Shining light on the beta-adrenoceptor signaling in the heart

Beta-blockade remains the main therapeutic approach to treat cardiac arrhythmia to date. However, beta-blockers have significant drawbacks, as they are unspecific towards cell type and target tissue, and are associated with major adverse effects, such as fatigue, dizziness, poor circulation, gastrointestinal symptoms, or weight gain. We here propose to use an optogenetic approach to study the complex down-stream signaling pathways of beta-adrenergic activation in the heart. Exploiting the precise molecular and spatiotemporal control offered by optogenetics, we aim to disentangle specific roles of the three beta-adrenoceptor (β-AR) subtypes expressed in cardiomyocytes. Understanding the distinct effects of the three β-AR signaling pathways in health and disease and, ultimately, becoming able to selectively activate the most cardio-protective components of adrenergic pathways using optogenetic manipulation, has great potential for developing more specific and potent therapies to treat arrhythmia.

To address this, we have designed and applied optimized, chimeric Opto-GPCR, which combine the extracellular domains of a light-sensitive rhodopsin GPCR with the intracellular domains of each of the three β-AR subtypes [1]. By rendering β-AR sensitive to different wavelengths of light, we can selectively activate the respective downstream signaling pathways of the individual receptors and assess both synergistic and. The constructs were first tested and electrophysiologically characterized in HEK293 and HL-1 cells, and their Gs-protein specificity confirmed in second messenger assays. Next, adenoviruses containing Opto-β-AR were used to virally transduce isolated ventricular cardiomyocytes from adult rabbits to study the expression and localization of the receptors, and to quantify the electromechanical effects of light stimulation, such as on lusitropy (assessed by action potential recordings) and inotropy (as assessed by sarcomere length measurements), and to compare them to pharmacological stimulation of β-AR.

We found that our engineered Opto-β-AR are expressed at similar density in the plasma membrane of HEK293, HL-1 cells and ventricular cardiomyocytes. Stimulation of the chimeric receptors results in a pronounced cytosolic cAMP elevation in HEK293 cells, as would be expected from the activation of the desired Gs-protein signaling pathway [1]. Further, preliminary results in isolated cardiomyocytes show action potential shortening and increased contractility following brief (500-2000 ms) light stimulation, comparable with the amplitudes and durations of pharmacological stimulation of AR with isoproterenol. Additionally, we observed an instantaneous increase of L-type Ca2+ current (I_Ca,L) following illumination. Finally, the differential assessment of receptor subtype stimulation is currently ongoing.

Opto-β-AR pose promising tools to study a vast variety of biological questions. The knowledge gained from this work and the selective activation of potentially anti-arrhythmic, cardioprotective beta-AR pathways has great potential to develop novel concepts for treatment of arrhythmogenic cardiac diseases, such as heart failure.

1.         Leemann, S. and S. Kleinlogel, Functional optimization of light-activatable Opto-GPCRs: Illuminating the importance of the proximal C-terminus in G-protein specificity. Front Cell Dev Biol, 2023. 11: p. 1053022.