Cardiomyopathy Associated with Worsening Intravascular Hemolysis in Transgenic Knock-in Mouse Model of Sickle Cell Disease

Cardiopulmonary complications are the leading cause of death in sickle cell disease (SCD). Cardiac complications, including left ventricular (LV) dysfunction, are prevalent in both young and adult SCD patients. Our studies in the Townes transgenic murine model indicate mice with homozygous SCD (SS) recapitulate the age-related clinical deterioration seen in humans. This is evidenced by the sudden decline in survival of SS mice during the transition from adolescence to adulthood (between the ages of three to six months); we have previously determined that the severity of intravascular hemolysis increases during this transition period in SS mice (Ghosh et al JCI Insight 2016; 1(4): e81090). Hitherto, the cardiac phenotype of this murine model of SCD, which appears the most versatile, has not been defined. In this study, we imaged the hearts of one, three, and six month old Townes SS mice and littermates with sickle cell trait (AS mice) using high resolution ultrasound (Vevo 2100, VisualSonics). Standard indices of LV systolic function, size, and mass, were derived from measurements made on short axis B-mode images of the heart. Ejection fraction and fractional shortening were similar for AS and SS mice, signifying preservation of systolic function in both groups up to 6 months of age. The LV mass, LV internal dimensions (LVID), systolic and diastolic volumes as well as stroke volume significantly increased with age in the SS but not in the AS. Major differences in the cardiac phenotype in SS compared to AS littermates were observed only at six-months. At six months, the SS mice had larger hearts ( 236.5 ± 20.1 mg, n=7, p=0.005) compared to AS littermates (180.2 ± 9.6 mg, n=6). The LVID in both systole (3.1 ± 0.2 mm versus2.1± 0.3, p=0.03) and diastole (4.7 ± 0.2 mmversus 3.9 ± 0.1 mm, p=0.002) were larger in the SS mice, accompanied by higher LV Mass Index (4.6 ± 0.2, n=7 versus 3.1 ± 0.3, n= 6 , p=0.03) indicating LV hypertrophy in the SS mice. RT-PCR analysis of hypertrophy markers in the LV revealed increased expression of Atrial Natriuretic Peptide (ANP, p=0.001) and β-Myosin Heavy Chain (p=0.03) genes in the SS mice compared to AS mice. In agreement with findings in humans, at 6 months, systolic volume (38.4± 6.6 µ L, n=7,versus 17.2±5.3 µ L, n=6, p=0.003) , diastolic volume (101.3± 8.4 µ L, n=7, versus 62.3 ± 4.6 µ L , n=6, p=0.0002), stroke Index (2.2± 0.2, n=7, versus 1.5±0.2, n=6, p=0.03)and cardiac index (1.0±0.1, n=7, versus 0.7±0.1, n=6, p=0.047)were all higher in the six-month SS mice compared to AS littermates. To evaluate vascular inflammation in the heart, P-selectin was measured by ELISA; Compared to AS littermates, SS mice hearts showed a 2-fold higher expression of P-selectin (p=0.009). The severity of anemia and intravascular hemolysis increased with aging in the SS mice. Our study shows progression of LV enlargement and dilation in the Townes SS mice concomitant with increased severity of intravascular hemolysis with aging. Future studies are aimed at delineating the specific molecules and cognate pathways downstream of intravascular hemolysis that promote cardiomyopathy in SCD.