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4  RESULTS

4.1 Generation and basic characterization of the transgenic rats

The construct shown in Figure 9 was used to generate transgenic rats of the inbred WKY strain. The presence of the hALC-1 transgene in the genome was verified by a specific PCR protocol (Fig. 10) and Southern blot using DNA isolated from the tails of the animals. To the best of my knowledge this is the first transgenic rat model made on an inbred WKY background. Transgenic animals were morphologically normal. In particular, the heart weight/body weight ratio was not significantly different between TGR/hALC-1 and WKY control rats (Fig.11) indicating that the hALC-1 gene did not induce hypertrophic alterations in the heart of the transgenic animals.

Figure 10 . Genotyping of transgenic rats.

Genomic DNA from tail biopsies of a TGR/hALC-1 (TGR) and a control (WKY) rat was analyzed by transgene-specific PCR yielding a fragment of 606 bp only in TGR/hALC-1.


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Figure 11 . Heart weight/body weight relationship (HW/BW).

The heart weight/body weight relationship was evaluated in 12 weeks-old TGR/hALC-1 (TGR) and WKY animals. Revealing no significant difference between both groups. Values were expressed as means ± SEM (n=7).


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4.2  Quantification of the expressed hALC-1

To determine transgene expression at the protein level, a unique peptide antibody against human ALC-1 sequence was developed. Figure 13 illustrates a typical immunoblot analysis of the total protein fractions extracted from the ventricles of WKY or TGR/hALC-1 rats. The anti-human ALC-1 antibody specifically recognized a 28-kDa protein in the TGR/hALC-1 animals, i.e. the expected molecular mass of the human ALC-1. This apparent molecular mass is consistent with the migration of the immunostained ALC-1 in human atrial tissue extracts (Fig. 14B). The antibody did not cross-react with the ventricular light chains of WKY rats or any other proteins on Western blots of total rat ventricular proteins (Fig. 14B).

Recombinant HIS-tagged hALC-1 migrating at 35 kDa was used as standard to quantify the amount of expressed hALC-1 by Western blot analysis (Fig.12A). There was a linear relationship between the optical density of ECL-signals and recombinant h-ALC-1 which was used as standard curve (Fig. 12B).

Densitometric analysis of proteins extracted from 12-week old rats revealed an average amount of 17 ± 4μg hALC-1 per mg of SDS-extracted ventricular proteins (n=7). Assuming myosin represents 45% of the whole SDS-soluble protein and the MLC-1 is 10% of the whole myosin (Swynghedauw 1986), therefore the expressed hALC-1 represents 37% of whole MLC-1. Moreover an age-dependent decline in transgene expression was observed in the same animal line.

By quantifying hALC-1 expression in 24 and 36-week old TGR/hALC-1, the average amount of hALC-1 expression in the 24 weeks-old rats declined significantly (p<0.05) to 9.5 ± 3μg hALC-1 per mg of whole SDS-soluble protein (n=8), which represents 21% of the whole MLC-1. In the 36-week old TGR/hALC-1 rats, hALC-1 expression was significantly reduced to 4.8 ±2μg hALC-1 per mg of whole SDS-soluble protein (n=10), which represents about 10% of the total MLC-1.


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Figure 12 . hALC-1 HIST Standard Curve .

(A) Different concentrations (1μg, 2μg, 3μg, 4μg & 5μg) of the hALC-1HIST were identified by 12% SDS gel and Western Blot using anti human ALC-1 antibodies.
(B) The hALC-1HISTsignals were scanned densitometrically then plotted against the concentration for obtaining a standard curve for the estimation of protein content expressed by the transgene.


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Figure 13 . Quantification of the expressed hALC-1.

Immunoblot analysis of the total protein fractions (80μg) extracted from the ventricles of WKY or TGR/hALC-1 . The anti-human ALC-1 antibody recognized specifically the hALC-1 (28-kDa) protein in the TGR/hALC-1 animals. The antibody did not cross-react with the ventricular light chains of WKY rats. Recombinant HIS-tagged hALC-1 migrating at 35 kDa was used as standard (1, 1.5 and 2μg) to quantify the amount of expressed hALC-1.


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4.3  MLC analysis

Figure 14A illustrates an SDS-PAGE gel for the separation of human atrial tissue SDS-extract (hAT) and left ventricular purified myosin preparations of TGR/hALC-1 and WKY animals, with assessment of the stoichiometry of the total MLC-1 isoforms (28 kDa) versus total MLC-2 isoforms (18kDa).

The bands were scanned densitometrically and the MLC-1/MLC-2 ratio was 1.2±0.1 (n= 4) and 1.3±0.03 (n= 4) respectively in the transgenic and WKY rats, showing no difference in MLC pattern in both animal groups.

Native hALC-1 (from human atrial tissue SDS-extract) as well as the rat VLC-1 co-migrated in SDS-PAGE (28 kDa) (Fig. 14A).

The anti-human ALC-1 antibody recognized specifically the hALC-1 in the myosin preparations of all TGR/hALC-1 animals (TGR/hALC-1) and the native hALC-1 in the human atrial tissue extract on immunoblot analysis (Fig. 14B) of human atrial tissue extract and left ventricular purified myosin preparations of TGR/hALC-1 and WKY rats. The TGR/hALC-1 revealed an identical molecular weight (28 kDa) to the native hALC-1.

To estimate the amount of replacement of the rat-endogenous VLC-1 by the hALC-1 left ventricular purified myosin preparations (20μg) of TGR/hALC-1 and WKY rat were separated by SDS-PAGE on a 12% resolving gel and transferred to a nitrocellulose membrane in a typical immunoblot analysis. hALC-1HIST was used as standard to quantify the amount of expressed hALC-1 in the myosin preparations. The amount of hALC-1 associated with the purified TGR/hALC-1 myosin represented 20±2% (n=4) of whole transgenic rat MLC-1.


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Figure 14 . MLC composition analysis.

(A) SDS-Page for the purified myosin of a TGR/hALC-1 (TGR), WKY rats and human atrial tissue SDS-extract (hAT). Note the co-migration of the native hALC-1 (of the human atrial tissue SDS-extract) (28 kDa) and the rat VLC-1 (28 kDa).
(B) Immunoblot analysis of the hAT and the purified myosin preparations of TGR/hALC-1 and WKY rats.


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4.4  Immunofluorescence Microscopy

Ventricular tissue sections from the transgenic animals were analysed using specific anti-hALC-1 antibodies and anti α-actinin antibodies. TGR/hALC-1 were investigated by double-labelling immunofluorescence microscopy, Z-lines were identified by alpha-actinin specific fluorescence, showing regular striated pattern that is consistent with labelling of the sarcomere (Fig. 15A).

The specific fluorescence of hALC-1 (Fig. 15B) in the sarcomere could be localized between the Z-lines by overlaying the hALC-1 and α-actinin labelling patterns, (Fig.15C). These observations confirm the appropriate placement of the hALC-1 into the contractile machine.

Furthermore, by double-labelling analysis using anti hALC-1 (Fig. 16B) and anti-MHC antibodies (Fig. 16A), mosaic patterns of transgene expression in cardiomyocytes were observed. The specific hALC-1 fluorescence signals of cardiomyocytes revealed different signal intensities, ranging from strong signals to undetectable levels of hALC-1.


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Figure 15 . Immunofluorescence localization of the transgene-expression.

Left ventricular tissue sections from the transgenic animals were analysed by double-labelling immunofluorescent microscopy. α-actinin was detected by using anti α-actinin antibodies demonstrating its binding to the Z-bands with regular striated pattern that is consistent with labelling of the sarcomere (A). The hALC-1 in the ventricles of the TGR/hALC-1 was detected by using specific anti-hALC-1 antibodies (B). The hALC-1 and α-actinin labelling patterns were overlaid, showing the localization of the hALC-1 in-between the Z-bands of the sarcomere, (C).


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Figure 16 . Expression-patterns of the transgene.

By double-labelling immunofluorescent analysis of the left ventricular tissue sections of the TGR/hALC-1, the tissues were incubated withanti-myosin heavy chain-antibodies demonstrating the myosin heavy chains (Green) (A), and the anti-hALC-1 antibodies demonstrated the hALC-1 (Red) (B). The specific hALC-1 fluorescence signals of cardiomyocytes revealed different signal intensities between very strong signals and undetectable levels of hALC-1.


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4.5  Comparative proteome analysis

In a 2D-PAGE comparative analysis of the whole rat ventricular protein expression-patterns (4 pairs of 12 weeks-old TGR/hALC-1 and WKY rats), I was able to resolve approximately 3000 protein spots in each pattern.

The whole protein expression patterns in both animal groups showed no differences with the exception of the transgenic spot (Fig.17A and B) that was absent in all the WKY rats and present in all the TGR/hALC-1, but other spot variations existed in between the same animal group. The transgenic protein spot was identified by molecular weight and mass spectrometry and proved to be hALC-1.


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Figure 17 . Analysis of the ventricular proteome by 2D-PAGE.

Shown are the representative silver-stained gels of both animal groups at protein loads of 120μg. (A) Silver stained 2-D gel for 12-week old TGR/hALC-1 (pH 3 to 10) with a magnified section showing the transgenic hALC-1 protein spot (arrow). (B) Silver stained 2-D gel for 12-week old WKY rat (pH 3 to 10) with a magnified section showing the absence of the transgenic hALC-1 protein spot (arrow).

4.6 Functional properties of the transgene

Cardiac functions in the TGR/hALC-1 and WKY rats were evaluated by examining the isolated perfused hearts. Hearts were obtained from 12-24-36-week old animals.

Left ventricular functions were evaluated and there was a statistically significant improvement (p<0.001) in the contractile functions of the 12-week old TGR/hALC-1 (n=7) compared to their age-matched control (WKY) (n=7) animals. This improvement is represented in an increase in: the developed left ventricular pressure (LVP) by 2.17-fold (Fig. 18), the maximum rate of pressure increase (contraction rate, +dP/dtmax) by 2.11- fold (Fig. 19) and the maximum rate of pressure decrease (relaxation rate, -dP/dtmax) by 1.91-fold (Fig. 20) in the TGR/hALC-1 compared to the age matched WKY rats.

While evaluating the contractile functions of the 24 and 36 weeks-old rats compared to the 12 weeks-old rats, there was an age-dependent decline of the improved contractility parameters tightly associated with a decline in transgene expression levels in the 24 and 36-week old TGR/hALC-1 (n=8 and 10-respectively) (Fig. 18, 19, 20).

In contrast, there was an age-dependent increase in contractility in the 24, 36 weeks-old WKY rats compared with 12 weeks old animals (Fig. 18 19, 20). Therefore, improved contractile functions of TGR/hALC-1 levelled-off to normal in 36 weeks-old animals.


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Figure 18 . Developed left ventricular pressure (LVP):

Showed significant increase in the 12 and 24 weeks-old TGR/hALC-1 (TGR) compared to the age-matching WKY rats, while the 36 weeks-old rats showed no significant difference between the TGR/hALC-1 and WKY groups p<0.001.


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Figure 19 . Maximum rate of pressure increase (+dP/dt max) (contraction rate):

showed significant increase in the 12 weeks-old TGR/hALC-1 (TGR) compared to the age-matching WKY rats, while the 24 and 36 weeks-old rats showed no significant difference between the TGR/hALC-1 and WKY groups p<0.001.


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Figure 20 . Maximum rate of pressure decrease (-dP/dt max) (relaxation rate):

showed significant increase in the 12 and 24 weeks-old TGR/hALC-1 (TGR) compared to the age-matching WKY rats, while the 36 weeks-old rats showed no significant difference between the TGR/hALC-1 and WKY groups p<0.001.


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