Human mutation in the anti-apoptotic heat shock protein 20 abrogates its cardioprotective effects.
The small heat shock protein Hsp20 protects cardiomyocytes against apoptosis, and phosphorylation at its Ser16 site enhances its cardioprotection. To determine whether genetic variants exist in human Hsp20, which may modify these beneficial effects, we sequenced the coding region of the Hsp20 gene in 1347 patients suffering from dilated cardiomyopathy and 744 subjects with no heart disease. We identified a C59T substitution in the human Hsp20 gene in one patient and three individuals without heart disease. All subjects were heterozygous for this mutation, which changes a fully conserved proline residue into leucine at position 20 (P20L), resulting in secondary structural alterations. To examine the potential functional significance of the P20L-Hsp20 human variant, adult rat cardiomyocytes were infected with Ad.GFP (where Ad is adenovirus and GFP is green fluorescent protein), Ad.WT-Hsp20 (where WT is wild-type), and Ad.P20L-Hsp20 and subjected to simulated ischemia/reperfusion injury. Expression of WT-Hsp20 resulted in significant attenuation of apoptosis compared with the GFP control. However, the P20L-Hsp20 mutant showed no protection against apoptosis, assessed by Hoechst staining and DNA fragmentation. The loss of cardioprotection by the mutant Hsp20 was associated with its diminished phosphorylation at Ser16 compared with WT-Hsp20. Furthermore, maximal stimulation of cardiomyocytes with isoproterenol or protein kinase A-mediated phosphorylation in vitro confirmed the impaired ability of the mutant Hsp20 to become phosphorylated at Ser16. In conclusion, we have identified a P20L substitution in human Hsp20, which is associated with diminished phosphorylation at Ser16 and complete abrogation of the Hsp20 cardioprotective effects which may adversely affect the ability of human carriers to cope with cellular stress.
Nicolaou
Persoulla
P
Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0575, USA.
Knöll
Ralph
R
Haghighi
Kobra
K
Fan
Guo-Chang
GC
Dorn
Gerald W
GW
Hasenfub
Gerd
G
Kranias
Evangelia G
EG
D019943:Amino Acid Substitution; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics*; D002311:Cardiomyopathy, Dilated / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D002316:Cardiotonic Agents / Q000494:pharmacology; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects; D005260:Female; D050886:HSP20 Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000235:genetics* / Q000378:metabolism; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D020125:Mutation, Missense; D032383:Myocytes, Cardiac / Q000378:metabolism / Q000473:pathology; D016366:Open Reading Frames / Q000235:genetics; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley
D002316:Cardiotonic Agents; D050886:HSP20 Heat-Shock Proteins; C494382:HSPB6 protein, human; D051034:Inhibitor of Apoptosis Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D007545:Isoproterenol
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