Elsevier

Analytical Biochemistry

Volume 408, Issue 1, 1 January 2011, Pages 132-135
Analytical Biochemistry

A high-performance liquid chromatography assay for quantification of cardiac myosin heavy chain isoform protein expression

https://doi.org/10.1016/j.ab.2010.08.041Get rights and content

Abstract

Stress signaling in the myocardium results in enhanced expression of fetal β-myosin heavy chain (β-MyHC) and reduced expression of adult α-myosin heavy chain (α-MyHC), with the net outcome of diminished myofibrillar ATPase activity and impaired contractility. Pharmacological approaches aimed at preventing this myosin isoform “switch” could provide therapeutic benefit to patients with heart failure. Myosin isoform protein expression is typically quantified using gel electrophoresis methods, which are time-consuming and prone to variability. Here we describe a facile, reversed-phase high-performance liquid chromatography (HPLC) method for rapidly determining the relative amounts of full-length α- and β-MyHC in rat hearts. The assay was validated using cardiac tissues from rats in which a key transcriptional regulator of MyHC expression, the thyroid hormone receptor, was pharmacologically manipulated. This novel assay should facilitate drug discovery efforts focused on the MyHC axis.

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.

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