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Ryanodine receptors (RyRs), the intracellular Ca2+ release channels, are expressed in T lymphocytes and other types of immune cells. Modulation of RyRs has been shown to affect T cell functions in vitro and immune responses in vivo. The effects of modulation of RyRs on the development of autoimmune diseases have not been investigated. Here we studied how modulation of RyRs through administration of RyR inhibitor dantrolene or introducing a gain-of-function RYR1-p.R163C mutation affects clinical progression of experimental autoimmune encephalomyelitis (EAE) in mice, a T cell-mediated autoimmune neuroinflammatory disease. We found that daily intraperitoneal administration of 5 or 10 mg/kg dantrolene beginning at the time of EAE induction significantly reduced the severity of EAE clinical symptoms and dampened inflammation in the spinal cord. The protective effect of dantrolene on EAE was reversible. Dantrolene administration elicited dose-dependent skeletal muscle weakness: mice that received 10 mg/kg dose developed a waddling gait, while 5 mg/kg dantrolene dose administration produced a reduction in four-limb holding impulse values. Mice bearing the gain-of-function RYR1-p.R163C mutation developed the EAE clinical symptoms faster and more severely than wild-type mice. This study demonstrates that RyRs play a significant role in EAE pathogenesis and suggests that inhibition of RyRs with low doses of dantrolene may have a protective effect against autoimmunity and inflammation in humans.

Ryanodine receptor (RYR) mutations confer stress-triggered malignant hyperthermia (MH) susceptibility. Dietary caffeine (CAF) is the most commonly consumed psychoactive compound by humans. CAF-triggered Ca2+ release and its influences on skeletal muscle contractility are widely used as experimental tools to study RYR function/dysfunction and diagnose MH susceptibility. We hypothesize that dietary CAF achieving blood levels measured in human plasma exacerbates the penetrance of RYR1 MH susceptibility mutations triggered by gaseous anesthetic, affecting both central and peripheral adverse responses. Heterozygous R163C-RYR1 (HET) MH susceptible mice are used to investigate the influences of dietary CAF on both peripheral and central responses before and after induction of halothane (HAL) maintenance anesthesia under experimental conditions that maintain normal core body temperature. HET mice receiving CAF (plasma CAF 893 ng/ml) have significantly shorter times to respiratory arrest compared with wild type, without altering blood chemistry or displaying hyperthermia or muscle rigor. Intraperitoneal bolus dantrolene before HAL prolongs time to respiratory arrest. A pilot electrographic study using subcutaneous electrodes reveals that dietary CAF does not alter baseline electroencephalogram (EEG) total power, but significantly shortens delay to isoelectric EEG, which precedes respiratory and cardiac arrest. CAF ± HAL are studied on RYR1 single-channel currents and HET myotubes to define molecular mechanisms of gene-by-environment synergism. Strong pharmacological synergism between CAF and HAL is demonstrated in both single-channel and myotube preparations. Central and peripheral nervous systems mediate adverse responses to HAL in a HET model of MH susceptibility exposed to dietary CAF, a modifiable lifestyle factor that may mitigate risks of acute and chronic diseases associated with RYR1 mutations. SIGNIFICANCE STATEMENT: Dietary caffeine at a human-relevant dose synergi

La hemorragia postparto es considerada una emergencia obstétrica y es una de las cinco principales causas de mortalidad materna a nivel mundial. La mortalidad depende tanto de la salud general de la mujer embarazada así como de los recursos para su tratamiento agudo. La incidencia depende de los criterios utilizados para su diagnóstico. La determinación de la causa e intervención temprana son de gran importancia ya que la mayoría de las muertes por sangrado postparto ocurren dentro de las primeras cuatro horas del puerperio y el monitoreo cercano es indispensable para evaluar y conocer la agresividad de la intervención que se dará a cada paciente., Postpartum hemorrhage is considered an obstetric emergency and is one of the five leading causes of maternal mortality worldwide. Mortality depends on both the general health of pregnant women and the resources for their acute treatment. The incidence will depend on the criteria used for its diagnosis. Detecting the main cause and having an early intervention is important since the majority of deaths from postpartum bleeding occur within the first four hours of the puerperium. Also, a close monitoring is essential to assess and know the aggressiveness of the intervention that will be given to each patient

Systemic delivery of antisense oligonucleotides (AO) for DMD exon skipping has proven effective for reframing DMD mRNA, rescuing dystrophin expression, and slowing disease progression in animal models. In humans with Duchenne muscular dystrophy treated with AOs, low levels of dystrophin have been induced, and modest slowing of disease progression has been observed, highlighting the need for improved efficiency of human skipping drugs. Here, we demonstrate that dantrolene and Rycals S107 and ARM210 potentiate AO-mediated exon skipping of exon 44 or exon 45 in patient-derived myotube cultures with appropriate mutations. Further, dantrolene is shown to boost AO-mediated exon skipping in patient-derived, induced cardiomyocyte cultures. Our findings further validate the ryanodine receptors (RyR) as the likely target responsible for exon skip boosting and demonstrate potential applicability beyond exon 51 skipping. These data provide preclinical support of dantrolene trial as an adjuvant to AO-mediated exon-skipping therapy in humans and identify a novel Rycal, ARM210, for development as a potential exon-skipping booster. Further, they highlight the value of mutation-specific DMD culture models for basic discovery, preclinical drug screening and translation of personalized genetic medicines.

Malignant hyperthermia (MH) is a rare and life-threatening pharmacogenetic disorder triggered by volatile anesthetics, the depolarizing muscle relaxant succinylcholine, and rarely by strenuous exercise or environmental heat. The exact prevalence of MH is unknown, and it varies from 1:16 000 in Denmark to 1:100 000 in New York State. The underlying mechanism of MH is excessive calcium release from the sarcoplasmic reticulum (SR), leading to uncontrolled skeletal muscle hyper-metabolism. Genetic mutations in ryanodine receptor type 1 ( RYR1) and CACNA1S have been identified in approximately 50% to 86% and 1% of MH-susceptible (MHS) individuals, respectively. Classic clinical symptoms of MH include hypercarbia, sinus tachycardia, masseter spasm, hyperthermia, acidosis, muscle rigidity, hyperkalemia, myoglobinuria, and etc. There are two types of testing for MH: a genetic test and a contracture test. Contracture testing is still being considered as the gold standard for MH diagnosis. Dantrolene is the only available drug approved for the treatment of MH through suppressing the calcium release from SR. Since clinical symptoms of MH are highly variable, it can be difficult to establish a diagnosis of MH. Nevertheless, prompt diagnosis and treatments are crucial to avoid a fatal outcome. Therefore, it is very important for anesthesiologists to raise awareness and understand the characteristics of MH. This review summarizes epidemiology, clinical symptoms, diagnosis and treatments of MH and any new developments.

Malignant hyperthermia (MH) is a rare and life-threatening pharmacogenetic disorder triggered by volatile anesthetics, the depolarizing muscle relaxant succinylcholine, and rarely by strenuous exercise or environmental heat. The exact prevalence of MH is unknown, and it varies from 1:16 000 in Denmark to 1:100 000 in New York State. The underlying mechanism of MH is excessive calcium release from the sarcoplasmic reticulum (SR), leading to uncontrolled skeletal muscle hyper-metabolism. Genetic mutations in ryanodine receptor type 1 ( RYR1) and CACNA1S have been identified in approximately 50% to 86% and 1% of MH-susceptible (MHS) individuals, respectively. Classic clinical symptoms of MH include hypercarbia, sinus tachycardia, masseter spasm, hyperthermia, acidosis, muscle rigidity, hyperkalemia, myoglobinuria, and etc. There are two types of testing for MH: a genetic test and a contracture test. Contracture testing is still being considered as the gold standard for MH diagnosis. Dantrolene is the only available drug approved for the treatment of MH through suppressing the calcium release from SR. Since clinical symptoms of MH are highly variable, it can be difficult to establish a diagnosis of MH. Nevertheless, prompt diagnosis and treatments are crucial to avoid a fatal outcome. Therefore, it is very important for anesthesiologists to raise awareness and understand the characteristics of MH. This review summarizes epidemiology, clinical symptoms, diagnosis and treatments of MH and any new developments.

Systemic delivery of antisense oligonucleotides (AO) for DMD exon skipping has proven effective for reframing DMD mRNA, rescuing dystrophin expression, and slowing disease progression in animal models. In humans with Duchenne muscular dystrophy treated with AOs, low levels of dystrophin have been induced, and modest slowing of disease progression has been observed, highlighting the need for improved efficiency of human skipping drugs. Here, we demonstrate that dantrolene and Rycals S107 and ARM210 potentiate AO-mediated exon skipping of exon 44 or exon 45 in patient-derived myotube cultures with appropriate mutations. Further, dantrolene is shown to boost AO-mediated exon skipping in patient-derived, induced cardiomyocyte cultures. Our findings further validate the ryanodine receptors (RyR) as the likely target responsible for exon skip boosting and demonstrate potential applicability beyond exon 51 skipping. These data provide preclinical support of dantrolene trial as an adjuvant to AO-mediated exon-skipping therapy in humans and identify a novel Rycal, ARM210, for development as a potential exon-skipping booster. Further, they highlight the value of mutation-specific DMD culture models for basic discovery, preclinical drug screening and translation of personalized genetic medicines.

Malignant hyperthermia (MH) is a rare and life-threatening pharmacogenetic disorder triggered by volatile anesthetics, the depolarizing muscle relaxant succinylcholine, and rarely by strenuous exercise or environmental heat. The exact prevalence of MH is unknown, and it varies from 1:16 000 in Denmark to 1:100 000 in New York State. The underlying mechanism of MH is excessive calcium release from the sarcoplasmic reticulum (SR), leading to uncontrolled skeletal muscle hyper-metabolism. Genetic mutations in ryanodine receptor type 1 ( RYR1) and CACNA1S have been identified in approximately 50% to 86% and 1% of MH-susceptible (MHS) individuals, respectively. Classic clinical symptoms of MH include hypercarbia, sinus tachycardia, masseter spasm, hyperthermia, acidosis, muscle rigidity, hyperkalemia, myoglobinuria, and etc. There are two types of testing for MH: a genetic test and a contracture test. Contracture testing is still being considered as the gold standard for MH diagnosis. Dantrolene is the only available drug approved for the treatment of MH through suppressing the calcium release from SR. Since clinical symptoms of MH are highly variable, it can be difficult to establish a diagnosis of MH. Nevertheless, prompt diagnosis and treatments are crucial to avoid a fatal outcome. Therefore, it is very important for anesthesiologists to raise awareness and understand the characteristics of MH. This review summarizes epidemiology, clinical symptoms, diagnosis and treatments of MH and any new developments.

Duchenne muscular dystrophy (DMD) is caused by mutations in DMD, resulting in loss of dystrophin, which is essential to muscle health. DMD "exon skipping" uses anti-sense oligo-nucleotides (AONs) to force specific exon exclusion during mRNA processing to restore reading frame and rescue of partially functional dystrophin protein. Although exon-skipping drugs in humans show promise, levels of rescued dystrophin protein remain suboptimal. We previously identified dantrolene as a skip booster when combined with AON in human DMD cultures and short-term mdx dystrophic mouse studies. Here, we assess the effect of dantrolene/AON combination on DMD exon-23 skipping over long-term mdx treatment under conditions that better approximate potential human dosing. To evaluate the dantrolene/AON combination treatment effect on dystrophin induction, we assayed three AON doses, with and without oral dantrolene, to assess multiple outcomes across different muscles. Meta-analyses of the results of statistical tests from both the quadriceps and diaphragm assessing contributions of dantrolene beyond AON, across all AON treatment groups, provide strong evidence that dantrolene modestly boosts exon skipping and dystrophin rescue while reducing muscle pathology in mdx mice (p < 0.0087). These findings support a trial of combination dantrolene/AON to increase exon-skipping efficacy and highlight the value of combinatorial approaches and Food and Drug Administration (FDA) drug re-purposing for discovery of unsuspected therapeutic application and rapid translation.

Duchenne muscular dystrophy (DMD) is caused by mutations in DMD, resulting in loss of dystrophin, which is essential to muscle health. DMD "exon skipping" uses anti-sense oligo-nucleotides (AONs) to force specific exon exclusion during mRNA processing to restore reading frame and rescue of partially functional dystrophin protein. Although exon-skipping drugs in humans show promise, levels of rescued dystrophin protein remain suboptimal. We previously identified dantrolene as a skip booster when combined with AON in human DMD cultures and short-term mdx dystrophic mouse studies. Here, we assess the effect of dantrolene/AON combination on DMD exon-23 skipping over long-term mdx treatment under conditions that better approximate potential human dosing. To evaluate the dantrolene/AON combination treatment effect on dystrophin induction, we assayed three AON doses, with and without oral dantrolene, to assess multiple outcomes across different muscles. Meta-analyses of the results of statistical tests from both the quadriceps and diaphragm assessing contributions of dantrolene beyond AON, across all AON treatment groups, provide strong evidence that dantrolene modestly boosts exon skipping and dystrophin rescue while reducing muscle pathology in mdx mice (p < 0.0087). These findings support a trial of combination dantrolene/AON to increase exon-skipping efficacy and highlight the value of combinatorial approaches and Food and Drug Administration (FDA) drug re-purposing for discovery of unsuspected therapeutic application and rapid translation.

Duchenne muscular dystrophy (DMD) is caused by mutations in DMD, resulting in loss of dystrophin, which is essential to muscle health. DMD "exon skipping" uses anti-sense oligo-nucleotides (AONs) to force specific exon exclusion during mRNA processing to restore reading frame and rescue of partially functional dystrophin protein. Although exon-skipping drugs in humans show promise, levels of rescued dystrophin protein remain suboptimal. We previously identified dantrolene as a skip booster when combined with AON in human DMD cultures and short-term mdx dystrophic mouse studies. Here, we assess the effect of dantrolene/AON combination on DMD exon-23 skipping over long-term mdx treatment under conditions that better approximate potential human dosing. To evaluate the dantrolene/AON combination treatment effect on dystrophin induction, we assayed three AON doses, with and without oral dantrolene, to assess multiple outcomes across different muscles. Meta-analyses of the results of statistical tests from both the quadriceps and diaphragm assessing contributions of dantrolene beyond AON, across all AON treatment groups, provide strong evidence that dantrolene modestly boosts exon skipping and dystrophin rescue while reducing muscle pathology in mdx mice (p < 0.0087). These findings support a trial of combination dantrolene/AON to increase exon-skipping efficacy and highlight the value of combinatorial approaches and Food and Drug Administration (FDA) drug re-purposing for discovery of unsuspected therapeutic application and rapid translation.

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene. Most deletions, duplications, or indels lead to shift of mRNA reading frame, which prevent the production of dystrophin protein. DMD is the leading fatal genetic disorder in childhood. One therapeutic strategy aims to skip one or more exons to restore reading frame to enable the production of internally truncated proteins with partial functionality. However, to date the efficiency of this strategy is suboptimal. Here we present methods for assessing exon skipping using AON alone or in combination with skip booster in the context of human DMD patient fibroblast derived myotubes and in the mdx mouse model of DMD.

In cardiac myocytes Calmodulin (CaM) bound to the ryanodine receptor (RyR2) constitutes a large pool of total myocyte CaM, but the CaM-RyR2 affinity is reduced in pathological conditions. Knock-in mice expressing RyR2 unable to bind CaM also developed hypertrophy and early death. However, it is unknown whether CaM released from this RyR2-bound pool participates in pathological cardiac hypertrophy. We found that angiotensin II (AngII) or phenylephrine (PE) both cause CaM to dissociate from the RyR2 and translocate to the nucleus. To test whether this nuclear CaM accumulation depends on CaM released from RyR2, we enhanced CaM-RyR2 binding affinity (with dantrolene), or caused CaM dissociation from RyR2 (using suramin). Dantrolene dramatically reduced AngII- and PE-induced nuclear CaM accumulation. Conversely, suramin enhanced nuclear CaM accumulation. This is consistent with nuclear CaM accumulation coming largely from the CaM-RyR2 pool. CaM lacks a nuclear localization signal (NLS), but G-protein coupled receptor kinase 5 (GRK5) binds CaM, has a NLS and translocates like CaM in response to AngII or PE. Suramin also promoted GRK5 nuclear import, and caused nuclear export of histone deacetylase 5 (HDAC5). Dantrolene prevented these effects. After 2-8 weeks of pressure overload (TAC) CaM binding to RyR2 was reduced, nuclear CaM and GRK5 were both elevated and there was enhanced nuclear export of HDAC5. Stress (acute AngII or TAC) causes CaM dissociation from RyR2 and translocation to the nucleus with GRK5 with parallel HDAC5 nuclear export. Thus CaM dissociation from RyR2 may be an important step in driving pathological hypertrophic gene transcription.

Byly porovnávány elektrochemické vlastnosti borem dopovaných diamantových elektrod v závislosti na různém obsahu boru. Aplikační možnosti těchto elektrod byly ověřeny při stanovené dnatrolenu., This study is focused on comparison of lab-made boron doped diamond electrodes (BDDE) with different content of boron prepared by hot filaments chemical vapor deposition. The set of electrodes with the range of B/C ratio in the gas phase from 2 000-20 000 ppm was tested. Their physical, chemical and electrochemical properties were investigated by using cyclic voltammetry with the redox markers [Fe(CN)6]3-/4- and [Ru(NH3)6]3+/2+. At the end, all BDDEs were applied for determination of drug dantrolene, to compare obtained statistical parameters and to find out as behavior of these electrodes exhibits some trends in relation to the boron content.

Voltammetric behavior of muscle relaxant dantrolene sodium (DAN) was studied and the voltammetric methods for its determination using polished and mercury meniscus modified silver solid amalgam electrodes (p-AgSAE and m-AgSAE) as well as using bismuth film electrode (BiFE, ex situ plating on GCE) have been proposed. These working electrodes represent the most commonly used alternatives to mercury ones which come wrongfully into disfavor because of alleged toxicity of mercury. Within this work, the obtained results of DAN determination have been completed by corresponding statistical parameters and also some electrochemical characteristics of AgSAEs and BiFE were assessed, especially in comparison with the mercury electrodes., Bylo studováno voltametrické chování dantrolenu na stříbrné pevné amalgámové a bismutové filmové elektrodě. Pro obě uvedené elektrody byla vyvinuta metoda stanovení analytu a dosažené výsledky byly porovnány.

Obsahem této bakalářské práce je studium voltametrického chování dantrolenu. Teoretická část je zaměřena na úvod do polarografických a voltametrických metod a na rozdělení a vlastnosti používaných pracovních elektrod. Pozornost byla také věnována novým elektrodovým materiálům a obzvláště pak borem dopované diamantové elektrodě. V práci je rovněž shrnuto elektrochemické chování dantrolenu na rtuťových elektrodách popsané v literatuře. V rámci experimentální části bylo studováno voltametrické chování dantrolenu na borem dopované diamantové elektrodě v závislosti na pH a rychlosti polarizace a byla navržena metoda stanovení této látky., The aim of this thesis is to study the voltammetric behavior of dantrolene. The theoretical part focuses on the introduction of the polarographic and voltammetric methods and on the classification and characterization of the commonly used working electrodes. The attention was also paid to the new electrode materials, especially, to boron doped diamond electrode. The electrochemical behavior of dantrolene on mercury electrodes described in literature is also summarized. In the experimental part, the voltammetric behavior of dantrolene on boron doped diamond electrode in dependence on pH and scan rate was studied. Finally, the new method for dantrolene determination was developed., Fakulta chemicko-technologická, Posluchačka seznámila komisi s obsahem své bakalářské práce a následně zodpověděla dotazy členů komise. Výtěžnost v tabulce je vyšší než je udáno, různé koncentrace. Použití reálných vod, koncentrace léčiva ve vodě, praktické využití.

Diastolic calcium (Ca) leak via cardiac ryanodine receptors (RyR2) can cause arrhythmias and heart failure (HF). Ca/calmodulin (CaM)-dependent kinase II (CaMKII) is upregulated and more active in HF, promoting RyR2-mediated Ca leak by RyR2-Ser2814 phosphorylation. Here, we tested a mechanistic hypothesis that RyR2 phosphorylation by CaMKII increases Ca leak by promoting a pathological RyR2 conformation with reduced CaM affinity. Acute CaMKII activation in wild-type RyR2, and phosphomimetic RyR2-S2814D (vs. non-phosphorylatable RyR2-S2814A) knock-in mouse myocytes increased SR Ca leak, reduced CaM-RyR2 affinity, and caused a pathological shift in RyR2 conformation (detected via increased access of the RyR2 structural peptide DPc10). This same trio of effects was seen in myocytes from rabbits with pressure/volume-overload induced HF. Excess CaM quieted leak and restored control conformation, consistent with negative allosteric coupling between CaM affinity and DPc10 accessible conformation. Dantrolene (DAN) also restored CaM affinity, reduced DPc10 access, and suppressed RyR2-mediated Ca leak and ventricular tachycardia in RyR2-S2814D mice. We propose that a common pathological RyR2 conformational state (low CaM affinity, high DPc10 access, and elevated leak) may be caused by CaMKII-dependent phosphorylation, oxidation, and HF. Moreover, DAN (or excess CaM) can shift this pathological gating state back to the normal physiological conformation, a potentially important therapeutic approach.

Diastolic calcium (Ca) leak via cardiac ryanodine receptors (RyR2) can cause arrhythmias and heart failure (HF). Ca/calmodulin (CaM)-dependent kinase II (CaMKII) is upregulated and more active in HF, promoting RyR2-mediated Ca leak by RyR2-Ser2814 phosphorylation. Here, we tested a mechanistic hypothesis that RyR2 phosphorylation by CaMKII increases Ca leak by promoting a pathological RyR2 conformation with reduced CaM affinity. Acute CaMKII activation in wild-type RyR2, and phosphomimetic RyR2-S2814D (vs. non-phosphorylatable RyR2-S2814A) knock-in mouse myocytes increased SR Ca leak, reduced CaM-RyR2 affinity, and caused a pathological shift in RyR2 conformation (detected via increased access of the RyR2 structural peptide DPc10). This same trio of effects was seen in myocytes from rabbits with pressure/volume-overload induced HF. Excess CaM quieted leak and restored control conformation, consistent with negative allosteric coupling between CaM affinity and DPc10 accessible conformation. Dantrolene (DAN) also restored CaM affinity, reduced DPc10 access, and suppressed RyR2-mediated Ca leak and ventricular tachycardia in RyR2-S2814D mice. We propose that a common pathological RyR2 conformational state (low CaM affinity, high DPc10 access, and elevated leak) may be caused by CaMKII-dependent phosphorylation, oxidation, and HF. Moreover, DAN (or excess CaM) can shift this pathological gating state back to the normal physiological conformation, a potentially important therapeutic approach.

Cílem této diplomové práce bylo studium voltametrického chování dantrolenu a vývoj metod jeho stanovení s využitím elektrod na bázi stříbrného pevného amalgámu a bismutového filmu jako nejlepších dostupných alternativ rtuťových elektrod. Získané výsledky byly porovnány a byly diskutovány elektrochemické vlastnosti použitých elektrodových materiálů, a to i v porovnání s elektrodou rtuťovou., The aim of this thesis was to study the voltammetric behavior of dantrolene and development of methods for its determination using electrodes based on a solid silver amalgam and bismuth film as the best available alternatives to mercury electrodes. The results were compared and the electrochemical properties of the used electrode materials were discused too, even in comparison with the mercury electrode., Fakulta chemicko-technologická

Oxidative stress may contribute to cardiac ryanodine receptor (RyR2) dysfunction in heart failure (HF) and arrhythmias. Altered RyR2 domain-domain interaction (domain unzipping) and calmodulin (CaM) binding affinity are allosterically coupled indices of RyR2 conformation. In HF RyR2 exhibits reduced CaM binding, increased domain unzipping and greater SR Ca leak, and dantrolene can reverse these changes. However, effects of oxidative stress on RyR2 conformation and leak in myocytes are poorly understood. We used fluorescent CaM, FKBP12.6, and domain-peptide biosensor (F-DPc10) to measure, directly in cardiac myocytes, (1) RyR2 activation by hydrogen peroxide (H2O2)-induced oxidation, (2) RyR2 conformation change caused by oxidation, (3) CaM-RyR2 and FK506-binding protein (FKBP12.6)-RyR2 interaction upon oxidation, and (4) whether dantrolene affects 1-3. H2O2 was used to mimic oxidative stress. H2O2 significantly increased the frequency of Ca(2+) sparks and spontaneous Ca(2+) waves, and dantrolene almost completely blocked these effects. H2O2 pretreatment significantly reduced CaM-RyR2 binding, but had no effect on FKBP12.6-RyR2 binding. Dantrolene restored CaM-RyR2 binding but had no effect on intracellular and RyR2 oxidation levels. H2O2 also accelerated F-DPc10-RyR2 association while dantrolene slowed it. Thus, H2O2 causes conformational changes (sensed by CaM and DPc10 binding) associated with Ca leak, and dantrolene reverses these RyR2 effects. In conclusion, in cardiomyocytes, H2O2 treatment markedly reduces the CaM-RyR2 affinity, has no effect on FKBP12.6-RyR2 affinity, and causes domain unzipping. Dantrolene can correct domain unzipping, restore CaM-RyR2 affinity, and quiet pathological RyR2 channel gating. F-DPc10 and CaM are useful biosensors of a pathophysiological RyR2 state.

Histamine, a neurotransmitter/neuromodulator implicated in the control of arousal state, exerts a potent phase-shifting effect on the circadian clock in the rodent suprachiasmatic nucleus (SCN). In this study, the mechanisms by which histamine resets the circadian clock in the mouse SCN were investigated. As a first step, Ca(2+) -imaging techniques were used to demonstrate that histamine increases intracellular Ca(2+) concentration ([Ca(2+) ]i ) in acutely dissociated SCN neurons and that this increase is blocked by the H1 histamine receptor (H1R) antagonist pyrilamine, the removal of extracellular Ca(2+) and the L-type Ca(2+) channel blocker nimodipine. The histamine-induced Ca(2+) transient is reduced, but not blocked, by application of the ryanodine receptor (RyR) blocker dantrolene. Immunohistochemical techniques indicated that CaV 1.3 L-type Ca(2+) channels are expressed mainly in the somata of SCN cells along with the H1R, whereas CaV 1.2 channels are located primarily in the processes. Finally, extracellular single-unit recordings demonstrated that the histamine-elicited phase delay of the circadian neural activity rhythm recorded from SCN slices is blocked by pyrilamine, nimodipine and the knockout of CaV 1.3 channel. Again, application of dantrolene reduced but did not block the histamine-induced phase delays. Collectively, these results indicate that, to reset the circadian clock, histamine increases [Ca(2+) ]i in SCN neurons by activating CaV 1.3 channels through H1R, and secondarily by causing Ca(2+) -induced Ca(2+) release from RyR-mediated internal stores.

Oxidative stress may contribute to cardiac ryanodine receptor (RyR2) dysfunction in heart failure (HF) and arrhythmias. Altered RyR2 domain-domain interaction (domain unzipping) and calmodulin (CaM) binding affinity are allosterically coupled indices of RyR2 conformation. In HF RyR2 exhibits reduced CaM binding, increased domain unzipping and greater SR Ca leak, and dantrolene can reverse these changes. However, effects of oxidative stress on RyR2 conformation and leak in myocytes are poorly understood. We used fluorescent CaM, FKBP12.6, and domain-peptide biosensor (F-DPc10) to measure, directly in cardiac myocytes, (1) RyR2 activation by hydrogen peroxide (H2O2)-induced oxidation, (2) RyR2 conformation change caused by oxidation, (3) CaM-RyR2 and FK506-binding protein (FKBP12.6)-RyR2 interaction upon oxidation, and (4) whether dantrolene affects 1-3. H2O2 was used to mimic oxidative stress. H2O2 significantly increased the frequency of Ca(2+) sparks and spontaneous Ca(2+) waves, and dantrolene almost completely blocked these effects. H2O2 pretreatment significantly reduced CaM-RyR2 binding, but had no effect on FKBP12.6-RyR2 binding. Dantrolene restored CaM-RyR2 binding but had no effect on intracellular and RyR2 oxidation levels. H2O2 also accelerated F-DPc10-RyR2 association while dantrolene slowed it. Thus, H2O2 causes conformational changes (sensed by CaM and DPc10 binding) associated with Ca leak, and dantrolene reverses these RyR2 effects. In conclusion, in cardiomyocytes, H2O2 treatment markedly reduces the CaM-RyR2 affinity, has no effect on FKBP12.6-RyR2 affinity, and causes domain unzipping. Dantrolene can correct domain unzipping, restore CaM-RyR2 affinity, and quiet pathological RyR2 channel gating. F-DPc10 and CaM are useful biosensors of a pathophysiological RyR2 state.

Histamine, a neurotransmitter/neuromodulator implicated in the control of arousal state, exerts a potent phase-shifting effect on the circadian clock in the rodent suprachiasmatic nucleus (SCN). In this study, the mechanisms by which histamine resets the circadian clock in the mouse SCN were investigated. As a first step, Ca(2+) -imaging techniques were used to demonstrate that histamine increases intracellular Ca(2+) concentration ([Ca(2+) ]i ) in acutely dissociated SCN neurons and that this increase is blocked by the H1 histamine receptor (H1R) antagonist pyrilamine, the removal of extracellular Ca(2+) and the L-type Ca(2+) channel blocker nimodipine. The histamine-induced Ca(2+) transient is reduced, but not blocked, by application of the ryanodine receptor (RyR) blocker dantrolene. Immunohistochemical techniques indicated that CaV 1.3 L-type Ca(2+) channels are expressed mainly in the somata of SCN cells along with the H1R, whereas CaV 1.2 channels are located primarily in the processes. Finally, extracellular single-unit recordings demonstrated that the histamine-elicited phase delay of the circadian neural activity rhythm recorded from SCN slices is blocked by pyrilamine, nimodipine and the knockout of CaV 1.3 channel. Again, application of dantrolene reduced but did not block the histamine-induced phase delays. Collectively, these results indicate that, to reset the circadian clock, histamine increases [Ca(2+) ]i in SCN neurons by activating CaV 1.3 channels through H1R, and secondarily by causing Ca(2+) -induced Ca(2+) release from RyR-mediated internal stores.

Mitochondrial Ca(2+) controls numerous cell functions, such as energy metabolism, reactive oxygen species generation, spatiotemporal dynamics of Ca(2+) signaling, cell growth and death in various cell types including neurons. Mitochondrial Ca(2+) accumulation is mainly mediated by the mitochondrial Ca(2+) uniporter (MCU), but recent reports also indicate that mitochondrial Ca(2+)-influx mechanisms are regulated not only by MCU, but also by multiple channels/transporters. We previously reported that ryanodine receptor (RyR), which is a one of the main Ca(2+)-release channels at endoplasmic/sarcoplasmic reticulum (SR/ER) in excitable cells, is expressed at the mitochondrial inner membrane (IMM) and serves as a part of the Ca(2+) uptake mechanism in cardiomyocytes. Although RyR is also expressed in neuronal cells and works as a Ca(2+)-release channel at ER, it has not been well investigated whether neuronal mitochondria possess RyR and, if so, whether this mitochondrial RyR has physiological functions in neuronal cells. Here we show that neuronal mitochondria express RyR at IMM and accumulate Ca(2+) through this channel in response to cytosolic Ca(2+) elevation, which is similar to what we observed in another excitable cell-type, cardiomyocytes. In addition, the RyR blockers dantrolene or ryanodine significantly inhibits mitochondrial Ca(2+) uptake in permeabilized striatal neurons. Taken together, we identify RyR as an additional mitochondrial Ca(2+) uptake mechanism in response to the elevation of [Ca(2+)]c in neurons, suggesting that this channel may play a critical role in mitochondrial Ca(2+)-mediated functions such as energy metabolism.

Setting: University hospital-based acute rehabilitation. Patient: 75-year-old woman with Stiff-Person Syndrome (SPS) with a recent fall and Colles fracture. Case Description: Four months prior to admission, the patient was diagnosed with SPS, negative for anti-GAD antibodies. Diagnosis was based on a 3-year history of progressive rigidity leading to frequent falls and fractures. Anxiety and fear of falling limited her mobility, and she sustained a sacral pressure ulcer during acute hospitalization. On admission, history was remarkable for unsteady gait and muscle cramps exacerbated when startled or excited. Examination was remarkable for rigidity in her axial and limb muscles. She presented at the maximal assist level for transfers and toileting and moderate assist level for grooming and ambulation using a platform walker (right arm in cast). She was unable to tolerate titration of diazepam due to sedation, or baclofen due to hypotension. Results: During acute rehabilitation, rigidity was treated with titration of dantrolene (from 25 to 50 mg four times daily) in addition to maximal tolerated doses of diazepam (1 mg qAM/2 mg qPM) and baclofen (20mg TID). The addition of dantrolene reduced rigidity and improved range of motion, both subjectively per patient and objectively by exam. Functional gains stalled with dose decrease and resumed with dose increase. She had pronounced gains in grooming to the supervision level, modest gains in transfers and toileting to the moderate assist level, but remained at the moderate assist level for ambulation. Progress was limited due to a change to non-weight bearing status of her right arm. Anxiety and depression were improved with buspirone, paroxetine, and psychological counseling. Discussion: SPS results in significant activity of daily life and ambulatory dysfunction as exemplified by her pressure ulcer and multiple falls. Although GABA agonists are the preferred treatment for SPS, the adverse effects of high doses can increase

Are United States Air Force hospitals prepared to treat a patient undergoing a malignant hyperthermia crisis? Malignant Hyperthermia (MH) is a life threatening syndrome that affects genetically susceptible individuals when exposed to volatile agents or succinylcholine. Patient survival is based on rapidly diagnosing MH, having a treatment plan in place, and having the necessary drugs and equipment available to treat the patient. The Malignant Hyperthermia Association of the United States (MHAUS) has advocated a five step readiness plan for hospitals to be prepared to treat an MH crisis. The results of two previous studies show a large number of hospitals are not fully prepared to handle an MH episode. A survey questionnaire was developed based on MHAUS guidelines perform a quantitative study of USAF hospitals. A telephone interview was conducted with all 39 USAF hospitals in the continental United States and Alaska that provide general surgery and anesthesia. The results showed 100% of the hospitals had a treatment plan in place and the emergency Hotline number to MHAUS was accessible. Also, 100% of the hospitals had an MH treatment cart. The results showed 38 out of 39 hospitals stocked the recommended supply of dantrolene. All of the hospitals fully stocked the ancillary drugs needed when treating MH. All of the hospitals had an ice source and equipment needed to cool the patient during an MH episode. An annual inservice on MH recognition and treatment was performed at 33 out of 39 hospitals. Overall, based on MHAUS standards, USAF hospitals are well prepared with the supplies, equipment and training to treat a patient experiencing an MH crisis.

Because little is known about the subject, the purpose of the study is to describe perioperative nurse preparedness to identify and manage MH. A description of nurse preparedness may serve to improve clinical practice and reduce the risks associated with MH for surgical patients. The specific aims of this study are as follows: (1) to describe the perioperative nurses' training, level of knowledge, and perceived educational needs regarding the identification and management of MH (nurse preparedness). (2) to describe the availability of MH supplies, cart, medications and institutional protocol necessary to maximize patient outcomes during MH crises (institutional preparedness).