Blockade of Angiotensin II Signaling Ameliorates Cardiac Fibrosis and Improves Diastolic Dysfunction in Mice with Sickle Cell Anemia

Introduction: Cardiopulmonary complications are the most common cause of death in adults with sickle cell disease (SCD). We previously reported a unique cardiomyopathy with restrictive physiology due to myocardial fibrosis, resulting in diastolic dysfunction and predisposition to arrhythmias, both in mice (Bakeer et al, PNAS 2016) and humans (Niss et al, Blood 2017) with SCD. However, the underlying mechanism resulting in myocardial fibrosis is unknown. We have also reported high Angiotensin-II (Ang-II) levels in mice and humans with SCD, in the absence of hypertension (Roy et al, AJH 2018). Herein, we hypothesized that increased Ang-II signaling promotes sickle cardiomyopathy and can be reversed by blocking this signaling in SCD mice.

Methods: We blocked Ang-II receptor-I (ATR₁) signaling in 5-8-week-old Townes SCD mice with losartan mixed in drinking water, or vehicle control for 3 months, 6 months, or 9 months in different experiments. Cardiac parameters were measured using echocardiograms and electrocardiograms. IL-18 and Galectin-3 levels were measured as plasma biomarkers of cardiomyopathy by ELISA, and qRT-PCR was performed on myocardial mRNA for candidate genes. At sacrifice, cardiac histopathology was performed using H&E staining, as well as staining for fibrosis using Mason-trichrome and Sirius red.

Results: Echocardiograms showed that there was improvement in diastolic function and restrictive physiology with losartan treatment. There was a significant reduction in left atrium size (P=0.04), and left ventricular hypertrophy (P=0.0003), and an increase in isovolumetric relaxation time (P=0.0006) in losartan-treated SCD mice compared to vehicle-control SCD mice. Electrocardiograms showed significantly shortened QRS duration (P=0.001) with losartan treatment. There was a significant reduction in CTGF (connective tissue growth factor) mRNA expression (P=0.03), and plasma levels of Galectin-3 (P=0.07) and IL-18 (P=0.0005). Histopathology confirmed reduced intramyocardial fibrosis in losartan-treated mice, which was quantified using Aperio ImageScope64 software (P=0.01). All of these occurred despite more anemia in losartan-treated vs. control SCD mice, as ATR₁ signaling enhances erythropoiesis. We also treated losartan and control groups of mice with biweekly blood transfusions and compared them to the corresponding non-transfused mice for 3 months. With this, we observed a similar reduction in IL-18 and Galectin-3, although the duration of treatment was not sufficient to show differences in myocardial fibrosis. Losartan treatment resulted in reduced oxidative stress, which may have contributed to the improved cardiomyopathy.

Conclusion: Ang-II signaling plays an important role in the development of cardiac fibrosis and its downstream complications in SCD patients, which can be reversed by blockade of ATR₁.