Effects of calmodulin and okadaic acid on myofibrillar Ca2+ sensitivity in cardiac myocytes.
Whereas it has been established that the phosphorylation of 20 kD regulatory myosin light chain (MLC20) is a key regulator of contraction in smooth muscle, troponin complex has been thought to be that of myofibrillar Ca2+ sensitivity in cardiac muscle. To elucidate the role of the phosphorylation of cardiac regulatory myosin light chain (MLC2) in the regulation of cardiac muscle contraction, we observed effects of calmodulin and okadaic acid, a protein phosphatase inhibitor, on myofibrillar Ca2+ sensitivity as estimated by pCa50 values obtained from pCa-tension relationships using beta-escin-skinned cardiomyocytes from Wistar rat hearts, in relation to changes in the phosphorylation of myofibrillar regulatory proteins. Whereas myofibrillar Ca2+ sensitivity tended to be progressively decreased by repeated Ca2+-activation in the absence of calmodulin (pCa50; from 5.91 to 5.86, n = 5), calmodulin (2.5 microM) significantly increased myofibrillar Ca2+ sensitivity (pCa50; from 5.92 to 6.03, n = 5, p < 0.05). Okadaic acid over 3 microM enhanced Ca2+-activated force, which was inhibited by 50 microM trifluoperazine, a calmodulin antagonist. Okadaic acid (3 microM) significantly increased myofibrillar Ca2+ sensitivity (pCa50; from 5.96 to 6.11, n = 6, p < 0.05). Whereas the phosphorylation level of troponin I was not changed by 3 microM okadaic acid, that of MLC2 was significantly increased by the same dose of okadaic acid (from 12 to 31%, n = 4, p < 0.05). These results suggest that MLC2 phosphorylation plays a partial role in the regulation of myofibrillar Ca2+ sensitivity in cardiac muscle.
Suematsu
Nobuhiro
N
Department of Cardiovascular Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
Satoh
Shinji
S
Ueda
Yasuko
Y
Makino
Naoki
N
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D066298:In Vitro Techniques; D008297:Male; D018485:Muscle Fibers, Skeletal / Q000187:drug effects / Q000378:metabolism; D009200:Myocardial Contraction / Q000187:drug effects / Q000502:physiology; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D009210:Myofibrils / Q000187:drug effects / Q000378:metabolism; D018994:Myosin Light Chains / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase; D019319:Okadaic Acid / Q000494:pharmacology*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D017208:Rats, Wistar; D019210:Troponin I / Q000378:metabolism
D002147:Calmodulin; D004791:Enzyme Inhibitors; D018994:Myosin Light Chains; D019210:Troponin I; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases; D043263:Myosin-Light-Chain Phosphatase; D002118:Calcium