Thermal manipulations in late-term chick embryos have immediate and longer term effects on myoblast proliferation and skeletal muscle hypertrophy

Abstract

We investigated the cellular and molecular bases for the promotion of muscle development and growth by temperature manipulations (TMs) during late-term chick embryogenesis. We show that incubation at 39.5 degrees C (increase of 1.7 degrees C from normal conditions) from embryonic days 16 to 18 (E16 to E18) for 3 or 6 h daily increased diameter of myofibers as of day 13 of age and enhanced absolute muscle growth relative to controls, until day 35 of age. TMs had immediate (E17) and later (up to 2 wk posthatch) effects in elevating muscle cell proliferation relative to controls. This was indicated by higher DNA incorporation of thymidine and a higher number of cells expressing PCNA in intact muscle, accompanied by higher Pax7 levels, all reflecting a higher number of myogenic cells, and suggesting that the increased hypertrophy can be attributed to a higher reservoir of myogenic progeny cells produced in response to the TM. IGF-I levels were higher in the TM groups than in controls, implying a mechanism by which heat manipulations in chicks affect muscle development, with locally secreted IGF-I playing a major role. Whereas hypertrophy was similar in both TM groups, cell proliferation and Pax7 levels were more robust in the 6-h muscle, mainly posthatch, suggesting a differential effect of various TM periods on cell reservoir vs. hypertrophy and a high sensitivity of myoblasts to relatively small changes in heat duration with respect to these processes, which is manifested in the short and long term.

Fig. 1.

Body weight (BW) (A) and pectoralis muscle as percentage of BW (B) of thermally manipulated (TM) [3 h daily (3H), 6 h daily (6H)] and control chickens on days 25 and 35 of age. Results are means ± SE; n = 30. ^a,bData with different letters differ significantly within the same age group (P < 0.05).

Fig. 2.

Myofiber diameter distribution in pectoralis muscle from control and TM (3H, 6H) groups at various days of age. Myofibers are clustered in bin intervals of 5 μm, and myofiber diameter values are ranked in ascending order within each treatment group. Results are presented as percentage of total fibers.

Fig. 3.

Thymidine incorporation by DNA (A) and number of muscle cells per gram of pectoralis muscle (B) in control and TM (3H, 6H) groups. Muscle was removed from embryos or posthatch chicks on various days and pooled within each group. Left: embryonic days (E) 16–19 and posthatch day 3; right: posthatch days 6–13. Myoblasts were isolated in parallel from each group and counted using a hemocytometer. Cells were incubated for 1 day, after which labeled thymidine was added for 2 h. Results are means ± SE (n = 6) of a representative of three independent experiments. ^a,b,cData with different letters differ significantly within the same age group (P < 0.05).

Fig. 4.

A: PCNA staining of pectoralis muscle cross sections prepared from the experimental chicks (control and TM: 3H, 6H) at 9 days of age. Magnification ×200. B: quantitation analysis of the PCNA-expressing cells. Results are means ± SE of PCNA-expressing cells within the myofibers and are presented as percentage of total myonuclei. Five sections were studied per five chicks (n = 5), monitoring five random fields per section (total of 1,000 nuclei per chick) (P < 0.05). Only nuclei within the myofiber perimeter were included in this analysis, and regions rich with connective tissue were not included. ^a,bData with different letters differ significantly (P < 0.05).

Fig. 5.

Pax7 levels in pectoralis muscle-derived myoblasts and muscle tissue are upregulated in response to TM (3H, 6H). Pectoralis muscle samples were removed in parallel from individual embryos or chicks from all groups (n = 4) at various days of age. On each day, samples derived from the control and TM groups were electrophoresed side by side on an SDS-polyacrylamide gel, and protein expression levels were evaluated by Western blot analysis. Bands were quantified by densitometry relative to α-tubulin, and results are means ± SE presented as fold induction relative to control. ^a,b,cData with different letters differ significantly within the same age group (P < 0.05).

Fig. 6.

Densitometric analysis of myogenin (A) and IGF-I (B) expression relative to α-tubulin protein levels in pectoralis muscle samples derived from control and TM (3H, 6H) embryos and chicks at various days of age. Sample collection and Western blot analysis were performed as described in Fig. 5 legend. Results are means ± SE and presented as fold induction of control (n = 4). ^a,bData with different letters differ significantly within the same age (P < 0.05). C: RT-PCR analysis for IGF-I mRNA expression levels in myoblasts derived from pectoralis muscle of the experimental chicks on E17 and day 3 posthatch. Equal samples were analyzed and electrophoresed as demonstrated by GAPDH mRNA levels.