title: Post-ischemic PKC inhibition impairs myocardial calcium handling and increases contractile protein calcium sensitivity.
abstract: OBJECTIVE
  Protein kinase C (PKC) activation impairs contractility in the normal heart but is protective during myocardial ischemia. We hypothesized that PKC remains activated post-ischemia and modulates myocardial excitation-contraction coupling during early reperfusion.
  
  
  METHODS
  Langendorff-perfused rabbit hearts where subjected to 25 min unmodified ischemia and 30 min reperfusion. Total PKC activity was measured, and the intracellular translocation pattern of PKC-alpha, -delta, -epsilon, and -eta assessed by immunohistochemistry and fractionated Western immunoblotting. The PKC-inhibitors chelerythrine and GF109203X were added during reperfusion and also given to non-ischemic hearts. Measurements included left ventricular function, intracellular calcium handling measured by Rhod-2 spectrofluorometry, myofibrillar calcium responsiveness in beating and tetanized hearts, and metabolic parameters.
  
  
  RESULTS
  Total PKC activity was increased at end-ischemia and remained elevated after 30 min of reperfusion. The translocation pattern indicated PKC-epsilon as the main active isoform during reperfusion. Post-ischemic PKC inhibition affected mainly diastolic relaxation, with lesser effect on contractility. Both PKC inhibitors increased the Ca(2+) responsiveness of the myofilaments as indicated by a leftward shift of the calcium-to-force relationship and increased maximum calcium activated tetanic pressure. Diastolic Ca(2+) removal was delayed and the post-ischemic [Ca(2+)](i) overload further exacerbated. Depressed systolic function was associated with a lower amplitude of [Ca(2+)](i) transients.
  
  
  CONCLUSIONS
  PKC is activated during ischemia and remains activated during early reperfusion. Inhibition of PKC activity post-ischemia impairs functional recovery, delays diastolic [Ca(2+)](i) removal, and increases Ca(2+) sensitivity of the contractile apparatus, resulting in impaired diastolic relaxation. Thus, post-ischemic PKC activity may serve to restore post-ischemic Ca(2+) homeostasis and attenuate contractile protein calcium sensitivity during the period of post-ischemic [Ca(2+)](i) overload.
authors.affiliation: Department of Cardiac Surgery, Children's Hospital and Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, USA.
authors.forename: C
authors.initials: C
authors.lastname: Stamm
authors.forename: I
authors.initials: I
authors.lastname: Friehs
authors.forename: D B
authors.initials: DB
authors.lastname: Cowan
authors.forename: H
authors.initials: H
authors.lastname: Cao-Danh
authors.forename: S
authors.initials: S
authors.lastname: Noria
authors.forename: M
authors.initials: M
authors.lastname: Munakata
authors.forename: F X
authors.initials: FX
authors.lastname: McGowan
authors.forename: P J
authors.initials: PJ
authors.lastname: del Nido
mesh_terms: D000470:Alkaloids; D000704:Analysis of Variance; D000818:Animals; D053119:Benzophenanthridines; D015153:Blotting, Western / Q000379:methods; D002118:Calcium / Q000378:metabolism*; D003285:Contractile Proteins / Q000378:metabolism*; D003971:Diastole; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007150:Immunohistochemistry; D007211:Indoles / Q000494:pharmacology; D007527:Isoenzymes / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008301:Maleimides / Q000494:pharmacology; D018613:Microscopy, Confocal; D015428:Myocardial Reperfusion Injury / Q000378:metabolism*; D009206:Myocardium / Q000201:enzymology*; D010477:Perfusion; D010617:Phenanthridines / Q000494:pharmacology; D011493:Protein Kinase C / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011817:Rabbits
chemical_list: D000470:Alkaloids; D053119:Benzophenanthridines; D003285:Contractile Proteins; D004791:Enzyme Inhibitors; D007211:Indoles; D007527:Isoenzymes; D008301:Maleimides; D010617:Phenanthridines; C016299:chelerythrine; D011493:Protein Kinase C; C070515:bisindolylmaleimide I; D002118:Calcium