Nonhematopoietic Nrf2 Deficiency Worsens Chronic Intravascular Hemolysis but Not Cardiomyopathy in Transgenic Sickle Cell Mice

Background: Cardiopulmonary complications are the leading causes of death in sickle cell disease (SCD). Cardiac complications in SCD include left ventricular (LV) hypertrophy and LV systolic or diastolic dysfunction. In SCD, intravascular hemolysis (IVH) contributes to multiorgan damage, but its role in cardiac injury is unknown. We previously reported that nonhematopoietic deficiency of nuclear factor erythroid-2-related factor 2 (Nrf2), a transcription factor that controls antioxidative and anti-inflammatory genes, worsens the severity of IVH in SCD mice. Here, we first sought to determine if Townes transgenic SCD mice develop an abnormal cardiac phenotype, utilizing a cross-sectional study of varying-aged mice to test the hypothesis that these mice develop an age-related cardiomyopathy. Second, we determined whether IVH associated with absence of Nrf2 in the nonhematopoietic system worsens SCD cardiomyopathy.

Methods: To identify a SCD cardiac phenotype, we performed echocardiography in a cross sectional cohort of Townes transgenic sickle cell mice (SS) and matched AA controls of varying ages (1-10 months). LV echocardiography dimensions were normalized to body weight. To determine the role of IVH and Nrf2 on the SCD cardiac phenotype, we transplanted whole bone marrow cells from SS mice to irradiated Nrf2^-/- mice or C57BL/6 (Nrf2^+/+) mice, to generate bone marrow chimeric sickle mice with Nrf2 deficiency in nonhematopoietic tissues (SS^NHNrf2-/-) or chimeric sickle controls (SS^Nrf2+/+), respectively. Serial echocardiography was performed for up to 10 months, and hearts were harvested post-mortem for histology and molecular studies.

Results: Cross-sectional study: Echocardiography showed cardiac abnormalities, most pronounced by 10 months, in the SS (n=8-9) compared to AA mice (n=9-13), respectively: LV hypertrophy was evidenced by a larger LV internal diameter (0.16 ± 0.01 vs. 0.13 ± 0.10 mm/g, p=0.005), higher LV mass index (5.2 ± 0.5 vs. 4.0 ±0.2 mg/g, p=0.02) and higher post-mortem heart weight (7.8 ± 0.3 vs. 5.2 ± 0.2 mg/g, p<0.0001) in SS mice. Compared to AA mice, SS mice had a lower ejection fraction (52 ± 4% vs 69 ± 2%, p=0.006) and fractional shortening (27 ± 2% vs. 39 ± 2%, p=0.001), consistent with impaired systolic function in SCD. Trichome-stained histological heart sections revealed myocardial fibrosis in SS but not in AA mice. Longitudinal study: Ten months after bone marrow transplant engraftment, SS^NHNrf2-/- mice (n=6) had a significantly higher total plasma heme (p=0.008), lactate dehydrogenase (p=0.038), and plasma hemoglobin (p=0.008), confirming an exacerbation of IVH compared to SS^Nrf2+/+ controls (n=3). Surprisingly, compared to SS^Nrf2+/+ mice, SS^NHNrf2-/- mice had a higher hematocrit (27.0 ± 1.0 vs. 23.1 ± 0.2%, p=0.035), lower reticulocyte count (57.5 ± 2.2% vs. 68.6 ± 4.8%, p=0.04), and a trend towards higher RBC count (p=0.05). Serial echo revealed that SS^NHNrf2-/- mice, compared to SS^Nrf2+/+ controls, had a lower LV internal diameter (0.17 ± 0.01 vs 0.21 ± 0.01 mm/g, p=0.001) and LV mass index (4.7 ± 0.2 vs. 6.8 ± 0.2 mg/g, p=0.0004), consistent with less LV hypertrophy in the SS^NHNrf2-/- mice. These findings were confirmed postmortem, where SS^NHNrf2-/- mice had lower heart weight (p=0.0002) and cardiac mRNA expression of BNP (p=0.045) compared to SS^Nrf2+/+ controls. There were no significant differences in systolic or diastolic function parameters between SS^Nrf2+/+ controls and SS^NHNrf2-/- mice. There was, however, a trend towards less myocardial fibrosis in SS^NHNrf2-/- mice based on cardiac mRNA expression of Collagens 1 and 3.

Conclusions: Townes transgenic sickle mice exhibit an age-dependent cardiomyopathy, with LV eccentric hypertrophy, systolic dysfunction, and myocardial fibrosis by 10 months of age. We reaffirm that Nrf2 deficiency in non-hematopoietic tissue worsens markers of IVH in SCD mice and unveil an unexpected decoupling phenomenon whereby despite increasing IVH in SS^NHNrf2-/- mice, anemia improves. The amelioration of the SCD cardiac phenotype in SS^NHNrf2-/- mice suggests that chronic IVH conferred by Nrf2 deficiency does not principally drive SCD-associated cardiomyopathy. While the improvement in anemia that we observed with Nrf2 deficiency may have played a role in amelioration of cardiomyopathy, further study is warranted to explore a possible cardiac protective effect of Nrf2 deficiency in SCD mice.