Introduction: 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.
Methods: 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.
Results: 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.
Conclusions: 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.
Keywords: Cardiomyocyte; ERK 1/2; Isoproterenol; ROCK; mTOR; β-adrenergic receptor stimulation.
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