Study on the effect of doxorubicin on expressions of genes encoding myocardial sarcoplasmic reticulum Ca^2＋ transport proteins and the effect of taurine on myocardial protection in rabbits

To investigate the effect of doxorubicin(DOX) on gene expression of the myocardial sarcoplasmic reticulum (SR)Ca^2 transport proteins and the mechanism of taurine(Tau) protecting cardiac muscle cells, 9 rabbits were injected with DOX , 8 rabbits with DOX and Tau, and 9 rabbits with normal saline. Cardiac function , concentration of calcium in cardiomyocytes ( Myo [ Ca^2 ]i ), activity of SR Ca^2 -ATPase (SERCA2a) , level of SERCA2a mRNA and Ca^2 released channels(RYR2) mRNA were detected. The left ventricle tissues were observed by electron microscopy. The results showed that cardiac index, left ventricular systolic pressure, activity of SR Ca^2 -ATPase and level of SERCA2a mRNA decreased , while Myo[ Ca^2 ]i increased in DOX-treated rabbits. DOX could not affect the level of RYR2 mRNA. Tau intervention could alleviate the increase of left ventricular diastolic pressure, Myo[ Ca^2 ] i and the decrease of SERCA2a mRNA induced by doxorubicin. Tile results suggested that downregulation of SERCA2a gene expression was an important mechanism of DOX-induced cardiomyopathy and that Tau could partially improve the heart function by reducing calcium overload and alleviating downregulation of SERCA2a mRNA.     

p53-induced inhibition of Hif-1 causes cardiac dysfunction during pressure overload

Cardiac hypertrophy occurs as an adaptive response to increased workload to maintain cardiac function. However, prolonged cardiac hypertrophy causes heart failure, and its mechanisms are largely unknown. Here we show that cardiac angiogenesis is crucially involved in the adaptive mechanism of cardiac hypertrophy and that p53 accumulation is essential for the transition from cardiac hypertrophy to heart failure. Pressure overload initially promoted vascular growth in the heart by hypoxia-inducible factor-1 (Hif-1)-dependent induction of angiogenic factors, and inhibition of angiogenesis prevented the development of cardiac hypertrophy and induced systolic dysfunction. Sustained pressure overload induced an accumulation of p53 that inhibited Hif-1 activity and thereby impaired cardiac angiogenesis and systolic function. Conversely, promoting cardiac angiogenesis by introducing angiogenic factors or by inhibiting p53 accumulation developed hypertrophy further and restored cardiac dysfunction under chronic pressure overload. These results indicate that the anti-angiogenic property of p53 may have a crucial function in the transition from cardiac hypertrophy to heart failure.     

Structural changes in the calcium pump accompanying the dissociation of calcium

In skeletal muscle, calcium ions are transported (pumped) against a concentration gradient from the cytoplasm into the sarcoplasmic reticulum, an intracellular organelle. This causes muscle cells to relax after cytosolic calcium increases during excitation. The Ca(2+) ATPase that carries out this pumping is a representative P-type ion-transporting ATPase. Here we describe the structure of this ion pump at 3.1 A resolution in a Ca(2+)-free (E2) state, and compare it with that determined previously for the Ca(2+)-bound (E1Ca(2+)) state. The structure of the enzyme stabilized by thapsigargin, a potent inhibitor, shows large conformation differences from that in E1Ca(2+). Three cytoplasmic domains gather to form a single headpiece, and six of the ten transmembrane helices exhibit large-scale rearrangements. These rearrangements ensure the release of calcium ions into the lumen of sarcoplasmic reticulum and, on the cytoplasmic side, create a pathway for entry of new calcium ions.     

Hsp72 preserves muscle function and slows progression of severe muscular dystrophy

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder caused by mutations in the dystrophin gene that result in the absence of the membrane-stabilizing protein dystrophin. Dystrophin-deficient muscle fibres are fragile and susceptible to an influx of Ca(2+), which activates inflammatory and muscle degenerative pathways. At present there is no cure for DMD, and existing therapies are ineffective. Here we show that increasing the expression of intramuscular heat shock protein 72 (Hsp72) preserves muscle strength and ameliorates the dystrophic pathology in two mouse models of muscular dystrophy. Treatment with BGP-15 (a pharmacological inducer of Hsp72 currently in clinical trials for diabetes) improved muscle architecture, strength and contractile function in severely affected diaphragm muscles in mdx dystrophic mice. In dko mice, a phenocopy of DMD that results in severe spinal curvature (kyphosis), muscle weakness and premature death, BGP-15 decreased kyphosis, improved the dystrophic pathophysiology in limb and diaphragm muscles and extended lifespan. We found that the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA, the main protein responsible for the removal of intracellular Ca(2+)) is dysfunctional in severely affected muscles of mdx and dko mice, and that Hsp72 interacts with SERCA to preserve its function under conditions of stress, ultimately contributing to the decreased muscle degeneration seen with Hsp72 upregulation. Treatment with BGP-15 similarly increased SERCA activity in dystrophic skeletal muscles. Our results provide evidence that increasing the expression of Hsp72 in muscle (through the administration of BGP-15) has significant therapeutic potential for DMD and related conditions, either as a self-contained therapy or as an adjuvant with other potential treatments, including gene, cell and pharmacological therapies.     

A forty-kilodalton protein of the inner membrane is the mitochondrial calcium uniporter

Mitochondrial Ca(2+) homeostasis has a key role in the regulation of aerobic metabolism and cell survival, but the molecular identity of the Ca(2+) channel, the mitochondrial calcium uniporter, is still unknown. Here we have identified in silico a protein (named MCU) that shares tissue distribution with MICU1 (also known as CBARA1), a recently characterized uniporter regulator, is present in organisms in which mitochondrial Ca(2+) uptake was demonstrated and whose sequence includes two transmembrane domains. Short interfering RNA (siRNA) silencing of MCU in HeLa cells markedly reduced mitochondrial Ca(2+) uptake. MCU overexpression doubled the matrix Ca(2+) concentration increase evoked by inositol 1,4,5-trisphosphate-generating agonists, thus significantly buffering the cytosolic elevation. The purified MCU protein showed channel activity in planar lipid bilayers, with electrophysiological properties and inhibitor sensitivity of the uniporter. A mutant MCU, in which two negatively charged residues of the putative pore-forming region were replaced, had no channel activity and reduced agonist-dependent matrix Ca(2+) concentration transients when overexpressed in HeLa cells. Overall, these data demonstrate that the 40-kDa protein identified is the channel responsible for ruthenium-red-sensitive mitochondrial Ca(2+) uptake, thus providing a molecular basis for this process of utmost physiological and pathological relevance.     

TRIC channels are essential for Ca2+ handling in intracellular stores

Cell signalling requires efficient Ca2+ mobilization from intracellular stores through Ca2+ release channels, as well as predicted counter-movement of ions across the sarcoplasmic/endoplasmic reticulum membrane to balance the transient negative potential generated by Ca2+ release. Ca2+ release channels were cloned more than 15 years ago, whereas the molecular identity of putative counter-ion channels remains unknown. Here we report two TRIC (trimeric intracellular cation) channel subtypes that are differentially expressed on intracellular stores in animal cell types. TRIC subtypes contain three proposed transmembrane segments, and form homo-trimers with a bullet-like structure. Electrophysiological measurements with purified TRIC preparations identify a monovalent cation-selective channel. In TRIC-knockout mice suffering embryonic cardiac failure, mutant cardiac myocytes show severe dysfunction in intracellular Ca2+ handling. The TRIC-deficient skeletal muscle sarcoplasmic reticulum shows reduced K+ permeability, as well as altered Ca2+ 'spark' signalling and voltage-induced Ca2+ release. Therefore, TRIC channels are likely to act as counter-ion channels that function in synchronization with Ca2+ release from intracellular stores.     

α2δ expression sets presynaptic calcium channel abundance and release probability

Synaptic neurotransmitter release is driven by Ca(2+) influx through active zone voltage-gated calcium channels (VGCCs). Control of active zone VGCC abundance and function remains poorly understood. Here we show that a trafficking step probably sets synaptic VGCC levels in rats, because overexpression of the pore-forming α1(A) VGCC subunit fails to change synaptic VGCC abundance or function. α2δs are a family of glycosylphosphatidylinositol (GPI)-anchored VGCC-associated subunits that, in addition to being the target of the potent neuropathic analgesics gabapentin and pregabalin (α2δ-1 and α2δ-2), were also identified in a forward genetic screen for pain genes (α2δ-3). We show that these proteins confer powerful modulation of presynaptic function through two distinct molecular mechanisms. First, α2δ subunits set synaptic VGCC abundance, as predicted from their chaperone-like function when expressed in non-neuronal cells. Second, α2δs configure synaptic VGCCs to drive exocytosis through an extracellular metal ion-dependent adhesion site (MIDAS), a conserved set of amino acids within the predicted von Willebrand A domain of α2δ. Expression of α2δ with an intact MIDAS motif leads to an 80% increase in release probability, while simultaneously protecting exocytosis from blockade by an intracellular Ca(2+) chelator. α2δs harbouring MIDAS site mutations still drive synaptic accumulation of VGCCs; however, they no longer change release probability or sensitivity to intracellular Ca(2+) chelators. Our data reveal dual functionality of these clinically important VGCC subunits, allowing synapses to make more efficient use of Ca(2+) entry to drive neurotransmitter release.     

有氧运动改善心肌梗塞大鼠血流动力学异常和自主神经功能紊乱

目的:观察8周有氧运动对心肌梗塞大鼠血流动力学和自主神经功能的影响并探讨其可能机制.方法:27只SD大鼠随机分为假手术组(S组,n=10)、心肌梗塞组(M组,n=9)和心肌梗塞运动组(ME组,n=8),心肌梗塞模型采用冠状动脉结扎术,ME组进行8周跑台训练.末次训练后48h处死动物,分别测定大鼠心脏结构与功能、血流动力学、心率变异性以及心肌神经生长因子(NGF)和酪氨酸羟化酶(TH)基因表达水平.结果:(1)与S组比较,M组大鼠左室扩张、心功能下降,血流动力学异常,心率变异性显示自主神经功能紊乱,心肌NGF和TH mRNA与蛋白表达下调(P0.05);(2)与M组比较,ME组心功能增强,血流动力学异常和自主神经功能紊乱得以改善,心肌NGF和TH mRNA与蛋白表达上调(P0.05).结论:长期有氧运动可能通过改善血流动力学异常和自主神经功能紊乱抑制心肌梗塞大鼠心脏重塑并提高心功能.     

A calcium sensor in the sodium channel modulates cardiac excitability.

Sodium channels are principal molecular determinants responsible for myocardial conduction and maintenance of the cardiac rhythm. Calcium ions (Ca2+) have a fundamental role in the coupling of cardiac myocyte excitation and contraction, yet mechanisms whereby intracellular Ca2+ may directly modulate Na channel function have yet to be identified. Here we show that calmodulin (CaM), a ubiquitous Ca2+-sensing protein, binds to the carboxy-terminal 'IQ' domain of the human cardiac Na channel (hH1) in a Ca2+-dependent manner. This binding interaction significantly enhances slow inactivation-a channel-gating process linked to life-threatening idiopathic ventricular arrhythmias. Mutations targeted to the IQ domain disrupted CaM binding and eliminated Ca2+/CaM-dependent slow inactivation, whereas the gating effects of Ca2+/CaM were restored by intracellular application of a peptide modelled after the IQ domain. A naturally occurring mutation (A1924T) in the IQ domain altered hH1 function in a manner characteristic of the Brugada arrhythmia syndrome, but at the same time inhibited slow inactivation induced by Ca2+/CaM, yielding a clinically benign (arrhythmia free) phenotype.     

Multiple transport modes of the cardiac Na+/Ca2+ exchanger

The cardiac Na+/Ca2+ exchanger (NCX1; ref. 2) is a bi-directional Ca2+ transporter that contributes to the electrical activity of the heart. When, and if, Ca2+ is exported or imported depends on the Na+/Ca2+ exchange ratio. Whereas a ratio of 3:1 (Na+:Ca2+) has been indicated by Ca2+ flux equilibrium studies, a ratio closer to 4:1 has been indicated by exchange current reversal potentials. Here we show, using an ion-selective electrode technique to quantify ion fluxes in giant patches, that ion flux ratios are approximately 3.2 for maximal transport in either direction. With Na+ and Ca2+ on both sides of the membrane, net current and Ca2+ flux can reverse at different membrane potentials, and inward current can be generated in the absence of cytoplasmic Ca2+, but not Na+. We propose that NCX1 can transport not only 1 Ca2+ or 3 Na+ ions, but also 1 Ca2+ with 1 Na+ ion at a low rate. Therefore, in addition to the major 3:1 transport mode, import of 1 Na+ with 1 Ca2+ defines a Na+-conducting mode that exports 1 Ca2+, and an electroneutral Ca2+ influx mode that exports 3 Na+. The two minor transport modes can potentially determine resting free Ca2+ and background inward current in heart.     

TMEM16A confers receptor-activated calcium-dependent chloride conductance

Calcium (Ca(2+))-activated chloride channels are fundamental mediators in numerous physiological processes including transepithelial secretion, cardiac and neuronal excitation, sensory transduction, smooth muscle contraction and fertilization. Despite their physiological importance, their molecular identity has remained largely unknown. Here we show that transmembrane protein 16A (TMEM16A, which we also call anoctamin 1 (ANO1)) is a bona fide Ca(2+)-activated chloride channel that is activated by intracellular Ca(2+) and Ca(2+)-mobilizing stimuli. With eight putative transmembrane domains and no apparent similarity to previously characterized channels, ANO1 defines a new family of ionic channels. The biophysical properties as well as the pharmacological profile of ANO1 are in full agreement with native Ca(2+)-activated chloride currents. ANO1 is expressed in various secretory epithelia, the retina and sensory neurons. Furthermore, knockdown of mouse Ano1 markedly reduced native Ca(2+)-activated chloride currents as well as saliva production in mice. We conclude that ANO1 is a candidate Ca(2+)-activated chloride channel that mediates receptor-activated chloride currents in diverse physiological processes.     

Gated regulation of CRAC channel ion selectivity by STIM1

Two defining functional features of ion channels are ion selectivity and channel gating. Ion selectivity is generally considered an immutable property of the open channel structure, whereas gating involves transitions between open and closed channel states, typically without changes in ion selectivity. In store-operated Ca(2+) release-activated Ca(2+) (CRAC) channels, the molecular mechanism of channel gating by the CRAC channel activator, stromal interaction molecule 1 (STIM1), remains unknown. CRAC channels are distinguished by a very high Ca(2+) selectivity and are instrumental in generating sustained intracellular calcium concentration elevations that are necessary for gene expression and effector function in many eukaryotic cells. Here we probe the central features of the STIM1 gating mechanism in the human CRAC channel protein, ORAI1, and identify V102, a residue located in the extracellular region of the pore, as a candidate for the channel gate. Mutations at V102 produce constitutively active CRAC channels that are open even in the absence of STIM1. Unexpectedly, although STIM1-free V102 mutant channels are not Ca(2+)-selective, their Ca(2+) selectivity is dose-dependently boosted by interactions with STIM1. Similar enhancement of Ca(2+) selectivity is also seen in wild-type ORAI1 channels by increasing the number of STIM1 activation domains that are directly tethered to ORAI1 channels, or by increasing the relative expression of full-length STIM1. Thus, exquisite Ca(2+) selectivity is not an intrinsic property of CRAC channels but rather a tuneable feature that is bestowed on otherwise non-selective ORAI1 channels by STIM1. Our results demonstrate that STIM1-mediated gating of CRAC channels occurs through an unusual mechanism in which permeation and gating are closely coupled.     

Yeast plasma membrane ATPase is essential for growth and has homology with (Na+ + K+), K+- and Ca2+-ATPases

The plasma membrane ATPase of plants and fungi is a hydrogen ion pump. The proton gradient generated by the enzyme drives the active transport of nutrients by H+-symport. In addition, the external acidification in plants and the internal alkalinization in fungi, both resulting from activation of the H+ pump, have been proposed to mediate growth responses. This ATPase has a relative molecular mass (Mr) similar to those of the Na+-, K+- and Ca2+-ATPases of animal cells and, like these proteins, forms an aspartylphosphate intermediate. We have cloned, mapped and sequenced the gene encoding the yeast plasma membrane ATPase (PMA1) and report here that it maps to chromosome VII adjacent to LEU1. The strong homology between the amino-acid sequence encoded by PMA1 and those of (Na+ + K+), Na+-, K+- and Ca2+- ATPases is consistent with the notion that the family of cation pumps which form a phosphorylated intermediate evolved from a common ancestral ATPase. The function of the PMA1 gene is essential because a null mutation is lethal in haploid cells.     

代谢综合征左心功能研究进展

 简述了代谢综合征(metalbolic syndrome,MS)的定义及临床诊断标准,综述了高血压(EH)、肥胖、血糖异常、血脂异常等危险因素与左室心功能间的关系,分析了多种危险因素聚集一体的MS 的心功能变化特点。MS 患者的左室功能降低是由多个心血管危险因素引起,会导致多个复杂的代谢反应,影响心肌的结构和代谢环境,也改变了心肌功能和心肌能量。因此MS 患者在有症状的心力衰竭发展之前,可能存在一段时间亚临床的左心室功能障碍。     

Oligomerization of STIM1 couples ER calcium depletion to CRAC channel activation

Ca(2+)-release-activated Ca(2+) (CRAC) channels generate sustained Ca(2+) signals that are essential for a range of cell functions, including antigen-stimulated T lymphocyte activation and proliferation. Recent studies have revealed that the depletion of Ca(2+) from the endoplasmic reticulum (ER) triggers the oligomerization of stromal interaction molecule 1 (STIM1), the ER Ca(2+) sensor, and its redistribution to ER-plasma membrane (ER-PM) junctions where the CRAC channel subunit ORAI1 accumulates in the plasma membrane and CRAC channels open. However, how the loss of ER Ca(2+) sets into motion these coordinated molecular rearrangements remains unclear. Here we define the relationships among [Ca(2+)](ER), STIM1 redistribution and CRAC channel activation and identify STIM1 oligomerization as the critical [Ca(2+)](ER)-dependent event that drives store-operated Ca(2+) entry. In human Jurkat leukaemic T cells expressing an ER-targeted Ca(2+) indicator, CRAC channel activation and STIM1 redistribution follow the same function of [Ca(2+)](ER), reaching half-maximum at approximately 200 microM with a Hill coefficient of approximately 4. Because STIM1 binds only a single Ca(2+) ion, the high apparent cooperativity suggests that STIM1 must first oligomerize to enable its accumulation at ER-PM junctions. To assess directly the causal role of STIM1 oligomerization in store-operated Ca(2+) entry, we replaced the luminal Ca(2+)-sensing domain of STIM1 with the 12-kDa FK506- and rapamycin-binding protein (FKBP12, also known as FKBP1A) or the FKBP-rapamycin binding (FRB) domain of the mammalian target of rapamycin (mTOR, also known as FRAP1). A rapamycin analogue oligomerizes the fusion proteins and causes them to accumulate at ER-PM junctions and activate CRAC channels without depleting Ca(2+) from the ER. Thus, STIM1 oligomerization is the critical transduction event through which Ca(2+) store depletion controls store-operated Ca(2+) entry, acting as a switch that triggers the self-organization and activation of STIM1-ORAI1 clusters at ER-PM junctions.     

Vanadate inhibits uncoupled Ca efflux but not Na--Ca exchange in squid axons.

Nerve cells can maintain a very low intracellular calcium concentration ([Ca2+]i) against large Ca2+ electrochemical gradients (see ref. 1 for review). The properties of the calcium efflux from these cells depend on [Ca2+]i (ref. 2), and within the physiological range, most Ca efflux depends on ATP (which stimulates with high affinity) and is insensitive to Na1, Na0 and Ca0 (uncoupled Ca efflux). When the [Ca2+]i is well above the physiological range, Ca efflux becomes only partially dependent on ATP (acting now with low affinity), is inhibited by Nai and is stimulated by Na0 and Ca0 (Na--Ca exchange). Orthovanadate, a powerful inhibitor of the (Na+ + K+)ATPase and the Na pump, also inhibits the Ca-stimulated ATPase activity, which is the enzymatic basis for the uncoupled Ca pump, in human red cells. The experiments reported here show that in squid axons the ATP-dependent uncoupled Ca efflux can be fully and reversibly inhibited by vanadate, whereas concentrations of vanadate 10 times higher have no effect on the Na--Ca exchange. This is another indication that the uncoupled Ca efflux represents an ATP-driven Ca pump, and supports the suggestion that the uncoupled Ca efflux and Na--Ca exchange are mediated by different mechanisms.     

Oestrogen protects FKBP12.6 null mice from cardiac hypertrophy

FK506 binding proteins 12 and 12.6 (FKBP12 and FKBP12.6) are intracellular receptors for the immunosuppressant drug FK506 (ref. 1). The skeletal muscle ryanodine receptor (RyR1) is isolated as a hetero-oligomer with FKBP12 (ref. 2), whereas the cardiac ryanodine receptor (RyR2) more selectively associates with FKBP12.6 (refs 3, 4, 5). FKBP12 modulates Ca2+ release from the sarcoplasmic reticulum in skeletal muscle and developmental cardiac defects have been reported in FKBP12-deficient mice, but the role of FKBP12.6 in cardiac excitation-contraction coupling remains unclear. Here we show that disruption of the FKBP12.6 gene in mice results in cardiac hypertrophy in male mice, but not in females. Female hearts are normal, despite the fact that male and female knockout mice display similar dysregulation of Ca2+ release, seen as increases in the amplitude and duration of Ca2+ sparks and calcium-induced calcium release gain. Female FKBP12.6-null mice treated with tamoxifen, an oestrogen receptor antagonist, develop cardiac hypertrophy similar to that of male mice. We conclude that FKBP12.6 modulates cardiac excitation-contraction coupling and that oestrogen plays a protective role in the hypertrophic response of the heart to Ca2+ dysregulation.