Clonal Hematopoiesis in Clinical and Experimental Heart Failure With Preserved Ejection Fraction

Abstract

Background: Clonal hematopoiesis (CH), which results from an array of nonmalignant driver gene mutations, can lead to altered immune cell function and chronic disease, and has been associated with worse outcomes in patients with heart failure (HF) with reduced ejection fraction. However, the role of CH in the prognosis of HF with preserved ejection fraction (HFpEF) has been understudied. This study aimed to characterize CH in patients with HFpEF and elucidate its causal role in a murine model.

Methods: Using a panel of 20 candidate CH driver genes and a variant allele fraction cutoff of 0.5%, ultradeep error-corrected sequencing identified CH in a cohort of 81 patients with HFpEF (mean age, 71±6 years; ejection fraction, 63±5%) and 36 controls without a diagnosis of HFpEF (mean age, 74±7 years; ejection fraction, 61.5±8%). CH was also evaluated in a replication cohort of 59 individuals with HFpEF.

Results: Compared with controls, there was an enrichment of TET2-mediated CH in the HFpEF patient cohort (12% versus 0%, respectively; P=0.02). In the HFpEF cohort, patients with CH exhibited exacerbated diastolic dysfunction in terms of E/e' (14.9 versus 11.7, respectively; P=0.0096) and E/A (1.69 versus 0.89, respectively; P=0.0206) compared with those without CH. The association of CH with exacerbated diastolic dysfunction was corroborated in a validation cohort of individuals with HFpEF. In accordance, patients with HFpEF, an age ≥70 years, and CH exhibited worse prognosis in terms of 5-year cardiovascular-related hospitalization rate (hazard ratio, 5.06; P=0.042) compared with patients with HFpEF and an age ≥70 years without CH. To investigate the causal role of CH in HFpEF, nonconditioned mice underwent adoptive transfer with Tet2-wild-type or Tet2-deficient bone marrow and were subsequently subjected to a high-fat diet/L-NAME (N_ω-nitro-l-arginine methyl ester) combination treatment to induce features of HFpEF. This model of Tet2-CH exacerbated cardiac hypertrophy by heart weight/tibia length and cardiomyocyte size, diastolic dysfunction by E/e' and left ventricular end-diastolic pressure, and cardiac fibrosis compared with the Tet2-wild-type condition.

Conclusions: CH is associated with worse heart function and prognosis in patients with HFpEF, and a murine experimental model of Tet2-mediated CH displays greater features of HFpEF.

Keywords: biomarkers; clonal hematopoiesis; heart failure; prognosis.

Figure 1.. Characterization of clonal hematopoiesis in the Alberta HEART patient cohort.

A. Abundance of the specified driver gene mutation in the patient cohort (n=58). B. Proportion of patients with the specified number of CH mutations (n=47). C. CH prevalence as a function of age in the patient cohort (n=117). D. VAF for the largest clone identified in patients with CH in control individuals and HFpEF patients. Statistical significance was determined by Mann-Whitney U test (n= 11 control individuals and 36 HFpEF patients). E. Prevalence of CH and driver gene mutations in control individuals and patients with HFpEF. Statistical significance was determined by Fisher’s Exact Test (n= 36 control individuals and 81 HFpEF patients).

Figure 2.. HFpEF patients with CH exhibit worse diastolic dysfunction and possess elevated levels of heart failure biomarkers at baseline.

A. Echocardiographic analysis of E/e’ in HFpEF patients with and without CH. Statistical significance was determined by Mann-Whitney U test (n= 35 HFpEF CH− patients and 27 HFpEF CH+ patients). B. Echocardiographic analysis of E/A in HFpEF patients with and without CH. Statistical significance was determined by Mann-Whitney U test (n= 27 HFpEF CH− patients and 22 HFpEF CH+ patients). C. Echocardiographic analysis of deceleration time in HFpEF patients with and without CH. Statistical significance was determined by Welch’s t test (n= 31 HFpEF CH− patients and 22 HFpEF CH+ patients). D. Plasma brain natriuretic peptide (BNP) levels for HFpEF patients with and without CH. Statistical significance was determined by Mann-Whitney U test (n= 45 HFpEF CH− patients and 36 HFpEF CH+ patients). E. Plasma N-terminal pro b-type natriuretic peptide (NT-proBNP) levels for HFpEF patients with and without CH. Statistical significance was determined by Mann-Whitney U test (n= 45 HFpEF CH− patients and 36 HFpEF CH+ patients).

Figure 3.. HFpEF patients with mutations in CH driver genes exhibit worse long-term prognosis.

A. Univariate and multivariate analysis of CV-related hospitalization for HFpEF patients and an age ≥ 70 years old based on CH status, DNMT3A/TET2-driven CH status, DNMT3A-driven CH status, and TET2-driven CH status. Statistical significance and hazards ratio were determined by Cox proportional hazards model. B. 5-year CV-related hospitalization based on clonal hematopoiesis status for HFpEF patients with an age ≥ 70 years old. Statistical significance and hazards ratio were determined by Cox proportional hazards model (n= 27 HFpEF CH− patients and 30 HFpEF CH+ patients). C. 5-year CV-related hospitalization for HFpEF patients with and without DNMT3A/TET2-driven CH and an age ≥ 70 years old. Statistical significance and hazards ratio were determined by Cox proportional hazards model (n= 36 HFpEF DNMT3A/TET2- patients and 21 HFpEF DNMT3A/TET2+ patients). D. 5-year CV-related hospitalization for HFpEF patients with and without DNMT3A-driven CH and an age ≥ 70 years old. Statistical significance and hazards ratio were determined by Cox proportional hazards model (n= 41 HFpEF DNMT3A- patients and 16 HFpEF DNMT3A+ patients). E. 5-year CV-related hospitalization for HFpEF patients with and without TET2-driven CH and an age ≥ 70 years old. Statistical significance and hazards ratio were determined by Cox proportional hazards model (n= 49 HFpEF TET2- patients and 8 HFpEF TET2+ patients).

Figure 4.. HFD/L-NAME treatment accelerates expansion of Tet2-deficient cells in peripheral blood.

A. Schematic of the experimental design. CD45.2 Tet2-sufficient or Tet2-deficient bone marrow was adoptively transferred to CD45.1 Pep Boy mice. One month after bone marrow transplantation, mice were started on either HFD/L-NAME combination treatment or continued on control diet and water. Serial measurements were taken at baseline, 5 weeks, and 12 weeks. B-G. Flow cytometric quantification of donor cell chimerism for neutrophils, Ly6C+ monocytes, Ly6C− monocytes, B cells, CD4 T cells, and CD8 T cells in the peripheral blood at baseline and after 5 weeks and 12 weeks of treatment. Statistical significance was determined by 2-way ANOVA with post hoc Sidak multiple comparison test (n= 13–26 per group).

Figure 5.. HFD/L-NAME treatment promotes expansion of Tet2-deficient cells in the hematopoietic stem and progenitor cells.

A-F. Flow cytometric quantification of donor cell chimerism in LSK cells, MMP4 cells, MMP3 cells, MMP2 cells, long-term hematopoietic stem cells (LT-HSC), and short-term hematopoietic stem cells (ST-HSC) of the bone marrow after 12 weeks of treatment. Statistical significance was determined by 2-way ANOVA with post hoc Sidak multiple comparison test (n=4–11 per group).

Figure 6.. Tet2-mediated clonal hematopoiesis exacerbates cardiomyopathy in a model of HFpEF.

A. Normalized change in body mass relative to baseline body mass measured at the initiation of treatment. Measurements were performed at baseline and after 5 and 12 weeks of treatment. Statistical significance was determined by 2-way ANOVA with post hoc Sidak multiple comparison test (n=14–17 per group). B. Systolic blood pressure measured by tail-cuff plethysmography at baseline and after 5 and 12 weeks of treatment. Statistical significance was determined by 2-way ANOVA with post hoc Sidak multiple comparison test (n=8–17 per group). C. Serial echocardiographic analysis after 5 and 12 weeks of treatment. Statistical significance was determined by 2-way ANOVA with post hoc Sidak multiple comparison test (n= 13–16 per group). D. Left ventricular end diastolic pressure was obtained via LV catheterization after 12 weeks of treatment. Statistical significance was determined by 2-way ANOVA with post hoc Sidak multiple comparison test (n=4–12 per group). E. Heart weight normalized to tibia length after 12 weeks of treatment. Statistical significance was determined by 2-way ANOVA with post hoc Sidak multiple comparison test (n=13–28 per group). F. Quantification of cardiomyocyte cross-sectional area from heat germ agglutinin (WGA) stained hearts after 12 weeks of treatment. Statistical significance was determined by 2-way ANOVA with post hoc Sidak multiple comparison test (n=7–9 per group). G. Representative images and analysis of Picrosirius Red/Fast Green staining of the hearts after 12 weeks of treatment. Scale bars are included in the lower right corner and correspond to 200 μm. Statistical significance was determined by 2-way ANOVA with post hoc Sidak multiple comparison test (n=5–11 per group).