Use of tetanus to investigate myofibrillar responsiveness to Ca(2+) in isolated mouse ventricular myocytes.
We used the relation between intracellular Ca(2+) concentration ([Ca(2+)](i)) and cell shortening during tetanus to evaluate the endogenous characteristics of Ca(2+) responsiveness of myofibrils in mouse ventricular myocytes. Enzymatically isolated myocytes were loaded with fura-2 AM (4 microM for 10 min), and the fura-2 fluorescence ratio at 340 and 380 nm excitation wave length [F(340)/F(380)] and cell length were measured simultaneously. Following treatment with thapsigargin (0.2 microM) (an inhibitor of the Ca(2+) pump of sarcoplasmic reticulum), myocytes were stimulated at 10 Hz for 10 s to produce a tetanic contraction and an instantaneous plot of the fluorescence ratio signal versus cell length (R-L trajectory) was constructed. An increase in the extracellular Ca(2+) concentration ([Ca(2+)](o)) from 0.5 to 2 mM extended the R-L trajectory without a substantial shift of the relation. The R-L trajectory was shifted rightward by the nonselective phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xantine (IBMX, 200 microM) (desensitization of the myofibrils to Ca(2+)), and shifted leftward by the Ca(2+) sensitizing thiadiazinone derivative, EMD-57033 (0.5 microM) (sensitization of the myofibrils to Ca(2+)). Beta-adrenergic stimulant, isoproterenol (5 nM), also shifted the R-L trajectory to the right, suggesting that the membrane receptor could be preserved. These results suggest that the R-L trajectory is a useful method to estimate the myofibrillar responsiveness to Ca(2+) in isolated mouse myocytes and can be applied to various mouse models of heart disease.
Hongo
Kenichi
K
Division of Cardiology, Department of Internal Medicine, The Jikei University School of Medicine, Minato-ku, Tokyo, 105-8461 Japan. hongo@jikei.ac.jp
Kusakari
Yoichiro
Y
Kawai
Makoto
M
Konishi
Masato
M
Kurihara
Satoshi
S
Mochizuki
Seibu
S
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000502:physiology*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002316:Cardiotonic Agents / Q000494:pharmacology; D002469:Cell Separation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005453:Fluorescence; D007545:Isoproterenol / Q000494:pharmacology; D051379:Mice; D009119:Muscle Contraction; D009206:Myocardium / Q000166:cytology; D009210:Myofibrils / Q000187:drug effects / Q000502:physiology*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011804:Quinolines / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology; D013829:Thiadiazines / Q000494:pharmacology; D016276:Ventricular Function / Q000187:drug effects
D000318:Adrenergic beta-Agonists; D002316:Cardiotonic Agents; D004791:Enzyme Inhibitors; D010726:Phosphodiesterase Inhibitors; D011804:Quinolines; D013829:Thiadiazines; C070609:EMD 53998; D019284:Thapsigargin; D000252:Calcium-Transporting ATPases; D007545:Isoproterenol; D002118:Calcium; D015056:1-Methyl-3-isobutylxanthine