Junctional Cleft Research Articles

PO-01-209 THE MECHANISMS OF IMPAIRED CALCIUM HANDLING UNDERLYING THE INCREASED SUSCEPTIBILITY TO ATRIAL FIBRILLATION IN METABOLIC SYNDROME

Atrial fibrillation (AF) is becoming increasingly common partly due to the prevalence of metabolic syndrome (MetS). Altered Ca2+-handling proteins on the atrial cellular membrane as well as across the sarcoplasmic reticulum (SR) network and axial tubules due to this condition can precipitate AF through elevation of Ca2+ in junctional clefts, triggering Ca2+ sparks. Through the stochastic recruitment of these sparks, a Ca2+ wave can manifest to activate neighbouring ryanodine receptors (RyRs), stimulating Na+/Ca2+ exchangers (INaCa) into potentially triggering arrhythmogenic spontaneous action potentials (SAPs). However, the mechanisms by which MetS can disrupt Ca2+ handling and trigger SAPs is not well understood. To investigate the mechanisms of impaired calcium handling underlying the increased susceptibility to AF in MetS. We utilized a 3D model of subcellular Ca2+ signalling by Zhang et al. 2022, which compromises of a network of Ca2+-release units (CRUs) and ion channel and exchanger formulations from the Grandi et al. common pool model. The Zhang et al. model far more accurately captures the processes by which Ca2+ sparks can occur and terminate through the inclusion of crucial membrane ion channels/transporters within the extensive network of axial tubules interwoven across the CRUs (Fig A). Based on experimental studies, MetS meditated functional change in Ca2+-handling proteins was replicated by increasing the conductivity of SERCA and RyR and reducing ICaL and INaCa. To evaluate the contribution of each individual remodeled Ca2+-handling component to the membrane potential and Ca2+ sparks, a series of simulations was conducted where we first incorporated each individual ionic remodeling into the control model, then from the MetS model, we reversed each remodeled Ca2+-handling component separately. We have demonstrated that SAPs were attributed to a decrease in INaCa and an increase in SERCA. For the former, this also corresponded to a much greater incidence of Ca2+ sparks compared to other cases. However, for the latter, increasing SERCA surprisingly had a negligible impact on the genesis of these sparks (Fig B). This suggests that an alternate pathway can potentially exist by which upregulated SERCA function can induce SAPs. Increased Ca2+ spark incidence due to functional changes in Ca2+-handling proteins is not the sole mechanism to SAP genesis. Our study therefore highlights that MetS-mediated AF can occur through different pathways.

Elevated Ca2+ at the triad junction underlies dysregulation of Ca2+ signaling in dysferlinopathy.

Dysferlin-null A/J myofibers generate abnormal Ca2+ transients that are slightly reduced in amplitude compared to controls. These are further reduced in amplitude by hypoosmotic shock and often appear as Ca2+ waves (Lukyanenko et al., J Physiol., 2017). Ca2+ waves are typically associated with Ca2+-induced Ca2+ release, or CICR, which can be myopathic. We tested the ability of a permeable Ca2+ chelator, BAPTA-AM, to inhibit CICR in injured dysferlin-null fibers and found that 10-50 nM BAPTA-AM suppressed all Ca2+ waves. The same concentrations of BAPTA-AM increased the amplitude of the Ca2+ transient in A/J fibers to wild type levels and protected transients against the loss of amplitude after hypoosmotic shock, as also seen in wild type fibers. Incubation with 10 nM BAPTA-AM led to intracellular BAPTA concentrations of ∼60 nM, as estimated with its fluorescent analog, Fluo-4AM. This should be sufficient to restore intracellular Ca2+ to levels seen in wild type muscle. Fluo-4AM was ∼10-fold less effective than BAPTA-AM; however, consistent with its lower affinity for Ca2+. EGTA, which has an affinity for Ca2+ similar to BAPTA, but with much slower kinetics of binding, was also ineffective when introduced as the -AM derivative. By contrast, a dysferlin variant with GCaMP6fu in place of its C2A domain accumulated at triad junctions, like wild type dysferlin, and suppressed all abnormal Ca2+ signaling; GCaMP6fu alone or as a Venus chimera did not accumulate at junctions and failed to suppress abnormal Ca2+ signaling. Our results suggest that leak of Ca2+ into the triad junctional cleft underlies dysregulation of Ca2+ signaling in dysferlin-null myofibers, and that dysferlin's C2A domain suppresses abnormal Ca2+ signaling and protects muscle against injury by binding Ca2+ in the cleft. Supported by the Jain Foundation, and NIH (RO1 AR064268).

Elevated Ca2+ at the triad junction underlies dysregulation of Ca2+ signaling in dysferlin-null skeletal muscle.

Dysferlin-null A/J myofibers generate abnormal Ca2+ transients that are slightly reduced in amplitude compared to controls. These are further reduced in amplitude by hypoosmotic shock and often appear as Ca2+ waves (Lukyanenko et al., J. Physiol., 2017). Ca2+ waves are typically associated with Ca2+-induced Ca2+ release, or CICR, which can be myopathic. We tested the ability of a permeable Ca2+ chelator, BAPTA-AM, to inhibit CICR in injured dysferlin-null fibers and found that 10-50nM BAPTA-AM suppressed all Ca2+ waves. The same concentrations of BAPTA-AM increased the amplitude of the Ca2+ transient in A/J fibers to wild type levels and protected transients against the loss of amplitude after hypoosmotic shock, as also seen in wild type fibers. Incubation with 10nM BAPTA-AM led to intracellular BAPTA concentrations of ∼60nM, as estimated with its fluorescent analog, Fluo-4AM. This should be sufficient to restore intracellular Ca2+ to levels seen in wild type muscle. Fluo-4AM was ∼10-fold less effective than BAPTA-AM, however, consistent with its lower affinity for Ca2+. EGTA, which has an affinity for Ca2+ similar to BAPTA, but with much slower kinetics of binding, was even less potent when introduced as the -AM derivative. By contrast, a dysferlin variant with GCaMP6fu in place of its C2A domain accumulated at triad junctions, like wild type dysferlin, and suppressed all abnormal Ca2+ signaling. GCaMP6fu introduced as a Venus chimera did not accumulate at junctions and failed to suppress abnormal Ca2+ signaling. Our results suggest that leak of Ca2+ into the triad junctional cleft underlies dysregulation of Ca2+ signaling in dysferlin-null myofibers, and that dysferlin's C2A domain suppresses abnormal Ca2+ signaling and protects muscle against injury by binding Ca2+ in the cleft.

Small-conductance Ca2+-activated K+ channels promote J-wave syndrome and phase 2 reentry

Small-conductance Ca2+-activated potassium (SK) channels play complex roles in cardiac arrhythmogenesis. SK channels colocalize with L-type Ca2+ channels, yet how this colocalization affects cardiac arrhythmogenesis is unknown. The purpose of this study was to investigate the role of colocalization of SK channels with L-type Ca2+ channels in promoting J-wave syndrome and ventricular arrhythmias. We carried out computer simulations of single-cell and tissue models. SK channels in the model were assigned to preferentially sense Ca2+ in the bulk cytosol, subsarcolemmal space, or junctional cleft. When SK channels sense Ca2+ in the bulk cytosol, the SK current (ISK) rises and decays slowly during an action potential, the action potential duration (APD) decreases as the maximum conductance increases, no complex APD dynamics and phase 2 reentry canbe induced by ISK. When SK channels sense Ca2+ in the subsarcolemmal space or junctional cleft, ISK can rise and decay rapidly during an action potential in a spike-like pattern because of spiky Ca2+ transients in these compartments, which can cause spike-and-dome action potential morphology, APD alternans, J-wave elevation, and phase 2 reentry. Our results can account for the experimental finding that activation of ISK induced J-wave syndrome and phase 2 reentry in rabbit hearts. Colocalization of SK channels with L-type Ca2+ channels so that they preferentially sense Ca2+ in the subsarcolemmal or junctional space may result in a spiky ISK, which can functionally play a similar role of the transient outward K+ current in promoting J-wave syndrome and ventricular arrhythmias.

Elevated local [Ca2+] and CaMKII promote spontaneous Ca2+ release in ankyrin-B-deficient hearts.

Loss-of-function mutations in the cytoskeletal protein ankyrin-B (AnkB) cause ventricular tachyarrhythmias in humans. Previously, we found that a larger fraction of the sarcoplasmic reticulum (SR) Ca(2+) leak occurs through Ca(2+) sparks in AnkB-deficient (AnkB(+/-)) mice, which may contribute to arrhythmogenicity via Ca(2+) waves. Here, we investigated the mechanisms responsible for increased Ca(2+) spark frequency in AnkB(+/-) hearts. Using immunoblots and phospho-specific antibodies, we found that phosphorylation of ryanodine receptors (RyRs) by CaMKII is enhanced in AnkB(+/-) hearts. In contrast, the PKA-mediated RyR phosphorylation was comparable in AnkB(+/-) and wild-type (WT) mice. CaMKII inhibition greatly reduced Ca(2+) spark frequency in myocytes from AnkB(+/-) mice but had little effect in the WT. Global activities of the major phosphatases PP1 and PP2A were similar in AnkB(+/-) and WT hearts, while CaMKII autophosphorylation, a marker of CaMKII activation, was increased in AnkB(+/-) hearts. Thus, CaMKII-dependent RyR hyperphosphorylation in AnkB(+/-) hearts is caused by augmented CaMKII activity. Intriguingly, CaMKII activation is limited to the sarcolemma-SR junctions since non-junctional CaMKII targets (phospholamban, HDAC4) are not hyperphosphorylated in AnkB(+/-) myocytes. This local CaMKII activation may be the consequence of elevated [Ca(2+)] in the junctional cleft caused by reduced Na(+)/Ca(2+) exchange activity. Indeed, using the RyR-targeted Ca(2+) sensor GCaMP2.2-FBKP12.6, we found that local junctional [Ca(2+)] is significantly elevated in AnkB(+/-) myocytes. The increased incidence of pro-arrhythmogenic Ca(2+) sparks and waves in AnkB(+/-) hearts is due to enhanced CaMKII-mediated RyR phosphorylation, which is caused by higher junctional [Ca(2+)] and consequent local CaMKII activation.

Junctional cleft [Ca²⁺]i measurements using novel cleft-targeted Ca²⁺ sensors.

Intracellular Ca(2+) concentration ([Ca(2+)]i) is regulated and signals differently in various subcellular microdomains, which greatly enhances its second messenger versatility. In the heart, sarcoplasmic reticulum Ca(2+) release and signaling are controlled by local [Ca(2+)]i in the junctional cleft ([Ca(2+)]Cleft), the small space between sarcolemma and junctional sarcoplasmic reticulum. However, methods to measure [Ca(2+)]Cleft directly are needed. To construct novel sensors that allow direct measurement of [Ca(2+)]Cleft. We constructed cleft-targeted [Ca(2+)] sensors by fusing Ca(2+)-sensor GCaMP2.2 and a new lower Ca(2+)-affinity variant GCaMP2.2Low to FKBP12.6, which binds with high affinity and selectivity to ryanodine receptors. The fluorescence pattern, affinity for ryanodine receptors, and competition by untagged FKBP12.6 demonstrated that FKBP12.6-tagged sensors are positioned to measure local [Ca(2+)]Cleft in adult rat myocytes. Using GCaMP2.2Low-FKBP12.6, we showed that [Ca(2+)]Cleft reaches higher levels with faster kinetics than global [Ca(2+)]i during excitation-contraction coupling. Diastolic sarcoplasmic reticulum Ca(2+) leak or sarcolemmal Ca(2+) entry may raise local [Ca(2+)]Cleft above bulk cytosolic [Ca(2+)]i ([Ca(2+)]Bulk), an effect that may contribute to triggered arrhythmias and even transcriptional regulation. We measured this diastolic standing [Ca(2+)]Cleft-[Ca(2+)]Bulk gradient with GCaMP2.2-FKBP12.6 versus GCaMP2.2, using [Ca(2+)] measured without gradients as a reference point. This diastolic difference ([Ca(2+)]Cleft=194 nmol/L versus [Ca(2+)]Bulk=100 nmol/L) is dictated mainly by the sarcoplasmic reticulum Ca(2+) leak rather than sarcolemmal Ca(2+) flux. We have developed junctional cleft-targeted sensors to measure [Ca(2+)]Cleft versus [Ca(2+)]Bulk and demonstrated dynamic differences during electric excitation and a standing diastolic [Ca(2+)]i gradient, which could influence local Ca(2+)-dependent signaling within the junctional cleft.

Ephaptic Coupling in Cardiac Myocytes

While it is widely believed that conduction in cardiac tissue is regulated by gap junctions, recent experimental evidence suggests that the extracellular space may play a significant role in action potential propagation. Cardiac tissue with low gap junctional coupling still exhibits conduction, with conflicting degrees of slowing that may be due to variations in the extracellular space. Inhomogeneities in the extracellular space caused by the complex cellular structure in cardiac tissue can lead to ephaptic, or field effect, coupling. Here, we present data from simulations of a cylindrical strand of cells in which we see the dramatic effect highly resistant extracellular spaces have on propagation velocity. We find that ephaptic effects occur in all areas of small extracellular spaces and are not restricted to the junctional cleft between cells. This previously unrecognized type of field coupling, which we call lateral coupling, can allow conduction in the absence of gap junctions. We compare our results with the classically used cable theory, demonstrating the quantitative difference in propagation velocity arising from the cellular geometry. Ephaptic effects are shown to be highly dependent upon parameter values, frequently enhancing, but sometimes decreasing propagation speed. Our mathematical analysis incorporates the inhomogeneities in the extracellular microdomains that cannot be directly measured by experimental techniques and will aid in optimizing cardiac treatments that require manipulation of the cellular geometry and understanding heart functionality.

Na+/K+-ATPase 2-isoform preferentially modulates Ca2+ transients and sarcoplasmic reticulum Ca2+ release in cardiac myocytes

Na(+)/K(+)-ATPase (NKA) is essential in regulating [Na(+)](i), and thus cardiac myocyte Ca(2+) and contractility via Na(+)/Ca(2+) exchange. Different NKA-α subunit isoforms are present in the heart and may differ functionally, depending on specific membrane localization. In smooth muscle and astrocytes, NKA-α2 is located at the junctions with the endo(sarco)plasmic reticulum, where they could regulate local [Na(+)], and indirectly junctional cleft [Ca(2+)]. Whether this model holds for cardiac myocytes is unclear. The ouabain-resistant NKA-α1 cannot be selectively blocked to assess its effect. To overcome this, we used mice in which NKA-α1 is ouabain sensitive and NKA-α2 is ouabain resistant (SWAP mice). We measured the effect of ouabain at low concentration on [Na(+)](i), Ca(2+) transients, and the fractional sarcoplasmic reticulum (SR) Ca(2+) release in cardiac myocytes from wild-type (WT; NKA-α2 inhibition) and SWAP mice (selective NKA-α1 block). At baseline, Na(+) and Ca(2+) regulations are similar in WT and SWAP mice. For equal levels of total NKA inhibition (~25%), ouabain significantly increased Ca(2+) transients (from ΔF/F(0)= 1.5 ± 0.1 to 1.8 ± 0.1), and fractional SR Ca(2+) release (from 24 ± 3 to 29 ± 3%) in WT (NKA-α2 block) but not in SWAP myocytes (NKA-α1 block). This occurred despite a similar and modest increase in [Na(+)](i) (~2 mM) in both groups. The effect in WT mice was mediated specifically by NKA-α2 inhibition because at a similar concentration ouabain had no effect in transgenic mice where both NKA-α1 and NKA-α2 are ouabain resistant. NKA-α2 has a more prominent role (vs. NKA-α1) in modulating cardiac myocyte SR Ca(2+) release.

Junctional Cleft [Ca]i Measurements using Novel Cleft-Targeted Ca Sensors

Junctional Cleft [Ca]i Measurements using Novel Cleft-Targeted Ca Sensors

Lethal Junctional Epidermolysis Bullosa with Pyloric Atresia due to Compound Heterozygosity for Two Novel Mutations in the Integrin β4 Gene

Junctional epidermolysis bullosa with pyloric atresia (JEB-PA) is a rare autosomal recessive disease with blister formation within the lamina lucida due to mutations in the integrin β4 (ITGB4) and α6 (ITGA6) genes. A female preterm infant, first child of healthy non-consanguineous parents, was born at 26 + 4 weeks of gestation by caesarean section, following polyhydramnion and abruption of placenta. She presented with extensive areas of denuded skin on both lateral sides of the head, neck and extremities. Auricles were hypoplastic. Abdominal ultrasound and X-ray were suggestive of pyloric atresia which was revised surgically on the 4th day of life. Further course was complicated by progressive skin detachment, sepsis, and renal insufficiency with fatal outcome at 18 days of age. Immunofluorescence mapping of cryopreserved skin showed junctional cleft formation with negative staining for integrin α6 and integrin β4. Mutational analysis disclosed compound heterozygosity for two novel nonsense mutations in the ITGB4 gene: c.600dupC/p.F201fsX14 and c.2533C>T/p.Q845X. 2 subsequent pregnancies were terminated following prenatal diagnosis disclosing the same ITGB4 mutations, a 4th pregnancy was unaffected. We describe a case of lethal JEB-PA with negative immunoreactivity to integrin α6 and integrin β4 predicting a poor outcome. Identification of compound heterozygosity for two novel ITGB4 mutations in the affected preterm infant permitted prenatal diagnosis and finally birth of a healthy sibling.

Na+currents are required for efficient excitation-contraction coupling in rabbit ventricular myocytes: a possible contribution of neuronal Na+channels

Ca2+ transients were activated in rabbit ventricular cells by a sequence of action potential shaped voltage clamps. After activating a series of control transients, Na+ currents (INa) were inactivated with a ramp from -80 to -40 mV (1.5 s) prior to the action potential clamp. The transients were detected with the calcium indicator Fluo-4 and an epifluorescence system. With zero Na+ in the pipette INa inactivation produced a decline in the SR Ca2+ release flux (measured as the maximum rate of rise of the transient) of 27 ± 4% (n = 9, P < 0.001) and a peak amplitude reduction of 10 ± 3% (n = 9, P < 0.05). With 5 mm Na+ in the pipette the reduction in release flux was greater (34 ± 4%, n = 4, P < 0.05). The ramp effectively inactivates INa without changing ICa, and there was no significant change in the transmembrane Ca2+ flux after the inactivation of INa. We next evoked action potentials under current clamp. TTX at 100 nm, which selectively blocks neuronal isoforms of Na+ channels, produced a decline in SR Ca2+ release flux of 35 ± 3% (n = 6, P < 0.001) and transient amplitude of 12 ± 2% (n = 6, P < 0.05). This effect was similar to the effect of INa inactivation on release flux. We conclude that a TTX-sensitive INa is essential for efficient triggering of SR Ca2+ release. We propose that neuronal Na+ channels residing within couplons activate sufficient reverse Na+-Ca2+ exchanger (NCX) to prime the junctional cleft with Ca2+. The results can be explained if non-linearities in excitation-contraction coupling mechanisms modify the coupling fidelity of ICa, which is known to be low at positive potentials.

Toward the roles of store-operated Ca2+ entry in skeletal muscle

Store-operated Ca(2+) entry (SOCE) has been found to be a rapidly activated robust mechanism in skeletal muscle fibres. It is conducted across the junctional membranes by stromal interacting molecule 1 (STIM1) and Orai1, which are housed in the sarcoplasmic reticulum (SR) and tubular (t-) system, respectively. These molecules that conduct SOCE appear evenly distributed throughout the SR and t-system of skeletal muscle, allowing for rapid and local control in response to depletions of Ca(2+) from SR. The significant depletion of SR Ca(2+) required to reach the activation threshold for SOCE could only be achieved during prolonged bouts of excitation-contraction coupling (EC coupling) in a healthy skeletal muscle fibre, meaning that this mechanism is not responsible for refilling the SR with Ca(2+) during periods of fibre quiescence. While Ca(2+) in SR remains below the activation threshold for SOCE, a low-amplitude persistent Ca(2+) influx is provided to the junctional cleft. This article reviews the properties of SOCE in skeletal muscle and the proposed molecular mechanism, assesses its potential physiological roles during EC coupling, namely refilling the SR with Ca(2+) and simple balancing of Ca(2+) within the cell, and also proposes the possibility of SOCE as a potential regulator of t-system and SR membrane protein function.

Upregulation of store-operated Ca2+ entry in dystrophic mdx mouse muscle

Store-operated Ca(2+) entry (SOCE) is an important mechanism in virtually all cells. In adult skeletal muscle, this mechanism is highly specialized for the rapid delivery of Ca(2+) from the transverse tubule into the junctional cleft during periods of depleting Ca(2+) release. In dystrophic muscle fibers, SOCE may be a source of Ca(2+) overload, leading to cell necrosis. However, this possibility is yet to be examined in an adult fiber during Ca(2+) release. To examine this, Ca(2+) in the tubular system and cytoplasm were simultaneously imaged during direct release of Ca(2+) from sarcoplasmic reticulum (SR) in skeletal muscle fibers from healthy (wild-type, WT) and dystrophic mdx mouse. The mdx fibers were found to have normal activation and deactivation properties of SOCE. However, a depression of the cytoplasmic Ca(2+) transient in mdx compared with WT fibers was observed, as was a shift in the SOCE activation and deactivation thresholds to higher SR Ca(2+) concentrations ([Ca(2+)](SR)). The shift in SOCE activation and deactivation thresholds was accompanied by an approximately threefold increase in STIM1 and Orai1 proteins in dystrophic muscle. While the mdx fibers can introduce more Ca(2+) into the fiber for an equivalent depletion of [Ca(2+)](SR) via SOCE, it remains unclear whether this is deleterious.

Na/K-ATpase α2-Subunit Preferentially Modulates Ca Transients and SR Ca Release in Cardiac Myocytes

Na/K-ATPase (NKA) is essential in regulating [Na]i, and thus cardiac myocyte Ca and contractility via Na/Ca exchange. Different NKAα-subunit isoforms are present in heart and may differ functionally, depending on differential membrane localization. In smooth muscle and astrocytes, NKA-α2 is located at the junctions with endo(sarco)plasmic reticulum, where they could regulate local Na, and indirectly junctional cleft [Ca]. In contrast, NKA-α1 is ubiquitously distributed and may regulate bulk [Na]i. It is controversial whether this model holds for cardiac myocytes. We measured the effect of selective NKA-α2/NKA-α1 inhibition with low concentrations of ouabain on [Na]i, Ca transients and fractional SR Ca release in cardiac myocytes from wild-type (WT) mice and transgenic mice in which NKA-α1 is ouabain-sensitive and NKA-α2 is ouabain-resistant (SWAP mice), respectively. Different [ouabain] (5 and 0.1 μM, respectively) were used to attain a similar level of total NKA inhibition in WT and SWAP mice. Ouabain increased Ca transients in WT (F/F0=1.84±0.13 vs. 1.54±0.12 under control conditions) but not in SWAP mice (1.49±0.15 vs. 1.46±0.12), despite a similar and modest increase in [Na]i (≤2 mM). Ca transient increase in WT mice was mediated specifically by NKA-α2 inhibition, because 5 μM ouabain had no effect in transgenic mice where both NKA-α1 and NKA-α2 are ouabain-resistant. Ouabain also significantly increased the fractional SR Ca release in WT mice (from 23±2% to 28±2%) but not SWAP mice (23±3% with and without ouabain). Dual-color immunofluorescence measurements coupled with spatial cross-correlation analysis revealed that NKA-α2 is co-localized to a significant extent with both Na/Ca exchanger (43% co-localization) and ryanodine receptors (43% co-localization) in cardiac myocytes. In conclusion, our data indicate that NKA-α2 has a more prominent role (vs. NKA-α1) in modulating Ca transients and SR Ca release in cardiac myocytes.

Commentaries on Viewpoint: The cardiac contraction cycle: Is Ca2+ going local?

Commentaries on Viewpoint: The cardiac contraction cycle: Is Ca²⁺ going local?

A protein diffusion model of the sealing effect.

Water transport across the arterial endothelium is believed primarily to occur through breaks in the tight junction strands at the cell periphery between neighboring cells. Additional proteins arriving at the tight junction can close these breaks, thereby attenuating this water flux. Motivated by evidence that the diffusion of presynthesized protein from the interior of the cell to and incorporation into the cell border is the mechanism of endothelial tight junctional sealing, we develop a diffusion-limited mathematical model of intercellular gap sealing. A single endothelial cell is represented as a thin, axisymmetric disk, initially containing a uniform distribution of junctional protein that does not interact with the apical or basal cell surfaces. Upon application of a transmural pressure gradient, water flows through the junctional cleft, and tight junction remodeling begins. We assume that proteins at the junction are instantaneously incorporated into its strand, dropping the free protein concentration at the cell periphery to zero. This sets the diffusion of intracellular proteins toward the junction in motion. The solution of this one-dimensional initial value problem provides excellent fits to current and previously published experimental data over a wide variety of conditions. It yields three physically meaningful parameters for each fit, including a protein diffusivity in the cytoplasm that varies little within experimental treatments. Statistical variation of these parameters allows rational comparison of experimental runs and identification of outlier runs.

The Ins and Outs of Calcium in Heart Failure

See related article, pages 1398–1405 Cardiac excitation–contraction coupling is akin to a biophysical juggling act that involves fast and slow ion movements overlapping in time and space across several compartments. In response to sarcolemmal depolarization, Ca2+ release from the large intracellular store in the sarcoplasmic reticulum (SR) is triggered by a rush of Ca2+ from the extracellular space through L-type Ca2+ channels into the very tiny junctional cleft (12 nm) to activate SR Ca2+ release channels in a tightly controlled signal amplification step. Local inactivation of the L-type Ca2+ channel mediated by the large junctional Ca2+ spike (peaking at >50 μmol/L) provides a mechanism of negative feedback on beat-to-beat Ca2+ influx; thus, an abrupt change in the size of cytosolic Ca2+ transient will impact the trigger for subsequent beats and reciprocally influence the SR Ca2+ content in a process known as autoregulation.1 Ca2+ removal processes, although kinetically slower, also play a major role in shaping the Ca2+ dynamics, and, ultimately, the contraction, of the myocyte. The 2 main pathways, Ca2+ reuptake into the SR by the Ca2+ ATPase (SERCA2A) and Ca2+ efflux across the sarcolemma by the Na+/Ca2+ exchanger (NCX), compete for the job of removing Ca2+ ions in a species-dependent manner. In ventricular myocytes from rats and mice, ≈90% of the Ca2+ decline during a transient is mediated by SERCA2A, whereas NCX contributes only ≈7% to Ca2+ removal (the remainder is taken up by mitochondria or extruded from the cell by the sarcolemmal Ca2+ ATPase).2 In most large animals (eg, rabbits, dogs, cats, ferrets), some small animals (eg, guinea pigs and hamsters), and humans,3 Ca2+ removal by SERCA2A accounts for only 60% to …

Effect of Focused Ultrasound Applied With an Ultrasound Contrast Agent on the Tight Junctional Integrity of the Brain Microvascular Endothelium

Previous studies have investigated a potential method for targeted drug delivery in the central nervous system that uses focused ultrasound bursts combined with an ultrasound contrast agent to temporarily disrupt the blood-brain barrier (BBB). The purpose of this work was to investigate the integrity of the tight junctions (TJs) in rat brain microvessels after this BBB disruption. Ultrasound bursts (1.5-MHz) in combination with a gas contrast agent (Optison) was applied at two locations in the brain in 25 rats to induce BBB disruption. Using immunoelectron microscopy, the distributions of the TJ-specific transmembrane proteins occludin, claudin-1, claudin-5, and of submembranous ZO-1 were examined at 1, 2, 4, 6 and 24 h after sonication. A quantitative evaluation of the protein expression was made by counting the number of immunosignals per micrometer in the junctional clefts. BBB disruption at the sonicated locations was confirmed by the leakage of i.v. administered horseradish peroxidase (HRP, m.w. 40,000 Da) and lanthanum chloride (La 3+, m.w. ∼ 139 Da). Leakage of these agents was observed at 1 and 2 h and, in a few vessels, at 4 h after ultrasound application. These changes were paralleled by the apparent disintegration of the TJ complexes, as evidenced by the redistribution and loss of the immunosignals for occludin, claudin-5 and ZO-1. Claudin-1 seemed less involved. At 6 and 24 h after sonication, no HRP or lanthanum leakage was observed and the barrier function of the TJs, as indicated by the localization and density of immunosignals, appeared to be completely restored. This study provides the first direct evidence that ultrasound bursts combined with a gas contrast agent cause disassembling of the TJ molecular structure, leading to loss of the junctional barrier functions in brain microvessels. The BBB disruption appears to last up to 4 h after sonication and permits the paracellular passage of agents with molecular weights up to at least 40 kDa. These promising features can be exploited in the future development of this method that could enable the delivery of drugs, antibodies or genes to targeted locations in the brain. (E-mail: njm@bwh.harvard.edu)

Ephaptic coupling of cardiac cells through the junctional electric potential

Cardiac cells are electrically coupled through gap junction channels, which allow ionic current to spread intercellularly from one cell to the next. In addition, it is possible that cardiac cells are coupled through the electric potential in the junctional cleft space between neighboring cells. We develop and analyze a mathematical model of two cells coupled through a common junctional cleft potential. Consistent with more detailed models, we find that the coupling mechanism is highly parameter dependent. Analysis of our model reveals that there are two time scales involved, and the dynamics of the slow subsystem provide new mathematical insight into how the coupling mechanism works. We find that there are two distinct types of propagation failure and we are able to characterize parameter space into regions of propagation success and the two different types of propagation failure.

Functional groups of ryanodine receptors in rat ventricular cells

Ryanodine receptors (RyR2s) are ion channels in the sarcoplasmic reticulum (SR) that are responsible for Ca2+ release in rat ventricular myocytes. Localization of RyR2s is therefore crucial for our understanding of contraction and other Ca2+-dependent intracellular processes. Recent results (e.g. circular waves and Ca2+ sparks in perinuclear area) raised questions about the classical views of RyR2 distribution and organization within ventricular cells. A Ca2+ spark is a fluorescent signal reflecting the activation of a small group of RyR2s. Frequency and spatio-temporal characteristics of Ca2+ sparks depend on the state of cytoplasmic and intraluminal macromolecular complexes regulating cardiac RyR2 function. We employed electron microscopy, confocal imaging of spontaneous Ca2+ sparks and immunofluorescence to visualize the distribution of RyR2s in ventricular myocytes and to evaluate the local involvement of the macromolecular complexes in regulation of functional activity of the RyR2 group. An electron microscopy study revealed that the axial tubules of the transverse-axial tubular system probably do not have junctions with the network SR (nSR). The nSR was found to be wrapped around intermyofibrillar mitochondria and contained structures similar to feet of the junctional cleft. Treatment of ventricular myocytes with antibodies against RyR2 showed that in addition to the junctional SR, a small number of RyR2s can be localized at the middle of the sarcomere and in the zone of perinuclear mitochondria. Recordings of spontaneous Ca2+ sparks showed the existence of functional groups of RyR2s in these intracellular compartments. We found that within the sarcomere about 20% of Ca2+ sparks were not colocalized with the zone of the junctional or corbular SR (Z-line zone). The spatio-temporal characteristics of sparks found in the Z-line and A-band zones were very similar, whereas sparks from the zone of the perinuclear mitochondria were about 25% longer. Analysis of the initiation sites of Ca2+ sparks within the same junctional SR cluster suggested that 18-25 RyR2s are in the functional group producing a spark. Because of the similarity of the spatio-temporal characteristics of sarcomeric sparks and ultrastructural characteristics of nSR, we suggest that the functional groups of RyR2s in the middle of the sarcomere are macromolecular complexes of approximately 20 RyR2s with regulatory proteins. Our data allowed us to conclude that a significant number of functional RyR2s is located in the middle of the sarcomere and in the zone of perinuclear mitochondria. These RyR2s could contribute to excitation-contraction coupling, mitochondrial and nuclear signalling, and Ca2+-dependent gene regulation, but their existence raises many additional questions.