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

Abstract

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 Ϸ200 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.

Effects of hypoxia on stress proteins in the piglet heart at birth

“…In the heart, Hsp20 is biochemically associated with ␣B-crystallin but localizes in distinct bands as does ␣B-crystallin and sarcomeric actin. This report appears to dissociate the effects of hypoxia on ␣B-crystallin and Hsp20 in the heart (Pipkin et al 2003). Hsp60 is a mitochondrial HSP associated with Hsp10.…”

Human Mutation in the Anti-apoptotic Heat Shock Protein 20 Abrogates Its Cardioprotective Effects

“…First, we have previously shown that Hsp20 confers protection via inhibition of the Bax/ caspase-3 pro-apoptotic pathway (16), which may be subject to regulation by its phosphorylation state. Second, pSer 16 Hsp20 may help maintain cardiomyocyte integrity via stabilization of the cytoskeleton (16,27). Finally, Hsp20 may form both homologous and heterologous macromolecular structures (3,27,28), which may be affected by its phosphorylation status.…”

“…Second, pSer 16 Hsp20 may help maintain cardiomyocyte integrity via stabilization of the cytoskeleton (16,27). Finally, Hsp20 may form both homologous and heterologous macromolecular structures (3,27,28), which may be affected by its phosphorylation status. Overall, loss of the Hsp20 ability to become phosphorylated may have detrimental effects on its cellular functions.…”

“…At basal levels, no phosphorylated Hsp20 could be detected, consistent with the low activity of PKA in isolated myocytes. To test the specificity of this antibody, peptide competition was performed with the anti- [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29] in human, dog, rat, and mouse shows that proline 20 is fully conserved. B, secondary structure predictions revealed alterations in the helical content around the Ser 16 phosphorylation site as well as around the site of the mutation.…”

Organelle Proteomics

“…Chromatography with molecular sieving columns revealed that HSP20 and ␣B-crystallin were associated in an aggregate of Ϸ200 kDa, and the phosphorylation of HSP20 was determined by 2D gel electrophoresis and immunoblotting. 33 Signal transduction protein complexes and their activation, eg, protein kinase C activation, were analyzed as described above by subcellular fractionation, detergent extraction, and Western blotting in cultures of neonatal rat ventricular myocytes after electric stimulation. 34 Caveolae are plasma membrane invaginations that are enriched in cholesterol, sphingolipids, and the marker protein caveolin.…”