Functional Calsequestrin-1 Is Expressed in the Heart and Its Deficiency Is Causally Related to Malignant Hyperthermia-Like Arrhythmia

Background: Calsequestrins (Casqs), comprising the Casq1 and Casq2 isoforms, buffer Ca 2+ and regulate its release in the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle, respectively. Human inherited diseases associated with mutations in CASQ1 or CASQ2 include malignant hyperthermia/environmental heat stroke (MH/EHS) and catecholaminergic polymorphic ventricular tachycardia. However, patients with an MH/EHS event often suffer from arrhythmia for which the underlying mechanism remains unknown. Methods: Working hearts from conventional ( Casq1 -KO) and cardiac-specific ( Casq1 -CKO) Casq1 knockout mice were monitored in vivo and ex vivo by electrocardiogram and electrical mapping, respectively. MH was induced by 2% isoflurane and treated intraperitoneally with dantrolene. Time-lapse imaging was used to monitor intracellular Ca 2+ activity in isolated mouse cardiomyocytes or neonatal rat ventricular myocytes (NRVMs) with knockdown, over-expression or truncation of the Casq1 gene. Conformational change in both Casqs was determined by crosslinking Western blot analysis. Results: Like MH/EHS patients, Casq1 -KO and Casq1 -CKO mice had faster basal heart rate, and ventricular tachycardia upon exposure to 2% isoflurane, which could be relieved by dantrolene. Basal sinus tachycardia and ventricular ectopic electrical triggering also occurred in Casq1 -KO hearts ex vivo . Accordingly, the ventricular cardiomyocytes from Casq1 -CKO mice displayed dantrolene-sensitive increased Ca 2+ waves and diastole premature Ca 2+ transients/oscillations upon isoflurane. NRVMs with Casq1-knockdown had enhanced spontaneous Ca2+ sparks/transients upon isoflurane, while cells over-expressing Casq1 exhibited decreased Ca2+ sparks/transients that were absent in cells with truncation of 9 amino acids at the C-terminus of Casq1. Structural evaluation showed that most of the Casq1 protein was present as a polymer and physically interacted with RyR2 in the ventricular SR. The Casq1 isoform was also expressed in human myocardium. Mechanistically, exposure to 2% isoflurane or heating at 41ºC induced Casq1 oligomerization in mouse ventricular and skeletal muscle tissues, leading to a reduced Casq1/RyR2 interaction and increased RyR2 activity in the ventricle. Conclusions: Casq1 is expressed in the heart, where it regulates SR Ca 2+ release and heart rate. Casq1 deficiency independently causes MH/EHS-like ventricular arrhythmia by trigger-induced Casq1 oligomerization and a relief of its inhibitory effect on RyR2-mediated Ca 2+ release, thus revealing a new inherited arrhythmia and a novel mechanism for MH/EHS arrhythmogenesis.

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Selective toxic effects of tamoxifen on osteoclasts: comparison with the effects of oestrogen

Journal of Endocrinology ◽

10.1677/joe.0.1490503 ◽

1996 ◽

Vol 149 (3) ◽

pp. 503-508 ◽

Cited By ~ 20

Author(s):

T R Arnett ◽

R Lindsay ◽

J M Kilb ◽

B S Moonga ◽

M Spowage ◽

...

Keyword(s):

Mononuclear Cells ◽

Time Lapse ◽

Bone Cell ◽

Neonatal Rat ◽

Pit Formation ◽

Cell Counts ◽

Bone Cell Cultures ◽

Dying Cells

Abstract We investigated the actions of the trans- and cis-isomers of tamoxifen on the function of neonatal rat osteoclasts in vitro. Both compounds inhibited resorption pit formation by osteoclast-containing mixed bone cell cultures incubated for 24 h on cortical bone slices. Cell counts revealed that the inhibition was closely related to a cytotoxic effect, to which osteoclasts appeared particularly sensitive. Partial inhibition of resorption was seen in the presence of 2 μm trans-tamoxifen, whereas complete abolition of resorption and osteoclast viability occurred with 10 μm trans-tamoxifen; survival of mononuclear cells was unimpaired at either concentration. Cis-tamoxifen appeared to be slightly more toxic, with significant inhibitions of osteoclast viability and thus resorption pit formation at a concentration of 2 μm, and also of mononuclear cell numbers at 10 μm. Time-lapse video observations indicated that osteoclast death occurred rapidly (within 2–3 h) following exposure to 10 μm of either trans-tamoxifen or cis-tamoxifen. The morphological appearance of the dying cells was consistent with apoptosis. These results may help to explain the anti-resorptive action of tamoxifen seen in vivo in rats and humans. In contrast, oestradiol-17β consistently exerted no significant effects on resorption pit formation by rat osteoclasts over 24 h, even at grossly supraphysiological concentrations (up to 10 μm). Journal of Endocrinology (1996) 149, 503–508

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Abstract 1079: Prevention of Fatal Arrhythmia in a Catecholaminergic Polymorphic Ventricular Tachycardia Mouse Model Carrying Calsequestrin-2 Mutation

Circulation ◽

10.1161/circ.116.suppl_16.ii_216-a ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Hiroko Wakimoto ◽

Ronny Alcalai ◽

Lei Song ◽

Michael Arad ◽

Christine E Seidman ◽

...

Keyword(s):

Ventricular Tachycardia ◽

Mouse Model ◽

Peak Height ◽

Mutant Mice ◽

Catecholaminergic Polymorphic Ventricular Tachycardia ◽

Polymorphic Ventricular Tachycardia ◽

Study Drug Administration ◽

Intracellular Ca

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmia syndrome caused by mutations in the ryanodine receptor (RyR2) or calsequestrin-2 (CASQ2) genes and characterized by exercise or emotional stress-induced sudden death. Beta-adrenergic blockers are only partially effective and other agents have not been widely tested. Recent studies have shown that CPVT is mediated by increased Ca 2+ leak through the RyR2 channel. Our aim was to determine whether agents that inhibit intracellular Ca 2+ leak can effectively prevent CPVT. Methods: The efficacy of intraperitoneal (IP) propranolol (1mcg/g), Mg 2+ (0.002mEq/g), verapamil (8 mcg/g) and diltiazem (8 mcg/g) were tested both in vivo and in vitro using CASQ2 mutant mouse CPVT model. In vivo studies included ambulatory ECG recordings at rest and following epinephrine stress (0.4 mcg/g IP) at baseline and after study drug administration. Experiments for each drug were performed on separate days to avoid confounding effects. In vitro studies included intracellular Ca 2+ transient analysis on isolated cardiomyocytes from mutant mice with and without epinephrine (5.5 μM). Results: All 4 drugs restored sinus rhythm and reduced the frequency of VT episodes induced by epinephrine in CASQ2 mutant mice. Only verapamil completely prevented epinephrine-induced VT in 87% of the mice (p<0.01). Cardiomyocyte studies in basal conditions revealed that Mg 2+ and verapamil inhibited sarcomere contraction and normalized the prolonged Ca 2+ reuptake period in CASQ2 mutants, but did not decrease baseline Ca 2+ peak height. Epinephrine-stressed mutant myocytes had increased diastolic Ca 2+ levels, lower Ca 2+ peak height and spontaneous SR Ca 2+ release events that were partially prevented by verapamil and Mg 2+ . Verapamil was more effective than Mg 2+ in reducing the frequency of spontaneous Ca 2+ releases induced by epinephrine. Conclusions: All 4 agents can inhibit ventricular arrhythmia in CPVT mouse model; however verapamil appears most effective in preventing arrhythmia in vivo and in modifying intracellular abnormal calcium handling. Calcium antagonists might have therapeutic value in CPVT and other RyR2-mediated arrhythmias and should be considered for human clinical studies.

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Abstract 9815: Nadolol Seems to Be Superior to Selective Beta Blockers in Patients With Catecholaminergic Polymorphic Ventricular Tachycardia: Is a Smaller Arrhythmic Window Part of the Explanation?

Circulation ◽

10.1161/circ.132.suppl_3.9815 ◽

2015 ◽

Vol 132 (suppl_3) ◽

Author(s):

Ida Skrinde Leren ◽

Jørg Saberniak ◽

Eman Majid ◽

Trine F Haland ◽

Thor Edvardsen ◽

...

Keyword(s):

Heart Rate ◽

Ventricular Tachycardia ◽

Beta Blockers ◽

Maximum Tolerated Dose ◽

Catecholaminergic Polymorphic Ventricular Tachycardia ◽

Polymorphic Ventricular Tachycardia ◽

Maximum Heart Rate ◽

Exercise Tests ◽

Severe Arrhythmia ◽

First Choice

Introduction: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inheritable arrhythmogenic disease, and typically presents as syncope or sudden cardiac death during exercise. Beta blockers are first choice therapy but little is known about antiarrhythmic effects of different beta blockers in CPVT. Nadolol has shown superior antiarrhythmic effect in other cardiomyopathies. Hypothesis: We hypothesized that nadolol is superior to selective beta blockers in arrhythmia protection in CPVT patients. Methods: We included 34 CPVT patients (age 34±19 yrs, 44% female, 88% RYR2 mutation positive). We serially performed 2 bicycle exercise tests in each patient; 1)>6 weeks on maximum tolerated dose of selective beta blockers. 2)>6 weeks on maximum tolerated dose of nadolol. We recorded resting and maximum heart rate (HR), HR at first arrhythmia and the most severe arrhythmia occurring. Arrhythmic window was defined as the difference between maximum HR and HR at first arrhythmia. Severity of arrhythmias was scored as arrhythmic score: no arrhythmias (0point), single ventricular extra systoles (1point), bigemini (2points), couplets (3points) and nonsustained VT (4points). Results: Resting HR was similar on nadolol and selective beta blockers (54±10bpm vs. 56±14bpm, p=0.50), while maximum heart rate was lower on nadolol (122±21bpm vs. 139±24bpm, p<0.01). First arrhythmias occurred at similar HR at both exercise tests (113±21bpm vs. 113±19bpm, p=1.0). Consequently, arrhythmic window was smaller during nadolol treatment (17±10bpm vs. 32±26bpm, p=0.03) (Figure) and also the arrhythmic score was lower than on selective beta blockers (1.1±1.2 vs. 2.4±0.9, p<0.01). Conclusion: Arrhythmic score was lower on nadolol compared to selective beta blockers. Also, arrhythmic window, representing the span of heart rates where arrhythmias may occur and progress in severity, was smaller. This suggests that nadolol should be the beta blocker of choice in CPVT patients.

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Abstract 15420: Resolving a Controversy: Inhibition of Cardiac Ryanodine Receptors is the Principal Mechanism of Antiarrhythmic Action of Flecainide in Catecholaminergic Polymorphic Ventricular Tachycardia

Circulation ◽

10.1161/circ.142.suppl_3.15420 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

Dmytro O Kryshtal ◽

Daniel J Blackwell ◽

Christian L Egly ◽

Abigail N Smith ◽

Suzanne M Batiste ◽

...

Keyword(s):

Ventricular Tachycardia ◽

Sodium Channel ◽

Sodium Channels ◽

Catecholaminergic Polymorphic Ventricular Tachycardia ◽

Polymorphic Ventricular Tachycardia ◽

Antiarrhythmic Action ◽

Channel Block ◽

Principal Mechanism ◽

Cardiac Sodium Channels

Rationale: The class Ic antiarrhythmic drug flecainide prevents ventricular tachyarrhythmia in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), a disease caused by hyperactive cardiac ryanodine receptor (RyR2) calcium (Ca) release. Although flecainide inhibits single RyR2 channels in vitro , reports have claimed that RyR2 inhibition by flecainide is not relevant for its mechanism of antiarrhythmic action and concluded that sodium channel block alone is responsible for flecainide’s efficacy in CPVT. Objective: To determine whether RyR2 block independently contributes to flecainide’s efficacy for suppressing spontaneous sarcoplasmic reticulum (SR) Ca release and for preventing ventricular tachycardia in vivo . Methods and Results: We synthesized N -methyl flecainide analogues (QX-FL and NM-FL) and showed that N -methylation reduces flecainide’s inhibitory potency on RyR2 channels but not on cardiac sodium channels. Antiarrhythmic efficacy was tested utilizing a calsequestrin knockout (Casq2-/-) CPVT mouse model. In membrane-permeabilized Casq2-/- cardiomyocytes — lacking intact sarcolemma and devoid of sodium channel contribution — flecainide, but not its analogues, suppressed RyR2-mediated Ca release at clinically relevant concentrations. In voltage-clamped, intact Casq2-/- cardiomyocytes pretreated with tetrodotoxin (TTX) to inhibit sodium channels and isolate the effect of flecainide on RyR2, flecainide significantly reduced the frequency of spontaneous SR Ca release, while QX-FL and NM-FL did not. In vivo , flecainide effectively suppressed catecholamine-induced ventricular tachyarrhythmias in Casq2-/- mice, whereas NM-FL did not, despite comparable sodium channel block. Conclusions: Flecainide remains an effective inhibitor of RyR2-mediated arrhythmogenic Ca release even when cardiac sodium channels are blocked. In mice with CPVT, sodium channel block alone was not enough to prevent arrhythmias. Hence, RyR2 inhibition by flecainide is critical for its mechanism of antiarrhythmic action.

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RYR2 p.R169L mutation and left ventricular hypertrophy in a child with emotion-triggered sudden death

Cardiology in the Young ◽

10.1017/s1047951120001316 ◽

2020 ◽

Vol 30 (7) ◽

pp. 1039-1042

Author(s):

Utkarsh Kohli ◽

Lisa Kuntz ◽

Hemal M. Nayak

Keyword(s):

Ventricular Tachycardia ◽

Left Ventricular Hypertrophy ◽

Ventricular Hypertrophy ◽

Left Ventricular ◽

Catecholaminergic Polymorphic Ventricular Tachycardia ◽

Polymorphic Ventricular Tachycardia ◽

Exercise Induced ◽

In Vivo Effects

AbstractCatecholaminergic polymorphic ventricular tachycardia is a rare (prevalence: 1/10,000) channelopathy characterised by exercise-induced or emotion-triggered ventricular arrhythmias. There is an overall paucity of genotype-phenotype correlation studies in patients with catecholaminergic polymorphic ventricular tachycardia, and in vitro and in vivo effects of individual mutations have not been well characterised. We report an 8-year-old child who carried a mutation in the coding exon 8 of RYR2 (p.R169L) and presented with emotion-triggered sudden cardiac death. He was also found to have left ventricular hypertrophy, a combination which has not been reported before. We discuss the association between genetic variation in RYR2, particularly mutations causing replacement of arginine at position 169 of RYR2 and structural cardiac abnormalities.

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Citrulline directly modulates muscle protein synthesis via the PI3K/MAPK/4E-BP1 pathway in a malnourished state: evidence from in vivo, ex vivo, and in vitro studies

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00203.2016 ◽

2017 ◽

Vol 312 (1) ◽

pp. E27-E36 ◽

Cited By ~ 16

Author(s):

Servane Le Plénier ◽

Arthur Goron ◽

Athanassia Sotiropoulos ◽

Eliane Archambault ◽

Chantal Guihenneuc ◽

...

Keyword(s):

Protein Synthesis ◽

Ex Vivo ◽

Muscle Protein ◽

Muscle Protein Synthesis ◽

Fold Increase ◽

Underlying Mechanism ◽

Muscle Homeostasis

Citrulline (CIT) is an endogenous amino acid produced by the intestine. Recent literature has consistently shown CIT to be an activator of muscle protein synthesis (MPS). However, the underlying mechanism is still unknown. Our working hypothesis was that CIT might regulate muscle homeostasis directly through the mTORC1/PI3K/MAPK pathways. Because CIT undergoes both interorgan and intraorgan trafficking and metabolism, we combined three approaches: in vivo, ex vivo, and in vitro. Using a model of malnourished aged rats, CIT supplementation activated the phosphorylation of S6K1 and 4E-BP1 in muscle. Interestingly, the increase in S6K1 phosphorylation was positively correlated ( P < 0.05) with plasma CIT concentration. In a model of isolated incubated skeletal muscle from malnourished rats, CIT enhanced MPS (from 30 to 80% CIT vs. Ctrl, P < 0.05), and the CIT effect was abolished in the presence of wortmannin, rapamycin, and PD-98059. In vitro, on myotubes in culture, CIT led to a 2.5-fold increase in S6K1 phosphorylation and a 1.5-fold increase in 4E-BP1 phosphorylation. Both rapamycin and PD-98059 inhibited the CIT effect on S6K1, whereas only LY-294002 inhibited the CIT effect on both S6K1 and 4E-BP1. These findings show that CIT is a signaling agent for muscle homeostasis, suggesting a new role of the intestine in muscle mass control.

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Ginseng Is Useful to Enhance Cardiac Contractility in Animals

BioMed Research International ◽

10.1155/2014/723084 ◽

2014 ◽

Vol 2014 ◽

pp. 1-9 ◽

Cited By ~ 3

Author(s):

Jia-Wei Lin ◽

Yih-Giun Cherng ◽

Li-Jen Chen ◽

Ho-Shan Niu ◽

Chen Kuei Chang ◽

...

Keyword(s):

Troponin I ◽

Ex Vivo ◽

Cardiac Contractility ◽

Cardiac Cells ◽

Neonatal Rat ◽

Peroxisome Proliferator ◽

Isolated Hearts ◽

Blood Pressure Data ◽

Peroxisome Proliferator Activated Receptors

Ginseng has been shown to be effective on cardiac dysfunction. Recent evidence has highlighted the mediation of peroxisome proliferator-activated receptors (PPARs) in cardiac function. Thus, we are interested to investigate the role of PPARδin ginseng-induced modification of cardiac contractility. The isolated hearts in Langendorff apparatus and hemodynamic analysis in catheterized rats were applied to measure the actions of ginsengex vivoandin vivo. In normal rats, ginseng enhanced cardiac contractility and hemodynamicdP/dtmaxsignificantly. Both actions were diminished by GSK0660 at a dose enough to block PPARδ. However, ginseng failed to modify heart rate at the same dose, although it did produce a mild increase in blood pressure. Data of intracellular calcium level and Western blotting analysis showed that both the PPARδexpression and troponin I phosphorylation were raised by ginseng in neonatal rat cardiomyocyte. Thus, we suggest that ginseng could enhance cardiac contractility through increased PPARδexpression in cardiac cells.

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Abstract 13914: Ischemic Preconditioning Induces MG53 Secretion From the Heart Through H 2 O 2 /PKC-delta Signaling

Circulation ◽

10.1161/circ.142.suppl_3.13914 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

Dan Shan ◽

Yan Zhang ◽

Rui-ping Xiao

Keyword(s):

Ischemic Preconditioning ◽

Cell Injury ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Embryonic Stem ◽

Underlying Mechanism ◽

Neonatal Rat ◽

Control Group ◽

Systemic Delivery

Introduction: Ischemic heart disease is the leading cause of morbidity and mortality worldwide. Ischemic preconditioning (IPC) is the most powerful intrinsic protection against cardiac ischemia/reperfusion (I/R) injury. Previous studies have shown that a multifunctional TRIM family protein, MG53 (or TRIM72), not only plays an essential role in IPC-mediated cardioprotection, but also as a myokine/cardiokine, can be secreted from the heart and skeletal muscle in response to metabolic stress in addition to its intracellular actions. Hypothesis: We hypothesized that IPC-mediated cardioprotection is causally related to MG53 secretion and figured out the underlying mechanism. Methods and Results: Using proteomic analysis in conjunction with genetic and pharmacological approaches, we examined MG53 secretion in response to IPC and explored the underlying mechanism using rodents in in vivo , isolated perfused hearts, and cultured neonatal rat ventricular cardiomyocytes. IPC profoundly increased perfusate MG53 levels in mouse hearts by 5.50 ± 0.32 and 4.26 ± 0.40 folds from baseline over 0-60 and 60-120 min of reperfusion, respectively. Mechanistically, IPC-induced MG53 secretion is dependent on H 2 O 2 -evoked, Src-mediated phosphorylation of PKC-δ-Y311. Functionally, systemic delivery of recombinant human MG53 proteins (rhMG53) to mimic elevated circulating MG53 not only restored IPC function in MG53-deficient mice, but also protected rodent hearts from I/R injury even in the absence of IPC. Treatment of rhMG53 overtly decreased the infarct size (IF/AAR) induced by I/R compared to the BSA-treated control group (11.9 ± 1.8% vs 27.3 ± 2.0%, P <0.01), and reduced the mortality from 44.7% to 5.3% in rats. Moreover, H 2 O 2 augmented MG53 secretion, and MG53 knockdown exacerbated H 2 O 2 -induced cell injury in human embryonic stem cell-derived cardiomyocytes. Conclusions: In conclusion, IPC and oxidative stress can trigger MG53 secretion from the heart via an H 2 O 2 -PKC-δ-dependent mechanism, and secreted MG53 acts as an essential factor conveying IPC-induced cardioprotection against ischemia/reperfusion injury. Recombinant MG53 proteins can be developed into a novel treatment for various diseases of human heart in which the endogenous MG53 is low.

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