Abstract
The cardiac Ca(2+) 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(2+) sparks showed that Ca(2+) sparks originated exclusively from RyR2 clusters. Ca(2+) sparks from RyR2 clusters induced no detectable changes in mitochondrial Ca(2+) level. These results reveal, for the first time, the distribution of RyR2 clusters and its functional correlation in living ventricular myocytes.
Highlights
Little is known about the distribution of cardiac ryanodine receptor (RyR2) and its functional correlation in living cells
Staining with di-8-ANEPPS revealed that most 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
Staining of Live Cardiomyocytes—Live ventricular myocytes were stained with 3 M di-8-ANEPPS (Invitrogen) or 2 M MitoTracker Red-FM (Invitrogen) in KRH buffer containing 0.5–1 mM Ca2ϩ for 5–20 min at room temperature
Summary
Little is known about the distribution of cardiac ryanodine receptor (RyR2) and its functional correlation in living cells. Results: Imaging live GFP-tagged RyR2 cardiomyocytes revealed Ca2ϩ sparks originated exclusively from RyR2 clusters distributed along z-lines and transverse tubules. AUGUST 14, 2015 VOLUME 290 NUMBER 33 detectable changes in mitochondrial Ca2؉ level These results reveal, for the first time, the distribution of RyR2 clusters and its functional correlation in living ventricular myocytes. A significant portion of Ca2ϩ sparks was detected in regions near the middle of mitochondria These indirect studies suggest that functional groups of RyR2s may exist between z-lines in the middle of sarcomere. Co-detection of GFP-RyR2 clusters and Ca2ϩ sparks revealed that Ca2ϩ sparks originate exclusively from RyR2 clusters These studies shed novel insights into the distribution of RyR2 clusters and its functional correlation in living cardiomyocytes
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
