When calcium turns arrhythmogenic: intracellular calcium handling during the development of hypertrophy and heart failure

Alterations of intracellular Ca2+ handling in hypertrophied myocardium have been proposed as a mechanism of ventricular tachyarrhythmias, which are a major cause of sudden death in patients with heart failure. In this review, alterations in intracellular Ca2+ handling and Ca2+ handling proteins in the development of myocardial hypertrophy and the transition to heart failure are discussed. The leading question is at what stage of hypertrophy or heart failure Ca2+ handling can turn arrhythmogenic. During the development of myocardial hypertrophy and the transition to failure, Ca2+ handling is progressively altered. Recordings of free myocyte Ca2+ concentrations during a cardiac cycle (Ca2+ transients) are prolonged early in the development of hypertrophy. However, resting (or diastolic) Ca2+ does not increase before end-stage heart failure has developed. These alterations are due to progressively defective Ca2+ uptake into the sarcoplasmic reticulum that seems to be caused by quantitative changes of gene expression of the Ca2+ ATPase of the sarcoplasmic reticulum. Increased expression and activity of the Na+/Ca2+ exchanger might compensate for this defective Ca2+ uptake, probably at the expense of increased arrhythmogenicity. When the Ca2+ handling proteins no longer efficiently counterbalance increasing intracellular Ca2+ - during stress conditions, resulting Ca2+ overload can lead to spontaneous intracellular Ca2+ oscillations, after depolarizations. Thus, after the transition to heart failure, Ca2+ overloaded sarcoplasmic reticulum, increasing resting intracellular Ca2+, and increased Na+/Ca2+ activity may all provoke afterdepolarizations, triggered activity, and finally, life-threatening ventricular arrhythmias. This increased susceptibility to ventricular arrhythmias in heart failure should not be treated with calcium antagonists.     

Ischemic myocardial cell injury. Prevention by chlorpromazine of an accelerated phospholipid degradation and associated membrane dysfunction.

Ligation of the left coronary artery of an adult rat heart results in the reproducible ischemic cell death of the entire free wall of the left ventricular myocardium. The time course of the development of the cellular changes is biphasic. The subendocardial and subepicardial cells die within the first few hours. The main mass of free-wall myocardium reacts more slowly, with morphologic evidence of irreversible cell injury developing after 12 hours. Measurement of the increases in total free wall Ca++ reflected this biphasic pattern. There was a rapid 3-fold rise in total Ca++ during the first 4 hours. Between 4 and 12 hours the Ca++ was constant. Between 12 and 30 hours there was a second increase that reached a level some 8-10 times the control value. Treatment with chlorpromazine before and subsequent to surgery prevented the appearance of ischemic cell death in the main portion of the free-wall myocardium for at least 24 hours without affecting the reaction of the subepicardial and subendocardial cells. Chlorpromazine also inhibited the second phase of Ca++ accumulation. An accelerated degradation of phospholipids was observed with a 33% decrease in total phospholipids by 12 hours. Phosphatidylethanolamine was reduced by 50% and phosphatidylcholine by 25% without increases in the corresponding lysophospholipids. Chlorpromazine prevented the accelerated degradation and consequent loss of phospholipid. Isolated sarcoplasmic reticulum showed a time-dependent loss of phospholipid with a parallel loss of active Ca++ uptake that reach 60% with a total lipid depletion from these membranes of 33% by 12 hours. Twelve-hour ischemic sarcoplasmic reticulum exhibited a 6--7-fold increase in passive permeability to Ca++. Chlorpromazine protected against the loss of phospholipids, the inhibition of Ca++ uptake, and the increased Ca++ permeability of the sarcoplasmic reticulum. These observations indicate that rat myocardial cells react to lethal doses of ischemia in a manner similar to the reaction of liver cells described previously. In both cases the evidence implies that a disturbance in phospholipid metabolism and its associated membrane dysfunction is the critical alteration that produces irreversible cell injury in ischemia.     

Excitation-contraction coupling in hypothermic ischemic myocardium

The excitation-contraction coupling system of the global ischemic hypothermic myocardium was studied by evaluating the functional integrity of the isolated sarcoplasmic reticulum (SR) and myofibrils and determining glycogen decay 30 and 60 min after the onset of surgically induced global ischemia. Calcium uptake by the SR from both the 30- and 60-min groups was depressed (control 0.940 +/- 0.05, 30 min 0.430 +/- 0.033, 60 min 0.535 +/- 0.033 mumol Ca2+ . mg-1 . min-1; P less than 0.001). In contrast SR Ca2+-ATPase activity was not different in the three groups (control 1.150 +/- 0.080, 30 min 1.468 +/- 0.025, 60 min 1.338 +/- 0.199 mumol Pi . mg-1 . min-1; P greater than 0.2). Glycogen decay in the hypothermic group was depressed compared to control (control 7.52 +/- 2.01, 30 min 6.152 +/- 1.16, 60 min 5.814 +/- 1.76 mumol glycogen/mg myocardium; P less than 0.05). Myofibrillar pCa-ATPase curves in both hypothermic ischemic groups were depressed (maximal ATPase activity; control 0.160 +/- 0.028, 30 min 0.1130 +/- 0.01, 60 min 0.127 +/- 0.008 mumol Pi . mg-1 . min-1; P less than 0.01). Kinetic analysis of the myofibrillar pCa-ATPase data, utilizing double-reciprocal plots, demonstrated an increase in Km for the hypothermic ischemic groups. It is concluded that the excitation-contraction coupling system of the hypothermic ischemic myocardium at 1 h is characterized by a defect in the calcium transport system of the sarcoplasmic reticulum with preservation of the Ca2+-ATPase, a depression of the myofibrillar ATPase activity, a decrease in affinity, and the preservation of adequate glycogen stores. It is hypothesized that these defects may explain an observed depression in myocardial function following reperfusion.     

Impaired Ca2+-sequestration in dilated cardiomyopathy (review)

Excitation-contraction coupling is the process by which depolarisation of the myocardial surface membrane leads to the release of Ca2+-ions from the sarcoplasmic reticulum, inducing cardiac muscle contraction. This process is made possible by an elaborate system of ion-release, uptake and sequestration that controls the contraction and relaxation cycle of heart muscle fibres. The free intracellular Ca2+-concentration determines the contractile state of the myocardium, and the sequestration of Ca2+-ions into the lumen of the sarcoplasmic reticulum by the Ca2+-ATPase pump units represents a critical step towards the maintenance of normal Ca2+-cycling. The Ca2+-ATPase pump activity is regulated by phospholamban, a small 52-amino acid protein whose phosphorylation state dictates its inhibitory action on the pump. A large body of evidence points to the central role of abnormal Ca2+-ATPase-phospholamban interactions in pathophysiological heart conditions, thereby compromising the contractile state of the cardiac muscle cell. It has been shown that alterations in the oligomeric status of the Ca2+-ATPase and modified interactions between the Ca2+-pump and its regulatory subunit phospholamban underlie the contractile dysfunction that characterises certain forms of dilated cardiomyopathy. Hence, elucidation of interactions within physiological Ca2+-ATPase pump units in normal and diseased myocardium is a vital link in the development of improved diagnostic and therapeutic techniques for dealing with this elusive condition.     

衰老大鼠心肌细胞器形态的定性定量改变及其调控钙离子能力分析

目的　探讨心肌衰老机制。　方法　采用 Wistar大鼠 ,分老龄组 (2 2月龄 )和成年组 (7月龄 )。用透射电镜观察心肌细胞器形态定性改变 ;用体视学方法测定心肌细胞器形态定量改变 ;用 X射线能谱仪分析心肌细胞器调控 Ca2 +能力。　结果　与成年组比较 ,老龄组大鼠心肌改变 :(1)核有切痕、肌原纤维不规整、闰盘离解、线粒体和肌浆网肿胀、脂褐素和残余体增多。(2 )在心肌组织内 ,非肌细胞所占体积份数增加 ,线粒体和肌浆网体密度减少 ,线粒体外膜比面积、内膜 +嵴比面积、肌浆网比面积减少。(3)在舒张状态下 ,肌原纤维和线粒体内 Ca2 +增加 ,肌浆网内 Ca2 +减少。　结论　衰老心肌的舒缩力减退 ,心肌细胞器形态呈明显改变 ,心肌衰老与线粒体和肌浆网的形态改变及其调控 Ca2 +能力有直接关系。     

Sodium-calcium exchange and sarcolemmal enzymes in ischemic rabbit hearts

We have investigated alterations in sarcolemmal function that occur during myocardial ischemia. Rabbit ventricles were incubated at 37 degrees C for time periods ranging from 5 min to 2 h. The ischemic tissue was homogenized, and activities of the sarcolemmal enzymes Na+-K+-ATPase, K+-p-nitrophenylphosphatase (K+-pNPPase), and adenylate cyclase were measured in the crude homogenate. Na+-K+-ATPase and K+-pNPPase were substantially inhibited after only 10 min of ischemia, and activities for all three enzymes declined progressively up to 1 h of ischemia, when activities were 37-59% of control. Highly purified sarcolemmal membranes prepared from control tissue and myocardium that had been made ischemic for 1 h showed similar purification of sarcolemmal enzymes, passive Ca2+ binding, and passive permeability to Ca2+. However, the velocity of Na+-Ca2+ exchange in ischemic sarcolemmal vesicles was reduced approximately 50% due to a reduction in Vmax. Although the parallel decline in activities of several sarcolemmal functions might suggest a change in membrane structure, phospholipid and cholesterol contents in ischemic sarcolemma were the same as control.     

Factors modifying contraction-relaxation cycle in vascular smooth muscles

Contraction-relaxation cycles in vascular smooth muscles are largely dependent on the regulation of free Ca2+ in the myoplasm, as is the case in skeletal and cardiac muscles. In this article we describe the varieties of contraction-relaxation cycles of vascular smooth muscles determined at cellular and subcellular levels. To discuss the excitation-contraction and pharmacomechanical coupling mechanisms in vascular tissues, passive and active membrane properties and ionic movements measured by various procedures are briefly introduced. In vascular smooth muscles the sources of Ca2+ contributing to the activation of contractile proteins are extra- and intracellular. Influxes of Ca2+ across the membrane are enhanced by the calcium spike and electrical and chemical depolarizations or activations of autonomic receptors.l However, the Ca2+ influx during the generation of action potential does not directly increase the free Ca2+ in the cell; rather, this ion is sequestered in the storage site and activates the calcium-induced calcium-release mechanism in the storage sites with a subsequent increase in the levels of free Ca2+. In some vascular tissues depolarizations induced by activations of autonomic receptors are not a prerequisite for generation of the contraction, as these mechanical responses appear with hyperpolarization of the membrane or without a change in the membrane potential. Possible functional links between the myoplasmic membrane where the receptors are distributed and the Ca2+ storage and releasing sites (mainly sarcoplasmic reticulum) in the cell are discussed. In addition, small arteries possess possibly more than three subtypes of alpha-adrenoceptors, including the presynaptic alpha 2-adrenoceptor. The roles of sarcoplasmic reticulum and the calcium receptor of contractile proteins (calmodulin or leiotonin C) from the chemically skinned muscles of vascular tissues were compared with those of intact muscles. The relaxation of vascular tissues as induced by activations of beta-adrenoceptors, nitrites, and other chemicals is also briefly introduced.     

Oxygen free radicals and excitation-contraction coupling

Oxygen free radicals (OFR) contribute to contractile failure, rigor, and calcium (Ca2+) overload in ischemic/reperfused myocardium. Using both multicellular and isolated single-cell preparations, our laboratory has identified two fundamental mechanisms contributing to the deleterious effects of OFR: (i) impaired myocardial metabolism, and (ii) altered myocardial calcium handling. Impaired metabolism leads to activation of metabolically sensitive K+ currents, which shorten the action potential, thereby decreasing the duration of systole. Ultimately, high-energy phosphate depletion secondary to metabolic failure results in rigor. Altered myocardial Ca2+ handling is evidenced by a decrease in Ca2+ entry via L-type Ca2+ channels [another cause of decreased action potential duration (APD)], a reduction in sarcoplasmic reticulum (SR) Ca2+ content, slowed Ca2+ uptake in diastole, and increased sodium-calcium exchange (NaCaX) activity. The increase in NaCaX activity may contribute to the early increase in developed tension frequently observed in multicellular preparations exposed to free radicals, as well as the SR depletion occurring early on in voltage-clamped isolated cell preparations. Increased NaCaX activity is likely to be a critical factor underlying the late Ca2+ overload that occurs in the setting of increased intracellular Na+, and which leads to irreversible injury. The extent to which free radical-mediated metabolic inhibition participates in the dysfunction of the L-type Ca2+ channel is uncertain. The altered activity of the SR Ca2+ pump and NaCaX are more likely caused by direct actions of OFR on these proteins.     

Immunoelectron microscopic studies on the localization of creatine kinase M in normal and ischemic myocardial cells

The localization of creatine kinase M (CK-M) in both normal and acute ischemic canine myocardial cells was studied by immunoelectron microscopy using the anti-CK-M Fab'-horseradish peroxidase conjugate. Myocardial ischemia was induced by occlusion of the left anterior descending coronary artery for 15, 60, or 180 minutes. In the normal myocardial cells, CK-M was localized mostly in the A-band and some in the Z-line, M-line, sarcolemmal membrane, and membrane of sarcoplasmic reticulum. Most CK-M in the A-band appeared to associate with thick fibers. This finding strongly suggests that the CK associated with thick fibers may be the enzyme to rephosphorylate ADP produced by myosin ATPase. In 15 minutes of myocardial ischemia, CK-M showed only minimal changes in its location, i.e., almost similar to normal, indicating that the CK in the A-band still has the ability to couple with myosin ATPase. However, in 60 and 180 minutes of ischemia, the A-band CK dissociated markedly from thick fibers, diffused to the I-band and leaked out to the intercellular spaces. These results suggest that the dissociation and disappearance of the A-band CK from thick fibers induced by progress of myocardial ischemia disrupt the myocardial energy transport system via CK reaction, and lead to the irreversible injury of myocardial cells.     

Calcium uptake and release through sarcoplasmic reticulum in the inferior oblique muscles of patients with inferior oblique overaction

We characterized and compared the characteristics of Ca2+ movements through the sarcoplasmic reticulum of inferior oblique muscles in the various conditions including primary inferior oblique overaction (IOOA), secondary IOOA, and controls, so as to further understand the pathogenesis of primary IOOA. Of 15 specimens obtained through inferior oblique myectomy, six were from primary IOOA, 6 from secondary IOOA, and the remaining 3 were controls from enucleated eyes. Ryanodine binding assays were performed, and Ca2+ uptake rates, calsequestrins and SERCA levels were determined. Ryanodine bindings and sarcoplasmic reticulum Ca2+ uptake rates were significantly decreased in primary IOOA (p < 0.05). Western blot analysis conducted to quantify calsequestrins and SERCA, found no significant difference between primary IOOA, secondary IOOA, and the controls. Increased intracellular Ca2+ concentration due to reduced sarcoplasmic reticulum Ca2+ uptake may play a role in primary IOOA.     

非创伤性预处理在离体心脏抗再灌注损伤中作用机制的探讨

目的:观察非创伤性肢体缺血预处理对大鼠离体再灌注心肌是否有保护作用。方法:实验采用体重(250±30)gSD雄性大鼠25只随机分成3组,在Langendorff装置上对大鼠离体心脏进行灌流。对照组(C,n=8):在灌注全程均用富氧K-H液(充以95%O₂+5%CO₂),在恒压(8.33kPa)、恒温(37℃)条件下灌注;缺氧/复氧组(A,n=8):预灌15min后,灌注心脏先全心缺血缺氧15min,随后15min复氧再灌注(37℃);非创伤性肢体缺血预处理组(N-WIP,n=9):先将大鼠双后肢捆绑5min,松开5min,反复4次后,随后的方法同R组。在相应时点分别测定冠脉流出液和心肌匀浆中超氧化物歧化酶(SOD)活性,Ca²⁺-Mg²⁺-ATP酶活性和丙二醛(MDA)含量,同时记录心肌细胞的单相动作电位(MAP)和心肌收缩张力曲线。结果:非创伤性肢体缺血预处理能使再灌注心律失常发生率显著低于A组;心肌组织中MDA含量显著低于A组,心肌组织中SOD活性显著高于A组,心肌细胞的膜电位、Ca²⁺-Mg²⁺-ATP酶活性及肌张力较稳定。结论:非创伤性肢体缺血预处理对大鼠离体再灌注心肌有明显的保护作用,可能是通过增强心肌的抗氧化能力、稳定心肌Ca²⁺-Mg²⁺-ATP酶活性和膜相结构等途径,提高心肌细胞对再灌注损伤的抵抗力。     

Effect of cyclopiazonic acid, an inhibitor of the sarcoplasmic reticulum Ca-ATPase, on skeletal muscles from normal and mdx mice

AIM: In this study, we investigated Ca2+ loading by the sarcoplasmic reticulum in skeletal muscle from mdx mice, an animal model of human Duchenne's muscular dystrophy, at two stages of development: 4 and 11 weeks. METHOD: Experiments were conducted on fast- (extensor digitorum longus, EDL) and slow- (soleus) twitch muscles expressing different isoforms of Ca2+-ATPase, which is responsible for the uptake of Ca2+ by the sarcoplasmic reticulum. RESULTS: In sarcoplasmic reticulum vesicles, the ATP-dependent activity and sensitivity to cyclopiazonic acid (CPA), an inhibitor of the sarcoplasmic reticulum Ca2+-ATPase, were similar in mdx and normal EDL muscle. Furthermore, in chemically-skinned fibres from both normal and mdx muscles, the presence of CPA induced a decrease in Ca2+ uptake by the sarcoplasmic reticulum. However, the sensitivity to CPA was lower in mdx EDL muscle than in normal muscle. In addition, in EDL muscle from 4-week-old mdx mice, the expression of the slow Ca2+-pump isoform (SERCA2a) was significantly increased, without any accompanying change in slow myosin expression. In contrast, the expression and function of the Ca2+-ATPase in mdx soleus muscles at 4- and 11-weeks of development did not differ from those in age-matched controls. CONCLUSION: These findings show that in dystrophic muscle, where the Ca2+ homeostasis was perturbed, the Ca2+ handling by the sarcoplasmic reticulum was altered in fast-twitch muscle, and this was associated with the expression of the slow isoform of SERCA. In these muscles, reduced Ca2+ uptake could then contribute to an elevated concentration of Ca2+ in the cytosol, and also to Ca2+ depletion of the sarcoplasmic reticulum.     

Kinetics of Calcium Accumulation in Acute Myocardial Ischemic Injury

The effect of ischemic injury on calcium uptake by dog myocardial cells was investigated in tissue damaged by transient or permanent occlusion of the circumflex branch of the left coronary artery. Tracer doses of ⁴⁵CaCl₂ were given at selected intervals before or after occlusion, and tissue uptake was measured in damaged and control left ventricular myocardium. No significant uptake of ⁴⁵Ca occurred after 60 minutes of ischemia produced by permanent occlusion of a coronary artery. However, 40 minutes of ischemia followed by 10 minutes of arterial reflow resulted in an 18-fold increase in Ca uptake in the injured tissue. Tissue ⁴⁵Ca increased linearly up through 10 minutes of arterial reflow but did not increase further with an additional 10 minutes of reflow. Myocardium reversibly injured by 10 minutes of ischemia followed by 20 minutes of arterial reflow did not accumulate excess ⁴⁵Ca. Calcium uptake is assumed to be an active process associated with mitochondrial accumulation of calcium into dense intramitochondrial granules of calcium phosphate. The uptake is a feature of irreversible cellular injury, but occurs only when arterial blood flow is present. The mechanism of the uptake has not been established. It appears to be related to defects in cellular permeability or mitochondrial function.     

Control of intracellular Ca2+ activity in rat myometrium.

Four fractions enriched, respectively, in plasma membrane (PM), smooth endoplasmic reticulum (SER), rough endoplasmic reticulum (RER), and mitochondria were isolated from estrogen-dominated rat myometrium. Ca2+ uptake by these fractions was studied in order to estimate the relative potential of the corresponding organelles for controlling intracellular Ca2+ activity. Ca2+ uptake properties of the PM, SER, and RER fractions were similar except that potentiation by oxalate was in the order RER greater than or equal SER greater than PM. However, studies with the ionophores X-537A and A23187 suggested that Ca2+ was transported into the lumen of membrane vesicles of all these fractions. Unlike that of skeletal muscle sarcoplasmic reticulum, Ca2+ uptake by the myometrial fractions was not supported by high-energy compounds other than ATP. Mitochondria took up much less Ca2+ at low, and much more Ca2+ at high, free Ca2+ concentrations than did the other fractions. The amount of Ca2+ taken up in 30 s from a 1 muM free Ca2+ solution in the presence of ATP was similar for all fractions. These results suggested that mitochondria may act as an important Ca2+ control system in rat myometrium when the intracellular Ca2+ concentration is near 1 muM or higher, whereas the PM, SER, and RER may be of major importance at Ca2+ levels of 0.3 muM or lower.     

感染性休克大鼠心肌肌浆网摄钙功能的变化

本工作观察大鼠感染性休克不同阶段心肌肌浆网(SR)摄钙功能的改变。结果表明在休克早期心肌SR的钙摄取功能尚无明显变化;SR Ca~(2+)-ATP酶活性抑制和钙摄取量降低出现在感染性休克的晚期阶段,可能是心功能障碍的重要原因。     

氟烷和七氟醚对缺血心肌功能和代谢及Ca2+-ATP酶活性的影响

目的: 研究氟烷、七氟醚(1.5MAC)对缺血心肌的影响。方法: 应用离体大鼠心脏Langendorff逆行灌注模型研究氟烷、七氟醚对心肌缺血前心率(HR)、左室舒张末期压力(LVEDP)、左室发展压(LVDP)、左室压力升高速率(+dp/dt)、左室压力下降速率(-dp/dt)和冠脉流量(CF)的影响,测定缺血前、缺血10min、缺血25min3个不同时间的心肌ATP含量、Ca²⁺-ATP酶活性,同时记录缺血间期左室内压的变化情况。结果: 七氟醚显著增加正常离体心脏的CF,氟烷、七氟醚均不同程度地抑制心肌收缩功能和Ca²⁺-ATP酶活性,能够增加正常心肌的能量贮备。缺血10min时,二药能够减缓心肌ATP含量及Ca²⁺-ATP酶活性的下降,氟烷的作用比较明显。缺血间期,氟烷明显推迟缺血性挛缩的起始时间,降低挛缩幅度。结论: 氟烷的抗缺血损伤作用优于七氟醚,延缓缺血期心肌ATP含量及Ca²⁺-ATP酶活性的下降可能是氟烷抗缺血损伤作用的重要机制之一。     

Phosphate transport into the sarcoplasmic reticulum of skinned fibres from rat skeletal muscle

The rate, magnitude and pharmacology of inorganic phosphate (Pi) transport into the sarcoplasmic reticulum were estimated in single, mechanically skinned skeletal muscle fibres of the rat. This was done, indirectly, by using a technique that measured the total Ca2+ content of the sarcoplasmic reticulum and by taking advantage of the 1:1 stoichiometry of Ca2+ and Pi transport into the sarcoplasmic reticulum lumen during Ca--Pi precipitation- induced Ca2+ loading. The apparent rate of Pi entry into the sarcoplasmic reticulum increased with increasing myoplasmic [Pi] in the 10 mm--50 mm range at a fixed, resting myoplasmic pCa of 7.15, as judged by the increase in the rate of Ca--Pi precipitation-induced sarcoplasmic reticulum Ca2+ uptake. At 20 mm myoplasmic [Pi] the rate of Pi entry was calculated to be at least 51 m s–1 while the amount of Pi loaded appeared to saturate at around 3.5 mm (per fibre volume). These values are approximations due to the complex kinetics of formation of different species of Ca--Pi precipitate formed under physiological conditions. Phenylphosphonic acid (PhPA, 2.5 mm inhibited Pi transport by 37% at myoplasmic pCa 6.5 and also had a small, direct inhibitory effect on the sarcoplasmic reticulum Ca2+ pump (16%). In contrast, phosphonoformic acid (PFA, 1 mm) appeared to enhance both the degree of Pi entry and the activity of the sarcoplasmic reticulum Ca2+ pump, results that were attributed to transport of PFA into the sarcoplasmic reticulum lumen and its subsequent complexation with Ca2+. Thus, results from these studies indicate the presence of a Pi transporter in the sarcoplasmic reticulum membrane of mammalian skeletal muscle fibres that is (1) active at physiological concentrations of myoplasmic Pi and Ca2+ and (2) partially inhibited by PhPA. This Pi transporter represents a link between changes in myoplasmic [Pi] and subsequent changes in sarcoplasmic reticulum luminal [Pi]. It might therefore play a role in the delayed metabolic impairment of sarcoplasmic reticulum Ca2+ release seen during muscle fatigue, which should occur abruptly once the Ca--Pi solubility product is exceeded in the sarcoplasmic reticulum lumen     

Functional properties of human embryonic stem cell-derived cardiomyocytes: intracellular Ca2+ handling and the role of sarcoplasmic reticulum in the contraction

Since cardiac transplantation is limited by the small availability of donor organs, regeneration of the diseased myocardium by cell transplantation is an attractive therapeutic modality. To determine the compatibility of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) (7 to 55 days old) with the myocardium, we investigated their functional properties regarding intracellular Ca2+ handling and the role of the sarcoplasmic reticulum in the contraction. The functional properties of hESC-CMs were investigated by recording simultaneously [Ca2+]i transients and contractions. Additionally, we performed Western blot analysis of the Ca2+-handling proteins SERCA2, calsequestrin, phospholamban, and Na+/Ca2+ exchanger (NCX). Our major findings are, first, that hESC-CMs displayed temporally related [Ca2+]i transients and contractions, negative force-frequency relations, and lack of post-rest potentiation. Second, ryanodine, thapsigargin, and caffeine did not affect the [Ca2+]i transient and contraction, indicating that at this developmental stage, contraction depends on transsarcolemmal Ca2+ influx rather than on sarcoplasmic reticulum Ca2+ release. Third, in agreement with the notion that a voltage-dependent Ca2+ current is present in hESC-CMs and contributes to the mechanical function, verapamil completely blocked contraction. Fourth, whereas hESC-CMs expressed SERCA2 and NCX at levels comparable to those of the adult porcine myocardium, calsequestrin and phospholamban were not expressed. Our study shows for the first time that functional properties related to intracellular Ca2+ handling of hESC-CMs differ markedly from the adult myocardium, probably due to immature sarcoplasmic reticulum capacity.     

The distribution of calcium in toad cardiac pacemaker cells during spontaneous firing

Isolated, spontaneously active pacemaker cells from the sinus venosus region of the toad heart were loaded with the calcium indicator fluo-3. The cells were examined with a confocal microscope to investigate the distribution of calcium during spontaneous activity. Three classes of calcium-related signals were present. First, intense, localised, time-invariant signals were detected from structures distributed across the cell interior. Based on the insensitivity to saponin and the distribution in the cell, these signals appear to arise from fluo-3 located in the sarcoplasmic reticulum and the nuclear envelope. Second, spatially uniform signals from the cytoplasm were present at rest and showed spontaneous increases in [Ca2+]i which propagated along the cell. These Ca2+ transients were uniform in intensity across the diameter of the cell and we could detect no significant delay in the middle of the cell compared to the edges. However, within the nucleus the Ca2+ transient showed a clear delay compared to the cytoplasm. Third, localised, transient increases in [Ca2+]i (Ca2+ sparks) which did not propagate were also detectable. These could be detected both near the surface membrane and in the interior of the cell and reduced in magnitude and increased in duration in the presence of ryanodine. The frequency of firing of Ca2+ sparks significantly increased in the 200-ms period preceding a spontaneous Ca2+ transient. These results suggest that pacemaker cells contain sarcoplasmic reticulum which is distributed across the cell. The Ca2+ transient is uniform across the cell indicating that near-synchronous release of Ca2+ from the sarcoplasmic reticulum is achieved. Ca2+ sparks occur in pacemaker cells though their role in pacemaker function remains to be elucidated.     

The composition and calcium transport activity of the sarcoplasmic reticulum from goats with and without heritable myotonia.

Studies were conducted on purified sarcoplasmic reticulum isolated from myotonic goats, an animal model of heritable myotonia. When compared to sarcoplasmic reticulum from normal goats, fragmented sarcoplasmic reticulum from the myotonic goat had (1) increased levels of calcium, (2) increased rates of calcium uptake and efflux, (3) an increased sialic acid content, and (4) an increased content of saturated fatty acids. These differences support the concept of a structural and functional defect as a basis for the abnormal contraction-relaxation characteristics of myotonia.