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Am J Physiol Heart Circ Physiol 278: H1840-H1847, 2000;
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Vol. 278, Issue 6, H1840-H1847, June 2000

Decreased passive stiffness of cardiac myocytes and cardiac tissue from copper-deficient rat hearts

Lois Jane Heller1, David E. Mohrman1, and Joseph R. Prohaska2

Departments of 1 Medical and Molecular Physiology and 2 Biochemistry and Molecular Biology, School of Medicine, University of Minnesota, Duluth, Duluth, Minnesota 55812


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Passive stiffness characteristics of isolated cardiac myocytes, papillary muscles, and aortic strips from male Holtzman rats fed a copper-deficient diet for ~5 wk were compared with those of rats fed a copper-adequate diet to determine whether alterations in these characteristics might accompany the well-documented cardiac hypertrophy and high incidence of ventricular rupture characteristic of copper deficiency. Stiffness of isolated cardiac myocytes was assessed from measurements of cellular dimensional changes to varied osmotic conditions. Stiffness of papillary muscles and aortic strips was determined from resting length-tension analyses and included steady-state characteristics, dynamic viscoelastic stiffness properties, and maximum tensile strength. The primary findings were that copper deficiency resulted in cardiac hypertrophy with increased cardiac myocyte size and fragility, decreased cardiac myocyte stiffness, and decreased papillary muscle passive stiffness, dynamic stiffness, and tensile strength and no alteration in aortic connective tissue passive stiffness or tensile strength. These findings suggest that a reduction of cardiac myocyte stiffness and increased cellular fragility could contribute to the reduced overall cardiac tissue stiffness and the high incidence of ventricular aneurysm observed in copper-deficient rats.

cardiac hypertrophy; myocyte dimensions; papillary muscles; osmotic stress


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

COPPER IS KNOWN TO BE AN ESSENTIAL nutrient for proper organ development and function. For example, dietary copper deficiency in growing rats leads to severe concentric cardiac hypertrophy (10, 12, 14-16) as well as cardiac wall weakness, which often leads eventually to ruptured ventricular aneurysm (2, 21). The dramatic cardiac effects of copper deficiency are puzzling for several reasons. For example, none of the usual suspected stimuli of cardiac growth, such as pressure or volume overload, catecholamines, or ANG II, appear to be responsible for the cardiac hypertrophy of copper deficiency (7, 8, 10, 12).

The heart contains an extensive extracellular collagen network to which myocytes are tethered. This extracellular matrix is instrumental in maintaining tissue architecture and chamber geometry (20). Myocardial tissue stiffness is reduced in copper deficiency (2, 17, 21). This is thought to be due to a reduction in the activity of the copper-dependent enzyme lysyl oxidase, which normally facilitates collagen and elastin cross-linking in the extracellular cardiac matrix (17, 20). Naturally, it has been argued that such structural abnormalities in the extracellular matrix are responsible for the high incidence of ventricular wall rupture that accompanies copper deficiency. Moreover, it has been argued that changes in the mechanical properties of the extracellular matrix could alter the loading of the myocytes in such a way as to stimulate their hypertrophy (10, 12). However, other studies have shown that treatment of rats with the lysyl oxidase inhibitor beta -amino proprionitrile decreases myocardial tissue stiffness but does not influence heart size or ventricular wall fragility (1, 13).

The known changes in the properties of the extracellular matrix alone do not seem sufficient to account for the cardiac abnormalities that are seen with copper deficiency. Consequently, this study was undertaken to look at whether the mechanical properties of other components of the myocardial wall and, in particular, myocytes themselves are altered by copper deficiency.

Information about overall cytoskeletal stiffness of individual cardiac myocytes was obtained from measurements of the dimensional changes in isolated cardiac myocytes evoked by altered tonicity of the bathing media (i.e., hypotonic swelling and hypertonic shrinking) (5). Information about intact cardiac tissue stiffness was obtained from measurements of passive and dynamic stress-strain characteristics of isolated papillary muscles, and information about extracellular connective tissue stiffness was inferred from passive stress-strain characteristics of isolated longitudinally oriented aortic strips. Data from isolated myocytes, cardiac tissue, and connective tissue of copper-deficient rats were compared with those of rats with adequate copper in their diets.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animal Preparation

Copper deficiency was produced by feeding weanling Holtzman male rats a purified diet low in copper (modified AIN-76A, Teklad Laboratories, Madison, WI) (14, 16). This diet contained the following components (g/kg diet): 500 sucrose, 200 casein, 150 cornstarch, 50 corn oil, 50 cellulose, 35 modified AIN-76 mineral mix, 10 AIN-76A vitamin mix, 3 DL-methionine, 2 choline bitartrate, and 0.01 ethoxyquin. Cupric carbonate was omitted from the AIN-76 mineral mix. For the experiments on cardiac myocytes (study 1), the basal concentration of copper in the diet was analyzed to be 0.30 mg/kg purified diet, and for the experiments on papillary muscles and aortic strips (study 2), the basal dietary copper concentration was 0.52 mg/kg purified diet. The rats in which copper deficiency was produced drank deionized water. Control rats for these studies were fed the same copper-deficient diet but received supplemental CuSO4 added to the drinking water (20 µg Cu/ml). The estimated daily copper intake of the copper-deficient rats is ~1% that of the copper-adequate rats.

Rats used in study 1 were started on diets at 19 days of age and killed in pairs (1 copper-adequate and 1 copper-deficient rat per day) after 30 ± 2 days. Rats used in study 2 were started on diets at 21 days of age and killed after 37 ± 1 days. Housing was provided in the American Association for Accreditation of Laboratory Animal Care-accredited university facility under control conditions of 24°C, 55% relative humidity, and 12:12-h light-dark cycle. All rats had free access to food and water as described throughout the experiment, and all protocols were approved by the University of Minnesota Institutional Animal Care and Use Committee.

Assessment of Copper Deficiency

Liver copper and iron concentrations were determined by flame atomic absorption spectroscopy. Blood Hb levels and plasma ceruloplasmin activity were determined by spectrophotometric techniques. Cardiac myocyte Cu/Zn superoxide dismutase activity was determined spectrophotometrically to assess copper status of the isolated cardiac cells. These biochemical protocols are described in detail elsewhere (14).

Heart Harvest and Determination of Cardiac Hypertrophy

Rats were given an injection of heparin (1 U/g body wt ip) 30 min before heart removal. This was done to minimize intracoronary clotting during the subsequent perfusion. Rats were then anesthetized with CO2 and decapitated, and hearts were rapidly removed and placed in iced physiological salt solution. After extraneous tissue was trimmed from the hearts and the hearts were lightly blotted, they were transferred to a preweighed beaker for weighing. Cardiac hypertrophy was assessed by comparison of heart weights and heart weight-to-body weight ratios between the copper-deficient and copper-adequate groups.

Cardiac Myocyte Studies

Preparation. Cardiac myocytes were isolated from hearts by use of standard enzymatic techniques (16, 18). Briefly, hearts were perfused at 37°C with Joklik's modified calcium-free MEM containing 0.7% collagenase (Worthington) until the vascular bed deteriorated (~20 min). The tissue was then coarsely minced, and digestion with the same collagenase-containing solution continued in a beaker placed in a gyrating water bath for an additional ~20 min in the presence of 1.0 µM CaCl2. The tissue digestate was then filtered through cheesecloth, and cells were allowed to settle out of the filtrate. The cells were resuspended in fresh collagenase-free Joklik's solution and resettled twice before the final resuspension.

The initial yield of myocytes in the low-calcium Joklik's solution was assessed at this point by determining the number of live rod-shaped cells obtained per heart. Viability of myocytes in low-calcium solution was determined as the percentage of rod-shaped cells in the total population that excluded the vital dye, trypan blue. Calcium was then added to the cell suspension (in several steps spread over a 30-min period) to a final concentration of 1.0 mM. Viability was reassessed at the end of this procedure.

Cells were then resettled and resuspended in a physiological salt solution (HEPES buffer) for subsequent visualization and dimension measurements. The 324 mosM (isotonic) solution contained (in mM) 135 NaCl, 4.9 KCl, 1.2 MgSO4, 1.2 NaH2PO4, 1.0 CaCl2, 15 glucose, and 20 HEPES. Insulin (10 U/l) was also included, and pH was adjusted to 7.4. The cells were stored at room temperature for >= 30 min before measurements were obtained, and all measurements were taken within the next 3 h.

Myocyte visualization, osmotic challenges, and dimension determination. An aliquot of the cardiac myocyte suspension in HEPES buffer was placed in a 35-mm tissue culture dish, and after 5 min cells had attached to the bottom of the dish. The dish was mounted on a moveable stage of an inverted microscope, and suffusion with the normal HEPES buffer at 27°C was instituted. A computerized, positional mapping program of the microscope stage allowed the identification of 10-20 cells at different areas of the dish, and video images were captured for later analysis. Cell selection for measurement was based on minimum criteria that cells had to be 1) freestanding with all edges visible, 2) rod shaped, with the length at least twice the width, and 3) similar in size to other cells in the dish. To evaluate the passive distensibility (or stiffness) of the isolated myocytes, cells were then subjected to changes in the osmolarity of the extracellular solution. Changes in cell size reflect the fluid movements evoked by the changes in osmotic pressure and the characteristics of the cytoskeletal structure of the cell (5). The suffusing solution was changed to one made hypertonic (~2 times isotonic) by addition of 300 mosM sucrose to the normal HEPES buffer (total osmolarity 624 mosM). After a 5-min equilibration period, the selected cells were revisited, and video images were again captured. The suffusing solution was then made hypotonic by addition of water to the normal HEPES buffer. Viability of the selected cells was assessed at 243 mosM (0.75 times isotonic), but dimension measurements were not made until the solution tonicity was reduced to 162 mosM (0.5 times isotonic). Video images of the selected cells were again captured after a 5-min incubation period.

The software program NIH Image was used to obtain dimension estimates from the digitized videotaped images of the myocytes under isotonic, hypertonic, and hypotonic conditions. Measurements of multiple cells from a single heart were averaged to obtain representative dimensions of cells from that heart. Data from individual hearts were then combined to obtain the group average. Unless otherwise indicated, the number of animals for the reported measurements in each group was eight.

Papillary Muscle and Aortic Strip Studies

Papillary muscles. After the heart was weighed, papillary muscles were removed from the wall of the left ventricle. The tissue was isolated to include a piece of mitral valve tissue at one end and a piece of ventricular wall at the other end. These ends were used to mount the tissue between a small clamp (at the mitral end) and a ligature (at the wall end). The clamp was connected by a noncompliant chain to a measuring arm of the strain gauge (model FT03C, Grass), and the ligature was tied to a post rigidly fixed to the casing of the strain gauge. The tissue was then submerged in a 37°C bath of physiological saline solution (0.9% NaCl). In the absence of calcium, magnesium, and glucose or external stimulation, muscles did not contract or develop contractures and remained in a relaxed state throughout the experiment.

Muscle length was controlled and measured electronically with a specially constructed apparatus based on the servo-position control of a pen motor from a Gould/Brush chart recorder. The device automatically compensated for the compliance of the strain gauge beam.

Muscle lengths were initially adjusted to those at which tension first appeared and were allowed to equilibrate for ~10 min. Three protocols were then conducted in sequential order to study the passive characteristics of the papillary muscles. Protocol A was carried out to determine the passive, steady-state length-tension characteristics of the muscle. Lengths were increased in five steps that each produced an initial 1.0-g increase in tension, muscles were allowed to equilibrate at each length for 1 min, and tension was determined after equilibration. This procedure was repeated three times, and the length changes were normalized to those determined at the beginning of the stretching sequence. Protocol B was conducted to assess the dynamic compliance of the tissue. The lengths of the papillary muscles were again increased in five steps, as described for protocol A, but at each length, small (~0.10-mm) 0.1-Hz square-wave perturbations in length were superimposed. The resulting change in tension per change in length was determined at the end of 1 min at each step length. Protocol C was carried out to determine the tensile strength of the tissue. The muscle lengths were again increased in steps to determine the tension at which the tissue broke or began to tear so that tension no longer increased with increasing length.

Aortic strips. Longitudinal aortic strips (rather than circular strips) were used for this study, because there is little or no longitudinal smooth muscle or elastin in the aorta, and longitudinal mechanics primarily reflect the longitudinally oriented collagen fibers. The descending thoracic aorta was removed from the rats, slit open lengthwise, and cut into strips ~10 mm long and 2 mm wide. The strips were mounted as described for papillary muscles. A 10-min equilibration period followed the setting of initial length to the point where tension first appeared. The protocol used for these studies consisted of stretching the strip in multiple steps from its initial length to the point at which it failed. Each length step was set to produce ~2.5-g initial changes in tension, and after 1 min of equilibration at each length, length and tension data were collected for determination of the passive length-tension relationship. Dynamic compliance was determined at the length producing ~10 g of tension by applying rapid square-wave perturbations in length (~0.1 mm at 0.1 Hz) and recording the change in tension per change in length. Tensile strength was determined by measuring the tension and length at which the strips broke.

At the end of the experiments, papillary muscles and aortic strips were removed from the bath and weighed so that average cross-sectional area could be determined. This value was used to normalize the tension measurements to stress values for these tissues.

Statistics

Values are means ± SE. Differences between copper-deficient and copper-adequate groups were assessed by Student's t-test, with significance declared at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of Copper Deficiency on Rats

Characteristics of the rats used for the experiments with isolated myocytes (study 1) and experiments with isolated papillary muscles and aortic strips (study 2) are shown in Table 1. In both studies the copper-deficient rats had significantly greater heart weights (absolute weight and relative to body weight), lower Hb levels, lower plasma ceruloplasmin activity, lower liver copper concentration, and higher liver iron concentration than the copper-adequate rats. This composite set of alterations is consistent with previously reported characteristics of rats significantly deficient in copper. (Because of the subsequent perfusion, hearts used for the myocyte isolations were not trimmed as thoroughly as in other studies, which accounts for the abnormally high absolute and relative heart weights of copper-adequate and copper-deficient rats in study 1.)

                              
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  		Table 1.   Characteristics of copper-adequate and copper-deficient rats used for studies 1 and 2 

The copper deficiency was more pronounced in the rats used for study 1 than in those used for study 2, as is evident from their lower body weights (which is characteristic of the growth retardation of severe copper deficiency) and lower liver copper concentration. This difference is probably a result of the lower basal dietary copper levels in study 1 than in study 2.

The Cu/Zn superoxide dismutase activity of isolated cardiac myocytes from the copper-deficient rats in study 1 (33 ± 4 U/mg) was significantly less (P < 0.01) than that from the copper-adequate rats (72 ± 3 U/mg), thus verifying the copper deficiency at the cellular level.

Isolated Cardiac Myocytes

The yield of viable rod-shaped myocytes isolated in the low-calcium media from hearts of copper-deficient rats (3.5 ± 0.3 × 106 cells) was significantly less than that of copper-adequate rats (5.6 ± 0.4 × 106 cells, P < 0.05). However, the percentage of viable, rod-shaped myocytes obtained from the copper-deficient rats in the final settling in the low-calcium media (78 ± 2%) was not significantly different from that of the myocytes obtained from the copper-adequate rats (84 ± 1%). Addition of calcium to the media to a final concentration of 1.0 mM resulted in a loss of viable cells in both groups. The decrease in viability of myocytes from copper-deficient rats (to 47 ± 3%) was significantly greater than that from copper-adequate rats (to 72 ± 1%).

Cardiac myocyte dimensions measured under isotonic conditions clearly demonstrate that the viable myocytes from hearts of the copper-deficient rats were significantly larger than those of the copper-adequate rats (Table 2). There was a greater increase in myocyte width than length, indicating that the hypertrophy is more pronounced in the lateral than in the longitudinal axis of the cell. Estimates of cardiac myocyte surface area and volume were calculated by assuming that the two-dimensional video image was actually the top view of a cylinder and that the width of the image was equal to the cell diameter. These estimates suggest that with copper deficiency the myocyte surface area was increased by ~36% and the myocyte volume was increased by ~69%.

                              
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  		Table 2.   Dimensions of isolated cardiac myocytes from copper-adequate and copper-deficient rats determined under isotonic conditions

Cardiac myocytes from both experimental groups tolerated the increase in tonicity with no loss of viability, and those from the copper-adequate group tolerated the decreases in tonicity with no loss of viability. However, cardiac myocytes from the copper-deficient rats were much less tolerant of the hypotonic conditions. Changing to the moderately hypotonic (243 mosM) environment resulted in death of 75% of the observed cells. A further decrease of osmolarity to 162 mosM did not result in additional cell mortality.

Changes in tonicity to 624 and 162 mosM altered the sizes of cardiac myocytes (Fig. 1). The hypertonic environment caused shrinking and the hypotonic environment caused swelling of all cells. Changes in myocyte diameter were more pronounced than changes in length, indicating that the cells were most distensible in the lateral axis and quite nondistensible in the longitudinal axis. The tonicity-induced changes in absolute values of diameter, surface area, and volume of the myocytes from hearts of copper-deficient rats were significantly greater than those of copper-adequate rats. When these greater absolute responses were expressed relative to the values in isotonic conditions, there were no significant differences in the changes in relative diameter or volume between myocytes of the copper-deficient and copper-adequate groups (Table 3). However, the increase in relative surface area of the copper-deficient myocytes was significantly greater than that of the copper-adequate myocytes.


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  		Fig. 1.   Dimensions of cardiac myocytes isolated from hearts of copper-adequate and copper-deficient rats under isotonic (324 mosM), hypertonic (624 mosM), and hypotonic (162 mosM) conditions. A: length; B: diameter; C: surface area; D: volume. Values are means ± SE of 7 rats/experimental group; 11 ± 2 and 17 ± 2 cells were measured per preparation in copper-adequate and copper-deficient groups, respectively, under each tonicity, except in copper-deficient group under hypotonic conditions, where surviving number of cells was 5 ± 1. * P < 0.05 compared with copper-adequate value.


                              
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  		Table 3.   Tonicity-induced changes in cardiac myocyte dimensions relative to dimensions under isotonic conditions

Papillary Muscle Studies

Although the initial lengths of papillary muscles from the copper-deficient rats as obtained at the beginning of protocol A (4.08 ± 0.31 mm) were not significantly different from those of the copper-adequate rats (4.13 ± 0.36 mm), the calculated cross-sectional area was significantly greater (2.72 ± 0.28 and 1.94 ± 0.13 mm2, respectively, P < 0.05). Steady-state, passive length-stress characteristics obtained during the three stretching sequences of protocol A are shown in Fig. 2. The resting length at which passive tension was first measurable became progressively longer with each successive stretch. The degree of this "slippage" in the papillary muscles between the first and the second stretch was significantly greater in the copper-deficient than in the copper-adequate preparations (14 ± 3 and 3 ± 1%, respectively, P < 0.05). Furthermore, the steepness of the slope of the relationship between passive length and stress in the copper-deficient preparations was much lower than that of the copper-adequate preparations. This difference in slope was most exaggerated during the first stretch but was still significant during the third stretch (1.60 ± 0.26 and 0.93 ± 0.14 g/mm2 per 10% change in length for copper-adequate and copper-deficient rats, respectively, P < 0.05). These data indicate that the steady-state passive stiffness of isolated papillary muscles of the copper-deficient rats is significantly less than that of the copper-adequate rats in the low range of strains tested in protocol A. 


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  		Fig. 2.   Passive length-stress relationships of papillary muscles from hearts of copper-adequate (A, n = 5) and copper-deficient (B, n = 5) rats obtained during 3 consecutive stretching procedures. Values are means ± SE. Increase in length of copper-deficient papillary muscles between 1st and 2nd stretches at which stress was first measurable was significantly greater (P < 0.05) than that of copper-adequate papillary muscle. Slopes of 3 length-stress relationships of copper-deficient papillary muscles were significantly less (P < 0.05) than those of copper-adequate papillary muscles.

The dynamic stiffness of the isolated papillary muscles was assessed during protocol B and was found to increase with increasing lengths in both experimental groups (Fig. 3). Although at short muscle lengths there were no significant differences in dynamic stiffness between copper-deficient and copper-adequate preparations, at the longer muscle lengths the dynamic stiffness of the copper-deficient muscles was significantly lower than that of the copper-adequate muscles.


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  		Fig. 3.   Dynamic stiffness of papillary muscles from hearts of copper-adequate (n = 5) and copper-deficient (n = 5) rats obtained from small-amplitude length oscillations superimposed on a passive stretch of muscles. Data were obtained at increasing lengths relative to initial length at beginning of stretching protocol. Values are means ± SE. * P < 0.05 vs. copper-adequate value.

The normalized lengths and stresses at which the papillary muscles failed during protocol C are shown in Fig. 4. The tissues from the copper-deficient rats broke at significantly lower stresses that averaged only 20% of those from the copper-adequate rats.


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  		Fig. 4.   Points describing lengths and stresses, where increasing stretch of papillary muscles from copper-adequate (n = 5) and copper-deficient (n = 5) rat hearts resulted in a failure to increase passive tension. Values are means ± SE. * P < 0.05 vs. copper-adequate value.

Aortic Strip Studies

There were no significant differences in the initial lengths (7.2 ± 0.7 and 8.5 ± 0.9 mm in copper-deficient and copper-adequate rats, respectively) or the normalized cross-sectional areas (0.39 ± 0.09 and 0.32 ± 0.07 mm2 for copper-deficient and copper-adequate rats, respectively) of the aortic strips used for these studies. Steady-state passive length-stress characteristics of the aortic strips are shown in Fig. 5. Lengths of the tissue were normalized to the length producing a stress of 15 g/mm2. These data indicate that the steady-state passive stiffness of aortic tissue from copper-deficient rats was not significantly different from that from copper-adequate rats. Dynamic stiffness of the aortic strips was measured at similar relative lengths (1.19 ± 0.06 and 1.13 ± 0.03 for copper-adequate and copper-deficient rats, respectively), and there was no significant difference between the groups (13 ± 1 and 12 ± 1 g/mm2 per 1% change in length for copper-adequate and copper-deficient rats, respectively).


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  		Fig. 5.   Passive length-stress relationships of aortic strips of copper-adequate (n = 4) and copper-deficient (n = 4) rats. Points at right indicate length and stress where increasing stretch failed to increase passive tension. +Cu, copper adequate; -Cu, copper deficient. Values are means ± SE. There were no significant differences between experimental groups.

The normalized lengths and stresses at which the aortic strips failed are also shown in Fig. 5. The values for the aortic strips of copper-deficient rats were not significantly different from those of copper-adequate rats.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The primary findings of this study are that copper deficiency results in 1) increased size of cardiac myocytes primarily due to an increased cellular diameter, 2) increased cardiac myocyte fragility, as suggested by high mortality in hypotonic conditions, 3) decreased cardiac myocyte stiffness, as demonstrated by increased hypotonic expansion of the cell surface area in surviving cells, 4) increased papillary muscle fragility, as demonstrated by stretch-induced tissue tearing at lower stresses, 5) decreased papillary muscle steady-state passive stiffness, 6) decreased papillary muscle dynamic stiffness, as demonstrated by reduced changes in tension per small rapid changes in length, and 7) no alterations in aortic longitudinal connective tissue passive length-tension relationship or tensile strength.

The increase in myocyte size induced by copper deficiency in this study is substantially greater than that reported to accompany pressure overload. In the present study the length and width were ~8% and 23% greater for the average isolated cardiac myocyte from the copper-deficient rats than from the copper-adequate rats, whereas abdominal aortic banding of rats for 15-18 wk is reported to produce an increase of ~7% in length and width of the average isolated cardiac myocytes measured under similar conditions (4). Thus the impressive increases in heart weight-to-body weight ratios reported to accompany copper deficiency (10, 12, 14-16) seem to be correlated with impressive increases in cardiac myocyte dimensions, particularly in the lateral axis, as is consistent with concentric hypertrophy. This observed increase in myocyte cross-sectional area is also consistent with reports that the hypertrophy of copper deficiency is a result primarily of increased mitochondrial content rather than increased myofibrillar content (3, 9, 11).

The findings of this study suggest that the high incidence of ventricular wall rupture in the copper-deficient rat model (2, 10, 12, 21) may be a result of an increased fragility of the cardiac myocytes themselves in addition to the well-documented defects in the extracellular matrix of the heart (2, 13, 17, 21). The intolerance of the cardiac myocytes to the hypotonic environment and cellular stretch may be correlated with the intolerance of the intact cardiac tissue (i.e., papillary muscle) to externally applied stretch. In addition, the increased calcium intolerance of the cardiac myocytes from copper-deficient rats found in this study, and described in detail elsewhere (16), further supports the possibility of increased fragility of these myocytes. Furthermore, the absence of any changes in the passive length-tension characteristics of the aorta suggests that the connective tissue's distensibility and stiffness may not be influenced by this degree of copper deficiency. Clearly, caution must be taken when findings from the aorta are generalized to the heart, because important regional differences within the heart itself in distribution of lysyl oxidase activity, as well as in collagen cross-linkages and ultrastructural morphology, have been shown (21). Also, a reduction in tensile strength of helically cut aortic strips has been reported to accompany severe copper deficiency (6).

The findings of this study suggest that reduction in myocyte stiffness might also account for the decrease in steady-state passive stiffness of the papillary muscle and the dynamic stiffness observed when rapid small length changes are applied. However, the length-tension protocol for assessing papillary muscle passive stiffness probably measures characteristics of the longitudinal axis of the cell, whereas the method for assessing myocyte passive stiffness used in this study only revealed differences in the lateral axis. Clearly, other methods of measuring myocyte passive stiffness characteristics need to be examined before firm conclusions can be made. Studies by Tagawa et al. (19) indicated that pressure-overload hypertrophy of cardiac myocyte increased cytoskeletal stiffness and apparent intracellular viscosity as measured by magnetic twisting cytometry. They proposed that this was a result of an increased density of the microtubule component of the cardiac myocyte's cytoskeleton and constituted an increased viscous load on the contractile apparatus. Although the present studies suggest that the hypertrophy of copper deficiency is associated with a decrease in cytoskeletal stiffness, at least in the lateral axis, the method used did not evaluate viscous characteristics of the isolated myocyte.

All conclusions about myocyte characteristics were made on only a small portion of the cells initially present in the heart. Cardiac myocytes from the copper-deficient rats were lost during several steps in the protocols in significantly greater numbers than those from the copper-adequate rats 1) during the enzymatic isolation, 2) after the reintroduction of calcium to 1.0 mM, and 3) on exposure to hypotonic conditions. Thus the surviving cardiac myocytes from the copper-deficient rats are probably those least altered by the copper deficiency and most similar to myocytes from copper-adequate rats. Therefore, we suggest that differences observed in this study between the experimental groups are probably underestimated and the actual in situ average myocyte stiffness and fragility in hearts of copper-deficient rats may be considerably different from that predicted by our in vitro measurements.

In summary, this study indicates that a reduction in passive stiffness of cardiac myocytes and an increase in their structural fragility may contribute to the reduction in overall cardiac tissue stiffness and high incidence of ruptured ventricular aneurysms observed in copper-deficient rats.


    ACKNOWLEDGEMENTS

The authors greatly appreciate the technical expertise of Bruce Brokate for animal preparations and biochemical assays, Juline Smith for myocyte isolations, and Ashley Becker (American Physiological Society Summer Research Teacher) and David VanDyke (American Heart Association Summer Student Research Fellow) for myocyte dimension determinations and data analysis.


    FOOTNOTES

This research was supported by National Research Initiative Competitive Grants Program/US Department of Agriculture Grant 96-35200-3138 and funds from the University of Minnesota, Duluth School of Medicine.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: L. J. Heller, Dept. of Medical and Molecular Physiology, School of Medicine, University of Minnesota, Duluth, 10 University Dr., Duluth, MN 55812 (E-mail: lheller{at}d.umn.edu).

Received 9 June 1999; accepted in final form 2 December 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bing, OHL, Fanburg BL, Brooks WW, and Matsushita S. The effects of the lathyrogen beta -amino proprionitrile (BAPN) on the mechanical properties of experimentally hypertrophied rat cardiac muscle. Circ Res 43: 632-637, 1978[Free Full Text].

2.   Borg, TK, Klevay LM, Gay RE, Siegel R, and Bergin ME. Alteration of the connective tissue network of striated muscle in copper-deficient rats. J Mol Cell Cardiol 17: 1173-1183, 1985[ISI][Medline].

3.   Dallman, PR, and Goodman JR. Enlargement of mitochondrial compartment in iron and copper deficiency. Blood 4: 496-505, 1970.

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Am J Physiol Heart Circ Physiol 278(6):H1840-H1847
0363-6135/00 $5.00 Copyright © 2000 the American Physiological Society


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