Cardiomyocyte Membrane Structure and cAMP Compartmentation Produce Anatomical Variation in β2AR-cAMP Responsiveness in Murine Hearts

Wright, Peter T.; Bhogal, Navneet; Diakonov, Ivan; Pannell, Laura; Perera, Ruwan K.; Bork, Nadja I.; Schobesberger, Sophie; Lucarelli, Carla; Faggian, Giuseppe; Alvarez-Laviada, Anita; Zaccolo, Manuela; Kamp, Timothy J.; Balijepalli, Ravi C.; Lyon, Alexander R.; Harding, Siân E.; Nikolaev, Viacheslav O.; Gorelik, Julia

doi:10.1016/j.celrep.2018.03.053

Cited by 50 publications

(59 citation statements)

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“…Cav1 and −3 contain a scaffolding domain (CSD) located in the N-terminal region suggested to interact with transmembrane adenylate cyclases (tmACs), to inhibit their activities and thus control intracellular cAMP levels (Toya et al, 1998). In cardiomyocytes, caveolae participate in the compartmentalization of intracellular cAMP which can regulate cell contractility in distal regions of the heart and, therefore, its function (Wright et al, 2014, 2018). The mechanoprotective role of caveolae is associated with the maintenance of plasma membrane integrity when both, cells and tissues, experience chronical mechanical stress (Cheng et al, 2015; Lo et al, 2015; Parton et al, 2017; Sinha et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

Caveolae coupling of melanocytes signaling and mechanics is required for human skin pigmentation

Domingues

Hurbain

Gilles-Marsens

et al. 2019

Preprint

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22Tissue homeostasis requires regulation of cell-cell communication, which relies on signaling 23 molecules and cell contacts. In skin epidermis, keratinocytes secrete specific factors transduced 24 by melanocytes into signaling cues to promote their pigmentation and dendrite outgrowth, while 25 melanocytes transfer melanin pigments to keratinocytes to convey skin photoprotection. How 26 epidermal cells integrate these functions remains poorly characterized. Here, we found that 27 caveolae polarize in melanocytes and are particularly abundant at melanocyte-keratinocyte 28 interface. Caveolae in melanocytes are sensitive to ultra-violet radiations and miRNAs released 29 by keratinocytes. Preventing caveolae formation in melanocytes results in increased production 30 of intracellular cAMP and melanin pigments, but decreases cell protrusions, cell-cell contacts, 31 pigment transfer and epidermis pigmentation. Altogether, our data establish that, in 32 melanocytes, caveolae serve as key molecular hubs that couple signaling outputs from 33 keratinocytes to mechanical plasticity. This process is crucial to maintain cell-cell contacts and 34 intercellular communication, skin pigmentation and tissue homeostasis. 84Epidermal melanocytes and keratinocytes are in constant communication, not only via secreted 85 factors and exosomes that modulate cellular responses, but also by the physical contacts they 86 establish to maintain the tissue homeostasis and pigmentation. Here, we report a new function 87 for caveolae, which, by integrating the biochemical and mechanical behavior of melanocytes, 88 control melanin transfer to keratinocytes and epidermis pigmentation. Altogether, this study 89 provides the first evidence for a physiologic role of caveolae as a molecular sensing platform 90 required for the homeostasis of the largest human tissue, the skin epidermis. 91 5 Results 92 Caveolae polarize in melanocytes and are positively-regulated by keratinocytes-secreted 93 factors 94Melanocytes and keratinocytes establish a complex intercellular dialogue required for skin 95 photoprotection. 2D-co-culture systems, where these two cell types share the same medium, 96have been widely used to study intercellular communication and pigment transfer between 97 epidermal cells (Hirobe, 2005; Lei et al., 2002). To evaluate the distribution of caveolae within 98 the epidermal unit in 2D, normal human melanocytes and keratinocytes were co-cultured and 99 labelled for the two constituents of caveolae, Cav1 or Cavin1. Immunofluorescence microscopy 100 revealed that both Cav1 and Cavin1, and therefore caveolae, were asymmetrically distributed in 101 melanocytes (Figures 1A and B), which were identified by the abundant staining of the 102 premelanosome protein PMEL [hereafter referred as melanin, see Experimental Procedures; 103 (Raposo et al., 2001)]. This polarization was not observed in keratinocytes.104 Cells can break their symmetry in response to local external chemical and/or mechanical cues 105 such as signaling molecules and/or cell-ce...

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Section: Introductionmentioning

confidence: 99%

Caveolae coupling of melanocytes signaling and mechanics is required for human skin pigmentation

Domingues

Hurbain

Gilles-Marsens

et al. 2019

Preprint

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“…PMU reduced the amplitude of β 2 AR-cAMP responses in the cytosol but not in the RII-PKA domains. This is an intriguing finding, as previous papers ( MacDougall et al, 2012 ; Wright et al, 2018 ) which have employed these two sensors have demonstrated that β 2 AR is a compartmentalized receptor in healthy cardiomyocytes, which will produce cAMP responses in the cell cytosol but not in the RII_PKA domains. The data we present suggest that the production of β 2 AR-cAMP is load-sensitive and may involve the loss of receptor subunits.…”

Section: Discussionmentioning

confidence: 76%

“…Cell imaging was performed with 60x oil immersion Zeiss lens, using inverted confocal laser scanning microscope (A Zeiss LSM-780), with argon laser beam at specific wavelengths for the secondary antibodies (Alexa Fluor Cav3 546 nm). Protein density and respective regularity of distribution were obtained with the same method used for TTs analysis described in more detail elsewhere ( Wright et al, 2018 ). PLA analysis was performed by automatically thresholding cell images in ImageJ (Image J Corp, United States) and utilizing default plugins to measure area covered by particles, size of particles, number of particles and number of particles per μm.…”

Section: Methodsmentioning

confidence: 99%

“…The localized signaling of LTCCs was studied with a modified SICM technique which allows the selective patching of specific membrane sub-domains such as TT openings or sarcolemmal crests ( Bhargava et al, 2013 ). βAR function was assessed by monitoring sub-cellular cAMP responses using FRET-based biosensors ( Nikolaev et al, 2004 ; Di Benedetto et al, 2008 ; Wright et al, 2018 ). These biosensors were delivered to either the cell cytosol or membrane regions using specific molecular tags.…”

Section: Introductionmentioning

confidence: 99%

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Partial Mechanical Unloading of the Heart Disrupts L-Type Calcium Channel and Beta-Adrenoceptor Signaling Microdomains

et al. 2018

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Introduction: We investigated the effect of partial mechanical unloading (PMU) of the heart on the physiology of calcium and beta-adrenoceptor-cAMP (βAR-cAMP) microdomains. Previous studies have investigated PMU using a model of heterotopic-heart and lung transplantation (HTHAL). These studies have demonstrated that PMU disrupts the structure of cardiomyocytes and calcium handling. We sought to understand these processes by studying L-Type Calcium Channel (LTCC) activity and sub-type-specific βAR-cAMP signaling within cardiomyocyte membrane microdomains.Method: We utilized an 8-week model of HTHAL, whereby the hearts of syngeneic Lewis rats were transplanted into the abdomens of randomly assigned cage mates. A pronounced atrophy was observed in hearts after HTHAL. Cardiomyocytes were isolated via enzymatic perfusion. We utilized Förster Resonance Energy Transfer (FRET) based cAMP-biosensors and scanning ion conductance microscopy (SICM) based methodologies to study localization of LTCC and βAR-cAMP signaling.Results: β2AR-cAMP responses measured by FRET in the cardiomyocyte cytosol were reduced by PMU (loaded 28.51 ± 7.18% vs. unloaded 10.84 ± 3.27% N,n 4/10-13 mean ± SEM ∗p < 0.05). There was no effect of PMU on β2AR-cAMP signaling in RII_Protein Kinase A domains. β1AR-cAMP was unaffected by PMU in either microdomain. Consistent with this SICM/FRET analysis demonstrated that β2AR-cAMP was specifically reduced in t-tubules (TTs) after PMU (loaded TT 0.721 ± 0.106% vs. loaded crest 0.104 ± 0.062%, unloaded TT 0.112 ± 0.072% vs. unloaded crest 0.219 ± 0.084% N,n 5/6-9 mean ± SEM ∗∗p < 0.01, ∗∗∗p < 0.001 vs. loaded TT). By comparison β1AR-cAMP responses in either TT or sarcolemmal crests were unaffected by the PMU. LTCC occurrence and open probability (Po) were reduced by PMU (loaded TT Po 0.073 ± 0.011% vs. loaded crest Po 0.027 ± 0.006% N,n 5/18-26 mean ± SEM ∗p < 0.05) (unloaded TT 0.0350 ± 0.003% vs. unloaded crest Po 0.025 N,n 5/20-30 mean ± SEM NS #p < 0.05 unloaded vs. loaded TT). We discovered that PMU had reduced the association between Caveolin-3, Junctophilin-2, and Cav1.2.Discussion: PMU suppresses’ β2AR-cAMP and LTCC activity. When activated, the signaling of β2AR-cAMP and LTCC become more far-reaching after PMU. We suggest that a situation of ‘suppression/decompartmentation’ is elicited by the loss of refined cardiomyocyte structure following PMU. As PMU is a component of modern device therapy for heart failure this study has clinical ramifications and raises important questions for regenerative medicine.

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“…The current dogma is that a larger cell demands a more extensive tubular system to function which sits uneasily with data concerning species differences, the loss of T-tubules in compensatory hypertrophy in disease and the lack of correlation between atrial cardiomyocyte size and TAT density (when excluding very small cells) [40]. We demonstrated intra-chamber variability in LV cardiomyocyte TAT organisation in adult rat cells [41]. Cells derived from the apical LV increased their contractility following β2-AR activation.…”

Section: Active Mechanismsmentioning

confidence: 78%

Studying signal compartmentation in adult cardiomyocytes

Judina

Gorelik

2020

Biochemical Society Transactions

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Multiple intra-cellular signalling pathways rely on calcium and 3′–5′ cyclic adenosine monophosphate (cAMP) to act as secondary messengers. This is especially true in cardiomyocytes which act as the force-producing units of the cardiac muscle and are required to react rapidly to environmental stimuli. The specificity of functional responses within cardiomyocytes and other cell types is produced by the organellar compartmentation of both calcium and cAMP. In this review, we assess the role of molecular localisation and relative contribution of active and passive processes in producing compartmentation. Active processes comprise the creation and destruction of signals, whereas passive processes comprise the release or sequestration of signals. Cardiomyocytes display a highly articulated membrane structure which displays significant cell-to-cell variability. Special attention is paid to the way in which cell membrane caveolae and the transverse-axial tubule system allow molecular localisation. We explore the effects of cell maturation, pathology and regional differences in the organisation of these processes. The subject of signal compartmentation has had a significant amount of attention within the cardiovascular field and has undergone a revolution over the past two decades. Advances in the area have been driven by molecular imaging using fluorescent dyes and genetically encoded constructs based upon fluorescent proteins. We also explore the use of scanning probe microscopy in the area. These techniques allow the analysis of molecular compartmentation within specific organellar compartments which gives researchers an entirely new perspective.

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Cardiomyocyte Membrane Structure and cAMP Compartmentation Produce Anatomical Variation in β2AR-cAMP Responsiveness in Murine Hearts

Cited by 50 publications

References 41 publications

Caveolae coupling of melanocytes signaling and mechanics is required for human skin pigmentation

Caveolae coupling of melanocytes signaling and mechanics is required for human skin pigmentation

Partial Mechanical Unloading of the Heart Disrupts L-Type Calcium Channel and Beta-Adrenoceptor Signaling Microdomains

Studying signal compartmentation in adult cardiomyocytes

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