Abstract
Reversible acetylation of lysine residues within a protein is considered a biologically relevant modification that rivals phosphorylation ( Kouzarides, T. (2000) EMBO J. 19, 1176-1179 ). The enzymes responsible for such protein modification are called histone acetyltransferases (HATs) and deacetylases (HDACs). A role of protein phosphorylation in regulating muscle contraction is well established ( Solaro, R. J., Moir, A. J., and Perry, S. V. (1976) Nature 262, 615-617 ). Here we show that reversible protein acetylation carried out by HATs and HDACs also plays a role in regulating the myofilament contractile activity. We found that a Class II HDAC, HDAC4, and an HAT, PCAF, associate with cardiac myofilaments. Primary cultures of cardiomyocytes as well as mouse heart sections examined by immunohistochemical and electron microscopic analyses revealed that both HDAC4 and PCAF associate with the Z-disc and I- and A-bands of cardiac sarcomeres. Increased acetylation of sarcomeric proteins by HDAC inhibition (using class I and II HDAC inhibitors or anti-HDAC4 antibody) enhanced the myofilament calcium sensitivity. We identified the Z-disc-associated protein, MLP, a sensor of cardiac mechanical stretch, as an acetylated target of PCAF and HDAC4. We also show that trichostatin-A, a class I and II HDAC inhibitor, increases myofilament calcium sensitivity of wild-type, but not of MLP knock-out mice, thus demonstrating a role of MLP in acetylation-dependent increased contractile activity of myofilaments. These studies provide the first evidence that HATs and HDACs play a role in regulation of muscle contraction.
Highlights
Histone deacetylases (HDACs)2 and acetyltransferases (HATs) constitute two separate families of enzymes, which were originally characterized as nuclear enzymes modifying histones [3, 4]
We found that a Class II HDAC, HDAC4, and an histone acetyltransferases (HATs), PCAF, associate with cardiac myofilaments
When myocytes were examined for HDAC4 localization, we found that the deacetylase was present both in the cytoplasm and in the nucleus of the cell
Summary
Plasmid Constructs—The constructs Myc-HDAC4 [11], pcGAL4-HDAC4 [11], GST-HDAC4-AA208-310 [12], GSTMLP full-length, GST-MLP-LIM1 or LIM2 [13], and FLAGhMLP [14] have been described elsewhere. For the deacetylation assay, acetylated FLAG-MLP bound to beads was washed as above and resuspended in 1ϫ HDAC buffer (Upstate Biotechnology). This acetylated FLAG-MLP substrate was either incubated just in 1ϫ HDAC buffer (control) or with 10 g of HeLa cell lysates or with equal amounts of Myc-HDAC4 or Myc tag protein immobilized onto Myc (9E10, Santa Cruz)-agarose beads in 1ϫ HDAC buffer. To obtain Myc tag proteins, COS7 cells were transfected with the respective plasmids and 24 h post-transfection proteins were immunoprecipitated using the Myc-agarose beads These beads were washed with 1ϫ RIPA buffer 4 –5 times, once with PBS, and once with a final wash of 1ϫ HDAC buffer and resuspended in 20 l of the same buffer. Statistical differences among groups were determined using either Student’s t test (for two groups) or one-way analysis of variance (for more than two groups) followed by Fisher’s least significant difference test for post hoc pairwise comparisons if necessary
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