The mitochondrial ryanodine receptor in rat heart: A pharmaco-kinetic profile

Altschafl, Beth A.; Beutner, Gisela; Sharma, Virendra K.; Sheu, Shey Shing; Valdivia, Héctor H.

doi:10.1016/j.bbamem.2007.04.011

Cited by 64 publications

(66 citation statements)

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“…Interestingly, our single channel studies identified at least four conductance levels for the mRyR, similar to those previously shown for the SR ryanodine receptor (44). We previously reported the molecular mass of mRyR under denaturing conditions to be ϳ500 -600 kDa, a size similar to that of each subunit in the tetrameric structure of ryanodine receptor in the SR (16,18). Thus, our results are consistent with mRyR being a homotetramer in which conformational changes in each subunit contribute equally to channel gating and conductance.…”

Section: Discussionsupporting

confidence: 88%

“…Ryanodine receptors purified from the SR membrane of skeletal and cardiac muscle exhibit large single channel conductances of 400 -750 pS in 250 mM K ϩ or Cs ϩ (30,41). Previously, we reported that ryanodine-sensitive activities purified from rat heart mitochondria and reconstituted into lipid bilayers exhibited single channel conductances of ϳ500 -800 pS with symmetrical 300 mM cesium methanesulfonate (18). Considering the reduced ionic strength in the present patch clamp experiments, we expected conductances of ϳ250 -400 pS but saw 225 pS of activity in symmetrical 150 mM CsCl conditions.…”

Section: Discussionmentioning

confidence: 99%

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Single Channel Characterization of the Mitochondrial Ryanodine Receptor in Heart Mitoplasts

Ryu

Beutner

Kinnally

et al. 2011

Journal of Biological Chemistry

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Heart mitochondria utilize multiple Ca 2؉ transport mechanisms. Among them, the mitochondrial ryanodine receptor provides a fast Ca 2؉ uptake pathway across the inner membrane to control "excitation and metabolism coupling." In the present study, we identified a novel ryanodine-sensitive channel in the native inner membrane of heart mitochondria and characterized its pharmacological and biophysical properties by directly patch clamping mitoplasts. Four distinct channel conductances of ϳ100, ϳ225, ϳ700, and ϳ1,000 picosiemens (pS) in symmetrical 150 mM CsCl were observed. The 225 pS cation-selective channel exhibited multiple subconductance states and was blocked by high concentrations of ryanodine and ruthenium red, known inhibitors of ryanodine receptors. Ryanodine exhibited a concentration-dependent modulation of this channel, with low concentrations stabilizing a subconductance state and high concentrations abolishing activity. The 100, 700, and 1,000 pS conductances exhibited different channel characteristics and were not inhibited by ryanodine. Taken together, these findings identified a novel 225 pS channel as the native mitochondrial ryanodine receptor channel activity in heart mitoplasts with biophysical and pharmacological properties that distinguish it from previously identified mitochondrial ion channels. Mitochondrial Ca2ϩ transport plays critical roles in the regulation of mitochondrial function under both physiological and pathological conditions (1-3). An increase of matrix Ca 2ϩ within the physiological range facilitates ATP production (4) by stimulating the activity of Ca 2ϩ -dependent dehydrogenases (5, 6) in the tricarboxylic acid cycle and the ATP synthase (7). Dysregulation of mitochondrial Ca 2ϩ handling and subsequent alterations in bioenergetics contribute to contractile dysfunction in heart failure (8, 9). Moreover, mitochondrial Ca 2ϩ overload during ischemia/reperfusion induces opening of the mitochondrial permeability transition pore (mPTP), 2 mitochondrial swelling, and an increase of reactive oxygen species generation, all of which lead to cardiac cell death (10, 11). Several Ca 2ϩ transport pathways have been identified. The mitochondrial Ca 2ϩ uniporter (MCU) is a large capacity, low affinity system that provides a slow Ca 2ϩ uptake mechanism (12). A highly Ca 2ϩ selective, inwardly rectifying conductance with biophysical properties similar to those expected of the MCU was characterized in elegant patch clamp studies of mitoplasts isolated from Cos-7 cells (13) and human cardiomyocytes (14). Recently, MICU1, a gene encoding an EF hand protein that regulates MCU, was identified (15). However, heart mitochondria are also known to exhibit rapid Ca 2ϩ uptake and efflux pathways that possess Ca 2ϩ sensitivity and pharmacology different from those of the MCU (3).We previously reported that a ryanodine-sensitive, fast Ca 2ϩ uptake pathway co-exists with MCU in heart mitochondria (16, 17). High affinity binding of [ 3 H]ryanodine, immunogold staining, and Western blot analysis revealed th...

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Section: Discussionsupporting

confidence: 88%

Section: Discussionmentioning

confidence: 99%

Single Channel Characterization of the Mitochondrial Ryanodine Receptor in Heart Mitoplasts

Ryu

Beutner

Kinnally

et al. 2011

Journal of Biological Chemistry

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“…64, 71, and 72). We first found that a low level of RyR1 is expressed at the mitochondrial inner membrane in cardiomyocytes, as shown by a combination of biochemical and cell biological experiments, and finally confirmed its role in cardiomyocytes as a fast Ca 2ϩ uptake pathway by electrophysiological experiments (1,7,8,72). Since mRyR1 shows a bellshaped Ca 2ϩ -dependent activation (bimodal activation) in a physiological range of [Ca 2ϩ ] c , this unique property places mRyR1 as an ideal candidate for sequestering Ca 2ϩ quickly and transiently under physiological [Ca 2ϩ ] c oscillation, such as during heart beats.…”

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confidence: 58%

Overexpression of ryanodine receptor type 1 enhances mitochondrial fragmentation and Ca²⁺-induced ATP production in cardiac H9c2 myoblasts

O‐Uchi

Jhun

Hurst

et al. 2013

American Journal of Physiology-Heart and Circulatory Physiology

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ϩ influx to mitochondria is an important trigger for both mitochondrial dynamics and ATP generation in various cell types, including cardiac cells. Mitochondrial Ca 2ϩ influx is mainly mediated by the mitochondrial Ca 2ϩ uniporter (MCU). Growing evidence also indicates that mitochondrial Ca 2ϩ influx mechanisms are regulated not solely by MCU but also by multiple channels/transporters. We have previously reported that skeletal muscle-type ryanodine receptor (RyR) type 1 (RyR1), which expressed at the mitochondrial inner membrane, serves as an additional Ca 2ϩ uptake pathway in cardiomyocytes. However, it is still unclear which mitochondrial Ca 2ϩ influx mechanism is the dominant regulator of mitochondrial morphology/dynamics and energetics in cardiomyocytes. To investigate the role of mitochondrial RyR1 in the regulation of mitochondrial morphology/function in cardiac cells, RyR1 was transiently or stably overexpressed in cardiac H9c2 myoblasts. We found that overexpressed RyR1 was partially localized in mitochondria as observed using both immunoblots of mitochondrial fractionation and confocal microscopy, whereas RyR2, the main RyR isoform in the cardiac sarcoplasmic reticulum, did not show any expression at mitochondria. Interestingly, overexpression of RyR1 but not MCU or RyR2 resulted in mitochondrial fragmentation. These fragmented mitochondria showed bigger and sustained mitochondrial Ca 2ϩ transients compared with basal tubular mitochondria. In addition, RyR1-overexpressing cells had a higher mitochondrial ATP concentration under basal conditions and showed more ATP production in response to cytosolic Ca 2ϩ elevation compared with nontransfected cells as observed by a matrix-targeted ATP biosensor. These results indicate that RyR1 possesses a mitochondrial targeting/retention signal and modulates mitochondrial morphology and Ca 2ϩ -induced ATP production in cardiac H9c2 myoblasts. fluorescence resonance energy transfer; mitochondrial Ca 2ϩ uniporter; mitochondria; mitochondrial morphology; ryanodine receptor type 1 MITOCHONDRIAL Ca 2ϩ is critical for the regulation of various cellular functions, including energy metabolism, ROS generation, spatiotemporal dynamics of Ca 2ϩ signaling, and cell growth/development and death (11,18,23). Historically, Ca 2ϩ was found to be accumulated by mitochondria over 5 decades ago (for reviews, see Refs. 24, 64, and 69), and, shortly thereafter, it was also recognized that Ca 2ϩ stimulates the oxidative phosphorylation [tricarboxylic acid (TCA) cycle] and electron transport chain activity, which results in the stimulation of ATP synthesis (for a review, see Ref. 23). Additionally, the coexistence of mitochondrial dysfunction and loss of cellular Ca 2ϩ homeostasis are frequently observed in various cardiovascular diseases, but it is still not clear how altered mitochondrial Ca 2ϩ handing and/or mitochondrial dysfunction are involved in the pathogenesis of each different disease setting (23,33,56).Although the basic functional and pharmacological properties of mitochondrial ...

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“…Although the majority of the RyR2 does not seem to directly face the mitochondria in the intact cardiac muscle, the RyR2s are sufficiently close (27-200 nm (9)) to the mitochondrial surface to expose the Ca 2ϩ uptake sites to an estimated 10 -20 M [Ca 2ϩ ] c during Ca 2ϩ release (36). Regarding the molecular mechanism of the Ca 2ϩ uptake by cardiac mitochondria, an interesting candidate is the mitochondrial mRyR1, recently described by Beutner, Sheu, and co-workers (22,37,38). The ruthenium derivative Ru360 has been reported highly specific to the MCU with no inhibitory effect on RyR2 in heart (21) or on RyR1 in skeletal muscle (39) (as opposed to ruthenium red that inhibits both MCU and RyR).…”

Section: Discussionmentioning

confidence: 98%

Physical Coupling Supports the Local Ca2+ Transfer between Sarcoplasmic Reticulum Subdomains and the Mitochondria in Heart Muscle

Garcı́a-Pérez

Hajnóczky

Csordás

2008

Journal of Biological Chemistry

133

112

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In many cell types, transfer of Ca 2؉ released via ryanodine receptors (RyR) to the mitochondrial matrix is locally supported by high [Ca 2؉ ] microdomains at close contacts between the sarcoplasmic reticulum (SR) and mitochondria. Here we studied whether the close contacts were secured via direct physical coupling in cardiac muscle using isolated rat heart mitochondria (RHMs). "Immuno-organelle chemistry" revealed RyR2 and calsequestrin-positive SR particles associated with mitochondria in both crude and Percoll-purified "heavy" mitochondrial fractions (cRHM and pRHM), to a smaller extent in the latter one. Mitochondria-associated vesicles were also visualized by electron microscopy in the RHMs. Western blot analysis detected greatly reduced presence of SR markers (calsequestrin, SERCA2a, and phospholamban) in pRHM, suggesting that the mitochondria-associated particles represented a small subfraction of the SR. signal propagation from the ryanodine receptors (RyR, the phylogenetic ancestors of the inositol 1,4,5-trisphosphate receptor) to the mitochondria in cardiac muscle cells (9, 10) and in skeletal muscle (11-13). However, whether the local Ca 2ϩ communication between the SR and mitochondria is supported by physical coupling is yet to be elucidated. Very recently, Protasi and co-workers (14, 15) have visualized tethering structures between SR and mitochondria in electron micrographs and tomographs of skeletal muscle, although those data did not examine the involvement of the tethers in the Ca 2ϩ communication between the organelles. A local metabolic triangle between the sarcomere, SR, and mitochondria that could be dissociated by limited proteolysis in permeabilized cardiomyocytes has also been described earlier (16). Here, we studied the preservation of the SR-mitochondrial associations and local Ca 2ϩ coupling between RyR and mitochondria by visualization of individual organelles and monitoring their function in mitochondria isolated from rat heart homogenates. MATERIALS AND METHODS Chemicals/ImmunochemicalsFluorescent probes and fluorescently labeled secondary antibodies were from Molecular Probes (Eugene, OR), except the Ca 2ϩ probes fura2 (K ϩ salt) and rhod2 acetoxymethylester (rhod2/AM), which were purchased from Teflabs (Austin, TX). Primary antibodies were obtained as follows: mouse monoclonal: anti-RyR2 from ABR (MA3-916), anti-phospholamban from Abcam (ab2865), and anti-cytochrome oxidase subunit 1 from Molecular Probes (A6403); rabbit polyclonal: anti-RyR2 * This work was supported by an American Heart Association Grant SDG 0435236N. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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The mitochondrial ryanodine receptor in rat heart: A pharmaco-kinetic profile

Cited by 64 publications

References 50 publications

Single Channel Characterization of the Mitochondrial Ryanodine Receptor in Heart Mitoplasts

Single Channel Characterization of the Mitochondrial Ryanodine Receptor in Heart Mitoplasts

Overexpression of ryanodine receptor type 1 enhances mitochondrial fragmentation and Ca²⁺-induced ATP production in cardiac H9c2 myoblasts

Physical Coupling Supports the Local Ca2+ Transfer between Sarcoplasmic Reticulum Subdomains and the Mitochondria in Heart Muscle

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The mitochondrial ryanodine receptor in rat heart: A pharmaco-kinetic profile

Cited by 64 publications

References 50 publications

Single Channel Characterization of the Mitochondrial Ryanodine Receptor in Heart Mitoplasts

Single Channel Characterization of the Mitochondrial Ryanodine Receptor in Heart Mitoplasts

Overexpression of ryanodine receptor type 1 enhances mitochondrial fragmentation and Ca2+-induced ATP production in cardiac H9c2 myoblasts

Physical Coupling Supports the Local Ca2+ Transfer between Sarcoplasmic Reticulum Subdomains and the Mitochondria in Heart Muscle

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Overexpression of ryanodine receptor type 1 enhances mitochondrial fragmentation and Ca²⁺-induced ATP production in cardiac H9c2 myoblasts