Identification of a Ryanodine Receptor in Rat Heart Mitochondria

Beutner, Gisela; Sharma, Virendra K.; Giovannucci, David R.; Yule, David I.; Sheu, Shey‐Shing

doi:10.1074/jbc.m101486200

Cited by 227 publications

(191 citation statements)

References 48 publications

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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: 99%

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

135

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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Section: Discussionmentioning

confidence: 99%

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

135

112

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“…However, in cardiac mitochondria, the RaM required > 30 s to become fully reactivated at extramitochondrial [Ca 2+ ] < 100 nmol/L [30] and thus, cannot be responsible for beat-to-beat mitochondrial Ca 2+ uptake. Furthermore, Beutner et al [18,19] have proposed that mitochondrial RyR (RyR1 subtype) channels may mediate Ca 2+ flux across the inner mitochondrial membrane. The binding of [ 3 H]ryanodine to this mRyR1 has a bellshaped Ca 2+ -dependence, with an optimum between 10-30 µM [19], the latter value comparable to the reported K 0.5 of the mCU for Ca 2+ [54,77].…”

Section: Molecular Identity Of the Mcumentioning

confidence: 99%

Excitation-contraction coupling and mitochondrial energetics

Maack

O’Rourke²

2007

Basic Res Cardiol

221

183

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Cardiac excitation-contraction (EC) coupling consumes vast amounts of cellular energy, most of which is produced in mitochondria by oxidative phosphorylation. In order to adapt the constantly varying workload of the heart to energy supply, tight coupling mechanisms are essential to maintain cellular pools of ATP, phosphocreatine and NADH. To our current knowledge, the most important regulators of oxidative phosphorylation are ADP, P i , and Ca 2+ . However, the kinetics of mitochondrial Ca 2+ -uptake during EC coupling are currently a matter of intense debate. Recent experimental findings suggest the existence of a mitochondrial Ca 2+ microdomain in cardiac myocytes, justified by the close proximity of mitochondria to the sites of cellular Ca 2+ release, i. e., the ryanodine receptors of the sarcoplasmic reticulum. Such a Ca 2+ microdomain could explain seemingly controversial results on mitochondrial Ca 2+ uptake kinetics in isolated mitochondria versus whole cardiac myocytes. Another important consideration is that rapid mitochondrial Ca 2+ uptake facilitated by microdomains may shape cytosolic Ca 2+ signals in cardiac myocytes and have an impact on energy supply and demand matching. Defects in EC coupling in chronic heart failure may adversely affect mitochondrial Ca 2+ uptake and energetics, initiating a vicious cycle of contractile dysfunction and energy depletion. Future therapeutic approaches in the treatment of heart failure could be aimed at interrupting this vicious cycle. Keywords calcium; sodium; microdomain; heart failure; adenosine triphosphate; adenosine diphosphate; respiration; tricarboxylic acid cycle Physiological aspectsThe most important function of the heart is to pump blood to supply the body with oxygen and substrates. In order to adapt cardiac output to the constantly changing demand of blood supply, the heart utilizes three major mechanisms to increase cardiac output: the force-frequency relationship (the Bowditch effect or Treppe; [22]), the Frank-Starling mechanism (sarcomere length-dependent activation of contractile force) [66,149,164], and sympathetic activation [28]. All of these mechanisms increase and accelerate myocardial force generation, and at the same time increase cellular energy demand. By these mechanisms, cardiac output during exertion can be increased more than five-fold compared to resting conditions. © Steinkopff Verlag 2007Correspondence to: Christoph Maack. NIH Public Access Excitation-contraction couplingOf fundamental importance for cardiac contraction and relaxation are the processes of excitation-contraction (EC) coupling (Fig. 1). During the action potential (AP), voltage-gated Na + -channels are activated, and the inward Na + -current (I Na ) induces a rapid depolarization of the cell membrane, facilitating voltage-dependent opening of L-type Ca 2+ -channels (I Ca,L ). The Ca 2+ influx triggers the opening of the ryanodine receptor (RyR2 subtype), inducing the release of even greater amounts of Ca 2+ from the sarcoplasmic reticulum (SR), a process te...

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“…After a few tentative identifications of putative RuR-binding glycoproteins in the 1970s (73), in a series of papers, Sheu and co-workers (74,75) proposed that mitochondrially sorted RyR1 drives mitochondrial Ca 2ϩ uptake in the heart. Although the tissue distribution and electrophysiological properties seem to exclude RyR1 as the MCU, the possibility that RyR is an additional uptake pathway cooperating with MCU in the heart is still open.…”

Section: The Discovery Of the Mcu: Finding The Needle In The Mitochonmentioning

confidence: 99%

The Mitochondrial Calcium Uniporter (MCU): Molecular Identity and Physiological Roles

Patrón

Raffaello

Granatiero

et al. 2013

Journal of Biological Chemistry

152

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The direct measurement of mitochondrial [Ca 2؉ ] with highly specific probes demonstrated that major swings in organellar [Ca 2؉ ] parallel the changes occurring in the cytosol and regulate processes as diverse as aerobic metabolism and cell death by necrosis and apoptosis. Despite great biological relevance, insight was limited by the complete lack of molecular understanding. The situation has changed, and new perspectives have emerged following the very recent identification of the mitochondrial Ca 2؉ uniporter, the channel allowing rapid Ca 2؉ accumulation across the inner mitochondrial membrane.

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Identification of a Ryanodine Receptor in Rat Heart Mitochondria

Cited by 227 publications

References 48 publications

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

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

Excitation-contraction coupling and mitochondrial energetics

The Mitochondrial Calcium Uniporter (MCU): Molecular Identity and Physiological Roles

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