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Pathological activation of CaMKII induces arrhythmogenicity through TRPM4 overactivation

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Abstract

TRPM4 is a Ca2+-activated nonselective cation channel involved in cardiovascular physiology and pathophysiology. Based on cellular experiments and numerical simulations, the present study aimed to explore the potential arrhythmogenicity of CaMKII-mediated TRPM4 channel overactivation linked to Ca2+ dysregulation in the heart. The confocal immunofluorescence microscopy, western blot, and proximity ligation assay (PLA) in HL-1 atrial cardiomyocytes and/or TRPM4-expressing TSA201 cells suggested that TRPM4 and CaMKII proteins are closely localized. Co-expression of TRPM4 and CaMKIIδ or a FRET-based sensor Camui in HEK293 cells showed that the extent of TRPM4 channel activation was correlated with that of CaMKII activity, suggesting their functional interaction. Both expressions and interaction of the two proteins were greatly enhanced by angiotensin II treatment, which induced early afterdepolarizations (EADs) at the repolarization phase of action potentials (APs) recorded from HL-1 cells by the current clamp mode of patch clamp technique. This arrhythmic change disappeared after treatment with the TRPM4 channel blocker 9-phenanthrol or CaMKII inhibitor KN-62. In order to quantitatively assess how CaMKII modulates the gating behavior of TRPM4 channel, the ionomycin-permeabilized cell-attached recording was employed to obtain the voltage-dependent parameters such as steady-state open probability and time constants for activation/deactivation at different [Ca2+]i. Numerical simulations incorporating these kinetic data into a modified HL-1 model indicated that > 3-fold increase in TRPM4 current density induces EADs at the late repolarization phase and CaMKII inhibition (by KN-62) completely eliminates them. These results collectively suggest a novel arrhythmogenic mechanism involving excessive CaMKII activity that causes TRPM4 overactivation in the stressed heart.

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Availability of data and material (data transparency)

The data described and materials used in this paper are available on request.

Funding

This work has been supported by a JSPS Grant-in-Aid for Young Scientists (B) (No. 17K15566) to Y.H., a JSPS Grant-in-Aid for Scientific Research (B) (No. 15H04678) to R.I., and Swiss National Science Foundation to H.A. (No. 310030_184783).

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Authors and Affiliations

  1. Department of Physiology, Fukuoka University School of Medicine, 7-45-1 Nanakuma, Jonan-ku, Fukuoka, 814-0180, Japan

    Yaopeng Hu, Takayuki Fujita & Ryuji Inoue

  2. Institute of Biochemistry and Molecular Medicine, National Center of Competence in Research NCCR TransCure, University of Bern, Bern, Switzerland

    Daniela Ross Kaschitza, Maria Essers, Prakash Arullampalam & Hugues Abriel

Authors
  1. Yaopeng Hu
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  5. Takayuki Fujita
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  7. Ryuji Inoue
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Contributions

Y.H., H.A., and R.I. conceived this study; Y.H. conducted patch clamp experiments and analyzed the obtained data; Y.H., DR.K., M.E., and P.A. contributed immunoblotting, immunofluorescence, and PLA experiments and analyzed the obtained data; R.I. and Y.H. conducted simulations on the HL-1 AP model. T.F. helped to design the work and commented on the draft. Y.H., R.I., and H.A. contributed funding acquisition; Y.H. and R.I. wrote the manuscript and all authors reviewed and commented it.

Corresponding authors

Correspondence to Yaopeng Hu or Ryuji Inoue.

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Consent to participate

All authors confirmed that their roles played in this work are properly appreciated, and approved the order of authorship. The authors gratefully acknowledge Jean-Sébastien Rougier for constructive comments during experiments.

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All authors agreed to submit/publish this paper after careful reading.

Code availability (software application or custom code)

The codes for modified HL-1 models incorporating TRPM4 kinetics with and without CaMKII inhibition (written in CellML) can be provided on request. The original HL-1 model was written in Visual C++ (Takeuchi et al., 2013) which was translated into CellML (by Hu and Inoue). The Luo-Rudy 2000 model is available from the Cor library (Oxford; URL: http://cor.physiol.ox.ac.uk/).

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Contributions to Special Issues

This article is published as part of the Special Issue on Calcium Signal Dynamics in Cardiac Myocytes and Fibroblasts: Mechanisms and Therapeutics.

This article is part of the special issue on Calcium Signal Dynamics in Cardiac Myocytes and Fibroblasts: Mechanisms in Pflügers Archiv—European Journal of Physiology

Supplementary information

Supplementary Fig. 1

Reconstructed rate constants and open probability for TRPM4 channel gating in the absence and presence of KN-62. The rate constants of opening (α) and closing (β) are calculated with respect to two variables, i.e. membrane potential (Vm) and intracellular Ca2+ concentration ([Ca2+]i), by using the equations in the main text. Open probability (Po) is then calculated by the formula: α/(α+β). In the absence of KN-62 (con), α and β show reciprocal voltage-dependency over a wide range of [Ca2+]i. These rate constants however become much less sensitive to Vm after CaMKII inhibition by KN-62 (KN62), making α and β stay at low and high values, respectively. As a result, (except for extremely high [Ca2+]i) the transition from the ‘O’ to ‘C’ states is decelerated, while that from the ‘O’ to ‘C’ states is accelerated by CaMKII inhibition to favor the ‘C’ state, which renders the deactivation of TRPM4 channel faster around the resting membrane potential. Accordingly, Po at given Vm and [Ca2+]i becomes smaller and less voltage-dependent. Supplementary Fig. 2 AP simulation with Luo-Rudy 2000 model. Luo-Rudy AP 2000 models with TRPM4 gating kinetics before and after application of KN-62. The density of TRPM4 channel is increased from normal (see the Methods) to several-fold levels. (PPTX 282 kb)

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Hu, Y., Kaschitza, D.R., Essers, M. et al. Pathological activation of CaMKII induces arrhythmogenicity through TRPM4 overactivation. Pflugers Arch - Eur J Physiol 473, 507–519 (2021). https://doi.org/10.1007/s00424-020-02507-w

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  • DOI: https://doi.org/10.1007/s00424-020-02507-w

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