Journal of the American College of Nutrition, Vol. 18, No. 1, 51-60 (1999)
Published by the American College of Nutrition
Heart Murmurs, Valvular Regurgitation and Electrical Disturbances in Copper-Deficient Genetically Hypertensive, Hypertrophic Cardiomyopathic Rats
Laura Jeanne Shiry, MS,
Denis M. Medeiros, PhD,
John D. Bonagura, DVM and
Sylvia A. McCune, PhD
Department of Human Nutrition and Food Management (L.J.S., D.M.M.), The Ohio State University, Columbus
Department of Food Science and Technology (S.A.V.), The Ohio State University, Columbus, Ohio
Department of Veterinary Medicine and Surgery (J.D.B.), Columbia University, Columbia, Missouri
Address reprint requests to: Denis M. Medeiros, PhD, Department of Human Nutrition and Food Management, 347 Campbell Hall, 1787 Neil Ave., The Ohio State University, Columbus, OH 43210-1295
 |
ABSTRACT
|
|---|
Objective: Rats with a genetic tendency to develop hypertensive, hypertrophic cardiomyopathy were fed copper-deficient diets and their cardiac responses were investigated.
Methods: Five male weanling rats of the Long-Evans and SHHF/Mcc-facp strains were randomly selected to receive diets containing either adequate quantities of copper (94.5 µmol Cu/kg diet) or reduced quantities of copper (<15.8 µmol Cu/kg diet) for 6 weeks, (n=5 within each group). Echocardiograms and electrocardiograms were recorded and analyzed at the end of the 6-week interval.
Results: Electrocardiograms from copper deficient groups showed longer Q-T intervals and increased QRS amplitudes than controls. Both the copper deficient and control SHHF groups demonstrated significant QRS complex prolongation compared to Long-Evans rats. Echocardiography analysis showed significant increases in left ventricular area, free wall dimension, and myocardial cross-sectional areas in rats fed a copper deficient diet. The frequency of systolic cardiac murmurs increased in copper deficient rats and were related to the presence of valvular regurgitation as determined from echocardiography.
Discussion: However, the data do not suggest that a copper-deficient diet fed to a strain of rats genetically susceptible to heart disease later in life, hastens or worsens the onset of cardiac disease. The genetic predisposition and copper-deficient states exert independent effects upon the heart.
Key words: cardiac hypertrophy, electrocardiogram, SHHF/Mcc-facp, cardiac murmurs, echocardiograms, valve regurgitation, copper-deficiency
|
INTRODUCTION
|
|---|
Copper deficient diets consumed from weaning produce such biochemical and physical changes as concentric cardiac hypertrophy, hemothorax, anemia, and decreased liver copper (Cu)-zinc (Zn) superoxide dismutase (SOD) and cardiac cytochrome c oxidase (CCO) activities [1–4]. Electrocardiograph abnormalities in rats subsequent to copper restriction include conduction disturbances, prolongation of the QT segment, supraventricular and ventricular ectopic beats, and increased R wave amplitude [5,6]. Histological abnormalities associated with copper deficiency reveal abnormal connective tissue within the vasculature and cardiac valves, vacuolated mitochondria with fragmented cristae, decreased myofibril volume density, increased mitochondria volume density, deposition of glycogen granules, and infiltration with lipid droplets [5,7]. It is unknown whether the reported increased thickness of the heart valves and appearance of less dense connective tissue in the valves from copper deficient rats result in physiological disturbances, such as valvular regurgitation or cardiac murmurs.
The development of cardiomyopathies may also have a familial basis. It is therefore reasonable to hypothesize that interaction of a nutrient factor known to cause heart disease may synergestically enhance the onset and severity of genetically based cardiomyopathy.
This study used a rat strain that develops hypertensive, hypertrophic cardiomyopathy as it ages. McCune [8] obtained breeders produced from the seventh backcross of Koltesky rats [9] heterozygous for the corpulent gene (cp) with spontaneously hypertensive rats (SHR). This colony of animals is maintained at The Ohio State University and is designated as SHHF/Mcc-facp (spontaneously hypertensive hypertrophic failure). The mating of heterozygotes yields the following three genotypes: homozygous corpulent (facp/facp), heterozygous lean (facp/+), and homozygous lean (+/+). Animals from this colony exhibit severe hypertension and eventually develop heart failure [8,10]. The physical and biochemical alterations reported in the SHHF rat include ventricular hypertrophy, pulmonary edema, and elevated plasma levels of norepinephrine, aldosterone, renin, and atrial natriuretic peptide [8,10]. Electrocardiograph abnormalities in this strain of rat consist of notching of the QRS complex, prolonged PQ and QT intervals, increased R wave amplitude, and premature supraventricular beats [11]. Myocyte enlargement, increased interstitial fibrosis, and an increased deposition of glycogen granules have been reported in this strain [2,12]. Echocardiography findings include detection of eccentric ventricular hypertrophy, increased diastolic chamber size, and increased diastolic thickness of the posterior wall and interventricular septum [13].
The main objectives of the present study were: 1) to determine whether a genetic tendency to the development of heart failure is hastened or exacerbated by feeding a purified diet deficient in copper; 2) to determine if there was evidence of valvular regurgitation in hearts from copper deficient rats; and 3) to characterize the type of hypertrophy which occurs when the two models (copper deficiency and genetic) are present simultaneously.
|
METHODS
|
|---|
Animals and Diets
Ten male weanling LE rats were purchased from Harlan Sprague Dawley (Indianapolis, IN) and 10 male weanling homozygous lean SHHF rats were obtained from the laboratory of Dr. Sylvia McCune. Five rats from each strain were randomly assigned to a copper deficient diet, and the remaining five rats from each strain served as the control groups. Initial weights were recorded at the beginning of the study, and weekly thereafter. For a 6-week period rats consumed a basal diet which followed the recommendations of the American Institute of Nutrition [14], consisting of (g/g diet by weight) 0.50 sucrose, 0.20 casein, 0.15 cornstarch, and 0.05 corn oil as energy sources. The control groups (LE-Cu+, SHHF-Cu+) received copper in the form of cupric carbonate at 94.5 µmol Cu/kg diet. The experimental groups (LE-Cu-, SHHF-Cu-) received feed with no added copper. Diets formulated to these specifications, as verified by flame atomic absorption spectrophotometry (Varian, Varian SpectrAA-5, Victoria, Australia), were purchased from Research Diets, Inc. (New Brunswick, NJ). The Institutional Laboratory Animal Care and Use Committee at The Ohio State University approved the protocol for this study.
Rats were singly housed in stainless steel cages in a controlled environment with a 12-hour light: dark cycle at a constant room temperature. All animals had free access to deionized-distilled water and food throughout the study. At the end of week 6, electrocardiograms and echocardiograms were obtained from each rat. Soon thereafter the study was concluded and samples were taken for biochemical assessment.
Electrocardiograms
At week 6, rats were anesthetized using an intraperitoneal injection of ketamine (85 mg/kg) and xylazine (15 mg/kg). Electrocardiogram leads I, aVF, and V3 were recorded at 100 mm/sec paper speed on a photographic oscillograph with frequency response flat to over 1000 Hz. All leads were established by surface electrodes to minimize injury to the animal. Upon completion of the ECG recordings, animals were returned to their cages and allowed to fully recover from the procedure.
Voltages of the P, QRS, and T waves, along with the duration of the P wave, QRS complex, and P-Q and Q-T intervals, were measured from lead I by one individual blinded to treatment group. Mean values for each rat were obtained by averaging measurements from three consecutive cardiac cycles. Without prior knowledge of strain or dietary treatment, a veterinary cardiologist subjectively interpreted the records from all three electrocardiograph leads, noting any alterations in electrical activity suggestive of cardiac pathology.
Doppler Echocardiography Methods and Imaging
Within 3 days rats were again anesthetized using an intraperitoneal injection of ketamine (50 mg/kg) and xylazine (10 mg/kg). Prior to echocardiography, a veterinary cardiologist performed auscultation of the heart using a stethoscope with a pediatric diaphragm placed over the left and right sternal borders at the level of the palpable apex beat. After recording the presence or absence of a grade 2 or louder systolic murmur, two dimensional (2D) echocardiography and color and spectral Doppler echocardiography were performed on each animal.
A phased array, color flow, Doppler echocardiography system (Sonos 1000, Hewlett-Packard, Inc., Watham, MA) equipped with a 7.5 megahertz (MHz) pediatric transducer was used for all studies. The transducer functioned in a dual frequency mode (7.5 MHz imaging/5.0 MHz Doppler) during Doppler studies. The digital zoom mode of the instrument permitted focus on areas of interest while concurrent 2D echocardiography images guided the Doppler recordings at a sweep speed of 100 mm/sec. Simultaneous recording of a lead II electrocardiogram was used to time cardiac events.
After clipping the fur covering the right and left precordium, rats were positioned in left lateral recumbency. The application of echocardiography coupling gel maximized contact between the transducer and the animal. The transducer, placed at the left caudal sternal edge and rotated approximately 90 degrees from the sagittal plane, recorded the short axis image of the left ventricle at the dorsal level of the papillary muscle. Apical placement of the transducer with dorsocranial angulation yielded modified 4 and 5-chamber apical images which were used to record transvalvular flow across the atrioventricular valves and aortic valve. Placement of the transducer cranial to this position, in conjunction with clockwise rotation of the sector by approximately 20 degrees and steep dorsal angulation of the axial beam, yielded an image of the tricuspid and pulmonic valves. Color coded Doppler recorded at a rate of 16–18 frame/s provided evidence regarding the presence or absence of valvular regurgitation. Pulsed wave and/or color M-mode echocardiography verified the location and timing of the flow pattern relative to the ECG in rats suspected of valvular regurgitation on the basis of two-dimensional color-coded Doppler studies. All studies were recorded on 1/2 inch VHS videotape and on an optical disk for later analysis.
Short axis tomograms provided a means for obtaining left ventricular dimension, volume, and area following the guidelines of the American Society of Echocardiography Committee on Standards [15]. Minimal and maximal left ventricular internal and external short axis areas were measured at the tips of the papillary muscle during systole and diastole. Analysis excluded papillary muscle projections. The short axis left ventricular chamber diameters in systole and diastole were back-calculated from short axis cavity areas as:
Myocardial thickness in systole and diastole were then back-calculated from short axis epicardial and cavity areas as:
Cross-sectional areas occupied by the myocardium in systole and diastole were determined as:
A global index of left ventricular systolic function was calculated as:
Echocardiography measurements were made from stored digital images using the optical disk and software analysis packages of the echocardiograph. Mean values for the above parameters were obtained by averaging the results of three consecutive cardiac cycles for each rat. Units of measurement for linear and area measurements are reported in mm and mm2, respectively.
Echocardiography images were reviewed for Doppler evidence of valvular regurgitation by one individual without prior knowledge of strain, dietary treatment, or results of cardiac auscultation. Patterns of blood flow across the cardiac valves were evaluated in slow motion using stored digital images and slow motion review of the recorded videotape images. Color Doppler echocardiograms were considered positive for mitral regurgitation or tricuspid regurgitation when a transvalvular jet and retrograde color flow pattern were consistently noted. Brief, regurgitant signals recorded immediately adjacent to the center of the valve were considered negative for significant regurgitation. Retrograde flow was considered abnormal when a regurgitant jet extended 1/2 of the distance between the valve and posterior atrial wall and persisted for at least 1/2 of the duration of systole as measured by pulsed wave or by color M-mode studies. Aortic regurgitation was diagnosed when a holodiastolic regurgitant jet was recorded in the left ventricular outflow tract by pulsed-wave Doppler.
Organ and Blood Sampling
Within 3 days upon completion of the functional cardiac tests, rats were anesthetized by CO2 inhalation. The thoracic cavities were opened by midline incision and a small sample of blood was obtained by cardiac puncture and placed in a heparinized tube for hematocrit determination and hearts were removed, rinsed in deionized distilled water, blotted dry and weighed. Livers were removed from all rats, rinsed in deionized distilled water, blotted dry, and frozen for subsequent determination of liver copper levels and liver Cu-Zn SOD activity.
Hematocrit
Heparinized blood was transferred to microhematocrit tubes and centrifuged in a microcapillary centrifuge for 2 minutes. Hematocrit was determined as the percentage of space occupied by packed red blood cells.
Superoxide Dismutase Activity and Liver Copper Determination
Liver cytoplasm Cu-Zn SOD, (EC 1.15.1.1) activity was determined spectrophotometrically to assess the relative copper status of rats in different treatment groups. This technique is based on the autoxidation of pyrogallol, as described by Marklund and Marklund [16] and modified by Prohaska [17]. Briefly, the quantity of pyrogallol required to yield a change in absorbance of 0.016/30 s was determined, after which the amount of liver sample necessary to reduce this change in absorbance to 0.008/30 s was determined. One unit of SOD was defined as the amount of activity which inhibits the autoxidation of pyrogallol by 50%, expressed as U/g wet weight of tissue.
To determine the µmol concentration of copper per g wet weight of liver tissue, 0.5 g samples were added to 10 ml 15.4 mol/L nitric acid and digested in a microwave oven (MDS-81D, CEM Corp., Matthews, NC) in three stages. The oven was programmed to run a 4 minute cycle at 65% power followed by a 2 minute cycle at 40% power. After cooling and venting of the vessels, a final 13 minute cycle at 40% power was run. Evaporation of approximately 9 ml nitric acid and reconstitution to the original volume with deionized-distilled water permitted sample analysis by flame atomic absorption spectrophotometry.
Statistical Analysis
Data were analyzed using the Statistical Analysis System [18]. For normally distributed data, the General Linear Models (GLM) procedure was used to determine significant differences between strain, dietary treatment, and the interaction of strain and dietary treatment. When significant F values were obtained, Fisher’s post-test procedure was used to determine which of the group means differed from one another. When data were not normally distributed, Kruskal-Wallis’ nonparametric test was used. Contingency tables were constructed to identify the frequencies of valvular regurgitation or the presence of a cardiac murmur in the different groups. Fisher’s exact test was used to identify significant differences from expected frequencies. The alpha level was set a priori at 0.05.
|
RESULTS
|
|---|
All rats survived through the electrocardiograph portion of the study. However, loss of one LE-Cu+ rat from anesthesia prior to echocardiography resulted in a decrease in sample size for the echocardiographic portion of the study. Adequate echocardiography images could not be obtained on one LE-Cu+ and one SHHF-Cu- rat, thus reducing these sample sizes to three and four, respectively.
Biochemical and Physical Measures
Independent of the effects of copper deficiency, SHHF rats as a group had significantly lower body weights (p
0.01) and higher heart to body weight ratios (p0.005) than LE rats (Table 1). Though not statistically significant, the rate of weight gain tended to decrease by 18% in the SHHF-Cu- group when compared to the SHHF-Cu+ group, and by 36% when compared to the LE-Cu+ group. A significant increase in heart weight (p<0.01) and the ratio of heart weight to body weight (p<0.01) in both copper deficient groups confirmed the presence of hypertrophy which was ventricular. The SHHF-Cu- group had significantly lower hematocrits than any of the other groups (p<0.005). After 6 weeks of dietary treatment, rats assigned to the Cu- groups possessed lower liver copper levels (p<0.01) and lower liver SOD activity (p<0.0005) than Cu+ rats.
Electrocardiograms
Electrocardiograms from rats fed a copper deficient diet showed significantly greater QRS complex amplitudes (p<0.005), and longer Q-T intervals (p<0.05) when compared with control groups (Table 2). Within the copper deficient groups, the SHHF rats showed a 25% increase in QRS complex amplitude when compared to LE rats. SHHF rats demonstrated greater QRS complex durations than LE rats with the deficient rats showing a 38% increase over controls (p<0.05). Although Cu- rats tended to display greater T wave amplitudes, these differences were not significant. No group showed significant differences in either the amplitude or duration of the P wave, or the duration of the P-Q segment.
Visual inspection of the traces confirmed quantitative analysis and provided additional information regarding the electrocardiograph aberrations associated with copper deficiency in the rat as well as abnormalities characteristic of the SHHF rat (Fig. 1). The most striking difference between groups is the increased amplitude of the QRS complex in the LE-Cu- and SHHF groups. Notching of the QRS complex was absent in all the LE-Cu+ rats, but evident in 80% of the recordings obtained from Cu- rats, and 60% of SHHF-Cu+ rats. The electrocardiogram obtained from one LE-Cu- rat showed a slurring of the baseline and primary S-T segment changes in all leads. Primary S-T segment changes also occurred in one of the SHHF control rats. All groups except the LE-Cu+ group showed inversion of either the P wave, the T wave, or the QRS complex to some degree. This finding was more common in the SHHF rats than in the LE-Cu- rats. While the sample sizes were limited here, we have reported similar results in separate studies for each of these rat strains fed copper deficient diets [2,3].
View larger version (21K):
[in this window]
[in a new window]
|
Fig. 1. Representative ECG tracings from LE-Cu+ (a), LE-Cu- (b), SHHF-Cu+ (c), and SHHF-Cu- (d) rats. Primary S-T segment changes occurred in one LE-Cu- animal (b) and one SHHF-Cu+ animal (not pictured). The LE-Cu- group (b) and the SHHF groups (c and d) showed a higher incidence of notching of the QRS complex (c, *) when compared to the LE-Cu+ group. The SHHF-Cu- (d) group had a significant increase in the duration of the QRS complex when compared to other groups. Vertical bars represent 1 mV; horizontal bars represent 100 ms.
|
|
Echocardiograms
Analysis of short axis 2D echocardiography measurements showed a non-normal distribution for left ventricular external areas in systole and diastole, and systolic left ventricular cross sectional area. Using the Kruskal-Wallis test, the left ventricular external area was significantly different between groups (p<0.05, p<0.01, Fig. 2a and 2b) with the SHHF-Cu- group showing a 10% increase in systolic dimension over the LE-Cu- rats. Similarly, systolic and diastolic myocardial cross-sectional areas (p<0.05, p<0.001) were significantly increased in Cu- rats when compared to controls (Table 3), with the SHHF-Cu- group showing a 10% increase in systolic dimension over the LE-Cu- rats. Calculations of myocardial thickness in both systole and diastole increased in copper deficiency (p<0.005, p<0.001). No group showed significant differences in left ventricular chamber area, diameter, or percent fractional shortening.
Except for two trivial cases of valvular regurgitation, subjective analysis of pulsed wave and color coded Doppler studies yielded unremarkable results in the LE-Cu+ and SHHF-Cu+ rats. However, two of the LE-Cu- rats showed evidence of mitral regurgitation. In addition, one of these animals displayed tricuspid and aortic regurgitation (Fig. 3a). Three animals belonging to the SHHF-Cu- group demonstrated tricuspid regurgitation as evidenced by pulsed wave Doppler (Fig. 3b) and color coded Doppler (Fig. 4a). Color coded Doppler also verified the presence of mitral regurgitation in the SHHF-Cu- rats (Fig. 4b).
View larger version (117K):
[in this window]
[in a new window]
|
Fig. 3. Doppler echocardiograms demonstrating valvular regurgitation. Recording from a SHHF-Cu- rat showing a black and white representation of a color Doppler mapped tricuspid regurgitant jet (*). The pulse-wave Doppler tracing (below) is characterized by a broad, holosystolic, turbulent flow pattern (a). Aortic regurgitation recorded from the left ventricular outflow tract of a LE-Cu- rat (b). The disturbed, holodiastolic signal (*) was recorded using pulsed-wave DE from a large angle which underestimates true maximal velocity. The triangular, normal systolic flow signal is also recorded. This rat also demonstrated mitral regurgitation (not pictured).
|
|
View larger version (77K):
[in this window]
[in a new window]
|
Fig. 4. Black and white representation of color coded Doppler echocardiograms demonstrating valvular regurgitation. A wide tricuspid regurgitant jet recorded from a parasternal image of the right ventricle and pulmonary artery of a SHHF-Cu- animal (a). Mitral regurgitant jet of narrow origin and wide distribution (dotted lines) recorded in the left atrium of a SHHF-Cu- animal from a parasternal long-axis image of the left ventricle (b). The aorta demonstrated a normal flow pattern. TR=tricuspid regurgitant jet; TV=tricuspid valve; MR=mitral regurgitant jet; RV=right ventricle; RA=right atrium; LV=left ventricle; LA=left atrium; AO=aorta; PA=pulmonary artery.
|
|
No significant differences were found in the frequencies of valvular regurgitation between strain. However, there was a trend for Cu- rats to show abnormal Doppler recordings when compared to Cu+ rats (p=0.06).
Eight of the 10 Cu- rats showed evidence of a grade 2 or louder systolic murmur upon cardiac auscultation, whereas none of the control rats showed no evidence of systolic murmurs (p<0.001). There was also a significant relationship between the auscultation of systolic cardiac murmurs and Doppler identification of mitral or tricuspid valvular regurgitation (p<0.005) by Fischer’s Exact test.
|
DISCUSSION
|
|---|
Results of the current study agree with data published by others that a diet deficient in copper induces physical [1], biochemical [5,19], electrocardiograph [3,6], and hematological [20] abnormalities in the rat.
Physical Measures of Copper-Deficiency
Both LE and SHHF rats showed a decrease in the rate of weight gain in states of copper deficiency [2]. In the present study, the SHHF rats showed this deceleration, regardless of dietary treatment. The significant increase in heart weight relative to body weight observed between strain and dietary treatment is one measure suggesting the presence of cardiac hypertrophy. The difference by strain suggests that the hearts of the SHHF rats began to increase at this early age due to their genetic predisposition, whereas the difference found by dietary treatment indicates hypertrophy resulting from copper deficiency. However, the hypertrophy observed in the SHHF-Cu- rats was neither purely eccentric or concentric, but rather a mixed hypertrophic pattern similar to that reported by Medeiros et al [2]. Also, previous studies [1,5] have shown that the hypertrophic pattern observed in the copper deficient rat precedes the development of anemia, suggesting that the hypertrophy is independent of the anemia. In the present study, only the SHHF-Cu- rats showed significantly depressed hematocrit levels despite the presence of ventricular hypertrophy in both Cu- groups and the SHHF control group. Anemia can lead to a volume overload which results in an eccentric cardiac hypertrophy [20,21]. Though the added effect of moderate anemia cannot be conclusively eliminated, our results support the hypothesis that cardiac hypertrophy may occur in the absence of anemia in copper deficiency. This finding may be due in part to the increased protein synthesis, myocyte enlargement, and mitochondria proliferation associated with a reduction in dietary copper [22,23].
Electrocardiograms
Electrocardiograph abnormalities associated with copper deficiency include S-T segment changes, increased P-R intervals, increased R wave duration and amplitude [6,24], a prolongation of the Q-T interval [3], and His-bundle electrogram aberrations [25]. Pocchiari et al [11] found significant increases in notching of the QRS complex, duration of P waves, P-Q intervals, QRS complexes, and Q-T intervals in SHHF rats when compared to controls. Medeiros et al [2] reported a greater QRS amplitude in SHHF rats fed a copper deficient diet when compared to rats fed a diet adequate in all minerals. This study provides additional verification that rats exhibit an increased incidence of notching and a greater amplitude of the QRS complex after consuming a diet deficient in copper. The prolonged Q-T interval observed in Cu- rats could be due to an impedance in the electrical conductance within the His-Purkinje system. The primary S-T segment changes found in one LE-Cu- rat and one SHHF-Cu+ rat suggests disruption of flow across ion sensitive channels within the heart.
The changes in the electrocardiograms in copper deficiency may be due to altered Na/K-ATPase isoforms. Huang et al [26] reported that in copper deficient rats, the mRNAs of 
1 and 2 isoforms and the 2 protein were significantly reduced. These are the catalytic subunits of the enzyme complex that maintain the Na and K electrical gradient and are more prevalent in the conducting system of the heart. The lower subunits may lead to the observed arrhythmias reported here.
Echocardiograms
By normalizing echocardiography variables to body weight, Wildman et al [27] reported a significant increase in left ventricular diastolic dimension as well as thickness of the diastolic posterior wall and interventricular septum of pigs fed a copper deficient diet from weaning. In another study, echocardiogram findings in the SHHF rat included an increased diastolic dimension of the posterior wall, interventricular septum, and left ventricular chamber diameter [13]. Using these measurements, these authors reported a significant increase in the calculated left ventricular mass in SHHF rats when compared to controls. The aforementioned authors used M-mode images to obtain measurements. Although this was not done in the present study, differences in echocardiography measurements were found between dietary groups using short axis 2D images. Significant increases in left ventricular circumference in both systole and diastole indicate an increase in left ventricular mass among Cu- groups. Detection of myocardial thickening and increases in cross-sectional areas in Cu- rats suggest that echocardiography techniques offer predictive value in diagnosing ventricular hypertrophy in the rat.
Valvular Disturbances
Using light microscopy, Medeiros et al [1] reported fragmented heart valves with decreased connective tissue in rats fed a diet deficient in copper from weaning. This may explain the increased incidence of systolic cardiac murmurs in copper deficient rats. These data revealed a significant relationship between the detection of cardiac murmurs by auscultation and Doppler evidence of mitral or tricuspid regurgitation. Cardiac auscultation may have predictive value in identifying valvular insufficiency in rats. Although the small size of the rat thorax makes precise localization of cardiac murmurs somewhat difficult, the detection of moderate to loud systolic murmurs was not difficult in the sedated rat. Mitral or tricuspid regurgitation can be associated with altered ventricular geometry. This could be occurring here since the incidence of aortic valve regurgitation was much less than that observed for the tricuspid and bicuspid valves.
Another possible mechanism for the valvular pathology may be the result of decreased crosslinking of elastin and collagen via decreased activity of the cupro-enzyme lysyl oxidase. This enzyme facilitates deamination of the epsilon amino group of lysine side chains to produce a reactive aldehyde that may condense to form several types of crosslinks to enhance physical structure [28]. Some of the pathology observed in copper deficiency is believed due to the decreased crosslinking, which could explain the greater incidence of aneurysm and hemothorax in copper deficient animals [29,30]. Valves are composed both of collagen and elastin; most of elastin is expressed in gestation with crosslinking occurring for the first few weeks of life in the rat. However, the turnover period for elastin is slow and approximates the life span of the animal [25,31]. If copper deficiency occurs at a time when elastin crosslinking normally occurs, the result is not likely to be reversible upon copper repletion after the window of opportunity is lost. This is significant in terms of human development and copper requirements in that a diet deficient in copper in a newborn could lead to valvular problems and cardiac murmurs and be irreversible in a practical sense. However, in copper-deficient pigs, while bicuspid and tricuspid valves demonstrated clear pathology in the hypertrophied hearts, stress-strain studies on the valves did not demonstrate any difference between copper-deficient and controls [32]. This suggested that while there was a decrease in valve crosslinks, the strength of the valve itself was unchanged. However, papillary muscles also control the valve function, which was not measured in that study. Shields [33] reported papillary muscle damage in copper deficient pigs and also provided the first evidence that the cardiac enlargement in copper deficiency was greater than the degree of anemia. Thus, while the potential decrease in collagen and elastin crosslinking leading to altered valve function cannot be ruled out, the change in ventricular geometry of the heart may be a more likely candidate for the cause of the murmurs based on available data.
|
CONCLUSIONS
|
|---|
The low in copper content of many human diets [34] served as one of the basis for conducting this study. Based on the objectives stated for this study it may be concluded that: 1) it does not appear with the data from this study that a copper-deficient data hastened or worsened the cardiac pathology or function in a strain of rats genetically susceptible to heart disease later in life; 2) this study documents the presence of systolic cardiac murmurs and valvular regurgitation in two strains of rats fed a copper deficient diet for 6 weeks from weaning; and 3) while there was a tendency for the SHHF strain to be more sensitive to the effects of copper deficiency than the LE strain, the presence of anemia may be a confounding factor. Overall, copper deficiency exerted distinct pathology and functional disturbances in both strains.
|
ACKNOWLEDGMENTS
|
|---|
Funding for this project was provided by The United States Department of Agriculture; Grant Number 93-37200-8757.
Received February 1, 1998.
Accepted June 1, 1998.
|
REFERENCES
|
|---|
- Medeiros DM, Bagby D, Ovecka G, McCormick R: Myofibrillar, mitochondrial, and valvular morphological alterations in cardiac hypertrophy among copper-deficient rats.
J Nutr
121:
815–824,
1991.
- Medeiros DM, Liao Z, Hamlin RL: Copper deficiency in a genetically hypertensive cardiomyopathic rat: electrocardiogram, functional and ultrastructural aspects.
J Nutr
121:
1026–1034,
1991.
- Medeiros DM, Liao Z, Hamlin RL: Electrocardiographic activity and cardiac function in copper-restricted rats.
Proc Soc Exp Biol Med
200:
78–84,
1992.[Abstract]
- Medeiros DM, Shiry L, Lincoln AJ, Prochaska L: Cardiac non-myofibrillar proteins in copper-deficient rats: amino acid sequencing and Western blotting of altered proteins.
Biol Trace Elem Res
36:
271–282,
1993.
- Davidson JA, Medeiros DM, Hamlin RL, Jenkins JE: Submaximal, aerobic exercise training exacerbates the cardiomyopathy of postweanling Cu-depleted rats.
Biol Trace Elem Res
38:
251–272,
1993.[Medline]
- Viestenz KE, Klevay LM: A randomized trial of copper therapy in rats with electrocardiographic abnormalities due to copper deficiency.
Am J Clin Nutr
35:
258–266,
1982.[Abstract/Free Full Text]
- Wildman REC, Medeiros DM, McCoy E: Cardiac changes with dietary copper, iron, or selenium restriction: organelle and basal laminae abberations, decreased ventricular function, and altered gross morphometry.
J Tr Elem Exp Med
8:
11–27,
1995.
- McCune SA, Baker PB, Stills HF: SHR/Mcc-cp rat. Model of obesity, non-insulin dependent diabetes and congestive heart failure. ILAR News
32:
22–27,
1990.
- Koletsky S: Pathologic findings and laboratory data in a new strain of obese hypertensive rats.
Am J Pathol
80:
129–142,
1995.[Abstract]
- McCune SA, Jenkins JE, Stills HF, Park S, Radin MJ, Jurin RR, Hamlin RL:
"Renal and heart function in the SHHF/Mcc-cp Rat. Frontiers in Diabetes Research. Lessons from Animal Diabetes III." London: Smith-Gordon, pp
397–401,
1990.
- Pocchiari RJ, Hamlin RL, McCune SA: Electrocardiographic findings in rats with cardiomyopathy.
Am J Vet Res
54:
607–611,
1993.[Medline]
- Rubin Z, Miller JE, Rohrbacher E, Walsh GM: A potential model for a human disease: spontaneous cardiomyopathy-congestive heart failure in SHR/N-cp rats.
Hum Pathol
15:
901–902,
1984.
- Haas GJ, McCune SA, Brown DM, Cody RJ: Echocardiographic characterization of left ventricular adaptation in a genetically determined heart failure rat model.
Am Heart J
130:
806–811,
1995.[Medline]
- American Institute of Nutrition: Report of the AIN Ad Hoc Committee on standards for nutritional studies.
J Nutr
107:
1340–1348,
1997.
- Schiller NB, Shah PM, Crawford M, DeMaria A, Devereaux R, Feigenbaum H, Gutgesell H, Reichek N, Sahn N, Schnittger I, Silverman NH, Tajik AJ: Recommendations for the quantitation of the left ventricle by two-dimensional echocardiography. American Society of Echocardiography Committee on Standards, Subcommittee on Quantitation of Two-Dimensional Echocardiograms.
J Am Soc Echocardiograph
2:
358–367,
1989.
- Marklund S, Marklund G: Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase.
Eur J Biochem
47:
469–474,
1974.[Medline]
- Prohaska J: Changes in tissue growth, concentrations of copper, iron, cytochrome oxidase and superoxide dismutase subsequent to dietary or genetic copper deficiency in mice.
J Nutr
113:
2048–2058,
1983.
- Statistical Analysis System. Cary, NC: SAS Institute Inc., 1988.
- Paynter DI, Moir RJ, Underwood EJ: Changes in activity of the Cu-Zn superoxide dismutase enzyme in tissues of the rat with changes in dietary copper.
J Nutr
109:
1570–1576,
1979.
- Wan Q, Yang BS, Kato N: Feeding of excessive cystine and cysteine enhances defects of dietary copper deficiency in rats by differential mechanisms involving altered iron status.
J Nutr Sci Vitaminol
42:
185–193,
1996.
- Datta BN, Silver MD: Cardiomegaly in chronic anemia in rats: an experimental study including ultrastructural, histometric, and stereologic observations.
Lab Invest
32(4):
503–514,
1975.[Medline]
- Medeiros DM, Shiry L, Samelman T: Cardiac nuclear encoded cytochrome c oxidase subunits are decreased with copper restriction but not from iron restriction: gene expression, protein synthesis and heat shock protein aspects.
Comp Biochem Physiol
117A:
77–87,
1997.
- Davidson J, Medeiros DM, Hamlin RL: Cardiac ultrastructural and electrophysiological abnormalities in postweanling copper-restricted and copper-repleted rats in the absence of hypertrophy.
J Nutr
122:
1566–1575,
1991.
- Kopp SJ, Klevay LM, Feliksik JM: Physiological and metabolic characterization of a cardiomyopathy induced by chronic copper deficiency.
Am J Physiol
245:
H855–H866,
1983.
- McCune SA, Jurin RR, Hansen T, Dunn RB, Chedid A, Fox E, Summers B, Wey TT: SHR:
N-Mcc-cp rat substrain: general characteristics and congestive heart failure syndrome. New Models of Genetically Obese Rats for Studies in Diabetes, Heart Disease and Complications of Obesity. Bethesda, MD: Veterinary Resources Branch, National Institutes of Health, pp
95–103,
1988.
- Huang W, Chih-Chia L, Wang Y, Askari A, Klevay LM, Askari A, Chiu TH: Altered expression of cardiac Na/K ATPase isoforms in copper deficient rats.
Cardiovasc Res
29:
563–568,
1995.[Medline]
- Wildman REC, Medeiros DM, Hamlin RL, Stills H, Jones DA, Bonagura JD: Aspects of cardiomyopathy in copper-deficient pigs.
Biol Trace Elem Res
55:
55–70,
1996.[Medline]
- Mecham RP, Heuse JE: The elastic fiber. In
"Cell Biology of Extracellular Matrix," 2nd ed. New York: Plenum Press, pp
79–109,
1991.
- Bird DW, Savage JE, O’Dell BL: Effect of copper deficiency and inhibitors on the amine oxidase activity of chick tissues.
Proc Soc Exp Biol Med
123:
250–254,
1966.[Medline]
- Borg TK, Klevay LM, Gay RE, Siegel R, Bergin ME: Alteration of the connective tissue network of striated muscle in copper deficient rats.
J Mol Cell Cardiol
17:
1173–1183,
1985.[Medline]
- Lefevre M, Rucker RB: Aorta elastin turnover in normal and hypercholesterolemic Japanese quail.
Biochim Biophys Acta
630(4):
519–529,
1980.[Medline]
- Vadlamudi RK, McCormick RJ, Medeiros DM, Vossoughi J, Failla ML: Copper deficiency alters collagen types and covalent cross-linking in swine myocardium and cardiac valves.
Am J Physiol
264(33):
H2154–H2161,
1993.[Abstract/Free Full Text]
- Shields GS, Coulson WF, Kimball DA, Carnes WH, Cartwright GE, Wintrobe MM: Studies on copper metabolism. XXXII. Cardiovascular lesions in copper-deficient swine.
Am J Pathol
41:
603–621,
1962.
- Klevay LM, Medeiros DM: Deliberations and evaluations of the approaches, endpoints and paradigms for dietary recommendations about copper.
J Nutr
126:
2419S–2426S,
1996.
TOP
ABSTRACT
INTRODUCTION
(left ventricular internal area/
). Myocardial thickness in diastole and systole (MTD, MTS) were calculated from left ventricular internal and external areas as: Myocardial thickness=