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Research Communication

Calcium Reintroduction Decreases Viability of Cardiac Myocytes from Copper-Deficient Rats¹

Departments of Biochemistry & Molecular Biology and ^* Medical and Molecular Physiology, School of Medicine, University of Minnesota, Duluth, MN 55812

²To whom correspondence should be addressed.

	ABSTRACT

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Copper deficiency leads to profound cardiac hypertrophy and failure. Myocytes were isolated from hearts of copper-deficient and copper-adequate male Holtzman rats to characterize size and function of the cells. Weanling rats were offered a semipurified diet low in copper in two separate experiments (Experiment 1, 0.45 mg Cu/kg and Experiment 2, 0.30 mg Cu/kg). Control (copper-adequate) rats drank water supplemented with cupric sulfate (20 mg Cu/L). Compared with copper-adequate rats, copper-deficient rats had lower hematocrits, liver copper concentrations and plasma ceruloplasmin activities, and higher heart weights and liver iron concentrations. When myocytes were isolated in low calcium media (1 µmol/L), cell viability was not affected by diet history. However, upon restoration to more physiologic levels of calcium (1 mmol/L), cells from copper-deficient rats were less viable, exhibiting an average loss of 34 and 40% in Experiments 1 and 2, respectively, compared with a 9.5 and 13% loss of cells, respectively, from the copper-adequate rats. Addition of the calcium channel blocker, verapamil, did not block this calcium-dependent loss of viability nor did the mitochondrial calcium channel blockers, ruthenium red and cyclosporin A. For comparison with another model of cardiac hypertrophy, the calcium sensitivity of myocytes from hypertrophic hearts of Sprague-Dawley rats with aortic constrictions was found not to differ from that of sham-operated rats. Thus, cardiac hypertrophy associated with postnatal copper deficiency results in a unique increased calcium intolerance of isolated myocytes.

KEY WORDS: • copper-deficient • cardiac myocyte • rats

	INTRODUCTION

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Copper is essential for the development and optimal function of biological systems. Many of the required processes can be linked with known cuproenzymes. However, lack of adequate dietary copper results in a mammalian phenotype in which alterations in several biological systems have yet to be linked with specific enzyme defects. Notable in this context is a pronounced cardiac hypertrophy. Previous studies on the cardiac hypertrophy of copper deficiency have been thoroughly reviewed (Medeiros et al. 1993 , Medeiros and Wildman 1997 ).

Several hypotheses surrounding changes in antioxidant systems via lower Cu,Zn-superoxide dismutase, connective tissue pathology via lower lysyl oxidase, and impaired energy metabolism via lower cytochrome c oxidase have been proposed. We tested the idea that overproduction of humoral growth factor such as angiotensin II might be involved, but we showed that this was unlikely (Lear et al. 1996 ). Typical physiologic reasons for developing hypertrophy such as hypertension and volume overload are not involved in the postnatal copper-deficient rat model. Thus, the specific trigger responsible for inducing cardiac hypertrophy in copper deficiency is not known.

To extend previous work, experiments designed to characterize the morphometric characteristic of cardiac myocytes were undertaken. It was discovered in a first experiment that viability of myocytes from copper-deficient rats was compromised by the restoration of physiologic levels of calcium (Heller et al. 1997 ). This preliminary observation prompted further research, which is reported here, including a second experiment assessing calcium sensitivity of copper deficiency and a third experiment assessing calcium sensitivity of cardiac myocytes from hypertrophied hearts of rats with arterial pressure overload.

	MATERIALS AND METHODS

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Animal care and diets.

Male weanling Holtzman rats were purchased commercially (Harlan Sprague Dawley, Indianapolis, IN). Rats were fed one of two dietary treatments, copper-deficient (-Cu)³ or copper-adequate (+Cu), consisting of a low copper purified diet (Teklad Laboratories, Madison, WI) and either low copper drinking water or copper-supplemented drinking water, respectively. The purified diet was a modified AIN-76A diet (Prohaska 1991 ) and contained the following components (g/kg diet): sucrose, 500; casein, 200; cornstarch, 150; corn oil, 50; cellulose, 50; modified AIN-76 mineral mix, 35; AIN-76A vitamin mix, 10, DL-methionine, 3; choline bitartrate, 2; and ethoxyquin, 0.01. Cupric carbonate was omitted from the AIN-76 mineral mix. The purified diet contained by chemical analysis 0.45 mg Cu/kg and 47 mg Fe/kg in Experiment 1 and 0.30 mg Cu/kg and 44 mg Fe/kg in Experiment 2. Holtzman male rats fed the -Cu treatment drank deionized water, whereas the +Cu treatment groups drank water that contained 20 mg Cu/L through the addition of CuSO₄ to the drinking water. Rats were given free access to diet and drinking water. All animals were maintained at 24°C with 55% relative humidity on a 12-h light cycle (0700–1900 h). All protocols were approved formally by the University of Minnesota Animal Care Committee.

A third study was done in which young adult male Sprague-Dawley rats were offered a nonpurified laboratory rat diet (5001 Ralston Purina, St. Louis, MO). This diet, which was evaluated previously, contains adequate copper (13 mg Cu/kg) (Prohaska and Hoffman 1996 ). These rats drank tap water.

In Experiment 1, rats began the treatments at age 21 d and were maintained for ~5 wk. Rats were killed in pairs (one +Cu and one -Cu) and averaged 55 d of age at the time of analysis (Table 1 ). In Experiment 2, rats began the treatments at age 20 d and were maintained for ~4 wk at which time rats were killed because growth impairment was detected (Table 1) .

Myocyte preparation.

Cardiac myocytes were isolated from hearts using standard techniques (Solem et al. 1996 ). Briefly, hearts were perfused at 37°C with Joklik's modified minimum essential medium containing collagenase (7g/L) (Worthington Biochemical, Lakewood, NJ) until the vascular bed deteriorated (~20 min). The tissue was then coarsely minced and digested with the collagenase-containing solution; the process was continued in a beaker placed in a gyrating water bath for an additional ~20 min in the presence of 1.0 µmol/L CaCl₂. The tissue digestate was then filtered through cheesecloth and cells allowed to settle out of the filtrate. This cell preparation was resuspended and resettled two times in collagenase-free Joklik's solution. Settling time was limited to promote recovery of live rod-shaped myoctes, which have a greater density than nonmyoctes and dead cells. A 100-µL sample was taken from the final suspension in low calcium for assessment of initial viability. Calcium was then added to the cell suspension (in several steps spread over a 30-min period) to a final concentration of 1.0 mmol/L at 37°C. Viability was reassessed at the end of this procedure.

Viability assessment.

Viability of myocytes was assessed by determining the percentage of live rod-shaped cells out of total cells in a given sample. A 100-µL sample of suspended cells was mixed with 50 µL of solution containing trypan blue (5 g/L) and gluteraldehyde (60 g/L). An aliquot of these fixed cells was placed on a microhematocytometer, and live cells were identified by their shape and ability to exclude trypan blue.

Copper analyses and ceruloplasmin assay.

Portions of liver and diet (~1 g) were weighed to the nearest milligram and wet-digested with 4 mL of concentrated HNO₃ (AR select grade, Mallinckrodt, St. Louis, MO); the residue was brought to 4.0 mL with 0.1 mol/L HNO₃. Samples were then analyzed for total copper and iron by flame atomic absorption spectroscopy (AAS) (Model 2380, Perkin-Elmer, Norwalk, CT). The method was checked with a certified standard, U.S. National Bureau of Standards 1577 bovine liver (Gaithersburg, MD).

Plasma calcium was determined by flame AAS at 425 nm according to the manufacturer's protocol. Samples of plasma were diluted with 49 vol of a solution containing 2.67 g/L LaCl₃ · 7H₂0. Standards were prepared in the same diluent.

Plasma ceruloplasmin activity was measured by its ability to oxidize o-dianisidine as described in detail elsewhere (Prohaska 1991 ).

Calcium antagonists.

In Experiment 1, three +Cu and three -Cu rat myocyte preparations were used to test the hypothesis that the enhanced sensitivity to calcium observed in -Cu preparations could be attenuated or blocked by the use of calcium transport antagonists (Smogorzewski et al. 1993 , Solem et al. 1996 ). Cells were incubated with buffer alone or buffer with the following drugs (final concentration): verapamil (40 µmol/L); ruthenium red (10 µmol/L); or cyclosporin A (5 µmol/L) for 5 min before calcium additions as described above.

Aortic constriction-induced cardiac hypertrophy.

Six young adult male Sprague-Dawley rats (7 wk old), maintained on a copper-sufficient diet, were divided randomly into two groups. Three rats served as sham-operated controls and three were surgically given aortic constrictions to increase upstream aortic pressure and cardiac afterload. After sedation with xylazine (6 mg/kg, intraperitoneal) and anesthesia with ketamine (30 mg/kg, intraperitoneal), a midline incision was made and a ligature passed under the subdiaphragmatic-suprarenal segment of the abdominal aortic. A blunted 20-gauge hypodermic needle was placed alongside the aorta and the ligature tightened over both. The needle was carefully withdrawn, and the position and integrity of the ligature knot verified by inspection. The abdominal incision was closed by suturing the muscle layers and using wound clips on the skin. Sham treatment in this model consisted of all surgical steps except for the tightening of the ligature. The development of cardiac hypertrophy in the aortic-constricted group determined at the time of killing verified the efficacy of the constriction and the presence of chronic elevation of aortic pressure. After an average of 13 ± 2 d, pairs were killed and cardiac myocytes harvested as described above. Cardiac myocyte viability was assessed.

Statistics.

Dietary treatment effects were evaluated by Student's t test after variance equality was tested; = 0.05 and 0.01. Data in the calcium antagonist study were analyzed by 2 x 4 factorial ANOVA. Data were analyzed using a personal computer and statistical software (Statview 4.5, Abacus Concepts, Berkeley, CA).

	RESULTS

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Restriction of dietary copper during the rapid growth phase of male Holtzman rats resulted in severe copper deficiency compared with rats drinking copper-supplemented water (Table 1) . In Experiment 1, in which the basal diet contained more copper (0.45 mg Cu/kg) than in Experiment 2 (0.30 mg Cu/kg), there was no growth impairment during the 5-wk feeding period. In contrast, in Experiment 2, growth was impaired after 3 wk of the treatment; by 4 wk, a 22% average difference in body weight was evident (Table 1) .

In both experiments, biochemical characteristics consistent with severe copper deficiency were evident in -Cu rats compared with +Cu rats, including a 90% reduction in liver copper concentration and near total loss of ceruloplasmin activity (Table 1) .

Cardiac hypertrophy was evident because absolute heart weight was elevated 60 and 80%, respectively, in Experiments 1 and 2 (Table 1) . Relative heart weight/body weight was also significantly elevated (P < 0.01). For example, in Experiment 1 -Cu rats averaged 7.8 ± 0.91 mg/g compared with 4.4 ± 0.32 mg/g for the +Cu rats.

After myocytes were isolated by collagenase perfusion in low calcium solutions (1 µmol/L), there was no difference in viability of the live rod-shaped cells. However, after calcium was reintroduced gradually to restore extracellular levels to 1 mmol/L, there was a pronounced loss of viability (Fig. 1 ). Experiment 2 was designed to verify the observations in Experiment 1, and indeed results were similar (Fig. 1) . On average, there was a loss of cells from +Cu rat hearts after calcium restoration of 9.5 and 13% in Experiment 1 and 2, respectively, compared with average losses of 34 and 40% for the -Cu cardiac myocytes, P < 0.01.

View larger version (26K): [in this window] [in a new window]   Figure 1. Cardiac myocyte viability and cell loss is enhanced in Cu-deficient myocytes following increase in calcium concentration. Bars represent means and error bars SEM for cells isolated from either n = 5 (Experiment 1) or n = 8 (Experiment 2) male rats fed either Cu-adequate or Cu-deficient diets. For Experiment 1 or 2, means were compared by Student's t test; *P < 0.05 compared with Cu-adequate group. Low calcium refers to 1 µmol/L. Cell loss with calcium addition is the percentage loss of viable cells when switching from media containing 1 µmol/L calcium to 1 mmol/L calcium.

View larger version (47K): [in this window] [in a new window]   Figure 2. Loss of cardiac myocyte viability in the presence of calcium antagonists after the addition of calcium is greater in Cu-deficient myocytes. Bars represent means and error bars SEM for n = 3 rats of each dietary treatment group in Experiment 1. Cells were pretreated with the appropriate drug for 5 min before calcium was gradually added to a final concentration of 1 mmol/L from an initial 1 µmol/L. Factorial ANOVA indicated a main effect of diet, P < 0.01.

A third study was done using subdiaphragmatic aortic constriction on male Sprague-Dawley rats fed a copper-adequate diet to determine whether cardiac myocytes from another hypertrophic model would also show such calcium intolerance. Surgical intervention resulted in rats with an elevated heart weight to body weight ratio due to the elevated aortic pressure above the constriction. The heart weight to body weight ratio of the constricted rats averaged 5.7 ± 0.1 mg/g compared with 4.3 ± 0.11 mg/g for the sham-operated group. The body weights of the constricted group, 228 ± 11 g (n = 3), were not different from those of the sham-operated group, 250 ± 12 g (n = 3). Cardiac myocytes harvested from the aortic-constricted group had an initial viability of 83 ± 2% compared with 88 ± 3% for cells from the sham-operated group. When calcium was restored to 1 mmol/L, viability decreased to a similar extent in both groups, aortic-constricted to 73 ± 4% and sham-operated to 79 ± 3%. These results in pressure-overload hypertrophy are clearly different from the results in the copper-deficient hypertrophy.

	DISCUSSION

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Copper deficiency results in a severe, unique cardiac enlargement that is characterized by a concentric hypertrophy without hypertension (Medeiros et al. 1993 ). Transmission electron microscopic data show enlarged myocytes with a pronounced increase in the ratio of mitochondrial to myofibrillar volume. Mitochondria are swollen and lipid droplets are evident in the cytoplasm. Results of the present experiments clearly demonstrate that the functional integrity of isolated myocytes was also altered in some way that made them highly susceptible to toxic effects of calcium. This conclusion is supported by the robust and reproducible cell loss that occurred upon reintroduction of calcium to the cell suspensions (Fig. 1) . It should be noted that many cells from Cu-deficient rats were still viable upon calcium addition. These cells remained viable for other studies lasting up to 5 h.

Another goal of this series of experiments was to characterize the morphometric properties of the isolated cardiac myocytes from -Cu hearts and to characterize their tolerance to distension and stretch. Results of these experiments, which are described elsewhere (Heller et al. 1999 ), clearly verify a myocyte enlargement and also identify an increase fragility to distension in hypotonic conditions.

The basis for the increased calcium sensitivity of the cardiac myocytes from -Cu rats is not clear. In an attempt to obtain information about this abnormality, we tried to interfere with calcium transport processes across the cell and/or mitochondrial membrane. Part of the approach was patterned after studies on a different rodent model of cardiac failure in which myocytes showed increased calcium sensitivity; i.e., adriamycin-induced cardiac failure (Solem et al. 1996 ). With this model, chronic treatment of rats with adriamycin, a cancer chemotherapeutic drug with cardioselective toxic effects, produces calcium intolerance in isolated myocytes that can be blocked acutely by the presence of cyclosporin A or ruthenium red in the media. Because these drugs interfere with mitochondrial calcium uptake and release, and prevent the mitochondrial permeability transition, it is likely that the calcium intolerance in this model is related to the mitochondrial abnormality. However, in the -Cu model of cardiac hypertrophy, even though mitochondria are abnormal in shape and function, the mitochondrial calcium blockers, ruthenium red and cyclosporin A, did not attenuate the cell loss of isolated myocytes. Furthermore, isolated mitochondria from -Cu rat hearts were able to accumulate calcium to the same extent as +Cu mitochondria, i.e., ~1060 nmol/mg protein (unpublished data). Thus, these data suggest that the basis for increased calcium sensitivity in the -Cu cardiac myocyte is not mitochondrial.

Another approach to identify the basis for the increased calcium sensitivity was to block calcium uptake at the cell membrane with the calcium channel antagonist, verapamil. Although the dose used was high enough to completely block movement of calcium into isolated rat cardiac myocytes via this route (Smogorzewski et al. 1993 ), verapamil had no effect on the calcium intolerance of the -Cu myocytes. Therefore, it is unlikely that the increased calcium sensitivity of these cells proceeds by a process involving the L-type calcium channel.

If the toxicity is a result of calcium accumulation within the myocyte, it is possible that calcium may enter the cell via an enhancement of the sodium/calcium exchanger. This protein, expressed in the sarcolemma, is up-regulated in end-stage heart failure (Studer et al. 1994 ). The increased diastolic calcium transient in isolated hypertrophic myocytes from failing hearts is thought to be due in part to the enhanced sodium/calcium exchanger protein content (Mittmann et al. 1998 ). Such a process might contribute to calcium accumulation and toxicity in the -Cu hearts. The observation that there is an impairment in the Na⁺/K⁺ ATPase in the -Cu heart (Huang et al. 1995 ) is consistent with an increase in intracellular sodium, which would enhance calcium uptake via the Na⁺/Ca²⁺ exchanger. This intriguing possibility requires further studies.

It should be emphasized that the observed calcium sensitivity in -Cu hypertrophic cardiac myocytes is not characteristic of all models of cardiac hypertrophy because it was not seen in the hypertrophic cardiac myocytes from the aortic-constricted rats. Thus, it is possible that a unique mechanism involving abnormal myocyte calcium handling exists as a consequence of dietary copper deficiency. Studies of intracellular calcium homeostasis as affected by copper deficiency are limited. Johnson and Dufault (1993) were able to demonstrate in -Cu platelets a diminished rise in cytosolic calcium upon agonist administration. It is not clear how that observation in platelets relates to enhanced toxicity of -Cu cardiac myocytes.

The relevance of this in vitro observation is not clear. Circulating levels of total plasma calcium that we measured (~2.4 mmol/L) would result in a free calcium ion concentration similar to that used in the cell exposures (1.0 mmol/L). Thus, the enzymatic dispersal techniques and/or the transient imposition of a low calcium environment probably reveal the calcium intolerance of the isolated -Cu myocyte. However, it is interesting to point out that apoptotic and necrotic processes can be triggered by rises in cellular calcium, and apopotosis is a feature of dietary copper deficiency in certain organs such as pancreas (Rao et al. 1993 ).

	ACKNOWLEDGMENTS

 
The skillful technical assistance of Juline Smith and Bruce Brokate is appreciated.

	FOOTNOTES

 
¹ Supported by grant 96–35200-3138 from the National Research Initiative Competitive Grants Program/U.S. Department of Agriculture and funds from the University of Minnesota, Duluth School of Medicine.

³ Abbreviations used: AAS, atomic absorption spectroscopy; -Cu, copper-deficient; +Cu, copper-adequate.

Manuscript received May 28, 1999. Revision accepted July 14, 1999.

	REFERENCES

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1. Heller L. J., Becker A., VanDyke D., Smith J., Brokate B., Prohaska J. R. Isolated cardiac myocytes of copper-deficient rats have altered dimensions and responses to osmotic stress. FASEB J 1999;13:A768(abs.)

2. Heller L. J., Smith J. A., Prohaska J. R. Cardiac myocytes isolated from hypertrophied hearts of copper-deficient rats are calcium intolerant. FASEB J 1997;11:A65(abs.)

3. Huang W., Lai C. C., Wang Y., Askari A., Klevay L. M., Askari A., Chiu T. H. Altered expressions of cardiac Na/K-ATPase isoforms in copper deficient rats. Cardiovasc. Res. 1995;29:563-568[Medline]

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11. Rao M. S., Yeldandi A. V., Subbarao V., Reddy J. K. Role of apoptosis in copper deficiency-induced pancreatic involution in the rat. Am. J. Pathol. 1993;142:1952-1957[Abstract]

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