Effects of calmodulin and okadaic acid on myofibrillar Ca²⁺ sensitivity in cardiac myocytes

Abstract

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 β-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 μM) significantly increased myofibrillar Ca²⁺ sensitivity (pCa₅₀; from 5.92 to 6.03, n = 5, p < 0.05). Okadaic acid over 3 μM enhanced Ca²⁺-activated force, which was inhibited by 50 μM trifluoperazine, a calmodulin antagonist. Okadaic acid (3 μM) 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 μM 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.