Angiotensin 1–7 prevents the excessive force loss resulting from 14- and 28-day denervation in mouse EDL and soleus muscle

Figures

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  Figure 1.. Ang 1–7/DIZE significantly reduced muscle weight loss at 14-d denervation for EDL and at 14- and 28-d denervation for soleus; an effect not observed in the presence of A779 or in MasR−/− soleus. Muscle weight expressed as a ratio of body weight of (A) wild type (CD-1) and (B) MasR−/− EDL, (C) wild type and (D) MasR−/− soleus. Ang 1–7 and A779 were delivered using an osmotic pump (100 ng/kg/min), while sucrose (1 g/kg/d) and DIZE (15 mg/kg/d) were given once daily by gavage. INN, innervated; DEN, denervated; SUC, sucrose. Vertical bars represent SE of 5–19 muscles. *, mean muscle weight significantly different from that of innervated muscles; §, mean muscle weight significantly different from that of denervated or denervated/sucrose muscles (ANOVA and LSD, P < 0.05).

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  Figure 2.. Ang 1–7 did not prevent the decrease in fiber CSA associated with 28-d denervation in EDL. (A) Mean fiber CSA values; fiber CSA distribution of (B) innervated, denervated EDL without or with Ang 1–7 treatment; (C) innervated, sucrose-treated, and DIZE-treated denervated EDL. CSA values were divided in bin of 100 µm2, and the number of fibers in each bin is presented as a percentage of the total number of fibers analyzed. For clarity, the CSA distributions of EDL from mice treated with the MasR antagonist, A779, is not shown, as it was similar to that of denervated/Ang 1–7 and denervated/DIZE. The CSA frequency distribution was first determined for each muscle, and then mean and SE (vertical bars) were calculated from four or five muscles (with a total number of fibers ranging between 2,728 and 4,797). *, CSA distribution was significantly shifted toward lower CSA when compared with innervated (ANOVA, and LSD, P < 0.05).

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  Figure 3.. Ang 1–7 did not prevent the decrease in fiber CSA associated with 28-d denervation, but A779 significantly worsened the decrease in CSA in soleus. (A) Mean fiber CSA values; fiber CSA distribution of (B) innervated, denervated soleus without or with Ang 1–7 treatment; (C) innervated, sucrose-treated, and DIZE-treated denervated soleus. CSA values were divided in bin of 100 µm2, and the number of fibers in each bin is presented as a percentage of the total number of fibers analyzed. The CSA frequency distribution was first determined for each muscle, and then mean and SE (vertical bars) were calculated from four or five muscles (with a total number of fibers ranging between 1,970 and 3,850). *, CSA distribution was significantly shifted toward lower CSA when compared with innervated; §, CSA distribution was significantly shifted toward lower CSA when compared with denervated or denervated/sucrose (ANOVA and LSD, P < 0.05).

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  Figure 4.. Ang 1–7 and DIZE significantly prevented the loss of tetanic force in CD-1 EDL and soleus associated with 14- and 28-d denervation periods. Tetanic force in N of EDL after (A) 2-wk and (B) 4-wk denervation; of soleus at (E) 2-wk and (F) 4-wk denervation, normalized to CSA in N/cm2 for EDL at (C) 2-wk and (D) 4-wk denervation, for soleus at (G) 2-wk and (H) 4-wk denervation. Tetanic force represents the maximum force a muscle can generate and was measured at 200 Hz. Vertical bars represent the SE of five or six muscles. *, Mean tetanic force significantly different from that of innervated muscles; §, mean tetanic force significantly different from that of denervated or denervated/sucrose muscles; †, mean tetanic force significantly different from that of CD-1 muscles (ANOVA and LSD, P < 0.05).

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  Figure 5.. Ang 1–7 and DIZE significantly increased twitch force of CD-1 EDL and soleus associated with 14- and 28-d denervation periods, but not in MasR−/− muscles. Twitch force in N of EDL after (A) 2-wk and (B) 4-wk denervation; of soleus at (E) 2-wk and (F) 4-wk denervation, normalized to CSA in N/cm2 for EDL at (C) 2-wk and (D) 4-wk denervation, for soleus at (G) 2-wk and (H) 4-wk denervation. Vertical bars represent the SE of five muscles. *, Mean twitch force significantly different from that of INN muscle; §, mean twitch force significantly different from that of denervated or denervated/sucrose muscle; †, mean twitch force significantly different from that of wild-type soleus (ANOVA and LSD, P < 0.05).

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  Figure 6.. In CD-1 EDL, Ang 1–7 did not prevent the shift in the force–frequency relationship toward lower stimulation frequencies that occurred after 14 and 28 d of denervation. (A and B) Force–frequency relationships at (A) 2-wk and (B) 4-wk denervation. Forces are expressed as a percentage of the force measured at 200 Hz. (C and D) Frequency 50 at (C) 2-wk and (D) 4-wk denervation. Frequency 50 is the stimulation frequency at which force was 50% of the maximum tetanic force (see Materials and methods for calculations). (E) Examples of twitch force traces from innervated and denervated muscles. Forces are plotted as a percentage of the maximum force during contraction. (F) Example of force traces elicited at 50 Hz from innervated and denervated muscle. Horizontal bars show the 200-ms portions of the contractions that are expanded below. Vertical bars (A and B) represent the SE of five muscles. *, Mean frequency 50 significantly different compared with innervated muscle (ANOVA and LSD, P < 0.05).

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  Figure 7.. In CD-1 soleus, Ang 1–7 did not prevent the shift of the force–frequency relationship toward lower stimulation frequencies following 14 and 28 d of denervation. (A and B) Force–frequency relationships at (A) 2-wk and (B) 4-wk denervation. Forces are expressed as a percent of the force at 200 Hz. (C and D) Frequency 50 at (C) 2-wk and (D) 4-wk denervation. Frequency 50 is the stimulation frequency at which force was 50% of the maximum tetanic force (see Materials and methods for calculations). Vertical bars represent SE of five muscles. *, Mean frequency 50 significantly different from that of innervated muscles; §, mean frequency 50 significantly different from that of denervated or denervated/sucrose muscle soleus (ANOVA and LSD, P < 0.05).

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  Figure 8.. Force–frequency relationships of MasR−/− EDL and soleus were significantly shifted toward lower stimulation frequencies after a 28 d denervation period. (A and C) Force-frequency relationships of (A) EDL and (C) soleus. Forces are expressed as a percentage of the force at 200 Hz. Frequency 50 of (B) EDL and (D) soleus. Frequency 50 is the stimulation frequency at which force was 50% of the maximum tetanic force (see Materials and methods for calculations). Vertical bars represent SE of five muscles. *, Mean frequency 50 significantly different compared with innervated muscle (ANOVA and LSD, P < 0.05).

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  Figure 9.. Ang 1–7 significantly improved membrane excitability in 28-d denervated EDL and soleus. (A) Fiber excitability, (B) resting EM, (C) action potential overshoot, (D) rate of depolarization in EDL fibers, (E) fiber excitability, (F) resting EM, (G) action potential overshoot, and (H) rate of depolarization in soleus fibers. Action potentials were triggered with a single stimulation. Excitability is defined as the number of fibers that generated an action potential upon stimulation and is expressed as a percentage of all tested fibers. Mean values from individual fibers were first averaged for each muscle, and mean and SE (vertical bars) were calculated from five or six muscles, with a total number of tested fibers ranging between 70 and 123. *, Mean value significantly different from that of innervated muscle; §, mean value significantly different from that of denervated or denervated/sucrose muscle; †, mean value for denervated MasR−/− muscles significantly different from mean value of denervated CD-1 muscles (ANOVA and LSD, P < 0.05).

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  Figure 10.. Ang 1–7 partially shifted the resting EM distribution of 28-d denervated CD-1 EDL and soleus toward that of innervated muscles. Frequency distribution of resting EM of (A) innervated, denervated, Ang 1–7 treated denervated EDL; (B) innervated, sucrose-treated, and DIZE-treated denervated EDL; (C) innervated, denervated, Ang 1–7 treated denervated soleus; (D) innervated, sucrose-treated, and DIZE-treated denervated soleus. Resting EM values were divided in bin of 5 mV; the number of fibers from all muscles was counted for each bin and is expressed as a percentage of the total number of fibers tested. Total number of samples used for the distribution was 70–123 fibers from five or six muscles. *, Resting EM distribution significantly shifted compared with that of innervated muscle; §, resting EM distribution significantly shifted compared with that of denervated or denervated/sucrose muscle (Fisher test, P < 0.05).

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  Figure 11.. Ang 1–7 significantly increased the Na+ K+ pump protein content in CD-1 EDL, but not the α-actin and myosin content after a 28-d denervation period. Protein content of (A) Na+-K+ pump α1, (B) Na+-K+ pump α2, (C) α-actin, and (D) myosin. Ponceau staining is from the 3-d Western blots to provide an example for total proteins. A, denervated Ang 1–7 treated; D, denervated; Di, denervated-DIZE treated; I, innervated; SOL, soleus. The same blot was used to determine soleus actin and myosin content at 3 d, so images of Ponceau staining are the same for both proteins. Vertical bars represent the SE of six to eight muscles. *, Mean value significantly different from that of INN muscle; §, mean value significantly different from that of denervated muscle (ANOVA and LSD, P < 0.05).

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  Figure 12.. Denervation significantly increased the expression of myosin I and IIA in CD-1 EDL, while Ang 1–7 partially reduced that effect. (A) Images of type I, IIA, and IIB fibers in innervated, denervated, and denervated Ang 1–7–treated EDL (DEN-ANG 1–7). (B–D) The number of fibers expressing myosin I (B), myosin IIA (C), and myosin IIB fibers (D) is expressed as a percentage of the total number of fibers. Percent values were calculated for each muscle, and then mean and SE (vertical bars) were calculated from four or five muscles. Scale bars, 100 µm. The total number of fibers analyzed for innervated, denervated, and DEN-Ang 1–7 were 3,293, 4,144, and 4,321, respectively. *, Mean value significantly different from that of innervated muscle; §, mean value significantly different from that of denervated muscle (ANOVA and LSD, P < 0.05).