Age-Dependent Maturation of iPSC-CMs Leads to the Enhanced Compartmentation of β2AR-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 (βAR) pathway, which is crucial in regulating contraction and signifying the health and maturity of myocytes. We explored the compartmentation of β2AR-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 βAR subtype-specific stimulation. Subsequently, the PDE2, PDE3, and PDE4 activity was investigated using specific inhibitors. Cells were treated with methyl-β-cyclodextrin (MβCD) to remove cholesterol as a method of decompartmentalising β2AR. 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 β2AR (but not β1AR). Pan-phosphodiesterase inhibition or caveolae depletion leads to an increase in the β2AR-stimulated cytoplasmic cAMP. Moreover, with time in culture, the increase in the βAR-dependent cytoplasmic cAMP becomes more sensitive to cholesterol removal. The regulation of the β2AR response by PDE2 and 4 is similarly increased with the time in culture. We conclude that both the β2AR 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.

Fluorescence Spectroscopy of Low-Level Endogenous β-Adrenergic Receptor Expression at the Plasma Membrane of Differentiating Human iPSC-Derived Cardiomyocytes

“…It shall be further noted that the fully differentiated hiPSC-CMs, even at day ~100, do not display a complete T-Tubular network, leaving open the question if the β 2 -AR, that in this work we observed on the basolateral membrane, would eventually segregate in these compartments [ 13 ]. Our observation of higher relative β 2 -AR expression levels in hiPSC-CMs appears to be in line with what was previously reported in at least three works [ 8 , 9 , 10 ]. However, we shall mention here that in recent measurements in hiPSC-derived engineered heart tissue, higher levels of β 1 -AR were observed throughout (private communication and [ 25 ]).…”

“…Wu et al observed mRNA levels and effects on the contractility of β-AR expression in differentiating hiPSC, in a model of dilated cardiomyopathy [ 8 ]. Jung et al studied adrenoreceptor mRNA and total protein levels within the context of functional remodeling [ 9 ], while Hasan et al examined how receptor-specific cAMP signaling evolves throughout hiPSC differentiation towards CMs [ 10 ]. Kondrashov et al studied the temporal dynamics of β 2 -AR expression in CRISPRed hiPSC [ 11 ].…”

Fluorescence spectroscopy of low-level endogenous β-adrenergic receptor expression at the plasma membrane of differentiating human iPSC-derived cardiomyocytes

“…It shall be further noted that the fully CM-differentiated hiPSCs, even at day ~100, do not display a complete T-Tubular network, leaving open the question if the β2-AR, that in this work we observed on the basolateral membrane, would eventually segregate in these compartments [13]. Our observation of higher relative β2-AR expression levels in hiPSC-CMs appears to be in line with what was previously reported in at least three works [6,9,10]. Although, we shall mention here, that in recent measurements in hiPSC-derived engineered heart tissue, higher levels of β1-AR were observed throughout (private communication and [23]).…”

“…Wu et al observed mRNA levels and effects on contractility of β-AR expression in differentiating hiPSC, in a model of dilated cardiomyopathy [6]. Jung et al studied adrenoreceptor mRNA levels within the context of functional remodelling [9], while Hasan et al monitored how receptor-specific cAMP signaling evolves throughout hiPSC differentiation towards CMs [10]. Kondrashov et al studied the temporal dynamics of β 2 -AR expression in CRISPRed hiPSC [11].…”

A detailed characterization of the hyperpolarization-activated “funny” current (If) in human-induced pluripotent stem cell (iPSC)–derived cardiomyocytes with pacemaker activity

“…The low level of cAMP may for example derive from the high activity of phosphodiesterases (PDEs). The role of PDEs and in particular PDE3 and PDE4 in hiPS-CM has been recently shown in various works in which their inhibition resulted in the increase of basal cAMP level [22,25,26]. We may speculate that the low level of cAMP and high PDE activity may also explain the significantly slower time constant of the isoproterenol-mediated rate acceleration (7.6 ± 1.0 s) than the time constant of the rate slowing due to muscarinic receptors activation (3.4 ± 0.8 s).…”