Stress-dependent and -independent expression of the myogenic regulatory factors and the MARP genes after eccentric contractions in rats

Abstract

The relationship between muscle mechanical conditions and gene expression was investigated by varying both stress and contraction mode imposed upon rat dorsiflexors (n= 25), activating them at high or low frequencies (150 Hz or 40 Hz) either eccentrically or isometrically. Muscle physiological, immunohistochemical and gene expression changes were then measured 24 h after the exercise bout. Peak stress was the best predictor of muscle injury, independent of contraction mode (i.e. eccentric or isometric). When peak stresses were matched, no physiological or immunohistochemical differences were detected between isometric and eccentric contractions. The expression of certain myogenic regulatory and muscle ankyrin repeat protein (MARP) genes (myoD, myogenin, MLP and CARP) depended both on peak muscle stress achieved during contraction and contraction mode. In contrast, Arpp/Ankrd2 was dramatically upregulated only by eccentric contractions, but not by isometric contractions, even though the stress level of the eccentric contractions varied over a three-fold range and overlapped with that of the isometric group. The role that Arpp/Ankrd2 upregulation plays in the biological response to eccentric contraction remains to be determined, as does the control mechanism whereby the expression of certain genes (such as myoD, myogenin, MLP and CARP) is sensitive to muscle stress while another (Arpp/Ankrd2) is sensitive only to contraction mode.

Figure 1. Determination of experimental parameters matching torque across contraction modes

A, torque–frequency relationship for muscles stimulated either isometrically (○) or eccentrically (•) across a range of frequencies. Data represent the average ± s.e.m. of n = 4 animal subjects. The crosses illustrate the points chosen to match stress between isometric and eccentric contractions (EC40 and ISO150). B, sample contractile records of muscles stimulated under the four different conditions described in Methods. Note that nearly identical peak torque is obtained when stimulating eccentrically at 40 Hz (EC40) and isometrically at 150 Hz (ISO150).

Figure 2. Torque production records for the four treatment groups

Each point represents the mean value for the group. s.e.m. bars are shown only for the final contraction for all groups for clarity. No change in data variability were seen as a function of contraction number. (○) EC150 (□) EC40 (•) ISO150 (▪) ISO40.

Figure 3. Peak torque measured during treatment in each of the various modes

The largest torque change 24 h after eccentric contraction was observed for the EC150 group (−32 ± 2%) while the ISO40 demonstrated almost no change (+3.4 ± 4.4%). Importantly, no significant difference was observed between the ISO150 (9.7 ± 6.1%) and EC40 (−14.4 ± 5.8%). Symbols at the top of the figure indicate significant differences between leftmost group (with arrowhead) and another group when connected with a vertical bar (P < 0.05). Error bars are s.e.m.

Figure 4. Expression of the myogenic regulatory factors 24 h after treatment

A, MyoD gene expression levels. B, myogenin gene expression levels. Control myogenin levels were finite and easily measurable (0.057 ± 004 fg. (μg RNA)⁻¹) but not visible on this graph's scale. Symbols at the top of the figure. indicate significant differences between leftmost group (with arrowhead) and another group when connected with a vertical bar (P < 0.05). Error bars are s.e.m.

Figure 5. Expression levels of various mechanically sensitive genes 24 h after treatment

A, CARP expression levels, B, Arpp/Ankrd2 expression levels, C, MLP expression levels. Symbols at the top of the figure. indicate significant differences between leftmost group (with arrowhead) and another group when connected with a vertical bar (P < 0.05). Error bars are s.e.m.