From the Laboratory of Experimental Cardiology, Department of Cardiovascular Sciences, KU Leuven, Belgium (E.D., V.B., I.L., G.A., K.R.S., N.M.); Biomedical Research Institute, University of Hasselt, Belgium (V.B.); Division of Cardiology, Medical University of Graz, Austria (G.A.); and Institute of Cardiovascular and Medical Sciences, University of Glasgow, United Kingdom (N.M.).
Circ Res. 2013 Nov 8;113(11):1242-52. doi: 10.1161/CIRCRESAHA.113.301896. Epub 2013 Sep 30.
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.
Our aim was to test the hypothesis that coupled and noncoupled RyRs have distinct microdomain-dependent modulation.
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.
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.
在低 T 管密度的大型哺乳动物的心室肌细胞中，大量的兰尼碱受体（RyR）与肌膜没有偶联；心脏重构增加了非偶联的 RyR。
我们的目的是检验以下假设，即偶联和非偶联 RyR 具有不同的微域依赖性调节。
我们研究了来自猪左心室的单个心肌细胞。通过三维（3D）分析 T 管网络，测量释放部位与膜的距离。钙瞬变的上升相与接近膜的程度相关（共聚焦成像、全细胞电压钳、K5fluo-4 作为钙指示剂）。因此，刺激后的钙火花被鉴定为来自偶联或非偶联 RyR。我们使用高频刺激作为已知的钙/钙调蛋白依赖性激酶 II 激活剂。与非偶联 RyR 相比，偶联 RyR 的火花频率显著增加。这种对偶联 RyR 的特异性调节被钙/钙调蛋白依赖性激酶 II 抑制剂 autocamtide-2 相关抑制肽和 KN-93 完全阻断，但 KN-92 则不然。偶联和非偶联部位的钙/钙调蛋白依赖性激酶 II 和 RyR 的共定位没有明显差异，但肌动蛋白破坏剂细胞松弛素 D 阻止了偶联 RyR 的特异性调节。二苯并碘二酮或 apocynin 抑制 NADPH 氧化酶 2 或整体活性氧物质清除也阻止了偶联 RyR 的调节。在刺激的钙瞬变过程中，仅在偶联 RyR 区域观察到频率依赖性增加钙上升的速率，并且被 autocamtide-2 相关抑制肽所消除。心肌梗死后，偶联 RyR 的选择性调节丧失。
偶联 RyR 具有由钙/钙调蛋白依赖性激酶 II 和活性氧物质引起的独特调节，依赖于完整的细胞骨架，与局部钙/活性氧物质微域一致，并受疾病的修饰。
