Background: Signaling by cAMP is organized in multiple distinct subcellular nanodomains regulated by cAMP-hydrolyzing PDEs (phosphodiesterases). Cardiac β-adrenergic signaling has served as the prototypical system to elucidate cAMP compartmentalization. Although studies in cardiac myocytes have provided an understanding of the location and properties of a handful of cAMP subcellular compartments, an overall view of the cellular landscape of cAMP nanodomains is missing. Methods: Here, we combined an integrated phosphoproteomics approach that takes advantage of the unique role that individual PDEs play in the control of local cAMP, with network analysis to identify previously unrecognized cAMP nanodomains associated with β-adrenergic stimulation. We then validated the composition and function of one of these nanodomains using biochemical, pharmacological, and genetic approaches and cardiac myocytes from both rodents and humans. Results: We demonstrate the validity of the integrated phosphoproteomic strategy to pinpoint the location and provide critical cues to determine the function of previously unknown cAMP nanodomains. We characterize in detail one such compartment and demonstrate that the PDE3A2 isoform operates in a nuclear nanodomain that involves SMAD4 (SMAD family member 4) and HDAC-1. Inhibition of PDE3 results in increased HDAC-1 phosphorylation, leading to inhibition of its deacetylase activity, derepression of gene transcription, and cardiac myocyte hypertrophic growth. Conclusions: We developed a strategy for detailed mapping of subcellular PDE-specific cAMP nanodomains. Our findings reveal a mechanism that explains the negative long-term clinical outcome observed in patients with heart failure treated with PDE3 inhibitors.

Osteoarthritis (OA), a progressive degenerative disease of cartilage in joints, is the most common cause of chronic disability in older adults. While OA is mostly considered an age-related pathology, women have a 1.5-fold higher risk of developing OA relative to men and experience more severe symptoms. Yet, they remain underrepresented in musculoskeletal research and clinical trials. Responsible for cartilage formation, articular chondrocytes experience physiological changes in OA, but the functional implications of such alterations remain largely unexplored due to difficulties in acquiring the data experimentally. Through reparameterization, we expand a mathematical chondrocyte model to investigate sex-specific OA pathogenesis. We performed sensitivity analysis to address the impact of ion channel activity in healthy and OA chondrocyte populations. Simulations show that in healthy female chondrocytes, the resting membrane potential is more depolarized than in healthy male chondrocytes, suggesting potential sex-specific emergent physiological differences in articular chondrocytes. In both sexes, the resting membrane potential of healthy chondrocytes is most sensitive to 𝐼𝐶𝑎−𝐴𝑇𝑃, 𝐼𝑁𝑎−𝑏, 𝐼𝑁𝑎𝐾 and 𝐼𝐾−𝑏, but in OA it depolarizes and becomes sensitive to 𝐼𝐾𝐷𝑅, 𝐼𝑁𝑎𝐾 and 𝐼𝐾−𝑏. Developed and evaluated against experimental data, our articular chondrocyte OA electrophysiological model can be used to further study OA pathology and sex-specific pathological OA changes.

Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): BHF Background Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) enable accessible human data-based cardiology studies. However, a caveat in hiPSC-CM-based studies is their immature electrophysiological and contractile phenotype. One of the modifications occurring during hiPSC-CM maturation is the change in myofilament calcium sensitivity, an important indicator of cardiac muscle function [1, 2]. In silico hiPSC-CM investigations could help improve understanding of the hiPSC-CM-specific contractile behaviour and its changes during maturation. Considering the growing use of hiPSC-CM, it is vital to enable investigations of hiPSC-CM-specific contractile features. Purpose To address the need of hiPSC-CM model with integrated contractile element, our goal is to develop an electromechanical human data-based iPSC-CM computer model. We aim to use the model to investigate the effects of the changes in myofilament calcium sensitivity on hiPSC-CM electrophysiology and contractility. Methods We coupled a published hiPSC-CM electrophysiological model [3] with a model of the human adult cardiomyocyte contractile machinery [4] by linking intracellular calcium and calcium-bound troponin dynamics. The established electromechanical hiPSC-CM model was calibrated using experimental hiPSC-CM active tension data and its simulated electromechanical biomarkers were also evaluated against experimental action potential and calcium transient data. We conducted a sensitivity analysis to investigate the effects of changes in myofilament calcium sensitivity on the electrophysiology and contractility of the cell. Results First, we demonstrated that the model successfully reproduces the hiPSC-CM contractile phenotype. Simulations showed a peak twitch tension of 0.44 kPa which takes 201 ms to peak and 164 ms to achieve 50% relaxation, which all agree with the experimental hiPSC-CM values. Simulated calcium transient and action potential biomarkers remain within the experimentally established ranges after electromechanical coupling. The sensitivity analysis of the hiPSC-CM model focused on the myofilament calcium sensitivity effects showed an increase in active tension amplitude with a decrease in calcium transient peaks upon increased myofilament calcium sensitivity. Large increases in myofilament calcium sensitivity result in depolarization failure with low amplitude fluctuations of membrane voltage, calcium transient and active tension. Altogether simulation results demonstrate the usability of the model for simulating and exploring not only physiological, but also pathological cardiac conditions. Conclusions We present a new electromechanical hiPSC-CM model for in silico hiPSC-CM-based studies. The model has been evaluated against experimental data and has demonstrated the capacity to generate key electrophysiological currents, active tension as well as myofilament calcium sensitivity-induced electromechanical abnormalities.

Cyclic adenosine monophosphate (cAMP) is a diffusible intracellular second messenger that plays a key role in the regulation of cardiac function. In response to the release of catecholamines from sympathetic terminals, cAMP modulates heart rate and the strength of contraction and ease of relaxation of each heartbeat. At the same time, cAMP is involved in the response to a multitude of other hormones and neurotransmitters. A sophisticated network of regulatory mechanisms controls the temporal and spatial propagation of cAMP, resulting in the generation of signaling nanodomains that enable the second messenger to match each extracellular stimulus with the appropriate cellular response. Multiple proteins contribute to this spatio-temporal regulation, including the cAMP-hydrolyzing phosphodiesterases. By breaking down cAMP to a different extent at different locations, these enzymes generate subcellular cAMP gradients. As a result, only a subset of the downstream effectors is activated and a specific response is executed. Dysregulation of cAMP compartmentalization has been observed in cardiovascular diseases, highlighting the importance of appropriate control of local cAMP signaling. Current research is unveiling the molecular organization underpinning cAMP compartmentalization, providing original insight into the physiology of cardiac myocytes and the alteration associated with disease, with the potential to uncover novel therapeutic targets. Here we present an overview of the mechanisms that are currently understood to be involved in generating cAMP nanodomains and we highlight the questions that remain to be answered.

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