Erythrocyte calcium influx is related to severity of ventricular arrhythmias in uraemic patients

Laura Soldati, Donatella Adamo, Paolo Manunta, Paola Stella, Daniele Ciurlino, Donatella Spotti, Giorgio Slaviero, Marco Melandri, Daniele Cusi, Giuseppe Bianchi and Giuseppe Vezzoli

Division of Nephrology, Dialysis and Hypertension, San Raffaele Scientific Institute, University of Milan, Milan, Italy



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 References
 
Background. Myocardial disorders are a remarkable cause of morbidity and mortality in chronic haemodialysed patients (HD). They could be favoured by alteration of cell Ca2+ handling. In previous studies we characterized an erythrocyte Ca2+ influx, sensitive to membrane potential and inhibited by Ca2+ antagonists. Since its maximal influx rate was decreased in HD patients, this study investigates if Ca2+ influx alterations are related to myocardial disorders in HD patients.

Methods. Voltage-sensitive erythrocyte Ca2+ influx was measured in 30 healthy controls and in 53 patients (47 HD patients and six patients with left ventricular hypertrophy and normal kidney function), using fura 2. In 29 HD patients and in six healthy subjects Ca2+ influx was also determined in the presence of parathyroid hormone (PTH) in vitro. Patients were classified according to Lown's ventricular arrhythmias classification after 24-h Holter electrocardiograph (ECG) monitoring. Forty-six patients underwent echocardiography.

Results. Voltage-sensitive erythrocyte Ca2+ influx was significantly reduced in HD patients. Maximal influx rate was significantly higher in HD patients of Lown's classes 3 and 4 (0.789±0.156 nmol/s, n=8; P<0.01) than in patients of classes 1 and 2 (0.499±0.055 nmol/s, n=15), or without ventricular arrhythmias (0.400±0.041 nmol/s, n=24). Maximal influx rate was directly correlated to left ventricular mass index (LVM) (r=0.353, P<0.05). Subjects with left ventricular hypertrophy and normal kidney function displayed erythrocyte Ca2+ influx similar to that of normal subjects. Multiple regression indicates that LVM and Ca2+ influx were independently related to severity of arrhythmias. When added to the influx assay, PTH increased the maximal influx rate only in patients with ventricular arrhythmias.

Conclusion. Myocardial dysfunction and altered ventricular excitability could be related in uraemic HD patients to alterations of calcium transport, as found in the erythrocyte model. Reduced resistance to PTH could contribute to this phenomenon.

Keywords: arrhythmias; Ca2+ influx; erythrocytes; left ventricular hypertrophy; parathyroid hormone; uraemia



   Introduction
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 Abstract
 Introduction
 Subjects and methods
 References
 
Ventricular arrhythmias are an important cause of morbidity and mortality in haemodialysed patients [1]. The risk of ventricular arrhythmias is increased in myocardial hypertrophy, ischaemic heart disease, and heart failure. It can also be related to electrolyte disorders such as intracellular Ca2+ accumulation and tissue deposition of Ca2+ salts that may favour myocardial impairment [2]. A deep derangement of cellular Ca2+ homeostasis has been observed in myocardiocytes of rats with chronic renal failure. Intracellular Ca2+ content and Ca2+ uptake were found to be increased, while sarcolemmal Ca2+ pump activity was found to be reduced, in relation to the state of hyperparathyroidism [3]. Therefore, it has been hypothesized that parathyroid hormone (PTH) can act as a uraemic toxin altering Ca2+ influx, thus contributing to intracellular Ca2+ accumulation in human myocardiocytes where Ca2+ content has been found to be abnormally high [4]. Through these alterations, PTH could negatively influence cardiac function, since Ca2+ transport is involved in myocardiocyte membrane depolarization and contraction. In addition to PTH, other uraemic toxins have been hypothesized to inhibit the capacity of plasma membrane Ca2+ pump to extrude Ca2+, as observed in human erythrocytes [5].

We recently characterized a Ca2+ influx in human erythrocytes. It displayed a sigmoidal kinetic, was modulated by membrane potential, and was inhibited by dihydropyridine Ca2+ antagonists [6]. The kinetic of this erythrocyte transport was altered in HD patients. Compared to healthy donors' erythrocytes, maximal influx rate was reduced and influx plateau was reached at lower intracellular free Ca2+ concentrations ([Ca2+]i). On the contrary, resting [Ca2+]i was higher in erythrocytes from HD patients. Circulating PTH levels were directly correlated with maximal Ca2+ influx rates [7]. In keeping with these findings, we suggested that voltage sensitive influx could be reduced as a reaction to intracellular Ca2+ accumulation.

Although erythrocytes are not excitable cells, voltage-sensitive erythrocyte influx shares some functional characteristics with myocardial L-type Ca2+ channels, like modulation by membrane potential and dihydropyridine inhibition. Therefore, we searched for alterations in voltage-sensitive Ca2+ influx and Ca2+ content in uraemic erythrocytes from patients with different degree of myocardial dysfunction and ventricular arrhythmias. The effect of PTH on Ca2+ influx was also evaluated.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 References
 
Subjects
Forty-seven uraemic patients (29 men/18 women, 60±2.0 years), under haemodialysis maintenance therapy, were enrolled in the study. The duration of dialysis therapy was 31±4.5 months. Table 1Go provides HD patient clinical data and biochemical parameters. Causes of chronic renal failure were: unknown (n=14), type 1 diabetes (n=10), chronic glomerulonephritis (n=9), type 2 diabetes (n=7), polycystic kidney disease (n=3), chronic pyelonephritis (n=2), angiosclerotic nephropathy and amyloidosis (n=2). Thirty-one patients (66%) were treated with antihypertensive drugs, alone or in combination (Ca2+ channel blockers, angiotensin-converting enzyme inhibitors, beta-blocker agents, clonidine). Other drugs used were erythropoietin (31 cases), 1,25(OH)2vitaminD3 (six cases), digoxin (five cases). Patients were dialysed with cuprophane, polymethylmethacrylate, or polyacrylonitrile membranes, three times per week (3–4 h sessions). Electrolyte dialysate composition was (in mmol/l) Na (140), K (3 or 2), Mg (0.5), Ca (1.5), HCO3 (34), Cl (109), acetate (3) and glucose (5.55). Blood was collected from each patient after overnight fast immediately before the first dialysis in a week. No patient showed signs of congestive heart failure or recent coronary attack.


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Table 1. Clinical data and blood biochemical parameters of HD patients

 
A control group, matched for age and sex with uraemic patients, was also studied: 30 healthy subjects (18 men/12 women, 55±1.9 years). In addition, six patients (4 men/2 women, 52±2.4 years) with left ventricular hypertrophy without arrhythmias and with normal kidney function were studied.

Subjects gave informed consent. The study was approved by the San Raffaele Hospital Ethics Committee.

Holter monitoring
Holter ECG was carried out for 24 h, including a dialysis session, by a micro recorder (AMP AM5600, SpaceLabs Medical, Redmond, Wa, USA). Records were analysed by the same experienced investigator who valued them for both the presence and frequency of complex ventricular arrhythmias and the occurrence of ST segment deviation. Patients suffering from ventricular arrhythmias were grouped according to Lown's and Wolf's classification: 0=no ventricular ectopic beats; 1=occasional, isolated ventricular premature beats (VPB); 2=frequent VPB; 3=multiform VPB; 4=repetitive VPB; 5=early VPB [8]. Those without arrhythmias were compared to patients of different Lown's classes.

Echocardiographic examinations
M-mode and two-dimensional echocardiography were carried out on 40 patients (SONOS 2500, Hewlett Packard). Measurements of left ventricular (LV) dimensions were done according to American Society of Echocardiography guidelines [9]. Left ventricular mass (LVM) was calculated at the end-diastole with the formula validated by Devereux and Reichek [10]. LV mass index was calculated by the ratio of LVM to body surface area. LV hypertrophy was defined as LVM index greater than 131 g/m2 in men and 108 g/m2 in women [11].

Biochemical analyses
Biochemical parameters (Ca, P, Na, K, HCO3, albumin, and cholesterol) were determined by autoanalyser (Hitachi 747; Hitachi, Tokyo, Japan). Haematocrit and haemoglobin were measured by STKS (Coulter Electronics, Hialeah, FL, USA). Serum intact PTH was determined by immunoradiometric assay (Nichols Institute Diagnostics, San Juan Capistrano, CA, USA). Kt/V (therapy adequacy index) and PCR (protein catabolic rate) were calculated according to the method previously described [12]; K is the dialyser urea clearance, t is the duration of dialysis, V is the body urea distribution volume (total body water) assessed by bioelectrical impedance [13].

Erythrocyte Ca2+ metabolism Preparation of erythrocytes
Blood (2 ml) was collected in tubes containing 1/10 volume of anticoagulant (2.73% citric acid, 4.48% trisodium citrate, 2% glucose). Cells were prepared immediately after blood sampling and maintained at room temperature. Blood was centrifuged and plasma and buffy-coat were discarded.

Intracellular free Ca2+ concentration in fresh erythrocytes
The measurement was carried out with the Ca2+-sensitive fluorescent dye fura 2 as previously described [6,7]. Erythrocytes were diluted to 1% haematocrit with a HEPES-buffered saline (HBS), pH 7.4 at 37°C. Erythrocytes were loaded with 1 µmol/l fura 2 AM by incubation for 45 min under shaking at 37°C. Erythrocytes were washed with HBS to eliminate extracellular fura 2 AM. Packed erythrocytes were suspended in HBS at 0.1% haematocrit and were transferred into a quartz cuvette for the fluorescence measurements with a luminescence spectrometer (Perkin-Elmer LS50; Perkin-Elmer, Norwalk, CT, USA). All experiments were carried out at 37°C using a magnetic stirrer. Data were analysed by the Intracellular Biochemistry application software package (ICBC) that allows a fluorescence measurement every 1.9 s. [Ca2+]i was determined using the two-wavelength method described by Grynkiewicz et al. [14]. [Ca2+]i was calculated according to the equation given by Poenie et al. [15]. Calibration parameters were determined by the in vitro calibration method described by David-Dufilho et al. [16]. The intra- and inter-assay variation coefficients of resting [Ca2+]i calculated in erythrocytes from a same donor were 2.7% (n=6) and 3.1% (n=7) respectively.

Erythrocyte voltage-sensitive Ca2+ influx
Ca2+ influx was measured as previously described [6,7]. Erythrocytes (1% haematocrit) were depleted of ATP by incubation for 1 h at 37°C under shaking in a medium containing (in mmol/l): NaCl (140), KCl (5), iodoacetate (1), inosine (10), pH 7.4 (medium A). 1 µmol/l fura 2 AM was added to the medium (45 min of loading) after 15 min from the beginning of the ATP-depletion procedure. Erythrocytes were washed with the medium A to eliminate extracellular fura 2 AM, diluted to 0.1% haematocrit in medium A plus 2 mmol/l EGTA-Tris, pH 7.2 and transferred into a quartz cuvette for the fluorescence measurements at 37°C under magnetic stirring. After the stabilization of temperature and fluorescence signal, 20 mmol/l CaCl2 were added. The Ca2+-EGTA binding in the unbuffered medium causes a membrane depolarization, due to a decrease of external pH (internal pH remains unchanged) [6]. In this assay, the membrane potential changed from -12 mV to 0 mV at the beginning of Ca2+ influx [6,7]. [Ca2+]i was calculated for every point of fluorescence measurement (1 every 1.9 s) as described in the previous paragraph. The Ca2+ influx time courses that display a sigmoidal shape were analysed by a logistic function [7]. The curve slope between the times when Ca2+ influx increases and decreases most rapidly (maximal influx rate) and the maximal Ca2+ concentration reached (plateau) were taken as indices of Ca2+ influx. The interassay variation coefficient of the maximal influx rate calculated in erythrocytes from a same donor was 5.6% (n=6).

Erythrocyte voltage-dependent Ca2+ influx in the presence of PTH
The measurement of Ca2+ influx was carried out as described in the previous paragraph, except for the presence of 50 nmol/l human intact (1–84) PTH. The hormone was added in the assay cuvette and erythrocytes were incubated at 37°C for 10 min before Ca2+ influx beginning. In preliminary experiments, the effects of various human intact PTH concentrations (10, 25, 50, 100 nmol/l) on Ca2+ influx were evaluated. The lowest concentration giving maximal effects (50 nmol/l) was chosen for following experiments.

Statistical analyses
Values are expressed as mean±SEM. Results in patient groups were compared by one-way ANOVA with Fisher post-hoc test for multiple comparisons between groups. Simple linear correlations were computed. Measurements of erythrocyte Ca2+ influx carried out with or without PTH were compared by Wilcoxon signed rank test. Multiple regression was used in HD patients to study relationships of arrhythmias classes (dependent variable) with erythrocyte maximal Ca2+ influx rate, LVM index, PTH, arterial blood pressure, Kt/V, erythrocyte [Ca2+]i, body weight, blood haemoglobin (independent variables).

Results
Ca2+ influx kinetics were different in uraemic and control erythrocytes. Uraemic erythrocytes showed a significant reduction of maximal Ca2+ influx rate (uraemics, 0.498±0.042 nmol/s; healthy controls, 1.037±0.141 nmol/s; P<0.001), and curve plateau (uraemics, 360±12.0 nmol/l; healthy controls, 594±53.4 nmol/l; P<0.001). In spite of influx rate reduction, erythrocyte [Ca2+]i values were significantly higher (uraemics, 101±1.8 nmol/l; healthy controls, 85±4.2 nmol/l; P<0.001). Resting [Ca2+]i in patients was not correlated with clinical parameters listed in Table 1Go or with the presence of ventricular arrhythmias or hypertrophy and was not influenced by the treatment with erythropoietin, antihypertensive drugs, or Vitamin D.

No significant influx differences were observed in patients suffering from different nephropathies, being treated with erythropoietin (treated, 0.477±0.05, n=22; untreated, 0.516±0.067, n=25, P=n.s.), Ca2+ channel blockers (treated, 0.476±0.058, n=21; untreated, 0.515±0.06, n=26, P=n.s.) or 1,25(OH)2 vitamin D3 (treated, 0.638±0.072, n=6; untreated, 0.477±0.046, n=41, P=n.s.).

Ca2+ influx kinetics were also studied in patients with cardiac hypertrophy (LVM index 144±8.9 g/m2) with normal kidney function; they had no cardiac arrhythmias. Their values of influx rate (1.041± 0.324 nmol/s), curve plateau (554±72.6 nmol/l) and erythrocyte [Ca2+]i (82±7.0 nmol/l) were not different from those in controls but were significantly different from those in uraemics (P<0.001).

Maximal influx rate was positively correlated with PTH plasma values only in patients not treated with Ca2+ channel blockers or 1,25(OH)2 vitamin D3 (r=0.495, n=21, P<0.025). Plateau values of erythrocyte Ca2+ influx were negatively correlated to patient Kt/V (r=-0.352, n=47, P<0.025) (Figure 1Go).



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Fig. 1. Significant correlations between calcium influx indices and clinical parameters of uraemic patients. (A) Correlation between maximal calcium influx and plasma PTH values in HD patients not treated with calcium channel blockers or 1,25(OH)2vitamin D (n=21). (B) Correlation between plateau values of calcium influx and Kt/V in HD patients (n=47).

 
Of the 40 uraemic patients submitted to echocardiographic analysis, 32 (80%) had LV hypertrophy. LVM index was positively correlated with maximal Ca2+ influx rate (r=0.353, P<0.05), but not with resting [Ca2+]i and plateau levels.

No other clinical parameters were correlated to the Ca2+ influx indices.

Arrhythmias and erythrocyte Ca2+ handling
All 47 uraemic patients were submitted to 24-h Holter ECG. Ventricular arrhythmias occurred in 23 patients (48.9%). Their maximal Ca2+ influx rate was significantly higher (0.600±0.060 nmol/s; P<0.02) compared to patients without arrhythmias (0.400±0.041 nmol/s). Patients with arrhythmias and those without arrhythmias were homogeneous for clinical and biochemical parameters. However, patients suffering from arrhythmias had LV end-systolic diameter, LV end-diastolic diameter, and LVM index significantly higher, as shown in Table 2Go.


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Table 2. Clinical and echocardiographic characteristics of HD patients with and without ventricular arrhythmias divided according to Lown's classification

 
Patients were classified according to Lown's ventricular arrhythmias classification: 12 were attributed to Lown's class 1; three to class 2; one to class 3, and seven to class 4. The maximal influx rate increased with the severity of ventricular arrhythmias. On the contrary, the plateau values were similar (Table 3Go). Figure 2Go shows the mean of measured Ca2+ influx time courses of the three groups of patients. The results did not change considering only the patients untreated with calcium channels blockers (Table 3Go).


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Table 3. Comparison of erythrocyte Ca2+ influx indices measured in uraemic patients and in uraemic patients not treated with calcium channel blockers, with and without arrhythmias

 


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Fig. 2. Erythrocyte Ca2+ influx. Figure shows the mean of Ca2+ fluxes in HD patients without arrhythmias (n=24), Lown's classes 1, 2 (n=15), and Lown's classes 3, 4 (n=8). The values (MV±SEM) of maximal influx rate and influx plateau are reported in the ‘Results’ section.

 
Erythrocyte [Ca2+]i values were similar in the three groups of patients (patients without arrhythmias 101±1.8 nmol/l; Lown's classes 1 and 2: 104±3.0 nmol/l; Lown's classes 3 and 4: 99±7.6 nmol/l; P ns).

Multiple regression analysis in uraemic patients showed that the development of ventricular arrhythmias was related to erythrocyte Ca2+ influx rate and LVM index (cumulative r=0.548).

PTH and erythrocyte Ca2+ influx
Erythrocyte Ca2+ influx was measured in 29 uraemic patients and in six healthy subjects in the presence or absence of PTH. Maximal Ca2+ influx rate measured in these two experimental conditions was positively correlated (r=0.780, P<0.001). Addition of PTH produced in uraemic cells an increase of maximal Ca2+ influx rate (0.476±0.042 vs 0.559±0.049 nmol/s in the presence of PTH, P<0.02), while influx plateau remained unchanged (357±15 vs 366±15 nmol/l in the presence of PTH, P=n.s.). Ca2+ influx was significantly increased by the presence of PTH in patients having arrhythmias, but not in those without arrhythmias (Table 4Go). On the contrary, PTH was not effective in healthy subjects (1.006±0.072 vs 1.019±0.045 nmol/s in the presence of PTH, P=n.s.).


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Table 4. Comparison of erythrocyte Ca2+ influx indices measured in the presence and absence of PTH in HD patients with and without arrhythmias

 

Discussion
The present work finds that alterations of erythrocyte voltage-sensitive Ca2+ influx observed in HD patients [7] are associated with the presence of ventricular arrhythmias and myocardial hypertrophy.

A large number of patients were treated with antihypertensive drugs, vitamin D and erythropoietin, but these therapies did not influence the Ca2+ influx. Particularly, erythropoietin therapy increases the percentage of young circulating erythrocytes, by a stimulation of erythropoiesis, but a difference of Ca2+ influx between young and old erythrocyte was previously excluded [7]. Ca2+ channel blockers did not affect the mean value of Ca2+ influx rate, although they may reduce the influence of PTH on cell Ca2+ uptake.

Although elevated in HD subjects, erythrocyte [Ca2+]i was not related to the presence of ventricular arrhythmias or hypertrophy. On the contrary, voltage-sensitive Ca2+ influx rate directly correlated to the degree of cardiac hypertrophy and the severity of ventricular arrhythmias. The relationship between Ca2+ influx and cardiac hypertrophy was not found in subjects with normal kidney function, so it appears to be a characteristic of uraemic patients.

Many factors have been known to increase Ca2+ content in erythrocytes of HD patients. The inhibition of Ca2+ pump activity by a circulating factor could be the main cause, due to the key role played by this carrier in controlling [Ca2+]i [5]. An enhanced passive Ca2+ uptake was also observed in human erythrocytes [18] and may be induced by the hyperparathyroidism that occurs in uraemic patients [19]. Since in the present work we did not study passive influx, but a specific Ca2+ influx pathway, these findings do not impinge on our results.

Provided that the alterations of Ca2+ influx rate and Ca2+ content found in uraemic erythrocytes are also expressed in myocardiocytes, they could influence membrane potential in its generation, thus enhancing the probability to generate autonomous electric signal by ventricular myocardiocytes or cells of conduction tissue. On the other hand, myocardial hypertrophy could be favoured by [Ca2+]i increase [2]. However, this effect could only occur in uraemic patients, because it was not observed in our patients with myocardial hypertrophy and normal kidney function.

Although erythrocyte Ca2+ influx cannot be compared or identified with the influx mediated by voltage-sensitive cardiac channels, we cannot exclude that erythrocyte and myocardiocyte voltage-sensitive Ca2+ influx carriers could share common molecular features. In keeping with this hypothesis, our findings suggest that cardiac manifestations of uraemia result from cell inability in handling Ca2+ overload. As previously suggested, the inhibition of voltage-sensitive Ca2+ influx could arise to compensate overload Ca2+ toxicity [7], while Ca2+ influx rate appears more indicative of cardiac damage in uraemic patients than resting cell Ca2+ concentration.

The appearance of arrhythmias can be consequent to hypertrophic cardiac dysfunction, alteration of cellular Ca2+ metabolism, anaemia, and possible uraemic cardiotoxic factors [2,3]. Therefore, the variables potentially able to affect myocardial cell function were analysed by multiple regression. This analysis suggests that only hypertrophy of LV mass and impairment of cell Ca2+ influx could independently influence the appearance of arrhythmias. They may create the conditions to make myocardial tissue prone to rhythm disorders.

Although no specific PTH receptors have so far been isolated from erythrocyte membranes [4], in our experimental conditions the hormone can stimulate Ca2+ influx rate, in agreement with the results obtained by Bogin et al. [19]. This in vitro activity of PTH was found only in patients with arrhythmias. This suggests that erythrocytes from patients without arrhythmias had a higher degree of resistance to PTH activity. The resistance to PTH usually develops in patients with chronic kidney failure. It produces a blunted cellular response to the hormone that could also involve erythrocytes. The relation between Ca2+ influx and PTH may also be influenced by drugs, like Ca2+ blockers or 1,25(OH)2vitaminD3 [20]. These drugs could reverse PTH activity on Ca2+ influx: the correlation between maximal Ca2+ influx rate and PTH was indeed found only in HD patients not treated with these two drugs.

Moreover, the correlation with Kt/V suggests that influx kinetic plateau has a specific biological meaning. It may be expression of cellular capacity to handle Ca2+ load and to maintain low levels of free Ca2+ in cytoplasm. A highly efficient dialytic therapy may be safe for these ion-buffering and transport systems.

In conclusion, myocardial dysfunction and altered ventricular excitability in uraemia could be due to the alterations of cell Ca2+ transport mediated by voltage-sensitive channels, as found in the erythrocyte model. Reduced resistance to PTH could contribute to this phenomenon.



   Acknowledgments
 
This research was supported by grants from the Italian Ministry of University for Scientific and Technologic Research (attributed to Laura Soldati: MURST ex 60% and MURST 40%) and from San Raffaele Hospital. The work was presented in Abstract form at the 32nd Annual Meeting of the American Society of Nephrology, 5–8 November 1999, Miami, FL, USA. We thank Dr Renato Spaventa for collaborative assistance.



   Notes
 
Correspondence and offprint requests to: Dr Laura Soldati, Divisione di Nefrologia, Ospedale San Raffaele, Via Olgettina 60, I-20132 Milano, Italy. Back



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Received for publication: 28. 2.99
Revision received 2. 8.00.



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