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(Circulation. 2003;107:469.)
© 2003 American Heart Association, Inc.

Basic Science Reports

Localization, Macromolecular Associations, and Function of the Small Heat Shock–Related Protein HSP20 in Rat Heart

Walter Pipkin, MD; John A. Johnson, PhD; Tony L. Creazzo, PhD; Jarrett Burch, BS; Padmini Komalavilas, PhD; Colleen Brophy, MD

From the Departments of Surgery (W.P.), Medicine (J.A.J.) (Institute of Molecular Medicine and Genetics), and Pharmacology and Toxicology (J.A.J.), Medical College of Georgia, Augusta; Pediatrics/Neonatology Division, Neonatal/Perinatal Research Institute (T.L.C., J.B.), Duke University Medical Center, Durham, NC; Carl T. Hayden, Phoenix VAMC (P.K., C.B.), Phoenix, Ariz; and the Department of Bioengineering (P.K., C.B.), Arizona State University, Tempe.

Correspondence to Colleen Brophy, MD, Research Professor of Bioengineering, Arizona State University, Vascular Surgeon, Phoenix VAMC CS112, 650 E Indian School Rd, Phoenix, AZ 85012. E-mail colleen.brophy{at}asu.edu

Background— The small heat shock proteins HSP20, HSP25, B-crystallin, and myotonic dystrophy kinase binding protein (MKBP) may regulate dynamic changes in the cytoskeleton. For example, the phosphorylation of HSP20 has been associated with relaxation of vascular smooth muscle. This study examined the function of HSP20 in heart muscle.

Methods and Results— Western blotting identified immunoreactive HSP20, B-crystallin, and MKBP in rat heart homogenates. Subcellular fractionation demonstrated that HSP20, B-crystallin, and MKBP were predominantly in cytosolic fractions. Chromatography with molecular sieving columns revealed that HSP20 and B-crystallin were associated in an aggregate of 200 kDa, and B-crystallin coimmunoprecipitated with HSP20. Immunofluorescence microscopy demonstrated that the pattern of HSP20, B-crystallin, and actin staining was predominantly in transverse bands. Treatment with sodium nitroprusside led to increases in the phosphorylation of HSP20, as determined with 2-dimensional immunoblots. Incubation of transiently permeabilized myocytes with phosphopeptide analogues of HSP20 led to an increase in the rate of shortening. The increased shortening rate was associated with an increase in the rate of lengthening and a more rapid decay of the calcium transient.

Conclusions— HSP20 is associated with B-crystallin, possibly at the level of the actin sarcomere. Phosphorylated HSP20 increases myocyte shortening rate through increases in calcium uptake and more rapid lengthening.

Key Words: myocytes • muscle, smooth • contractility

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