A simple adeno-associated virus-based approach for the generation of cardiac genetic models in rats

Figures

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  Figure 1.. Rats injected with AAV9-cTnT-eGFP show no signs of toxicity. A. Gravimetric analysis show no difference in body weight between rats injected with ‘high dose’ or ‘low dose’ AAV9-cTnT-eGFP and control saline injected rats. Black bars show average and each dot represent a measurement from one rat. N.S = not statistically significant from control by Student t-test. B. Gravimetric analysis shows no difference in heart weight normalized to body weight between rats injected with ‘high dose’ or ‘low dose’ AAV9-cTnT-eGFP and control saline injected rats. Black bars show average and each dot represent a measurement from one rat. N.S = not statistically significant from control by Student t-test. C. Representative hematoxylin & eosin stained cardiac sections at low (top) and high (bottom) magnification, showing normal histology with no foci of necrosis or inflammatory cell infiltrates even in rats injected with ‘high dose’ AAV9-cTnT-eGFP (right). Control Saline injected rat heart is shown on the left. Bar = 100 µm.

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  Figure 2.. Rats injected with AAV9-cTnT-eGFP have normal cardiac dimensions and function. A. Representative echocardiographic images in two-dimensional short axis view (top) and M-mode (bottom) magnification, showing normal cardiac dimensions, wall thickness and contractile function even in rats injected with ‘high dose’ AAV9-cTnT-eGFP (right). Control saline injected rat heart is shown on the left. B. Echocardiographic measurements of left ventricular internal diameter in end diastole (LVIDd) showing no significant changes between rats injected with ‘high dose’ or ‘low dose’ AAV9-cTnT-eGFP and control saline injected rats. Black bars show average and each dot represent measurement from one rat. N.S = not statistically significant from control by Student t-test. C. Echocardiographic measurements of left ventricular fractional shortening (FS%) showing no decrement in cardiac function between rats injected with ‘high dose’ or ‘low dose’ AAV9-cTnT-eGFP and control saline injected rats. Black bars show average and each dot represent measurement from one rat. N.S = not statistically significant from control by Student t test.

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  Figure 3.. High cardiac expression of GFP in adult rats injected with AAV9-cTnT-eGFP. A– C. Low magnification images of cardiac sections of rats with GFP fluorescence (green) and DAPI nuclear stain (blue) showing no green GFP fluorescence in control saline injected rats ( A), few transduced GFP positive cardiomyocytes in the low dose AAV9-cTnT-eGFP injected rats ( B), and robust high GFP expression in the high dose AAV9-cTnT-eGFP injected rats ( C). Each image was taken from a different rat. Bar = 5 mm. D– F. High magnification images from the same rats shown in A– C, showing no green GFP fluorescence in control saline injected rats ( D), few transduced GFP positive cardiomyocytes in the low dose AAV9-cTnT-eGFP injected rats ( E), and robust high GFP expression in the high dose AAV9-cTnT-eGFP injected rats ( F). Each image was taken from a different rat. Bar = 200 µm.

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  Figure 4.. Cardiomyocyte specific expression of GFP in adult rats injected with AAV9-cTnT-eGFP. A– B. Representative images of cardiac sections of control rats ( A – low magnification, Bar= 100 µm; B- High magnification Bar= 20 µm). C– D. Representative images of cardiac sections of ‘high dose’ AAV injected rats ( C – low magnification, Bar= 100 µm; D- High magnification Bar= 20 µm). For all panels ( A– D) staining with sarcomeric α actinin immunostaining (red), eGFP fluorescence (green) and DAPI nuclear stain (blue). Left column of panels shows a composite of actinin (red) and DAPI (blue) channels, middle column shows a composite of GFP (green) and DAPI (blue) channels, and right column of panels shows a composite of all three actinin (red), GFP (green) and DAPI (blue) channels. No green GFP fluorescence is seen in cardiomyocytes from control saline injected rats ( A– B), and ~100% of cardiomyocytes show green GFP fluorescence in the high dose AAV9-cTnT-eGFP injected rats ( C– D). Importantly, non-cardiomyocyte cells, likely cardiac fibroblasts, identified by the lack of sarcomeric α actinin immunostaining do not show GFP fluorescence even in the high dose AAV9-cTnT-eGFP injected rats,…

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  Figure 5.. Quantification shows high transduction efficiency. A. A violin scatter plot of mean cardiomyocyte GFP intensity measured from random high power magnification of cardiac sections. Each point represents measurements from one cardiomyocytes in the control, low dose, and high dose AAV9-cTnT-eGFP injected rats, black bars show the mean intensity (N=3 rats, n=~900 cardiomyocytes, in each group). B. Quantitative reverse transcriptase PCR of normalized GFP expression showing very modest expression in the low dose AAV9-cTnT-eGFP rat hearts and high expression in the high dose AAV9-cTnT-eGFP rat hearts. No GFP expression was detectable in the liver or kidney even in the high dose AAV9-cTnT-eGFP rats. N=5 rats. Bars show the average ± standard error. * Student t-test vs. control p<0.05.