Estimation of myofibrillar responsiveness to Ca2+ in isolated rat ventricular myocytes.
To estimate myofibrillar responsiveness to Ca2+, we used the relation between cell length and intracellular [Ca2+] ([Ca2+]i) during tetanic contractions of isolated ventricular myocytes. Enzymatically isolated rat ventricular myocytes were loaded with fura-2 AM (4 microM for 10 min) and excited alternately at 340 nm and 380 nm. The ratio (R) of fura-2 fluorescence at these wavelengths [F(340)/F(380), an index of [Ca2+]i] and cell length (L) were measured simultaneously. Following treatment with thapsigargin (0.2 microM), myocytes were stimulated at 10 Hz for 10 s to produce a tetanic contraction every min and an instantaneous plot of R vs L (R-L trajectory) was constructed. The R-L trajectory followed the same path during cell shortening and re-lengthening, suggesting that dynamic equilibrium between R and L was achieved during tetanus. Increasing the extracellular [Ca2+] from 1 to 8 mM extended the R-L trajectory without a substantial shift of the relation. The Ca2+-sensitizing thiadiazinone derivative, EMD57033 (1 microM), shifted the R-L trajectory to the left (sensitization of the myofibrils to Ca2+), whereas the non-selective phosphodiesterase inhibitor, 3-isobutyl-1-methylxantine (200 microM), shifted the R-L trajectory to the right (desensitization of the myofibrils to Ca2+), in agreement with previous results obtained using skinned preparations. We conclude that the R-L trajectory is useful for estimating the myofibrillar responsiveness to Ca2+ in isolated myocytes and may be beneficial for the evaluation of inotropic agents.
Department of Internal Medicine IV, The Jikei University, School of Medicine, 3-25-8 Nishishinbashi, Minato-ku, Tokyo 105-8461, Japan.
K
K
Hongo
Y
Y
Kusakari
M
M
Konishi
S
S
Kurihara
S
S
Mochizuki
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D048429:Cell Size / Q000187:drug effects; D002478:Cells, Cultured; D004558:Electric Stimulation; D005453:Fluorescence; D016257:Fura-2 / Q000032:analysis / Q000378:metabolism; D006352:Heart Ventricles / Q000166:cytology / Q000187:drug effects*; D008297:Male; D009200:Myocardial Contraction / Q000187:drug effects / Q000502:physiology; D009206:Myocardium / Q000166:cytology*; D009210:Myofibrils / Q000187:drug effects* / Q000502:physiology*; D011804:Quinolines / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013746:Tetany / Q000139:chemically induced; D019284:Thapsigargin / Q000494:pharmacology; D013829:Thiadiazines / Q000494:pharmacology; D016276:Ventricular Function
D011804:Quinolines; D013829:Thiadiazines; C070609:EMD 53998; D019284:Thapsigargin; D002118:Calcium; D015056:1-Methyl-3-isobutylxanthine; D016257:Fura-2