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Analysis of ryanodine receptor clusters in rat and human cardiac myocytes.

ABSTRACT: Single rat ventricular myocytes and human ventricle tissue sections were labeled with antibodies against the ryanodine receptor (RyR) and alpha-actinin to examine the 3D distribution of RyRs with confocal microscopy. Image contrast was maximized by refractive index matching and deconvolution. The RyR label formed discrete puncta representing clusters of RyRs or "couplons" around the edges of the myofilaments with a nearest-neighbor spacing of 0.66 +/- 0.06 microm in rat and 0.78 +/- 0.07 microm in human. Each bundle of myofibrils was served by approximately six couplons, which supplied a cross-sectional area of approximately 0.6 microm(2) in rat and approximately 0.8 microm(2) in human. Although the couplons were in reasonable registration with z-lines, there were discontinuities in the longitudinal position of sarcomeres so that dislocations in the order of RyR clusters occurred. There was approximately 53% longitudinal registration of RyR clusters, suggesting a nonrandom placement of couplons around the sarcomere. These data can explain the spherical propagation of Ca(2+) waves and provide quantitative 3D data sets needed for accurate modeling of cardiac Ca(2+)-induced Ca(2+) release. By quantifying labeling intensity in rat ventricular myocytes, a lower limit of 78 RyRs per cluster (on average) was obtained. By modeling the couplon as a disk wrapping around a t-tubule and fitting cluster images, 95% of couplons contained between 120 and 260 RyRs (assuming that RyRs are tight packed with a spacing of 29 nm). Assuming similar labeling efficiency in human, from the fluorescence intensity alone we estimate that human ventricular myocytes contain approximately 30% fewer RyRs per couplon than rat.

SUBMITTER: Soeller C

PROVIDER: S-EPMC1986595 | biostudies-literature | 2007 Sep

REPOSITORIES: biostudies-literature

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Analysis of ryanodine receptor clusters in rat and human cardiac myocytes.

Soeller Christian C Crossman David D Gilbert Ray R Cannell Mark B MB

Proceedings of the National Academy of Sciences of the United States of America 20070911 38

Single rat ventricular myocytes and human ventricle tissue sections were labeled with antibodies against the ryanodine receptor (RyR) and alpha-actinin to examine the 3D distribution of RyRs with confocal microscopy. Image contrast was maximized by refractive index matching and deconvolution. The RyR label formed discrete puncta representing clusters of RyRs or "couplons" around the edges of the myofilaments with a nearest-neighbor spacing of 0.66 +/- 0.06 microm in rat and 0.78 +/- 0.07 microm ...[more]

PMID: 17848521

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Project description:Key pointsUsing 3D direct stochastic optical reconstruction microscopy (dSTORM), we developed novel approaches to quantitatively describe the nanoscale, 3D organization of ryanodine receptors (RyRs) in cardiomyocytes. Complex arrangements of RyR clusters were observed in 3D space, both at the cell surface and within the cell interior, with allocation to dyadic and non-dyadic pools. 3D imaging importantly allowed discernment of clusters overlapping in the z-axis, for which detection was obscured by conventional 2D imaging techniques. Thus, RyR clusters were found to be significantly smaller than previous 2D estimates. Ca2+ release units (CRUs), i.e. functional groupings of neighbouring RyR clusters, were similarly observed to be smaller than earlier reports. Internal CRUs contained more RyRs in more clusters than CRUs on the cell surface, and yielded longer duration Ca2+ sparks.AbstractCardiomyocyte contraction is dependent on Ca2+ release from ryanodine receptors (RyRs). However, the precise localization of RyRs remains unknown, due to shortcomings of imaging techniques which are diffraction limited or restricted to 2D. We aimed to determine the 3D nanoscale organization of RyRs in rat cardiomyocytes by employing direct stochastic optical reconstruction microscopy (dSTORM) with phase ramp technology. Initial observations at the cell surface showed an undulating organization of RyR clusters, resulting in their frequent overlap in the z-axis and obscured detection by 2D techniques. Non-overlapping clusters were imaged to create a calibration curve for estimating RyR number based on recorded fluorescence blinks. Employing this method at the cell surface and interior revealed smaller RyR clusters than 2D estimates, as erroneous merging of axially aligned RyRs was circumvented. Functional groupings of RyR clusters (Ca2+ release units, CRUs), contained an average of 18 and 23 RyRs at the surface and interior, respectively, although half of all CRUs contained only a single 'rogue' RyR. Internal CRUs were more tightly packed along z-lines than surface CRUs, contained larger and more numerous RyR clusters, and constituted ∼75% of the roughly 1 million RyRs present in an average cardiomyocyte. This complex internal 3D geometry was underscored by correlative imaging of RyRs and t-tubules, which enabled quantification of dyadic and non-dyadic RyR populations. Mirroring differences in CRU size and complexity, Ca2+ sparks originating from internal CRUs were of longer duration than those at the surface. These data provide novel, nanoscale insight into RyR organization and function across cardiomyocytes.

Project description:Ryanodine receptors (RyRs) regulate contractility in resistance-size cerebral artery smooth muscle, yet their molecular identity, subcellular location, and phenotype in this tissue remain unknown. Following rat resistance-size cerebral artery myocyte sarcoplasmic reticulum (SR) purification and incorporation into POPE-POPS-POPC (5:3:2; wt/wt) bilayers, unitary conductances of 110 +/- 8, 334 +/- 15, and 441 +/- 27 pS in symmetric 300 mM Cs(+) were usually detected. The most frequent (34/40 bilayers) conductance (334 pS) decreased to <or=100 pS when Cs(+) was replaced with Ca(2+). The predominant conductance displayed 66 bursts/min with at least three open and three closed states. The steady-state activity (NP(o))-voltage curve was bell shaped, with NP(o) drastically decreasing when voltage was switched from -30 to -40 mV. NP(o) increased when intracellular calcium (Ca(2+)(i)) was raised within 0.1-100 microM to abruptly diminish with higher Ca(2+)(i). Thus maximal activity occurred within the Ca(2+)(i) range found in rat cerebral artery myocytes under physiological conditions. NP(o) was reduced by ruthenium red (80 muM), increased monotonically by caffeine (0.1-5 mM) or ryanodine (0.05-5 microM), and unaffected by heparin (2 mg/ml). This phenotype resembles that of cardiac RyR and recombinant RyR2. RT-PCR detected RyR1, RyR2, and RyR3 transcripts in cerebral artery myocytes. However, real-time PCR indicated that RyR2 was 4 and 1.5 times more abundant than RyR1 and RyR3, respectively. Consistently, Western blotting showed that the RyR2 product was very abundant. Immunofluorescence showed that each RyR isoform distributed differentially among subcellular compartments. In particular, RyR2 was drastically stronger in the subplasmalemma than in other compartments, underscoring the predominance of RyR2 in a compartment where SR is abundant. Consistently, RyR from SR-enriched membranes displayed pharmacological specificity typical of RyR2, being activated by digoxin (1 muM), resistant to dantrolene (100 muM), and shifted to a subconductance by neomycin (100 nM). Therefore, RyR2 is the predominant molecular and functional RyR that is expressed in the SR membrane of rat resistance-size cerebral artery myocytes.

Dataset Information

Analysis of ryanodine receptor clusters in rat and human cardiac myocytes.

Publications

Analysis of ryanodine receptor clusters in rat and human cardiac myocytes.

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