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* Department of Cardiothoracic Surgery, National Heart and Lung Institute, Imperial College School of Medicine, Royal Brompton and Harefield Trust, Harefield Hospital, Middlesex, UK;
Bristol Heart Institute, University of Bristol, Bristol Royal Infirmary, Bristol, England UK; and
London School of Hygiene and Tropical Medicine, London, UK
1Correspondence: Department of Cardiothoracic Surgery, National Heart and Lung Institute, Imperial College School of Medicine, Royal Brompton and Harefield Trust, Harefield Hospital, Middlesex UB9 6JH, UK. E-mail: rwarraich{at}lycos.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: dilated cardiomyopathy • antimyosin autoantibodies • myocyte contractility
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Class G-Igs, particularly against isoforms of cardiac myosin heavy chain (CM), are frequently described in clinical DCM (5
, 6)
. These Igs are detected with a similar frequency in heart failure (HF) patients of ischemic (IHD) origin (7)
and occur in response to myocardial injury (8)
. Similarly, Ig reactivity against other autoantigens (heat shock proteins and ß1-adrenoreceptors) in DCM is observed in HF patients of other etiologies (9
10
11)
. Nevertheless, Igs of the total G class represent a relatively nonspecific measure of humoral immunity.
In DCM, a multi-specificity of autoantigens are targets of humoral immunity. It may be that through the process of structural homology and epitope spreading multiple reactivities are seen. Hence, in disease entities whereby multiple autoantigens gain recognition by the humoral immune system, the relative importance of autoantibodies against a particular autoantigen may come into disrepute.
We previously demonstrated heterogeneity in subclass Igs with etiology of HF (7)
. Igs of the G3 subclass against CM were selectively raised in patients with DCM (7)
. G2-Igs were raised in DCM and HF patients with IHD (7)
. The differential distribution of subclass Igs is suggestive of distinct immune mechanisms regulating the humoral effector system in disease. Igs of the G3 subclass, high-affinity molecules with proinflammatory characteristics (12)
, correlated with high-grade cardiac allograft rejection, indices of left ventricular dysfunction and severity of disease (13
14
15)
. Hence, the functional effects exerted by regulatory components of the Ig Fc domains are independent of the antigen specificities of the Ig variable regions. The findings emphasize the relative impact of the underlying disorder on humoral immunity as opposed to the autoantigen recognized in these entities. Hence, the contribution by antigen-specific Ig segments would be of further interest.
Thus, given the distinct functional properties of subclasses and the clinical relevance of humoral immunity in DCM, this study sought to evaluate effects of autoantibodies against CM purified by immunoaffinity column chromotography from patients with DCM and IHD on excitation-contraction coupling in isolated rat myocytes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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No patient with DCM had a clinical history of specific heart muscle disease causing left ventricular dysfunction, hypertension (>160/100 mmHg), systemic diseases, or chronic excess alcohol consumption. Coronary artery disease (CAD) or valvular heart disease was excluded by selective coronary angiography. No patient was a recipient of immune-modulatory therapy. Clinical examination included a 12-lead electrocardiogram, chest X-ray, left heart catheterization, and transthoracic (M-mode) and Doppler echocardiography (Table 1
). Patients with IHD had secondary dilation and depressed ventricular function, defined as a documented history of myocardial infarction or CAD (by angiography) and prior revascularization (Table 1)
. Patients with IHD were comparable in severity of disease clinically and hemodynamically to patients with DCM. All patients were recipients of conventional drug therapy for heart failure.
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Serum from healthy blood donors, n=65 (41 males), median age 42 years (33–49), served as controls.
Antigen preparation and ELISA
Procedures used for antigen (
-, atrial-specific; ß-, ventricular-specific isoforms of human cardiac myosin heavy chain [CD]) extraction, purification (in accordance with institutional guidelines for human subject research), isoform specificity, and the ELISAs for immunoglobulin (Ig) class G and subclasses (G1, G2, and G3 [Binding Site, Birmingham, UK]) have been described (7)
. Ig reactivity was the optical density (OD) registered at 490 nm (Titertek Plus MS2 plate reader). Ig frequency (threshold for Ig positivity) was reported as the mean Ig response +2 SD above levels of the corresponding control Igs.
Preparation of the cardiac myosin immunoaffinity column
A 5 mL affinity column containing 5 mg/mL of human ß-CM was prepared using cyanogen bromide (CNBr) -activated Sepharose-4B (Pharmacia, Milton Keynes, UK) according to the manufacturer’s instructions. Cardiac myosin was prepared as described (7), then dialyzed overnight against phosphate-buffered saline (PBS) at 4°C to remove the protein diluent (0.5M NaCl containing 50% glycerol). This protein was then placed in PBS washed dialysis tubing (D 9277, Sigma, Poole, Dorset, UK) and dialyzed under reduced pressure (4°C) against 3x 500 mL changes of coupling buffer (0.1 M NaHC03 containing 0.2 5M NaCl, pH 8.5). The contents of a 9 mL PD-10 Sephadex G-25M polypropylene column (containing frits and caps) (Pharmacia) were removed and thoroughly washed with PBS. The bottom frit was then replaced and 1.43 g of freeze-dried CNBr-activated Sepharose 4B (1.43 g) was added to the column and washed with 500 mL of ice-cold 0.1M HCl. The bottom cap was added to the column and the dialyzed cardiac myosin contained in coupling buffer was added. After the top cap was added, the column was placed on an electrical blood mixer and the antigen was allowed to couple to the activated Sepharose by constant inversion overnight at 4°C. The coupling buffer was then collected and replaced with an equal volume of 10 mM Na2HPO4 containing 0.2M glycine pH 8.0 and incubated for 2 h at 25°C to block any excess coupling sites on the activated Sepharose. The column was washed with PBS and subjected to five high (0.2M glycine/NaOH pH 9.0) and low (0.1 M sodium acetate buffer containing 0.5 M NaCl pH 4.0) pH oscillations to remove any uncoupled cardiac myosin. The column was then washed with elution buffer (30 mM diethylamine in PBS pH 11.6), then thoroughly washed with PBS and stored at 4°C in PBS containing 0.02% w/v NaN3. The coupling efficiency was assessed by performing protein determinations using a bichinoninic acid (BCA) reagent (Pierce, Rockford, IL, USA) on serial 10-fold dilutions of the pre- and postcoupling solutions in PBS in 96-well ELISA plates (Immulon 1, Dynatech, Vienna, VA, USA). Ten mL of each serial dilution was transferred to wells containing 200 mL of the BCA reagent. After sealing, the plate was incubated at 37°C for 1 h, then read at a wavelength of 570 nm (MR700, Dynatech), and the percentage of coupling was calculated to be >99.9%.
Immunoaffinity purification
CM autoantibodies were purified by immunoaffinity chromatography from patients’ serum samples (4 mL/patient) with end-stage heart failure. For this procedure, each 4 mL serum sample was diluted in 20 mL of PBS and slowly cycled (2.5 mL/h) through the PBS-equilibrated immunoaffinity column at 4°C using a peristaltic pump. The column was then washed with 40 volumes (200 mL) of PBS at 4°C. The column was slowly eluted (8 mL/h) with 30 mM diethylamine in PBS pH 11.6 and ten 0.9 mL fractions were immediately neutralized with 0.1 mL of 0.1M Tris/HCl pH 7.2. Each fraction was tested for the presence of anti-CM IgG antibodies against the - and ß-isoforms of the protein in an ELISA as described previously (7)
. The fractions from each patient of the highest level of anti-CM reactivity were aliquoted in 50 mL volumes, frozen, and stored at –80°C until use. The preparation and use of immunoblot (Western blots) with lanes containing the - and ß-CM and whole heart homogenates have been described (7)
. As the protein concentration in these fractions was below the detection range of the protein assay (Bio-Rad, Hemel Hempstead, UK), we opted, for the contractility assays, to compare the affinity-purified (AF) fraction dilutions with the volume of myocyte cell suspension.
Incubation of myocytes with affinity-purified fractions
Single adult rat ventricular myocytes were enzymatically isolated as described previously (16)
. Aliquots (0.5 mL) of myocyte cell suspension were incubated with serial dilutions of 0.5, 1, 5, and 10% volume/volume (v/v) of the AF fractions for 1 to 3 h (to achieve the optimal concentration and time-related effects) and total human purified IgG and IgM (5% v/v of known mg/mL). Serum from patients with heart failure and healthy blood donors was used at 10% v/v.
Ab-treated cells were placed in a chamber on the stage of an inverted microscope (Nikon Diaphot 300) and continuously superperfused at 37°C as described previously (16)
. Cells were electrically field-stimulated to contract at the stated rates. Cell morphology (% yield of rod shaped myocytes) and the capacity to respond when electrically stimulated were assessed via a TV camera connected to a monitor. Cells were counted as "responding" if they contracted in time to the stimulus with no spontaneous contractions. Approximately 20 cells in at least 5 different fields of view were studied for each incubation.
Measurement of intracellular calcium and cell length
Total intracellular calcium ([Ca2+]i) was determined using indo-1/AM (Molecular Probes Inc., Junction City, OR, USA [3 mL cell suspension incubated with 10 µM indo-1/AM for 10 min at 37°C]) as described previously (17)
. Indo-1 was excited at 340–390 nm and emission detected at 410 ± 5 nm and 490 ± 5 nm, corresponding to the peak emissions of the Ca2+-bound and Ca2+-free forms of the indicator, respectively. Fluorescent light was detected and collected on-line by a Newcastle Photometric Systems (Newcastle, UK) at a rate of up to one data point/10 ms from a single myocyte after subtraction of background fluorescence. Simultaneous acquisition of fluorescence signal and cell shortening (using a Crescent Electronics Video Edge Motion Detector) were recorded.
Measurement of L-type Ca2+ currents
Selective recording conditions for measurement of cardiac L-type calcium currents (ICa,L) in ventricular myocytes were as described by Witchel et al. (18)
. All K+ in the pipette solution was replaced with Cs+ (to abolish K currents) and 5 mM BAPTA was added to buffer intracellular Ca2+ and attenuate Ca-activated currents. The whole cell patch clamp recordings of ICa,L were made at 37°C. ICa,L was elicited by a two-step protocol applied from a holding potential of –80 mV: an initial step to –40 mV for 100 ms to activate and inactivate fast sodium current; a second step +10 mV for 600 ms to elicit ICa,L.
Immunofluorescence studies
Adult rat cardiac myocytes were allowed to attach to laminin-coated coverslips for 1 h. Cells were then incubated with 0, 0.5, 1, or 5% primary Ab (affinity-purified Ab or mouse anti-cardiac myosin monoclonal Ab [Abcam]) for 2 h prior to fixing with 4% para-formaldehyde at 4°C overnight. All washing was with PBS. To detect primary Ab attached to the outside of the cells, after washing, cells were incubated with 1:200 Alexa 488 conjugated (green) anti-human secondary-Ab (Molecular Probes) for 1 h. The cells were then washed briefly. To detect primary Ab that may have entered the cells, cells were first permeabilized using 0.5% Triton X-100, washed, then incubated with 1:200 Alexa 568 conjugated (red) anti-human secondary Ab (Molecular Probes) for 1 h. Cells were then washed and mounted onto microscope slides in mowiol.
In the positive control samples, cells were not incubated with primary Ab but were fixed and permeabilized as described above, then incubated with primary Ab (1:10 affinity-purified Ab or 1:200 anticardiac myosin), followed by washing and incubation with either the red or green secondary Ab as described above. Imaging was performed using a Leica TCS-NT confocal microscope with a 63x objective.
Statistics
Comparison of Ig reactivity (after categorical distribution) was computed as medians and interquartile range (Mann-Whitney U test). Ig frequency, qualitative data, differences in cell morphology, number of rod-shaped or responding myocytes were determined using 
2. Changes in [Ca2+]i and cell length of individual cells were analyzed using Student’s t test (paired or unpaired as appropriate) or repeated measured ANOVA indicated. P < 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Subsequent dialysis and reconstitution of the AF fractions was avoided in an effort to conserve the eluted material (protein content below assay detection range). The eluting solution (constant per elute per patient) constituted 90% of the eluted material. However, a 30 mM solution of diethylamine in PBS was of relatively low stringency since elution has been reported to use concentrations of much higher molarity (19)
. Nevertheless, this concentration (30 mM) in the working solutions, diluted to 5% and 1% v/v (20- and 100-fold lower, respectively) for the functional assays, was negligible. However, reactivity of the AF-Igs (fraction of the highest reactivity) following serial dilutions correlated with serum IgG reactivity: r = 0.66 (P=0.0001, Fig. 1
).
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Although a single-step procedure for antibody desorption was used (as an increase in the concentration of the eluting substance allows desorption of antibodies with different affinities [stepwise procedure]), the eluted fraction (from a total of 10 fractions per patient) of the highest IgG reactivity differed in the two groups. In DCM patients (n=19), the fraction (median) of the highest reactivity eluted off relatively later; 8 (7–8.5) compared with IHD (n=19) patients; 5.3 (5
6)
(P<0.0001). A difference in the elution profiles of the AF-Abs may reflect antibodies of distinct affinities in the two groups.
Autoantibody specificity
SDS-polyacrylamide gel electrophoresis (PAGE) and ELISA
SDS-PAGE, immunoblotting, and immunoprobing of nitrocellulose membranes of interest were carried out as described (7)
. Protein bands corresponding to Ig heavy and lights chains for the AF fractions were not detected by SDS-PAGE. With an increase in loading volume (10–50 µL) of the fraction with the highest IgG reactivities, no bands were detected after staining of the polyacrylamide gels with Coomassie blue stain. Immunoprobing of nitrocellulose membranes carrying separate lanes of - and ß-CM (1 µg/lane) detected both isoforms (Fig. 2
), showing that ß-CM-specific antibodies cross-react with the -specific isoform.
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The potency of ß-CM AF-Abs to recognize the -isoform by ELISA, a more definitive measure of Ig reactivity, varied with patients. The level of variation was independent of Ig titer or the patient group sought (data not shown). Furthermore, there were no differences in IgG titer of the AF-Abs from DCM and IHD patients (Fig. 3
a, b1, b2). This finding invalidates the concept of a dilution criterion as a limiting factor or a measure to discriminate humoral immune responses against CM in different clinical disease states (Table 2
). However, of the Ig subclasses, frequency of IgG3 differed between the groups (DCM; 21% 
and IHD; 5% 
) and correlated with serum IgG3 reactivity (data not shown).
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Functional assays
Effect of affinity-purified Abs on cell contraction induced by electrical field stimulation
The capacity of isolated myocytes to respond to field stimulation (0.5 Hz) was reduced by the AF-Abs. Percentage inhibition of cell contractility (determined as the % inhibition of cells beating as a % of the untreated viable myocytes [12 field counts]) was concentration and time dependent (data not shown as this represented the preliminary findings to achieve the optimal assay conditions for subsequent studies). The decline in the capacity of Ab-treated myocytes to respond to field stimulation varied between patients and the study groups, but there was no further decline incubation with >5% v/v AF-Abs for 2 h.
Percentage inhibition of myocytes after incubation (2 h) with a 1% v/v of the AF-Abs induced a significant inhibitory effect in only 3 of the 19 DCM-Abs (16%) and 2 of the 19 IHD-Abs (11%, P=NS). However, when the effect of all the AF-Abs was considered collectively, the overall % inhibition in contractility of the beating myocytes was significantly greater by Abs from DCM patients (n=19) compared with IHD (n=19, P<0.005). Incubation with a 5% v/v of the AF fractions induced a greater inhibition than the 1% v/v in 11 of the 19 (53%) DCM-Abs compared with 4 of the 19 (21%) IHD-Abs (P=0.004, shown in Fig. 4
). Percentage inhibition was also significantly altered by AF-Abs positive for IgG3 reactivity. This was not observed for the other subclasses. Unlike the potent inhibitory effect of the AF fractions rich in G3 Igs, the level of total IgG (from either group) did not correlate with the degree of % inhibition in contractility of the cells (data not shown).
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Incubation with sera (10%) reduced the capacity of myocytes to respond when field stimulated in 5 of the 70 (8%) sera from patients with heart failure (Fig. 4)
. This was not observed with healthy blood donors (n=6), total (commercial) human-IgG, or IgM (5% v/v of known mg/mL [data not shown]).
Effect of affinity-purified Abs on percentage cell shortening
Percentage cell shortening was determined in "responding" cells preincubated with AF-Abs from patients that reduced the capacity of myocytes to contract when field-stimulated.
The effects of the AF-Abs on cell contraction in the preliminary work were measured at 0.2, 0.5, 1, 2, 3, and 4 Hz. There were no significant differences in cell amplitude at stimulation frequencies of 1 to 4 HZ (data not shown). Therefore, cell shortening in subsequent samples; untreated cells (controls) and cells treated with AF-Abs from 9 patients (at random); 4 DCM and 5 IHD (conducted blindly), was determined at two different frequencies of 0.2 Hz and 3 Hz only (Fig. 5
). These were the stimulation rates at which subsequent measurements of peak systolic and diastolic levels of [Ca2+]i were recorded.
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With an increase in the stimulation frequency of 0.2 Hz to 3 Hz, the significant difference in cell shortening in untreated myocytes (P=0.0001 [paired t test]) was relatively less pronounced with the AF-Abs from patients with DCM (P=0.03) and IHD (0.001).
At 0.2 Hz, cell shortening by DCM and IHD-Abs compared with untreated cells differed significantly (Fig. 5)
The effect at 3 Hz was less prominent than at 0.2 Hz, with the AF-Abs.
Effect of affinity-purified Abs on calcium transients
Although a subsequent increase in the stimulation frequency of 0.2 Hz to 3.0 Hz did not result in a further significant decrease in cell shortening by Abs from DCM patients (Fig. 5)
, levels of [Ca2+]i were significantly altered by these Abs at the higher stimulation frequency. The frequency-dependent effect of CM-Abs on [Ca2+]i at 3.0 Hz compared with 0.2 Hz is shown in Fig. 6
a and b, respectively. Changes in peak systolic and diastolic levels of [Ca2+]i in myocytes treated with DCM-Abs (n=4) were significantly higher vs. untreated cells (P=0.0046 and P=0.0003, respectively) or cells treated with Abs from IHD patients (P=0.0012 and P=0.0001, respectively). This effect was independent of IgG reactivity or titer of the affinity-purified Abs. Cells treated with Abs from patients with IHD did not significantly differ from controls (systolic, P=0.290 and diastolic, P=0.052).
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Hence, the observed discordance between the negative force-frequency relationship shown in Fig. 5
and the increased calcium amplitudes by AF-Abs from DCM patients (Fig. 6b
) is suggestive of dissociation between the contracting filaments and their sensitivity to [Ca2+]i at the higher stimulating frequency.
Effect of affinity-purified Abs on calcium currents
Affinity-purified Abs from patients with DCM in this study altered levels of [Ca 2+]i. We therefore sought to determine whether this effect was mediated by modulation of the sarcolemmal Ca channels. L-type Ca currents were measured in ventricular myocytes in untreated (controls), sera-treated, and cells treated with affinity-purified DCM antibodies. The data in Fig. 7
show these antibodies did not significantly alter the activity of the Ca channels, suggesting a possible intracellular effect of the antibodies. Therefore, the possibility that the Abs were internalized was investigated using immunofluorescence.
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Immunofluorescence (IF) studies
IF with affinity-purified (AF-Abs)
Detection of IgG: For intracellular localization, fixed myocytes preincubated with the AF-Abs were permeabilized. No cellular entry of the AF-Abs was detected as shown in Fig. 8
a, where there is no fluorescence above that of the secondary Ab with AF-Abs from a DCM patient that altered levels of [Ca2+]i. For cell surface detection, the cells were not permeabilized and no fluorescence was detected (Fig. 8b
). This is likely to indicate no extracellular expression of CM on cardiac myocytes and no strong binding of the AF-Abs to the cellular membrane of myocytes.
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In the positive control experiments, cells were incubated with AF-Abs after fixation. Increased fluorescence was observed in permeabilized (Fig. 8d
) and nonpermeabilized cells (Fig. 8e
), demonstrating the efficacy of the AF-Abs for standard IF assays.
IF with commercial human myosin Abs
For comparison, studies with commercially available human cardiac myosin Abs were performed. No fluorescence (above the secondary Abs) was noted in impermeabilized cells or with permeabilization (Fig. 9
a). However, enhanced fluorescence was seen in cells incubated with commercial Abs after cell fixation (positive control cells), demonstrating the efficacy of the Abs (Fig. 9b1, b2
), for the assays used in this study.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CM-Abs have been rendered as serological markers of disease-specific humoral autoimmunity in clinical DCM (5
, 20)
. However, 1) their purification from patients with HF of an ischemic origin and 2) the inability to distinguish the Igs by total class G reactivity or titer negate disease specificity of CM-Abs and the concept of a dilution criterion a threshold to discriminate CM reactivity in clinical DCM (21)
. Purification of CM-Abs from patients with end stage HF does not support the notion that these Igs become undetectable with progression of disease (22)
. These findings have important implications for studies reported by others (5
, 20
, 21)
.
Igs purified against CM from patients with DCM and IHD differed in their capacity to alter contractility of isolated myocytes. Affinity-purified fractions positive for G3-Igs, higher in DCM patients, significantly increased % inhibition of the contracting myocytes, providing direct evidence of their capacity to impair cellular contractility. Hence, the Ig Fc domains that specify the subclass isotype are independent of the Ig variable regions and therefore the autoantigens recognized by subclass Igs. This may in part contribute to the disparity in contractility by AF-Abs from the two study groups.
In addition, only Abs from DCM patients affected levels of [Ca2+]i of the isolated myocytes. The Abs raised levels of diastolic Ca2+, suggesting their ability to impair removal of [Ca2+]i. Hence, disparity observed between the reduced contractility and increased Ca2+ amplitudes by DCM-Abs may show altered sensitivity of the myofilaments to Ca2+ at the higher stimulating frequency. Although this is known to occur secondary to phosphorlylation by protein kinase A, some agents have been shown to have direct Ca2+-sensitizing effects (23)
, and this may represent one mechanism of immune-mediated functional effects at the cellular level in clinical DCM.
The increase in diastolic Ca2+ may reflect a capacity of these Igs to indirectly impair regulatory components of the contractile apparatus to extrude [Ca2+]i either by reuptake into the sarcoplasmic reticulum (SR) or efflux across the sarcolemmal membrane. The inability to extrude [Ca2+]i, may also in part account for the raised systolic calcium as reported. This is supported by the fact that the AF-Abs had no effect on L-type Ca2+ currents.
It is clear that cellular calcium homeostasis in isolated rat ventricular myocytes is altered by the affinity-purified Abs against CM from DCM patients. However, the precise mechanisms for the antibody-mediated Ca2+ impairment are not known. Studies with immunoflorescence do not demonstrate internalization of these Igs. It may be that the Igs initiate an intracellular signal as a result of interaction with a surface site. Intracellular signaling may involve organelles such as the SR, possibly the mitochondria or other membrane-bound domains. However, more studies are required to elucidate 1) whether the Igs directly alter SR and mitochondrial activity and 2) the relevance of the Igs to mobilize Ca2+ in disease.
Ca2+ accumulation at the SR has been described in early stages of DCM (24)
. Autoimmune inhibition of the SR-Ca2+ ATPase enzyme in the SR has been shown to induce myocardial lesions consistent with the diagnosis of myocarditis (25)
, and Igs against the SR-Ca2+-ATPase and ADP/ATP have been shown to impair cell contractility in experimental models of myocarditis (26)
.
However, unlike previous studies that have used only the total IgG fraction or products of hyper-immunization, this is the first report to provide evidence of 1) Ig-mediated functional impairment of myocytes by cardiac specific Abs from DCM patients and 2) to demonstrate comparative effects of CM-Abs from HF patients of different etiologies. These findings may reflect the need to identify the type of immune responses evolving in clinical DCM that give rise to Igs that contribute to contractile dysfunction.
In conclusion, the differential capacity of myosin autoantibodies to impair cell contractility supports a different category of Igs arising in patients with DCM compared with IHD. The findings reflect the importance of the underlying disorder on humoral immunity (attributed in part by differences in the subclass Igs), evolving in disease as opposed to the autoantigens recognized in this entity. Hence, these avenues of investigation may facilitate appropriate therapeutic strategies in clinical practice.
Study limitations
Although the current study has used rat rather than human myocytes, intracellular signaling pathways (adenylyl cyclase/Camp/PKA, the NO/cGMP, MAP kinases, ERK, Jun, and PI3 kinase) are similar in both species and driven by similar membrane currents (27)
(which differ subtly), rendering rat myocytes a reliable assay system to study effects of positive and negative inotropic agents.
In addition, although the source of human myocytes would be from explanted hearts, the Ca2+ handling and ß-adrenoceptor responses in myocytes from tissue of these patients with HF are severely compromised. Other signaling pathways are also likely to be altered (28)
and influenced by etiology of disease, age, and drug treatment. Donor tissue, although rarely available, has also been reported to show signs of acute failure (29)
.
Received for publication September 6, 2004. Accepted for publication December 8, 2005.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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- and ß- myosin heavy chain isoforms as major autoantigens in dilated cardiomyopathy. Circulation 85,1734-1742-myosin in a subset of patients with idiopathic dilated cardiomyopathy. Br. Heart J. 74,598-603
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