Local Control Models of Cardiac Excitation–Contraction Coupling

Self Cite

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178

Intracellular Ca 2؉ release in many types of cells is mediated by ryanodine receptor Ca 2؉ release channels (RyRCs) that are assembled into two-dimensional paracrystalline arrays in the endoplasmic͞sarcoplasmic reticulum. However, the in situ operating mechanism of the RyRC array is unknown. Here T he ryanodine receptor Ca 2ϩ release channel (RyRC) is a prototypical member of the Ca 2ϩ release channel superfamily located in the endoplasmic reticulum͞sarcoplasmic reticulum (SR) of eukaryotic cells and plays a pivotal role in intracellular Ca 2ϩ signaling (1-5). Instances of local Ca 2ϩ release, in the form of ''Ca 2ϩ sparks'' or their equivalents, constitute the elementary Ca 2ϩ signaling events in heart, brain, and muscle cells (6-12). Intriguingly, RyRCs in intact cells are almost exclusively found at discrete spark-generating sites, where Ϸ100 channels are assembled into two-dimensional paracrystalline arrays (13)(14)(15). This pattern of RyRC organization seems to be highly conserved from crustaceans to vertebrates (13,15,16), suggesting that array formation is critical to RyRC-mediated Ca 2ϩ signaling in vivo. Thus, understanding array-based RyRC behavior is of fundamental importance for elucidation of intracellular Ca 2ϩ signaling mechanism.Despite a wealth of information on behavior of RyRCs in planar lipid bilayers and other cell-free systems (5,(17)(18)(19)(20), little is known on how RyRCs operate in situ; many fundamental issues regarding the genesis and termination of Ca 2ϩ sparks remain unanswered (5,21,22). Based on in vitro properties of RyRCs, the classic Ca 2ϩ -induced Ca 2ϩ release (CICR) mechanism (23) would predict an all-or-none activation of the RyRC array and an everlasting local Ca 2ϩ release, which cannot explain the prompt termination of Ca 2ϩ sparks and the microstability of intracellular signaling (24, 25). The relatively constant Ca 2ϩ spark rise time (Ϸ10 ms in the heart) indicates a stereotypical open time of RyRCs (25) whereas RyRCs in vitro always follow exponential open-time distributions (5,17,20). The brief spark duration also contrasts sharply with the coupled gating (19) kinetics of RyRCs, in which the open time of physically linked channels acting in unison is prolonged by orders of magnitude (up to 2,500 ms) (20). These paradoxical observations underscore that in vivo operation of RyRCs may involve gating mechanisms that are apparently absent in cell-free systems. Alternatively, the formation of RyRC array may endow the channels with new regulatory mechanisms that are unshared by a corresponding set of solitary RyRCs.In the present study, we sought to investigate RyRC operation in their native arrays. Because intracellular location of RyRC arrays makes them inaccessible to electrophysiological means, we devised an optical approach to analyze the Ca 2ϩ release flux underlying the spark (I spark ) in intact cardiac myocytes. By splitting I spark from individual RyRC arrays into single-channel components, we provided a view of the in situ gating of a single RyRC and demonstrat...

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

The quantal nature of Ca ²⁺ sparks and in situ operation of the ryanodine receptor array in cardiac cells

Wang

Stern

Rı́os

et al. 2004

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119

178

“…Therefore, even though the involvement of multiple channels is strongly supported here, their structural location remains to be established. Because Ca 2ϩ release during sparks starts and ends abruptly (23), an allosterically connected cluster (17,44) may be at work. These and other issues must be clarified to base a cellwide picture of Ca 2ϩ release on the properties of its channels and their sensors.…”

Section: Discussionmentioning

confidence: 99%

Involvement of multiple intracellular release channels in calcium sparks of skeletal muscle

Gonzalez

Kirsch

Shirokova

et al. 2000

110

100

In many types of muscle, intracellular Ca 2؉ release for contraction consists of brief Ca 2؉ sparks. Whether these result from the opening of one or many channels in the sarcoplasmic reticulum is not known. Examining massive numbers of sparks from frog skeletal muscle and evaluating their Ca 2؉ release current, we provide evidence that they are generated by multiple channels. A mode is demonstrated in the distribution of spark rise times in the presence of the channel activator caffeine. This finding contradicts expectations for single channels evolving reversibly, but not for channels in a group, which collectively could give rise to a stereotyped spark. The release channel agonists imperatoxin A, ryanodine, and bastadin 10 elicit fluorescence events that start with a spark, then decay to steady levels roughly proportional to the unitary conductances of 35%, 50%, and 100% that the agonists, respectively, promote in bilayer experiments. This correspondence indicates that the steady phase is produced by one open channel. Calculated Ca 2؉ release current decays 10-to 20-fold from spark to steady phase, which requires that six or more channels be open during the spark. (4, 5), and smooth muscle (6). Sparks are fundamental in health and disease (7), but their mechanism, especially whether one or many channels are involved in the spark generator or ''release unit,'' remains unclear (8-10). Answering this question, which pervades the field since its inception (2), will help understand how the channels are coaxed by their agonists (Ca, membrane voltage; reviewed in refs. 11 and 12) and restrained by their antagonists (Ca, Mg) to shape these events.Here we improve a technique for detection of massive numbers of sparks (13) Materials and MethodsExperiments were carried out at 17°C in cut skeletal muscle fibers from Rana pipiens semitendinosus muscle, stretched at 3-3.5 m͞sarcomere, either voltage-clamped in a two-Vaseline gap chamber or permeabilized and immersed in internal solution, on an inverted microscope. Adult frogs anaesthetized in 15% ethanol were killed by pithing. The external solution contained 10 mM Ca(CH 3 SO 3 ) 2 , 130 mM tetraethylammonium-CH 3 SO 3 , 5 mM Tris maleate, 1 mM 3,4 diaminopyridine, and 1 M tetrodotoxin. The internal solution contained 110 mM Cs-glutamate, 1 mM EGTA, 5 mM glucose, 5 mM Mg-ATP, 5 mM phosphocreatine, 10 mM Hepes, 0.2 mM fluo-3, with 100 nM free [Ca 2ϩ ] and 1.8 mM [Mg 2ϩ ]. For permeabilized cells the internal solution contained 0.05 mM fluo-3 and 0.34 mM [Mg 2ϩ ] and included 4% 10-kDa dextran. Solutions were adjusted to pH 7 and 270 mosmol͞kg. The scanning microscope (MRC 1000, Bio-Rad) was in fluo-3 configuration (14) using a 40ϫ, 1.2 numerical aperture water immersion objective (Zeiss). Images shown are of fluorescence determined at 2-ms intervals (4.3 ms in toxin experiments) along a parallel to the fiber axis. Fluorescence F(x,t) is presented normalized to its average F 0 (x) before the voltage pulse. Sparks are located on a spatially filtered version of...

“…They may be functionally distinct from those created by long-term pathology. Because tight coupling between most RyR clusters and LCCs is essential to maintain local control of CICR (48,49) TT Imaging and Analysis. Myocytes were loaded with the lipophilic membrane marker Di-8-ANEPPS (10 M in Tyrode solution for 10 min).…”

Section: Tt Remodeling Of Fetal Phenotype: a Unique Finding From Shr mentioning

confidence: 99%

Orphaned ryanodine receptors in the failing heart

Song

Sobie²,

McCulle³

et al. 2006

Self Cite

406

469

Heart muscle is characterized by a regular array of proteins and structures that form a repeating functional unit identified as the sarcomere. This regular structure enables tight coupling between electrical activity and Ca 2؉ signaling. In heart failure, multiple cellular defects develop, including reduced contractility, altered Ca 2؉ signaling, and arrhythmias; however, the underlying causes of these defects are not well understood. Here, in ventricular myocytes from spontaneously hypertensive rats that develop heart failure, we identify fundamental changes in Ca 2؉ signaling that are related to restructuring of the spatial organization of the cells. Myocytes display both a reduced ability to trigger sarcoplasmic reticulum Ca 2؉ release and increased spatial dispersion of the transverse tubules (TTs). Remodeled TTs in cells from failing hearts no longer exist in the regularly organized structures found in normal heart cells, instead moving within the sarcomere away from the Z-line structures and leaving behind the sarcoplasmic reticulum Ca 2؉ release channels, the ryanodine receptors (RyRs). These orphaned RyRs appear to be responsible for the dyssynchronous Ca 2؉ sparks that have been linked to blunted contractility and, probably, Ca 2؉ -dependent arrhythmias in diverse models of heart failure. We conclude that the increased spatial dispersion of the TTs and orphaned RyRs lead to the loss of local control and Ca 2؉ instability in heart failure.calcium signaling ͉ local control ͉ dyssynchrony ͉ heart failure ͉ transverse tubules C a 2ϩ sparks, the elementary Ca 2ϩ release events during the excitation-contraction (EC) coupling process in heart (1-5), are triggered by the opening of L-type Ca 2ϩ channels (LCCs) and sum to produce the [Ca 2ϩ ] i transient (6). Each Ca 2ϩ spark arises from a nanometer-sized structural and functional unit or ''couplon,'' which includes both (i) LCCs in the sarcolemma or transverse tubules (TTs), and (ii) a cluster of ryanodine receptors (RyRs) that reside in the sarcoplasmic reticulum (SR) membrane and face the LCCs across a 15-nm gap (7-9). During the cardiac action potential (AP), the nearly simultaneous opening of LCCs synchronizes the triggering of Ca 2ϩ sparks from the RyR clusters (also referred to as Ca 2ϩ release units, or CRUs). Ca 2ϩ signaling in heart failure is characterized by contractile dysfunction (10, 11) (24). In tachycardia-induced HF, Kamp and colleagues (25, 26) have described a regional loss of TTs within ventricular myocytes, but other evidence suggests that TTs remain normal. † † Additionally, many other factors may contribute to arrhythmogenesis, including reduced K ϩ channel expression, increased Na ϩ ͞Ca 2ϩ exchanger activity (21, 27), and altered Ca 2ϩ channel gating (28); therefore, possible links between structural changes and dysfunctional Ca 2ϩ signaling in this HF model remain unclear. In a cell culture investigation using pig ventricular myocytes, dyssynchronous Ca 2ϩ release was observed as the cells in culture dedifferentiated and lost TTs (18)...