Xuchen Zhang, Qiang Xie, Duanjun Tan, Patty J Lee, Felicitas L Lacbawan, Jenny Libian. State University of New York, Downstate Medical Center, Brooklyn; Yale University School of Medicine, New Haven, CT
Background: Transthyretin (TTR) is associated with two forms of cardiac amyloidosis: familial (mutant TTR) and systemic senile amyloidosis (wildtype TTR). Approximately 4% of African Americans are heterozygous for V122I variant TTR, a mutation associated with familial amyloidotic cardiomyopathy. The mechanisms of TTR-induced cardiac injury remain elusive. Markers of oxidative stress have been associated with TTR amyloid deposits in peripheral nerve and we previously reported that oxidative stress and ERK1/2 activation mediate cell death in lung epithelium. We investigated the potential role of oxidative stress and ERK1/2 activation in mediating TTR-induced cardiomyocyte injury.
Design: Cases of TTR cardiac amyloidosis and age-matched controls were identified from 2007-2010 autopsy records. TUNEL and 8-OH-dG staining was performed on formalin-fixed paraffin-embedded sections from left ventricle. The TTR gene was sequenced using genomic DNA extracted from the paraffin blocks. Cultured rat cardiomyocytes were exposed to TTR fibrils formed by incubating wildtype TTR under acidic conditions. Apoptosis was assessed by TUNEL staining and Annexin V flow cytometry. Oxidative stress was examined by Western blot for heme oxygenase-1 (HO-1), reactive oxygen species (ROS) production, and 8-OH-dG staining. ERK1/2 activation was measured by Western blot of phospho-ERK1/2.
Results: Four cases of TTR cardiac amyloidosis (average age 82.8 years; range 80 to 87 years) were identified. All patients had presented with chronic heart failure and arrhythmia. TTR gene sequencing identified mutant TTR (V122I) in 3 cases and wildtype TTR in 1 case of cardiac amyloidosis. Positive staining of TUNEL and 8-OH-dG was more prominent in cases of cardiac amyloidosis than in control heart tissue. Rat cardiomyocytes treated with TTR fibrils showed more ROS production, HO-1 expression, phospho-ERK1/2, and apoptosis than untreated cardiomyocytes. Inhibition of ERK1/2 activation by PD98059 or ROS production by diphenylene iodonium ameliorated TTR fibril-induced oxidative injury and apoptosis.
Conclusions: Apoptosis and oxidative injury are increased in hearts of patients with cardiac amyloidosis compared with age-matched controls. Oxidative stress, ERK1/2 activation, and apoptosis are involved in TTR-induced injury of cultured rat cardiomyocytes. Inhibition of oxidative stress and ERK1/2 activation may provide a potential mechanism for prevention and treatment of cardiac injury associated with TTR cardiac amyloidosis.