The regulation of the calcium sensitivity of the contractile system in mammalian cardiac muscle.

McClellan, George; Winegrad, Saul

doi:10.1085/jgp.72.6.737

Cited by 148 publications

(100 citation statements)

References 46 publications

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“…For example, this could arise if the high permeability to proteins were caused not by the presence of EGTA (or the absence of Cat+), but by an excess of ATP over Mg. In all these studies (McClellan and Winegrad, 1978;Sutherland et al ., 1980;Morgan and Morgan, 1982) and in the present experiments, the disruption solution contained 5 mM total ATP and 2 mM total Mg. The amphibian cells, at least, could be resealed by increasing the [Mg] to 10 mM (and thereby decreasing the [free ATP]), even though EGTA 'was still present (Sutherland et al, 1980;Morgan and Morgan, 1982).…”

Section: Discussionmentioning

confidence: 94%

“…This implies that the detergent disrupted or removed a functional obstacle to the diffusion of Ca" or CaEGTA2-into the EGTA-treated muscles . This barrier could have been a physical structure such as the sarcolemma, which in EGTAtreated muscles appears abnormal but intact (McClellan and Winegrad, 1978) and which is impermeable to extracellular La" (Elder et al, 1981). By contrast, if living cardiac muscle is skinned with 1 % Triton X-100, the sarcolemma is disrupted to such an extent that it becomes permeable to large molecules such as ferritin (Kentish, 1982).…”

Section: Discussionmentioning

confidence: 99%

“…Alternatively, a functional barrier to Ca" or Ca-EGTA2-diffusion would have been the result if Ca" were bound or taken up by intracellular organelles before reaching the myofibrils, i.e., if there were a powerful Ca 21 "sink" in the muscle cells. Both the sarcoplasmic reticulum (SR) and mitochondria have Ca 2+ -sequestering activity and both may be active under certain conditions in the EGTA-treated muscles (McClellan and Winegrad, 1978;Weisberg et al, 1983). The action of Triton to accelerate the development of Ca 2+…”

Section: Discussionmentioning

confidence: 99%

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Some characteristics of Ca2+- regulated force production in EGTA-treated muscles from rat heart.

Kentish¹,

Jewell²

1984

The Journal of General Physiology

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McClellan and Winegrad (1980, J. Gen. Physiol., 75:283-295) have reported that in rat ventricular muscles that have reportedly been made "hyperpermeable" to small ions such as Ca2+, CaEGTA2-, and MgATP2- by a soak in EGTA, the maximum Ca2+-regulated force can be permanently increased by a short exposure to positively inotropic drugs, such as epinephrine or cAMP plus theophylline, in the presence of the detergent Triton X-100. The experiments reported here were begun as an attempt to repeat and extend this important observation. However, no evidence could be found for a potentiation of force that was not merely produced by Triton alone. In addition, the thickest muscles used (250-440 microns diameter) exhibited very low values for force per unit cross-sectional area, which suggested that either Ca2+ reached only a fraction of the myofibrils or the myofibrils were in a state of low contractility. The results of further experiments that were designed to test the permeability characteristics of these EGTA-treated muscles indicated that the movement of certain ions into these preparations was restricted, even in thin muscles (80-200 microns diameter). The rate of development of Ca2+-regulated force was slow (t1/2 approximately equal to 1-3 min), but was greatly accelerated after the muscles had been superfused with Triton X-100 (t1/2 approximately equal to 10-20 s). Removal of creatine phosphate (CP) in the presence of MgATP produced a partial rigor contracture in the EGTA-treated muscles. The results were consistent with the suggestion that the EGTA-treated muscles were permeable to some extent to Ca2+ and HCP2- ions but not to CaEGTA2- and MgATP2-. Thus, it would seem unlikely that the [Ca2+], [MgATP2-], and [Mg2+] in the immediate vicinity of the myofibrils in these preparations can be adequately controlled by the solution bathing the muscles.

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Section: Discussionmentioning

confidence: 94%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Some characteristics of Ca2+- regulated force production in EGTA-treated muscles from rat heart.

Kentish¹,

Jewell²

1984

The Journal of General Physiology

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“…Reports have shown, however, that a long exposure of heart cells to Ca 2+ -free condition degrades their surface glycocalix, and also that it permeabilizes the sarcolemmal membrane to ions and small molecules [14,15,28,29]. Alternatively, the administration of 10-20 mM BDM in the media for cell isolation is also widely used for reducing cell damage [12,30].…”

Section: Comparison With Other Heart Cell Isolation Techniquesmentioning

confidence: 99%

A Simple Technique for Isolating Healthy Heart Cells from Mouse Models

Shioya

2007

J. Physiol. Sci

128

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Single heart cells of mouse models provide powerful tools for heart research. However, their isolation is not easy, and it imposes a significant bottleneck on their use in cellular studies of the heart. Aiming to overcome this problem, this report introduces a novel technique that reproducibly isolates healthy heart cells from mouse models. Using simple devices that ensure easy handling and the rapid aortic cannulation of a small mouse heart, cell isolation was done under physiological conditions without using the "KB" medium or 2,3-butanedione monoxime (BDM). The isolated cells consistently had a healthy appearance and a high viability of 75 ± 5% (mean ± SD) in Tyrode solution containing 1.8 mM Ca 2+ . After 8 h of storage at 37°C, they still had a viability of 45 ± 12%. The cells showed normal contraction properties when field-stimulated, and they generated normal action potentials and membrane currents under the whole-cell clamp condition. The β-adrenergic signal transduction of the cells was also normal when it was examined with the isoproterenol enhancement of the L-type Ca 2+ current.Key words: mouse, cardiac myocyte, contraction, action potential, ionic current.Single heart cells of mouse models provide powerful tools for heart research. In these cells, one can take advantage of the versatility of recent genetic engineering technology [1] and also of the accuracy of single-cell measurement techniques such as patch-clamp and Ca 2+ imaging. So far, molecular mechanisms of fundamental heart functions, such as excitation-contraction coupling, action potential shaping, and β-adrenergic signaling, have been successfully unveiled by using single heart cells of mouse models [2][3][4][5][6][7]. Moreover, these cells also provide a convenient platform for analyzing pathogenic mechanisms and the pathophysiology of hereditary heart diseases in molecular detail [8,9].For such single-cell studies, healthy heart cells are absolutely necessary; however, their isolation from mouse models is not easy. Surgery and aortic cannulation of a small mouse heart are hard to do and require a long time, and during that time, the heart suffers from complete ischemia. Consequently, the cells isolated from these hearts show various signs of ischemic damages, such as bizarre appearance, low viability levels, and abnormalities in their excitation and contraction properties. Cells of these kinds are hard to use for the experiment, and the data they provide are often difficult to interpret. This issue imposes a significant bottleneck on the use of mouse heart cells and is especially problematic in genetically engineered mouse models that are usually available only in limited amounts.To solve this problem, two major work-arounds have been devised and used for heart cell isolation from mouse models. One is to incubate the cells in high-K + , Ca 2+ -free "KB" medium at 4°C [10,11], and the other is to administer 10-20 mM 2,3-butanedione monoxime (BDM) in the media for cell isolation and storage [12,13]. Both workarounds proved to be...

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“…Phosphorylation of the inhibitory subunit of troponin (TNI) can occur and is associated with the increased contractility produced by catecholamines (England, 1976;Solaro et al, 1976), but phosphorylation of TNI only increases the concentrations of Ca required for activation of the ATPase in isolated myofibrils without enhancing ATPase activity (Ray and England, 1976). Analogous regulation of the Ca sensitivity without change in contractility exists in the hyperpermeable rat heart (McClellan and Winegrad, 1978). Under certain circumstances a close relation between contractility and the ratio of cAMP to cGMP can be demonstrated in the intact frog heart (Singh et al, 1978), but it is not clear that this is due to a change in the contractile proteins.…”

Section: Introductionmentioning

confidence: 99%

Cyclic nucleotide regulation of the contractile proteins in mammalian cardiac muscle.

McClellan¹,

Winegrad²

1980

The Journal of General Physiology

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A B ST R A C T The contractile system of rat cardiac muscle that has been made hyperpermeable by soaking the tissue in EGTA (McClellan and Winegrad. 1978. J. Gen. Physiol. 72:737-764) can be probed directly with Ca buffer from the bathing solution without significant interference from either sarcoplasmic reticulum or mitochondria on the Ca concentration. Changes in Ca-activated force are due therefore to changes in the properties of the contractile system itself and not to regulation of Ca concentration. The addition of cAMP, cGMP, and GTP, guanylyl imidodiphosphate (GMP-PNP), or epinephrine to the bath does not alter maximum Ca-activated force, but when these drugs are added with 1% nonionic detergent to the bath, contractility increases by as much as 180%. An inhibitor of phosphodiesterase must be present for the inotropic effect of cAMP but not cGMP, GTP, GMP-PNP, or epinephrine. The inotropic response to cAMP is independent of the Ca sensitivity of the contractile system, but guanine nucleotides enhance contractility only when Ca sensitivity is not high. The inotropic effect of epinephrine is inhibited to a large extent by cGMP but not by GMP-PNP. These data can be explained by a model in which contractility is enhanced by a cAMP-regulated phosphorylation that can be controlled through the/~-receptor adenylate cyclase complex in the sarcolemma. The regulation involves two reactions, one a phosphorylation and a second that occurs in the presence of detergent. Phosphorylation of neither the myosin light chain nor the inhibitory subunit of troponin appears to be involved in this mechanism for regulating contractility.

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The regulation of the calcium sensitivity of the contractile system in mammalian cardiac muscle.

Cited by 148 publications

References 46 publications

Some characteristics of Ca2+- regulated force production in EGTA-treated muscles from rat heart.

Some characteristics of Ca2+- regulated force production in EGTA-treated muscles from rat heart.

A Simple Technique for Isolating Healthy Heart Cells from Mouse Models

Cyclic nucleotide regulation of the contractile proteins in mammalian cardiac muscle.

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