t-Tubules and sarcoplasmic reticulum function in cardiac ventricular myocytes

Although the existence of t-tubules in mammalian cardiac ventricular myocytes has been recognized for a long time, it now appears that their structure and function are more complex than previously believed. Recent work has provided evidence that many of the key proteins underlying excitation-contraction coupling are located predominantly at the t-tubules. L-type Ca(2+) current (I(Ca)) flowing across the t-tubule membrane provides a rapidly inactivating Ca(2+) influx that triggers Ca(2+) release from the sarcoplasmic reticulum (SR), thereby allowing rapid and synchronous Ca(2+) release throughout the cell; I(Ca) at the t-tubules also appears to be more sensitive than that at the surface membrane to regulation by beta-adrenergic stimulation and intracellular Ca(2+). In contrast, although its density is lower, I(Ca) flowing across the surface membrane inactivates slowly, and thus may help load the SR with Ca(2+). There is also increasing evidence that many of the mechanisms that remove Ca(2+) from the cytoplasm are located predominantly at the t-tubules, which therefore play an important role in determining cellular, and hence SR, Ca(2+) content. Thus, the t-tubules appear to play a central role in the increase and subsequent decrease of Ca(2+) during the systolic Ca(2+) transient. Remodelling of the t-tubules has been reported in cardiac pathologies, and may play a role in the altered cellular, and hence cardiac, function observed in such conditions.     

Isolation and characterization of beta 1-adrenergic receptors from adult, rat cardiac myocytes

The beta-receptors were isolated from rat cardiac myocytes and characterized. Isolated myocytes were prepared from adult rat hearts and characterized for viability. Membrane proteins were solubilized from myocytes with 1% Triton X-102. The solubilized membrane proteins were fractionated by DEAE-Sephacel ion exchange column chromatography. Two major protein peaks were obtained. The second protein peak sample was found to contain beta-receptors to which 125I-15-(4'-azido-3'-iodobenzyl)-carazolol (125I-ABC) was specifically bound. This sample was labeled covalently with 125I-ABC by UV irradiation. The radiolabeled sample was applied to a Sepharose CL-6B gel column. Two radiolabeled protein peaks, one with a molecular weight of approximately 570,000 and the other with a molecular weight of approximately 95,000 were found. When the 570,000-dalton complex was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, it was dissociated into a component with a molecular weight of 66,000. The 95,000-dalton complex was dissociated into a 58,000-dalton component upon SDS-PAGE under reducing conditions. An excess amount of isoproterenol and propranolol decreased photolabeling of the beta-receptors with 125I-ABC by 60% and 40%, respectively.     

Contractile properties of ventricular myocytes isolated from spontaneously hypertensive rat.

OBJECTIVE: To determine whether there are any differences in contractile properties of individual cardiac myocytes isolated from the spontaneously hypertensive rat (SHR) in comparison with its normotensive control--the Wistar-Kyoto (WKY) rat. DESIGN: The effects of cardiac hypertrophy upon individual myocytes from SHR have not been studied previously. Isolated cardiac myocytes do not suffer from a number of problems inherent in experiments on multicellular preparations. METHODS: Seven SHR and eight WKY animals were studied. Age-matched animals were compared at 60 and 100 days old. Ventricular myocytes were isolated enzymatically. Myocyte length and width was measured. The cells were stimulated with extracellular electrodes and contraction was measured optically. The effects of altering stimulus rate and extracellular calcium concentration upon contraction were studied. RESULTS: SHR myocytes were found to be significantly wider than WKY myocytes. The contraction (i.e. unloaded cell shortening) of SHR myocytes at stimulation rate of 0.3, 1, 2 and 3 Hz was significantly increased. The time-course of contraction was altered, with SHR myocytes having an increased maximal velocity of shortening and relaxation. The response to changes in bathing calcium was similar in both strains. CONCLUSIONS: Individual cardiac myocytes isolated from SHR have an increased contraction. This indicates that cardiac hypertrophy, at least in the early stages, is a protective adaptation allowing the heart to overcome the increased afterload resulting from hypertension.     

Spatial and temporal inhomogeneities during Ca2+ release from the sarcoplasmic reticulum in pig ventricular myocytes

The [Ca2+]i transient of ventricular myocytes during normal excitation-contraction coupling is the summation of primary Ca2+ release events, which originate at the junction of the sarcoplasmic reticulum (SR) and the T-tubular system. Studies in small mammals have shown a high density of release sites, but little is known of larger mammals. We have studied the spatial distribution of SR Ca2+ release in pig ventricular myocytes using a confocal microscopy. In 69 of 107 cells, large inhomogeneities of Ca2+ release were observed along the longitudinal scan line. Areas where the increase of [Ca2+]i was delayed (time to 50% of peak F/F0 [where F indicates fluorescence intensity, and F0 indicates F at rest] was 26+/-1 ms in delayed areas versus 11+/-2 ms in early areas) and smaller (peak F/F0 was 2.27+/-0.10 for delayed areas versus 2.69+/-0.13 for early areas; n=13 cells, P<0.05) could be up to 26 microm wide. The sum of all delayed areas could make up to 55% of the line scan. The spatial pattern was constant during steady-state stimulation and was not altered by enhancing Ca2+ channel opening or SR Ca2+ content (Bay K8644, isoproterenol). Imaging of sarcolemmal membranes revealed several areas devoid of T tubules, but SR Ca2+ release channels were homogeneously distributed. In contrast, compared with pig myocytes, mouse myocytes had a very dense T-tubular network, no large inhomogeneities of release, and a faster rate of rise of [Ca2+]i. In conclusion, in pig ventricular myocytes, areas of delayed release are related to regional absence of T tubules but not ryanodine receptors. This lower number of functional couplons contributes to a slower overall rate of rise of [Ca2+]i.     

Elevated sarcoplasmic reticulum Ca2+ leak in intact ventricular myocytes from rabbits in heart failure

Altered sarcoplasmic reticulum (SR) Ca2+-ATPase and Na+-Ca2+ exchange (NCX) function have been implicated in depressing SR Ca2+ content and contractile function in heart failure (HF). Enhanced diastolic ryanodine receptor (RyR) leak could also lower SR Ca2+ load in HF, but direct cellular measurements are lacking. In this study, we measure SR Ca2+ leak directly in intact isolated rabbit ventricular myocytes from a well-developed nonischemic HF model. Abrupt block of SR Ca2+ leak by tetracaine shifts Ca2+ from the cytosol to SR. The tetracaine-induced decline in [Ca2+]i and increase total SR Ca2+ load ([Ca2+]SRT) directly indicate the SR Ca2+ leak (before tetracaine). Diastolic SR Ca2+ leak increases with [Ca2+]SRT, and for any [Ca2+]SRT is greater in HF versus control. Mathematical modeling was used to compare the relative impact of alterations in SR Ca2+ leak, SR Ca2+-ATPase, and Na+-Ca2+ exchange on SR Ca2+ load in HF. We conclude that increased diastolic SR Ca2+ leak in HF may contribute to reductions in SR Ca2+ content, but changes in NCX in this HF model have more impact on [Ca2+]SRT.     

Primary culture of purified Leydig cells isolated from adult rat testes

Methods for isolating highly purified Leydig cells permit the study of acute responses and biochemical properties of Leydig cells independent of other testicular cell types. The present study describes the development of a primary culture system for purified Leydig cells from adult rats in which the cells retain their ability to secrete testosterone for at least 72 h in culture. When Leydig cells were cultured in tissue culture medium 199--0.1% BSA (M199-BSA), basal testosterone secretion declined by 72 h, whereas hCGB-stimulated testosterone secretion was reduced by 48 h. Changing the culture medium twice daily or adding 0.5% fetal calf serum (fcs) enhanced basal and gonadotropin-stimulated testosterone secretion at 72 h in culture, although responsiveness to hCG was reduced to 57% of that in freshly isolated cells. Incubation of Leydig cells in the defined culture medium Dulbecco's Modified Eagles-Ham's F-12 (1:1, vol/vol) supplemented with 15 mM Hepes buffer, transferrin, insulin, and epidermal growth factor (DHG:F12 + Hepes + TIE) in either the presence or absence of 0.5% fcs yielded functional Leydig cells for longer intervals in culture. Furthermore, testosterone secretion was greater in DHG:F12 + Hepes + TIE than in M199-BSA at all time intervals tested. In DHG:F12 + Hepes + TIE, basal and gonadotropin-stimulated testosterone production by Leydig cells were maintained for 72 h in culture. Degenerative changes in morphology were apparent in some cells at 72 h, but not at earlier times in culture. This primary culture system for isolated Leydig cells provides a valuable tool to examine the temporally regulated events in Leydig cell function.     

Ultrastructural studies of metabolically active isolated adult rat heart myocytes.

Myocytes from adult rat heart were isolated by a method recently developed by us in which hearts were pre-perfused with calcium-free phosphate-buffered saline medium containing collagenase and hyaluronidase. This was followed by incubation in the enzyme medium and cellular sedimentation through 3% Ficoll. Myocytes isolated in this manner were metabolically active and morphologically intact. In the present study, scanning and transmission electron microscopy of isolated myocytes showed long cylindrical cells with transverse microridges which corresponded directly with sarcomere lengths. Most cells appeared to be in a fully contracted state. Contractile elements and associated membranes, other intracellular compartments and sarcolemmae were indistinguishable from their in vivo counterparts. Although myocytic basal laminae and other structurally identifiable surface coats were removed by our isolation procedure, sarcolemmae remained remarkably unaffected. Cyclic AMP assays in control and epinephrine- or glucagon-stimulated cells strongly suggested that membranebound receptors were present and the functional integrity of the sarcolemmae was maintained in our preparations.     

Single calcium channels in native sarcoplasmic reticulum membranes from skeletal muscle.

Electrical properties of native sarcoplasmic reticulum membranes from rabbit skeletal muscle were investigated using the patch-clamp technique. Bilayers were assembled at the tip of patch pipettes from monolayers formed at the air-water interface of sarcoplasmic reticulum membrane suspensions. The membranes were found to contain a spontaneously active cation channel of small conductance (5 pS in 200 mM CaCl2, symmetrical solutions) that was selective for Ca2+ and Ba2+. Between 50 and 200 mM CaCl2 (symmetrical) the increase in conductance as a function of [Ca2+] fit a hyperbola (K0.5, 83 mM, and gamma max, 7.9 pS) that extrapolated to a single-channel conductance of 0.5 pS at physiological Ca2+ levels. The channel opened in bursts followed by long silent periods of up to a minute. During a burst the channel fluctuated very rapidly with time constants in the millisecond range. The mean burst duration was voltage dependent, increasing from 1.8 s at a pipette voltage of +60 mV to 4.1 s at +80 mV. Over this range, burst frequency decreased with increasing voltage such that the fraction of time spent in the open state (fb) remained constant. Application of 1.6 mM caffeine resulted in activation of the channel that appeared as an increase in mean burst duration. In contrast, 50 microM dantrolene significantly decreased burst frequency, whereas 10 microM nitrendipine had no effect. The functional and pharmacological properties of this Ca2+ channel suggest that it may be important in mediating Ca2+ release from the sarcoplasmic reticulum during excitation-contraction coupling.     

Mitochondrial free calcium regulation during sarcoplasmic reticulum calcium release in rat cardiac myocytes

Cardiac mitochondria can take up Ca²⁺, competing with Ca²⁺ transporters like the sarcoplasmic reticulum (SR) Ca²⁺-ATPase. Rapid mitochondrial [Ca²⁺] transients have been reported to be synchronized with normal cytosolic [Ca²⁺]_i transients. However, most intra-mitochondrial free [Ca²⁺] ([Ca²⁺]_mito) measurements have been uncalibrated, and potentially contaminated by non-mitochondrial signals. Here we measured calibrated [Ca²⁺]_mito in single rat myocytes using the ratiometric Ca²⁺ indicator fura-2 AM and plasmalemmal permeabilization by saponin (to eliminate cytosolic fura-2). The steady-state [Ca²⁺]_mito dependence on [Ca²⁺]_i (with 5 mM EGTA) was sigmoid with [Ca²⁺]_mito < [Ca²⁺]_i for [Ca²⁺]_i below 475 nM. With low [EGTA] (50 μM) and 150 nM [Ca²⁺]_i (± 15 mM Na⁺) cyclical spontaneous SR Ca²⁺ release occurred (5–15/min). Changes in [Ca²⁺]_mito during individual [Ca²⁺]_i transients were small ( 2–10 nM/beat), but integrated gradually to steady-state. Inhibition SR Ca²⁺ handling by thapsigargin, 2 mM tetracaine or 10 mM caffeine all stopped the progressive rise in [Ca²⁺]_mito and spontaneous Ca²⁺ transients (confirming that SR Ca²⁺ releases caused the [Ca²⁺]_mito rise). Confocal imaging of local [Ca²⁺]_mito (using rhod-2) showed that [Ca²⁺]_mito rose rapidly with a delay after SR Ca²⁺ release (with amplitude up to 10 nM), but declined much more slowly than [Ca²⁺]_i (time constant 2.8 ± 0.7 s vs. 0.19 ± 0.06 s). Total Ca²⁺ uptake for larger [Ca²⁺]_mito transients was  0.5 μmol/L cytosol (assuming 100:1 mitochondrial Ca²⁺ buffering), consistent with prior indirect estimates from [Ca²⁺]_i measurements, and corresponds to  1% of the SR Ca²⁺ uptake during a normal Ca²⁺ transient. Thus small phasic [Ca²⁺]_mito transients and gradually integrating [Ca²⁺]_mito signals occur during repeating [Ca²⁺]_i transients.     

Functional consequences of detubulation of isolated rat ventricular myocytes

OBJECTIVE: Recent work has suggested that Na(+)/Ca(2+) exchange (NCX) and L-type Ca(2+) current (I(Ca)) are located predominantly in the t-tubules of cardiac ventricular myocytes, which therefore represent a microdomain for the regulation of intracellular Na(+) (Na(i)) and Ca(2+) (Ca(i)). The aim of this study was to investigate the role of the t-tubules in the response of Ca(i) and contraction to interventions that alter the transsarcolemmal Na(+)gradient. METHODS: Enzymatically isolated and detubulated Wistar rat ventricular myocytes were investigated using fluorescence microscopy and optical detection of cell length. RESULTS: In unstimulated cells, spontaneous contractile activity increased when extracellular [Na(+)] was decreased or strophanthidin (100 microM) was added to the bathing solution, but the increase was significantly smaller in detubulated cells than in control cells. In electrically stimulated cells, strophanthidin increased Na(i) to a similar extent in normal and detubulated cells, although the associated increase in Ca(2+) transient amplitude and contraction were significantly smaller in detubulated cells. Similarly, tetrodotoxin (TTX, 10 microM) attenuated the Ca(2+) transient and contraction less in detubulated than in control cells. Increasing stimulation rate (0.05-1 Hz) caused little change or a small increase in contraction amplitude in control cells, but a significant decrease in contraction amplitude in detubulated cells, although the change of Na(i) caused by increasing stimulation rate from 0 to 1 Hz was not significantly different in the two cells types. CONCLUSION: It is concluded that although some Na/K ATPase, NCX and Na(+)channel activity is present on the surface membrane, the t-tubules play a major role in the modulation of contraction via NCX, allowing changes of the transsarcolemmal Na(+)gradient to be translated into changes of Ca(i).     

Altered cardiac sarcoplasmic reticulum function of intact myocytes of rat ventricle during metabolic inhibition

Changes in the behavior of the sarcoplasmic reticulum (SR) in rat ventricular myocytes were investigated under conditions of metabolic inhibition using laser-scanning confocal microscopy to measure intracellular Ca(2+) and the perforated patch-clamp technique to measure SR Ca(2+) content. Metabolic inhibition had several effects on SR function, including reduced frequency of spontaneous releases of Ca(2+) (sparks and waves of Ca(2+)-induced Ca(2+) release), increased SR Ca(2+) content (79.4+/-5.7 to 115.2+/-6.6 micromol/L cell volume [mean+/-SEM; P:<0.001]), and, after a wave of Ca(2+) release, slower reuptake of Ca(2+) into the SR (rate constant of fall of Ca(2+) reduced from 8.5+/-1.1 s(-)(1) in control to 5.2+/-0.4 s(-)(1) in metabolic inhibition [P:<0.01]). Inhibition of L-type Ca(2+) channels with Cd(2+) (100 micromol/L) did not reproduce the effects of metabolic inhibition on spontaneous Ca(2+) sparks. These results are evidence of inhibition of both Ca(2+) release and reuptake mechanisms. Reduced frequency of release could be attributable to either of these effects, but the increased SR Ca(2+) content at the time of reduced frequency of spontaneous release of Ca(2+) shows that the dominant effect of metabolic inhibition is to inhibit release of Ca(2+) from the SR, allowing the accumulation of greater than normal amounts of Ca(2+). In the context of ischemia, this extra accumulation of Ca(2+) would present a risk of potentially arrhythmogenic, spontaneous release of Ca(2+) on reperfusion of the tissue.     

Synchronous occurrence of spontaneous localized calcium release from the sarcoplasmic reticulum generates action potentials in rat cardiac ventricular myocytes at normal resting membrane potential

Under certain conditions, spontaneous release of Ca2+ from the sarcoplasmic reticulum occurs in resting mammalian myocardium. In single rat ventricular myocytes, such spontaneous Ca2+ release appears localized rather than homogeneous. When the increase in cytosolic Ca2+ is present in a single locus within a cell, it causes a small depolarization, which, at the normal resting potential, is subthreshold for generating an action potential. However, when spontaneous Ca2+ release occurs simultaneously at more than a single discrete locus, the resultant sarcolemmal depolarization is augmented to levels that can induce an action potential, even when this depolarization begins at the normal resting membrane potential. Thus, the synchronous occurrence of multifocal localized increases in cytosolic Ca2+ due to spontaneous Ca2+ release from the sarcoplasmic reticulum within ventricular myocytes is a mechanism for "abnormal automaticity."     

Calcium current is increased in isolated adult myocytes from hypertrophied rat myocardium

To study the effects of myocardial hypertrophy resulting from chronic pressure overload on excitation-contraction coupling, the cardiac transmembrane L-type calcium current (ICa) was investigated in the Goldblatt renovascular hypertensive (HBP) rat. ICa was measured in single myocytes enzymatically isolated from control (CTRL) and HBP rat hearts using the whole-cell, patch-clamp method. The peak ICa and ICa density (obtained by normalizing ICa to the average cell capacitative surface area) were larger in HBP cells (n = 15) than in CTRL cells (n = 10) at membrane potentials of -20 to 50 mV (p less than 0.01). The maximal peak ICa increased from 0.9 +/- 0.5 nA (mean +/- SD) in CTRL cells to 2.8 +/- 1.0 nA in HBP cells (p less than 0.001). The corresponding ICa density increased from 5.3 +/- 2.7 to 16.2 +/- 6.0 microA/cm2 (p less than 0.001). There was no shift in the current-voltage relation between CTRL and HBP cells. The time course of decay of HBP ICa in response to clamp steps to the plateau range of the action potential (membrane potential, Vm = -10 to 30 mV) was delayed when compared with that of CTRL ICa. The inactivation time constants (biexponential) for the maximal ICa were 6.9 +/- 1.9 and 36.0 +/- 9.3 msec for CTRL cells and 6.7 +/- 1.4 and 49.5 +/- 12.9 msec for HBP cells (p less than 0.05 for the slower component of the maximal ICa). There was no difference in the steady-state inactivation of ICa (f infinity) for the CTRL and HBP cells. From the maximal peak ICa, cytoplasmic free Ca2+ was estimated to reach a pCa of 6.95 +/- 0.07 for CTRL cells and 6.64 +/- 0.13 for HBP cells. It is concluded that ICa is increased with myocardial hypertrophy. The lengthening of the action potential in hypertrophied rat myocardium is due to an increase in peak current density and to the slower inactivation of the maximal ICa. The increased transmembrane flux of Ca2+ via ICa in HBP cells is inadequate to achieve a myoplasmic free Ca2+ level sufficient for direct partial activation of the contractile myofilaments. However, in the scheme of the calcium-triggered calcium release hypothesis such an increase could provide an increased amount of activator calcium and/or serve to amplify the release of Ca2+ from sarcoplasmic reticulum, thereby contributing to preserved peak developed tension in hypertrophied rat myocardium.     

Loading of calcium and strontium into the sarcoplasmic reticulum in rat ventricular muscle

Previous work suggests that strontium ions (Sr(2+)) are less effective than calcium ions (Ca(2+)) at supporting excitation-contraction (EC) coupling in cardiac muscle. We therefore tested whether this was due to differences in the uptake and release of Ca(2+)and Sr(2+)by the sarcoplasmic reticulum (SR) of rat ventricular trabeculae and myocytes at 22-24 degrees C. In permeabilized trabeculae, isometric contractions activated by exposure to Ca(2+)- and Sr(2+)-containing solutions produced similar maximal force, but were four times more sensitive to Ca(2+)than to Sr(2+). The rate of loading and maximal SR capacity for caffeine-releasable Ca(2+)and Sr(2+)were similar. In isolated, voltage-clamped ventricular myocytes, the SR content was measured as Na(+)-Ca(2+)exchange current during caffeine-induced SR cation releases. The SR Ca(2+)load reached a steady maximum during a train of voltage clamp depolarizations. A similar maximal Sr(2+)load was not observed, suggesting that the SR capacity for Sr(2+)exceeds that for Ca(2+). Therefore, the relative inability of Sr(2+)to support cardiac EC coupling appears not to be due to failure of the SR to sequester Sr(2+). Instead, increases in cytosolic [Sr(2+)] seem to poorly activate Sr(2+)release from the SR.     

Spraque-Dawley大鼠心室肌细胞分离和胞内钙离子浓度测定

目的 探讨Spraque-Dawley大鼠心室肌细胞分离和胞内钙离子浓度测定方法.方法 采用三步法行逆行主动脉灌流获得单个耐钙心肌细胞,然后采用胞内钙荧光测定技术测定细胞内钙离子浓度.结果 在分离过程中如整个心脏保持红润,则细胞数量在90%以上,耐钙细胞KB液中孵育后在70%左右;在分离过程中心脏局部保持红润部位的细胞数量在80%以上,耐钙细胞在60%左右,而苍白区细胞数量变异较大,但一般均在50%以下,且耐钙细胞较少;在分离过程中如整个心脏始终苍白,则细胞数量不仅低于30%,且几乎没有耐钙细胞.采用荧光探针Fura-2/AM,可准确测定细胞内钙离子浓度.结论 通过本研究采用的方法,不仅可获得大量耐钙心肌细胞,而且可准确测定细胞内钙离子浓度.     

Wistar大鼠乳鼠与成年鼠心室肌细胞的分离与性质鉴定

目的 探索和优化稳定的适于电生理实验研究的乳鼠及成年大鼠心室肌细胞分离方法。方法 切碎乳鼠心室肌,胰蛋白酶消化,差速贴壁2 h纯化心室肌细胞,台盼蓝染色判定心肌细胞活力,体外培养48 h后分别行倒置显微镜观察细胞形态,免疫组化鉴定,微电极阵列记录细胞搏动频率和场电位。采用Langendorff灌流成年大鼠心脏,主动脉逆行插管,胶原酶域反复灌流消化约30 min,无钙台氏液冲洗心脏5 min,剪下心室肌组织,台氏液中室温下剪碎,吹打,孵育5 min后,用200目筛网过滤,将细胞悬液用逐步复钙法复钙后,室温静置1 h,用于膜片钳记录。结果 经4 -6次消化后,乳鼠心室组织消化完全,细胞存活率大于80%。倒置显微镜下观察,细胞呈梭形、多角形。 12 h有少部分细胞搏动,48 h细胞交织成网,搏动呈同步性,搏动频率30 - 80次/分。 琢鄄辅肌动蛋白（琢鄄actin）经免疫组化检测,纯度达96%。 Langendorff灌流酶解法可获得形态呈杆状、横纹清晰、膜周边光滑完整、立体感强的单个成年鼠心肌细胞,存活率85%,复钙后存活率50%,可用于膜片钳记录。结论 采用本方法可以获得高产量与高质量的用于电生理检测的心室肌细胞。