Sweroside Effectively Protects Against Ischemia/Reperfusion Injury by Regulating Excitation-contraction Coupling / 中国实验方剂学杂志

ObjectiveTo explore the effect of sweroside on the protection of cardiac systolic/diastolic function during ischemia/reperfusion （I/R） injury. MethodTwenty-four healthy male SD rats were randomly divided into control group， model group， 10 μmol·L-1 sweroside group and 1 μmol·L-1 digoxin group. The I/R injury was modeled by Langendorff and ligation of the left anterior descending coronary artery. The infarct size in each group was measured by 2，3，5-triphenyltetrazolium chloride （TTC） staining and hemodynamic parameters such as left ventricular diastolic pressure （LVDP）， left ventricular end-diastolic pressure （LVEDP）， left ventricular end-systolic pressure （LVESP）， maximum rate of rising of left ventricular pressure （+dp/dtmax） and maximum rate of decreasing of left ventricular pressure （-dp/dtmax） of rat isolated heart were detected by Powerlab. In addition， neonatal rat cardiomyocytes （NRCMs） were isolated and randomly divided into control group， model group， 1 μmol·L-1 sweroside group and 10 μmol·L-1 sweroside group. Hypoxia/reoxygenation （H/R） injury model was established. Cardiac systolic function and calcium transients were examined by multi-functional cell imaging analyzer and laser confocal microscope. Furthermore， real-time polymerase chain reaction（Real-time PCR） was used to verify the mRNA expression of excitation-contraction coupling genes such as L-type calcium channel （Cacnb2）， cytochrome c oxidase subunit 6A2 （Cox6a2）， troponin （Tnnc1， Tnni3， Tnnt2）， actin （Actc1）， and myosin （Myh6， Myl2， Myl4） according to the results of previous transcriptome sequencing and literature investigation. Differentially expressed genes were subjected to cluster analysis. ResultCompared with the conditions in the control group， increased cardiac infarction size （P<0.01） and LVEDP （P<0.01） and decreased LVDP （P<0.01） and LVESP （P<0.05） were observed in the model group， with +dp/dtmax of increasing trend while -dp/dtmax decreasing. Moreover， the cell viability， heart rate and contraction amplitude of NRCMs was reduced （P<0.01）， while the contraction duration， time to peak and relaxation time was elevated （P<0.01） in the model group. Interestingly， sweroside could reverse these indicators （P<0.05）. In addition， the expression of Cacnb2， Cox6a2， Tnnc1， Tnni3， Tnnt2， Actc1， and Myh6， Myl2， and Myl4 was down-regulated in the model group （P<0.05， P<0.01）， but sweroside could up-regulate the expression of the above genes （P<0.05）. ConclusionSweroside effectively regulated Ca2+ level in NRCMs， enhanced cardiac systolic function， and protected against H/R injury by regulating excitation-contraction coupling.

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.

JPH2 gene expression in persistent atrial fibrillation with mitral valve disease / 中华胸心血管外科杂志

Objective This research is to explore the Junctophilin-2 ( JPH2 ) expression in persistent atrial fibrillation with mitral valve disease.Methods The left atrial tissue samples were taken from 34 patients with mitral valve disease who underwent cardiac surgery.16 patients were in sinus rhythm, 18 patients had persistent atrial fibrillation.Western blot tech-nique was used to test the JPH2 expression, and the RT-PCR method was used to test the JPH2 gene expression.Results The Westernblot results showed JPH2 expression down-regulated in persistent atrial fibrillation group (0.94 ±0.29 vs.1.53 ± 0.61,P0.05).Expression of miRNA-24 was significantly up-regulated in patients with persistent atrial fibrillation(4.49 ±4.30 vs.1.72 ±1.08, P<0.05).There was no significant difference in mean age, gender, ejection fraction and NYHA among the two groups.But the left atrial diameter was significant larger in patients with persistent atrial fibrillation compared to those in sinus rhythm(P=0.02).Conclusion Junctophilin-2 protein down-regulation may be associated with atrial fibrillation , cause left atrial remodeling and contraction dysfunction .

Isolation and culture of adult rat cardiomyocytes and characteristics of excitation-contraction coupling / 中国比较医学杂志

Objective To compare two separation medium of isolation of adult rat cardiomyocytes , and to observe the characteristics of excitation-contraction coupling of cardiomyocytes .Methods The isolated adult rat heart was hanged on to the Langendorff apparatus for aortic counter-current perfusion and collagenase digestion using two different separation medium.The single cardiomyocytes were cultured and infected with adenovirus . The morphological features of cardiomyocytes were observed with microscope and fluorescent microscope . The shortening-re-lengthening features of sarcomere and the intake-discharge features of calcium were simultaneously recorded by IonOptix equipment .Results 70%rod-shaped with clear-striation adult rat cardiomyocytes could be obtained with the stated two separation medium and cultured in serum-free medium for more than 7 days.GFP could express more than 7 days when the cardiomyocytes were infected with adenovirus .Cardiomyocytes obtained by the first separation medium could not contract with the electrical stimulation, while cardiomyoctyes obtained by the second separation medium could be used for the detection of excitation -contraction coupling .The shortening fraction of sarcomere was 11.61%±2.15% and the relaxing time was ( 0.177 ± 0.031) s.The amplitude of calcium transient was 30.79% ±9.74 % and the decaying time of calcium transient was (0.300 ±0.074) s.Conclusion With the stated two separation medium , adult rat cardiomyocytes can be well isolated , cultured and infected with adenovirus .The second separation medium can be used for the detection of excitation-contraction coupling characteristics .

JP-2 gene expression affects excitation-contraction coupling in the dilated cardiomyopathy heart failure / 中华胸心血管外科杂志

ObjectiveHeart failure is closely associated with a defected calcium-induced calcium release (CICR) between the transverse tubular (TT) invagination of plasma membrane and terminal cistemae of sarcoplasmic reticulum (SR) in cardiac myocytes.The underlying cause of this defect is not well understood.Any factors impacting the TT and SR connecting may reduce the excitation-contraction coupling efficiency.Junctophilin 2 (Jph 2) is a cardiac protein anchoring SR to TT.This research is to explore the JP-2 expression in the cardiomyopathy heart failure myocytes.Objective Myocardium specimens of the lateral segments of left ventricule were collected from cardiomyopathy heart failure patients and the transplantation donors.MethodsGroup A:heart failure cases diagnosised as cardiomyopathy and samples were collected from the left ventricle lateral wall.Group B:control samples from the transplantation donors which not used for the recepients reason.Electron microscopy technique was used to test the mean junctional distance between TT and SR.Westernblot technique was used to test the Junctophilin 2 expression and the RT-PCR method was used to test the JP-2 gene expression.The data were analysises with the SPSS 12.0 software,P ＜ 0.05 was accepted as different significantly.ResultsSamples were collected from 14 patients with severe heart failure and 6 control cases.Electron microscopy ultrastructure results showed in an average 100 μm2 of the myocardial cells area the coupling numbers of the control group and heart failure group were 60 and 112 (P ＜0.001 ).The electrical micrography mean junctional distance between TT and SR was significantly increased from the control group ( 16.2 ± 3.2) nm to the ( 19.3 ±4.3 ) am in the heart failure group( P ＜0.001 ) ). The Westernblot results showed Junctophilin 2 versus GAPDH expression down regulated in the heart failure group comparing to the control group (7.2％ vs 15.3 ％,P ＜ 0.05 ).The RT-PCR implied the JP-2 gene versus GAPDH expression also down regulated in the heart failure group comparing to the control group (37.5％ vs 98.8％,P ＜ 0.01 ).ConclusionConclusion JP-2 gene down-regulation may be one of the earliest change in the heart failure molecular mechanisms.

Differences in Ca2+-management between the ventricle of two species of neotropical teleosts: the jeju, Hoplerythrinus unitaeniatus (Spix & Agassiz, 1829), and the acara, Geophagus brasiliensis (Quoy & Gaimard, 1824)

This study analyzed the physiological role of the cardiac sarcoplasmic reticulum (SR) of two neotropical teleosts, the jeju, Hoplerythrinus unitaeniatus (Erythrinidae), and the acara, Geophagus brasiliensis (Cichlidae). While the in vivo heart frequency (fH - bpm) of acara (79.6 ± 6.6) was higher than that of the jeju (50.3 ± 2.7), the opposite was observed for the ventricular inotropism (Fc - mN/mm²) at 12 bpm (acara = 28.66 ± 1.86 vs. jeju = 36.09 ± 1.67). A 5 min diastolic pause resulted in a strong potentiation of Fc (≅ 90 percent) of strips from jeju, which was completely abolished by ryanodine. Ryanodine also resulted in a ≅ 20 percent decrease in the Fc developed by strips from jeju at both subphysiological (12 bpm) and physiological (in vivo) frequencies. However, this effect of ryanodine reducing the Fc from jeju was completely compensated by adrenaline increments (10-9 and 10-6 M). In contrast, strips from acara were irresponsive to ryanodine, irrespective of the stimulation frequency, and increases in adrenaline concentration (to 10-9 and 10-6 M) further increased Fc. These results reinforce the hypothesis of the functionality of the SR as a common trait in neotropical ostariophysian (as jeju), while in acanthopterygians (as acara) it seems to be functional mainly in 'athletic' species.(AU)

O presente estudo analisou o papel fisiológico desempenhado pelo retículo sarcoplasmático (RS) de duas espécies de teleósteos neotropicais, o jeju, Hoplerythrinus unitaeniatus (Erythrinidae), e o acará, Geophagus brasiliensis (Cichlidae). Enquanto a frequência cardíaca registrada in vivo (fH - bpm) para o acará (79.6 ± 6.6) foi superior àquela observada para o jeju (50.3 ± 2.7), resposta inversa foi verificada para o inotropismo ventricular (Fc - mN/mm²) na frequência de estimulação de 12 bpm (acará = 28.66 ± 1.86 vs. jeju = 36.09 ± 1.67). Uma pausa diastólica de 5 min resultou em uma expressiva potenciação da Fc (≅ 90 por cento) das tiras de jeju, a qual foi completamente abolida pela rianodina. A rianodina também resultou em um decréscimo de ≅ 20 por cento na Fc desenvolvida pelas tiras de jeju tanto a frequências sub-fisiológicas (12 bpm) quanto fisiológicas (in vivo). No entanto, o decréscimo da Fc promovido pela rianodina foi completamente compensado pela adição de adrenalina (10-9 e 10-6 M). Em contraste, as tiras de acará foram irresponsivas à rianodina, independentemente da frequência de estimulação utilizada, fazendo com que a adição de adrenalina (10-9 e 10-6 M) resultasse em incrementos ainda maiores da Fc. Esses resultados reforçam a hipótese de que a funcionalidade do RS seja uma característica comum aos ostariofíseos neotropicais (como o jeju), enquanto nos acantopterígios (como o acará) esta organela parece ser funcional principalmente em espécies ativas.(AU)