Propofol increases phosphorylation of troponin I and myosin light chain 2 via protein kinase C activation in cardiomyocytes.
BACKGROUND
Troponin I (TnI) and myosin light chain 2 (MLC2) are important myofibrillar proteins involved in the regulation of myofilament calcium (Ca2+) sensitivity and cardiac inotropy. The objectives of this study were to determine the role of protein kinase C (PKC) in mediating propofol-induced changes in actomyosin adenosine triphosphatase activity in cardiac myofibrils and to examine the extent to which propofol alters the phosphorylation of TnI and MLC2 in cardiomyocytes.


METHODS
Freshly isolated adult rat ventricular myocytes were used for the study. Cardiac myofibrils were extracted for assessment of actomyosin adenosine triphosphatase activity and phosphorylation of TnI and MLC2. Western blot analysis for PKC-alpha was performed on cardiomyocyte subcellular fractions. Simultaneous measurement of intracellular free Ca2+ concentration ([Ca2+](i)) and myocyte shortening was assessed using fura-2 and video edge detection, respectively.


RESULTS
Propofol (30 microM) reduced the Ca2+ concentration required for activation of actomyosin adenosine triphosphatase activity, and this effect was abolished by bisindolylmaleimide I. In addition, propofol stimulated dose-dependent phosphorylation of TnI and MLC2. PKC activation with phorbol myristic acetate also stimulated an increase in phosphorylation of TnI and MLC2. The actions of propofol and phorbol myristic acetate together on phosphorylation of TnI and MLC2 were not additive. PKC inhibition with bisindolylmaleimide I attenuated phorbol myristic acetate- and propofol-induced phosphorylation of TnI and MLC2. Propofol stimulated translocation of PKC-alpha from cytosolic to membrane fraction. Propofol caused a shift in the extracellular Ca2+-shortening relationship, and this effect was abolished by bisindolylmaleimide I.


CONCLUSIONS
These results suggest that propofol increases myofilament Ca2+ sensitivity via a PKC-dependent pathway involving the phosphorylation of MLC2.
Kanaya
Noriaki
N
Center for Anesthesiology Research, Division of Anesthesiology and Critical Care Medicine, Cleveland Clinic Foundation, Ohio 44195, USA.
Gable
Brad
B
Murray
Paul A
PA
Damron
Derek S
DS
D008841:Actin Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D000205:Actomyosin / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D018686:Anesthetics, Intravenous / Q000494:pharmacology*; D000818:Animals; D015153:Blotting, Western; D024722:Cardiac Myosins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066298:In Vitro Techniques; D032342:Muscle Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D009200:Myocardial Contraction / Q000187:drug effects; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D018994:Myosin Light Chains / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D015742:Propofol / Q000494:pharmacology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D013347:Subcellular Fractions / Q000187:drug effects; D019210:Troponin I / Q000378:metabolism*
D018686:Anesthetics, Intravenous; D004791:Enzyme Inhibitors; D018994:Myosin Light Chains; D010710:Phosphates; D019210:Troponin I; C107184:myosin light chain 2; D000205:Actomyosin; D011493:Protein Kinase C; D000251:Adenosine Triphosphatases; D024722:Cardiac Myosins; D015742:Propofol