Exenatide exerts a PKA-dependent positive inotropic effect in human atrial myocardium: GLP-1R mediated effects in human myocardium
Introduction
The prevalence of type 2 diabetes mellitus (T2DM) is estimated to increase dramatically from 382 M (2013) to 592 M by 2035 (International Diabetes Federation, 2014). It is well known that subjects with T2DM carry an up to 5-fold greater mortality risk when compared with non-diabetics, and this is primarily driven by cardiovascular disease (CVD). [1] Even after adjusting for modifiable risk factors such as hyperglycaemia, hypertension, cholesterol or smoking, cardiovascular risk remains doubled in subjects with T2DM. [2] Furthermore, recent data demonstrated that subjects with T2DM face a 2-fold higher incidence of heart failure compared to those without diabetes, [3], [4] and that heart failure mortality is significantly increased in this population. [3], [5] Several antidiabetic medications, such as thiazolidinediones [6], [7] or the dipeptidyl-peptidase-4 (DPP-4) inhibitor saxagliptin [8] have been associated with increased risk for hospitalization due to heart failure. Therefore, new antidiabetic drugs which not only lower glucose but also beneficially influence cardiovascular outcome and cardiac function are urgently needed.
Glucagon-like peptide-1 receptor (GLP-1R) agonists are an emerging class of drug molecules for treating T2DM. Glucagon-like peptide-1 (GLP-1) is an incretin peptide hormone primarily synthesized by intestinal L cells. [9] Its release into the circulation in response to food intake leads to glucose-dependent insulin release and glucagon suppression. GLP-1(7–36)NH2, with a half-life of 2 min, is the primary active isoform that is rapidly degraded by DPP-4 to GLP-1(9–36)NH2, [10] a GLP-1R antagonist. [11] Marketed GLP-1R agonists which include exenatide (Byetta® and Bydureon®) [12] reduce HbA1c in T2DM patients while providing modest weight-loss. In addition, human data suggest that this drug class improves cardiac function in patients with congestive heart failure, ameliorates endothelial dysfunction or reduces the infarct size after ST-segment-elevation myocardial infarction [13], [14], [15]. The GLP-1R is a seven transmembrane, class B G protein-coupled receptor (GPCR). The GLP-1R is positively coupled to adenylate cyclase through Gαs-containing G proteins, which catalyze the conversion of ATP to cyclic adenosine monophosphate (cAMP). Increased cytosolic cAMP leads to activation of second messenger pathways including protein kinase A (PKA), cAMP-activated guanine nucleotide exchange factor (Epac) and the mitogenic kinase, extracellular signal-regulated kinase (ERK)-1/2. [16] Epac acts in a PKA-independent manner and therefore represents a novel mechanism for governing signaling specificity within the cAMP cascade. [17] A recent study showed that GLP-1R activation in mouse atrial cardiomyocytes increased cAMP levels, promoted Epac2 translocation to the membrane and increased atrial natriuretic peptide (ANP) secretion. [18].
Interestingly, GPCRs may also regulate cardiac function and increase cardiac contractility via G protein-independent signaling, especially with respect to β-arrestin-mediated processes. [19], [20], [21] The GLP-1R can signal via β-arrestins to activate kinases such as ERK1/2 and have been shown to play a role in GLP-1R signaling in pancreatic β-cells. [22], [23] However, the role of β-arrestin in GLP-1R effects on G protein-independent cardiac contractility remains unknown.
Although several beneficial cardiovascular effects have been reported in human and animal studies, the underlying mechanisms of GLP-1 action in the heart, especially in human myocardium, are yet to be resolved. These studies aimed to elucidate the GLP-1R-mediated effects on: 1) developed force in human atrial and ventricular myocardium, 2) underlying mechanisms of human cardiac contractility, and 3) translocation of the downstream molecular targets Epac2 and the glucose transporters 1 and 4 (GLUT-1/4).
Section snippets
Methods
A detailed description of the methods is provided in the Methods section of online-only Data Supplement.
Effects of GLP-1R-agonists on developed force in atrial myocardium
The direct functional effects of GLP-1R agonism on human atrial myocardium were evaluated in 126 muscle strips that were isolated from 65 patients' atrial appendages. Muscle strips were superfused with Tyrode solution, stimulated at 1 Hz and stretched to an optimal length, the point where the muscle strips exerted a maximal developed force. In the control Tyrode solution group we observed a decrease in developed force over time (Ctrl, n = 10) which is a well-established physiological rundown. [24]
Discussion
Our data demonstrate that GLP-1R activation exerts a concentration-dependent positive inotropic effect in non-failing human atrial myocardium. This positive inotropy does not require β-arrestin recruitment, but requires activation of PKA, the regulation of intracellular Ca2 + via SERCA and involves phosphorylation of PLB. GLP-1R activation also induces the translocation of Epac2 and GLUT-1 to the cell membrane in atrial cardiomyocytes. These data provide the first evidence that GLP-1R activation
Conclusion
To the best of our knowledge this is the first report of GLP-1R-agonist-mediated functional effects in non-failing human atrial tissue. We demonstrate that the GLP-1R-agonists exenatide, GLP-1(7–36)NH2, PF-06446009 and PF-06446667 significantly increased contractility in human atrial myocardium in a concentration-dependent manner directly through the GLP-1R/cAMP/PKA signaling pathway. In contrast, the metabolite GLP-1(9–36)NH2 failed to increase contractility. We further show that exenatide
Funding sources
This study was supported by Pfizer Inc., New York, NY, USA. PF-06446009 and PF-06446667 were provided by Pfizer Inc.
Disclosures
JBK and DAG are employees of Pfizer Inc.; NJE is employed by AstraZeneca.
Acknowledgements
We thank Chris Limberakis for writing the experimental procedures used to synthesize PF-06446009 and PF-06446667 and David R. Derksen for determining the agonist pharmacology in Table 1 and the writing of the corresponding assay methods. We would also like to thank DDr. Friederike von Lewinski, Dr. Ingeborg Klymiuk and Michael A. Volkert for their advice, which was instrumental in revising the manuscript.
Glossary
- cAMP
- cyclic adenosine monophosphate
- cTnI
- troponin I
- CVD
- cardiovascular disease
- DPP-4
- dipeptidyl-peptidase-4
- Epac
- cAMP-activated guanine nucleotide exchange factor
- ERK
- extracellular signal-regulated kinase
- GLP-1
- glucagon-like peptide-1
- GLP-1R
- glucagon-like peptide-1 receptor
- GLUT-1/4
- glucose transporter 1/4
- GPCR
- G protein-coupled receptor
- HFpEF
- heart failure with preserved ejection fraction
- HFrEF
- heart failure with reduced ejection fraction
- LA
- left atrium
- NO
- nitric oxide
- PAC
- pulmonary arterial compliance
- PKA
- protein kinase A
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