title: Increased Ca2+-sensitivity of the contractile apparatus in end-stage human heart failure results from altered phosphorylation of contractile proteins.
abstract: OBJECTIVE
  The alterations in contractile proteins underlying enhanced Ca(2+)-sensitivity of the contractile apparatus in end-stage failing human myocardium are still not resolved. In the present study an attempt was made to reveal to what extent protein alterations contribute to the increased Ca(2+)-responsiveness in human heart failure.
  
  
  METHODS
  Isometric force and its Ca(2+)-sensitivity were studied in single left ventricular myocytes from non-failing donor (n=6) and end-stage failing (n=10) hearts. To elucidate which protein alterations contribute to the increased Ca(2+)-responsiveness isoform composition and phosphorylation status of contractile proteins were analysed by one- and two-dimensional gel electrophoresis and Western immunoblotting.
  
  
  RESULTS
  Maximal tension did not differ between myocytes obtained from donor and failing hearts, while Ca(2+)-sensitivity of the contractile apparatus (pCa(50)) was significantly higher in failing myocardium (deltapCa(50)=0.17). Protein analysis indicated that neither re-expression of atrial light chain 1 and fetal troponin T (TnT) nor degradation of myosin light chains and troponin I (TnI) are responsible for the observed increase in Ca(2+)-responsiveness. An inverse correlation was found between pCa(50) and percentage of phosphorylated myosin light chain 2 (MLC-2), while phosphorylation of MLC-1 and TnT did not differ between donor and failing hearts. Incubation of myocytes with protein kinase A decreased Ca(2+)-sensitivity to a larger extent in failing (deltapCa(50)=0.20) than in donor (deltapCa(50)=0.03) myocytes, abolishing the difference in Ca(2+)-responsiveness. An increased percentage of dephosphorylated TnI was found in failing hearts, which significantly correlated with the enhanced Ca(2+)-responsiveness.
  
  
  CONCLUSIONS
  The increased Ca(2+)-responsiveness of the contractile apparatus in end-stage failing human hearts cannot be explained by a shift in contractile protein isoforms, but results from the complex interplay between changes in the phosphorylation status of MLC-2 and TnI.
authors.affiliation: Laboratory for Physiology, Institute for Cardiovascular Research (ICaR-VU), VU University Medical Center, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands. j.van_der_velden.physiol@med.vu.nl
authors.forename: J
authors.initials: J
authors.lastname: van der Velden
authors.forename: Z
authors.initials: Z
authors.lastname: Papp
authors.forename: R
authors.initials: R
authors.lastname: Zaremba
authors.forename: N M
authors.initials: NM
authors.lastname: Boontje
authors.forename: J W
authors.initials: JW
authors.lastname: de Jong
authors.forename: V J
authors.initials: VJ
authors.lastname: Owen
authors.forename: P B J
authors.initials: PB
authors.lastname: Burton
authors.forename: P
authors.initials: P
authors.lastname: Goldmann
authors.forename: K
authors.initials: K
authors.lastname: Jaquet
authors.forename: G J M
authors.initials: GJ
authors.lastname: Stienen
mesh_terms: D000328:Adult; D000368:Aged; D024743:Atrial Myosins / Q000378:metabolism; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D024722:Cardiac Myosins / Q000378:metabolism; D016022:Case-Control Studies; D002478:Cells, Cultured; D003285:Contractile Proteins / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D006333:Heart Failure / Q000378:metabolism*; D006801:Humans; D008297:Male; D008875:Middle Aged; D032383:Myocytes, Cardiac / Q000378:metabolism*; D018994:Myosin Light Chains / Q000378:metabolism; D010766:Phosphorylation; D020033:Protein Isoforms / Q000378:metabolism; D019210:Troponin I / Q000378:metabolism; D020107:Troponin T / Q000378:metabolism
chemical_list: D003285:Contractile Proteins; D018994:Myosin Light Chains; D020033:Protein Isoforms; D019210:Troponin I; D020107:Troponin T; C107184:myosin light chain 2; C076659:myosin light chain I; D024743:Atrial Myosins; D024722:Cardiac Myosins; D002118:Calcium