Abstract
Here we investigate how ß-adrenergic stimulation of the heart alters regulation of ryanodine receptors (RyRs) by intracellular Ca2+ and Mg2+ and the role of these changes in SR Ca2+ release. RyRs were isolated from rat hearts, perfused in a Langendorff apparatus for 5 min and subject to 1 min perfusion with 1 µM isoproterenol or without (control) and snap frozen in liquid N2 to capture their phosphorylation state. Western Blots show that RyR2 phosphorylation was increased by isoproterenol, confirming that RyR2 were subject to normal ß-adrenergic signaling. Under basal conditions, S2808 and S2814 had phosphorylation levels of 69% and 15%, respectively. These levels were increased to 83% and 60%, respectively, after 60 s of ß-adrenergic stimulation consistent with other reports that ß-adrenergic stimulation of the heart can phosphorylate RyRs at specific residues including S2808 and S2814 causing an increase in RyR activity. At cytoplasmic [Ca2+] <1 µM, ß-adrenergic stimulation increased luminal Ca2+ activation of single RyR channels, decreased luminal Mg2+ inhibition and decreased inhibition of RyRs by mM cytoplasmic Mg2+. At cytoplasmic [Ca2+] >1 µM, ß-adrenergic stimulation only decreased cytoplasmic Mg2+ and Ca2+ inhibition of RyRs. The Ka and maximum levels of cytoplasmic Ca2+ activation site were not affected by ß-adrenergic stimulation.Our RyR2 gating model was fitted to the single channel data. It predicted that in diastole, ß-adrenergic stimulation is mediated by 1) increasing the activating potency of Ca2+ binding to the luminal Ca2+ site and decreasing its affinity for luminal Mg2+ and 2) decreasing affinity of the low-affinity Ca2+/Mg2+ cytoplasmic inhibition site. However in systole, ß-adrenergic stimulation is mediated mainly by the latter.
Highlights
Exercise and stress induce the sympathetic ‘‘fight or flight’’response that increases heart rate and contractility
Increased intracellular [Ca2+] causes Ca2+/calmodulin dependent protein kinase II (CaMKII) autophosphorylation so that it remains active at low [Ca2+] [1,2]
We measured the effects of isoproterenol perfusion on RyR2 phosphorylation to confirm that RyR2 had been subjected to normal ß-adrenergic signalling processes in the heart. ß-adrenergic stimulation of rat heart was correlated with an increased phosphorylation at S2814 and S2808
Summary
Response that increases heart rate and contractility. During this response, increased catecholamine concentrations stimulate cardiac b-adrenergic receptors, resulting in adenylyl cyclase activation, increased cyclic AMP and increased activity of cyclic AMPdependent protein kinase A (PKA). Increased intracellular [Ca2+] causes Ca2+/calmodulin dependent protein kinase II (CaMKII) autophosphorylation so that it remains active at low [Ca2+] [1,2]. PKA and CaMKII phosphorylation increase Ca2+-transport activity that underlies contraction and pacemaking in cardiac cells [1,3,4,5,6]. Systolic contraction follows activation of sarcolemmal voltagegated L-type Ca2+ channels during an action potential resulting in Ca2+-influx, which activates cardiac ryanodine receptor (RyR2). Calcium release channels on the sarcoplasmic reticulum (SR, major intracellular Ca2+ store). Diastolic relaxation occurs with cessation of Ca2+ release and Ca2+ sequestration by the SR Ca2+ uptake transporter
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