Dataset Information

Age-Dependent Maturation of iPSC-CMs Leads to the Enhanced Compartmentation of ?2AR-cAMP Signalling.

ABSTRACT: The ability to differentiate induced-pluripotent stem cells into cardiomyocytes (iPSC-CMs) has opened up novel avenues for potential cardiac therapies. However, iPSC-CMs exhibit a range of somewhat immature functional properties. This study explored the development of the beta-adrenergic receptor (?AR) pathway, which is crucial in regulating contraction and signifying the health and maturity of myocytes. We explored the compartmentation of ?2AR-signalling and phosphodiesterases (PDEs) in caveolae, as functional nanodomains supporting the mature phenotype. Förster Resonance Energy Transfer (FRET) microscopy was used to study the cyclic adenosine monophosphate (cAMP) levels in iPSC-CMs at day 30, 60, and 90 following ?AR subtype-specific stimulation. Subsequently, the PDE2, PDE3, and PDE4 activity was investigated using specific inhibitors. Cells were treated with methyl-?-cyclodextrin (M?CD) to remove cholesterol as a method of decompartmentalising ?2AR. As iPSC-CMs mature with a prolonged culture time, the caveolae density is increased, leading to a reduction in the overall cytoplasmic cAMP signal stimulated through ?2AR (but not ?1AR). Pan-phosphodiesterase inhibition or caveolae depletion leads to an increase in the ?2AR-stimulated cytoplasmic cAMP. Moreover, with time in culture, the increase in the ?AR-dependent cytoplasmic cAMP becomes more sensitive to cholesterol removal. The regulation of the ?2AR response by PDE2 and 4 is similarly increased with the time in culture. We conclude that both the ?2AR and PDEs are restricted to the caveolae nanodomains, and thereby exhibit a tighter spatial restriction over the cAMP signal in late-stage compared to early iPSC-CMs.

SUBMITTER: Hasan A

PROVIDER: S-EPMC7601768 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Age-Dependent Maturation of iPSC-CMs Leads to the Enhanced Compartmentation of β<sub>2</sub>AR-cAMP Signalling.

Hasan Alveera A Mohammadi Neda N Nawaz Aisha A Kodagoda Thusharika T Diakonov Ivan I Harding Sian E SE Gorelik Julia J

Cells 20201012 10

The ability to differentiate induced-pluripotent stem cells into cardiomyocytes (iPSC-CMs) has opened up novel avenues for potential cardiac therapies. However, iPSC-CMs exhibit a range of somewhat immature functional properties. This study explored the development of the beta-adrenergic receptor (βAR) pathway, which is crucial in regulating contraction and signifying the health and maturity of myocytes. We explored the compartmentation of β<sub>2</sub>AR-signalling and phosphodiesterases (PDEs) ...[more]

PMID: 33053822

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Project description:Objective3',5'-Cyclic adenosine monophosphate (cAMP) is a central second messenger governing brown adipocyte differentiation and function. β-adrenergic receptors (β-ARs) stimulate adenylate cyclases which produce cAMP. Moreover, cyclic nucleotide levels are tightly controlled by phosphodiesterases (PDEs), which can generate subcellular microdomains of cAMP. Since the spatio-temporal organisation of the cAMP signalling pathway in adipocytes is still unclear, we sought to monitor real-time cAMP dynamics by live cell imaging in pre-mature and mature brown adipocytes.MethodsWe measured the real-time dynamics of cAMP in murine pre-mature and mature brown adipocytes during stimulation of individual β-AR subtypes, as well as its regulation by PDEs using a Förster Resonance Energy Transfer based biosensor and pharmacological tools. We also correlated these data with β-AR stimulated lipolysis and analysed the expression of β-ARs and PDEs in brown adipocytes using qPCR and immunoblotting. Furthermore, subcellular distribution of PDEs was studied using cell fractionation and immunoblots.ResultsUsing pre-mature and mature brown adipocytes isolated from transgenic mice expressing a highly sensitive cytosolic biosensor Epac1-camps, we established real-time measurements of cAMP responses. PDE4 turned out to be the major PDE regulating cytosolic cAMP in brown preadipocytes. Upon maturation, PDE3 gets upregulated and contributes with PDE4 to control β1-AR-induced cAMP. Unexpectedly, β3-AR initiated cAMP is resistant to increased PDE3 protein levels and simultaneously, the control of this microdomain by PDE4 is reduced upon brown adipocyte maturation. Therefore we postulate the existence of distinct cAMP pools in brown adipocytes. One cAMP pool is formed by β1-AR associated with PDE3 and PDE4, while another pool is centred around β3-AR and is much less controlled by these PDEs. Functionally, lower control of β3-AR initiated cAMP by PDE3 and PDE4 facilitates brown adipocyte lipolysis, while lipolysis activated by β1-AR and is under tight control of PDE3 and PDE4.ConclusionsWe have established a real-time live cell imaging approach to analyse brown adipocyte cAMP dynamics in real-time using a cAMP biosensor. We showed that during the differentiation from pre-mature to mature murine brown adipocytes, there was a change in PDE-dependent compartmentation of β1-and β3-AR-initiated cAMP responses by PDE3 and PDE4 regulating lipolysis.

Project description:Subcellular compartmentation of the ubiquitous second messenger cAMP has been widely proposed as a mechanism to explain unique receptor-dependent functional responses. How exactly compartmentation is achieved, however, has remained a mystery for more than 40 years. In this study, we developed computational and mathematical models to represent a subcellular sarcomeric space in a cardiac myocyte with varying detail. We then used these models to predict the contributions of various mechanisms that establish subcellular cAMP microdomains. We used the models to test the hypothesis that phosphodiesterases act as functional barriers to diffusion, creating discrete cAMP signaling domains. We also used the models to predict the effect of a range of experimentally measured diffusion rates on cAMP compartmentation. Finally, we modeled the anatomical structures in a cardiac myocyte diad, to predict the effects of anatomical diffusion barriers on cAMP compartmentation. When we incorporated experimentally informed model parameters to reconstruct an in silico subcellular sarcomeric space with spatially distinct cAMP production sites linked to caveloar domains, the models predict that under realistic conditions phosphodiesterases alone were insufficient to generate significant cAMP gradients. This prediction persisted even when combined with slow cAMP diffusion. When we additionally considered the effects of anatomic barriers to diffusion that are expected in the cardiac myocyte dyadic space, cAMP compartmentation did occur, but only when diffusion was slow. Our model simulations suggest that additional mechanisms likely contribute to cAMP gradients occurring in submicroscopic domains. The difference between the physiological and pathological effects resulting from the production of cAMP may be a function of appropriate compartmentation of cAMP signaling. Therefore, understanding the contribution of factors that are responsible for coordinating the spatial and temporal distribution of cAMP at the subcellular level could be important for developing new strategies for the prevention or treatment of unfavorable responses associated with different disease states.

Dataset Information

Age-Dependent Maturation of iPSC-CMs Leads to the Enhanced Compartmentation of ?2AR-cAMP Signalling.

Publications

Age-Dependent Maturation of iPSC-CMs Leads to the Enhanced Compartmentation of β<sub>2</sub>AR-cAMP Signalling.

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