Oral administration of TRPV4 inhibitor improves atrial calcium handling abnormalities in sterile pericarditis rats / 生理学报

Atrial Ca2+ handling abnormalities, mainly involving the dysfunction of ryanodine receptor (RyR) and sarcoplasmic reticulum Ca2+-ATPase (SERCA), play a role in the pathogenesis of atrial fibrillation (AF). Previously, we found that the expression and function of transient receptor potential vanilloid subtype 4 (TRPV4) are upregulated in a sterile pericarditis (SP) rat model of AF, and oral administration of TRPV4 inhibitor GSK2193874 alleviates AF in this animal model. The aim of this study was to investigate whether oral administration of GSK2193874 could alleviate atrial Ca2+ handling abnormalities in SP rats. A SP rat model of AF was established by daubing sterile talcum powder on both atria of Sprague-Dawley (SD) rats after a pericardiotomy, to simulate the pathogenesis of postoperative atrial fibrillation (POAF). On the 3rd postoperative day, Ca2+ signals of atria were collected in isolated perfused hearts by optical mapping. Ca2+ transient duration (CaD), alternan, and the recovery properties of Ca2+ transient (CaT) were quantified and analyzed. GSK2193874 treatment reversed the abnormal prolongation of time to peak (determined mainly by RyR activity) and CaD (determined mainly by SERCA activity), as well as the regional heterogeneity of CaD in SP rats. Furthermore, GSK2193874 treatment relieved alternan in SP rats, and reduced its incidence of discordant alternan (DIS-ALT). More importantly, GSK2193874 treatment prevented the reduction of the S2/S1 CaT ratio (determined mainly by RyR refractoriness) in SP rats, and decreased its regional heterogeneity. Taken together, oral administration of TRPV4 inhibitor alleviates Ca2+ handling abnormalities in SP rats primarily by blocking the TRPV4-Ca2+-RyR pathway, and thus exerts therapeutic effect on POAF.

Positive inotropic effect of phosphodiesterase type 9 inhibitor PF-04449613 in rats and its underlying mechanism / 生理学报

This study aimed to explore the positive inotropic effect of phosphodiesterase type 9 (PDE9) inhibitor PF-04449613 in ratsand its cellular and molecular mechanisms. The heart pressure-volume loop (P-V loop) analysis was used to detect the effects of PF-04449613 on rat left ventricular pressure-volume relationship, aortic pressures and peripheral vessel resistance in healthy rats. The Langendorff perfusion of isolated rat heart was used to explore the effects of PF-04449613 on heart contractility. The cardiomyocyte sarcoplasmic reticulum (SR) Ca

Intracoronary sarcoplasmic reticulum calcium-atpase gene therapy in advanced heart failure patients with reduced ejection fraction: a prospective cohort study

OBJECTIVE: Heart failure is a progressive and debilitating disease. Intracoronary sarcoplasmic reticulum calcium-ATPase gene therapy may improve the function of cardiac muscle cells. This study aimed to test the hypothesis that intracoronary sarcoplasmic reticulum calcium-ATPase gene therapy can improve outcomes and reduce the number of recurrent and terminal events in advanced heart failure patients with reduced ejection fraction. METHODS: A total of 768 heart failure patients with reduced ejection fraction and New York Heart Association classification II to IV were included in this prospective cohort study. Patients either underwent intracoronary sarcoplasmic reticulum calcium-ATPase gene therapy (CA group, n=384) or received oral placebo (PA group; n=384). Data regarding recurrent and terminal event(s), treatment-emergent adverse effects, and outcome measures were collected and analyzed. RESULTS: After a follow-up period of 18 months, intracoronary sarcoplasmic reticulum calcium-ATPase gene therapy reduced the number of hospital admissions (p=0.001), ambulatory treatments (p=0.0004), and deaths (p=0.024). Additionally, intracoronary sarcoplasmic reticulum calcium-ATPase gene therapy improved the left ventricular ejection fraction (p<0.0001) and Kansas City Cardiomyopathy Questionnaire score (p<0.0001). The number of recurrent and terminal events/patients were higher in the PA group than in the CA group after the follow-up period of 18 months (p=0.015). The effect of the intracoronary sarcoplasmic reticulum calcium-ATPase gene therapy was independent of the confounding variables. No new arrhythmias were reported in the CA group. CONCLUSIONS: Intracoronary sarcoplasmic reticulum calcium-ATPase gene therapy reduces the number of recurrent and terminal events and improves the clinical course of advanced heart failure patients with reduced ejection fraction.

Deficiency of Anoctamin 5/TMEM16E causes nuclear positioning defect and impairs Ca²⁺ signaling of differentiated C2C12 myotubes

Anoctamin 5 (ANO5)/TMEM16E belongs to a member of the ANO/TMEM16 family member of anion channels. However, it is a matter of debate whether ANO5 functions as a genuine plasma membrane chloride channel. It has been recognized that mutations in the ANO5 gene cause many skeletal muscle diseases such as limb girdle muscular dystrophy type 2L (LGMD2L) and Miyoshi muscular dystrophy type 3 (MMD3) in human. However, the molecular mechanisms of the skeletal myopathies caused by ANO5 defects are poorly understood. To understand the role of ANO5 in skeletal muscle development and function, we silenced the ANO5 gene in C2C12 myoblasts and evaluated whether it impairs myogenesis and myotube function. ANO5 knockdown (ANO5-KD) by shRNA resulted in clustered or aggregated nuclei at the body of myotubes without affecting differentiation or myotube formation. Nuclear positioning defect of ANO5-KD myotubes was accompanied with reduced expression of Kif5b protein, a kinesin-related motor protein that controls nuclear transport during myogenesis. ANO5-KD impaired depolarization-induced [Ca²⁺]i transient and reduced sarcoplasmic reticulum (SR) Ca²⁺ storage. ANO5-KD resulted in reduced protein expression of the dihydropyridine receptor (DHPR) and SR Ca²⁺-ATPase subtype 1. In addition, ANO5-KD compromised co-localization between DHPR and ryanodine receptor subtype 1. It is concluded that ANO5-KD causes nuclear positioning defect by reduction of Kif5b expression, and compromises Ca²⁺ signaling by downregulating the expression of DHPR and SERCA proteins.

Effect of Sphingosine-1-Phosphate on Intracellular Free Ca2+ in Cat Esophageal Smooth Muscle Cells

A comprehensive collection of proteins senses local changes in intracellular Ca²⁺ concentrations ([Ca²⁺](i) and transduces these signals into responses to agonists. In the present study, we examined the effect of sphingosine-1-phosphate (S1P) on modulation of intracellular Ca²⁺ concentrations in cat esophageal smooth muscle cells. To measure [Ca²⁺](i) levels in cat esophageal smooth muscle cells, we used a fluorescence microscopy with the Fura-2 loading method. S1P produced a concentration-dependent increase in [Ca²⁺](i) in the cells. Pretreatment with EGTA, an extracellular Ca²⁺ chelator, decreased the S1P-induced increase in [Ca²⁺](i), and an L-type Ca²⁺-channel blocker, nimodipine, decreased the effect of S1P. This indicates that Ca²⁺ influx may be required for muscle contraction by S1P. When stimulated with thapsigargin, an intracellular calcium chelator, or 2-Aminoethoxydiphenyl borate (2-APB), an InsP₃ receptor blocker, the S1P-evoked increase in [Ca²⁺](i) was significantly decreased. Treatment with pertussis toxin (PTX), an inhibitor of G(i)-protein, suppressed the increase in [Ca²⁺](i) evoked by S1P. These results suggest that the S1P-induced increase in [Ca²⁺](i) in cat esophageal smooth muscle cells occurs upon the activation of phospholipase C and subsequent release of Ca²⁺ from the InsP₃-sensitive Ca²⁺ pool in the sarcoplasmic reticulum. These results suggest that S1P utilized extracellular Ca²⁺ via the L type Ca²⁺ channel, which was dependent on activation of the S1P₄ receptor coupled to PTX-sensitive G(i) protein, via phospholipase C-mediated Ca²⁺ release from the InsP₃-sensitive Ca²⁺ pool in cat esophageal smooth muscle cells.

A focus on extracellular Ca²⁺ entry into skeletal muscle

The main task of skeletal muscle is contraction and relaxation for body movement and posture maintenance. During contraction and relaxation, Ca²⁺ in the cytosol has a critical role in activating and deactivating a series of contractile proteins. In skeletal muscle, the cytosolic Ca²⁺ level is mainly determined by Ca²⁺ movements between the cytosol and the sarcoplasmic reticulum. The importance of Ca²⁺ entry from extracellular spaces to the cytosol has gained significant attention over the past decade. Store-operated Ca²⁺ entry with a low amplitude and relatively slow kinetics is a main extracellular Ca²⁺ entryway into skeletal muscle. Herein, recent studies on extracellular Ca²⁺ entry into skeletal muscle are reviewed along with descriptions of the proteins that are related to extracellular Ca²⁺ entry and their influences on skeletal muscle function and disease.

El Diazepam altera la ultraestructura del atrio fetal de ratón / Diazepam alters the ultrastructure of fetal atrium in rat

El diazepam (DZ) es un tranquilizante menor sintético, utilizado en pacientes con trastornos psicológicos y psiquiátricos. Es sedante, miorrelajante, anticonvulsionante y antipsicótico. El DZ atraviesa la barrera placentaria humana y la del ratón. Mujeres jóvenes que son adictas al fármaco, si se embarazan y continúan utilizándolo, sobre todo durante el primer trimestre, exponen a sus hijos a presentar alteraciones psicomotoras. El propósito de este trabajo fue investigar si el DZ administrado durante la gestación,induce alteraciones ultraestructurales del miocardio fetal de ratón. El grupo (DZ) de hembras gestantes deratón de la cepa CD-1 fue tratado con dosis únicas diarias de 1,0 mg/kg/pc/sc del día 6 al 17 y un grupo (C)que recibió solución salina. El día 18 las hembras de ambos grupos se anestesiaron, los fetos se perfundieron por vía intracardiaca con paraformaldehído al 1 % y glutaraldehido al 2,5 %, se les extrajo el corazón, se disecó el atrio, se fijó en OsO4 al 1 % y se incluyó en resina epóxica. Los cortes finos se contrastaron conacetato de uranilo y citrato de plomo y se observaron en un microscopio electrónico de transmisión. En los miocitos de los fetos del grupo DZ las sarcómeras del miocardio compacto tenían menor longitud que las del grupo C. Se observaron zonas con miofibrillas desorganizadas. El retículo sarcoplásmico de algunos miocitos presentaba cisternas distendidas y fragmentadas, mitocondrias alteradas y se observaron abundantes polirribosomas. Los cambios podrían deberse al efecto del DZ sobre la síntesis de actina y miosina pesada y sobre los organelos citoplásmicos, mediados por receptores benzodiazepínicos periféricos presentes en la membrana externa de las mitocondrias y asociados a canales de calcio dependientes de voltaje. Las alteraciones ultraestructurales del miocardio atrial de fetos de ratones expuestos in utero a DZ podrían tener efectos posnatales.

Diazepam (DZ) is a syntheticminor tranquilizer, used in patients with psychologicaland psychiatric disorders. It is a relaxing sedative,anticonvulsant and antipsychotic. DZ crosses thehuman placental barrier in mouse. Young women who are addicted to the drug, if they become pregnantand continue to use it, particularly during the firsttrimester, expose their children to psychomotor disorders. The purpose of this study was to investigate whether DZ administered during pregnancy induces ultrastructural alterations of fetal mouse myocardium.The group (DZ) of pregnant female mice of the CD-1strain was treated with a single daily dose of 1.0 mg/ kg / pc / sc of day 6 to 17 and a group (C) that received saline solution. On day 18 females of bothgroups were anesthetized, the fetuses were perfusedby intracardiac route with 1 % paraformaldehyde and 2.5 % glutaraldehyde, the heart was removed, theatrium was dissected, fixed in 1 % OsO4, it wasimmersed in epoxy resin. The fine sections werecontrasted with uranyl acetate and lead citrate and observed in a transmission electron microscope. Inthe myocytes of the fetuses of the DZ group, the sarcomers of the compact myocardium were shorter than those of the C group. Areas with disorganized myofibrils were observed. The sarcoplasmic reticulumof some myocytes had distended and fragmented 996cisterns, altered mitochondria, and abundant polyribosomes were observed. The changes may bedue to the effect of DZ on the synthesis of actin and heavy myosin and on cytoplasmic organelles mediatedby peripheral benzodiazepine receptors present onthe outer membrane of the mitochondria and associated with voltage-dependent calcium channels.Ultrastructural alterations of the atrial myocardium of fetuses of mice exposed to DZ in utero may have postnatal effects.

Lowered sarcoendoplasmic reticulum calcium uptake and diaphragmatic SERCA1 expression contribute to diaphragmatic contractile and relaxation dysfunction in septic rats / 南方医科大学学报

<p><b>OBJECTIVE</b>The explore the mechanism responsible for diaphragmatic contractile and relaxation dysfunction in a rat model of sepsis.</p><p><b>METHODS</b>Thirty-six adult male Sprague-Dawley rats were randomized equally into a sham-operated group and two model groups of sepsis induced by cecal ligation and puncture (CLP) for examination at 6 and 12 h following CLP (CLP-6 h and CLP-12 h groups). The parameters of diaphragm contractile and relaxation were measured, and the calcium uptake and release rates of the diaphragmatic sarcoendoplasmic reticulum (SR) and the protein expressions of SERCA1, SERCA2 and RyR in the diaphragmatic muscles were determined.</p><p><b>RESULTS</b>The half-relaxation time of the diaphragm was extended in both the CLP-6 h and CLP-12 h groups with significantly reduced maximum tension declinerate and the peek uptake rate of SERCA (P<0.01). Diaphragmatic maximum twitch force development rate, the maximal twitch, tetanus tensions and the peek release rate of SR decreased only at 12h after CLP (P<0.01). The expression levels of SERCA1 protein decreased significantly in the diaphragmatic muscles at 12h following CLP (P<0.01) while SERCA2 expression level and SERCA activity showed no significant changes.</p><p><b>CONCLUSION</b>In the acute stage of sepsis, both the contractile and relaxation functions of the diaphragm are impaired. Diaphragmatic relaxation dysfunction may result from reduced calcium uptake in the SR and a decreased level of SERCA1 in the diaphragmatic muscles.</p>

Sarcoplasmic reticulum Ca²⁺ ATPase 2 (SERCA2) reduces the migratory capacity of CCL21-treated monocyte-derived dendritic cells

The migration of dendritic cells (DCs) to secondary lymphoid organs depends on chemoattraction through the interaction of the chemokine receptors with chemokines. However, the mechanism of how lymphoid chemokines attract DCs to lymphoid organs remains unclear. Here, we demonstrate the mechanism of DC migration in response to the lymphoid chemokine CCL21. CCL21-mediated DC migration is controlled by the regulation of sarcoplasmic reticulum Ca²⁺ ATPase 2 (SERCA2) expression rather than through the activation of mitogen-activated protein kinases CCL21-exposed mature DCs (mDCs) exhibited decreased SERCA2 expression but not decreased phospholamban (PLB) or Hax-1 expression, which are known to be SERCA2-interacting proteins. In addition, CCL21 did not affect the mRNA levels of SERCA2 or its interacting protein Hax-1. Interestingly, SERCA2 expression was inversely related to DC migration in response to chemokine stimulation. The migratory capacity of CCL21-treated mDCs was decreased by the phospholipase C inhibitor U73122 and by the protein kinase C inhibitor BAPTA-AM. The migratory capacities of mDCs were increased in response to SERCA2 siRNA expression but were decreased by SERCA2 overexpression. In addition, DCs treated with a SERCA2-specific inhibitor (cyclopiazonic acid) had significantly increased migratory capacities as mDCs regardless of SERCA2 expression. Moreover, SERCA2 expression was dependent on DC maturation induced by cytokines or Toll-like receptor agonists. Therefore, the migratory capacities differed in differentially matured DCs. Taken together, these results suggest that SERCA2 contributes to the migration of CCL21-activated DCs as an important feature of the adaptive immune response and provide novel insights regarding the role of SERCA2 in DC functions.

Cardiac misconceptions among healthy adults: implications for the promotion of health in the community / Crenças erróneas sobre as doenças cardíacas em adultos saudáveis: implicações para a promoção da saúde na comunidade

This study sought to confirm the structure and to investigate the psychometric properties of an experimental Portuguese version of the York Cardiac Beliefs Questionnaire (YCBQ) in a general population sample. It also set out to identify the prevalent misconceptions in the community and to assess the differences according to socio-demographic characteristics. It involved a cross-sectional survey in which both test and validation samples were collected (n = 476), including participants aged between 18 and 40, recruited via e-mail and social networks. The Confirmatory Factor Analysis on both samples suggested a shorter, three factor version of the YCBQ. Also, misconceptions differed significantly according to sociodemographic variables. The validation of the YCBQ for samples in the community constitutes an important starting point to promote research on misconceptions held in the community by specific groups, as well as to provide key points for health promotion.

Este estudo teve como objetivo confirmar a estrutura e investigar as propriedades psicométricas de uma versão experimental portuguesa do York Cardiac Beliefs Questionnaire numa amostra da população geral; identificar as crenças erróneas mais fortes na comunidade; e avaliar as diferenças de acordo com características sociodemográficas. Trata-se de um estudo transversal com uma amostra de teste e outra de validação, incluindo um total de 476 participantes, com idade entre 18 e 40 anos, recrutados via e-mail e nas redes sociais. A Análise Fatorial Confirmatória em ambas as amostras indicou uma versão reduzida do YCBQ de três factores. As crenças erróneas diferiram significativamente de acordo com as variáveis sociodemográficas. A validação do YCBQ para amostras da comunidade constitui um importante ponto de partida para promover a investigação sobre crenças erróneas em grupos específicos da comunidade, assim como fornecer indicadores relevantes para a promoção da saúde.

Function and Dysfunction of Human Sinoatrial Node

Sinoatrial node (SAN) automaticity is jointly regulated by a voltage (cyclic activation and deactivation of membrane ion channels) and Ca2+ clocks (rhythmic spontaneous sarcoplasmic reticulum Ca2+ release). Using optical mapping in Langendorff-perfused canine right atrium, we previously demonstrated that the beta-adrenergic stimulation pushes the leading pacemaker to the superior SAN, which has the fastest activation rate and the most robust late diastolic intracellular calcium (Cai) elevation. Dysfunction of the superior SAN is commonly observed in animal models of heart failure and atrial fibrillation (AF), which are known to be associated with abnormal SAN automaticity. Using the 3D electroanatomic mapping techniques, we demonstrated that superior SAN served as the earliest atrial activation site (EAS) during sympathetic stimulation in healthy humans. In contrast, unresponsiveness of superior SAN to sympathetic stimulation was a characteristic finding in patients with AF and SAN dysfunction, and the 3D electroanatomic mapping technique had better diagnostic sensitivity than corrected SAN recovery time testing. However, both tests have significant limitations in detecting patients with symptomatic sick sinus syndrome. Recently, we reported that the location of the EAS can be predicted by the amplitudes of P-wave in the inferior leads. The inferior P-wave amplitudes can also be used to assess the superior SAN responsiveness to sympathetic stimulation. Inverted or isoelectric P-waves at baseline that fail to normalize during isoproterenol infusion suggest SAN dysfunction. P-wave morphology analyses may be helpful in determining the SAN function in patients at risk of symptomatic sick sinus syndrome.

Decreased amplitude of Ca²⁺i elevation induced by menthol in pulmonary arterial smooth muscle cells of pulmonary hypertensive rats / 生理学报

The study was designed to explore the alteration of intracellular calcium concentration ([Ca²⁺]i), induced by transient receptor potential melastatin 8 (TRPM8) channel-specific agonist menthol, in pulmonary arterial smooth muscle cells (PASMCs) between control and pulmonary hypertensive (PH) rats. PH rat models were established by means of chronic hypoxia (CH) and monocrotaline (MCT) injection, respectively. PASMCs from control and PH rats were cultured. The change of [Ca²⁺]i in PASMCs induced by menthol, and the effect of TRPM8 channel-specific antagonist BCTC on the change of [Ca²⁺]i, were observed. Cellular localization of TRPM8 was examined by using immunohistochemistry. Results showed that menthol increased [Ca²⁺]i in the control PASMCs both in Ca²⁺ -normal and Ca²⁺ - free Tyrode's solutions, and at the same time BCTC could inhibit these two kinds of elevations. Compared with the control group, elevations of [Ca²⁺]i were decreased notably in CH- and MCT-pretreated PASMCs superfused with 2 mmol/L Ca²⁺ - or 0 Ca²⁺ -Tyrode's solutions. Immunohistochemical localization experiments showed that the whole PASMCs were dyed brown except for the nucleus. This study verified that TRPM8 exists both in membrane and sarcoplasmic reticulum of PASMCs. In addition, CH- and MCT-pretreatment could independently down-regulate the Ca²⁺ influx and Ca²⁺ release mediated by TRPM8 channel.

Abnormal calcium re-uptake in myocardium sarcoplasmic reticulum in rabbits with heart failure and the influencing factors / 生理学报

The aim of the present study was to investigate the abnormal calcium re-uptake function of myocardium sarcoplasmic reticulum (SR) in rabbits with heart failure, as well as potential mechanisms. Heart failure model was established in rabbits through aortic insufficiency and constriction of abdominal aorta. The SR Ca(2+) re-uptake function was measured with a calcium imaging device. The activity of myocardium SR calcium adenodine triphosphatase 2a (SERCA2a) was measured by inorganic phosphate. The protein expressions of SERCA2a, CaMKII, PKA, PP1α, phospholamban (PLB), PLB-Ser(16) and PLB-Thr(17) were evaluated by Western blot. The activities of PKA and CaMKII were detected by γ-(32)P substrate incorporation. The results showed that, compared with the sham operation group, the heart failure group exhibited reduced Ca(2+) re-uptake amount (P < 0.01) and the expression and activity of SERCA2a (P < 0.05 or P < 0.01), decreased expression of PLB and its phosphorylation status in sites of Ser(16) and Thr(17) (P < 0.05), increased expressions and activities of PKA and CaMKII (P < 0.05 or P < 0.01), and increased expression of PP1α (P < 0.05). These results suggest that the abnormal Ca(2+) re-uptake function in heart failure is related with reduced expression and activities of SERCA2a, as well as reduced expression of PLB and its phosphorylation status. Both PLB-Ser(16) and -Thr(17) may be involved in the regulation of myocardium SR calcium pump activity in heart failure.

Mepivacaine-induced intracellular calcium increase appears to be mediated primarily by calcium influx in rat aorta without endothelium / 대한마취과학회지

BACKGROUND: Mepivacaine induces contraction or decreased blood flow both in vivo and in vitro. Vasoconstriction is associated with an increase in the intracellular calcium concentration ([Ca2+]i). However, the mechanism responsible for the mepivacaine-evoked [Ca2+]i increase remains to be determined. Therefore, the objective of this in vitro study was to examine the mechanism responsible for the mepivacaine-evoked [Ca2+]i increment in isolated rat aorta. METHODS: Isometric tension was measured in isolated rat aorta without endothelium. In addition, fura-2 loaded aortic muscle strips were illuminated alternately (48 Hz) at two excitation wavelengths (340 and 380 nm). The ratio of F340 to F380 (F340/F380) was regarded as an amount of [Ca2+]i. We investigated the effects of nifedipine, 2-aminoethoxydiphenylborate (2-APB), gadolinium chloride hexahydrate (Gd3+), low calcium level and Krebs solution without calcium on the mepivacaine-evoked contraction in isolated rat aorta and on the mepivacaine-evoked [Ca2+]i increment in fura-2 loaded aortic strips. We assessed the effect of verapamil on the mepivacaine-evoked [Ca2+]i increment. RESULTS: Mepivacaine produced vasoconstriction and increased [Ca2+]i. Nifedipine, 2-APB and low calcium attenuated vasoconstriction and the [Ca2+]i increase evoked by mepivacaine. Verapamil attenuated the mepivacaine-induced [Ca2+]i increment. Calcium-free solution almost abolished mepivacaine-induced contraction and strongly attenuated the mepivacaineinduced [Ca2+]i increase. Gd3+ had no effect on either vasoconstriction or the [Ca2+]i increment evoked by mepivacaine. CONCLUSIONS: The mepivacaine-evoked [Ca2+]i increment, which contributes to mepivacaine-evoked contraction, appears to be mediated mainly by calcium influx and partially by calcium released from the sarcoplasmic reticulum.

Effect of Hypoxic Paracrine Media on Calcium-Regulatory Proteins in Infarcted Rat Myocardium

BACKGROUND AND OBJECTIVES: An increase in intracellular calcium concentration due to loss of Ca2+ homeostasis triggers arrhythmia or cardiac cell death in the heart. Paracrine factors released from stem cells have beneficial cardioprotective effects. However, the mechanism of modulation of Ca2+ homeostasis by paracrine factors in ischemic myocardium remains unclear. MATERIALS AND METHODS: We isolated rat bone marrow-derived mesenchymal stem cells (MSCs), and prepared paracrine media (PM) from MSCs under hypoxic or normoxic conditions (hypoxic PM and normoxic PM). We induced rat myocardial infarction by left anterior descending ligation for 1 hour, and treated PM into the border region of infarcted myocardium (n=6/group) to identify the alteration in calcium-regulated proteins. We isolated and stained the heart tissue with specific calcium-related antibodies after 11 days. RESULTS: The hypoxic PM treatment increased Ca2+-related proteins such as L-type Ca2+ channel, sarcoplasmic reticulum Ca2+ ATPase, Na+/K+ ATPase, and calmodulin, whereas the normoxic PM treatment increased those proteins only slightly. The sodium-calcium exchanger was significantly reduced by hypoxic PM treatment, compared to moderate suppression by the normoxic PM treatment. CONCLUSION: Our results suggest that hypoxic PM was significantly associated with the positive regulation of Ca2+ homeostasis in infarcted myocardium.

Tetanic contraction induces enhancement of fatigability and sarcomeric damage in atrophic skeletal muscle and its underlying molecular mechanisms / 中国应用生理学杂志

Muscle unloading due to long-term exposure of weightlessness or simulated weightlessness causes atrophy, loss of functional capacity, impaired locomotor coordination, and decreased resistance to fatigue in the antigravity muscles of the lower limbs. Besides reducing astronauts' mobility in space and on returning to a gravity environment, the molecular mechanisms for the adaptation of skeletal muscle to unloading also play an important medical role in conditions such as disuse and paralysis. The tail-suspended rat model was used to simulate the effects of weightlessness on skeletal muscles and to induce muscle unloading in the rat hindlimb. Our series studies have shown that the maximum of twitch tension and the twitch duration decreased significantly in the atrophic soleus muscles, the maximal tension of high-frequency tetanic contraction was significantly reduced in 2-week unloaded soleus muscles, however, the fatigability of high-frequency tetanic contraction increased after one week of unloading. The maximal isometric tension of intermittent tetanic contraction at optimal stimulating frequency did not alter in 1- and 2-week unloaded soleus, but significantly decreased in 4-week unloaded soleus. The 1-week unloaded soleus, but not extensor digitorum longus (EDL), was more susceptible to fatigue during intermittent tetanic contraction than the synchronous controls. The changes in K+ channel characteristics may increase the fatigability during high-frequency tetanic contraction in atrophic soleus muscles. High fatigability of intermittent tetanic contraction may be involved in enhanced activity of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) and switching from slow to fast isoform of myosin heavy chain, tropomyosin, troponin I and T subunit in atrophic soleus muscles. Unloaded soleus muscle also showed a decreased protein level of neuronal nitric oxide synthase (nNOS), and the reduction in nNOS-derived NO increased frequency of calcium sparks and elevated intracellular resting Ca2+ concentration ([Ca2+]i) in unloaded soleus muscles. High [Ca2+]i activated calpain-1 which induced a higher degradation of desmin. Desmin degradation may loose connections between adjacent myofibrils and further misaligned Z-disc during repeated tetanic contractions. Passive stretch in unloaded muscle could preserve the stability of sarcoplasmic reticulum Ca2+ release channels by means of keeping nNOS activity, and decrease the enhanced protein level and activity of calpain to control levels in unloaded soleus muscles. Therefore, passive stretch restored normal appearance of Z-disc and resisted in part atrophy of unloaded soleus muscles. The above results indicate that enhanced fatigability of high-frequency tetanic contraction is associated to the alteration in K+ channel characteristics, and elevated SERCA activity and slow to fast transition of myosin heavy chain (MHC) isoforms increases fatigability of intermittent tetanic contraction in atrophic soleus muscle. The sarcomeric damage induced by tetanic contraction can be retarded by stretch in atrophic soleus muscles.

Effects of buyang huanwu decoction on the sarcoplasmic reticulum calcium uptake in abdominal aortic constriction induced myocardial hypertrophic rats / 中国中西医结合杂志

<p><b>UNLABELLED</b>objective: To investigate effects of buyang huanwu decoction (BYHWD) on the rats' myocardial hypertrophic model induced by abdominal aortic constriction, and to clarify the molecular regulatory mechanisms for sarcoplasmic reticulum calcium uptake.</p><p><b>METHODS</b>Hypertrophic myocardium rat model was induced by abdominal aorta constriction (AAC). Four weeks after modeling, rats were randomly divided into the sham-operation group (Group S), the AAC model group (Group M), the Enalapril group (Group E), and the BYHWD treatment group (Group BYHWD), respectively. The left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), + dp/dtmax,-dp/dtmax, cardiac output (CO), heart mass index (HMI), and left ventricular mass index (LVMI) were observed in each group after 12-week medication. The serum contents of the atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) were detected using ELISA. The SERCA2a activity, the ex- pressions of SERCA2a, phospholamban (PLN), and PLN phosphorylation were observed finally.</p><p><b>RESULTS</b>Compared with Group S, LVSP and LVEDP significantly increased,-dp/dtmax and CO obviously decreased, the myocardial tissue was obviously thickened, the serum contents of ANP and BNP increased, the activity and expression of SERCA2a decreased, the SERCA2a/PLN ratio and PLN phosphorylation degree decreased in Group M (all P <0.05). Compared with Group M, LVEDP obviously decreased, -dp/dtmax and CO obviously increased, the hypertrophy myocardial tissue was obviously lessened, the serum contents of ANP and BNP decreased, the activity of SERCA2a increased, the relative expression contents of SERCA2a, Ser16, and Thrl7 were elevated in Group BYHWD (all P <0.05). BYHWD had significant roles in elevating the SERCA2a/PLN ratio and PLN phosphorylation degree (P <0.05).</p><p><b>CONCLUSION</b>BYHWD could significantly improve hemodynamics of heart failure rats, elevate CO, lessen cardiac hypertrophy, and improve the capabilities for sarcoplasmic reticulum calcium uptake.</p>

Diaphragm dysfunction and expressions of calcium regulatory proteins in diabetic rats / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the changes in diaphragmatic function and gene expressions of calcium regulatory proteins in diabetic rats and explore the mechanism of diaphragm dysfunction in diabetes mellitus.</p><p><b>METHODS</b>SD rats were randomly divided into normal control group and diabetic (induced by intraperitoneal STZ injection) group. After 4 and 8 weeks, the body weight and diaphragm to body weight ratio were measured, and the activities of succinic dehydrogenase (SDH) in the diaphragm and blood glucose were assayed. The diaphragm contractility was assessed and the alterations of diaphragm ultrastructure were observed. RT-PCR was used to detect the changes in sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) and phospholamban (PLB) mRNA expressions in the diaphragm.</p><p><b>RESULTS</b>The diabetic rats showed a significant weight loss with a lowered diaphragm to body weight ratio (P<0.01) and SDH activity (P<0.01). The peak twitch tension and maximum tetanic tension of the diaphragm were significantly lowered and the time to peak contraction and half relaxation time significantly prolonged (P<0.01) in the diabetic rats, which also exhibited a lowered tetanic force in response to stimulus (P<0.01). Transmission electron microscopy revealed obvious ultrastructural changes of the diaphragm in diabetic rats. RT-PCR showed significantly decreased SERCA and increased PLB mRNA expressions in diabetic rat diaphragm (P<0.01), and these changes intensified with time (P<0.01).</p><p><b>CONCLUSION</b>Diabetes can cause impairment of diaphragmatic ultrastructure, mitochondrial injuries, and lowered SDH activity and ATP production. Decreased SERCA and increased PLB mRNA expressions in diabetes result in reduced Ca(2+) uptake by the diaphragm sarcoplasmic reticulum to induce diaphragm dysfunction.</p>

Influences of quercetin on contraction of small intestine smooth muscle of rabbits in vitro and its mechanism / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To observe the influences of quercetin (Que) on the contraction of small intestine smooth muscle of rabbits in vitro and explore the mechanism.</p><p><b>METHODS</b>With the isothermal perfusion of small intestine in vitro. The influences of quercetin on the spontaneous contraction of small intestine and contraction induced by Ach, histamine and Bacl2 were observed and the mechanism of quercetin was studied.</p><p><b>RESULTS</b>Quercetin reduced the tension of contraction of small intestine smooth muscle in rabbits in a dose-depended manner. Quercetin could completely block the contraction of Bay K8644. Heparin could also block the inhibition of quercetin on small intestine smooth muscle but ruthenium red (RR) had no effect on the relaxation of quercetin. Nitro-L-arginine methylester(L-NAME) inhibited the relaxation of quercetin.</p><p><b>CONCLUSION</b>Quercetin inhibits the contraction of small intestine smooth muscle of rabbits in vitro. The mechanism may be related to increase NO concentration in small intestine smooth muscle so that it inhibits extracellular Ca2+ inflowing via cell membrane. And quercetin has effect on intracellular Ca2+ releasing via IP3 of sarcoplasmic reticulum.</p>

Mercury Promotes Catecholamines Which Potentiate Mercurial Autoimmunity and Vasodilation: Implications for Inositol 1,4,5-Triphosphate 3-Kinase C Susceptibility in Kawasaki Syndrome

Previously, we reviewed biological evidence that mercury could induce autoimmunity and coronary arterial wall relaxation as observed in Kawasaki syndrome (KS) through its effects on calcium signaling, and that inositol 1,4,5-triphosphate 3-kinase C (ITPKC) susceptibility in KS would predispose patients to mercury by increasing Ca2+ release. Hg2+ sensitizes inositol 1,4,5-triphosphate (IP3) receptors at low doses, which release Ca2+ from intracellular stores in the sarcoplasmic reticulum, resulting in delayed, repetitive calcium influx. ITPKC prevents IP3 from triggering IP3 receptors to release calcium by converting IP3 to inositol 1,3,4,5-tetrakisphosphate. Defective IP3 phosphorylation resulting from reduced genetic expressions of ITPKC in KS would promote IP3, which increases Ca2+ release. Hg2+ increases catecholamine levels through the inhibition of S-adenosylmethionine and subsequently catechol-O-methyltransferase (COMT), while a single nucleotide polymorphism of the COMT gene (rs769224) was recently found to be significantly associated with the development of coronary artery lesions in KS. Accumulation of norepinephrine or epinephrine would potentiate Hg2+-induced calcium influx by increasing IP3 production and increasing the permeability of cardiac sarcolemma to Ca2+. Norepinephrine and epinephrine also promote the secretion of atrial natriuretic peptide, a potent vasodilator that suppresses the release of vasoconstrictors. Elevated catecholamine levels can induce hypertension and tachycardia, while increased arterial pressure and a rapid heart rate would promote arterial vasodilation and subsequent fatal thromboses, particularly in tandem. Genetic risk factors may explain why only a susceptible subset of children develops KS although mercury exposure from methylmercury in fish or thimerosal in pediatric vaccines is nearly ubiquitous. During the infantile acrodynia epidemic, only 1 in 500 children developed acrodynia whereas mercury exposure was very common due to the use of teething powders. This hypothesis mirrors the leading theory for KS in which a widespread infection only induces KS in susceptible children. Acrodynia can mimic the clinical picture of KS, leading to its inclusion in the differential diagnosis for KS. Catecholamine levels are often elevated in acrodynia and may also play a role in KS. We conclude that KS may be the acute febrile form of acrodynia.