Moreau Steven, DaSilva Jean N, Valdivia Ana, Fernando Pasan
National Cardiac PET Centre, University of Ottawa Heart Institute, 40 Ruskin St., Ottawa, ON, Canada K1Y 4W7; Division of Cardiology and Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, ON K1H 8M5.
National Cardiac PET Centre, University of Ottawa Heart Institute, 40 Ruskin St., Ottawa, ON, Canada K1Y 4W7; Division of Cardiology and Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, ON K1H 8M5; Department of Radiology, Radio-Oncology and Nuclear Medicine, University of Montreal; University of Montreal Hospital Research Centre (CRCHUM), 900 rue Saint-Denis, Montréal, QC H2X 0A9.
Nucl Med Biol. 2015 Feb;42(2):192-7. doi: 10.1016/j.nucmedbio.2014.09.008. Epub 2014 Sep 30.
Pathologic cardiac hypertrophy is one of the leading causes of sudden death from cardiac disease and involves a complex network of bio-signaling mechanisms. To date, the clinical detection and pathologic progression of hypertrophy remains elusive. Here we tested whether imaging Rho kinase activity would serve an accurate proxy for detecting hypertrophy. Specifically, we examine the use of the N-[(11)C]-methylated derivative of hydroxyfasudil, a Rho kinase inhibitor, as a biomarker for accurate identification of cardiomyocyte hypertrophy.
Both transformed and primary neonatal cardiomyocytes were treated with isoproterenol to induce β-adrenergic receptor stimulation and hypertrophy. Phenotypic hypertrophy was verified using cytochemical evaluation of cell and nuclear size. Western blot and activity assays were used to detect ERK 1/2 mTOR and Rho kinase activation. N-[(11)C]-methyl-hydroxyfasudil binding was verified using in vitro binding assays with isoproterenol stimulated cells.
Isoproterenol induced a rapid and distinct activation of ERK 1/2, mTOR and Rho kinase with negligible cytotoxicity. Subsequent expansion in cell and nuclear size that is typically associated with hypertrophy was also observed. Enhanced retention of N-[(11)C]-methyl-hydroxyfasudil observed after ISO-induced Rho kinase activation in hypertrophic cells was prevented by pre-treatment with unlabeled hydroxyfasudil.
N-[(11)C]-methyl-hydroxyfasudil is able to measure increased Rho kinase activity via specific binding in hypertrophied cardiomyocytes and demonstrates the potential for molecular imaging of altered Rho kinase activity in diseases such as cardiac hypertrophy.
病理性心脏肥大是心脏病猝死的主要原因之一，涉及复杂的生物信号机制网络。迄今为止，肥大的临床检测和病理进展仍不明确。在此，我们测试了成像Rho激酶活性是否可作为检测肥大的准确指标。具体而言，我们研究了使用Rho激酶抑制剂羟基法舒地尔的N-[(11)C]-甲基化衍生物作为生物标志物来准确识别心肌细胞肥大。
用异丙肾上腺素处理转化的和原代新生心肌细胞，以诱导β-肾上腺素能受体刺激和肥大。使用细胞和细胞核大小的细胞化学评估来验证表型肥大。采用蛋白质免疫印迹法和活性测定法检测ERK 1/2、mTOR和Rho激酶的激活情况。使用异丙肾上腺素刺激的细胞进行体外结合试验来验证N-[(11)C]-甲基羟基法舒地尔的结合情况。
异丙肾上腺素诱导ERK 1/2、mTOR和Rho激酶迅速且明显激活，细胞毒性可忽略不计。随后还观察到通常与肥大相关的细胞和细胞核大小增加。在肥大细胞中，ISO诱导Rho激酶激活后观察到的N-[(11)C]-甲基羟基法舒地尔保留增强，可通过用未标记的羟基法舒地尔预处理来防止。
N-[(11)C]-甲基羟基法舒地尔能够通过在肥大心肌细胞中的特异性结合来测量增加的Rho激酶活性，并证明了在心脏肥大等疾病中对改变的Rho激酶活性进行分子成像的潜力。
