Sarcoplasmic Reticulum Network Research Articles

Dietary Magnesium Deficiency in Rats Enhances Free Radical Production in Skeletal Muscle

Recent studies suggest that free radicals may be involved in tissue injuries induced by magnesium deficiency. The aim of the present study was to assess the effect of magnesium deficiency on free radical production of skeletal muscle tissue. Male Wistar rats were pair-fed from weaning for 12 d either control or Mg-deficient diets containing 960 or 40 mg magnesium/kg diet, respectively. In the Mg-deficient rats, hypomagnesemia was accompanied by significantly lower magnesium and greater calcium concentrations in skeletal muscle tissue. Electron microscopy of skeletal muscle tissue revealed ultrastructural changes, including swelling mitochondria and disorganization of the sar-coplasmic reticulum network. Using the spin-trapping technique, we showed that significantly more hydroxyl radicals were generated in muscle homogenates of Mg-deficient rats. Moreover, the amount of spin trap adducts was increased in the presence of exogenous iron in both groups. In agreement with these observations, a greater concentration of thiobarbituric acid-reactive substances and a lower concentration of thiol groups were found in skeletal muscle of the Mg-deficient group compared with controls. These results strongly support the hypothesis that free radical-mediated injury could contribute to skeletal muscle lesions resulting from magnesium deficiency.

Exploring Impaired SERCA Pump-Caused Alternation Occurrence in Ischemia.

Impaired sarcoplasmic reticulum (SR) calcium transport ATPase (SERCA) gives rise to Ca2+ alternans and changes of the Ca2+release amount. These changes in Ca2+ release amount can reveal the mechanism underlying how the interaction between Ca2+ release and Ca2+ uptake induces Ca2+ alternans. This study of alternans by calculating the values of Ca2+ release properties with impaired SERCA has not been explored before. Here, we induced Ca2+ alternans by using an impaired SERCA pump under ischemic conditions. The results showed that the recruitment and refractoriness of the Ca2+ release increased as Ca2+ alternans occurred. This indicates triggering Ca waves. As the propagation of Ca waves is linked to the occurrence of Ca2+ alternans, the “threshold” for Ca waves reflects the key factor in Ca2+ alternans development, and it is still controversial nowadays. We proposed the ratio between the diastolic network SR (NSR) Ca content (Cansr) and the cytoplasmic Ca content (Cai) (Cansr/Cai) as the “threshold” of Ca waves and Ca2+ alternans. Diastolic Cansr, Cai, and their ratio were recorded at the onset of Ca2+ alternans. Compared with certain Cansr and Cai, the “threshold” of the ratio can better explain the comprehensive effects of the Ca2+ release and the Ca2+ uptake on Ca2+ alternans onset. In addition, these ratios are related with the function of SERCA pumps, which vary with different ischemic conditions. Thus, values of these ratios could be used to differentiate Ca2+ alternans from different ischemic cases. This agrees with some experimental results. Therefore, the certain value of diastolic Cansr/Cai can be the better “threshold” for Ca waves and Ca2+ alternans.

Small Ca2+ releases enable hour-long high-frequency contractions in midshipman swimbladder muscle.

Type I males of the Pacific midshipman fish (Porichthys notatus) vibrate their swimbladder to generate mating calls, or "hums," that attract females to their nests. In contrast to the intermittent calls produced by male Atlantic toadfish (Opsanus tau), which occur with a duty cycle (calling time divided by total time) of only 3-8%, midshipman can call continuously for up to an hour. With 100% duty cycles and frequencies of 50-100 Hz (15°C), the superfast muscle fibers that surround the midshipman swimbladder may contract and relax as many as 360,000 times in 1 h. The energy for this activity is supported by a large volume of densely packed mitochondria that are found in the peripheral and central regions of the fiber. The remaining fiber cross section contains contractile filaments and a well-developed network of sarcoplasmic reticulum (SR) and triadic junctions. Here, to understand quantitatively how Ca2+ is managed by midshipman fibers during calling, we measure (a) the Ca2+ pumping-versus-pCa and force-versus-pCa relations in skinned fiber bundles and (b) changes in myoplasmic free [Ca2+] (Δ[Ca2+]) during stimulated activity of individual fibers microinjected with the Ca2+ indicators Mag-fluo-4 and Fluo-4. As in toadfish, the force-pCa relation in midshipman is strongly right-shifted relative to the Ca2+ pumping-pCa relation, and contractile activity is controlled in a synchronous, not asynchronous, fashion during electrical stimulation. SR Ca2+ release per action potential is, however, approximately eightfold smaller in midshipman than in toadfish. Midshipman fibers have a larger time-averaged free [Ca2+] during activity than toadfish fibers, which permits faster Ca2+ pumping because the Ca2+ pumps work closer to their maximum rate. Even with midshipman's sustained release and pumping of Ca2+, however, the Ca2+ energy cost of calling (per kilogram wet weight) is less than twofold more in midshipman than in toadfish.

Simulations of Subcellular Heterogeneity in Calcium Blink Kinetics

In cardiac myocytes, calcium (Ca2+) release from the sarcoplasmic reticulum (SR) depends on local calcium concentrations in both the cytosol and the SR. The local dynamics of SR Ca2+ depletion have been visualized experimentally through the recording of Ca2+ “blinks.” Specifically, it has been shown that blink nadir is relatively reproducible between local Ca2+ release sites whereas blink recovery kinetics are highly variable. We applied a spatially-detailed, stochastic mathematical model to gain insight into the mechanisms underlying this heterogeneity.Novel features of our model include: (1) calculation of Ca2+ fluxes between locations within the junctional SR (JSR) and dyadic spaces; (2) variable numbers and locations of connections between the JSR and the SR network; (3) variable and irregular clusters of ryanodine receptor (RyR) channels in the JSR membrane; (4) spatially-defined localization of SR Ca2+ pumps (SERCA) in JSR and network SR membranes. Using this model, we investigated how different parameters influenced Ca2+ blink amplitude and kinetics. From these simulations we determined that the number of connections between the JSR and the SR network was the most important parameter controlling Ca2+ blink recovery kinetics. For instance, 80% recovery of pre-release JSR [Ca2+] decreased from 400 ms with 1 connection to 125 ms with 5 connections.SERCA density also influenced Ca2+ blink recovery, but the quantitative strength of this effect was relatively smaller (126 ms at nominal SERCA versus 209 ms with SERCA reduced by 80%). The remaining model parameters had only minor effects on blink kinetics. These results indicate the importance of SR Ca2+ diffusion in release kinetics and suggest mechanisms underlying Ca2+ blink heterogeneity.

Sarcoplasmic Reticulum Structure and Functional Properties that Promote Long-Lasting Calcium Sparks

Calcium (Ca) sparks are the fundamental sarcoplasmic reticulum (SR) Ca release events in cardiac myocytes, and they have a typical duration of 20–40 ms. However, when a fraction of ryanodine receptors (RyRs) are blocked by tetracaine or ruthenium red, Ca sparks lasting hundreds of milliseconds have been observed experimentally. The fundamental mechanism underlying these extremely prolonged Ca sparks is not understood. In this study, we use a physiologically detailed mathematical model of subcellular Ca cycling to examine how Ca spark duration is influenced by the number of functional RyRs in a junctional cluster (which is reduced by tetracaine or ruthenium red) and other SR Ca handling properties. One RyR cluster contains a few to several hundred RyRs, and we use a four-state Markov RyR gating model. Each RyR opens stochastically and is regulated by cytosolic and luminal Ca. We varied the number of functional RyRs in the single cluster, diffusion within the SR network, diffusion between network and junctional SR, cytosolic Ca diffusion, SERCA uptake activity, and RyR open probability. For long-lasting Ca release events, opening events within the cluster must occur continuously because the typical open time of the RyR is only a few milliseconds. We found the following: 1) if the number of RyRs is too small, it is difficult to maintain consecutive openings and stochastic attrition terminates the release; 2) if the number of RyRs is too large, the depletion of Ca from the junctional SR terminates the release; and 3) very long release events require relatively small-sized RyR clusters (reducing flux as seen experimentally with tetracaine) and sufficiently rapid intra-SR Ca diffusion, such that local junctional intra-SR [Ca] can be maintained by intra-SR diffusion and overall SR Ca reuptake.

The ESCRT-II proteins are involved in shaping the sarcoplasmic reticulum in C. elegans.

The sarcoplasmic reticulum is a network of tubules and cisternae localized in close association with the contractile apparatus, and regulates Ca(2+)dynamics within striated muscle cell. The sarcoplasmic reticulum maintains its shape and organization despite repeated muscle cell contractions, through mechanisms which are still under investigation. The ESCRT complexes are essential to organize membrane subdomains and modify membrane topology in multiple cellular processes. Here, we report for the first time that ESCRT-II proteins play a role in the maintenance of sarcoplasmic reticulum integrity inC. elegans ESCRT-II proteins colocalize with the sarcoplasmic reticulum marker ryanodine receptor UNC-68. The localization at the sarcoplasmic reticulum of ESCRT-II and UNC-68 are mutually dependent. Furthermore, the characterization of ESCRT-II mutants revealed a fragmentation of the sarcoplasmic reticulum network, associated with an alteration of Ca(2+)dynamics. Our data provide evidence that ESCRT-II proteins are involved in sarcoplasmic reticulum shaping.

Identification of Small Ankyrin 1 as a Novel Sarco(endo)plasmic Reticulum Ca2+-ATPase 1 (SERCA1) Regulatory Protein in Skeletal Muscle

Small ankyrin 1 (sAnk1) is a 17-kDa transmembrane (TM) protein that binds to the cytoskeletal protein, obscurin, and stabilizes the network sarcoplasmic reticulum in skeletal muscle. We report that sAnk1 shares homology in its TM amino acid sequence with sarcolipin, a small protein inhibitor of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA). Here we investigate whether sAnk1 and SERCA1 interact. Our results indicate that sAnk1 interacts specifically with SERCA1 in sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle, and in COS7 cells transfected to express these proteins. This interaction was demonstrated by co-immunoprecipitation and an anisotropy-based FRET method. Binding was reduced ~2-fold by the replacement of all of the TM amino acids of sAnk1 with leucines by mutagenesis. This suggests that, like sarcolipin, sAnk1 interacts with SERCA1 at least in part via its TM domain. Binding of the cytoplasmic domain of sAnk1 to SERCA1 was also detected in vitro. ATPase activity assays show that co-expression of sAnk1 with SERCA1 leads to a reduction of the apparent Ca(2+) affinity of SERCA1 but that the effect of sAnk1 is less than that of sarcolipin. The sAnk1 TM mutant has no effect on SERCA1 activity. Our results suggest that sAnk1 interacts with SERCA1 through its TM and cytoplasmic domains to regulate SERCA1 activity and modulate sequestration of Ca(2+) in the sarcoplasmic reticulum lumen. The identification of sAnk1 as a novel regulator of SERCA1 has significant implications for muscle physiology and the development of therapeutic approaches to treat heart failure and muscular dystrophies linked to Ca(2+) misregulation.

Calcium homeostasis in a local/global whole cell model of permeabilized ventricular myocytes with a Langevin description of stochastic calcium release.

Population density approaches to modeling local control of Ca(2+)-induced Ca(2+) release in cardiac myocytes can be used to construct minimal whole cell models that accurately represent heterogeneous local Ca(2+) signals. Unfortunately, the computational complexity of such "local/global" whole cell models scales with the number of Ca(2+) release unit (CaRU) states, which is a rapidly increasing function of the number of ryanodine receptors (RyRs) per CaRU. Here we present an alternative approach based on a Langevin description of the collective gating of RyRs coupled by local Ca(2+) concentration ([Ca(2+)]). The computational efficiency of this approach no longer depends on the number of RyRs per CaRU. When the RyR model is minimal, Langevin equations may be replaced by a single Fokker-Planck equation, yielding an extremely compact and efficient local/global whole cell model that reproduces and helps interpret recent experiments that investigate Ca(2+) homeostasis in permeabilized ventricular myocytes. Our calculations show that elevated myoplasmic [Ca(2+)] promotes elevated network sarcoplasmic reticulum (SR) [Ca(2+)] via SR Ca(2+)-ATPase-mediated Ca(2+) uptake. However, elevated myoplasmic [Ca(2+)] may also activate RyRs and promote stochastic SR Ca(2+) release, which can in turn decrease SR [Ca(2+)]. Increasing myoplasmic [Ca(2+)] results in an exponential increase in spark-mediated release and a linear increase in nonspark-mediated release, consistent with recent experiments. The model exhibits two steady-state release fluxes for the same network SR [Ca(2+)] depending on whether myoplasmic [Ca(2+)] is low or high. In the later case, spontaneous release decreases SR [Ca(2+)] in a manner that maintains robust Ca(2+) sparks.

Activation and propagation of Ca2+ release from inside the sarcoplasmic reticulum network of mammalian skeletal muscle.

Skeletal muscle fibres are large and highly elongated cells specialized for producing the force required for posture and movement. The process of controlling the production of force within the muscle, known as excitation-contraction coupling, requires virtually simultaneous release of large amounts of Ca(2+) from the sarcoplasmic reticulum (SR) at the level of every sarcomere within the muscle fibre. Here we imaged Ca(2+) movements within the SR, tubular (t-) system and in the cytoplasm to observe that the SR of skeletal muscle is a connected network capable of allowing diffusion of Ca(2+) within its lumen to promote the propagation of Ca(2+) release throughout the fibre under conditions where inhibition of SR ryanodine receptors (RyRs) was reduced. Reduction of cytoplasmic [Mg(2+)] ([Mg(2+)]cyto) induced a leak of Ca(2+) through RyRs, causing a reduction in SR Ca(2+) buffering power argued to be due to a breakdown of SR calsequestrin polymers, leading to a local elevation of [Ca(2+)]SR. The local rise in [Ca(2+)]SR, an intra-SR Ca(2+) transient, induced a local diffusely rising [Ca(2+)]cyto. A prolonged Ca(2+) wave lasting tens of seconds or more was generated from these events. Ca(2+) waves were dependent on the diffusion of Ca(2+) within the lumen of the SR and ended as [Ca(2+)]SR dropped to low levels to inactivate RyRs. Inactivation of RyRs allowed re-accumulation of [Ca(2+)]SR and the activation of secondary Ca(2+) waves in the persistent presence of low [Mg(2+)]cyto if the threshold [Ca(2+)]SR for RyR opening could be reached. Secondary Ca(2+) waves occurred without an abrupt reduction in SR Ca(2+) buffering power. Ca(2+) release and wave propagation occurred in the absence of Ca(2+)-induced Ca(2+) release. These observations are consistent with the activation of Ca(2+) release through RyRs of lowered cytoplasmic inhibition by [Ca(2+)]SR or store overload-induced Ca(2+) release. Restitution of SR Ca(2+) buffering power to its initially high value required imposing normal resting ionic conditions in the cytoplasm, which re-imposed the normal resting inhibition on the RyRs, allowing [Ca(2+)]SR to return to endogenous levels without activation of store overload-induced Ca(2+) release. These results are discussed in the context of how pathophysiological Ca(2+) release such as that occurring in malignant hyperthermia can be generated.

Ca handling during excitation–contraction coupling in heart failure

In the heart, coupling between excitation of the surface membrane and activation of contractile apparatus is mediated by Ca released from the sarcoplasmic reticulum (SR). Several components of Ca machinery are perfectly arranged within the SR network and the T-tubular system to generate a regular Ca cycling and thereby rhythmic beating activity of the heart. Among these components, ryanodine receptor (RyR) and SR Ca ATPase (SERCA) complexes play a particularly important role and their dysfunction largely underlies abnormal Ca homeostasis in diseased hearts such as in heart failure. The abnormalities in Ca regulation occur at practically all main steps of Ca cycling in the failing heart, including activation and termination of SR Ca release, diastolic SR Ca leak, and SR Ca uptake. The contributions of these different mechanisms to depressed contractile function and enhanced arrhythmogenesis may vary in different HF models. This brief review will therefore focus on modifications in RyR and SERCA structure that occur in the failing heart and how these molecular modifications affect SR Ca regulation and excitation-contraction coupling.

Role of the Inter-RyR Coupling in Cardiac Intracellular Calcium “Clock”

Cardiac rhythm is generated by the sinoatrial node in the normal heart. It has been recently shown that spontaneous local Ca2+ releases from sarcoplasmic reticulum (SR) through Ryanodine Receptors (RyRs) occur during late diastolic depolarization and may contribute to the periodic excitations of pacemaker cells [Vinogradova et al., Circ. Res. 02]. Such intracellular Ca2+ oscillations, referred to as Ca2+-“clock” [Maltsev, Lakatta, Am J Physiol Heart Circ Physiol, 2009], interact with the classic sarcolemmal voltage oscillator (membrane clock) by activating Na2+-Ca2+ exchange current.We developed an Electron-Conformational Model (ECM) of the stochastic RyR-channel activity, which accounted for cooperative effects of interaction between coupled RyR-channels within a Ca2+ release unit (RU) in cardiac pacemaker cells [Moskvin et al., PBMB 2006]. The RU includes a junctional Ca2+ release compartment of the SR network, a cluster of coupled RyR-channels, and adjacent sarcolemmal subspace. Inter-channel conformational coupling between nearest RyR neighbors is accounted for the energy potential of the cluster. The RU activity is described along with intracellular Ca2+ cycling between the subspace, cytosol, and the SR network.Computer simulations of a 9x9 RyR square lattice within the framework of ECM demonstrate that the Ca2+ SR “clock” activity is regulated by the interaction between RyR channels. Stability of oscillatory dynamics, frequency and amplitude of Ca2+ auto-oscillations depend essentially on the RyRs coupling force. We found that a combination of rather low Ca2+ release rate with strong enough RyR-RyR coupling may trigger sudden break of Ca2+ oscillations by forming of stable clusters of opened RyRs and thus providing with the steady-state Ca2+ leakage from the SR.Supported by Grants 12-П-4-1067, 12-04-32059-МОЛ_A_2013, 11-4-НП-673.

Three-Dimensional Reconstruction of Cardiac Sarcoplasmic Reticulum Reveals a Continuous Network Linking Transverse-Tubules

Rationale: The organization of the transverse-tubular (t-t) system and relationship to the sarcoplasmic reticulum (SR) underpins cardiac excitation–contraction coupling. The architecture of the SR, and relationship with the t-ts, is not well characterized at the whole-cell level. Furthermore, little is known regarding changes to SR ultrastructure in heart failure. Objective: The aim of this study was to unravel interspecies differences and commonalities between the relationship of SR and t-t networks within cardiac myocytes, as well as the modifications that occur in heart failure, using a novel high-resolution 3-dimensional (3D) imaging technique. Methods and Results: Using serial block face imaging coupled with scanning electron microscopy and image analysis, we have generated 3D reconstructions of whole cardiomyocytes from sheep and rat left ventricle, revealing that the SR forms a continuous network linking t-ts throughout the cell in both species. In sheep, but not rat, the SR has an intimate relationship with the sarcolemma forming junctional domains. 3D reconstructions also reveal details of the sheep t-t system. Using a model of tachypacing-induced heart failure, we show that there are populations of swollen and collapsed t-ts, patches of SR tangling, and disorder with rearrangement of the mitochondria. Conclusions: We provide the first high-resolution 3D structure of the SR network showing that it forms a cell-wide communication pipeline facilitating Ca 2+ diffusion, buffering, and synchronicity. The distribution of the SR within the cell is related to interspecies differences in excitation–contraction coupling, and we report the first detailed analysis of SR remodeling as a result of heart failure.

Waves of Calcium Depletion in the Sarcoplasmic Reticulum of Vascular Smooth Muscle Cells: An Inside View of Spatiotemporal Ca2+ Regulation

Agonist-stimulated smooth muscle Ca2+ waves regulate blood vessel tone and vasomotion. Previous studies employing cytoplasmic Ca2+ indicators revealed that these Ca2+ waves were stimulated by a combination of inositol 1,4,5-trisphosphate- and Ca2+-induced Ca2+ release from the endo/sarcoplasmic reticulum. Herein, we present the first report of endothelin-1 stimulated waves of Ca2+ depletion from the sarcoplasmic reticulum of vascular smooth muscle cells using a calsequestrin-targeted Ca2+ indicator. Our findings confirm that these waves are due to regenerative Ca2+-induced Ca2+ release by the receptors for inositol 1,4,5-trisphosphate. Our main new finding is a transient elevation in SR luminal Ca2+ concentration ([Ca2+]SR) both at the site of wave initiation, just before regenerative Ca2+ release commences, and at the advancing wave front, during propagation. This strongly suggests a role for [Ca2+]SR in the activation of inositol 1,4,5-trisphosphate receptors during agonist-induced calcium waves. In addition, quantitative analysis of the gradual decrease in the velocity of the depletion wave, observed in the absence of external Ca2+, indicates continuity of the lumen of the sarcoplasmic reticulum network. Finally, our observation that the depletion wave was arrested by the nuclear envelope may have implications for selective Ca2+ signalling.

Abstract 190: Protein Phosphatase 2Ce Is a Novel Phospholamban Phosphatase that Exacerbates Cell Death and Suppresses Cardiac Contractility

Regulation of sarcoplasmic reticulum (SR) calcium-ATPase (SERCA) activity is critical for calcium homeostasis in cardiomyocytes, and has a major impact on contractility and cellular viability of cardiomyocytes. The key regulators for SERCA activity include protein kinases, cAMP dependent protein kinase A and calcium/calmodulin dependent protein kinase II, and protein phosphatase 1. In this report, we have discovered that protein phosphatase 2Ce (PP2Ce) is a novel serine/threonine protein phosphatase specifically targeted to SR membrane in cardiomyocytes. PP2Ce was detected to interact with phosphlamban in heart. Recombinant PP2Ce protein showed a potent and specific activity towards the calcium/calmodulin dependent protein kinase II dependent phospholamban phosphorylation at threonin 17 site with no significant activity to cAMP dependent protein kinase A dependent phospholamban phosphorylation at serine 16 site. Expression of PP2Ce blunted β-adrenergic stimulated increase of phospholamban phosphorylation without affecting phosphorylation of ryanodine recepter or troponin I. PP2Ce expression reduced β-adrenergic stimulated intracellular calcium transient in isolated adult rabbit ventricular myocytes, and promoted hydrogen peroxide induced cell death in cultured neonatal rat ventricular myocytes. Transgenic mice with cardiac specific expression of PP2Ce showed no significant basal phenotype. However, in isolated perfusion heart preparation, β-adrenergic stimulated contractility was significant reduced in PP2Ce transgenic hearts comparing to wild type controls. Furthermore, we observed significantly larger infarct sizes and more impaired functional recovery following global ischemia/reperfusion injury in the transgenic hearts comparing to wild type controls. Therefore, PP2Ce is a novel component of SR calcium regulatory network that has a potentially important role in cell death regulation and cardiac contractility.

Integrity of the network sarcoplasmic reticulum in skeletal muscle requires small ankyrin 1.

Small ankyrin 1 (sAnk1; Ank1.5) is a ~20 kDa protein of striated muscle that concentrates in the network compartment of the sarcoplasmic reticulum (nSR). We used siRNA targeted to sAnk1 to assess its role in organizing the sarcoplasmic reticulum (SR) of skeletal myofibers in vitro. siRNA reduced sAnk1 mRNA and protein levels and disrupted the organization of the remaining sAnk1. Sarcomeric proteins were unchanged, but two other proteins of the nSR, SERCA and sarcolipin, decreased significantly in amount and segregated into distinct structures containing sarcolipin and sAnk1, and SERCA, respectively. Exogenous sAnk1 restored SERCA to its normal distribution. Ryanodine receptors and calsequestrin in the junctional SR, and L-type Ca(2+) channels in the transverse tubules were not reduced, although their striated organization was mildly altered. Consistent with the loss of SERCA, uptake and release of Ca(2+) were significantly inhibited. Our results show that sAnk1 stabilizes the nSR and that its absence causes the nSR to fragment into distinct membrane compartments.

Ca2+ on the move: what is better – fire and forget, or fire–diffuse–fire?

Ca²⁺ on the move: what is better – fire and forget, or fire–diffuse–fire?

The ontogeny of muscle structure and locomotory function in the long-finned squidDoryteuthis pealeii

Understanding the extent to which changes in muscle form and function underlie ontogenetic changes in locomotory behaviors and performance is important in understanding the evolution of musculoskeletal systems and also the ecology of different life stages. We explored ontogenetic changes in the structure, myosin heavy chain (MHC) expression and contractile properties of the circular muscles that provide power for jet locomotion in the long-finned squid Doryteuthis pealeii. The circular muscle fibers of newly hatched paralarvae had different sizes, shapes, thick filament lengths, thin:thick filament ratio, myofilament organization and sarcoplasmic reticulum (SR) distribution than those of adults. Viewed in cross section, most circular muscle cells were roughly triangular or ovoid in shape with a core of mitochondria; however, numerous muscle cells with crescent or other unusual cross-sectional shapes and muscle cells with unequal distributions of mitochondria were present in the paralarvae. The frequency of these muscle cells relative to 'normal' circular muscle cells ranged from 1:6 to 1:10 among the 19 paralarvae we surveyed. The thick filaments of the two types of circular fibers, superficial mitochondria-rich (SMR) and central mitochondria-poor (CMP), differed slightly in length among paralarvae with thick filament lengths of 0.83+/-0.15 microm and 0.71+/-0.1 microm for the SMR and CMP fibers, respectively (P 0.05; ANOVA). During ontogeny the thick filament lengths of both the CMP and SMR fibers increased significantly to 1.78+/-0.27 microm and 3.12+/-0.56 microm, respectively, in adults (P<0.0001 for both comparisons; ANOVA with Tukey's highly significant difference post hoc tests). When sectioned parallel to their long axes, the SMR and CMP fibers of both paralarvae and adults exhibited the myofilament arrangements typical of obliquely striated muscle cells but the angle of obliquity of the dense bodies was 22.8+/-2.4 deg. and 4.6+/-0.87 deg. for paralarvae and adults, respectively. There were also differences in the distribution of the anastomosing network of SR. In paralarvae, the outer and central zones of SR were well developed but the intramyoplasmic zone was greatly reduced in some cells or was scattered non-uniformly across the myoplasm. Whereas in adults the intramyoplasmic SR region was composed primarily of flattened tubules, it was composed primarily of rounded vesicles or tubules when present in the paralarvae. The ontogenetic differences in circular muscle structure were correlated with significant differences in their contractile properties. In brief tetanus at 20 degrees C, the mean unloaded shortening velocity of the paralarval circular muscle preparations was 9.1 L(0) s(-1) (where L(0) was the preparation length that generated the peak isometric stress), nearly twice that measured in other studies for the CMP fibers of adults. The mean peak isometric stress was 119+/-15 mN mm(-2) physiological cross section, nearly half that measured for the CMP fibers of adults. Reverse transcriptase-polymerase chain reaction analysis of paralarval and adult mantle samples revealed very similar expression patterns of the two known isoforms of squid MHC. The ontogenetic differences in the structure and physiology of the circular muscles may result in more rapid mantle movements during locomotion. This prediction is consistent with jet pulse durations observed in other studies, with shorter jet pulses providing hydrodynamic advantages for paralarvae.

Developmental Changes of the Sarcoplasmic Reticulum

In ventricular myocytes, excitation-contraction coupling (ECC), or the process by which an action potential leads to myocyte contraction, is reliant upon external influx of calcium (Ca+2) via sarcolemmal voltage-gated L-type Ca+2 channels to trigger Ca+2 release through nearby ryanodine receptors located in the sarcoplasmic reticulum (SR) via the mechanism of Ca+2-induced Ca+2 release. This causes a global increase in intracellular in Ca+2 concentration ([Ca+2]i) that activates contractile filaments. The level of expression of proteins and cellular organelles involved in cardiac ECC changes during development. These differences arise from adult myocytes being larger, having smaller surface-to-volume ratio and a developed transverse (T)-tubule system that tightly couples to the junctional SR. To investigate ECC developmental changes, we developed an adenovirus encoding a tagged red fluorescent protein with a retention signal for the sarcoplasmic reticulum (tRFP-SR). This approach allowed us for the first time to track in real-time SR structure and [Ca+2]i dynamics in neonatal and adult ventricular myocytes. Using confocal and total internal reflection fluorescence (TIRF) microscopy, we found that the SR within neonatal and adult ventricular cardiomyocytes forms a vast network that spans virtually the entire cell. Interestingly, we found that the SR is a highly dynamic network with relatively rapid changes in morphology in neonatal myocytes. Calcium sparks were observed from these SR structures, suggesting they harbor functional ryanodine receptors. In contrast to neonatal myocytes, the SR of adult ventricular myocytes is highly stable. Our findings suggest, that the fidelity of the [Ca+2]i transient during ECC in neonatal myocytes is not simply the result of ryanodine receptors in a static SR network. Instead, our data indicate it is the result of the activation of numerous couplings that form and dissolve rapidly between the sarcoplasmic reticulum and plasma membrane.

Termination of Cardiac Ca 2+ Sparks

Ca(2+) release from cardiac sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs) is regulated by dyadic cleft [Ca(2+)] and intra-SR free [Ca(2+)] ([Ca(2+)](SR)). Robust SR Ca(2+) release termination is important for stable excitation-contraction coupling, and partial [Ca(2+)](SR) depletion may contribute to release termination. Here, we investigated the regulation of SR Ca(2+) release termination of spontaneous local SR Ca(2+) release events (Ca(2+) sparks) by [Ca(2+)](SR), release flux, and intra-SR Ca(2+) diffusion. We simultaneously measured Ca(2+) sparks and Ca(2+) blinks (localized elementary [Ca(2+)](SR) depletions) in permeabilized ventricular cardiomyocytes over a wide range of SR Ca(2+) loads and release fluxes. Sparks terminated via a [Ca(2+)](SR)-dependent mechanism at a fixed [Ca(2+)](SR) depletion threshold independent of the initial [Ca(2+)](SR) and release flux. Ca(2+) blink recovery depended mainly on intra-SR Ca(2+) diffusion rather than SR Ca(2+) uptake. Therefore, the large variation in Ca(2+) blink recovery rates at different release sites occurred because of differences in the degree of release site interconnection within the SR network. When SR release flux was greatly reduced, long-lasting release events occurred from well-connected junctions. These junctions could sustain release because local SR Ca(2+) release and [Ca(2+)](SR) refilling reached a balance, preventing [Ca(2+)](SR) from depleting to the termination threshold. Prolonged release events eventually terminated at a steady [Ca(2+)](SR), indicative of a slower, [Ca(2+)](SR)-independent termination mechanism. These results demonstrate that there is high variability in local SR connectivity but that SR Ca(2+) release terminates at a fixed [Ca(2+)](SR) termination threshold. Thus, reliable SR Ca(2+) release termination depends on tight RyR regulation by [Ca(2+)](SR).

Ultrastructure of the sarcoplasmic reticulum in cardiac myocytes from Pacific bluefin tuna

Pacific bluefin tuna are active teleost fish with a large capacity for heat conservation and endothermy. They have a high metabolism, and hence the myocardium must be capable of sustaining elevated levels of cardiac output over a wide range of temperatures. To examine the way that the myocardial cells of bluefin tuna respond to their unique cardiac physiology, we have studied the ultrastructure of the internal membrane system and mitochondria of atrial and ventricular myocytes by light and electron microscopy. Our results reveal that cardiomyocytes of juvenile bluefin tuna posses a relatively high content of sarcoplasmic reticulum (SR), together with a large volume of mitochondria within the two (compact and spongy) ventricular compartments and in the atrial myocardium. The mitochondrial structure and distribution in bluefin tuna myocardium follow specific metabolic zonation resulting in a higher volume and lower cristae density in the compact ventricular layer than in atrium and spongy layer. The presence of junctional SR profiles and an extensive network of free SR within cells may ensure a rapid delivery of Ca(2+) to the myofibrils. This, in conjunction with transarcolemmal Ca(2+) entry, might contribute to a faster excitation-contraction-relaxation cycle and thus enhance cardiac performance, cardiac output, and the maintenance of excitability at low temperatures. We propose that the mitochondrial configuration together with the developed SR ultrastructure of bluefin tunas myocardium are important evolutionary steps for the maintenance of high heart rates and endothermy in this teleost fish.