Key result
FRET analysis of rat aortic smooth muscle contracted with phorbol 12,13-dibutyrate showed dynamic remodeling, with significant increases in alpha-actin-myosin at initiation (79%) and plateau (67%).
FRET analysis provides direct evidence of dynamic remodeling of contractile proteins during vascular smooth muscle contraction.
Advances understanding of vascular smooth muscle mechanics in animal models; leaves open whether similar dynamics occur in human vessels or influence therapy.
We examined the interaction of smooth muscle myosin with alpha-actin and beta-actin isoforms during the contraction of A7r5 smooth muscle cells and rat aortic smooth muscle. The techniques of confocal microscopy and fluorescence resonance energy transfer (FRET) analysis were utilized in examining A7r5 cells and rat aortic rings contracted with phorbol 12,13-dibutyrate. Visual evaluation of confocal images of A7r5 smooth muscle cells contracted by phorbol 12,13-dibutyrate indicated significant disassociation of myosin from alpha-actin but not beta-actin. Whole-cell FRET analysis confirmed these observations (alpha-actin-myosin -67%, beta-actin-myosin -2%). Time course studies further showed that alpha-actin-myosin complex increased significantly (40%) within 1.5 min after the addition of phorbol 12,13-dibutyrate and then declined as contraction progressed. FRET analysis of rat aortic rings at different intervals of contraction indicated significant increases in alpha-actin-myosin at the initiation (79%) and plateau (67%) in force development, but not during the intermediate period of slowly developing tension (-4%). By comparison, beta-actin-myosin complex was unchanged except during slow force development, in which the association was significantly decreased (-30%). Similar to that of alpha-actin-myosin, Alexa 488 - phalloidin staining fluorescence indicated increased tissue F-actin content at the initiation (21%) and plateau (62%) in force. FRET images indicated the development of thickened cables and patches of alpha-actin-myosin in tissue throughout the interval of contraction. The results provide direct evidence of dynamic remodeling of the contractile protein during vascular smooth muscle contraction and suggest that FRET analysis may be a powerful tool for assessment of tissue protein-protein associations.
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Black et al. (2009) studied this question. Phorbol 12,13-dibutyrate was evaluated on Interaction of smooth muscle myosin with alpha-actin and beta-actin isoforms. FRET analysis of rat aortic smooth muscle contracted with phorbol 12,13-dibutyrate showed dynamic remodeling, with significant increases in alpha-actin-myosin at initiation (79%) and plateau (67%).
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