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Cardiovascular Research 2002 53(2):439-450; doi:10.1016/S0008-6363(01)00500-4
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Copyright © 2001, European Society of Cardiology

Hearts from mice lacking desmin have a myopathy with impaired active force generation and unaltered wall compliance

J Balogha, M Merisckayb, Z Lib, D Paulinb and A Arnera,*

aDepartment of Physiological Sciences, Lund University, BMC F11, Tornavägen 10, SE-221 84, Lund, Sweden
bUniversity Paris VII, Paris, France

* Corresponding author. Tel.: +46-46-222-7758; fax: +46-46-222-7765 anders.arner{at}mphy.lu.se

Received 6 March 2001; accepted 4 October 2001


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Desmin intermediate filaments are key structures in the cytoskeleton of cardiac muscle. Since they are associated with Z-discs and intercalated discs, they may have a role in sarcomere alignment or force transmission. We have explored the mechanical function of the desmin filaments in the cardiac wall by comparing desmin-deficient (Des–/–) and wild-type (Des+/+) mice. Methods: The Langendorff technique was used to examine the contractility of the whole heart. Rate of force generation, Ca2+-sensitivity and force per cross-sectional area were measured in skinned ventricle muscle preparations. Results: Des–/– mice have a cardiomyopathy with increased heart weight. Diastolic pressure was increased at all filling volumes in the Des–/– group. Since passive wall stress (i.e. force per area) was unchanged, the alteration in diastolic pressure is a consequence of the thicker ventricle wall. Developed pressure, rate of pressure increase and developed wall stress were significantly reduced, suggesting that active force generation of the contractile apparatus is reduced in Des–/–. Concentrations of actin and myosin in the ventricle were unaltered. Measurements in skinned muscle preparations showed a lower active force development with unaltered Ca2+-sensitivity and rate of tension development. Conclusion: It is suggested that the intermediate filaments have a role in active force generation of cardiac muscle, possibly by supporting sarcomere alignment or force transmission. The desmin filaments do not contribute the passive elasticity of the ventricle wall. Des–/– mice provide a model for genetic cardiomyopathy where the main factor contributing to altered cardiac performance is a decrease in active force generation, possibly in combination with a loss of functional contractile units.

KEYWORDS Cardiomyopathy; Contractile apparatus; Contractile function; Heart failure; Mechanotransduction


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Contraction of cardiac muscle is the result of interaction between the main motor proteins, actin and myosin. Extensive research has characterized the molecular mechanisms involved in this biological energy conversion, and its regulation via the thin filament associated proteins. However, in the organized contractile apparatus in the cardiac muscle cells, the contractile sarcomere units have a complex mechanical interaction with the cytoskeleton. In the heart, especially in the Purkinje fibres the cytoskeletal intermediate filaments are abundant [1,2]. The intermediate filament system, composed of the protein desmin in striated muscle, has been found to integrate with the Z-disc structures of the sarcomere units [2,3]. The detailed mechanical functions of the intermediate filaments are not fully understood, but it has been suggested that they have a role in the alignment of sarcomeres [2,3] in addition to other possible cellular functions, e.g. modulating mitochondrial metabolism [4]. In the heart, intermediate filaments are found in the vicinity of the Z-discs and also in association with the intercalated discs [1]. Interestingly, abnormalities in the intermediate desmin filaments are found in several human cardiomyopathies. It has been shown that desmin filaments increase during cardiac hypertrophy and that some forms of myopathy are associated with increased amount of non-functional desmin filaments in inclusion bodies [5–7]. Mutations in the desmin gene have been described in some patients with desmin-related cardiomyopathy [8–10] and recently a mutation in the tail region of the desmin gene was found in a case study of a family with dilated cardiomyopathy [11]. However, the desmin-related myopathies are not always associated with mutations in the desmin gene and mutations in genes encoding associated proteins preventing the formation of desmin filaments also cause desmin-related myopathy [12].

The development of a transgenic mouse with a null mutation in the desmin gene has recently introduced a new approach to study the function of desmin in muscle tissue [13,14]. These animals develop normally but exhibit a cardiomyopathy associated with degeneration of cardiomyocytes, fibrosis and calcification [13–15]. These results suggest that the intermediate filament system is important for the structural integrity of the cardiomyocytes, a loss of desmin in the region of the intercalated discs has been suggested to lead to lysis and apoptosis of the cardiomyocytes [15]. The cardiac alterations in the desmin-deficient mice involves cardiac myocyte hypertrophy and an expression pattern of cellular markers similar to those of pressure overload hypertrophy [14]. A change in expression of myosin heavy chains toward slower isoforms has been found in heart and soleus muscle of desmin-deficient mice [16]. Echocardiography in anesthetized animals has revealed an impaired systolic function in older desmin-deficient mice [14], which may relate to a mechanical dysfunction of the cardiac muscle. Force generation of skeletal muscles has been shown to be decreased in the desmin-deficient mice [17]. Smooth muscle from these mice has an impaired active force generation suggesting that the intermediate filament system in the smooth muscle has a role in the mechanical performance of the contractile machinery or in the cellular force transmission [18]. It has been suggested that post-ischemic stunning of the heart involves proteolytic degradation of cytoskeletal proteins including desmin [19] and proteolytic degradation of desmin in cardiac muscle cells from rat has been shown to be associated with a decrease of active force [20]. It is thus possible that desmin in the cardiac muscle has a mechanical function in active force generation similar to that proposed for skeletal and smooth muscle. At present very limited mechanical data is available from cardiac muscle from desmin-deficient mice.

The aim of our study was to determine if desmin intermediate filaments have a role in maintenance of passive, diastolic, tension and in the generation of active, systolic, tension in the cardiac wall. The results could further explain the functional alterations in desmin-related cardiomyopathies. We have examined active and passive mechanical properties of heart muscle from normal and desmin-deficient mice. To study the performance of the intact heart we used the Langendorff retrograde perfusion technique and determined diastolic and systolic pressures at different filling volumes and beating frequencies. To assess the calcium sensitivity, contractile kinetics and force generation of the contractile system, without possible influence of other cellular processes, we also performed measurements on chemically skinned cardiac muscle preparations. Parts of these results have been presented in preliminary form [21].


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Animals
Transgenic desmin-deficient mice (Des–/–) were obtained of the strain C57BL/6J in the laboratory of Drs Paulin, Li and co-workers at University Paris VII [13]. A desmin gene construct was inserted in frame in a vector, which was transferred to embryonic stem cells via electroporation. Cells, with one mutated desmin gene inserted via homologous recombination, were micro injected into 3.5-days-old blastocysts of C57BL/6J mice. The homozygous Des–/– mice were obtained from back-crossing of the chimera mice. The animals used in the present investigation were all adult female mice, aged between 5 and 6 months. The animals were kept in the university animal facilities with free access to food and water according to regulations of the local animal ethics committee. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.2 Collagen staining
Hearts were fixed in 4% formaldehyde for 2 h and embedded in paraffin. Sections (5-µm-thick) were stained for 30 min in 0.1% Sirius Red solution.

2.3 Determination of contractile protein contents
Ventricular tissue from Des+/+ and Des–/– mice was rapidly frozen in liquid N2. The samples were weighed and homogenized (50 µl/mg wet weight) in a solution containing: 25 mM Tris–HCl (pH 6.8), 10% glycerol, 5% mercaptoethanol, 1 mg/ml Bromophenol Blue and 2% sodium dodecyl sulfate (SDS). The homogenates were boiled for 2 min, centrifuged and separated on 8% polyacrylamide gels using a BioRad MiniGel system (BioRad, Richmond, CA, USA). Three different volumes of sample and four concentrations of actin standard (skeletal actin) were loaded on the gel. The gels were stained with Coomassie Blue, destained, scanned and evaluated using a GS-710 calibrated imaging densitometer and Quantity One software (BioRad). The areas of the peaks corresponding to myosin heavy chain and actin were evaluated.

2.4 Langendorff heart preparation
The Langendorff technique [22] was performed essentially as described by Brooks and Apstein [23]. The animals were weighed and anesthetized with an intraperitoneal injection of pentobarbital sodium 60 mg/ml (0.13 mg/g body weight). The heart was excised and immersed in ice-cold Krebs–Henseleit solution (in mmol/l): 4.74 KCl, 25 NaHCO3, 1.19 MgCl2, 1.19 KH2PO4, 11 glucose, 118 NaCl, 2.5 CaCl2. While still in ice-cold solution the hearts were mounted on a catheter, PE60 tube, inserted into the aorta just below the aortic arch. The heart was perfused with Krebs–Henseleit solution (filtered through Sterile Acrodisc, Gelman, 0.2 µm pore size) and oxygenated with O2/CO2 (95%/5% resulting in a pH 7.4) at 37°C. The perfusion pressure was adjusted to 80 mmHg according to previous studies [23].

When the retrograde perfusion was established a balloon (polyethylene) approximately 10 µm thick, tied to a PE50 catheter connected to a pressure transducer, was inserted into the left ventricle through the left atrioventricular opening via an enlargement of the pulmonary vein. The balloon was made as small as possible to obtain a low pressure when filled to maximal volume. Fig. 2 shows that pressure from the balloon alone was negligible when filled to half of the maximal volume and contributed with about 30% of the total diastolic pressure at maximal filling volume. The balloon was filled with a saline solution (0.9% NaCl) to a maximum volume of 39.2 µl with increments of 8.5 µl. The hearts were electrically stimulated using a Grass stimulator and platinum electrodes against the cardiac wall. Stimulation voltage was set to 20% above threshold and impulse duration to 4 ms.


Figure 2
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  		Fig. 2 Original pressure recordings from the left ventricle of a Des+/+ (A) and a Des–/– (B) heart. The hearts were stimulated at 3 Hz and filled with 8.5 µl volume increments (balloon volume is indicated above the records). The pressure recordings show steady-state total pressure (balloon+heart) for 2.5 s preceding the next increase in volume. The straight lines in the diagrams show the pressure recorded from the balloon without the heart at each volume.

 
The pressure recordings from the ventricle, (systolic and diastolic) were used to calculate the wall stress according to Strömer et al. [24]. The internal radius (r) of the left ventricle was derived from the balloon volume (Vb) assuming a spherical shape and using the cubic formula:

Formula

The wall thickness (d) was calculated from the total left ventricle volume:

Formula
where the volume of the ventricle (Vwall) was obtained from the left ventricle weight (LVW, including free wall and septum) and the density ({rho}, 1.05 g/ml):

Formula

Formula

Left ventricle wall stress ({sigma}) was calculated according to Laplace's law from the intraluminal pressure (P):

Formula

2.5 Skinned cardiac muscle preparations
Muscle preparations from the heart (thin strips of trabecular tissue) were chemically skinned for 12 h at 4°C in a solution containing (in mmol/l): 5.5 ATP, 5.0 EGTA, 20 N-tris(Hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), 6.13 MgCl2, 0.11 CaCl2, 121.8 K–methane sulfonate, 30 phosphocreatine (PCr), 10 Dithioerythritol (DTE) and 10 µg/ml leupeptin, 50% glycerol and 1% Triton as described by Morano et al. [25]. The preparations were thereafter stored at –20°C in a solution as above, but without added Triton.

2.6 Determination of the active force development and Ca2+-sensitivity
Cardiac tissue was gently dissected from the skinned preparations and mounted between a force transducer and a carbon rod using cellulose acetate glue. The preparations were held at 22°C in a relaxing solution with low free [Ca2+] (pCa=9) with the following composition (in mmol/l): [10 ATP, 12.5 MgCl2, 5 EGTA, 20 imidazole (pH 6.7), 12 PCr and creatine kinase 0.5 mg/ml]. In preliminary experiments we stretched the skinned muscles to different passive tensions and activated at optimal [Ca2+] (pCa=4.3, replacing EGTA in the relaxing solution with CaEGTA). We found that maximal force was developed at a length where passive tension just became noticeable. In further control experiments we activated the preparations at this length and stretched or shortened by 10% to confirm that force was measured at optimal length.

For determination of active force development and Ca2+-sensitivity the skinned trabecular muscle was mounted at optimal length and first activated at pCa=4.3 to determine maximal active force. The muscles were then relaxed in pCa=9 solution and subsequently activated at increasing [Ca2+] to determine the Ca2+-sensitivity. The free Ca2+ concentrations were calculated using a program and stability constants presented by Fabiato and Fabiato (1979) and Fabiato (1981) [26,27]. At the end of the experiments the preparations were fixed using 1% glutaraldehyde in relaxing solution, then embedded and sectioned for light microscopy for determination of cross-sectional area. Maximal stress (i.e. maximal force/cross-sectional area) was calculated from the maximal force at pCa=4.3 and the cross-sectional area.

2.7 Determination of the rate of force development
The preparations were mounted as described above. After an initial contraction the preparations were held in a rigor solution containing 100 mmol/l 2,3-butanedione monoxime (BDM) to obtain a rigor tension below 30% of the maximal force [25]. The rigor solution was then changed to a Ca2+-containing rigor solution and finally to a Ca2+-rigor solution with 2 mM p3-1-(2-nitrophenyl)-ethyladenosine-5'-triphosphate (caged ATP) in a 50 µl bath with a quartz window. ATP was released using an UV-flash with duration of 1 ms from a xenon-lamp (Dr G. Rapp Optoelektronik, Hamburg, Germany). The ATP release resulted in a rapid force development and the rate of contraction was analyzed from the digitized force transients.

2.8 Statistics
All values are given as mean±S.E.M., with the number of observations within parenthesis. All curve fitting was performed using non-linear routines implemented in Sigmaplot for Windows (SPSS Inc., Chicago, IL, USA). Statistical comparisons were made using Student's t-test for unpaired observations.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Characteristics of animals and cardiac structure
The desmin-deficient animals (Des–/–) had similar body weights as the wild-type control animals (Des+/+) as shown in Table 1. We observed, during dissection, an increased heart size with calcifications in the cardiac wall in the majority of the Des–/– mice. The Des–/– mice had a significantly increased heart and left ventricle weight (Table 1). Fig. 1 shows pictures of cardiac sections stained with Sirius Red to detect collagen. In the wild-type mouse, localized regions with collagen are present, mainly in the perivascular space. In the heart of the Des–/– mouse deposits of collagen were found in the right ventricle, left ventricle and inter-ventricular septum. Larger deposits of collagen were found in the calcified regions.


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  		Table 1 Characteristics of desmin-deficient (Des–/–) and wild-type (Des+/+) mice

 

Figure 1
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  		Fig. 1 Longitudinal sections of hearts stained by Sirius Red. Darker (i.e. red) staining indicates the presence of collagen. (A) shows a Des+/+ heart where collagen staining was present only in the vessels. (B) shows a Des–/– heart where abundant collagen was present in the degenerated calcified regions. Enlargements of the fields indicated in B are shown in (C–G). Scale bar=500 µm.

 
The contents of actin and myosin heavy chain in the ventricular tissue, determined using quantitative electrophoresis, are shown in Table 1. We determined the actin concentration using skeletal actin as standard and calculated the myosin concentration by multiplying the actin content with the myosin/actin ratio. No significant differences were observed in the concentrations of the contractile proteins between the Des+/+ and Des–/– groups.

3.2 Pressure/volume relationships
Using the Langendorff technique, reproducible pressure volume recordings could be obtained from the hearts of the two groups of mice. The perfusion pressure applied (80 mmHg) gave a similar coronary flow in the two groups Des+/+: 1.7±0.3; Des–/–: 1.5±0.2 ml/min, n=6 both groups). The spontaneous beating frequency of the isolated heart in vitro was not different, 5.4±0.3 (n=5) and 4.3±1.5 (n=5) Hz in the Des+/+ and Des–/– groups, respectively. To obtain stable conditions during the pressure–volume determinations we used electrical stimulation at 3, 5, and 7.5 Hz. Fig. 2 shows original recordings of total pressure in the left ventricle. The pressure recorded from the balloon itself, without heart, indicated as a straight line in the diagram, was subtracted to obtain the diastolic and systolic pressures of the heart for further analysis. After each increase in volume the diastolic and systolic pressures were allowed to stabilize (usually within 5 s) and the diagram shows the pressure immediately before the next increase in volume. As seen in Fig. 2 the Des–/– heart had lower developed (i.e. systolic minus diastolic) pressure and increased diastolic pressures at all volumes except in the highest volume range. For each frequency, we recorded the pressure in the left ventricle at five different filling volumes and Figs. 3 and 4Go show the summarized data for the diastolic and developed pressures. As seen in Fig. 3, higher diastolic pressures were recorded in the Des–/– compared to Des+/+ hearts at similar filling volume in the two groups. As seen in Fig. 4 the developed pressure was significantly reduced in the Des–/– hearts. The developed pressure at optimal volume decreased with increasing stimulation frequency in a similar manner in both groups. To determine the optimal filling volume, we fitted a Gaussian function to the developed pressure–volume relationships of each heart preparation and estimated the optimal filling volume (i.e. the mean value of the Gaussian function, Table 2). Table 2 shows the mean values for the developed and diastolic pressures at optimal volume. The significantly lower developed pressure was associated with a lower rate of pressure change (dP/dt) during the rising and falling phases of contraction (Table 2).


Figure 3
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  		Fig. 3 Diastolic pressure at different filing volumes of the left ventricle of Des+/+ (open circles, n=7) and Des–/– (filled circles, n=8) hearts stimulated at 3 (A), 5 (B) and 7.5 Hz (C). Significant differences are indicated in the diagram (*P<0.05).

 

Figure 4
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  		Fig. 4 Developed pressure at different filling volumes of the left ventricle of Des+/+ (open circles, n=7) and Des–/– (filled circles, n=8) hearts stimulated at 3 (A), 5 (B) and 7.5 Hz (C). Significant differences are indicated in the diagram (*P<0.05; **P<0.01).

 

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  		Table 2 Mechanical characteristics of left ventricles of desmin-deficient (Des–/–) and wild-type (Des+/+) mice

 
The mean wall thickness of the left ventricle was calculated from the volume measurements and the ventricle weight assuming a spherical shape of the ventricle. The wall thickness at optimal volume was significantly increased in the Des–/– left ventricles (Table 2). Diastolic and developed wall stress were calculated at each filling volume from the wall thickness, volume and pressure data as described in Methods according to Strömer et al. [24]. As seen in A–C of Fig. 5 the diastolic wall stress–volume relationship was not significantly different between the Des+/+ and Des–/– hearts. The developed wall stress was significantly decreased at all volumes in the Des–/– hearts (D–F of Fig. 5).


Figure 5
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  		Fig. 5 Diastolic (A–C) and developed (D–F) wall stress at different filling volumes of the left ventricle of Des+/+ (open circles, n=7, except for the highest filling volume n=3–6) and Des–/– (open circles, n=8, except for the highest filling volume n=3–6) hearts stimulated at 3 Hz (panels A, D), 5 Hz (panels B, E) and 7.5 Hz (panels C, F). Significant differences are indicated are indicated in the diagram (*P<0.05; **P<0.01; ***P<0.001).

 
3.3 Force development of isolated skinned trabecular preparations
To explore if the lower developed wall stress of the Des–/– heart determined in the Langendorff preparation experiments was due to alterations in calcium sensitivity or in the mechanical properties of the cardiac muscle, we performed measurements on chemically skinned trabecular muscle strips. The muscles were stretched to a length where passive tension had just become noticeable. Since sarcomere inhomogeneities have been reported for Des–/– muscles [15] we confirmed that this length was optimal for active force development by activating at different lengths. In preparations stretched by 10% from this length active force was for Des+/+: 1.04±0.02 and for Des–/–: 1.03±0.03, relative to the force at the length used in the experiments. Corresponding data for muscle preparations shortened by 10% were Des+/+: 0.80±0.05 and for Des–/–: 0.75±0.02. Similar data were obtained using activation at suboptimal [Ca2+]. These data show that the experiments were performed at the plateau of the length–tension relationships for both groups of preparations. Fig. 6A shows that the active stress generated at maximal [Ca2+] was lower in the Des–/– group. Fig. 6B shows that the Ca2+-sensitivity of force was essentially unchanged in the Des–/– cardiac muscle compared to the Des+/+ group. The active force obtained during the contraction at maximal [Ca2+] at the end of the determination of Ca2+-sensitivity was slightly lower (legend Fig. 6) than the initial response at maximal [Ca2+] in the Des–/– group, which could suggest that the Des–/– skinned trabecular muscle lost some mechanical property during the course of the experiment, although this effect was very small.


Figure 6
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  		Fig. 6 (A) Active stress at pCa=4.3 (i.e. maximal Ca2+-activated force per preparation cross-sectional area) in skinned trabecular preparations from Des+/+ (open bar, n=6) and Des–/– mice (filled bar, n=8, *P<0.05). (B) Ca2+-dependence of active force in skinned preparations from Des+/+ (open circles, n=7) and Des–/– mice (filled circles, n=7) recorded after the initial activation at pCa=4.3, used for determination of active stress (A). Force in B is normalized to the force obtained at maximal [Ca2+] for each preparation. This force at pCa=4.3 at the end of the determination of the Ca2+-dependence of active force relative to the initial response at pCa=4.3 was for Des+/+: 93.9±2.7% and for Des–/–: 81.4±5.4%, n=6.

 
The rate of active force development was determined using photolytic release of ATP from caged-ATP. Release of ATP in the presence of Ca2+ resulted in a rapid force development as shown in Fig. 7A, B. We determined the rate constant by fitting a mono-exponential function to the force development data and (C) shows the summarized results. The average rate of force development was slightly lower in the Des–/– group although not significantly.


Figure 7
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  		Fig. 7 (A, B) Tension transients after photolytic release of ATP in skinned trabecular preparations from Des+/+ (A) and Des–/– (B) animals. Force is normalized to the maximal force reached after the flash. (C) Apparent rate constant for the increase in force after photolytic release of ATP (determined by fitting a mono-exponential function to the tension transients) in skinned trabecular preparations from Des+/+ (open bar, n=11) and Des–/– (filled bar, n=11) animals.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Consistent with previous observations [14,15], we observed that the desmin-deficient mice (Des–/–) have a pronounced cardiomyopathy. Information on cardiac properties from heterozygous (Des+/–) mice is sparse. It has been reported that heart weights and force from smooth and skeletal muscles are similar to the Des+/+ mice [18,17], suggesting that one desmin gene is sufficient to maintain muscle function. In the Des–/– mice we observe hypertrophy of the ventricle wall as well as macroscopic alterations with calcifications. Our histological investigation with Sirius Red shows that a collagen fibrosis is present in the calcified regions of cardiac tissue of the Des–/– mice. This is consistent with previous observations of calcifications in the ventricle wall of Des–/– hearts [13,15,28]. The structural data show that desmin is required for maintenance or development of normal structure of the cardiac muscle. The cause for the hypertrophy and the structural changes is unknown. It is possible that lack of desmin is associated with alterations in cellular physiology leading to cell degeneration. It has been suggested that tissues without intermediate filaments are mechanically unstable and therefore unable to resist physical stress and that this mechanism could be responsible for the cell degeneration in the Des–/– hearts [29]. Possibly the mechanical stability of the intercalated discs is impaired [15] leading to cardiomyocyte degeneration and increased production of collagen by cardiac fibroblasts and cardiomyocytes. The cause for the focal nature of the fibrosis is unknown, but the structural alterations in the Des–/– mice hearts appear regional in nature [15], possibly reflecting that parts of the wall are more exposed to stress in vivo. The structural changes in Des–/– hearts are generally similar to those observed in other forms of cardiomyopathy, as pointed out by Thornell et al. [15], and the myopathy in the Des–/– mice could be the result of a common response to cardiac cell dysfunction. In desmin deficiency the initiating event could be a mechanical failure as discussed below.

We present here functional data from isolated hearts of desmin-deficient mice. We observed two main alterations in the cardiac performance: (i) an increased diastolic pressure and (ii) a lower developed pressure. The weight of the left ventricle and the calculated wall thickness at optimal filling volume were increased which suggest that the functional alterations can be due to a structural remodeling of the ventricle wall. Our measurements of the wall thickness relate to the situation at optimal filling volume. The actual wall thickness in vivo might be different depending on the filling of the ventricle in the beating heart. The relation between diastolic pressure and volume appeared shifted toward lower volume in the Des–/– mice. The calculated diastolic stress (i.e. diastolic force per wall area) was however unchanged at all volumes. This suggests that the alterations in passive properties are due to a thicker wall rather than to a change in the wall compliance. Fibrosis in the cardiac wall might to some extent compensate for loss of cardiomyocytes and contribute to the stability of the wall, but the extent of the fibrosis appears too low to markedly affect the elasticity of the wall. The unaltered volume–passive stress relationship makes it unlikely that a change in wall compliance is responsible for the lower developed pressure in the Des–/– heart. The unaltered diastolic stress–volume relationship of the desmin-deficient hearts enables us to conclude that the desmin intermediate filaments do not contribute to a major extent to the passive elastic properties of the cardiac muscle.

The systolic dysfunction observed in the isolated heart suggests that the desmin-deficient mice would have a compensatory dilatation in vivo in order to maintain systolic pressure, a situation similar to the dilated cardiomyopathy observed in man [30]. We find an increased heart weight suggesting hypertrophy, possibly secondary as a compensatory phenomenon in response to the decreased force. No difference in the spontaneous beating frequency was noted in the Des–/– hearts, consistent with studies using Doppler flow measurements on living anesthetized animals [14]. The lowered developed pressure in the desmin-deficient hearts was associated with a lower active stress which shows that the contractile dysfunction is due to an altered force development of the muscle components in the ventricle wall. The unaltered coronary blood flow makes it unlikely that major alterations in the cardiac vasculature cause the impaired contractile function observed in the isolated hearts. Since pressure did not decrease more in the desmin-deficient hearts compared to the controls when the pacing frequency was increased, the lower developed pressure is not due to a fatigue phenomenon. As discussed in the Results section, sarcomere length distribution might be inhomogeneous in the Des–/– cardiac muscle and we therefore chose to determine the Ca2+-sensitivity in chemically skinned trabecular preparations stretched to optimal length for active force. At this length, the Ca2+-sensitivity was unaltered in the Des–/– preparations excluding major alterations in the thin filament regulatory system as a cause for the lowered active force in vivo. Since sarcomere length was not determined we cannot exclude a small change in the sarcomere length dependency of Ca2+-sensitivity. We have no information regarding the influx or release of Ca2+ in the intact cardiac muscle from desmin-deficient mice. However, the active force of skinned preparations at maximal [Ca2+] was lower in the desmin-deficient hearts. Although it should be considered that the skinned preparations represent trabecular muscle and not the whole ventricular wall, the data suggest that a lower force generation from cardiac cells, independently of the level of activation, contributes to the lower force of the intact cardiac wall in the Des–/– heart.

Structural studies [15] have revealed degeneration of muscle cells in the cardiac ventricle wall of Des–/– mice. It has been observed that the content of myosin heavy chain per mg tissue is lower in Des–/– skeletal muscle [17]. It is thus possible that a part of the lower active stress is due to a loss of contractile cardiac muscle tissue in the Des–/– hearts. We have therefore used quantitative electrophoresis and determined the concentration of actin and myosin in the mouse ventricular tissue. Our measurements agree with previous data from the dog heart [31], although the myosin concentration was slightly lower in the mouse. We could not detect any alteration in the concentrations of these contractile proteins between the Des–/– and Des+/+ mouse hearts. It is possible that the limited extent of cardiac fibrosis might contribute to the lower active force to a small degree. The unaltered contractile protein concentrations suggest, however, that although degeneration of ventricular tissue is present, a decreased quantity of muscle cells and contractile components in the ventricle wall are not the main factors involved in the lower active stress. A second, more likely, explanation for the lower force output in desmin-deficient hearts could be a failure in generation or transmission of active force. Desmin intermediate filaments are anchored in the Z-discs and it has been suggested that intermediate filaments have a role in the alignment of sarcomere units [3]. Also, the intercalated discs of cardiac muscle in Des–/– mice have altered structure [15], possibly suggesting alterations in the mechanical cell–cell coupling. Force output of skeletal and smooth muscle is also significantly reduced in the desmin-deficient animals [16–18]. Our data might therefore be consistent with a model where a lack of alignment of sarcomeres or altered mechanical cell–cell coupling are factors contributing to the lower active force output from the contractile apparatus in the Des–/– hearts.

In the whole heart preparation, during retrograde perfusion, the rate of pressure increase (dP/dt) was significantly reduced. This decrease was proportional to the decrease in active pressure which suggests that the alterations in dP/dt was a consequence of the lower active pressure. However, the lower dP/dt could also be due to alterations in the kinetics of active force generation of the cardiac muscle. In a study by Agbulut et al. [16] it was found that the relative amount of the slower myosin isoform (β-heavy chain) was increased in cardiac muscle from desmin-deficient animals. This isoform switch towards higher expression of the slower myosin isoform could tentatively lead to a slower tension development and increased force due to a slower cross-bridge turnover. The rate of force increase in the presence of Ca2+ after photolytical release of ATP, has been considered to reflect the rate of cross-bridge transition into force generating states. This parameter is correlated with the distribution of myosin isoforms in the cardiac muscle [25]. We therefore examined the rate of tension development in the skinned cardiac muscles and found that the rate was not significantly decreased in the desmin-deficient muscle. This result shows that the re-expression of β-myosin, reported by Agbulut et al. [16] in the Des–/– mouse heart does not influence the cross-bridge cycling rate to an extent that the rate of tension development is significantly influenced. Thus, the low rate of pressure increase in the isolated heart of Des–/– mice is not a consequence of slow cross-bridge turnover, but the result of an impaired absolute force generation.

In conclusion, the desmin-deficient mice provide an animal model for genetic cardiomyopathy with structural and functional alterations similar to those observed in desmin-related cardiomyopathies in man. The results from isolated hearts show that the desmin intermediate filaments do not contribute to the passive elastic properties of the ventricle wall to a major extent. The slow rate of systolic pressure development (dP/dt) in Des–/– hearts is not the result of slow cross-bridge kinetics, but rather a consequence of impaired force generation. The low active force is not due to less contractile components or altered Ca2+-sensitivity, but reflects a structural change in the ventricular wall and a defect in cellular force transmission or in sarcomere alignment.

Time for primary review 23 days.


   Acknowledgements
 
This work was supported by grants from the Swedish Medical Research Council (04X-8268), the Medical Faculty Lund University and AFM (French association against myopathies). We thank Dr Lacolley Patrick for helpful discussions.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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