Distribution and function of cardiac ryanodine receptor clusters in live ventricular myocytes
Hiess, Florian (University of Calgary)
Vallmitjana, Alexander (Universitat Politècnica de Catalunya)
Wang, Ruibu (University of Calgary)
Cheng, Hongqiang (University of California)
Ter Keurs, Henk E.D.J. (University of Calgary)
Chen, Ju (University of California)
Hove-Madsen, Leif (Institut d'Investigació Biomèdica Sant Pau)
Benítez, Raúl (Universitat Politècnica de Catalunya)
Chen, S.R.Wayne (University of Calgary)
Universitat Autònoma de Barcelona

Data:	2015
Resum:	The cardiac Ca release channel (ryanodine receptor, RyR2) plays an essential role in excitation-contraction coupling in cardiac muscle cells. Effective and stable excitation-contraction coupling critically depends not only on the expression of RyR2, but also on its distribution. Despite its importance, little is known about the distribution and organization of RyR2 in living cells. To study the distribution of RyR2 in living cardiomyocytes, we generated a knock-in mouse model expressing a GFP-tagged RyR2 (GFP-RyR2). Confocal imaging of live ventricular myocytes isolated from the GFP-RyR2 mouse heart revealed clusters of GFP-RyR2 organized in rows with a striated pattern. Similar organization of GFP-RyR2 clusters was observed in fixed ventricular myocytes. Immunofluorescence staining with the anti-α-actinin antibody (a z-line marker) showed that nearly all GFP-RyR2 clusters were localized in the z-line zone. There were small regions with dislocated GFP-RyR2 clusters. Interestingly, these same regions also displayed dislocated z-lines. Staining with di-8-ANEPPS revealed that nearly all GFP-RyR2 clusters were co-localized with transverse but not longitudinal tubules, whereas staining with MitoTracker Red showed that GFP-RyR2 clusters were not co-localized with mitochondria in live ventricular myocytes. We also found GFP-RyR2 clusters interspersed between z-lines only at the periphery of live ventricular myocytes. Simultaneous detection of GFP-RyR2 clusters and Ca sparks showed that Ca sparks originated exclusively from RyR2 clusters. Ca sparks from RyR2 clusters induced no detectable changes in mitochondrial Ca level. These results reveal, for the first time, the distribution of RyR2 clusters and its functional correlation in living ventricular myocytes.
Ajuts:	Ministerio de Ciencia e Innovación SAF2011-30312
Drets:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.
Llengua:	Anglès
Document:	Article ; recerca ; Versió publicada
Matèria:	Animals ; Calcium ; Cardiac ryanodine receptor ; Confocal imaging ; Excitation - contraction couplings ; Functional correlation ; Green Fluorescent Proteins ; Heart Ventricles ; Immunofluorescence staining ; Mice ; Mice, Transgenic ; Myocytes, Cardiac ; Ryanodine receptors ; Ryanodine Receptor Calcium Release Channel ; Simultaneous detection ; Ventricular myocytes
Publicat a:	Journal of biological chemistry, Vol. 290 Núm. 33 (14 2015) , p. 20477-20487, ISSN 1083-351X

DOI: 10.1074/jbc.M115.650531
PMID: 26109063

11 p, 3.9 MB

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