Targeted Genetic Reduction of Mutant Huntingtin Lessens Cardiac Pathology in the BACHD Mouse Model of Huntington's Disease

Figure 1. Schematic representation of the breeding strategy used in the present study. Genetic reduction of mHTT expression in the heart of BACHD mice was achieved by crossing this transgene (hemizygous) with mice hemizygous for the cardiomyocyte-specific Myh6-Cre transgene. The full-length mHTT with two loxP sites enables cell specific reduction of mHTT using Cre-recombinase (Cre). Approximately 25% of the offspring were double transgenics (BACHD; Myh6-Cre), BMYO, with the mHTT floxed out in the cardiomyocytes.

Figure 2. Genetic targeting of human mHTT elicits a selective reduction of its transcript and protein expression levels in the heart, but not the striatum of 3 months old BMYO mice. (A) Quantification of the human mHTT transcript levels in the heart and striatum shows a significant and selective decrease in the heart of the BMYO line [F(2, 8) = 125.5, P < 0.001]; while, striatal levels were comparable in the two mutant lines [F(2, 8) = 87.706, P < 0.001]. (B) The levels of the endogenous mouse Htt were unchanged among genotypes in both the heart [F (2, 8) = 2.045, P = 0.210] and striatum [F (2, 8) = 0.852, P = 0.473]. (C) PolyQ protein expression levels were detected in the BACHD heart and were greatly reduced in the BMYO [F (2, 8) = 162.549, P < 0.001], but not in the striatum of both mutants. These data demonstrate that the genetic cross was successful, and the mHTT levels were selectively reduced in the BMYO heart, while maintaining “normal” expression in the striatum. Representative immunoblots for the heart (D) and (E) striatum. Values derived from the densitometric analysis were corrected for the background and normalized to GAPDH. The vertical bar plots represent the group means and…

Figure 3. Echocardiographic measurements of BACHD, BMYO, and WT animals at 6 months of age. Two-dimensional, M-mode echocardiography and spectral Doppler images enabled measurement of heart dimension and function including Left ventricle mass (LV mass), fractional shortening (FS%), Lv Ejection Fraction (Lv EF), and heart rate. Most parameters did not vary between the genotypes (Table 1). (A) Lv mass was increased in both the BACHD and BMYO mice compared to WT controls, while (B) the ejection fraction was reduced in BACHD compared to BMYO and WT hearts. (C) The fractional shortening followed a similar pattern although the differences were not significant by our criterion. The selective reduction of mHtt from the heart did not reduce the hypertrophy in the BMYO heart but seemed to improve the ejection fraction. The vertical bar plots show group means and SEM. The symbols show the values from individual animals (n = 7–8 per genotype). Asterisks represent significant differences (P < 0.05) compared to WT controls while crosshatch indicates significant differences (P < 0.05) between BMYO and BACHD. One way ANOVA followed by Tukey's multiple comparison test was used to evaluate the possible…

Figure 4. Gene expression levels of molecular markers for cardiovascular injury and repair were significantly lowered by the specific removal of mHtt from the heart. (A) The expression levels of collagen 1a (Col1a) were significantly lower in the BMYO animals compared to the BACHD, while (B) the expression of ventricular/slow myosin heavy chain isoform (Myh7) did not significantly change. (C) GATA binding protein 4 (GATA4) and (D) brain natriuretic peptide (BNP) were both significantly diminished in the BMYO heart at 6 months of age as compared to BACHD (see also Table 2 for values derived from the statistical analyses). Together these data suggest that changes in molecular markers of CV injury were reversed by selective removal of the mHTT in the BMYO line. The vertical bar plots show group means and SEM. The symbols show the values from individual animals (n = 3–4 per genotype). Asterisks represent significant differences (P < 0.05) compared to WT controls. One way ANOVA followed by Tukey's multiple comparison test was used to evaluate the possible significance of the findings. If normality or equal variance tests failed, then Kruskal-Wallis one-way ANOVA on ranks followed by Dunn's…

Figure 5. The expression of fibrotic markers was diminished by the selective removal of human mHTT from the cardiomyocytes. (A) Representative images of immunostained mid-ventricular coronal heart sections showing stronger and diffuse expression of Collagen I in 6 months-old BACHD, but not in age-matched BMYO and WT mice (n = 2 per genotype). The white rectangles delineate the areas where the enlarged images were obtained: (a) left ventricular wall-papillary muscle; (b) right ventricular wall. Intense collagen I immunoreactivity can be observed around the cardiomyocytes in the BACHD heart (left panels), but at a lower level in the BMYO and WT (middle and right panels, respectively). Lv, left ventricle; Rv, right ventricle. Scale bars = 200 μm. (B) Representative western blotting of whole-tissue lysates of hearts from 6 months-old BACHD, BMYO and WT mice. The total protein levels of fibronectin displayed a significant increase in the BACHD mice as compared to WT. Each value derived from the densitometric analysis was corrected for background, and then divided by the sum of the background-corrected signals obtained in the same immunoblot. These values were then normalized to the respective…

Figure 6. Selective removal of the human mHtt lessened the fibrosis in the hearts of 6 months old BMYO mice. Representative photomicrographs of mid-ventricular heart sections stained with Masson's trichrome at different magnifications. For all three genotypes, a representative stained section is shown at three progressively higher magnifications. (A) Lower magnification images showing the enlarged BACHD heart, such phenotype was less evident in the BMYO mice (scale bar = 200 μm). The white rectangles delineate the areas where the higher magnification images were acquired. (B,C) Higher magnification images showing pockets of fibrotic tissue of variable extension in the septal part of the right ventricular wall of BACHD hearts. Some fibrotic areas could be also seen along the outer surface of the ventricular walls near the interventricular septum. Conversely, such areas were qualitatively judged to be almost inexistent in the BMYO (Table 3). At this age, WT hearts did not show strong evidence of fibrosis. Scale bars = 100 and 50 μm in the middle and lower panels, respectively. n = 3 per genotype. Lv, left ventricle; Rv, right ventricle.

Figure 7. Behavioral biomarkers of cardiovascular function were improved in the BMYO line. Behavior measures including grip strength, and diurnal rhythms of activity of the three lines were measured between 6 and 7 months of age. (A) Grip strength was reduced in the BACHD compared to BMYO and WT lines. (B) The average total activity over 24 h as reduced in both the BACHD and BMYO compared to WT. (C) Power, one measure of circadian rhythmicity, was reduced in both the BACHD and BMYO compared to WT controls. (D) The % of activity measured in the light was reduced in the BMYO. The vertical bar plots show group means and SEM. The symbols show the values from individual animals (n = 8 per genotype) in each group. Asterisks represent significant differences (P < 0.05) compared to WT mice while crosshatch indicates significant differences (P < 0.05) between BMYO and BACHD mice. One way ANOVA followed by Holm-Sidak or Tukey's multiple comparison test was used to evaluate the possible significance of the findings. If normality or equal variance tests failed, then Kruskal-Wallis one way ANOVA on ranks followed by Dunn's multiple comparison test was used instead (see Table 5).