Abstract
Calcium (Ca2+) and 3’,5’-cyclic adenosine monophosphate (cAMP) play a critical role for cardiac excitation-contraction-coupling. Both second messengers are known to interact with each other, for example via Ca2+-dependent modulation of phosphodiesterase 1 (PDE1) and adenylyl cyclase 5/6 (AC 5/6) activities, which is supposed to occur especially at the local level in distinct subcellular microdomains. Currently, many studies analyze global and local cAMP signaling and its regulation in resting cardiomyocytes devoid of electrical stimulation. For example, Förster resonance energy transfer (FRET) microscopy is a popular approach for visualization of real time cAMP dynamics performed in resting cardiomyocytes to avoid potential contractility-related movement artifacts. However, it is unknown whether such data are comparable with the cell behavior under more physiologically relevant conditions during contraction. Here, we directly compare the cAMP-FRET responses to AC stimulation and PDE inhibition in resting vs. paced adult mouse ventricular cardiomyocytes for both cytosolic and subsarcolemmal microdomains. Interestingly, no significant differences in cAMP dynamics could be detected after β-adrenergic (isoproterenol) stimulation, suggesting low impact of rapidly changing contractile Ca2+ concentrations on cytosolic cAMP levels associated with AC activation. However, the contribution of the calcium-dependent PDE1, but not of the Ca2+-insensitive PDE4, to the regulation of cAMP levels after forskolin stimulation was significantly increased. This increase could be mimicked by pretreatment of resting cells with Ca2+ elevating agents. Ca2+ imaging demonstrated significantly higher amplitudes of Ca2+ transients in forskolin than in isoproterenol stimulated cells, suggesting that forskolin stimulation might lead to stronger activation of PDE1. In conclusion, changes in intracellular Ca2+ during cardiomyocyte contraction dynamically interact with cAMP levels, especially after strong AC stimulation. The use of resting cells for FRET-based measurements of cAMP can be justified under β-adrenergic stimulation, while the reliable analysis of PDE1 effects may require electric field stimulation.
Highlights
A total number of 35 animals was sacrificed for this study. 3–4 month old CAG-Epac1-camps mice ubiquitously expressing the cytosolic cyclic adenosine monophosphate (cAMP) biosensor Epac1-camps under the control of cytomegalovirus enhancer/chicken β-actin promoter [20] or 3–4 month old pmEpac1 mice expressing membrane targeted cAMP sensor in adult cardiomyocytes under the control of α-myosin heavy chain promoter [8] were euthanized by cervical dislocation, and primary adult cardiomyocytes were isolated exactly as previously described [21]
We demonstrate that even if such rapid changes exist during contraction of isolated cardiomyocytes, they do not translate into sustained changes of cytosolic and submembrane adenylyl cyclases (ACs) and PDE activities under basal or cAMP-stimulating conditions in isolated mouse ventricular cardiomyocytes detected by Forster resonance energy transfer (FRET)
In sharp contrast, when paced cardiomyocytes are treated with the direct AC activator forskolin, this leads to a significant increase of phosphodiesterase 1 (PDE1) associated cAMP hydrolysis, as revealed by the use of the PDE1 inhibitor 8-MMX
Summary
3’,5’-cyclic adenosine monophosphate (cAMP) is a universal second messenger which regulates a plethora of cellular functions [1]. Besides its crucial role in contractility, Ca2+ is known for dynamically regulating intracellular cAMP levels This occurs either via the stimulation of the Ca2+/calmodulin-dependent PDE1 [13] or by inhibition of cardiac AC 5/6 activities [14], all these enzymes are abundantly expressed in the heart [15,16]. To avoid movement artifacts and to increase cell survival during the measurements, the majority of current FRET studies use resting cardiomyocytes to analyze intracellular cAMP dynamics [2,3,4,5,6,7,8,9,10] It is still unclear whether or not the rapid fluctuations in intracellular Ca+ levels during contraction are able to directly affect global and local cAMP levels in the cell. The cAMP dynamics under β-AR stimulation can be reliably studied in resting cells, while the exact analysis of PDE1-associated effects requires paced cardiomyocytes
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