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European Journal of Heart Failure 2004 6(4):389-398; doi:10.1016/j.ejheart.2003.07.010
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© 2004 European Society of Cardiology

Desmin-free cardiomyocytes and myocardial dysfunction in end stage heart failure

S. Di Sommaa,d,*, M.P. Di Benedettob, G. Salvatoreb, L. Agozzinoc, F. Ferrantia, S. Espositoc, P. La Doganad, M.I. Scaranod, G. Caputob, M. Cotrufoe, L.De Santoe and O. de Divitiisb,d

a Medical Department, II Medical School University, ‘La Sapienza’, Sant'Andrea Hospital Via di Grottarossa 1035-1039, 00186 Roma, Italy
b Department of Experimental and Clinical Medicine, University Federico II Naples, Italy
c Department of Health Medicine, Section of Pathology, Research Centre for Cardiovascular Diseases, II University of Naples Italy
d Inter-University Centre for Cardiovascular Research (G.I.M.E.C.), University of Naples Italy
e Cardiac Surgery, II University of Naples Italy

* Corresponding author. Tel.: +39-06-80345275; fax: +39-06-80345001. E-mail address: sdisomma{at}unina.it


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Statistical analysis
 4. Results
 5. Discussion
 References
 
Our aim was to evaluate the desmin content in the myocardial tissue of patients with end-stage heart failure of ischaemic origin and to assess its role on cardiac function. We studied 18 explanted hearts from patients transplanted for end-stage heart failure due to ischaemic cardiomyopathy (ICM). Control myocardial tissue was obtained from the cardiac biopsies of six women with breast cancer taken prior to commencing chemotherapy with anthracyclines, four male donors for heart transplantation and two autoptic hearts from patients who died due to non-cardiac events. Myocardial tissue, obtained from the left ventricle (remote zone from infarcted area), was analyzed by light and confocal immunochemistry (desmin) microscopy. The desmin content of myocardial tissue was obtained by real-time PCR. Cardiac function was evaluated by echocardiographic and right heart catheterization data, obtained before heart transplantation. Confocal microscopy evaluation showed a significant decrease in the number of desmin-positive myocytes (P<0.01) in ICM hearts compared to controls. At real-time PCR evaluation, there was a reduction (P<0.01) in desmin content in the ICM patients compared to controls. A negative correlation was found between desmin-free cardiomyocytes and ejection fraction (EF) (r=–0.834; P<0.02) on echocardiogram. A negative relationship (r=–0.688) was also found between desmin-negative myocytes and capillary wedge pressure. In conclusion, the myocardial tissue of patients with end-stage heart failure of ischaemic origin, shows a decreased number in desmin-positive myocytes at immunochemistry evaluation compared to normal individuals. This deficiency in cytoskeletal intermediate filament content is associated with reduced cardiac function.

Key Words: Heart failure • Ischaemic cardiomyopathy • Myocardium cytology • Cytoskeleton • Desmin • Myocardial function

Received November 1, 2002; Revised July 7, 2003; Accepted July 30, 2003


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Statistical analysis
 4. Results
 5. Discussion
 References
 
The cytoskeleton of myocardial cells is a well organized structure, with a complex array of proteins that maintain the internal organization of cellular organelles and transmit the mechanical forces within the cell to and from the adjacent cells and the extracellular matrix [1]. The myocyte cytoskeleton is a major determinant of the intrinsic function of the myocyte and it might be considered as the primary site of cellular damage in the clinical syndrome of heart failure due to both coronary artery disease and dilated cardiomyopathy [2–5]. Desmin [6,7] and titin [3] act as the major stress-bearing elements in the sarcomere [7], and an increase in desmin content has been described in myocyte hypertrophy [6–8] and in desmin-related cardiomyopathy [9]. Moreover, in the transition from compensated ventricular hypertrophy to heart failure, Collins et al. [6] have shown a progressive increase in myocyte levels of desmin. Schaper et al. [10] investigated the changes in the cytoskeleton and associated proteins in idiopathic dilated cardiomyopathy (DCM) and found increased amounts of desmin, tubulin, vinculin and vimentin. Thus, the role of cytoskeletal proteins in dilated cardiomyopathy as a final common pathway hypothesis of cardiovascular disease has been well established [11]. Recent endomyocardial biopsy data from our laboratory has shown that, compared to normal human tissue, the desmin alterations in the myocardium of patients with DCM are more related to its irregular distribution and disarray than to the actual levels, and are related to myocyte dysfunction [12], thus confirming the importance of the cytoskeleton in muscle cells in relation to function [13].

Furthermore, it has been suggested that cytoskeletal injury may be associated with irreversible myocardial damage in coronary artery disease [1]. Although the cellular basis of ICM has been well described [14–16], there is currently no data on the desmin content of myocardial tissue from patients with heart failure due to coronary artery disease and its role in cardiac function in this disease.

The aim of this study was to evaluate the content of cytoskeletal intermediate desmin filaments in end-stage dilated cardiomyopathy of ischaemic origin, compared to the myocardial tissue of a normal heart, and to evaluate whether these alterations could be associated with cardiac dysfunction.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Statistical analysis
 4. Results
 5. Discussion
 References
 
The study protocol was approved by the ethical review committee of the ‘Federico II’ University Medical School in Naples (Italy). The investigation conforms with the principles outlined in the Declaration of Helsinki.

We studied 18 explanted hearts, whose morphological characteristics are shown in Table 1.


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  		Table 1 Morphometric evaluation of explanted hearts from patients with ischemic cardiomyopathy (n=18)

 
Eighteen patients (12 males and six females) were transplanted for end-stage failure due to post-infarction dilated cardiomyopathy (ICM). The diagnosis of heart failure was made according to the criteria reported in the guidelines of the European Society of Cardiology [17].

The diagnosis of coronary artery disease was performed before heart transplantation, according to the guidelines of the European Society of Cardiology [18].

The control group consisted of myocardial tissue obtained from 12 patients (six women and six men). The six women were from a population at high risk of relapsing breast cancer, who were enrolled in a surveillance program that included endomyocardial biopsy in order to evaluate possible cardiac toxicity due to anthracycline treatment. In these female subjects, the cardiac biopsy was performed on the basis of specific alterations detected on ECG or echocardiographic evaluation, but in which the immuno-histochemical findings showed the cardiac tissue to be normal compared to identical patterns of myocardium obtained from Sprague–Dawley rats.

The myocardial samples from the six males were obtained from four heart transplant donors who had undergone cardiac biopsy at the time of heart explantation and two autoptic hearts from patients who had died from non-cardiac causes. All participants, except the donors and the autoptic cases, gave written informed consent to be enrolled in the study. The histological findings of the myocardial tissue from the controls reproduced the well known normal ultrastructure of the cardiac muscle both on light (LM) and confocal microscopy.

Doppler-echocardiographic evaluation was performed in all patients before heart transplantation and in all controls. M-mode, two-dimensional and spectral Doppler-echocardiographic evaluation was performed using an ultrasound mechanical system equipped with a 3.5 Hz transducer. System recordings were made with the patients in the left lateral position according to the standardized methodology of the American Society of Echocardiography. Measurements included inter-ventricular septum end-diastolic dimension (IVSD), left ventricular posterior end-diastolic wall dimension (LPWD), left ventricular end-diastolic dimension (LVEDD), left ventricular end-systolic dimension (LVESD). Left ventricular ejection fraction (LVEF) was calculated as follows: LVEF=(LVEDD3–LVESD3)/LVEDD3 [19].

The left ventricular mass (LVM) was calculated according to Devereux et al. [20], and LVM was indexed by body surface area (LVMi, g/m2).

Right heart catheterization: Right atrium, right ventricle, pulmonary arterial and capillary wedge pressures, cardiac output, and systemic vascular resistances were obtained during intra-cardiac right heart catheterization performed few days before heart transplantation.

Cardiac biopsy: After isolation of the right internal jugular vein by a 9.0 F introducer system, a right ventricular cardiac biopsy was carried out. The procedure was performed using a Sholten byotome (8 F) in cat-lab, with continuous EKG monitoring, under fluoroscopic and echocardiographic control, aimed at taking the endomyocardial specimens from the 1/3 medium of the interventricular septum. At least three samples were collected from each patient for light microscopy (LM), immuno-histochemistry, and molecular biology evaluation.

Pathology: Sections of the explanted hearts were taken as follows: three specimens (3 cm) taken from the anterior wall of the left ventricle approximately 3 cm distal to the scar; three in the middle third of the septum; and three in the free-wall of the right ventricle, in all recipients hearts. Some were fixed in formalin, others frozen at –80 °C.

Histological criteria for recruitment: Light microscopy examination excluded the presence of inflammatory cells in the myocardial interstitium, and of sites of myocytolytic and contraction band necrosis.

Light microscopy: The myocardial samples were immediately fixed in phosphate buffered formalin and embedded in paraffin. Serial sections (4-µm thick) were mounted on poly-L-lysine coated slides. Approximately 20 slides were obtained from each block. Non-consecutive sections were stained with hematoxylin–eosin and Picro-sirius red. At least five stained slides were used for morphology and stereologic evaluation.

Immunochemistry: Sections of specimens routinely processed for each case were dewaxed and rehydrated. Slides were incubated in 0.1% sodium azide containing 3% hydrogen peroxide to quench endogenous peroxidase, then treated with 5% normal goat serum in phosphate buffered saline (PBS) for 15 min, to block non-specific binding.

The primary antibody was desmin monoclonal mouse antibody (clone 33, Biogenex, San Ramon, CA), at a dilution of 1:160.

The primary antibodies were applied to sections in 1% BSA, 0.05 M Tris–HCI, pH 7.4; the slides were then incubated for 60 min in a moist chamber at 25 °C.

After three 5 min washes in PBS, the slides were treated with secondary biotinylated goat anti-mouse antibody (Biogenex, San Ramon, CA), at a dilution of 1:50 for 30 min.

After three 5 min washes in PBS, peroxidase-conjugated streptavidin (Biogenex, San Ramon, CA) diluted at 1:50 was applied for 45 min.

In order to check for peroxidase activity, the sections were treated for 10 min with 0.05% freshly made and filtered solution of 3-3'-diaminobenzidine tetrahydrochloride (DAB) (Sigma Chemical Company, St. Louis, Missouri) in 10 ml of 0.05 M Tris–HCI, pH 7.6, to which 0.03% hydrogen peroxide was added.

The correction for the signal of negative control was not performed because of the possibility that the substitution of the mouse monoclonal anti-desmin antibody by goat serum could act as an inhibitor of unspecific binding.

So we know the limitation inherent to this approach.

Positive control sections were obtained from Sprague–Dawley rat hearts.

2.1. Immunofluorescence and confocal microscopy evaluation
2.1.1. Immunofluorescence
The slides were treated with 0.1% Triton X-100 in Tris–HCl buffer for 5 h, washed in buffer and subsequently exposed to normal horse serum 1:50 dilution for 10 h, 1:200 dilution of monoclonal primary antibody to human desmin (klon D33, Dakopatts, Denmark) for 1 h and then FITC conjugated secondary antibody for 2 h in the dark.

The slides were observed with laser scanning microscope LSM5 Pascal (Zeiss).

2.1.2. Double staining
The slides were treated with 0.1% Triton X-100 in Tris–HCl buffer for 5 h, washed in buffer and subsequently exposed to normal horse serum 1:50 dilution for 10 h. The specimens were first exposed to the 1:200 dilution of monoclonal primary antibody to human desmin (clone D33, Dakopatts, Denmark) for 1 h and then FITC conjugated secondary antibody for 2 h in the dark. Then, specimens washed in Tris–HCl buffer, were exposed to the mouse sarcomeric actin antibody (Dako Co., Carpinteria, CA, USA) at a dilution of 1:50. The immunoreactivity was visualized using TRICT-conjugated rabbit anti-mouse immunoglobulin (Dako) at dilution of 1:50 for 3 h. The specimens were mounted with aqueous mounting medium with anti-fading agents (Biomega Corp., Foster City, CA, USA).

The double staining was observed and evaluated with laser scanning microscope LSM5 Pascal (Zeiss).

2.1.3. Imaging, image analysis and data processing
Each section was observed with a Zeiss Axioskop photomicroscope, equipped with a stabilized power supply, a 12 V, 100 W tungsten halogen lamp, a 1.4 NA condenser and a standard set of plan neofluar objectives. Microscopic images were detected with a high resolution CCD digital camera operating on a dynamic range of 12 bits per pixel (Lumina, Leaf Systems Inc.), transduced to a Power Mac Apple personal computer (Apple Computer Inc., Cupertino, CA) and saved as 8-bit uncompressed files.

Image analysis was, therefore performed on monochrome, 256 gray level images, with the public domain NIH Image 1.59 software.

The microscopic images were digitized at a resolution of 600 ppi (corresponding to a final resolution of 2.11 pixels per micrometer, with the 20/0.5 obj), then were spatially calibrated using images of a standard micrometer object as reference.

For morphometric evaluation, at least three microscopical fields from each H/E and Picro-sirius stained section were digitized with the 20/0.5 plan-neofluar objective, serial images were then taken with the 40/0.75 objective. Therefore, a minimum of 15 fields was taken from each bioptic sample, which corresponded to a tissue area of 3.3 mm2 with the 20/0.5 objective, with a similar number of near longitudinal and near transversal oriented myocytes.

In order to evaluate the antigen distribution and the immunostain intensity in the tissue, a minimum of three stained sections for each case was observed, and at least 12 microscopical fields with the x20/0.5 objective for each case were digitized and analyzed corresponding to a tissue area of 2.6 mm2.

The guidelines reported by Chieco [21], relative to absorbency image cytometry, were observed in the setting of the hardware and in the calibration steps.

A glass interference filter (Oriel Corporation, Stratford, CT, USA; cod. 53850), transmitting at 480 nm with a bandwidth of 10 nm at half-peak transmission, was positioned in the field diaphragm plane to obtain monochromatic light at a wavelength corresponding to the absorbency maximum of the DAB [22].

The sampling methods and stereologic formulas applied for calculation were derived from Loud and Anversa [23–25].

The estimation of the fractional area (AA), equal to the myocyte fraction volume (VV) in the bioptic samples, was performed with a stereologic square lattice point grid, by counting the fraction of points overlying the myocyte compartments of the tissue. The same method was used to evaluate the percentage of myocardium volume occupied by interstitial tissue. The direct count of the number of nuclear profiles on the area measured was used to calculate the mean number of myocyte nuclei per unit area (mm2) of myocytes, N (n)A. Mean nuclear diameter D, was calculated from the area of the nuclear profiles, assuming a spherical shape and estimating the particle diameter to be D=4/µ d (d, profile diameter) [24]. Myocyte diameter was measured orthogonal to cell profiles and estimated, assuming a cylindrical shape, to be D=4/µ d (d, profile diameter). Due to the small number of longitudinally sectioned myocyte nuclei it was necessary to have both diameter estimates.

2.1.4. Evaluation of the immunostaining
The antigen distribution was evaluated on density calibrated monochrome images. Each image was converted in pseudocolors by fixing the color palette at the staining intensity of the tissue background. All the points of a morphometric grid overlying the stained regions of the myocyte compartment were counted. This point fraction was related to the points of the overall myocyte compartment, and expressed as percent staining of myocyte cytoplasm.

Staining intensity was measured on the same compartment as absorbency at 480 nm of image pixels. The results for each individual case were the average of values obtained from each image processed. The results are presented as mean (S.D.) of all the cases included in the study group.

2.1.5. Test specimens
A test system was designed and evaluated as described by Huang [26], to assess the reliability of densitometry in order to detect changes in tissue antigen content as a function of staining intensity.

2.1.6. Reverse polymerase chain reaction
Total RNA was isolated by lysing the frozen heart tissue samples (150–300 mg) in Trizol solution (Life Technologies, GIBCO BRL) according to the supplier's protocol. RNA was precipitated and quantitated by spectroscopy. 2 µg of total RNA of each sample was reversely transcribed using the First-Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech) according to the protocol supplied by the manufacturer. The random hexamer primers provided in the kit were used.

Three microliters of cDNA was amplified using desmin-specific 5' sense (ACA ACC TGC TCG ACG ACC T) and 3' antisense (GGC AGT GAG GTC TGG CTT AG) cDNA primers, or GAPDH-specific 5' sense (TTC ACC ACC ATG GAG AAG GCT) and 3' antisense (ACA GCC TTG GCA GCA CCA GT) primers, as a control. Semiquantitative polymerase chain reaction (PCR) was performed using the following conditions: 95 °C, 3 min initial denaturing phase; 95 °C, 40 s; 62 °C, 20 s; 72 °C, 30 s for 25 cycles; 72 °C, 5 min, final extension. The reaction was carried out in a total volume of 50 µl containing 3 µl of cDNA, 10–20 pmol of each primer, 200 µM each of dNTP, 1.5 mM MgCl2 and 1 unit of Taq polymerase with the reaction buffer supplied with the kit.

Twelve microliters of the amplified products were analyzed by electrophoresis in a 2% agarose gel containing ethidium bromide, followed by photography under ultraviolet illumination. The levels of desmin mRNA were estimated by densitometric scanning and normalized against GAPDH loading controls.


   3. Statistical analysis
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Statistical analysis
4. Results
 5. Discussion
 References
 
All data were collected blind. Results are shown as mean (S.D.) of the number of cases in the study groups, obtained from the average measurements of each biopsy sample. Comparisons between the means were performed with the non-parametric Wilcoxon two-sample test. Correlation coefficients between each set of data were determined by the Spearman rank regression analysis. Values of P<0.05 were considered as significant.


   4. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Statistical analysis
 4. Results
5. Discussion
 References
 
The baseline characteristics of the patients with ICM was comparable to the control group for sex, age, body surface-area, blood pressure and heart rate.

The 18 patients with ICM, were all in NYHA class IV. All patients were treated with digitalis, diuretics, ACE-inhibitors and beta-blockers. At EKG, 12 patients presented with signs of previous anterior MI, five patients showed atrial fibrillation, four had a complete left branch block, 11 had ST-T changes, nine patients had left ventricular hypertrophy, and two patients had a I degree AV block. Coronary angiography confirmed the presence of significant coronary lesions in all ICM patients, and in particular, the presence of occlusion of the left anterior descendent coronary artery in each patient.

At echo-Doppler examination, LVEF was significantly lower (P<0.01) in the ICM group than in the control group (26.3±11.4 % vs. 53.5±8.6%, respectively). In the ICM group there was a 1.8-fold increase in left ventricular mass as compared to the control group; the left ventricular mass-to-body surface area was increased by 109%.

The transverse left ventricular chamber diameter at end-diastole was increased by 43, and the computed chamber volume expanded 3.8-fold; this reduced both the LV mass-to-LV volume ratio and LV wall thickness-to-chamber radius ratio.

4.1. Pathology
At gross examination, the hearts obtained from ICM patients appeared dilated and had a mean weight of 478.3±171.7 g (range 300–750 g) 700 g. The firm scar was evident in the anterior wall of the left ventricle of all infarcted hearts. The morphological measurements are listed in Table 1.

4.2. Light microscopy
Light microscopy evaluation of left ventricular myocardial tissue-specimens showed hypertrophic changes in myocytes, with nuclear enlargement (Fig. 1b) in ICM patients but not in controls (Fig. 1a). In the ICM group, the interstitial compartment appeared expanded, mainly because of interstitial fibrosis, as confirmed by Picro-sirius images (Fig. 2).


Figure 1
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  		Fig. 1 Immuno-histochemistry for desmin myocyte content (confocal microscopy 40X) showing normal desmin distribution in control patients (a) and altered desmin distribution in ICM patients (b). In (b), a desmin-free myocyte is present (lower arrow) and a myocyte with increased desmin content (upper arrow). Enlargement of myocytes is also evident in (b) compared to (a).

 


Figure 2
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  		Fig. 2 Accumulation of connective tissue (Picro-sirius red) from myocardial tissue of ICM patients.

 
The number of myocyte nuclei per unit area of myocytes was reduced by 38% (P<0.001) in ICM patients compared to controls. The average myocyte nuclear area in ICM patients increased by 53% as compared to controls (P<0.005).

There was a significant increase in the myocyte diameter in ICM compared to controls; the increase in diameter of myocytes was 41.5% (P<0.002).

4.3. Immunocytochemistry
4.3.1. Desmin
In cardiac myocytes from the control group, the immunostaining against desmin was localized to Z lines, resulting in a regular pattern of cross-striation (Fig. 1a). Furthermore, in many cells, an increase in desmin staining at the cell ends labeled the intercalated disks. In myocytes from ICM patients, the irregular pattern of desmin staining resulted in a scattered aspect (Fig. 1b). In fact, cell staining was either reduced or absent in some cytoplasmic regions, which contrasted with marked staining in both the perinuclear region and intermyofibrillar spaces where clumps of filaments were present (Fig. 1b).There was a significant reduction in the number of desmin-positive myocytes in ICM as compared to controls. This was due to the presence of a high number of desmin-negative cells in the ICM group (Fig. 3) compared to controls (Fig. 1a) (4.5+2.7% vs. 0.3+02%, P<0.001). Furthermore the distribution of the desmin-free myocytes was scattered with some areas of myocardium completely negative for desmin (Fig. 3a,b). A double staining for desmin (immunofluorescence, green) and actin (immunofluorescence, red) at confocal image confirms that this was not a fixation artifact (Fig. 4)


Figure 3
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  		Fig. 3 (a) Immuno-histochemistry for desmin (light microscopy 40X) showing altered desmin distribution in ICM patients with desmin-free myocyte (arrow). In (b), areas of desmin-free myocytes are present.

 


Figure 4
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  		Fig. 4 Confocal image of double immunofluorescence stained human myocytes. Actin filaments are marked red (TRITC) and desmin filaments are marked green (FITC). Free desmin myocyte is indicated by white arrow. Normal red and green marked myocyte is indicated by black arrow.

 
When the staining distribution was evaluated by morphometric sampling, the percentage of the area of the myocytic cytoplasm reacting with monoclonal desmin antibody was 98% in the control group and 52% in ICM patients (P<0.002).

In summary, in ICM, the myocyte content of desmin intermediate filaments was reduced compared to controls, and irregularly distributed.

There was a negative correlation between the amount of myocardial desmin and the ejection fraction (r=–0.773; P<0.02), whereas a positive relationship was found between desmin distribution and EF (r=0.764; P<0.02). Furthermore a negative relationship was found between the number of desmin-free myocytes and capillary wedge pressure at right ventricle heart catheterization (r=–0.62; P<0.01. Moreover, at real-time PCR, the desmin content on myocardial tissue of patients with ICM was significantly reduced compared to controls (0.96±0.45 vs. 2.073±0.31; P<0.001) (Fig. 5).


Figure 5
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  		Fig. 5 Comparison of desmin PCR between controls and ICM group. *=P<0.001. ICM group vs. controls=0.96±0.45 vs. 2.073±0.31 OD (mean±S.D.).

 

   5. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Statistical analysis
 4. Results
 5. Discussion
References
 
The main histological finding of our study on myocardial tissue from patients with end stage heart failure due to ICM compared with normal tissue, was a significant decrease in the amount of desmin in diseased myocardial compared to normal tissue. This result was confirmed both at computer confocal microscopy evaluation and at real time PCR. This result is quite new and does not confirm previous results obtained by other authors [2,10], which have mainly shown an increase in desmin and other cytoskeletal protein tissue in end stage heart failure. However, these studies are mostly in patients with DCM [27], who may have a desmin mutation as the basic pathology of their disease [28–30]. Nevertheless, in these studies in DCM, it was clearly reported that mutations in the desmin gene could be responsible for a skeletal and cardiac myopathy, characterized by fragility of the myofibrils due to a deficit in cytoskeletal protein. In particular, Dalakas et al. [29] concluded that a defect in the function of the desmin determines a reduction in the effect of this protein in protecting the structural integrity of the myofibrils during repeated muscle contraction over time.

Therefore it seems that in the myocardium of DCM patients it is possible to reproduce a situation comparable to that which we have found in our study of ICM patients in areas of myocardium where desmin is absent.

In fact, we have described areas of total desmin absence, which represent, in our opinion, the most important result of this study. To our knowledge, this is the first study to report an absence of desmin in the myocytes of myocardium with end-stage heart failure. It is possible that the late phase of chronic ischaemic disease could be associated with an inability of the cell to ensure a ‘compensatory’ growth in desmin content adequate for the increased cell size. In other words, it seems that the desmin protein has exhausted its compensatory reserve.

In normal hearts, desmin forms an interconnected network that, through longitudinal and transverse elements, keeps the myofibrils at Z disk in axial register [31]. Morphological studies have shown that desmin is strictly connected with sarcomere at the Z disks, and that the intermediate filaments play a ‘regulatory’ role [32,33] on the normal contractile function of the myofibrils. In other words, we can assume that there is a myofibril–desmin relationship in the sarcomere, whose integrity is essential for the normal contraction of the myocyte. Furthermore, this hypothesis is strengthened in our study by the positive correlation between desmin distribution and ejection fraction at echocardiographic evaluation. Cytoskeletal alterations have been reported to be responsible for the cellular contractile dysfunction in cardiac hypertrophy in response to pressure loading [6,34]; the persistence of this alteration could be significant for the deterioration of initial compensatory cellular mechanism leading to heart failure [5]. Studies of the intermediate filaments in hypertrophic myocardium have shown an increase in the amount of desmin, in an attempt to keep the newly formed sarcomeres aligned, and to keep the myofibrils in register during the contraction–relaxation process [35]. Moreover, Collins et al. have shown a further increase in desmin in the transition from compensated left ventricular hypertrophy to heart failure [6]. The importance of a regular desmin distribution for normal heart function has also been demonstrated by our group [12] in patients with DCM. In that study, we also showed an irregular distribution of myocardial desmin content responsible for actin–myosin interaction alteration. In this study, we confirmed that desmin content is altered in the myocardial tissue of patients with end stage heart failure; but these alterations seem to be different according to the aetiology of the disease. Therefore, it seems possible to distinguish two different histological situations in the myocardium of the two diseases: (1) in DCM the desmin distribution is irregular and increased; (2) however, in heart failure due to ICM there is a percentage of cardiomyocyte completely free of desmin, while the irregularity of the distribution is confirmed in the other hypertrophic cardiomyocytes. It should be interesting in the future to speculate on the reasons for this difference.

The decrease in desmin content was confirmed in our study by confocal microscopy evaluation both at immunocytochemistry and double staining with actin to exclude artifacts of fixation and by quantitative PCR real time technique. Furthermore, as we have already shown in DCM, in end stage heart failure due to chronic coronary artery disease, the desmin alteration is responsible for left ventricular dysfunction as demonstrated by the negative relationship between the number of desmin free myocytes and EF.

This result confirms the importance of this intermediate filament on normal contractile function of the myofibrils.

The presence in our study of a positive relationship between desmin distribution and LVEF seems to emphasize the pathophysiological significance of the intermediate filament alterations in ICM. In fact, we can speculate that the degree of intermediate filament involvement in cardiac tissue specimens could be used as a marker of unfavorable evolution of cardiac myopathy, and the endomyocardial biopsy could represent a useful monitoring tool. This theory increases the diagnostic value of the endomyocardial biopsy in cardiomyopathies, as many authors have recently suggested [36], by complementing the histological data with immunochemical and molecular biology studies [37–39]. For example, in the future, evaluation of the desmin content of samples, obtained ‘in vivo’ through the endomyocardial biopsy, could be routinely used in the decision-making process for patients with ICM who are on the waiting list for heart transplantation. The number of desmin-free myocytes could be used as additive criteria for the ‘selection’ of the patients. In our opinion, the clinical relevance of our results comes from the negative correlation observed between capillary wedge pressure on right cardiac catheterization and the number of desmin-free myocytes. In other words, it seems that the deficiency in desmin content in the myocardium could lead to an increase in left ventricular diastolic pressure due to reduced left ventricular contractility.

In conclusion, the changes in intermediate cytoskeletal filaments observed in the myocardial tissue from patients with ICM are represented by a decreased number of myocytes positive for desmin as compared to normal human tissue.

The increase in myocyte dimension and the increase in the number of desmin-free cells with consequent desmin disarray lead to the reduction in EF, confirming the inability of the myocyte to provide a valid contraction in the absence of a normal distribution of this cytoskeletal protein.

The clinical implication of these results is that the ‘in vivo’ evaluation of the integrity of intermediate filaments of the myocyte could represent a marker of the severity of cellular dysfunction in dilated cardiomyopathy due to coronary artery disease. This desmin deficiency could represent a late stage of myocyte degeneration with consequent inability of the cells to contract.

A future therapeutic target will be to preserve cytoskeletal intermediate filament integrity to prevent the progression towards irreversible myocyte damage. Future studies evaluating desmin content from cardiac biopsies may be helpful in decision-making for patients who are on the waiting list for heart transplantation.


   Acknowledgements
 
We are grateful to Dr A. Luongo for technical support in collecting and preparing the histological samples. This study was supported by a grant from The Italian National Ministry of Research.


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Statistical analysis
 4. Results
 5. Discussion
 References
 

  1. Ganote C, Armstrong S. Ischaemia and the myocyte cytoskeleton: review and speculation. Cardiovasc Res (1993) 27:1387–1403.[Free Full Text]
  2. Schaper J, Speiser B, Brand T. The cytoskeleton and extracellular matrix in human hearts with dilated cardiomyopathy. In: Idiopathic dilated cardiomyopathy (1993) Berlin, Germany: Springer-Verlag. 75–80.
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