INTRODUCTION

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COPPER IS A COFACTOR in over 20 enzymes in the body, including lysyl oxidase, copper/zinc superoxide dismutase, and ceruloplasmin (17). Through these enzymes copper may contribute to the normal function of the heart. Deficiency of this important cation has been implicated in a number of cardiovascular disorders such as cardiac hypertrophy, abnormal electrocardiograms, cardiac fibrosis, distorted cardiac myofibrils, and an increase in the heart mitochondria volume fraction (see Ref. 26 for review). Copper deficiency causes concentric enlargement of the heart (11, 15) that occurs in both the atria and ventricles (13). Connective tissue of these hearts is also altered in copper deficiency (27) as is suggested upon gross examination by the presence of softened texture and ventricular aneurysms (19) and via microscopic study of changes in the structure of the basement membrane, perimysial and endomysial connective tissue (2, 4, 5), and valves (14).

Copper deficiency is also implicated in the impairment of energy metabolism (26) presumably through the depression of energy production by decreased oxidative phosphorylation. Huang and colleagues (10) showed a depression of mRNA for ₁- and ₂-isoforms and protein levels of the ₂-isoform of Na⁺-K⁺-ATPase in myocytes of copper-deficient rat hearts. Impairment of the transport of Na⁺ and K⁺ could result in reduction of cardiac function via a decrease in ion movement.

Cardiac contractile function as measured in the whole isolated heart is reduced by dietary copper deficiency (1, 18). However, studying the role of copper deficiency in cardiac electromechanical functions in multicellular preparations has limitations; because these preparations encompass a heterogeneous population of cells, functional changes of single myocytes may not be accurately represented. Thus the action of copper on cardiac electromechanical function may be influenced by nonmyocyte factors such as interstitial connective tissue or fibrous growth. For example, increased ventricular stiffness may reflect a greater amount of interstitial fibrosis and a shift in collagen content rather than a direct effect on the mechanical properties of myocytes specifically. In contrast, studies with isolated myocytes can utilize cell biological and biophysical techniques to evaluate the actions of copper in a homogeneous population of cells. To our knowledge, no study has utilized isolated cells to investigate the role of copper deficiency in ventricular contractile function. The aim of the present study was to determine the impact of copper deficiency on cardiac contractile function at the single myocyte level.

	MATERIALS AND METHODS

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Animals and diets. All animal experimentation was conducted in accordance with humane animal care standards as outlined in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 85-23, Revised 1996). The procedures were approved by the Animal Investigation Committee of the University of North Dakota and the Grand Forks Human Nutrition Research Center Animal Care Committee.

Liver copper analysis. Livers were removed and snap-frozen in liquid nitrogen for subsequent assessment of copper content. Copper concentration was determined by lyophilizing and digesting liver samples with nitric acid and hydrogen peroxide (16) and measuring the copper content via inductively coupled argon plasma-emission spectroscopy (Fisons-ARL model 3560B; Thermo Jarrell Ash; Franklin, MA). Parallel assays of NIST reference samples (1477a, bovine liver) yielded values within the specified range thus validating our copper assay.

Isolation of ventricular myocytes. Single ventricular myocytes were isolated from CuA or CuD rat hearts as described previously (20). One animal was killed each day and the isolations were alternated between the control and experimental groups. In brief, hearts were rapidly removed and perfused (at 37°C) with oxygenated (5% CO₂-95% O₂) perfusion buffer that contained (in mM) 118 NaCl, 4.7 KCl, 1.25 CaCl₂, 1.2 MgSO₄, 1.2 KH₂PO₄, 25 NaHCO₃, 10 HEPES, and 11.1 glucose (pH 7.4). Hearts were subsequently perfused with a nominally Ca²⁺-free perfusion buffer for 2-3 min (until spontaneous contractions ceased) and then were perfused for 20 min with Ca²⁺-free perfusion buffer containing 223 U/ml of collagenase (Worthington Biochemical; Freehold, NJ) and 0.1 mg/ml of hyaluronidase (Sigma Chemical; St. Louis, MO). After perfusion, the left ventricle was removed, minced, and incubated with fresh enzyme solution (Ca²⁺-free perfusion buffer containing 223 U/ml of collagenase) for 3-5 min. The cells were further digested with 0.02 mg/ml of trypsin (Sigma) before being filtered through a 300-µm nylon mesh and collected by centrifugation (60 g for 30 s). Myocytes were resuspended in a sterile filtered Ca²⁺-free perfusion buffer containing (in mM) 131 NaCl, 4 KCl, 1 MgCl₂, 10 HEPES, and 10 glucose, supplemented with 2% BSA (pH 7.4; 37°C). Cells were initially washed with Ca²⁺-free perfusion buffer to remove remnant enzyme, and extracellular Ca²⁺ was added incrementally to achieve a 1.25 mM concentration. Isolated myocytes were maintained at 37°C in a serum-free medium consisting of medium 199 (Sigma) with Earle's balanced salts containing 25 mM HEPES and NaHCO₃ supplemented with 2 mg/ml of BSA, 2 mM L-carnitine, 5 mM creatine, 5 mM taurine, 5 mM glucose, 0.1 µM insulin, 100 U/ml of penicillin, 100 mg/ml of streptomycin, and 100 mg/ml of gentamicin. Mechanical properties remained relatively stable in myocytes maintained for 12-24 h in the serum-free medium. Cells that had any obvious sarcolemmal blebs or spontaneous contractions were not used; only rod-shaped myocytes with distinctly clear edges were selected for recording of mechanical properties or intracellular Ca²⁺ transients as previously described (21).

Cell shortening and relengthening measurements. Mechanical properties of ventricular myocytes were assessed using a video-based edge-detection system (IonOptix; Milton, MA) as previously described (20). In brief, coverslips with cells attached were placed in a chamber mounted on the stage of an inverted microscope (Olympus X-70) and superfused (~2 ml/min at 25°C) with a buffer containing (in mM): 131 NaCl, 4 KCl, 1 CaCl₂, 1 MgCl₂, 10 glucose, and 10 HEPES (pH 7.4). The cells were field stimulated at a frequency of 0.5 Hz for 3 ms in duration using a pair of platinum wires placed on opposite sides of the chamber connected to an FHC stimulator (FHC; Brunswick, ME). The polarity of the stimulating electrodes was reversed periodically to avoid potential buildup of electrolysis byproducts. The myocyte being studied was displayed on a computer monitor using an IonOptix MyoCam camera, which rapidly scans the image area every 8.3 ms such that the amplitude and velocity of shortening or relengthening are recorded with good fidelity. Changes in cell length (CL) during shortening and relengthening were captured and converted into an analog voltage signal. Cell shortening and relengthening were assessed using indexes of peak shortening (PS), time to 90% PS (TPS), time to 90% relengthening (TR₉₀), and maximal velocities of shortening (+dL/dt) and relengthening (dL/dt), respectively (21).

Ca²+ dose response. A separate cohort of cells from both the CuA and CuD groups was initially perfused with a perfusion buffer containing 0.5 mM CaCl₂ for 10 min. The concentration of Ca²⁺ was increased to 1, 2, and 3 mM, and recordings of the same cell were taken at each of the doses to show the effect of increased extracellular Ca²⁺ on the contractile response of the cells. Cells were abandoned if spontaneous contractions developed at any time during this process.

Intracellular Ca²+ transient measurement. A separate group of myocytes was loaded with the Ca²⁺-sensitive indicator fura 2-acetoxymethyl ester (fura 2-AM; 0.5 µmol/l; Molecular Probes; Eugene, OR) for 15 min at 25°C. Fluorescence measurements were recorded with a dual-excitation single-emission fluorescence photomultiplier system (IonOptix). Myocytes were placed on an inverted microscope and imaged through an Olympus Fluor ×40 oil objective. Myocytes were exposed to light emitted by a 75-W halogen lamp through either a 360- or 380-nm filter while being stimulated to contract at 0.5 Hz. Fluorescence emissions were detected between 480 and 520 nm by a photomultiplier tube after initial illumination at 360 nm for 0.5 s and then at 380 nm for the duration of the recording protocol. The 360-nm excitation scan was repeated at the end of the protocol and qualitative changes in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) were inferred from the ratio of the fura fluorescence intensity (FFI) at both wavelengths. The fluorescence decay time () was also measured as an indication of the intracellular Ca²⁺ clearing rate (20).

Data analysis. For each experimental series, data are presented as means ± SE. Statistical significance (P < 0.05) for each variable was estimated by two-way ANOVA or t-test where appropriate.

	RESULTS

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General features of CuA and CuD animals. The CuD rats exhibited significantly lower body weights and liver copper concentrations with significant cardiac and renal hypertrophy and hepatomegaly compared with the age-matched CuA animals (see Table 1). When found in animals fed a CuD diet, these signs are regarded as evidence of copper deficiency (24-26).

PS, TPS, and TR₉₀ for cardiac myocytes isolated from CuA and CuD animals. There were no recognizable differences in myocyte yield, field-stimulation threshold, spontaneous twitch, Ca²⁺ intolerance, or viability between the CuA and CuD groups. Approximately 20 cells were randomly selected for mechanical studies from each animal. Copper deficiency did not affect resting CL. The average resting CL values for ventricular myocytes used in this study were 163 ± 4 and 172 ± 5 µm in CuA (n = 223 cells/group from 11 animals) and CuD (n = 193 cells/group from 9 animals), respectively. The PS amplitude normalized to CL was significantly enhanced in myocytes isolated from CuD hearts (see Fig. 1A). Cells from the CuD group exhibited significantly enhanced changes in PS relative to the CuA group (11.0 and 16.0% for CuA and CuD groups, respectively; see Fig. 1B). Myocytes under sustained copper deficiency also exhibited a normal TPS value and significantly reduced TR₉₀ measurement (see Fig. 1, C and D, respectively). The enhanced myocyte shortening and reduced TR₉₀ values are associated with significantly enhanced +dL/dt and dL/dt (see Fig. 2).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Peak cell shortening (PS) of cardiac ventricular myocytes isolated from rat hearts fed with either a copper-adequate (CuA) or copper-deficient (CuD) diet: representative traces depicting cell shortening (A); summary of PS amplitudes (B); duration of PS (TPS; C); and time to 90% relengthening (TR90; D). Myocyte shortening was recorded at 25°C. Cells were stimulated to contract at 0.5 Hz. Values are means ± SE; n = 223 cells from 11 rats (CuA) and 193 cells from 9 rats (CuD); *P < 0.05 vs. CuA group (t-test).

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Maximal velocities of cell shortening (+dL/dt; A) and relengthening (dL/dt; B) in ventricular myocytes isolated from CuA or CuD rat hearts. Values are means ± SE; n = 223 cells from 11 rats (CuA) and 193 cells from 9 rats (CuD); *P < 0.05 vs. CuA group (t-test).

Effect of stimulation frequency on myocyte shortening. Rat hearts normally contract at very high frequencies (300 beats/min at 37°C), whereas our baseline studies were conducted at 0.5 Hz. To assess for copper status-dependent differences in cardiac excitation-contraction (E-C) coupling at higher frequencies, we increased the stimulation frequency incrementally to 5 Hz (300 beats/min) and recorded steady-state PS. Cells maintained at 25°C were initially stimulated to contract at 0.5 Hz for 5 min to ensure that steady state was attained before the frequency study was started. Figure 3 shows a negative staircase effect in PS with increasing stimulation frequency that did not differ between CuA and CuD groups. Changes in the stimulation frequency (0.1-0.5 Hz) did not affect the prolongation in TPS and TR₉₀ values in CuD and CuA rats (data not shown). One CuD cell did not respond at 5 Hz and therefore the data from that cell was omitted. Unstimulated (resting) CL measurements obtained before initiation of the 0.1-Hz stimulus were used as the CL measurements for the entire frequency sequence. All PS values were normalized to the PS at 0.1 Hz (as control). These data suggest that intracellular Ca²⁺ storage and release are likely preserved in copper deficiency.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   PS amplitude of ventricular myocytes isolated from CuA and CuD rat hearts at different stimulus frequencies. Each point represents PS amplitude relative to that at 0.1 Hz (control). Baseline PS values at 0.1 Hz were 17.8 ± 2.0 and 21.8 ± 1.6% in CuA and CuD groups, respectively. Values are means ± SE; numbers in parentheses are number of frequency alterations. Two-way ANOVA indicates frequency effect (P < 0.05) but no diet or interaction effects.

Effect of extracellular Ca²+ on myocyte shortening. The effect of extracellular Ca²⁺ on myocyte shortening is shown in Fig. 4. Increases in extracellular Ca²⁺ concentration from 0.5-3 mM caused no significant effect on myocyte shortening in either the CuA or CuD groups. Note that the absence of a significant difference between the CuD and CuA cells at 1 mM Ca²⁺ (where Fig. 1B suggests that there should be a difference) is likely due to the lower sample number required to test for the effect of Ca²⁺.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Ca2+ dose-response curve for myocytes isolated from either CuA or CuD rat hearts. Myocyte shortening was recorded at 25°C. Cells were stimulated to contract at 0.5 Hz. Values are means ± SE; numbers in parentheses are number of dose responses. Two-way ANOVA indicates no diet, Ca2+, or interaction effects.

Intracellular Ca²+ transients. We used the membrane-permeant form of fura 2-AM to evaluate the properties of intracellular Ca²⁺ transients in myocytes from CuA and CuD rats. The time course of the fluorescent signal decay was well described by a single exponential equation, and  was used as a measure of the rate of decline of free cytoplasmic Ca²⁺. The fluorescence measurements revealed that the resting Ca²⁺ level was unchanged by the copper status. However, the intracellular Ca²⁺ transients decayed at a much faster rate in myocytes from the CuD group than from the CuA group (see Fig. 5). The traces and graphs in Fig. 5 were chosen to illustrate that copper deficiency caused no change in the resting Ca²⁺ ratio other than a smaller  value.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Typical experiments showing intracellular Ca2+ transient properties in myocytes isolated from CuA or CuD rat hearts (A); Ca2+ transient decay rate (; B); increase in fura 2-acetoxymethyl ester fluorescence intensity in response to electrical stimulus (C); and baseline intracellular Ca2+ levels (D). Intracellular properties were recorded at 25°C. Cells were stimulated to contract at 0.5 Hz. Values are means ± SE; n = 69 cells from 7 rats (CuA) and 67 cells from 4 rats (CuD); *P < 0.05 vs. CuA group (t-test).

	DISCUSSION

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This study reports for the first time the direct impact of copper deficiency on ventricular function at the single myocyte level. Our results indicate that compromised ventricular function in copper deficiency is not likely due to depressed contractile performance of the single myocyte. The major mechanical alterations observed in CuD cardiac myocytes are characterized by enhanced cell shortening and decreased duration, as well as increased velocities of both shortening and relengthening. Furthermore, the increased rate of Ca²⁺ decay is consistent with the enhanced cell shortening and decreased duration of relengthening (which are described by TR₉₀ values). Altered E-C coupling could contribute to these alterations in several ways, including increased sarcolemmal Ca²⁺, sarcoendoplasmic reticulum ATPase, Na⁺/Ca²⁺ exchange, or myofilament Ca²⁺ sensitivity.

Despite the improved function of individual cells, impaired mechanical function is an important characteristic of CuD hearts. Overt mechanical abnormalities of copper deficiency are commonly seen in whole hearts and the severity of the dysfunctions increases with time. Among the mechanisms that have been postulated for cardiac dysfunction from dietary copper deficiency is the reduced activity of copper-dependent lysyl oxidase. Lysyl oxidase initiates cross linking of elastin and collagen (22), which are involved in the structural integrity of the musculature. Reduced activity of this enzyme is associated with morphological and functional changes such as tissue flaccidity, aneurisms, and rupture of the heart, which are seen in whole hearts of CuD animals and may thus counteract the improved performance of individual cells.

The reduction of several copper-dependent and -independent antioxidant enzymes in copper deficiency has suggested that oxidative stress may contribute to altered cardiac function (23). Visual indications of damage and enhancement of lipid peroxidation have been observed in CuD hearts (3, 19). However, because peroxidation is generally a destructive process, it is difficult to visualize it as a direct cause of the improved contractile function observed in single myocytes. Indeed, in another prooxidant environment (diabetes), contractile function of individual cardiomyocytes is decreased (21). Thus although oxidative damage may explain structural changes in the CuD heart, it is necessary to look elsewhere for the cause of enhanced function of individual CuD myocytes.

Recent evidence has also suggested that titin may play a role in the alteration of myocardial function and development of heart dysfunction (9). Decreased internal stiffness of CuD cardiac myocytes along with an increase in myocyte diameter (7), presumably via an alteration in actin, myosin, titin, or other intracellular proteins, may allow a greater myocyte contraction to occur. Therefore it would be intriguing to determine the relative abundance of actin, myosin, and titin, which is an endosarcomeric elastic protein that potentially functions as a bidirectional spring in both shortening and relengthening of unloaded cardiac myocytes (8), and to evaluate the roles of these proteins in the altered mechanical function of CuD rat hearts.

Measurement of contractile performance in isolated myocytes has been established to provide a fundamental assessment of cardiac contractile function in pathological states; in this case, dietary copper deficiency. However, as in any study of this nature, caution must be taken when correlating our present cellular findings to whole heart function, because the latter is composed of heterogeneous cell types (including nerve terminals and fibroblasts) as well as the connective tissue alluded to earlier. Although we do not believe that copper deficiency led to skewed selection of cells, such factors as altered self-attachment of cells to the coverslips and differential sensitivity of the myocardium to collagenase digestion should be considered as possible contributing factors that may affect the results of this study.