Localization, macromolecular associations, and function of the small heat shock-related protein HSP20 in rat heart.
BACKGROUND
The small heat shock proteins HSP20, HSP25, alphaB-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.


RESULTS
Western blotting identified immunoreactive HSP20, alphaB-crystallin, and MKBP in rat heart homogenates. Subcellular fractionation demonstrated that HSP20, alphaB-crystallin, and MKBP were predominantly in cytosolic fractions. Chromatography with molecular sieving columns revealed that HSP20 and alphaB-crystallin were associated in an aggregate of approximately 200 kDa, and alphaB-crystallin coimmunoprecipitated with HSP20. Immunofluorescence microscopy demonstrated that the pattern of HSP20, alphaB-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 alphaB-crystallin, possibly at the level of the actin sarcomere. Phosphorylated HSP20 increases myocyte shortening rate through increases in calcium uptake and more rapid lengthening.
Pipkin
Walter
W
Department of Surgery, Medical College of Georgia, Augusta, USA.
Johnson
John A
JA
Creazzo
Tony L
TL
Burch
Jarrett
J
Komalavilas
Padmini
P
Brophy
Colleen
C
D000818:Animals; D000918:Antibody Specificity; D002118:Calcium / Q000032:analysis; D002478:Cells, Cultured; D050886:HSP20 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000032:analysis* / Q000378:metabolism / Q000502:physiology*; D008856:Microscopy, Fluorescence; D009200:Myocardial Contraction; D009206:Myocardium / Q000737:chemistry* / Q000378:metabolism*; D009712:Nucleotides, Cyclic / Q000378:metabolism; D010748:Phosphopeptides / Q000494:pharmacology; D010750:Phosphoproteins / Q000032:analysis* / Q000378:metabolism / Q000502:physiology*; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction
D050886:HSP20 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D009712:Nucleotides, Cyclic; D010748:Phosphopeptides; D010750:Phosphoproteins; D002118:Calcium