Hematopoietic loss of Y chromosome leads to cardiac fibrosis and heart failure mortality

Abstract

Hematopoietic mosaic loss of Y chromosome (mLOY) is associated with increased risk of mortality and age-related diseases in men, but the causal and mechanistic relationships have yet to be established. Here, we show that male mice reconstituted with bone marrow cells lacking the Y chromosome display increased mortality and age-related profibrotic pathologies including reduced cardiac function. Cardiac macrophages lacking the Y chromosome exhibited polarization toward a more fibrotic phenotype, and treatment with a transforming growth factor β1-neutralizing antibody ameliorated cardiac dysfunction in mLOY mice. A prospective study revealed that mLOY in blood is associated with an increased risk for cardiovascular disease and heart failure-associated mortality. Together, these results indicate that hematopoietic mLOY causally contributes to fibrosis, cardiac dysfunction, and mortality in men.

Keywords: clonal hematopoiesis.

Fig. 1.. Y chromosome deficiency in hematopoietic cells shortens life span and accelerates age-related cardiac dysfunction.

(A) Schematic of this study. Lethally irradiated male C57BL6/J mice were reconstituted with hematopoietic stem cells transduced with lentivirus encoding Y chromosome targeting gRNA (LOY-gRNA) or control gRNA and designated as mLOY and control mice, respectively. Phenotypic differences between mLOY and control mice during the natural aging process were analyzed. (B) Efficiency of Y chromosome ablation analyzed by FISH. tRFP⁺ peripheral blood cells were collected from mice reconstituted with bone marrow cells transduced with lentivirus encoding a control gRNA or either of two different LOY-gRNAs: LOY-gRNA1 or LOY-gRNA2. The percentages of Y chromosome–deficient (LOY) cells and Y chromosome–sufficient cells (XY) in total blood cells are shown. Approximately 200 cells were analyzed for each condition. Unless otherwise indicated, all subsequent studies were performed with hematopoietic stem cells transduced with LOY-gRNA1. (C) Representative images of FISH analysis of peripheral blood cells collected from mLOY and control mice. Green and red fluorescence indicate the X and Y chromosomes, respectively. (D) Karyotype analysis of LOY and control cells. Hematopoietic stem cells collected from mLOY and control mice were immortalized by lentivirus-mediated HoxB8 transduction and subjected to karyotype analysis. (E) mRNA expression of genes on the Y chromosome in RFP⁺ peripheral blood leukocytes in mLOY and control mice (n = 3 per group). (F) Kaplan-Meier survival curve for mLOY and control mice after BMT. The x axis indicates time after BMT (day) (control, n = 37; mLOY, n = 38). (G) Sequential echocardiographic analysis of mLOY and control mice after BMT at the indicated time points (month) (n = 8 to 10 per group). (H) Quantitative analysis of fibrotic area in heart section at 15 months after BMT (n = 8 to 9 per group). (I) Flow cytometric analysis of fibroblast counts in heart tissue at 15 months after BMT (n = 6 to 7 per group). The absolute numbers of cells were normalized by tissue weight. Dots in all panels represent individual samples. Data are shown as mean ± SEM. Statistical analyses were performed using unpaired Student’s t test [(E) and (H)], Student’s t test with Welch’s correction (I), log-rank test (F), and two-way repeated-measures ANOVA with Sidak’s multiple-comparisons tests (G). FS, fractional shortening; RFP; red fluorescent protein, Con; control. *P < 0.05, **P < 0.01, ****P < 0.0001.

Fig. 2.. Mosaic LOY in leukocytes is associated with all-cause mortality and death caused by cardiovascular diseases in men.

(A) Results from the primary multivariable-adjusted Cox proportional hazards regression for mLOY and mortality from diseases of the circulatory system (ICD10 codes I00 to I99) modeled with a penalized spline approach and mLOY as a continuous variable. The solid red line represents the strength of association over the spectra of LOY mosaicism, and the dotted black lines denote the 95% confidence limits of the model. A linear regression analysis supported a positive slope of the association (P value for β = 0.0009). The analyzed dataset was extracted from the UK Biobank with an average follow-up time of 11.5 years, and the model was adjusted for confounding effects from age, smoking, alcohol consumption, body mass index, ancestry, blood pressure, total cholesterol, and diabetes. (B) Results from exploratory analyses using analogous multivariable adjusted Cox models with mLOY as a binary variable (at a 40% threshold). Shown are results from analyses of all-cause mortality (ICD10 codes A00 to U99), all diseases of the circulatory system (ICD10 codes I00 to I99), as well as specific cardiovascular causes of death with at least 200 events and significant association with mLOY at the 0.05 α level. n, number of events.

Fig. 3.. Y chromosome deficiency in hematopoietic cells accelerates cardiac dysfunction in response to pressure overload.

(A) Schematic of experimental study for assessing cardiac dysfunction of mLOY mice in the pressure overload model. mLOY mice were generated using LOY-gRNA1. At 4 weeks after BMT, mLOY mice or control mice were subjected to TAC. (B) Sequential echocardiographic analysis of mLOY and control mice after TAC at the indicated time points (control, n = 6; LOY, n = 7). (C) Heart weight (HW) and lung weight (LW) relative to tibial length (TL) in control sham mice and mice 4 weeks after TAC (control sham, n = 6; control mLOY, n = 6; control TAC, n = 6; mLOY TAC, n = 7). (D) Gene expression of the heart failure markers Nppa and b/a-MHC in heart tissue in sham mice and mice 4 weeks after TAC (control sham, n = 6; control mLOY, n = 6; control TAC, n = 6; mLOY TAC, n = 7). (E) Representative images and quantitative analysis (control sham, n = 6; control mLOY, n = 6; control TAC, n = 6; mLOY TAC, n = 7) of fibrotic area in heart sections in sham and 4-week after TAC. Scale bar, 100 μm. (F and G) Flow cytometric analysis of fibroblast (F) and endothelial cell (G) counts in heart tissue in sham mice and mice 4 weeks after TAC. Fibroblasts and endothelial cells are defined as CD45⁻ CD31⁻MEF-SK4⁺ and CD45⁻CD31⁺, respectively. The absolute numbers of cells were normalized by tissue weight (n = 3 to 4 per group in sham mice; n = 6 to 12 per group in TAC mice). (H) Mice with mLOY show comparable hypertrophic response of cardiac myocytes after TAC. Quantitative analysis of cross-sectional area of myocytes (CSA) in heart section at 4 weeks after TAC (n = 6 to 7 per group). Dots in all panels represent individual samples. Data are shown as mean ± SEM. Statistical analyses were performed using two-way repeated-measures ANOVA with Sidak’s multiple-comparisons tests (B). Statistical analyses were performed using two-way ANOVA with Tukey’s multiple-comparisons tests [(C), (D), (E), (F), and (G). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 4.. Inhibition of TGFβ1 reverses cardiac dysfunction in mLOY mice after TAC.

(A) Single-cell RNA sequencing from CD45⁺RFP⁺ cardiac cells 7 days after TAC shown by uniform manifold approximation and projection (UMAP) dimensionality reduction, with inflammatory and fibrotic macrophages and expression of Il1b and Lyve1 highlighted. (B) PHATE dimensionality reduction showing cells separated from control (gray) or mLOY (red) samples, with nonactivated, inflammatory, and fibrotic phenotypes labeled. Quantification of the relative percentage of control or mLOY cells contained in the inflammatory or fibrotic macrophage clusters. (C) Heatmap of transcription factor regulons within inflammatory or fibrotic macrophages related to Il1b or Tgfb1 expression, respectively, that was generated using SCENIC analysis of control and mLOY cells. (D) Activation of TGFβ1 signaling in the heart accessed by immunofluorescent staining of phosphorylated SMAD2 (pSMAD2). The number of pSMAD2⁺ cells per view field and the percentage of pSMAD2⁺ fibroblasts (right) are shown. Fibroblasts are defined as vimentin⁺ cells (n = 6 fields per group). pSMAD2⁺ and pSMAD2⁻ fibroblasts are indicated by green and orange arrows, respectively. Scale bar, 50 μm. (E) At 4 weeks after BMT, mLOY mice or control mice were subjected to TAC. Anti-TGFβ1 antibody or isotype control was intraperitoneally injected every 3 days for 4 weeks. Shown is sequential echocardiographic analysis of mLOY and control mice after TAC operation at the indicated time points (n = 6 to 7 per group). (F) Representative images and quantitative analysis of fibrotic area in heart section at 4 weeks after TAC procedure (n = 6 to 8 per group). Scale bar, 1000 μm. (G) Flow cytometric analysis of fibroblast content in heart tissue at 4 weeks after TAC. The absolute numbers of cells were normalized by tissue weight (n = 7 to 8 per group). Dots in all panels represent individual samples. Data are shown as mean ± SEM. Statistical analyses were performed using chi-square test (B), unpaired Student’s t test (D), two-way repeated-measures ANOVA with Sidak’s multiple-comparisons test (E), and one-way ANOVA with Tukey’s multiple-comparisons test [(F) and (G)]. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.