Takotsubo syndrome associated miR-16 and miR-26a reduce basal contractility of cardiomyocytes in vitro by an inhibitory G-protein dependent mechanism

L. Couch1, A. A. Derda2, T. Thum2, C. Terracciano1, S. E. Harding1. 1National Heart and Lung Institute, Imperial College London, London, UNITED KINGDOM, 2Institute of Molecular and Translational Therapeutic Strategies, Hannover Medical School, Hannover, GERMANY.

Introduction: The pleiotropic β2AR signals via stimulatory (Gs) and inhibitory (Gi) G-proteins, whereas β1AR acts solely through Gs. Whilst βAR-Gs increases cardiac output, it concomitantly decreases survival at cellular and organ level. The duality of β2AR acts as an intrinsic homeostatic mechanism to limit the cardiotoxicity of short-term adrenergic stimulation by facilitating a switch to β2AR-Gi, serving as cardioprotective despite being cardiodepressive. This is dysregulated in Takotsubo syndrome (TTC), a severe but reversible acute heart failure affecting post-menopausal women, wherein ventricular apical akinesis results from supraphysiological adrenaline levels arising in situations of stress. Stimulus trafficking to β2AR-Gi results and causes excess negative inotropy. It is not understood what predisposes this patient demographic to develop TTC, however a microRNA (miR) profile of increased levels of miR-16 and miR-26a, has recently been identified as a biomarker(1). Given the demonstrated importance of miRs in other cardiac diseases and that TTC is thought to be causally related to β2AR-Gi signalling, we hypothesised that this miR profile could predispose to, or exacerbate the cardiodepression in TTC.

Method: miR levels were manipulated in adult rat apical cardiomyocytes in vitro with blinded transfection of RNA oligonucleotides using Lipofectamine 3000. Percentage shortening of cardiomyocytes was measured using an Ionoptix Myocyte Contractility System, and pharmacological protocols applied. N numbers are displayed as n/N, where n=cell number and N=number of rats.

Results: Upregulation of miR-16 significantly reduced basal contractility with respect to control (miR-16=3.52±0.34% versus control=4.91±0.46%; n/N=30/6; p<0.05), whereas downregulation had no effect. Similarly, increased miR-26a significantly reduced basal contractility compared to control (miR-26a=2.77±0.21% versus control=4.30±0.43%; n/N=50/10; p<0.01), and downregulation remained unchanged from control. miR-16 and miR-26a manipulation did not alter β2AR response. Inhibiting Gi using pertussis toxin (PTX) pre-treatment prevented the effect of both miR-16 and miR-26a on basal contractility (miR-16 untreated=5.08±0.49%, n=22/4; versus miR-16 PTX-treated=8.82±0.63%, n=20/4; p<0.001; and miR-26a untreated=3.20±0.29% versus miR-26a PTX-treated=5.05±0.48%; n/N=30/6; p<0.05 respectively). PTX-treatment did not change basal contractility of control transfected cells. Preliminary data suggests that the reduction in basal contractility may be by a unified mechanism, as no synergistic effect was observed with dual miR-16/-26a transfection. Conclusion: Increased miR-16 and miR-26a reduce basal contractility of cardiomyocytes in vitro, possibly through a shared Gi-dependent mechanism. This strengthens the notion that these miRs may be mechanistically involved in TTC, but further work is needed to investigate their specific mechanistic and temporal involvement.

References:

1. Jaguszewski M et al. (2014). European Heart Journal 35: 999-1006