The smooth muscle myosin seven amino acid heavy chain insert's kinetic role in the crossbridge cycle for mouse bladder

Abstract

The seven amino acid insert in the smooth muscle myosin heavy chain is thought to regulate the kinetics of contraction, contributing to the differences between fast and slow smooth muscle. The effects of this insert on force and stiffness were determined in bladder tissue of a transgenic mouse line expressing the insert SMB at one of three levels: an SMB wild type (+/+), an SMA homozygous type (-/-) and a heterozygous type (+/-). For skinned muscle, an increase in MgADP or inorganic phosphate (Pi) should shift the distribution of crossbridges in the actomyosin ATPase (AMATPase) to increase the relative population of the crossbridge state prior to ADP release and Pi release, respectively. Exogenous ADP increased force and stiffness in a manner consistent with increasing the Ca2+ concentration in both the +/+ and +/- mouse types. However, the -/- type showed a significantly greater increase in force than in stiffness suggesting that immediately prior to ADP release, the AMATPase either has an additional force producing isomerization state or a slower ADP dissociation rate for the -/- type compared to the +/+ or +/- types. Exogenous Pi led to a significantly greater decrease in stiffness than in force for all three mouse types suggesting that there is a force producing state prior to Pi release. In addition, the increase in Pi showed similar changes in the +/+ and -/- types whereas in the +/- type the decreases in both force and stiffness were greater than the other two mouse types indicating that the insert can affect the cooperativity between myosin heads. In conclusion, the seven amino acid insert modulates the kinetics and/or states of the AMATPase, which could lead to differences in the kinetics of contraction between fast and slow smooth muscle.

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Figure 1. Histological staining of wild-type and SMB −/− bladder

Sections were stained with haematoxylin and eosin, × 40 magnification.

Figure 2. Western blot analysis of light chain isoform expression

Three different concentrations of total proteins extracted from SMB+/+ (WT, wild-type), SMB+/−, and SMB−/− mouse bladder were resolved on a 12 % SDS-PAGE and immunoblotted with LC20, smooth muscle actin or LC17-specific antibody.

Figure 3. The force-stiffness relationship

Five different force and corresponding stiffness values were observed at pCa values of 5.8, 5.4, 5.0, 4.4 and 4.0 (n= 10) and fit to a line, r²= 0.99 (black line, +). Force vs. stiffness changes: ▾=+/+, ▴=+/−, ×=−/−; P_i= black, ADP = grey. Force and stiffness in pCa4 were defined as 100 %. The relationship between steady state force and steady state stiffness during Ca²⁺ activation is linear. In the ADP condition, only the −/− mouse type significantly deviated from the force vs. stiffness regression line at various pCa concentrations (P < 0.05). All three mouse types significantly deviated from the line in the P_i condition (P < 0.05).

Figure 4. Force traces during Ca²⁺ activation

A, force trace during Ca²⁺ activation in 5 mm MgADP. Tissue was allowed to relax in pCa9 (9) before being shifted to pCa4 (4), pCa4 with 5 mm MgADP (ADP) and then back into pCa9. Ca²⁺ activation results in an increase in steady state force, which is further increased with MgADP. B, force trace during Ca²⁺ activation in 10 mm inorganic P_i. Tissue was allowed to relax in pCa9 before being shifted to pCa4 (4), pCa4 with 10 mm P_i (P_i), and then back into pCa9. Ca²⁺ activation results in an increase in steady state force, which is decreased by P_i.

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