Effects of thyroxine on myosin isoform expression and mechanical properties in guinea-pig smooth muscle

Abstract

Information on the effects of thyroid hormone on smooth muscle contractile protein expression and mechanical properties is sparse. We have addressed the following questions. (1) Can thyroxine hormone alter myosin isoform composition in smooth muscle? (2) Can a change in myosin isoform composition lead to altered mechanical properties in smooth muscle? (3) Are alterations, if occurring, equal in fast and slow smooth muscle types? Guinea-pigs were treated with thyroxine (T(4)) for 12 days. Control animals were given physiological saline solution. Maximal unloaded shortening velocity (V(max)) was measured in chemically skinned, maximally activated muscle preparations from the aorta and the taenia coli. V(max) increased following thyroxine treatment, by approximately 20 % in the taenia coli. In the aorta, no significant increase in V(max) could be detected. The sensitivity of isometric force to inorganic phosphate (P(i)) was increased in the taenia coli following thyroxine treatment. The expression of mRNA (determined with RT-PCR) for the myosin heavy chain with the seven amino acid insert increased by approximately 70 % in the aorta and about 25 % in the taenia coli following thyroxine treatment. Western blot analysis showed an increase in the inserted myosin heavy chain form in the taenia coli. Expression of mRNA for the myosin essential light chains and the corresponding proteins did not change significantly in either muscle type. No alterations in non-muscle myosin heavy chain isoforms could be detected after thyroxine treatment. In conclusion, thyroxine treatment alters the isoform composition of myosin in fast and slow smooth muscles in vivo. This change is sufficient to increase shortening velocity and sensitivity of isometric force to P(i) in the fast, but not in the slow, smooth muscle type.

Figure 1. Expression of mRNA for myosin heavy chain

A, an original photograph of an agarose-ethidium bromide gel of RT-PCR products showing the myosin heavy chains (HC). The left lanes show data from a control animal (C) and the right lanes show data from a thyroxine-treated animal (T₄). PCR products for the inserted and non-inserted myosin heavy chains had gel mobilities with a predicted difference in size of 21 bp. B (taenia coli) and C (aorta) show the mean values of the relative expression of mRNA for the myosin heavy chain with the seven amino acid insert. □, control experiments; ▪, experiments on muscles from thyroxine-treated animals. **P < 0.01 and ***P < 0.001 (n = 6–9).

Figure 2. Expression of mRNA for LC₁₇

A, an original photograph of an agarose-ethidium bromide gel of RT-PCR products showing the myosin essential light chains (LC). The left lanes show data from a control animal (C) and the right lanes show data from a thyroxine-treated animal (T₄). The PCR products for the different isoforms of myosin essential light chains (LC_17a and LC_17b) had mobilities corresponding to 451 and 496 bp, respectively. B (taenia coli) and C (aorta) show the mean values of the relative expression of mRNA for the myosin essential light chain 17b (LC_17b). □, results from control experiments; ▪, results from experiments on muscles from thyroxine-treated animals (n = 8–9).

Figure 3. Force-velocity relationships and maximal unloaded shortening velocity (V_max) from quick-release experiments performed on taenia coli (A) and aorta (B)

The left panels show original plots of afterload (P/P_o) and velocity (in muscle lengths s⁻¹) from a control animal (○) and a thyroxine-treated animal (•). The right panels show mean values of V_max from control (□) and thyroxine-treated (▪) animals. *P < 0.05 (n = 9–11).

Figure 4. Effects of P_i on isometric force

A-D, original recordings from experiments investigating the effects of P_i on active isometric force in taenia coli (A and C) and aorta (B and D) preparations from control (A and B) and thyroxine-treated (C and D) animals. In these panels, three contractions at different concentrations (0, 5 and 10 mm) of P_i are shown. Between the contractions there are periods of treatment with ATP-γ-S and subsequent rinsing periods in rigor solution. Mean values of force at different [P_i] are shown in E (taenia coli) and F (aorta) for control (○) and thyroxine-treated (•) animals. The force from the T₄-treated preparations was significantly lower than that from the controls at all concentrations of added P_i (P < 0.05, Bonferroni test) in the taenia coli (E). (n = 5–6).