A high-performance liquid chromatography assay for quantification of cardiac myosin heavy chain isoform protein expression
Section snippets
Materials
Sprague Dawley rats were obtained from Charles River Laboratories. HPLC-grade water and HPLC-grade acetonitrile were purchased from Fisher. All other reagents were purchased from Sigma–Aldrich.
In vivo study
Male Sprague Dawley rats (7 weeks old) were fed iodine-deficient chow containing 0.15% propylthiouracil (PTU) for 3–4 weeks to render animals hypothyroid. Age-matched naive rats (fed normal chow) served as controls. After 4 weeks on the PTU diet, treatment groups received once daily intraperitoneal
Chromatographic separation
Reversed-phase HPLC of rat cardiac myosin resulted in five major peaks, as shown in Fig. 1A. Comparing the chromatograms of cardiac myosin samples derived from naive adult and PTU-treated rats, significant differences were observed only in peaks 3 and 4 (Fig. 1). Given that the thyroid axis regulates cardiac MyHC isoform expression [8], [9], [10], [11], [12], these results suggested that peaks 3 and 4 consisted of α- and β-MyHC, respectively, and that intact α- and β-MyHC proteins could be
Conclusions
We have demonstrated a new HPLC-based assay for cardiac MyHC composition in rats. The assay is rapid and capable of greater throughput than gel-based methods. The assay, which directly measures intact α- and β-MyHC protein with high accuracy and precision, should facilitate drug discovery focused on cardiac MyHC and/or TR signaling. So far, the method has been used only for rat cardiac samples. Future studies will address whether this assay can be translated to human heart and endomyocardial
Acknowledgment
We thank E.W. Bush, R.J. Gorczynski, and colleagues at the former Myogen (Westminster, CO, USA) for helpful discussions during the course of this work.
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- 1
Present address: Department of Medicine, Division of Cardiology, University of Colorado Denver, Aurora, CO 80045, USA.
- 2
Present address: Aerotek, Professional Services, Denver, CO 80222, USA.
- 3
Present address: Plato BioPharma, Westminster, CO 80021, USA.