Cardiomyocyte-Specific Telomere Shortening is a Distinct Signature of Heart Failure in Humans

Abstract

Background: Telomere defects are thought to play a role in cardiomyopathies, but the specific cell type affected by the disease in human hearts is not yet identified. The aim of this study was to systematically evaluate the cell type specificity of telomere shortening in patients with heart failure in relation to their cardiac disease, age, and sex.

Methods and results: We studied cardiac tissues from patients with heart failure by utilizing telomere quantitative fluorescence in situ hybridization, a highly sensitive method with single-cell resolution. In this study, total of 63 human left ventricular samples, including 37 diseased and 26 nonfailing donor hearts, were stained for telomeres in combination with cardiomyocyte- or α-smooth muscle cell-specific markers, cardiac troponin T, and smooth muscle actin, respectively, and assessed for telomere length. Patients with heart failure demonstrate shorter cardiomyocyte telomeres compared with nonfailing donors, which is specific only to cardiomyocytes within diseased human hearts and is associated with cardiomyocyte DNA damage. Our data further reveal that hypertrophic hearts with reduced ejection fraction exhibit the shortest telomeres. In contrast to other reported cell types, no difference in cardiomyocyte telomere length is evident with age. However, under the disease state, telomere attrition manifests in both young and older patients with cardiac hypertrophy. Finally, we demonstrate that cardiomyocyte-telomere length is better sustained in women than men under diseased conditions.

Conclusions: This study provides the first evidence of cardiomyocyte-specific telomere shortening in heart failure.

Keywords: cardiomyocyte; cardiomyopathy; telomere length.

Figure 1

Patients with hypertrophic cardiomyopathy have short cardiomyocyte telomeres. A, Representative images of quantitative fluorescence in situ hybridization (Q‐FISH) analysis, immunostained for the cardiomyocyte‐specific marker, cardiac troponin T (cTnT, green), telomeric probe (red), and 4′,6‐diamidino‐2‐phenylindole (DAPI) for nuclei (blue). White dotted lines mark the area used for telomere analysis within the nucleus of each cardiomyocyte. Scale bar, 10 μm. B, Telomere length distribution histogram of individual cardiomyocytes from nonfailing donors (NFDs) and hypertrophic cardiomyopathy (HCM) patient cardiac samples. Data are presented as percentage of cells within the patients' spectrum of the telomeric length range. Black and red vertical lines were drawn at the median value of the histogram obtained for NFDs and patients with HCM, respectively. The shift in telomeric length from NFD to HCM histogram median value is shown by the red horizontal line (Wilcoxon rank sum test, P=0.016). N indicates the number of cardiomyocytes scored (see also Table 2). The percentage of cells with short and long telomeres is shown in the graph. C, Boxplot analysis shows average telomere length measurements in NFDs and patients with HCM (Mann–Whitney test, P=0.003). A total of 26 NFDs and 17 patients with HCM were analyzed.

Figure 2

Cardiac smooth muscle cells have comparable telomere lengths in nonfailing donor (NFD) and hypertrophic cardiomyopathy (HCM) hearts. A, Representative picture of left ventricular tissues subjected to quantitative fluorescence in situ hybridization (Q‐FISH) analysis and immunostained for smooth muscle actin cell marker (α‐smooth muscle actin [α‐SMA]), telomeres, and 4′,6‐diamidino‐2‐phenylindole (DAPI) shown in green, red, and blue, respectively. Scale bar, 20 μm. Arrowheads indicate representative analyzed α‐SMA ⁺ cells. Insert shows a higher magnification of an α‐SMA ⁺ cell. B, Telomere length distribution histogram analysis of α‐SMA ⁺ cells from NFDs and HCM patient cardiac samples. Data are presented as percentage of cells within the patients' spectrum of telomeric length range. A black vertical line was drawn at the median value of the histogram obtained for NFDs. Statistical comparison of NFDs and patients with HCM, Wilcoxon rank sum test, P=0.322. N indicates the number of cardiomyocytes scored (see also Table 2). The percentage of cells with short and long telomeres is shown in the graph. C, Boxplot analysis shows average telomere length measurements between NFDs and patients with HCM (Mann–Whitney test, P=0.798). A total of 26 NFDs and 17 patients with HCM were analyzed.

Figure 3

Activated DNA damage response in hypertrophic cardiomyopathy (HCM) cardiomyocytes. A, Representative immunofluorescence image: γH2AX in green, 4′,6‐diamidino‐2‐phenylindole (DAPI)‐stained nuclei in blue, and cardiac troponin T (cTnT) in red. Scale bar, 5 μm. B, Representative immunofluorescence image of 53BP1 in green, DAPI‐stained nuclei in blue, and cTnT in red. Scale bar, 5 μm. Quantification of DNA damage as detected by (C) γH2AX ⁺ and (D) by 53BP1⁺ cardiomyocytes. Data are represented as mean±SEM. P<0.05 were considered significant; statistical comparison of mean values (2‐tailed, unpaired Student t test) from nonfailing donor and HCM groups for γH2AX and p53‐binding protein 1 (53BP1) are P=0.016 and P<0.0001, respectively.

Figure 4

Correlation between telomere length and ejection fraction. A, Cardiomyocyte telomere length distribution of nonfailing donors (NFDs) and patients with hypertrophic cardiomyopathy (HCM): HCM heart failure with preserved ejection fraction (HFpEF) and HCM heart failure with reduced ejection fraction (HFrEF). Black and red vertical lines are drawn at median value of the histogram obtained for NFDs and patients with HCM, respectively. Shifts in patients' telomeric length distribution median values are shown in brown and green horizontal lines for HCM HFpEF and HCM HFrEF, respectively. Wilcoxon rank sum analysis between NFDs and patients with HCM HFpEF and HCM HFrEF: P=0.004 and P=0.034, respectively. N indicates the number of cardiomyocytes scored (see also Table 3). The percentage of cells with short and long telomeres is shown in graphs. B, Boxplot analysis shows average telomere length measurements (per patient) between NFDs and patients with HCM HFpEF and HCM HFrEF (Kruskal‐Wallis test, P=0.164 and P=0.02, respectively). C, Distribution of telomere length (per patient) and cardiac function as measured by ejection fraction in NFDs (black circles), patients with HCM HFrEF (green triangles), and patients with HCM HFpEF (brown squares). Patients with shorter average telomere length are presented below the horizontal black line. A total of 20 NFD, 8 HCM HFpEF, and 9 HCM HFrEF patient samples with available ejection fraction data were analyzed. Q‐FISH indicates quantitative fluorescence in situ hybridization.

Figure 5

Effect of age in telomere shortening in cardiac tissues. A through D, Histograms presenting distribution of the percentage of cells within the patients' range of cardiomyocyte telomeric length. A, young and (B) old nonfailing donors (NFDs) and (C) young (green, aged 21–46 years) and (D) old (blue, aged 50–75 years) patients with hypertrophic cardiomyopathy (HCM). N indicates the number of cardiomyocytes scored. The percentage of cells with short and long telomeres is shown in the graph. Black and red vertical lines were drawn at the median value of the histogram obtained for NFDs and patients with HCM, respectively. The shift in telomeric length distribution from young patients with HCM to young NFDs is shown with the horizontal line green (Wilcoxon rank sum test, P=0.012); the shift in telomeric length distribution from old patients with HCM to NFDs is shown with the blue horizontal line (Wilcoxon rank sum test, P=0.036); P=0.874 (Wilcoxon rank sum test) for comparison of young NFDs and old NFDs. E, Boxplot analysis shows average telomere length measurements (per patient) between young and old NFDs and young and old patients with HCM (Kruskal‐Wallis test, P=0.850, 0.004, and 0.102). A total of 9 young NFDs, 15 old NFDs, 5 young patients with HCM, and 11 old patients with HCM were analyzed. Q‐FISH indicates quantitative fluorescence in situ hybridization.

Figure 6

Sex‐dependent telomere shortening in response to hypertrophic cardiomyopathy (HCM) disease environment. A through D, Distribution of the percentage of cells within the patients' range of cardiomyocyte telomeric length is presented as histograms in nonfailing donor (NFD; A) males and (B) females as well as (C) male and (D) female patients with HCM. N indicates the number of cardiomyocytes scored. The percentage of cells with short and long telomeres is shown in the graph (see also Table 3). Black and red vertical lines are drawn at the median value of the histogram for NFDs and patients with HCM, respectively. The shift in telomeric length distribution in male patients with HCM from male NFDs is shown with horizontal dark green line (Wilcoxon rank sum test, P=0.007) for comparison of male NFDs and female NFDs; the shift in telomeric length distribution in female patients with HCM to female NFDs is shown with the light blue horizontal line (Wilcoxon rank sum test, P=0.132); P=0.996 (Wilcoxon rank sum test). E, Boxplot analysis shows average telomere length measurements (per patient) between male (dark green) and female (light blue) NFDs and male and female patients with HCM (Kruskal‐Wallis test, P>0.999, P=0.045 and P>0.999). A total of 14 male NFDs, 10 female NFDs, 3 male patients with HCM, and 13 female patients with HCM were analyzed. Q‐FISH indicates quantitative fluorescence in situ hybridization.