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bioRxiv · 10.1101/2024.03.28.583721

Activation of IP3R in atrial cardiomyocytes leads to generation of cytosolic cAMP

Akerman, E. C.·Read, M. J.·Bose, S. J.·Koschinski, A.·Capel, R. A.·Chao, Y.-C.·Folkmanaite, M.·Hester, S.·Fischer, R.·Ayagama, T.·Broadbent, S. D.·Ahamed, R.·Simon, J. N.·Terrar, D. A.·Zaccolo, M.·Burton, R.-A. B.
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Abstract

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia. Excessive stimulation of the IP3 signaling pathway has been linked to AF through abnormal calcium handling. However, little is known about the mechanisms involved in this process. We expressed Fluorescence resonance energy transfer (FRET) based cytosolic cAMP sensor EPAC-SH187 in neonatal rat atrial myocytes (NRAMs) and neonatal rat ventricular myocytes (NRVMs). In NRAMs, addition of the alpha ()-1 agonist phenylephrine (PE, 3{micro}M) resulted in a bi-phasic FRET change (R1) 21.20 {+/-} 7.43% and (R2) 9.67 {+/-} 4.23% and addition of membrane permeant IP3 derivative, 2,3,6-tri-O-Butyryl-myo-IP3(1,4,5)-hexakis(acetoxymethyl)ester (IP3-AM, 20M) resulted in a peak of 20.31 {+/-} 6.74%. These FRET changes imply an increase in cAMP. Prior application of IP3 receptor (IP3R) inhibitors 2-Aminoethyl diphenylborinate (2-APB, 2.5M) or Xestospongin-C (0.3M) significantly inhibited the change in FRET in NRAMs in response to PE. Xestospongin-C (0.3M) significantly inhibited the change in FRET in NRAMs in response to IP3-AM. The FRET change in response to PE in NRVMs were not inhibited by 2-APB or Xestospongin-C. Finally, the localisation of cAMP signals was tested by expressing the FRET-based cAMP sensor, AKAP79-CUTie, which targets the intracellular surface of the plasmalemma. We found in NRAMs that PE led to FRET change corresponding to an increase in cAMP that was inhibited by 2-APB and Xestospongin C. This data support further investigation of the pro-arrhythmic nature and components of IP3 induced cAMP signalling to identify potential pharmacological targets. NEW & NOTEWORTHYThis study shows that indirect activation of the IP3 pathway in atrial myocytes using phenylephrine and direct activation using IP3-AM leads to an increase in cAMP and is in-part localized to the cell membrane. These changes can be pharmacologically inhibited using IP3R inhibitors. However, the cAMP rise in ventricular myocytes is independent of IP3R calcium release. Our data support further investigation into the pro-arrhythmic nature of IP3-induced cAMP signaling.

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Akerman, E. C., Read, M. J., Bose, S. J., Koschinski, A., Capel, R. A., Chao, Y.-C., Folkmanaite, M., Hester, S., Fischer, R., Ayagama, T., Broadbent, S. D., Ahamed, R., Simon, J. N., Terrar, D. A., Zaccolo, M., Burton, R.-A. B.. 2024-03-30. Activation of IP3R in atrial cardiomyocytes leads to generation of cytosolic cAMP. https://doi.org/10.1101/2024.03.28.583721

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