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5  DISKUSSION

Myosin is a motor protein, which acts as the cross-bridge in the contractile apparatus of all muscle types. It interacts cyclically with the thin (actin) filament thus producing force and shortening while consuming ATP. Myosin cross-bridge action therefore represents an important process determining cardiac systolic and diastolic function (Lowey et al. 1971). Two types of myosin light chains (MLC), essential and regulatory, are associated with the neck region of the myosin heavy chains (MHC) (Moncrief et al. 1990).

Expression of the atrial-specific essential MLC (ALC-1) is tissue specific and developmentally regulated. ALC-1 is expressed in large amounts in the whole heart of human embryos (Barton et al. 1985), then is only restricted to the atrium in early postnatal development (Fallot 1963). The hALC-1 gene is located on chromosome 17q21b and encodes a protein of 196 amino acids (Seharaseyon et al. 1990). Regulation of cardiac contractility in the human ventricles by ALC-1 provided an alternative to the MHC regulatory mechanisms seen in a rodent-overloaded heart that responds by a MHC isoform shift from α-MHC (high ATPase activity) to β-MHC (low ATPase activity) (Morano et al. 1997; Morano et al. 1996; Sutsch et al. 1992). It was previously reported that human ventricular skinned fibers revealed an increase in maximal shortening velocity, rate of tension development, isometric force generation, and calcium sensitivity of isometric force generation upon partial replacement of VLC-1 by ALC-1 (Morano et al. 1997; Morano et al. 1996). Furthermore, there was a significant positive correlation between ALC-1 expression and dP/dtmax in patients with hypertrophic obstructive cardiomyopathy (HOCM) (Ritter et al. 1999).

The functional properties of the human atrial essential myosin light chain (hALC-1) has never been evaluated in an intact heart preparation at the whole organ level. Only correlations between the hALC-1 and hemodynamic data have been reported, such as systolic, end-diastolic pressure, end-diastolic volume index, mean pressure gradient and wall stress (Brodie et al. 1977; Grossman et al. 1972). Previous research has identified the beneficial functional effects of hALC-1 in-vitro cardiac preparations (human ventricular skinned fibers) of patients expressing hALC-1 (Morano et al. 1997; Morano et al. 1996; Ritter et al. 1999). Cardiac-specific overexpression of mouse ALC-1 was accomplished in transgenic mice. The changes at the motor protein and fiber levels [page 66↓]were translated into changes in the contractile function of the whole mouse heart, represented by an increase in the contractile parameters. At the skinned fiber level of this transgenic mouse model, 95% replacement of the mouse VLC-1 by the mouse ALC-1 was accompanied by 1.78-fold increase in maximal shortening velocity (Vmax) compared to the control group (Fewell et al. 1998). On the other hand, it was previously observed that around 20% replacement of the human VLC-1 by the human ALC-1 increased Vmax of skinned fibres 1.88-fold (Morano et al. 1996).

This functional discrepancy between the human and mouse ALC-1 was supported by a considerable amino acid sequence variation of the atrial essential myosin light chains in both species (Fig. 21). Interestingly, the human-mouse amino acid differences are mostly located at the N-termini of both sequences, with a close similarity between the mouse and rat ALC-1. It has been demonstrated that the amino-terminal domain of the MLC-1 isoforms interacts with the carboxyl-terminal domain of actin (Stepkowski 1995; Sutoh 1982; Timson et al. 1998). This interaction could be of functional importance since inhibition of this interaction using synthetic peptides increased the force production and the shortening velocity of human heart fibers (Morano et al. 1995), which may explain the functional variation in both the human and mouse ALC-1.

Besides the main interest in the molecular therapy of human heart disease, the considerable difference between the human and mouse ALC-1 sequences and function, provided the initiative to undertake this study aiming to characterize, for the first time the effect of human ALC-1 at the intact whole heart contraction level. To achieve this aim, transgenesis was used to direct an essential light chain isoform switch in the rat heart.

Analyzing the morphological data of the transgenic model, no significant difference in the heart weight/bodyweight relationship between the TGR/hALC-1 and control animals was observed, which suggests the absence of hypertrophic response in the hearts of the rat models. Moreover, to confirm the absence of hypertrophy, the expression of alpha-myosin heavy chains, which is essentially down-regulated during hypertrophy (Swynghedauw 1986) was studied by using quantitative Western blot analysis. No change in the expression levels of alpha-MHC in TGR/hALC-1 compared to WKY could be detected.


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Figure 21 . Amino acid sequence comparison of the human, mouse and rat atrial essential myosin light chains (ALC-1):

Note the significant variability between the human and mouse sequences at the amino termini, which may explain the attenuated ALC-1 function in the mouse compared to the hALC-1. Also a close similarity of the mouse and rat sequences was observed.


[page 68↓]

A previously published study showed that transgenic overexpression of MLC at high-level results in severe cardiac pathology (James et al. 1999). These findings are in agreement with mine due to the moderate expression levels that were obtained in my TGR/hALC-1 model. However these results stand in opposition to the same author’s previous findings, which confirmed the absence of any hypertrophy in the transgenic mice with 95% replacement of the endogenous light chains by the transgene (Fewell et al. 1998).

I observed a significant improvement in the contractile parameters of the 12-24-week old TGR/hALC-1 compared with age-matched WKY animals. Interestingly, aged TGR/hALC-1, especially 36-week old animals, revealed no significant differences in their contractile parameters when compared to age-matched WKY. This normalization of heart function with age could be explained, firstly by the age-dependent attenuation of transgene expression discussed below, and secondly by the age-dependent increase in contractility in the WKY rats, which is in accordance with the findings of previous studies (Nair et al. 2001; Qi et al. 1997).

The 12 weeks old TGR/hALC-1 expressed a reasonable amount of hALC-1 in their ventricles, but a decline in the transgene expression upon aging was observed, that was closely associated with a decline in the improved contractility seen in young TGR/hALC-1. This confirms that the improved contractile functions were associated with the hALC-1 expression. The phenomenon of age-dependent transgene down-regulation was also observed in a previous study using the same α-MHC promoter (Hoffmann et al. 2001). This observation could be due to the attenuation of alpha-MHC expression in WKY rats with age (Morano et al. 1988). I suggest that the improved contractile functions observed in the transgenic rat model were due to the hALC-1 expression rather than to abnormal gene modification(s) caused by random transgene integration. Except for the transgene, there were no obvious changes in the expression levels of the proteins in TGR/hALC-1 compared to the WKY rats as revealed by 2D-proteome analyses. The hALC-1 transgenic protein was localized by immunofluorescence analysis, which showed that hALC-1 was correctly incorporated in-between the Z lines of the sarcomere of the ventricular contractile apparatus.


[page 69↓]

Furthermore, to confirm the incorporation of the transgenic protein into the ventricular myosin, myosin was purified from the ventricles of transgenic and control rats. An immunoblot for these pure myosin preparations was performed using specific anti-hALC-1 antibodies. The antibodies recognized the transgenic hALC-1 proteins within the purified myosin of the transgenic rat. The persistent existence of the transgenic hALC-1 proteins within the myosin preparation after high ionic strength purification and subsequent ammonium sulfate precipitation suggests that hALC-1 remained incorporated within the myosin. This confirms the correct association of hALC-1 and binding to the ventricular myosin heavy chains.

In a different set of analytical studies using immunofluorescence microscopy, a mosaic pattern of expression for hALC-1 was observed in the cardiomyocytes. These variegated expression patterns have been commonly observed in various transgenic studies in mammals (Dobie et al. 1996; Opsahl et al. 2002).

The MLC-1/ MLC-2 ratio was similar in WKY and TGR/hALC-1 ventricular purified myosin preparations, thus the ectopically expressed hALC-1 replaces the endogenously expressed rat VLC-1 with no net increase in the whole MLC-1 content. These data are compatible with previous results in which different myosin light chains were expressed in human ventricles (Morano et al. 1996) and transgenic mice (Gulick et al. 1997). In 12 weeks-old TGR/hALC-1, I observed around 20% replacement of the rat left ventricular VLC-1 by hALC-1 in purified myosin preparations. Meanwhile, hALC-1 represented about 37% of rat left ventricular whole MLC-1 in whole SDS-soluble transgenic ventricular tissue extracts. Therefore, I suggest that a fraction of the expressed transgene (about 1/3) seems to exist in a free non-bound state in cardiomyocytes.

The moderate replacement of rat VLC-1 by hALC-1 in the 12 weeks-old rat model was accompanied by marked alterations in whole heart mechanics, represented by significant increase in the pressure development, contraction rate, and relaxation rate.These data are consistent with the previous idea that partial replacement of endogenous MLC-1 by hALC-1 is sufficient to improve the contractile functions of the heart (Morano et al. 1996). Furthermore, it is demonstrated that the hALC-1 in rat is more functionally efficient than the corresponding mouse essential myosin light chain isoform in mouse: maximal contraction and relaxation rates were increased by around [page 70↓]100% upon only 20% replacement of endogenous MLC-1 with hALC-1. In contrast, the same functional parameters were only increased by 40-50% upon 95% replacement of mouse VLC-1 by mouse ALC-1. Hence, a moderate expression of hALC-1 could generate a much stronger increase of contractility of perfused intact hearts than an excessive expression of the corresponding mouse ALC-1.


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