ABSTRACT
AIM:Nitroxyl ( HNO) increases myofilament Ca 2+responsiveness relative to increases in intracel-lular Ca2+in cardiac muscle.In this study, we further investigated this effect of HNO on trabecular muscles from phospho-lamban knockout ( PLB-KO) and wide-type ( WT ) mice using a novel HNO donor , 1-nitrosocyclohexyl acetate ( NCA ) . METHODS:Trabecular muscles were dissected from the right ventricles of the rat hearts and mounted between a force transducer and a motor arm.The muscles were superfused with K-H solution (pH 7.4) at room temperature.Fura-2 was loaded into the trabecular muscles via electrophoresis .The length of the sarcomere was set to 2.2~2.3μm.During stead-y-state activations, the maximal Ca2+-activated force and Ca2+required for 50% activation were measured.RESULTS:The intracellular Ca 2+transients and force of the PLB-KO muscles at baseline were higher than those of the WT muscles and exhibited a negative force-frequency relationship (FFR).NCA (2.5μmol/L) increased systolic force in both PLB-KO group and WT group at any given [Ca2+]o.However, there was more dramatic increase in the force development due to moderate increases in the intracellular Ca 2+transients in the WT muscles when external Ca 2+increased from 1.5 to 4.5 mmol/L under NCA.NCA did not affect the negative FFR in PLB-KO muscle.Steady-state force-Ca2+relations obtained from skinned muscles were not different between the 2 groups, while NCA increased Ca2+responsiveness in skinned mus-cles from both PLB-KO and WT mice.CONCLUSION:HNO increases force development in both PLB-KO and WT mus-cles as a result of increases in myofilament Ca 2+responsiveness .The increased intracellular Ca 2+transients are accompa-nied by greater force development in WT mice , suggesting that HNO improves Ca 2+activation and establishes HNO as a positive inotropic agent with novel mechanisms .
ABSTRACT
OBJECTIVE: To test the effect of 1-nitrosocyclohexyl acetate (NCA), a newly developed nitroxyl (HNO) donor, on myofilaments and its action mechanism for the thiol in tropomyosin. METHODS: Trabeculae were dissected from the right ventricule of rat heart and mounted between a force transducer and a motor arm. The muscles were superfused with K-H solution (pH 7.4) at room temperature. Fura-2 salt was loaded into the trabeculae via electrophoresis, and the sarcomere length was set at 2.2-2.3 μm. Western blotting tested the change of tropomyosin. RESULTS: Twitch force increased in a dose dependent manner in the presence of NCA in the range of 20-100 μmol·L-1. Force increased (P0.05) with different frequencies (0.5-3.0 Hz) with NCA of 20 μmol·L-1 and Ca2+ of 0.5 μmol·L-1. Skinned force increased as compared to the control (P<0.05). The effect of NCA had characteristics of structure uniqueness. A cross-linking band in tropomyosin was seen by Western blotting under non-reducing condition. The thiol reducing agent dithiothreitol (DTT) both prevented and reversed HNO action. CONCLUSION: The donor of HNO, NCA, represents a new class of agents capable of directly sensitizing cardiac myofilaments to Ca2+. Post-translational thiol modification of the key regulatory myofilament protein (i.e. tropomyosin) may underlie the effect of NCA.