases (PDEs) hydrolyze the phosphodiester bond in cAMP or cyclic guanosine monophosphate for conversion to their respective 5′ monophosphates (2). In the failing heart, an increase in cAMP to more normal levels could potentially be achieved by inhibition of PDEs (3). However, several clinical trials have shown increased mortality (4,5) or lack of efficacy (6) in adult patients with advanced heart failure (HF) with reduced ejection fraction (HFrEF) treated with PDE3 inhibitors (PDE3i). The increase in mortality appears to be due to proarrhythmic effects interacting with other clinical variables to produce an increase in sudden death (7,8), whereas the lack of effectiveness when a PDE3i was used with a β-blocker appeared to be due in part to response heterogeneity (6) or an attenuation of initial effectiveness (9,10).
Effects of cAMP in the heart are classically attributed to the phosphorylation of cardiac myocyte proteins that affect excitation/contraction coupling, including L-Type Ca2+ channels, the sarcoplasmic reticulum (SR) ATPase 2 (SERCA2) regulatory protein phospholamban (PLN), ryanodine receptor 2, phosphatase 1 inhibitor, and various contractile proteins (11). In order to regulate localized cAMP-mediated signaling PDEs are compartmentalized intracellularly (12), and PDE3A protein is present in a microdomain that includes SERCA2, PLN, PKA-RII, and AKAP7/18 (13). Three isoforms of PDE3A have been identified in human myocardium, resulting from alternative transcriptional or translational start sites (14). PDE3A1, the longest isoform, is localized to microsomal fractions, whereas 2 shorter isoforms, PDE3A2 and PDE3A3, are present in microsomal and/or cytosolic fractions (14). Therefore, by virtue of its SR compartmentation, PDE3A1 in the human heart is a major regulator of PLN and SERCA2 activity.
Here, we show that a 29-nucleotide (nt) insertion (INS) or deletion (DEL) (indel) polymorphism in the PDE3A promoter regulates transcription via a downstream cAMP response element (CRE). In HFrEF patients, treatment with PDE3 inhibitors results in increased myocardial PDE3A1 mRNA expression and PDE3A catalytic activity in DEL homozygotes, but not in INS homozygotes. The INS site binds the transcription factor ATF3, which produces a repressive effect on the increase in PDE3A transcriptional activity mediated by increased cAMP levels. These results suggest that PDE3 inhibitor treatment may be more efficacious in INS homozygote patients, where tolerance resulting from up-regulation of PDE3A gene expression would not be present, and efficacy would be sustained.
Human subjects with end-stage HF were males and females of all ages, races, and ethnic backgrounds who gave written consent to donate their hearts to the institutional review board–approved cardiac transplant tissue bank at the University of Colorado. Nonfailing hearts that could not be used for transplant due to ABO or body size mismatch and had echocardiographic shortening fractions of ≥25% were obtained from organ donors, with consent for research use given by family members. Further details are described in the Online Appendix.
DNA extraction and genotyping
Genomic DNA was isolated using a modification of Chomczynski's method (Online Reference 1). Briefly, frozen tissue was digested with proteinase K followed by phenol/chloroform extraction and isopropanol precipitation. Polymerase chain reaction (PCR) was performed using the BioReady Taq DNA polymerase (Bulldog Bio, Portsmouth, New Hampshire). The resulting PCR product was separated in a 3% agarose gel. The following primers were used for genotype purposes:
PDE3A F: 5′ CCACTGCCATTGACTAGCTG
PDE3A R: 5′ GCCAAAAGGAGATCCTTGAGAT
mRNA extraction and reverse transcription PCR
RNA was extracted from left ventricular (LV) mid–free-wall 1-g aliquots of 44 nonfailing and 98 nonischemic or ischemic cardiomyopathy failing adult hearts. Tissue aliquots from ischemic cardiomyopathy hearts used for RNA extraction or biochemical measurements were from infarct-free areas. Tissue mRNA extraction was performed using the mirVana kit (Ambion, Thermo Fisher Scientific, Waltham, Massachusetts) according to manufacturer’s recommendation. Neonatal rat ventricular myocytes (NRVMs) were extracted using TRIzol (Thermo Fisher Scientific) and reverse transcription PCR was performed as previously described (Online Reference 2) using PDE3A1-, PLN-, or ATF3-specific primers:
Human PDE3A1 F: 5′ GCTCCGGAGCTCTCGGAAA
Human PDE3A1 R: 5′ CCAGCAGCGCCAGCAGAAA
Rat ATF3 F: 5′ CTGCCAAGTGTCGAAACAAG
Rat ATF3 R: 5′ GCAGGTTGAGCATGTAAATCAG
Human PLN F: 5′ CCAATACCTCACTCGCTCAG
Human PLN R: 5′ GATTCTGTAGCTTTTGACGTGC
Total β (β1, β2, and β3)-AR density and β1, β2 were measured in crude LV membranes by 125[I]CYP binding and displacement of the β1-AR fraction by CGP 20172a, by either of previously described methods (Online References 3, 4).
NRVMs were prepared from the hearts of 2-day-old rat pups by enzymatic digestion as previously described (Online Reference 2).
Construct design and cloning
Cloning of the PDE3A promoter region was performed using PCR primers that amplified the −1,434/+440 region of the human promoter that contains the INS/DEL genotype. The resulting fragment was cloned in the Promega pGL3-basic vector using MluI and BglII sites as previously described (Online Reference 5). Cloning of the construct that lacks CRE was done by amplifying the −1,066/+440 region of the PDE3A promoter downstream from the CRE site. Mutations were generated in the 29-nt INS region by PCR amplification of overlapping fragments, randomly designed as:
Wild type 5′TTCTCATATCTACTTATGTCATAATATTA
Transfection and luciferase activity assays
Firefly luciferase plasmid DNA was transfected using Lipofectamine 3000 (Thermo Fisher Scientific). Briefly, 1.5 μl of Lipofectamine 3000 and 2 μl of p300 reagents were transfected with 1 μg of plasmid DNA/160,000 cells. Twenty-four hours after transfection, cells were treated with 300 μmol/l N6-benzoyladenosine-3′,5′-cyclic monophosphate (6-Benz-cAMP), sodium salt, or 5 μmol/l enoximone for 48 h. Luciferase activity was determined as previously described (Online Reference 5). siRNA transfection was performed using Lipofectamine RNAiMAX (Thermo Fisher Scientific) with minor modifications. Briefly, 1.5 μl of Lipofectamine RNAiMax and 24 μmol/l scrambled (Dharmacon GE Healthcare Catalog # D-001810-10-05 ON-TARGET plus Non-targeting Pool; Dharmacon, Lafayette, Colorado) or ATF3 (Dharmacon GE Healthcare catalog # L-080117-02-0005 ON-TARGET plus Rat Atf3  siRNA SMART pool) siRNA were transfected in a 12-well dish (160,000 cells/well) 18 h after cells were plated. cDNA transfection was performed 24 h after siRNA transfection, and treated with 300 μmol/l 6-Benz-cAMP, sodium salt 4 h after transfection. Cells were harvested 48 h after treatment.
Electrophoretic mobility shift assay
Electrophoretic mobility shift assay (EMSA) was performed as previously described (Online Reference 5). Briefly, 29 base pairs (bp) of the double-stranded oligonucleotides of the wild-type INS or mutant were labeled by T4 polynucleotide kinase. The reaction was performed using 100,000 counts/min of wild-type or mutant probe in a 15-μl binding reaction containing HeLa or human heart nuclear extracts.
Nuclear extract preparation
Human heart nuclear extracts were prepared as previously described (Online Reference 5). HeLa cell nuclear extracts were purchased from Active Motif (Carlsbad, California). YY1 and ATF3 antibodies were purchased from Santa Cruz Biotechnology (Dallas, Texas).
Preparation of subcellular fractions from human LV myocardium
Approximately 150 mg of −80°C frozen LV mid–free-wall myocardium immediately frozen upon collection was homogenized and separated into nuclear-, cytosolic-, and SR-enriched microsomal fractions by differential sedimentation by a protocol adapted from previously published methods (Online Reference 6). Further details can be found in the Online Appendix.
PDE3 activity assay
Identification of an INS/DEL polymorphism in the promoter region of the PDE3A gene
In order to determine whether there are polymorphisms in the PDE3A promoter region that could affect mRNA expression and response to PDE3i treatment, PDE3A1 genomic DNA region including the upstream 10-Kb promoter region was sequenced in 60 samples of failing LV myocardium from the explanted hearts of transplant recipients that were outliers in a PDE3A1 mRNA expression screen. As shown in Figure 1, this resulted in the identification of a 29-nt DEL/INS polymorphism beginning at c.-2214(c.-2214_-2215, INS TTCTCATATCTACTTATGTCATAATATTA; rs145697127). Polymorphism distribution in the study populations and the enrichment of the DEL homozygous genotype failing versus nonfailing LVs is presented and discussed in the Online Appendix.
Polymorphism effects on PDE3A promoter activity
To determine the effect of the INS/DEL polymorphism on PDE3A expression in response to agents that act through cAMP, the promoter region containing the indel polymorphism was cloned in a PGL3 basic vector and transfected into NRVMs treated with 6-Benz-cAMP or the PDE3i enoximone. As shown in Figure 2A, treatment of this cAMP biosensor with 6-Benz-cAMP or enoximone resulted in increased luciferase activity of the DEL promoter construct, whereas the INS construct exhibited no evidence of up-regulation of PDE3A expression. Higher luciferase activity of the DEL promoter construct in response to increased cAMP levels suggested that the PDE3A promoter contains a CRE that activates PDE3A expression in the presence of cAMP. Further analysis of the PDE3A promoter region revealed the presence of a CRE (−5′TAAGTCATCT) 51 bp downstream from the INS region. The PDE3A DEL construct containing the CRE resulted in increased luciferase activity in response to cAMP or enoximone, whereas deletion of the CRE sequence attenuated cAMP-induced increases in promoter activity (Figure 2B). The increased cAMP-mediated luciferase activity in the CRE DEL construct may be due to other putative binding sites downstream from the indel site (data not shown).
The transcription factor ATF3 binds to the 29-nt INS sequence and regulates cAMP-dependent PDE3A promoter activity
To determine whether a specific transcription factor interacted with the INS sequence, an EMSA was performed using HeLa cell nuclear extracts and the 29-nt INS sequence as a probe. As shown in Figure 3A, a prominent band was observed in the EMSA using the wild-type INS probe and HeLa nuclear extracts. Analysis of the INS region using the TRANSFAC database showed potential binding sites for the transcription factors ATF3, GATA4, and EVI-1, but EMSA using a GATA4 antibody in NRVM nuclear extracts or EVI-1 in vitro translated protein failed to show interaction of these transcription factors with the INS region (data not shown). However, as shown in Figure 3A, addition of an ATF3 antibody reduced binding to the wild-type probe. The ATF3 antibody binds to the c-terminus of the protein where the DNA binding domain is located, and the addition of the antibody likely prevented ATF3 binding to the DNA. In addition, a YY1 antibody was used as negative control, and no decrease in binding was observed. To determine specificity of binding, a cold competitor (wild-type unlabeled probe) was added to the reaction and completely abolished binding. Furthermore, binding was not observed when a mutant probe was used (Figure 3A). Binding was also observed when human heart nuclear extract was used, and the addition of the ATF3 antibody abolished binding (Figure 3B).
To determine whether abolishing binding had an effect on luciferase activity, the mutant construct was transfected into NRVMs where treatment with 6-Benz-cAMP was associated with an increase in luciferase activity (Figure 3C).
Down-regulation of ATF3 prevents cAMP-mediated repression of the PDE3 promoter
We next determined whether down-regulation of ATF3 had an effect on INS promoter activity. As shown in Figure 4A, NRVMs transfected with an ATF3 siRNA had a 5-fold down-regulation of ATF3 mRNA. Importantly, in NRVMs incubated with 6-Benz-cAMP, down-regulation of ATF3 converted the INS promoter construct to a luciferase activity level comparable to the DEL construct, where down-regulation of ATF3 had no effect (Figure 4B).
Myocardial PDE3A1 mRNA abundance is increased in failing LVs, preferentially in DEL polymorphism genotypes
Demographics and other characteristics of hearts used in gene expression and biochemical experiments are given in Online Table 1. For data used in molecular/biochemistry assays, there were no clinically meaningful differences in ages (range 47.6 to 51.5 years) among the nonfailing, failing untreated, and failing treated with PDE3i clinical/treatment phenotypes. Because they originated from organ donors, nonfailing hearts were nearly sex balanced (52.2% male) compared with failing LVs (79.1% males; 86.8% males in PDE3i treated and 74.5% males in untreated patients). The LV ejection fractions exhibited severe dysfunction in the failing groups (17% in PDE3i treated, 18.7% in untreated) compared with nonfailing (63.0%).
PDE3i treatment is associated with increased PDE3A1 mRNA abundance in the failing hearts of patients with the DEL promoter polymorphism
As described in the Methods (Online Appendix), an initial 2-way analysis of variance (ANOVA) was performed on the PDE3A1 mRNA abundance data, which revealed evidence of an effect by clinical/treatment phenotype (nonfailing, failing untreated with PDE3i, failing treated with PDE3i) as well as by genotype. Accordingly, in order to determine whether treatment of HFrEF patients with PDE3 inhibition affects PDE3A1 mRNA levels, transcript abundance was analyzed in nonfailing LVs (n = 41), and in failing hearts from patients who had (n = 30) or had not (n = 66) been treated with the PDE3i enoximone or milrinone. There were no differences in baseline characteristics including LV ejection fraction between PDE inhibitor–treated and untreated patients (Online Table 1). In the failing-treated group, DEL homozygotes and DEL carriers exhibited respective increases in PDE3A1 abundance (by 45% and 33%; p = 0.046 and 0.025, respectively) compared with failing untreated patients (Figure 5). In addition, failing LVs from DEL/DEL or DEL carrier patients treated with PDE3i had higher mRNA abundance compared with nonfailing LVs (by respective values of 65% [p = 0.031] and 71% [p = 0.0007]) (Figure 5). By contrast, no significant differences in PDE3A1 mRNA abundance was observed across the 3 clinical/treatment phenotypes LVs that were INS homozygote (ANOVA p = 0.43) or heterozygotes (p = 0.052) (Figure 5).
PDE3i treatment is associated with increased microsomal PDE3 enzyme activity in DEL carrier failing hearts
PDE3 enzyme activity was measured in microsomal fractions prepared from 20 nonfailing and 47 failing hearts, 22 of which had been treated with PDE inhibitors (Figure 6). In the LVs of the 17 HFrEF patients treated with PDE3i, enzyme activity was increased in DEL homozygotes when compared with patients not treated with PDE3i (by 56%; p = 0.020). DEL carrier failing LVs from patients treated with PDE3i exhibited a trend for higher enzyme activity, by 32% (p = 0.056) versus untreated patients. For comparisons of failing LVs from PDE3i-treated patients to nonfailing LVs, DEL homozygotes had 27% (p = 0.092) and DEL carriers 31% (p = 0.026) higher enzyme activity. By contrast, INS homozygotes (ANOVA p = 0.97) or heterozygotes (p = 0.68) did not exhibit differences in PDE3 enzyme activity among the 3 clinical/treatment phenotypes (Figure 6).
PLN mRNA expression is affected by PDE3A indel genotypes
The PDE3A1 mRNA data support a PDEA promoter indel effect on PDE3A gene transcription in response to localized cAMP levels that are regulated by the same gene’s protein product, giving rise to a cAMP-PDE3A transcriptional negative feedback loop in DEL carriers that is inhibited in INS homozygotes. This feedback loop is most apparent in PDE3i-treated failing hearts, which in DEL carriers, exhibit an increase in PDE3A1 mRNA abundance and SR-enriched microsomal PDE3 enzyme activity. This implies that a PDE3i-induced increase in cAMP is sensed in the nucleus, leading to higher levels of PDE3A1 transcription in DEL and lower levels in INS alleles. We next investigated whether the expression of another cAMP-responsive gene exhibiting coregulation by ATF3 might be affected by cAMP levels modulated by PDE3i and polymorphisms of PDE3A. We chose PLN, whose promoter activity is negatively regulated by ATF3 and positively influenced by a CRE (15). In NRVMs, overexpression of adenylate cyclase 6 (ADCY6) was previously shown to repress PLN expression in an ATF3-dependent manner (15), presumably related to ADCY6-generated cAMP and PKA activation up-regulating the protein expression of ATF3. Heterozygotes and DEL carriers treated with a PDE3i displayed increased PLN mRNA abundance when compared with nonfailing hearts (respective p values = 0.0007 and 0.0008 for abundances 165% and 81% higher), but untreated failing hearts were not different from the nonfailing group (respective p values = 0.12 and 0.07) (Figure 7). In heterozygotes and DEL carriers, PDE3i-treated failing hearts also exhibited greater PLN mRNA abundance than untreated failing hearts (by respective amounts of 51% [p = 0.035] and 40% [p = 0.023]), whereas a trend for increased PLN mRNA abundance was observed in DEL homozygotes (by 36%; p = 0.15). PLN expression was not different in INS homozygotes (ANOVA p = 0.74).
Because PLN and PDE3A gene transcription are coregulated by the ATF3 repressor and enhanced through a CRE, it would be expected that the steady-state mRNA abundances of each would be related across PDE3A genotypes. Online Figure 1 demonstrates that is the case, with a significant positive correlation between PLN and PDE3A mRNA expression (r2 = 0.28; p < 0.0001) (Online Figure 1).
We identified a 29-nt indel polymorphism in the human PDE3A promoter region at c.-2214 that regulates gene transcription in response to cAMP (Central Illustration). In DEL promoter constructs, a cAMP-generated molecularly positive, but pharmacologically negative, feedback loop is created by increasing PDE3A gene expression that would subsequently limit or reduce increases in cAMP in microdomains where the increased levels of PDE3A1 protein are localized by membrane attachment. In the absence of the INS, the cAMP-mediated effects on PDE3A promoter activity are mediated by a CRE present 51 bp downstream from where the INS begins. In human failing LVs, PDE3A1 mRNA was increased in DEL genotypes compared with INS homozygotes, driven by effects in hearts that had been treated with PDE3i. In failing LVs treated with PDE3i, microsomal PDE3 enzyme activity was also increased in DEL genotypes, proportional to gene dose. In HeLa cell or human LV nuclear extracts, the INS binds to the transcription factor ATF3. In INS promoter constructs and in failing LVs explanted from INS homozygotes, there was complete prevention of the negative feedback loop of increases in cAMP or PDE3 inhibition leading to up-regulation in PDE3A gene expression and microsomal PDE3 enzyme activity. Importantly, down-regulation of ATF3 prevented the inhibitory effect of the INS sequence in response to cAMP stimulation.
We identified ATF3 as a transcription factor that binds to the PDE3A promoter 29-nt insertion. ATF3 is a transcription factor involved in the stress response (16) that can activate transcription when coexpressed with its heterodimeric partners, whereas it represses transcription as a homodimer. In NRVMs, Gao et al. (15) showed that overexpression of ADCY6 results in up-regulation of ATF3 and repression of PLN expression, presumably mediated through cAMP generation and PKA activation. Importantly, the putative ATF3 binding site in the PDE3A promoter 29-nt INS sequence is similar to the one identified in the rat or human PLN promoter (INS 5′ TATGACATAA 3′; rat PLN 5′ TGACATCACAT 3′; human PLN TGATGTCACAT; complementary sequence depicted) (15). We showed that PLN mRNA expression is also affected by the PDE3A indel polymorphism. Decreased cAMP levels or PKA signaling in a nuclear microdomain in DEL carrier patients treated with a PDE3i may be responsible for increased PLN mRNA abundance. Although ATF3 expression was unchanged in human failing hearts in response to enoximone or milrinone treatment, as previously shown by others (17), up-regulation of ATF3 occurs in a short-term and transient manner, suggesting that the increase in cAMP in response to PDE3A inhibition may result in a transient increase in ATF3 expression. In DEL carriers, this would result in increased PDE3A gene transcription, and through decreased levels of cAMP, an up-regulation of PLN gene expression.
What could be the biological signaling implications and consequences of the PDE3A promoter INS/DEL polymorphism? In the PDE3A-SERCA2-PLN microdomain, SR-attached PDE3A1 and to a lesser extent PDE3A2 (14) regulate cAMP level and therefore PKA activation and PLN phosphorylation (12). The SR PDE3A-SERCA2-PLN microdomain is the site of a fundamental defect in contractile function in the failing human heart (1,3,18,19), where the local concentration of cAMP and therefore PKA activity and PLN phosphorylation are regulated by SR membrane–bound PDE3A isoforms (12–14). For these reasons, we measured PDE3 activity in SR-enriched microsomes, as well as mRNA expression of the transcript encoding for the PDE3A1 isoform that is restricted to microsomal fractions (14,20). In LV free-wall aliquots of failing human hearts, we found that across all genotypes, microsomal fraction PDE3 enzyme activity and PDE3A1 mRNA were unchanged, but were increased in DEL homozygotes treated with PDE3i. It would be predicted that in DEL homozygotes, increased diffusible cAMP such as from endogenous β-adrenergic stimulation or by therapeutic administration of β-agonists or PDE3i would be met with activation of the CRE-mediated up-regulation in PDE3A gene transcription and an increase in PDE3A1 and possibly other PDE3A transcripts, creating a pharmacologically negative feedback loop resulting in lack of PDE3i effectiveness in DEL genotypes. Alternatively, in HF patients increased cAMP levels in INS homozygotes could result in increased SR-Ca2+ cycling and improved myocyte survival and cardiac function, as previously shown in models of PLN down-regulation (18).
What are the implications of the PDE3A promoter polymorphism findings for treatment with PDE3i? Treatment with this drug class has been evaluated in multiple clinical trials in acute and chronic HF, and it is currently only used in patients with decompensated HF who are refractory to diuretics and vasodilators (21). PDE3i have also been or are used as a bridge to transplantation (22,23, Online Reference 3) and in weaning from cardiopulmonary bypass. Although in adults with advanced HF an initial response is typically hemodynamically beneficial, long-term treatment with PDE3i may lead to adverse effects that include arrhythmias and sudden cardiac death (4,5), attenuation of favorable hemodynamic or clinical effects, and overt progression of HF (10,24–26). In adult HF patients, administration of PDE3i can result in tachyphylaxis (9,11) that can be observed as early as 72 h into continuous infusion (9). By contrast, in children with HF, PDE3i may have better safety and efficacy profiles (23) that could relate to differences in β-AR signaling (27,28) and/or PDE3A activity (28). Thus, there is ample evidence that treatment with PDE3i may be accompanied by loss of effectiveness in some patients, which may result in a shift of β-adrenergic signaling to PKA-independent pathways that have greater adverse effects on myocyte biology (1) or allow the Ca2+ mobilizing or other pharmacologic properties of milrinone (11,29) or other compounds to predominate.
In addition, PDE inhibitor-induced up-regulation in PDE expression resulting in PDE inhibitor loss of effectiveness has been observed for other PDEs (30,31), and cAMP-induced up-regulation in PDE3A gene expression has been reported in porcine ovarian cumulus cells (32). However, this is the first report to our knowledge of a genetic polymorphism in a PDE gene where one variant can mediate cAMP up-regulation in transcriptional activity and the counterpart variant cannot.
In summary, the data from the current study suggest that patients who are INS homozygotes would have less loss of effectiveness to PDE3i administration, due to a lack of up-regulation in PDE3A gene transcription and increased PDE3 activity. To be clinically relevant, this possibility would need to be tested in a prospective clinical trial with results stratified by PDE3A genotype, to determine the pharmacogenetic consequences of the PDE3A promoter indel polymorphism. In view of the demonstration that in adult HF patients receiving a background of β-blocker therapy, PDE3i are safe and have evidence of an efficacy signal in subpopulations (6), a pharmacogenetic clinical trial of a PDE3 inhibitor would seem justified.
The clinical component of the work was conducted entirely as a retrospective analysis in explanted human hearts, in tissue available from our human heart biobank. Although 166 end-stage LVs were available, fewer than one-half (n = 41) were from patients who had received PDE3i. The sample size was limited to the number of PDE3i-exposed hearts in the biobank, and for some genetic subsets, the number was relatively small (e.g., n = 5 for PDE3i-treated INS/INS). No prospective clinical study or trial data are included, and the findings in explanted hearts should be considered hypothesis generating for a subsequent trial or trials in advanced HFrEF patients stratified by INS/DEL genotype.
This study is the first to identify a polymorphic region in the PDE3A promoter that regulates gene transcriptional activity in response to changes in cAMP concentration or with PDE inhibitor treatment. The DEL allele of this polymorphism may be a genetic biomarker for the development or progression of HF (see the Online Appendix). In addition, on theoretical grounds, HF patients who are INS homozygotes may experience less tolerance to PDE3i, and PDE3i may exhibit greater effectiveness in this genetic subpopulation, a hypothesis that will need to be tested in a prospective clinical trial.
COMPETENCY IN MEDICAL KNOWLEDGE: PDE3 inhibitors produce short-term hemodynamic benefit in patients with heart failure, but long-term use has been ineffective. A common genetic variant in the promoter region of the PDE3A gene that regulates transcriptional activity may mediate tolerance to this line of therapy.
TRANSLATIONAL OUTLOOK: Clinical studies are needed to test the hypothesis that heart failure patients who are homozygous for the PDE3A insertion promoter variant and possibly those who are heterozygous gain sustained hemodynamic benefit from PDE3 inhibitor drugs.
The authors thank Laura Hofstatter and Rachel Rosenberg for assistance in manuscript preparation and submission.
This work was supported by Leducq Foundation Transatlantic Networks of Excellence grant FLQ-06 CVD 02 to Drs. Bristow and Movsesian; National Institutes of Health (NIH) grant 2R01 HL48013 to Dr. Bristow; NIH grant R21 HL097123 and American Heart Association (AHA) grants 11IRG5070006 and 13GRNT16950045 to Dr. Sucharov; NIH grant T32 HL007822 and American Academy of Pediatric fellowship grant to Dr. Nakano; NIH (HL116848 and HL127240) and American Heart Association (16SFRN31400013) grants to Dr. McKinsey; the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development (Merit Review grants CARA-029-09F and CARA-027-12S to Dr. Movsesian), and an American Heart Association (grant-in-aid to Dr. Movsesian); and ARCA Biopharma. Intellectual property relevant to this work has been licensed by the University of Colorado to ARCA Biopharma. Dr. Carroll is an employee of ARCA Biopharma. Dr. Taylor has been a consultant for Rocket Pharma, Protalix Pharma, Genzyme/Sanofi Pharma, Pfizer Pharma, Valerion Pharma, Allomek Pharma, Array Biopharma, and ARCA Biopharma. Dr. Bristow is an officer, director, and equity holder in ARCA Biopharma. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose.
Listen to this manuscript's audio summary by Editor-in-Chief Dr. Valentin Fuster on JACC.org.
- Abbreviations and Acronyms
- N6-benzoyladenosine-3′,5′-cyclic monophosphate
- analysis of variance
- β-adrenergic receptor
- base pair(s)
- cyclic adenosine monophosphate
- cAMP response element
- electrophoretic mobility shift assay
- heart failure
- heart failure with reduced left ventricular ejection fraction
- insertion/deletion polymorphism
- left ventricle/ventricular
- neonatal rat ventricular myocyte
- polymerase chain reaction
- phosphodiesterase 3 inhibitor(s)
- protein kinase A
- sarcoplasmic reticulum ATPase 2
- sarcoplasmic reticulum
- Received October 23, 2018.
- Revision received November 20, 2018.
- Accepted December 10, 2018.
- 2019 American College of Cardiology Foundation
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