Calcium elevation in cultured heart cells: its role in cell injury

Inhibition of the Na+-K+ pump in cultured embryonic chick heart cells promotes the elevation of intracellular Ca2+ and is a useful manipulation to study the relationship between Ca2+ and myocardial cell injury. One hour of Na+-K+ pump inhibition resulted in a fourfold increase in cell Na+, a 50% decline in cell K+, and a 5- to 10-fold increase in cell Ca2+, 45% of which is mitochondrial. The degree of cell injury induced by Ca2+ loading was evaluated by monitoring the content of adenosine 5'-triphosphate (ATP) and the release of the intracellular enzyme lactate dehydrogenase (LDH). Under these conditions ATP content declined by 25-30% and LDH release increased from 1 to 1.4% of the total LDH. Furthermore, cells subjected to 1 h of Na+-K+ pump inhibition and returned to control solution for 5 h showed that Ca2+ decreased to near control levels and ATP content was restored. Although inhibition of Na+-K+ transport caused a large increase in cell Ca2+, neither Na+-K+ pump inhibition nor elevation in total cell Ca2+ per se resulted in irreversible myocardial cell injury.

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Biochemical and structural changes in cultured heart cells induced by metabolic inhibition

AJP Cell Physiology ◽

10.1152/ajpcell.1987.253.5.c700 ◽

1987 ◽

Vol 253 (5) ◽

pp. C700-C706 ◽

Cited By ~ 19

Author(s):

E. Murphy ◽

A. LeFurgey ◽

M. Lieberman

Keyword(s):

Structural Changes ◽

Cell Injury ◽

Myocardial Cell ◽

Structural Defects ◽

Heart Cells ◽

Apparent Lack ◽

Late Event ◽

Myocardial Cell Injury ◽

Chick Heart ◽

Ldh Release

We examined the relationship between ionic homeostasis, ATP, and irreversible cell injury in cultured embryonic chick heart cells treated with rotenone (10(-4) M) alone or in combination with iodoacetate (IAA) (10(-3) M), in the presence of extracellular calcium (Ca0) (2.7 mM) and its nominal absence. Changes in Na, K, and total cell Ca content did not correlate with parameters indicative of irreversible injury, i.e., ultrastructural damage or lactate dehydrogenase (LDH) release. Because structural defects in the plasma membrane occurred without a significant release of LDH after exposure to rotenone plus IAA for 1 h, LDH release appears to be a relatively late event in cell injury. In addition, cells exposed to rotenone in the presence of Ca0 for 2.5 h showed a significant fall in ATP and a rise in LDH release. This response was attenuated in the nominal absence of Ca0, and the addition of rotenone caused an eightfold increase in intracellular sodium (Nai), whereas in the presence of Ca0, Nai increased only threefold in 2.5 h. Thus Ca0 appears to promote Nai-Ca0 exchange and lead to an increase in cell Ca that can then stimulate ATP breakdown by Ca-activated ATPases. Of the measured variables associated with myocardial cell injury, a decline in ATP correlates best with changes in either LDH or morphology. The apparent lack of correlation between changes in intracellular ion content, LDH release, and morphology supports the conclusion that myocardial cell injury is a multifactorial process.

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Ultrastructural response of cultured heart cells to calcium depletion and repletion

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100103851 ◽

1988 ◽

Vol 46 ◽

pp. 358-359

Author(s):

A. LeFurgey ◽

B. Wagenknecht ◽

P.J. Schreiner ◽

L.A. Hawkey ◽

M. Lieberman

Keyword(s):

Cell Injury ◽

Heart Cells ◽

Calcium Depletion ◽

Solution Ph ◽

Mitochondrial Uncoupling ◽

Carbonyl Cyanide ◽

Chick Heart ◽

Embryonic Chick Heart ◽

Series Of Experiments ◽

Cultured Heart Cells

Calcium-free incubation followed by exposure to Ca produces ultrastructural damage in the intact myocardium—a phenomenon known as the Ca paradox. Controversy exists as to whether the observed Ca overload during Ca repletion is a primary cause for, or a secondary consequence of, sarcolemmal disruption and cell injury. In these experiments cultured embryonic chick heart cells have been subjected to Ca depletion/repletion to explore the structural, transport and metabolic mechanisms by which cells may be irreversibly damaged.Monolayer muscle enriched cell cultures were prepared by serial trypsin-disaggregation of 11-day old chick embryo hearts. Cells were incubated for 10 or 120 min in Ca-free (1 mM EGTA) Hepes-buffered salt solution (pH 7.4) followed by incubation for 60 or 180 min in the same solution containing 1 mM Ca. In another series of experiments, the mitochondrial uncoupling agent carbonyl cyanide chlorophenylhydrazone (CCCP, 10 μM ) was added during depletion and repletion.

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Co-culture of embryonic chick heart cells and ciliary ganglia induces parasympathetic responsiveness in embryonic chick heart cells

Biochemical Journal ◽

10.1042/bj2920395 ◽

1993 ◽

Vol 292 (2) ◽

pp. 395-399 ◽

Cited By ~ 5

Author(s):

J V Barnett ◽

M Taniuchi ◽

M B Yang ◽

J B Galper

Keyword(s):

G Proteins ◽

Fold Increase ◽

Heart Cell ◽

Heart Cells ◽

Embryonic Chick ◽

In Ovo ◽

Chronotropic Response ◽

Muscarinic Stimulation ◽

Chick Heart ◽

Embryonic Chick Heart

We have developed a system for the co-culture of embryonic chick heart cells obtained from embryos at 3.5 days in ovo with ciliary ganglia from chick embryos at 7 days in vivo. After 3 days of co-culture, removal of the ciliary ganglia resulted in complete degeneration of axons within 6-8 h, leaving the post-innervated heart cell culture devoid of neurons. Embryonic chick heart cells at 3.5 days in ovo are unresponsive to muscarinic stimulation. However, following 3 days of co-culture with ciliary ganglia, the heart cells developed a negative chronotropic response to muscarinic stimulation (paired t test, P < 0.02) which persisted for at least 24 h after removal of the ciliary ganglion. The development of muscarinic responsiveness was associated with an increase in the levels of specific alpha-subunits of the guanine nucleotide binding proteins (G-proteins), with a 3-fold increase in the level of alpha 39 (39 kDa subunit) and a 2.5-fold increase in the level of alpha 41. The level of the G-protein subunit alpha s remained unchanged. Culture of embryonic chick heart cells at 3.5 days in ovo with medium conditioned by the growth of embryonic chick heart cells and ciliary ganglia had an effect on the chronotropic response to muscarinic stimulation and on alpha 39 and alpha 41 levels identical to that of co-culture. These data suggest that a soluble factor released during the co-culture of embryonic chick heart cells and ciliary ganglia is capable of inducing muscarinic responsiveness. These studies suggest that innervation of the heart may induce parasympathetic responsiveness by increasing the availability of G-proteins which couple the muscarinic receptor to a physiological response.

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Coupled sodium-calcium transport in cultured chick heart cells

AJP Cell Physiology ◽

10.1152/ajpcell.1986.250.3.c442 ◽

1986 ◽

Vol 250 (3) ◽

pp. C442-C452 ◽

Cited By ~ 22

Author(s):

E. Murphy ◽

D. M. Wheeler ◽

A. LeFurgey ◽

R. Jacob ◽

L. A. Lobaugh ◽

...

Keyword(s):

Fold Increase ◽

Total Cell ◽

Heart Cells ◽

Electrochemical Gradient ◽

X Ray ◽

Sodium Calcium ◽

Ca Uptake ◽

Chick Heart ◽

Embryonic Chick Heart ◽

Intracellular Organelles

In cultured embryonic chick heart cells, alterations of extracellular Na (Nao) and Ca (Cao), intracellular Na (Nai) and Ca, extracellular pH, and membrane potential resulted in changes in Na and Ca contents that were consistent with sarcolemmal Na-Ca exchange. 24Na efflux measurements revealed a large ouabain-insensitive component, one-third of which was inhibited by removal of Cao. Incubating the cells in Na-free solution resulted in a rapid, 1.5- to 2-fold increase in total cell Ca that remained elevated for at least 15 min. Cells exposed for 15 min to Nao less than or equal to 20 mM became maximally loaded with Ca, whereas Ca loading fell off sharply at values of Nao greater than 20 mM. The movement of Na against its electrochemical gradient was shown to be associated with Ca accumulation. During Na-K pump inhibition (in 10(-4) M ouabain), Na initially rose 2- to 3-fold to a level below its equilibrium value; then, lowering Cao for 30 min from 1.25 to 0.75 mM caused a 26% elevation in Nai, whereas raising Cao from 1.25 to 2.7 mM resulted in a 25% fall in Nai against its electrochemical gradient. These data are consistent with Nai being maintained by a Na-Ca exchange during Na-K pump inhibition. In the presence of ouabain (10(-4) M), Ca uptake into intracellular organelles, e.g., mitochondria, was suggested by an increase in total cell Ca as well as the occurrence of mitochondrial matrix granules, which were shown qualitatively by X-ray analysis to contain Ca. Although matrix granules also occurred in mitochondria during Na-free incubation, they did not contain detectable amounts of Ca when examined under identical conditions of fixation and analysis.

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Cytosolic free magnesium concentration in cultured chick heart cells

AJP Cell Physiology ◽

10.1152/ajpcell.1989.257.1.c141 ◽

1989 ◽

Vol 257 (1) ◽

pp. C141-C146 ◽

Cited By ~ 48

Author(s):

S. Rotevatn ◽

E. Murphy ◽

L. A. Levy ◽

B. Raju ◽

M. Lieberman ◽

...

Keyword(s):

Salt Solution ◽

Significant Proportion ◽

Fold Increase ◽

Magnesium Concentration ◽

Heart Cells ◽

Ion Gradient ◽

Chick Heart ◽

Embryonic Chick Heart ◽

Free Magnesium ◽

Solution Changes

Cytosolic free magnesium (Mgi) was measured in embryonic chick heart cells loaded with one of two newly developed 19F nuclear magnetic reasonance (NMR)-sensitive magnesium chelators, 4-methyl,5-fluoro-2-aminophenol-N,N,O-triacetate (MF-APTRA) and 5-fluoro-2-aminophenol-N,N,O-triacetate (5F-APTRA). The cells, embedded in strands of collagen, were superfused at a rate that allowed for solution changes in 2 min. In this preparation 19F- and 31P-NMR spectra were stable for at least 3.5 h. Because Na-coupled Mg countertransport may be a possible mechanism of Mg transport, in some experiments extracellular Na was reduced to 1 mM (choline substituted). This manipulation caused a 2.5-fold increase in Mgi from the basal level of 0.56 mM. A significant proportion of this increase in Mgi could be secondary to an increase in Cai that occurs with low extracellular Na (Nao) perfusion (Nai-Cao exchange). Perfusing cells with nominally Ca-free, 1 mM Na salt solution substantially attenuated the increase in Mgi that occurred with Ca present (1.25 mM) in the low Na (1 mM) solution. Furthermore, perfusion with 1 mM Na, Mg-free salt solution caused a 1.5-fold increase in Mgi, which cannot be attributable to Nai-Mgo exchange. Therefore attempts to describe the regulation of Mgi in heart cells must differentiate between the effects of Nai-Mgo exchange and competition for binding sites that are secondary to stimulation of ion gradient-coupled mechanisms.

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Techniques for cryoultramicrotomy of propane jet frozen biological samples

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010014292x ◽

1986 ◽

Vol 44 ◽

pp. 260-261

Author(s):

B. Craig ◽

L. Hawkey ◽

A. LeFurgey

Keyword(s):

Freeze Fracture ◽

Freeze Substitution ◽

Heart Cells ◽

Crystal Formation ◽

X Ray ◽

Transmission Electron ◽

Scanning Transmission ◽

Chick Heart ◽

Embryonic Chick Heart ◽

A New Technique

Ultra-rapid freezing followed by cryoultramicrotomy is essential for the preservation of diffusible elements in situ within cells prior to scanning transmission electron microscopy and quantitative energy dispersive x-ray microanalysis. For cells or tissue fragments in suspension and for monolayer cell cultures, propane jet freezing provides cooling rates greater than 30,000°C/sec with regions up to 40μm in thickness free of significant ice crystal formation. While this method of freezing has frequently been applied prior to freeze fracture or freeze substitution, it has not been widely utilized prior to cryoultramicrotomy and subsequent x-ray microanalytical studies. This report describes methods devised in our laboratory for cryosectioning of propane jet frozen kidney proximal tubule suspensions and cultured embryonic chick heart cells, in particular a new technique for mounting frozen suspension specimens for sectioning. The techniques utilize the same specimen supports and sample holders as those used for freeze fracture and freeze substitution and should be generally applicable to any cell suspension or culture preparation.

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