Normal Sarcoplasmic Reticulum Research Articles

Evidence of functional ryanodine receptors in rat mesenteric collecting lymphatic vessels.

In the current study, the potential contributions of ryanodine receptors (RyRs) to intrinsic pumping and responsiveness to substance P (SP) were investigated in isolated rat mesenteric collecting lymphatic vessels. Responses to SP were characterized in lymphatic vessels in the absence or presence of pretreatment with nifedipine to block L-type Ca2+ channels, caffeine to block normal release and uptake of Ca2+ from the sarcoplasmic reticulum, ryanodine to block all RyR isoforms, or dantrolene to more selectively block RyR1 and RyR3. RyR expression and localization in lymphatics was also assessed by quantitative PCR and immunofluorescence confocal microscopy. The results show that SP normally elicits a significant increase in contraction frequency and a decrease in end-diastolic diameter. In the presence of nifedipine, phasic contractions stop, yet subsequent SP treatment still elicits a strong tonic contraction. Caffeine treatment gradually relaxes lymphatics, causing a loss of phasic contractions, and prevents subsequent SP-induced tonic contraction. Ryanodine also gradually diminishes phasic contractions but without causing vessel relaxation and significantly inhibits the SP-induced tonic contraction. Dantrolene treatment did not significantly impair lymphatic contractions nor the response to SP. The mRNA for all RyR isoforms is detectable in isolated lymphatics. RyR2 and RyR3 proteins are found predominantly in the collecting lymphatic smooth muscle layer. Collectively, the data suggest that SP-induced tonic contraction requires both extracellular Ca2+ plus Ca2+ release from internal stores and that RyRs play a role in the normal contractions and responsiveness to SP of rat mesenteric collecting lymphatics.NEW & NOTEWORTHY The mechanisms that govern contractions of lymphatic vessels remain unclear. Tonic contraction of lymphatic vessels caused by substance P was blocked by caffeine, which prevents normal uptake and release of Ca2+ from internal stores, but not nifedipine, which blocks L-type channel-mediated Ca2+ entry. Ryanodine, which also disrupts normal sarcoplasmic reticulum Ca2+ release and reuptake, significantly inhibited substance P-induced tonic contraction. Ryanodine receptors 2 and 3 were detected within the smooth muscle layer of collecting lymphatic vessels.

Measuring Local Gradients of Intramitochondrial [Ca 2+ ] in Cardiac Myocytes During Sarcoplasmic Reticulum Ca 2+ Release

Rationale: Mitochondrial [Ca 2+ ] ([Ca 2+ ] mito ) regulates mitochondrial energy production, provides transient Ca 2+ buffering under stress, and can be involved in cell death. Mitochondria are near the sarcoplasmic reticulum (SR) in cardiac myocytes, and evidence for crosstalk exists. However, quantitative measurements of [Ca 2+ ] mito are limited, and spatial [Ca 2+ ] mito gradients have not been directly measured. Objective: To directly measure local [Ca 2+ ] mito during normal SR Ca release in intact myocytes, and evaluate potential subsarcomeric spatial [Ca 2+ ] mito gradients. Methods and Results: Using the mitochondrially targeted inverse pericam indicator Mitycam, calibrated in situ, we directly measured [Ca 2+ ] mito during SR Ca 2+ release in intact rabbit ventricular myocytes by confocal microscopy. During steady state pacing, Δ[Ca 2+ ] mito amplitude was 29±3 nmol/L, rising rapidly (similar to cytosolic free [Ca 2+ ]) but declining much more slowly. Taking advantage of the structural periodicity of cardiac sarcomeres, we found that [Ca 2+ ] mito near SR Ca 2+ release sites (Z-line) versus mid-sarcomere (M-line) reached a high peak amplitude (37±4 versus 26±4 nmol/L, respectively P <0.05) which occurred earlier in time. This difference was attributed to ends of mitochondria being physically closer to SR Ca 2+ release sites, because the mitochondrial Ca 2+ uniporter was homogeneously distributed, and elevated [Ca 2+ ] applied laterally did not produce longitudinal [Ca 2+ ] mito gradients. Conclusions: We developed methods to measure spatiotemporal [Ca 2+ ] mito gradients quantitatively during excitation–contraction coupling. The amplitude and kinetics of [Ca 2+ ] mito transients differ significantly from those in the cytosol and are respectively higher and faster near the Z-line versus M-line. This approach will help clarify SR-mitochondrial Ca 2+ signaling.

The Rapid Repolarization of the Action Potential in Twitch Skeletal Muscle Fibers from Frog is due Almost Entirely to a Calcium-Activated K Current

Action potentials (aps) in cut fibers mounted in a double Vaseline-gap chamber were generated by short current-clamp pulses superimposed on a holding current that maintained the resting potential at −90 mV. Ca2+ release from the sarcoplasmic reticulum (SR) was assessed with either the EGTA/phenol red method or the SR [Ca2+] indicator, tetramethylmurexide. In contrast to the rapid rate of repolarization of the ap with normal SR Ca contents, in the near absence of Ca, the time course of repolarization was approximately exponential with an average exponential time constant of 12.5 ms. Removal of K from the internal and external solutions eliminated the effect of SR Ca2+ release on the ap indicating that the current is carried by K+ ions. The time course of total current during the repolarization was assessed from the rate of change of voltage. Taking into account a small Ca-insensitive background current and the relationship between K current and permeability (PK) on voltage using the Goldman-Hodgkin-Katz equation, the time course of PK is seen to closely match that of the myoplasmic Ca transient. At physiological SR Ca load, the Ca-activated K current, denoted IK(Ca), at its peak was ∼80% of the total current. Since almost all of the background current was associated with recharging the membrane capacitance via the holding current passing through ohmic pathways in the cut-fiber preparation, the peak percentage of current responsible for repolarization in an intact fiber carried by IK(Ca) should be significantly greater than 90%. While results with voltage-clamp stimulation indicate a complex dependence of IK(Ca) on calcium and voltage similar to that displayed by BKCa channels, the IK(Ca) current was insensitive to the BKCa channel inhibitor charybdotoxin.

ß-Adrenergic-Mediated Inotropy and Lusitropy in Conditional Cardiac SERCA2 Ablated Mice

Proper contraction and relaxation of the heart depends on highly orchestrated and rapid release and reuptake of Ca2+ from the sarcoplasmic reticulum (SR). Normal SR Ca2+ reuptake in the rodent heart is accomplished predominately by the cardiac Ca-ATPase, SERCA2a, which in the failing heart exhibits diminished activity and expression. We have utilized a mouse model of inducible cardiac Ca2+ dysregulation, the Serca2fl/fl mouse, to investigate the relationship between diminished SR Ca2+ flux and heart dysfunction. Conditional deletion of Serca2 initiates the progressive loss of SERCA2 protein, allowing us to examine in fine detail the relationship between titrated loss of SR Ca2+ flux and heart dysfunction. By four weeks post-knockout, SERCA2 protein levels are below 5% of baseline and isolated 4-week KO cardiomyocytes have greatly diminished contractility and Ca2+ handling. The in vivo phenotype at this time, however, is unexpectedly mild, and although all KO mice succumb to congestive heart failure, they do so from 7-10 weeks after knockout. This disconnect between the mild in vivo phenotype and severe ex vivo phenotype is intriguing and prompts the hypothesis that a systemic signaling pathway, such as adrenergic signaling, is responsible for sustaining function in KO animals. We found that in isolated Serca2 KO hearts, s-adrenergic stimulation elicits a robust inotropic and lusitropic response despite the near-complete lack of SERCA2 protein. This finding will be discussed as it is at odds with prevailing views of Ca2+ regulation of heart function.

Enhanced binding of calmodulin to the ryanodine receptor corrects contractile dysfunction in failing hearts

The channel function of the cardiac ryanodine receptor (RyR2) is modulated by calmodulin (CaM). However, the involvement of CaM in aberrant Ca(2+) release in diseased hearts remains unclear. Here, we investigated the pathogenic role of defective CaM binding to the RyR2 in the channel dysfunction associated with heart failure. The involvement of CaM in aberrant Ca(2+) release was assessed in normal and pacing-induced failing canine hearts. The apparent affinity of CaM for RyR2 was considerably lower in failing sarcoplasmic reticulum (SR) compared with normal SR. Thus, the amount of CaM bound to RyR2 was markedly decreased in failing myocytes. Expression of the CaM isoform Gly-Ser-His-CaM (GSH-CaM), which has much higher binding affinity than wild-type CaM for RyR1, restored normal CaM binding to RyR2 in both SR and myocytes of failing hearts. The Ca(2+) spark frequency (SpF) was markedly higher and the SR Ca(2+) content was lower in failing myocytes compared with normal myocytes. The incorporation of GSH-CaM into the failing myocytes corrected the aberrant SpF and SR Ca(2+) content to normal levels. Reduced CaM binding to RyR2 seems to play a critical role in the pathogenesis of aberrant Ca(2+) release in failing hearts. Correction of the reduced CaM binding to RyR2 stabilizes the RyR2 channel function and thereby restores normal Ca(2+) handling and contractile function to failing hearts.

FKBP12.6 overexpression does not protect against remodelling after myocardial infarction

Reducing the open probability of the ryanodine receptor (RyR) has been proposed to have beneficial effects in heart failure. We investigated whether conditional FKBP12.6 overexpression at the time of myocardial infarction (MI) could improve cardiac remodelling and cell Ca(2+) handling. Wild-type (WT) mice and mice overexpressing FKBP12.6 (Tg) were studied on average 7.5 ± 0.2 weeks after MI and compared with sham-operated mice for in vivo, myocyte function and remodelling. At baseline, unloaded cell shortening in Tg was not different from WT. The [Ca(2+)](i) transient amplitude was similar, but sarcoplasmic reticulum (SR) Ca(2+) content was larger in Tg, suggesting reduced fractional release. Spontaneous spark frequency was similar despite the increased SR Ca(2+) content, consistent with a reduced RyR channel open probability in Tg. After MI, left ventricular dilatation and myocyte hypertrophy were present in both groups, but more pronounced in Tg. Cell shortening amplitude was unchanged with MI in WT, but increased with MI in Tg. The amplitude of the [Ca(2+)](i) transient was not affected by MI in either genotype, but time to peak was increased; this was most pronounced in Tg. The SR Ca(2+) content and Na(+)- Ca(2+) exchanger function were not affected by MI. Spontaneous spark frequency was increased significantly after MI in Tg, and larger than in WT (at 4 Hz, 2.6 ± 0.4 sparks (100 μm)(-1) s(-1) in Tg MI versus 1.6 ± 0.2 sparks (100 μm)(-1) s(-1) in WT MI; P < 0.05). We conclude that FKPB12.6 overexpression can effectively reduce RyR open probability with maintained cardiomyocyte contraction. However, this approach appears insufficient to prevent and reduce post-MI remodelling, indicating that additional pathways may need to be targeted.

Role of Junctin Protein Interactions in Cellular Dynamics of Calsequestrin Polymer upon Calcium Perturbation

Calsequestrin (CSQ), the major intrasarcoplasmic reticulum calcium storage protein, undergoes dynamic polymerization and depolymerization in a Ca(2+)-dependent manner. However, no direct evidence of CSQ depolymerization in vivo with physiological relevance has been obtained. In the present study, live cell imaging analysis facilitated characterization of the in vivo dynamics of the macromolecular CSQ structure. CSQ2 appeared as speckles in the presence of normal sarcoplasmic reticulum (SR) Ca(2+) that were decondensed upon Ca(2+) depletion. Moreover, CSQ2 decondensation occurred only in the stoichiometric presence of junctin (JNT). When expressed alone, CSQ2 speckles remained unchanged, even after Ca(2+) depletion. FRET analysis revealed constant interactions between CSQ2 and JNT, regardless of the SR Ca(2+) concentration, implying that JNT is an essential component of the CSQ scaffold. In vitro solubility assay, electron microscopy, and atomic force microscopy studies using purified recombinant proteins confirmed Ca(2+) and JNT-dependent disassembly of the CSQ2 polymer. Accordingly, we conclude that reversible polymerization and depolymerization of CSQ are critical in SR Ca(2+) homeostasis.

Dynamic Calcium Movement Inside Cardiac Sarcoplasmic Reticulum During Release

Intra-sarcoplasmic reticulum (SR) free [Ca] ([Ca](SR)) provides the driving force for SR Ca release and is a key regulator of SR Ca release channel gating during normal SR Ca release or arrhythmogenic spontaneous Ca release events. However, little is known about [Ca](SR) spatiotemporal dynamics. To directly measure local [Ca](SR) with subsarcomeric spatiotemporal resolution during both normal global SR Ca release and spontaneous Ca sparks and to evaluate the quantitative implications of spatial [Ca](SR) gradients. Intact and permeabilized rabbit ventricular myocytes were subjected to direct simultaneous measurement of cytosolic [Ca] and [Ca](SR) and FRAP (fluorescence recovery after photobleach). We found no detectable [Ca](SR) gradients between SR release sites (junctional SR) and Ca uptake sites (free SR) during normal global Ca release, clear spatiotemporal [Ca](SR) gradients during isolated Ca blinks, faster intra-SR diffusion in the longitudinal versus transverse direction, 3- to 4-fold slower diffusion of fluorophores in the SR than in cytosol, and that intra-SR Ca diffusion varies locally, dependent on local SR connectivity. A computational model clarified why spatiotemporal gradients are more detectable in isolated local releases versus global releases and provides a quantitative framework for understanding intra-SR Ca diffusion. Intra-SR Ca diffusion is rapid, limiting spatial [Ca](SR) gradients during excitation-contraction coupling. Spatiotemporal [Ca](SR) gradients are apparent during Ca sparks, and these observations constrain models of dynamic Ca movement inside the SR. This has important implications for myocyte SR Ca handling, synchrony, and potentially arrhythmogenic spontaneous contraction.

Muscle weakness in Ryr1I4895T/WT knock-in mice as a result of reduced ryanodine receptor Ca2+ ion permeation and release from the sarcoplasmic reticulum

The type 1 isoform of the ryanodine receptor (RYR1) is the Ca2+ release channel of the sarcoplasmic reticulum (SR) that is activated during skeletal muscle excitation–contraction (EC) coupling. Mutations in the RYR1 gene cause several rare inherited skeletal muscle disorders, including malignant hyperthermia and central core disease (CCD). The human RYR1I4898T mutation is one of the most common CCD mutations. To elucidate the mechanism by which RYR1 function is altered by this mutation, we characterized in vivo muscle strength, EC coupling, SR Ca2+ content, and RYR1 Ca2+ release channel function using adult heterozygous Ryr1I4895T/+ knock-in mice (IT/+). Compared with age-matched wild-type (WT) mice, IT/+ mice exhibited significantly reduced upper body and grip strength. In spite of normal total SR Ca2+ content, both electrically evoked and 4-chloro-m-cresol–induced Ca2+ release were significantly reduced and slowed in single intact flexor digitorum brevis fibers isolated from 4–6-mo-old IT/+ mice. The sensitivity of the SR Ca2+ release mechanism to activation was not enhanced in fibers of IT/+ mice. Single-channel measurements of purified recombinant channels incorporated in planar lipid bilayers revealed that Ca2+ permeation was abolished for homotetrameric IT channels and significantly reduced for heterotetrameric WT:IT channels. Collectively, these findings indicate that in vivo muscle weakness observed in IT/+ knock-in mice arises from a reduction in the magnitude and rate of RYR1 Ca2+ release during EC coupling that results from the mutation producing a dominant-negative suppression of RYR1 channel Ca2+ ion permeation.

Dissociation of calmodulin from cardiac ryanodine receptor causes aberrant Ca2+ release in heart failure

Calmodulin (CaM) is well known to modulate the channel function of the cardiac ryanodine receptor (RyR2). However, the possible role of CaM on the aberrant Ca(2+) release in diseased hearts remains unclear. In this study, we investigated the state of RyR2-bound CaM and channel dysfunctions in pacing-induced failing hearts. The characteristics of CaM binding to RyR2 and the role of CaM on the aberrant Ca(2+) release were assessed in normal and failing canine hearts. The affinity of CaM binding to RyR2 was lower in failing sarcoplasmic reticulum (SR) than in normal SR. Addition of FK506, which dissociates FKBP12.6 from RyR2, to normal SR reduced the CaM-binding affinity. Dantrolene restored a normal level of the CaM-binding affinity in either FK506-treated (normal) SR or failing SR, suggesting that the defective inter-domain interaction between the N-terminal domain and the central domain of RyR2 (the therapeutic target of dantrolene) is involved in the reduction of the CaM-binding affinity in failing hearts. In saponin-permeabilized cardiomyocytes, the frequency of spontaneous Ca(2+) sparks was much more increased in failing cardiomyocytes than in normal cardiomyocytes, whereas the addition of a high concentration of CaM attenuated the aberrant increase of Ca(2+) sparks. The defective inter-domain interaction between N-terminal and central domains within RyR2 reduces the binding affinity of CaM to RyR2, thereby causing the spontaneous Ca(2+) release events in failing hearts. Correction of the defective CaM binding may be a new strategy to protect against the aberrant Ca(2+) release in heart failure.

Defective Interaction of Calmodulin With RyR2 Plays a Key Role in the Pathogenesis of Leaky Channel in Failing Hearts

Background: We previously reported that interaction between N-terminal1-600 and central domains2000-2500 of ryanodine receptor (RyR2) is defective (i.e. domain unzipping) in failing hearts. Here, we investigated the pathogenic role of calmodulin (CaM), one of the accessory proteins in RyR2, on Ca2+ release in pacing-induced failing hearts. Methods & Results: Cardiomyocytes were isolated from dog LV muscles {normal; 4-weeks rapid RV pacing (heart failure) }. To assess CaM binding to RyR2, sarcoplasmic reticulum (SR) was mixed with CaM-SANPAH conjugate (16 nM-1μM), followed by UV photolysis. Then, the RyR2-bound CaM was detected by Western blotting using anti-CaM antibody. The affinity of CaM binding to RyR2 was lower in failing SR than normal SR. In normal SR, the decreased CaM binding seen in failing SR was reproduced by addition of FK506, which was found to induce domain unzipping mimicking failing SR. In normal cardiomyocytes, Ca2+ spark frequency (CaSF) was markedly increased by adding FK506, but this increase was inhibited by addition of exogenous CaM. In failing cardiomyocytes, CaSF was higher than normal at baseline, but it was again reduced by exogenous CaM. Conclusion: The defective inter-domain interaction within RyR2 seems to increase spontaneous Ca2+ release events in failing SR, via the reduced CaM-binding to RyR2.

Triadin, not essential, but useful

Triadin, not essential, but useful

Abstract 5251: Defective Inter-domain Interaction May Cause Spontaneous Ca 2+ Release Via Reduced Affinity of Calmodulin Binding to RyR2 in Failing Hearts

We previously reported that interaction between N-terminal 1–600 and central domains 2000–2500 of ryanodine receptor (RyR2), harboring many mutation sites in CPVT, is defective (i.e. domain unzipping) in failing hearts. Here, we investigated the pathogenic role of calmodulin (CaM), one of the accessory proteins in RyR2, on Ca 2+ release in failing hearts. Sarcoplasmic reticulum (SR) vesicles were isolated from dog LV muscles {normal (N), n=6; 4-weeks rapid RV pacing (HF: n=6). To assess CaM binding to RyR2, SR was mixed with CaM-SANPAH conjugate (16nM-1μM), followed by UV photolysis. Then, the RyR2-bound CaM was detected by Western blotting using anti-CaM antibody. The affinity of CaM binding to RyR2 was lower in failing SR than normal SR (Kd: 47nM in HF: 19nM in N , p&lt;0.01). To assess the possible relationship between domain unzipping and CaM dissociation from RyR2, RyR2 was also fluorescently labeled with methylcoumarin acetamido (MCA) using DP 2460–2495 (DPc10), which harbors a mutation site in CPVT; R2474S, as a site-directing carrier. In failing SR, domain unzipping was already taken place, as evidenced by an increased accessibility of the bound MCA to a large-size fluorescence quencher. Interestingly, addition of FK506 (10 μM), which was found to dissociate FKBP12.6 from RyR2 and to induce domain unzipping mimicking failing SR, to normal SR indeed reduced the CaM binding affinity to RyR2. In saponin-permeabilized, failing cardiomyocytes ([Ca 2+ ]=buffered at 75 nM), the frequency of Ca 2+ sparks was markedly increased (SpF; s −1 ·100μm −1 : 13.9±0.63 in HF; 7.3±0.6 in N, p&lt;0.01). Addition of CaM (1 μM) in the presence of KN-93 (CaMKII inhibitor) inhibited the increase in SpF (7.9 ±0.41 , p&lt;0.01). This CaM’s effect was, however, markedly inhibited by co-addition of CaM-binding domain peptide within RyR2 (3583–3603) (10.4±0.65, p=ns), strongly suggesting that the re-binding of CaM to RyR2 corrects the abnormal Ca 2+ release in failing cardiomyocytes. In conclusion, the defective inter-domain interaction between N-terminal and central domains within RyR2 seems to increase spontaneous Ca 2+ release events in failing SR, via the reduced CaM-binding to RyR2. Fixing CaM binding to RyR2 may be protective against the diastolic Ca 2+ release in failing hearts.

Ca 2+ Influx Through T- and L-Type Ca 2+ Channels Have Different Effects on Myocyte Contractility and Induce Unique Cardiac Phenotypes

T-type Ca(2+) channels (TTCCs) are expressed in the developing heart, are not present in the adult ventricle, and are reexpressed in cardiac diseases involving cardiac dysfunction and premature, arrhythmogenic death. The goal of this study was to determine the functional role of increased Ca(2+) influx through reexpressed TTCCs in the adult heart. A mouse line with cardiac-specific, conditional expression of the alpha1G-TTCC was used to increase Ca(2+) influx through TTCCs. alpha1G hearts had mild increases in contractility but no cardiac histopathology or premature death. This contrasts with the pathological phenotype of a previously studied mouse with increased Ca(2+) influx through the L-type Ca(2+) channel (LTCC) secondary to overexpression of its beta2a subunit. Although alpha1G and beta2a myocytes had similar increases in Ca(2+) influx, alpha1G myocytes had smaller increases in contraction magnitude, and, unlike beta2a myocytes, there were no increases in sarcoplasmic reticulum Ca(2+) loading. Ca(2+) influx through TTCCs also did not induce normal sarcoplasmic reticulum Ca(2+) release. alpha1G myocytes had changes in LTCC, SERCA2a, and phospholamban abundance, which appear to be adaptations that help maintain Ca(2+) homeostasis. Immunostaining suggested that the majority of alpha1G-TTCCs were on the surface membrane. Osmotic shock, which selectively eliminates T-tubules, induced a greater reduction in L- versus TTCC currents. These studies suggest that T- and LTCCs are in different portions of the sarcolemma (surface membrane versus T-tubules) and that Ca(2+) influx through these channels induce different effects on myocyte contractility and lead to distinct cardiac phenotypes.

Reduced Threshold for Luminal Ca2+ Activation of RyR1 Underlies a Causal Mechanism of Porcine Malignant Hyperthermia

Naturally occurring mutations in the skeletal muscle Ca(2+) release channel/ryanodine receptor RyR1 are linked to malignant hyperthermia (MH), a life-threatening complication of general anesthesia. Although it has long been recognized that MH results from uncontrolled or spontaneous Ca(2+) release from the sarcoplasmic reticulum, how MH RyR1 mutations render the sarcoplasmic reticulum susceptible to volatile anesthetic-induced spontaneous Ca(2+) release is unclear. Here we investigated the impact of the porcine MH mutation, R615C, the human equivalent of which also causes MH, on the intrinsic properties of the RyR1 channel and the propensity for spontaneous Ca(2+) release during store Ca(2+) overload, a process we refer to as store overload-induced Ca(2+) release (SOICR). Single channel analyses revealed that the R615C mutation markedly enhanced the luminal Ca(2+) activation of RyR1. Moreover, HEK293 cells expressing the R615C mutant displayed a reduced threshold for SOICR compared with cells expressing wild type RyR1. Furthermore, the MH-triggering agent, halothane, potentiated the response of RyR1 to luminal Ca(2+) and SOICR. Conversely, dantrolene, an effective treatment for MH, suppressed SOICR in HEK293 cells expressing the R615C mutant, but not in cells expressing an RyR2 mutant. These data suggest that the R615C mutation confers MH susceptibility by reducing the threshold for luminal Ca(2+) activation and SOICR, whereas volatile anesthetics trigger MH by further reducing the threshold, and dantrolene suppresses MH by increasing the SOICR threshold. Together, our data support a view in which altered luminal Ca(2+) regulation of RyR1 represents a primary causal mechanism of MH.

Unexpected Structural and Functional Consequences of the R33Q Homozygous Mutation in Cardiac Calsequestrin

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disorder characterized by life threatening arrhythmias elicited by physical and emotional stress in young individuals. The recessive form of CPVT is associated with mutation in the cardiac calsequestrin gene (CASQ2). We engineered and characterized a homozygous CASQ2(R33Q/R33Q) mouse model that closely mimics the clinical phenotype of CPVT patients. CASQ2(R33Q/R33Q) mice develop bidirectional VT on exposure to environmental stress whereas CASQ2(R33Q/R33Q) myocytes show reduction of the sarcoplasmic reticulum (SR) calcium content, adrenergically mediated delayed (DADs) and early (EADs) afterdepolarizations leading to triggered activity. Furthermore triadin, junctin, and CASQ2-R33Q proteins are significantly decreased in knock-in mice despite normal levels of mRNA, whereas the ryanodine receptor (RyR2), calreticulin, phospholamban, and SERCA2a-ATPase are not changed. Trypsin digestion studies show increased susceptibility to proteolysis of mutant CASQ2. Despite normal histology, CASQ2(R33Q/R33Q) hearts display ultrastructural changes such as disarray of junctional electron-dense material, referable to CASQ2 polymers, dilatation of junctional SR, yet normal total SR volume. Based on the foregoings, we propose that the phenotype of the CASQ2(R33Q/R33Q) CPVT mouse model is portrayed by an unexpected set of abnormalities including (1) reduced CASQ2 content, possibly attributable to increased degradation of CASQ2-R33Q, (2) reduction of SR calcium content, (3) dilatation of junctional SR, and (4) impaired clustering of mutant CASQ2.

Triadin is a critical determinant of cellular Ca cycling and contractility in the heart

Triadin is involved in the regulation of cardiac excitation-contraction coupling. However, the extent of its contribution to the regulation of sarcoplasmic reticulum (SR) Ca release remains unclear, because overexpression of triadin in single-transgenic mice was associated with the downregulation of its homologous protein, junctin. In the present study, this problem was circumvented by cross-breeding of mice with heart-directed overexpression of triadin and junctin (JxT). This resulted in a stable approximately threefold expression of total triadin but unchanged junctin protein. Transgenic mice exhibited cardiac hypertrophy and structural abnormalities of myofibrils. Measurement of cardiac function by echocardiography and edge detection in myocytes revealed an impaired relaxation in JxT mice. The stimulation of beta-adrenergic receptors resulted in a depressed contractility and an impaired relaxation in catheterized hearts and myocytes of JxT mice. The use of a maximum stimulation frequency (5 Hz) was associated with both a lower shortening and relengthening in isolated myocytes of JxT mice. The contractile effects in JxT myocytes were paralleled by similar changes of the intracellular Ca concentration ([Ca](i)) peak amplitude and Ca transient decay kinetics at basal conditions, under administration of isoproterenol, and with high-frequency stimulation. Finally, we found a higher caffeine-induced [Ca](i) peak amplitude in JxT myocytes. Our data show that the stable expression of triadin, independent of junctin expression, resulted in cardiac hypertrophy, prolonged basal relaxation, a depressed response to beta-adrenergic agonists, and altered Ca transients. Thus the maintenance of triadin expression is essential for normal SR Ca cycling and contractile function.

Correction of Defective Interdomain Interaction Within Ryanodine Receptor by Antioxidant Is a New Therapeutic Strategy Against Heart Failure

Defective interdomain interaction within the ryanodine receptor (RyR2) seems to play a key role in the pathogenesis of heart failure, as shown in recent studies. In the present study we investigated the effect of oxidative stress on the interdomain interaction, its outcome in the cardiac function in heart failure, and the possibility of preventing the problem with antioxidants. Sarcoplasmic reticulum (SR) vesicles were isolated from dog left ventricular (LV) muscle (normal or rapid ventricular pacing for 4 weeks with or without the antioxidant edaravone). In the edaravone-treated paced dogs (EV+), but not in the untreated paced dogs (EV-), normal cardiac function was restored almost completely. In the SR vesicles isolated from the EV-, oxidative stress of the RyR2 (reduction in the number of free thiols) was severe, but it was negligible in EV+. The oxidative stress of the RyR2 destabilized interdomain interactions within the RyR2 (EV-), but its effect was reversed in EV+. Abnormal Ca2+ leak through the RyR2 was found in EV- but not in EV+. The amount of the RyR2-bound FKBP12.6 was less in EV- than in normal dogs, whereas it was restored almost to a normal amount in EV+. The NO donor 3-morpholinosydnonimine (SIN-1) reproduced, in normal SR, several abnormal features seen in failing SR, such as defective interdomain interaction and abnormal Ca2+ leak. Both cell shortening and Ca2+ transients were impaired by SIN-1 in isolated normal myocytes, mimicking the pathophysiological conditions in failing myocytes. Incubation of failing myocytes with edaravone restored the normal properties. During the development of heart failure, edaravone ameliorated the defective interdomain interaction of the RyR2. This prevented Ca2+ leak and LV remodeling, leading to an improvement of cardiac function and an attenuation of LV remodeling.

Can Novel Therapies for Arrhythmias Caused by Spontaneous Sarcoplasmic Reticulum Ca 2+ Release be Developed Using Mouse Models?

See related article, pages e77–e82 Ventricular arrhythmias are a significant cause of premature death in Western society and occur both in the absence and presence of heart disease. For example, in heart failure close to 50% of patients die of lethal forms of ventricular arrhythmias. In spite of a large amount of basic and clinical investigations, current pharmacotherapy for ventricular arrhythmias is inadequate, demonstrating the need for novel therapeutic approaches. Abnormal automaticity is responsible for induction and maintenance of different types of ventricular tachycardias. The form of abnormal automaticity that results from abnormal release of Ca2+ from a Ca2+ overloaded sarcoplasmic reticulum (SR), referred to as a “triggered arrhythmia”, is the topic of this editorial. In vitro studies have shown that increasing the SR Ca2+ load beyond a critical level causes spontaneous opening of the SR Ca2+ release channel (ryanodine receptor; RYR) and results in spontaneous SR Ca2+ release.1 The subsequent increase in cytosolic [Ca] activates an inward current through the sarcolemmal Na+/ Ca2+ exchanger which causes membrane depolarization.1,2 Spontaneous SR Ca2+ release during the diastolic interval causes depolarization (delayed after depolarizations; DADs) which has been shown to result in action potentials in single cells and in the intact heart when the aberrant release is adequately synchronized within and among cardiac myocytes.3,4 Factors that lead to DADs include increased heart rate and catecholamine stress, which both induce triggered arrhythmias by increasing SR Ca2+ loading.3 Whereas many molecules are involved in SR Ca2+ overload (DAD) related arrhythmias, abnormal opening …

FKBP12.6-Mediated Stabilization of Calcium-Release Channel (Ryanodine Receptor) as a Novel Therapeutic Strategy Against Heart Failure

The development of heart failure is tightly correlated with a decrease in the stoichiometric ratio for FKBP12.6 binding to the ryanodine receptor (RyR) in the sarcoplasmic reticulum (SR). We report that a new drug, the 1,4-benzothiazepine derivative JTV519, reverses this pathogenic process. JTV519 is known to have a protective effect against Ca2+ overload-induced myocardial injury. Heart failure was produced by 4 weeks of rapid right ventricular pacing, with or without JTV519; SR were then isolated from dog left ventricular (LV) muscles. First, in JTV519-treated dogs, no signs of heart failure were observed after 4 weeks of chronic right ventricular pacing, LV systolic and diastolic functions were largely preserved, and LV remodeling was prevented. Second, JTV519 acutely inhibited both the FK506-induced Ca2+ leak from RyR in normal SR and the spontaneous Ca2+ leak in failing SR. Third, there was no abnormal Ca2+ leak in SR vesicles isolated from JTV519-treated hearts. Fourth, in JTV519-treated hearts, both the stoichiometry of FKBP12.6 binding to RyR and the amount of RyR-bound FKBP12.6 were restored toward the values seen in normal SR. Fifth, in JTV519-untreated hearts, RyR was PKA-hyperphosphorylated, whereas it was reversed in JTV519-treated hearts, returning the channel phosphorylation toward the levels seen in normal hearts. During the development of experimental heart failure, JTV519 prevented the amount of RyR-bound FKBP12.6 from decreasing. This in turn reduced the abnormal Ca2+ leak through the RyR, prevented LV remodeling, and led to less severe heart failure.