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.

Cyclic nucleotide-dependent relaxation of vascular smooth muscle is associated with increases in the phosphorylation of the small heat shock-related protein, HSP20. To determine whether phosphorylated HSP20 directly mediates relaxation, we used gene transfection and protein transduction of HSP20 analogues. Rat mesangial cells were transfected with constructs containing wild-type HSP20-enhanced green fluorescent protein (EGFP), phosphorylation site mutated HSP20 (S16A-HSP20-EGFP), or EGFP alone. Contractile properties were determined on a silicone polymer substrata. In the presence of serum, EGFP-vector transfected control cells and S16A-HSP20 transfected cells formed wrinkles on the polymer (contracted). Activation of cyclic nucleotide signaling pathways in the EGFP-vector transfected control cells led to a time-dependent decrease in the wrinkles (relaxation). The S16A-HSP20 transfected cells were refractory to cyclic nucleotide-dependent relaxation. Cells overexpressing the wild-type HSP20 did not form wrinkles on the polymer in response to serum (refractory to contraction). Treatment of precontracted strips of intact bovine carotid artery smooth muscle with synthetic peptides containing HIV-trans-activating transcriptional activator and a phosphopeptide motif of HSP20 led to dose-dependent relaxation. These data provide evidence that phosphorylated HSP20 has a direct role in smooth muscle relaxation and that small phosphopeptide motifs of HSP20 can mimic the effects of the entire molecule.

Cyclic nucleotide-dependent vascular relaxation is associated with increases in the phosphorylation of a small heat shock protein (HSP), HSP20. An increase in phosphorylation of another small HSP, HSP27, is associated with impaired cyclic nucleotide-dependent vascular relaxation. Expression of HSPs is altered by exposure to several types of cellular stress in vitro. To determine if behavioral stress in vivo alters vascular expression and phosphorylation of the small HSPs and cyclic nucleotide-dependent vascular relaxation, borderline hypertensive rats were stressed by restraint and exposure to air-jet stress 2 h/day for 10 days or remained in their home cage. Stress impaired relaxation of aorta to forskolin, which activates adenylyl cyclase, and sodium nitroprusside, which activates guanylyl cyclase. This was associated with an increase in the aortic expression and phosphorylation of HSP27, which was localized to the vascular smooth muscle, but a decrease in the amount of phosphorylated (P)-HSP20. To determine if P-HSP27 inhibits phosphorylation of HSP20, P-HSP27 was added to a reaction mixture containing recombinant HSP20 and the catalytic subunit of cAMP-dependent protein kinase. P-HSP27 inhibited phosphorylation of HSP20 in a concentration-dependent manner. These data demonstrate that P-HSP27 can inhibit phosphorylation of HSP20. The increase in P-HSP27 and decrease in P-HSP20 were associated with reduced cyclic nucleotide-dependent vascular smooth muscle relaxation in response to behavioral stress in vivo, an effect similar to that observed previously in response to cellular stress in vitro.

Percutaneous femoral artery closure devices are being used routinely after cardiac catheterizations. The use of these devices has been advocated to decrease length of stay, promote early ambulation, and prevent bleeding. We reviewed the use of these devices in our institution and report three cases of infectious complications (two pseudoaneurysms and one infected hematoma). Reports of infected pseudoaneurysms after cardiac catheterization before the implementation of these devices are rare. The use of these devices may be associated with an increased incidence of infected femoral pseudo-aneurysms.