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Selective modulation of coupled ryanodine receptors during microdomain activation of calcium/calmodulin-dependent kinase II in the dyadic cleft.

Abstract：

RATIONALE:In ventricular myocytes of large mammals with low T-tubule density, a significant number of ryanodine receptors (RyRs) are not coupled to the sarcolemma; cardiac remodeling increases noncoupled RyRs. OBJECTIVE:Our aim was to test the hypothesis that coupled and noncoupled RyRs have distinct microdomain-dependent modulation. METHODS AND RESULTS:We studied single myocytes from pig left ventricle. The T-tubule network was analyzed in 3-dimension (3D) to measure distance to membrane of release sites. The rising phase of the Ca(2+) transient was correlated with proximity to the membrane (confocal imaging, whole-cell voltage-clamp, K5fluo-4 as Ca(2+) indicator). Ca(2+) sparks after stimulation were thus identified as resulting from coupled or noncoupled RyRs. We used high-frequency stimulation as a known activator of Ca(2+)/calmodulin-dependent kinase II. Spark frequency increased significantly more in coupled than in noncoupled RyRs. This specific modulation of coupled RyRs was abolished by the Ca(2+)/calmodulin-dependent kinase II blockers autocamtide-2-related inhibitory peptide and KN-93, but not by KN-92. Colocalization of Ca(2+)/calmodulin-dependent kinase II and RyR was not detectably different for coupled and noncoupled sites, but the F-actin disruptor cytochalasin D prevented the specific modulation of coupled RyRs. NADPH oxidase 2 inhibition by diphenyleneiodonium or apocynin, or global reactive oxygen species scavenging, also prevented coupled RyR modulation. During stimulated Ca(2+) transients, frequency-dependent increase of the rate of Ca(2+) rise was seen in coupled RyR regions only and abolished by autocamtide-2-related inhibitory peptide. After myocardial infarction, selective modulation of coupled RyR was lost. CONCLUSIONS:Coupled RyRs have a distinct modulation by Ca(2+)/calmodulin-dependent kinase II and reactive oxygen species, dependent on an intact cytoskeleton and consistent with a local Ca(2+)/reactive oxygen species microdomain, and subject to modification with disease.

journal_name

Circ Res

journal_title

Circulation research

authors

Dries E,Bito V,Lenaerts I,Antoons G,Sipido KR,Macquaide N

doi

10.1161/CIRCRESAHA.113.301896

subject

Has Abstract

pub_date

2013-11-08 00:00:00

pages

1242-52

issue

eissn

0009-7330

issn

1524-4571

pii

CIRCRESAHA.113.301896

journal_volume

113

pub_type

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Selective modulation of coupled ryanodine receptors during microdomain activation of calcium/calmodulin-dependent kinase II in the dyadic cleft.