Abstract
ABSTRACTHere, we report the impact of redox potential on isolated cardiac ryanodine receptor (RyR2) channel activity and its response to physiological changes in luminal [Ca2+]. Basal leak from the sarcoplasmic reticulum is required for normal Ca2+ handling, but excess diastolic Ca2+ leak attributed to oxidative stress is thought to lower the threshold of RyR2 for spontaneous sarcoplasmic reticulum Ca2+ release, thus inducing arrhythmia in pathological situations. Therefore, we examined the RyR2 response to luminal [Ca2+] under reducing or oxidising cytoplasmic redox conditions. Unexpectedly, as luminal [Ca2+] increased from 0.1 to 1.5 mM, RyR2 activity declined when pretreated with cytoplasmic 1 mM DTT or buffered with GSH∶GSSG to a normal reduced cytoplasmic redox potential (−220 mV). Conversely, with 20 µM cytoplasmic 4,4′-DTDP or buffering of the redox potential to an oxidising value (−180 mV), RyR2 activity increased with increasing luminal [Ca2+]. The luminal redox potential was constant at −180 mV in each case. These responses to luminal [Ca2+] were maintained with cytoplasmic 2 mM Na2ATP or 5 mM MgATP (1 mM free Mg2+). Overall, the results suggest that the redox potential in the RyR2 junctional microdomain is normally more oxidised than that of the bulk cytoplasm.
Highlights
There is much evidence linking excess diastolic Ca2+ release with the onset of delayed afterdepolarisations (DADs) and arrhythmia in a number of pathological conditions (Fauconnier et al, 2011; Marks, 2013; Pogwizd and Bers, 2004; Shannon et al, 2002).This excessive Ca2+ leak has been linked to enhanced b adrenergic activity that leads to hyperphosphorylation of RyR2 channels and to oxidative stress
The results suggest that the redox potential in the RyR2 junctional microdomain is normally more oxidised than that of the bulk cytoplasm
The findings are difficult to reconcile with measurements in intact cardiac myocytes showing an increase in Ca2+ leak through RyR2 when sarcoplasmic reticulum load increases, indicating an increase in Po as luminal [Ca2+] increases (Lukyanenko et al, 1996; Lukyanenko et al, 2001; Satoh et al, 1997; Shannon et al, 2003)
Summary
There is much evidence linking excess diastolic Ca2+ release with the onset of delayed afterdepolarisations (DADs) and arrhythmia in a number of pathological conditions (Fauconnier et al, 2011; Marks, 2013; Pogwizd and Bers, 2004; Shannon et al, 2002) This excessive Ca2+ leak has been linked to enhanced b adrenergic activity that leads to hyperphosphorylation of RyR2 channels and to oxidative stress. RyR2 sensitivity to luminal [Ca2+] in canine heart failure (Belevych et al, 2009; Shan et al, 2012; Terentyev et al., 2008) This was elegantly illustrated by the finding of a significantly greater open probability of RyR2 channels from. The open probability of RyR2 channels from healthy heart tissue was increased to levels seen in RyR2 from failing hearts by the addition of 4,49-DTDP (Terentyev et al, 2008)
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