Ryanodine receptor modification and regulation by intracellular Ca2+ and Mg2+ in healthy and failing human hearts

K Walweel; P Molenaar; M S Imtiaz; A Denniss; C Dos Remedios; D F van Helden; A F Dulhunty; D R Laver; N A Beard

doi:10.1016/j.yjmcc.2017.01.016

Ryanodine receptor modification and regulation by intracellular Ca²⁺ and Mg²⁺ in healthy and failing human hearts

J Mol Cell Cardiol. 2017 Mar:104:53-62. doi: 10.1016/j.yjmcc.2017.01.016. Epub 2017 Jan 26.

Authors

K Walweel¹, P Molenaar², M S Imtiaz³, A Denniss⁴, C Dos Remedios⁵, D F van Helden¹, A F Dulhunty⁶, D R Laver¹, N A Beard⁷

Affiliations

¹ School of Biomedical Sciences and Pharmacy, University of Newcastle and Hunter Medical Research Institute, Callaghan, NSW 2308, Australia.
² Biomedical Sciences, Queensland University of Technology, Brisbane, QLD, 4000, Northside Clinical School, School of Clinical Medicine, University of Queensland and Critical Care Research Group, The Prince Charles Hospital, Chermside, QLD, 4032, Australia.
³ Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales 2010, Australia.
⁴ Health Research Institute, Faculty of Education Science and Mathematics, University of Canberra, Bruce, ACT 2617, Australia.
⁵ Bosch Institute, Discipline of Anatomy, University of Sydney, Sydney, New South Wales 2006, Australia.
⁶ John Curtin School of Medical Research, Australian National University, Canberra, ACT, 0200, Australia.
⁷ Health Research Institute, Faculty of Education Science and Mathematics, University of Canberra, Bruce, ACT 2617, Australia; John Curtin School of Medical Research, Australian National University, Canberra, ACT, 0200, Australia. Electronic address: nicole.beard@canberra.edu.au.

PMID: 28131631
DOI: 10.1016/j.yjmcc.2017.01.016

Abstract

Rationale: Heart failure is a multimodal disorder, of which disrupted Ca²⁺ homeostasis is a hallmark. Central to Ca²⁺ homeostasis is the major cardiac Ca²⁺ release channel - the ryanodine receptor (RyR2) - whose activity is influenced by associated proteins, covalent modification and by Ca²⁺ and Mg²⁺. That RyR2 is remodelled and its function disturbed in heart failure is well recognized, but poorly understood.

Objective: To assess Ca²⁺ and Mg²⁺ regulation of RyR2 from left ventricles of healthy, cystic fibrosis and failing hearts, and to correlate these functional changes with RyR2 modifications and remodelling.

Methods and results: The function of RyR2 from left ventricular samples was assessed using lipid bilayer single-channel measurements, whilst RyR2 modification and protein:protein interactions were determined using Western Blots and co-immunoprecipitation. In all failing hearts there was an increase in RyR2 activity at end-diastolic cytoplasmic Ca²⁺ (100nM), a decreased cytoplasmic [Ca²⁺] required for half maximal activation (K_a) and a decrease in inhibition by cytoplasmic Mg²⁺. This was accompanied by significant hyperphosphorylation of RyR2 S²⁸⁰⁸ and S²⁸¹⁴, reduced free thiol content and a reduced interaction with FKBP12.0 and FKBP12.6. Either dephosphorylation of RyR2 using PP1 or thiol reduction using DTT eliminated any significant difference in the activity of RyR2 from healthy and failing hearts. We also report a subgroup of RyR2 in failing hearts that were not responsive to regulation by intracellular Ca²⁺ or Mg²⁺.

Conclusion: Despite different aetiologies, disrupted RyR2 Ca²⁺ sensitivity and biochemical modification of the channel are common constituents of failing heart RyR2 and may underlie the pathological disturbances in intracellular Ca²⁺ signalling.

Keywords: Heart failure; Lipid bilayer; Mg(2+) and Ca(2+) signalling; Phosphorylation; Regulatory proteins; Ryanodine receptor.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Calcium / metabolism*
Calcium Signaling
Case-Control Studies
Heart Failure / metabolism*
Heart Failure / pathology
Heart Failure / physiopathology
Heart Ventricles / metabolism
Humans
Intracellular Space / metabolism
Magnesium / metabolism*
Myocardium / metabolism*
Myocytes, Cardiac / metabolism
Phosphorylation
Protein Binding
Protein Processing, Post-Translational
Ryanodine Receptor Calcium Release Channel / metabolism*
Sarcoplasmic Reticulum / metabolism
Tacrolimus Binding Proteins / metabolism

Substances

Ryanodine Receptor Calcium Release Channel
Tacrolimus Binding Proteins
Magnesium
Calcium