Selected Pathway Analyses to Gain Mechanistic Insights into the Pathogenesis of Feline Hypertrophic Cardiomyopathy

Figure 1. Relative transcription levels of markers for myocardial growth, hypertrophy and fetal genes, and markers of vascularization and intercellular signaling in the myocardium of control cats. (A) Values in young control cats (1.5 years; n = 16; each 8 female and 8 male). (B) Values in older adult control cats (n = 14; 4 female, 10 male). Dots represent individual animals; RT-qPCRs were performed in duplicates and expression values were normalized against the reference gene GAPDH. ∆∆CT values are calculated by using the 2−∆∆CT method. Data are shown as mean ± standard deviation.

Figure 2. Evaluation of gene expression by qRT-PCR in the myocardium of young control cats (1.5 years; n = 16) and older adult control cats (n = 14). Relative transcription levels of markers for myocardial growth (A), hypertrophy (B), fetal genes (C), vascularization (D) and intercellular signaling (E). All markers expressed by cardiomyocytes show significantly higher expression levels (all adjusted p ≤ 0.001) in the young control cats. For ITGAM, expressed by leukocytes, the difference is not significant (adjusted p = 0.964). Dots represent individual animals; RT-qPCRs were performed in duplicates and expression values are normalized against the reference gene GAPDH. ∆∆CT values are calculated by using the 2−∆∆CT method followed by statistical analyses in R including testing for normality (Shapiro–Wilk test), log10 transformation, one-way ANOVA and Tukey post hoc test. Data are shown as mean ± standard deviation. Asterisks indicate the significance level: adjusted p ≤ 0.001 (***); ns = non-significant. Detailed information on the statistical results is provided in Table S2.

Figure 3. Evaluation of gene expression by qRT-PCR in the myocardium of female (F) young control cats (n = 8) and male (M) young control cats (n = 8) as well as female (F) older adult control cats (n = 4) and male (M) older adult control cats (n = 10). Relative transcription levels of markers for myocardial growth (A), hypertrophy (B), fetal genes (C), vascularization (D) and intercellular signaling (E). Within both groups, there were no significant differences in the transcription level of any marker between male and female cats (not shown in graph). Almost all markers, except for MFN2, GATA4, and the intercellular signaling factors ITGAM and ITGA10, show significantly higher transcription levels in both female and male young control cats. Dots represent individual animals; RT-qPCRs were performed in duplicates and expression values are normalized against the reference gene GAPDH. ∆∆CT values are calculated by using the 2−∆∆CT method followed by statistical analyses in R including testing for normality (Shapiro–Wilk test), log10 transformation, two-way ANOVA and Tukey post hoc test. Data are shown as mean ± standard deviation. Asterisks indicate the significance level: adjusted p ≤ 0.05…

Figure 4. Relative transcription levels of markers for myocardial growth, hypertrophy, fetal genes, vascularization and intercellular signaling in the myocardium of cats with HCM. All markers are transcribed. Dots represent individual animals; RT-qPCRs were performed in duplicates and expression values are normalized against the reference gene GAPDH. ∆∆CT values are calculated by using the 2−∆∆CT method. Data are shown as mean ± standard deviation.

Figure 5. Evaluation of gene expression by qRT-PCR in the myocardium of young control cats (1.5 years; n = 16) and older adult control cats (n = 14) in comparison to cats with HCM (Group 3, n = 15). Relative transcription levels of markers for myocardial growth (A), myocardial hypertrophy (B), myocardial fetal genes (C), vascularization (D) and intercellular signaling (E). Dots represent individual animals; RT-qPCRs were performed in duplicates and expression values are normalized against the reference gene GAPDH. ∆∆CT values are calculated by using the 2−∆∆CT method followed by statistical analyses in R including testing for normality (Shapiro–Wilk test), log10 transformation, one-way ANOVA and Tukey post hoc test. Data are shown as mean ± standard deviation. Asterisks indicate the significance level: adjusted p ≤ 0.05 (*), adjusted p ≤ 0.01 (**) and adjusted p ≤ 0.001 (***); ns = non-significant. Detailed information on the statistical results is provided in Table S2.

Figure 6. Evaluation of gene expression by qRT-PCR in the myocardium of female (F; n = 4) and male (M; n = 11) cats with HCM. Relative transcription levels of markers for myocardial growth (A), myocardial hypertrophy (B), myocardial fetal genes (C), vascularization (D), and intercellular signaling (E) in the myocardium of female (F; n = 4) and male (M; n = 11) cats with HCM. All markers show higher transcription levels in male cats with HCM; for some the difference is significant. Dots represent individual animals; RT-qPCRs were performed in duplicates and expression values are normalized against the reference gene GAPDH. ∆∆CT values are calculated by using the 2−∆∆CT method followed by statistical analyses in R including testing for normality (Shapiro–Wilk test), log10 transformation, two-way ANOVA and Tukey post hoc test. Data are shown as mean ± standard deviation. Asterisks indicate the significance level: adjusted p ≤ 0.05 (*), adjusted p ≤ 0.01 (**); ns = non-significant. Detailed information on the statistical results is provided in Table S2.

Figure 7. Relative transcription levels of markers for myocardial growth (A), myocardial hypertrophy (B), myocardial fetal genes (C), vascularization (D), and intercellular signaling (E) in the myocardium of male young control cats (n = 8) and older adult control cats (n = 10) and in male cats with HCM (n = 11). Dots represent individual animals; RT-qPCRs were performed in duplicates and expression values are normalized against the reference gene GAPDH. ∆∆CT values are calculated by using the 2−∆∆CT method followed by statistical analyses in R including testing for normality (Shapiro–Wilk test), log10 transformation, two-way ANOVA and Tukey post hoc test. Data are shown as mean ± standard deviation. Asterisks indicate the significance level: adjusted p ≤ 0.05 (*), adjusted p ≤ 0.01 (**) and adjusted p ≤ 0.001 (***); ns = non-significant. Detailed information on the statistical results is provided in Table S2.

Figure 8. Relative transcription levels of markers for myocardial growth (A), myocardial hypertrophy (B), myocardial fetal genes (C), vascularization (D), and intercellular signaling (E) in the myocardium of female young control cats (n = 8) and older adult control cats (n = 4) and female cats with HCM (n = 4). All markers show no difference in the relative expression levels when comparing the older adult control animals and those with HCM. Dots represent individual animals; RT-qPCRs were performed in duplicates and expression values are normalized against the reference gene GAPDH. ∆∆CT values are calculated by using the 2−∆∆CT method followed by statistical analyses in R including testing for normality (Shapiro–Wilk test), log10 transformation, two-way ANOVA and Tukey post hoc test. Data are shown as mean ± standard deviation. Asterisks indicate the significance level: adjusted p ≤ 0.05 (*) and adjusted p ≤ 0.001 (***); ns = non-significant. Detailed information on the statistical results is provided in Table S2.