Calponin — knocked out but not down!

Smooth muscle contraction is regulated primarily by the reversible phosphorylation of myosin II (Somlyo & Somlyo, 1994), but may also be modulated by a thin filament-associated mechanism (Walsh, 1990). Calponin is a candidate for such a modulator (Winder et al. 1998). Calponin was first isolated from chicken gizzard smooth muscle as a 34 kDa actin- and calmodulin-binding protein (Takahashi et al. 1986) and later shown to exist as multiple isoforms. h1- (α or basic), h2- (neutral) and acidic calponins are the products of three distinct genes; β-calponin is an alternatively spliced variant of h1-calponin. In adult vertebrates, h1-calponin expression is restricted to differentiated smooth muscle cells where it is localized to the contractile and cytoskeletal actin filaments. Although its physiological functions are not clear, h1-calponin has been implicated in the regulation of both contractile and non-contractile functions (adhesion, migration and proliferation). h1-Calponin expression is down-regulated when vascular smooth muscle cells re-enter the cell cycle and proliferate, changing from a contractile to a synthetic phenotype (as occurs in response to vascular injury), and h1-calponin is a useful marker of the contractile phenotype of smooth muscle cells. During embryonic development, this isoform is expressed in other tissues, including the heart, but disappears during late fetal development. h1-Calponin has been implicated in cardiac myofibrillar development and regulation of embryonic heart contraction. Its expression has also been reported in some transformed cells, e.g. osteosarcomas, and a role for the protein in tumour suppression has been proposed. Neutral (h2-) and acidic isoforms of calponin are expressed in smooth muscle and non-muscle cells, but at much lower levels, and less is known about their potential physiological roles. The acidic isoform has been implicated in neurite outgrowth and navigation in the rat and in early embryonic development in Xenopus. β-Calponin has been detected at the protein level only in smooth muscle cells of the urogenital tract. Most work to date has focused on the possibility that h1-calponin regulates smooth muscle contraction via its interaction with actin, as suggested by the inhibition by calponin of the actin-activated myosin MgATPase activity of phosphorylated myosin, the movement of actin filaments over immobilized phosphorylated myosin in the in vitro motility assay, isometric force and shortening velocity in permeabilized smooth muscle strips. These inhibitory effects are alleviated by protein kinase C (PKC)-catalysed phosphorylation of calponin (Winder et al. 1998). A powerful experimental approach to the physiological function of a specific protein is to knock out its gene and observe the resultant phenotype. The h1-calponin knockout mouse was developed by Yoshikawa et al. (1998). They confirmed the absence of h1-calponin and demonstrated that its absence was not compensated for by the over-expression or ectopic expression of other calponin isoforms. Surprisingly, the only obvious phenotypic change was observed in bone, and this initial characterization suggested that h1-calponin acts as a negative regulator of bone formation. Furthermore, h1-calponin is highly expressed in the developing skeleton and in undifferentiated osteoblasts of wild-type mice, consistent with this proposed role. Given the wealth of in vitro data implicating h1-calponin in the regulation of smooth muscle contraction, one might have anticipated that knockout of the h1-calponin gene would have adverse physiological effects on smooth muscle function. In this issue of The Journal of Physiology, Matthew et al. (2000) report a very thorough comparison of the contractile properties of bladder and vas deferens smooth muscles of h1-calponin knockout and wild-type mice. The key functional difference is a faster unloaded shortening velocity (Vus) in the knockout mouse, due to a difference in crossbridge cycling. Exogenous calponin reduced Vus of the calponin-negative muscle to that of wild-type. This is consistent with in vitro studies showing a reduction in Vus by addition of exogenous calponin to permeabilized smooth muscle containing the normal complement of calponin (e.g. Obara et al. 1996). As the authors emphasize, these observations are consistent with, but do not unequivocally establish, a physiological role for calponin in the regulation of smooth muscle contraction, presumably by modulating crossbridge interaction kinetics. Intriguingly, Matthew and co-workers also show a reduction in actin content (≈50%) in the calponin knockout mouse. The high Vus in the calponin knockout mouse may therefore be due to the reduction in actin content, resulting in shorter mini-sarcomeres, although it was not possible to determine from morphological studies whether shorter rather than fewer actin filaments are present. Knockout of calponin had no effect on other parameters tested: isometric force, Ca2+ sensitivity of force development or Ca2+ sensitization of contraction by activation of RhoA or PKC pathways. Another intriguing difference between the wild-type and knockout mice involves h-caldesmon, another thin filament-associated protein that has been implicated in the regulation of contraction (Walsh, 1990). The electrophoretic mobility of this smooth muscle-specific isoform is slightly decreased in the calponin knockout mouse. This may reflect a switch to an alternatively spliced variant, although other explanations are also feasible. Nevertheless, it will be interesting to define the basis of this electrophoretic mobility shift, which could conceivably underlie compensation for the lack of h1-calponin expression. Finally, given that the two smooth muscle tissues studied by Matthew and co-workers are phasic smooth muscles, could it be that calponin plays a more important role in the regulation of contraction of tonic smooth muscles?