Occult Myopathic Effects of K118c Mutation in Cardiac Troponin I Detected in Isolated and Cultured Adult Mouse Cardiomyocyte

Abstract

Isolated adult cardiomyocytes provide a valuable system for cellular level phenotype studies. However, an N-terminal truncation of cardiac troponin T (cTnT-ND) found in ischemia-reperfusion with functional consequences also occurs during the perfusion-based cell isolation. Here we utilized short-term culture to allow isolated cardiomyocytes to recover for the study of a cardiac troponin I mutation (cTnI-K118C). Adult cardiomyocytes were isolated from wild type and cTnl-K118C transgenic mice. Myofilament proteins were monitored using SDS-PAGE, Western blot and Pro-Q phosphoprotein staining. Paced contraction was examined on day 0 and day 2 of culture using edge detection. The cultured adult mouse cardiomyocytes maintained normal contents of myosin, tropomyosin, cTnT and cTnI in culture for over 4 days. cTnT-ND declined from ∼7.7% of total cTnT at day 0 to ∼2.9% after 2 days of culture. The PKA-dependent phosphorylation of cTnI was preserved in 2 day-cultured cells. The amplitude of shortening in wild type myocytes on day 0 and day 2 were 3.05±0.53% and 3.79±0.68%, respectively, without statistical difference. The amplitude of shortening and the velocities of shortening/re-lengthening were decreased in day 0 and 2-day-cultured cTnI-K118C cardiomyocytes as compared with wild type controls, but only the 2-day-cultured group showed statistical significance (P<0.05). The slack length of the cardiomyocytes was 119.4±2.0 µm for wild type and 146.1±2.9 µm for cTnI-K118C groups (P<0.01) with no increase in sarcomere length or cell diameter. The data demonstrated that 2-day-cultured adult mouse cardiomyocytes were effectively recovering from the proteolytic modification of cTnT induced during isolation while maintaining the original level of contractility. The study of isolated cardiomyocytes from young adult transgenic mice without heart failure revealed that cTnI-118C decreased contractility and caused cellular remodeling toward dilated cardiomyopathy.