Muscle-specific expression of IGF-1 blocks angiotensin II–induced skeletal muscle wasting (original) (raw)
Angiotensin II–induced protein degradation in vivo involves activation of caspase-3 and actin cleavage. We infused 12- to 16-week-old C57BL/6 mice with 500 ng/kg/min angiotensin II or vehicle (n = 6 per group) for 7 days, and sham-infused mice were pair fed. Angiotensin II infusion increased blood pressure (Figure 1A) and reduced body weight (Figure 1B), which was similar to our previous findings in rats. Gastrocnemius and soleus muscle from angiotensin II–infused mice at 7 days weighed less than those from pair-fed, sham-infused controls (Figure 1, C and D). We have recently shown that actomyosin cleavage by recombinant caspase-3 increases proteolysis via the Ub-P’some system. We found a 6-fold increase in caspase-3 activity in gastrocnemius muscle (P < 0.01) after 7 days of angiotensin II infusion (Figure 2A). Activation of caspase-3 requires proteolytic processing of its inactive zymogen into active p17 and p12 subunits. We detected significant accumulation of cleaved p17 subunit of caspase-3 in the muscle of angiotensin II–infused mice (Figure 2B). Furthermore, angiotensin II infusion markedly increased levels of the characteristic footprint of caspase-3 activation, a 14-kDa actin fragment (Figure 2C), and this increase was blunted by the cell-permeable caspase inhibitor DEVD-CHO (Figure 2D), which indicates that caspase-3 activation was responsible for the accumulation of cleaved actin fragment in muscles of angiotensin II–infused animals. We have previously shown that accumulation of cleaved actin is associated with proteolysis through the Ub-P’some system (10). Evidence for involvement of the Ub-P’some system in animal models of catabolic conditions includes increased mRNA levels of the ubiquitin ligases atrogin-1/muscle atrophy F-box (atrogin-1/MAFbx) and MuRF-1 (7, 13). Using real-time PCR we found that angiotensin II caused a 24-fold and a 5-fold increase of atrogin-1 and MuRF-1 mRNA levels, respectively (Supplemental Figure 1; supplemental material available online with this article, doi:10.1172/JCI200522324DS1). Furthermore, there was a marked increase in ubiquitinated proteins in muscle from angiotensin II–infused mice (Figure 2E). Since there are a number of reports suggesting that the calpain/calpastatin system (14–16) is important in muscle wasting, we measured calpain activity in our model using gastrocnemius muscle lysates from angiotensin II–infused and pair-fed mice. Our data showed that there was no significant difference in calpain activity between angiotensin II and pair-fed animals. Furthermore, DEVD-CHO had no effect on calpain activity (Supplemental Figure 2). Thus, angiotensin II–induced muscle loss involves activation of caspase-3, actin cleavage, and activation of the Ub-P’some system.
Angiotensin II produces muscle wasting in mice. C57BL/6 mice were either angiotensin II infused or sham infused and pair fed. Angiotensin II (A) markedly increased blood pressure (*P < 0.001, angiotensin II–infused vs. pair-fed), (B) produced relative weight loss (**P < 0.01 angiotensin II–infused vs. pair-fed), and (C and D) reduced muscle mass (***P < 0.05, angiotensin II–infused vs. pair-fed). A, angiotensin II–infused; P, pair-fed.
Angiotensin II decreases Akt phosphorylation and activates caspase-3, producing actin cleavage and increased protein ubiquitinization in skeletal muscle. Mice were either angiotensin II infused or sham infused and pair fed (n = 5 per group, 7 days) and gastrocnemius lysates assessed for (A) caspase-3 activity (*P < 0.01 angiotensin II– vs. sham-infused); (B) levels of activated (17-kDa) caspase-3 by Western blotting; (C) actin cleavage as determined by accumulation of 14-kDa actin fragment; (D) accumulation of 14-kDa actin fragment with or without caspase-3 inhibitor DEVD-CHO; (E) expression of proteins conjugated to ubiquitin (Ub); (F) expression levels of phospho–Akt (p-Akt), total Akt (t-Akt), phospho- and total Foxos, and phospho-GSK3β and total GSK3β; and (G) Ser307 phosphorylation of IRS-1 after immunoprecipitation with anti–IRS-1 antibody and SDS-PAGE and Western blotting. A/DE and P/DE, angiotensin II–infused and pair-fed with caspase-3 inhibitor DEVD-CHO, which was added to muscle lysates; A/DC and P/DC, angiotensin II–infused and pair-fed with caspase inhibitor Z-Asp-2,6-dichlorobenzoyloxymethylketone, which was administered to mice for 7 days.
Angiotensin II infusion in mice induces serine phosphorylation of insulin receptor substrate–1 and reduces Akt, GSK3β and Foxo phosphorylation in skeletal muscle. We examined the mechanisms whereby caspase-3 became activated by angiotensin II. It has been shown that diminished Akt signaling promotes the cleavage and activation of caspase-3 (17). Using anti–phospho-Akt antibody (Ser473), we found that expression of phospho-Akt, but not total Akt, was reduced by angiotensin II infusion (Figure 2F). We next investigated the upstream signals that lead to the downregulation of Akt. There have been several reports that angiotensin II can inhibit both IGF-1 and insulin-stimulated PI3K activity and Akt activation, potentially via serine phosphorylation of insulin receptor substrate–1 (IRS-1) (18–20). We found that angiotensin II markedly stimulated Ser307 phosphorylation of IRS-1, which was not detected in muscle from control mice (Figure 2G).
Downstream effectors of Akt that can mediate hypertrophy include the negative regulator glycogen synthase kinase 3β (GSK3β; phosphorylated and inactivated by Akt) and positive regulators mammalian target of rapamycin (mTOR) and p70S6K. Additionally, the forkhead transcription factor Foxo1 is phosphorylated and inactivated by insulin and IGF-1 through an Akt-dependent mechanism, and this phosphorylation has been shown to be critical for insulin/IGF-1 antiapoptotic effects, potentially through a caspase-3–dependent mechanism (21). Furthermore, it has been shown that IGF-1 and insulin inhibit the expression of atrogin-1/MuRF-1 by inactivating Foxos (6). We found that angiotensin II infusion reduced phosphorylation of Foxo1, Foxo3, Foxo4, and GSK3β (Figure 2F).
To demonstrate that caspase-3 activation is required in angiotensin II–induced muscle wasting, we administered a caspase inhibitor Z-Asp-2,6-dichlorobenzoyloxymethylketone to mice. This inhibitor has been shown to inhibit caspase-3 activation and ameliorate apoptosis and cardiac remodeling in rats with myocardial infarction (22). Our data showed that the caspase inhibitor markedly blunted the wasting effect of angiotensin II. Thus, there was no statistically significant difference in body and muscle weight between angiotensin II–infused and pair-fed groups treated with caspase inhibitor after 5 days (Supplemental Figure 3, A–D). We further analyzed caspase-3 activity, actin cleavage, and protein ubiquitinization in mice receiving the caspase inhibitor. As shown in Figure 2A, caspase-3 activity was completely inhibited both in sham-infused, pair-fed and in angiotensin II–infused groups, and no actin cleavage was detected (Supplemental Figure 3E). Ubiquitinization was also minimal, and there was no difference between angiotensin II and pair-fed groups (Supplemental Figure 3F). Therefore, our data suggest that caspase-3 activation is required for actin cleavage and for increased protein ubiquitinization. Our data show that the expression levels of both atrogin-1 and MuRF-1, measured by real-time PCR, remain elevated in the angiotensin II–group compared with the pair-fed group (Supplemental Figure 1). However, the degree of angiotensin II induction of atrogin-1/MuRF-1 expression was blunted in the presence of caspase-3 inhibition. Thus, the angiotensin II–induced fold increases in atrogin-1 and MuRF-1 were 12.2 ± 1.2 and 1.4 ± 0.3, respectively, with caspase inhibition, as opposed to 24 ± 0.5 and 5 ± 0.3 without caspase inhibition. This finding suggests that angiotensin upregulation of ubiquitin ligases is in part caspase-3 dependent.
Angiotensin II–induced muscle wasting is not due to reduced potassium levels and is glucocorticoid dependent. It is possible that angiotensin II infusion, by raising aldosterone levels, could produce hypokalemia and tissue potassium depletion, which could contribute to muscle wasting. We thus measured circulating plasma K+ levels in angiotensin II–infused and pair-fed mice at day 7 (n = 5 per group) and did not find a significant difference (pair-fed, 4.01 ± 0.14 mM; angiotensin II–infused, 4.07 ± 0.22 mM; P = 0.59). Because K+ serum homeostasis tends to be maintained at the expense of the intracellular compartment, measuring serum levels may not be adequate; therefore, we also measured muscle K+ content. However, we did not find a difference in gastrocnemius muscle K+ content (pair-fed, 104.9 ± 1.3 μmol/g wet weight; angiotensin II–infused, 105.5 ± 1.6 μmol/g wet weight; n = 5, P = 0.53).
The muscle-wasting effect of angiotensin II could be mediated via a direct interaction between angiotensin II and its receptors on skeletal muscle tissue; however, muscle angiotensin II receptor expression decreases at the end of gestation to very low levels in differentiated muscle (23, 24). Alternatively, the angiotensin II–induced decrease in IGF-1 and muscle loss could be mediated by intermediate factors that are regulated by angiotensin II, such as glucocorticoids. Indeed, we have shown previously that there is an increase in urinary corticosterone levels with angiotensin II infusion in rats (25). It has been shown that catabolic doses of glucocorticoids injected in the rat activate the ubiquitin pathway of protein degradation (26) and reduce skeletal muscle expression of IGF-1 (27). We also found that glucocorticoids are necessary for activation of the Ub-P’some system in acidosis or diabetes (28–30). We tested an inhibitor of glucocorticoids, RU486 (2 mg/kg/d, a dose shown to be effective in mice; ref. 31), and found that it blunted the angiotensin II–induced relative weight loss and reduction in muscle mass (Figure 3, A–C).
Angiotensin II–induced muscle wasting in mice is in part glucocorticoid dependent. Mice were either angiotensin II infused or sham infused with daily injections of RU486 or vehicle and pair fed. RU486 significantly inhibited the angiotensin II–induced (A) relative loss of body weight (P < 0.01, angiotensin II–infused with RU486 [RA] vs. angiotensin II–infused), and (B and C) decrease in muscle mass as assessed at 7 days. RP, pair-fed with RU486.
Targeted expression of IGF-1 transgene in skeletal muscle completely inhibits angiotensin II–induced weight loss and muscle wasting. We have recently demonstrated that angiotensin II–induced muscle wasting is associated with a reduction in circulating and skeletal muscle levels of IGF-1; however, systemic administration of IGF-1 with angiotensin II did not prevent muscle wasting in rats, which suggests that the autocrine IGF-1 system was involved (25). In our present study, we found that angiotensin II infusion reduced muscle IGF-1 mRNA levels (measured by real-time PCR) by 37% ± 6% at 7 days (P < 0.05, angiotensin II–infused vs. pair-fed mice). To determine whether IGF-1 overexpression in muscle could prevent angiotensin II–induced wasting, we used MLC/mIgf-1 mice, which express a local IGF-1 isoform in muscle under control of the myosin light chain (MLC) promoter (32).
We infused WT (FVB) mice or transgenic MLC/mIgf-1 mice with angiotensin II and sham-infused pair-fed mice for 7 days. The hypertensive response to angiotensin II at 7 days was virtually identical in transgenic and WT mice (58 ± 6.7 mmHg increase vs. 54.8 ± 12.1 mmHg increase, respectively), and the baseline blood pressure was the same in both groups. The angiotensin II–induced relative weight loss in WT mice (Figure 4A) was completely prevented in MLC/mIgf-1 transgenics (Figure 4B), as was the reduction in gastrocnemius and extensor digitorum longus (EDL) muscle weight (Figure 4, C and D). However, soleus muscle weight was reduced in response to angiotensin II infusion in both WT and transgenic mice (Figure 4E), consistent with the preferential expression of IGF-1 in fast fibers. We then tested whether IGF-1 overexpression suppressed angiotensin II induction of ubiquitin ligases. In contrast to what was found in the WT FVB mice, angiotensin II infusion did not increase expression of atrogin-1 and MuRF-1 in the gastrocnemius muscle of the MLC/mIgf-1 mice (Supplemental Figure 1). Indeed, in transgenic mice, angiotensin II infusion caused a small decrease in atrogin-1 (fold decrease of 0.65 ± 0.08, angiotensin II–infused vs. pair-fed; P < 0.05) and MuRF-1 (fold decrease of 0.62 ± 0.05, angiotensin II–infused vs. pair-fed; P < 0.01) mRNA levels.
Skeletal muscle–specific expression of an IGF-1 transgene blocks angiotensin II–induced wasting. WT or transgenic (Tg) mice were either angiotensin II infused or sham infused and pair fed for 7 days, and daily weights were measured (A and B) and muscle weights obtained at 7 days (C–E). The relative weight loss in angiotensin II–infused WT mice (A; *P < 0.01 WT/angiotensin II–infused vs. WT/pair-fed) was blocked in transgenic mice (B) with preferential fast-fiber effect (C and D).
We next examined the signal pathways that mediate IGF-1’s anabolic effect in the MLC/mIgf-1 mice. IGF-1 induces muscle hypertrophy by activating the type 1 IGF-1 receptor (IGF-1R), which then activates multiple signaling pathways, including the PI3K and MAPK pathways. There were no significant differences in IGF-1R, phospho- or total Akt, and phospho- or total MAPK between MLC/mIgf-1 transgenic and WT mice at basal levels (Figure 5A). However, the expression level of phospho-mTOR and phospho-p70S6K was increased at basal levels in the MLC/mIgf-1 mice (Figure 5B). Furthermore, phospho-mTOR and phospho-p70S6K levels were reduced in angiotensin II–infused WT but maintained in the angiotensin II–infused MLC/mIgf-1 mice (Figure 5B). The angiotensin II–induced reduction in Akt phosphorylation that occurred in WT mice was completely blocked in the MLC/mIgf-1 mice (Figure 5C). Consequently, caspase-3 activation (Figure 5C) and actin cleavage (Figure 5D) were prevented in the angiotensin II–infused MLC/mIgf-1 mice. These data demonstrate that the Akt/mTOR/p70S6K pathway remains active in the MLC/mIgf-1 mice receiving angiotensin II.
Involvement of Akt/mTOR/p70S6K kinases in the ability of the MLC/mIgf-1 transgene to prevent angiotensin II–induced muscle loss. (A) Basal characterization of signaling pathways in MLC/mIgf-1 mice. Gastrocnemius lysates from WT or MLC/mIgf-1 mice were subjected to SDS-PAGE and Western blotting with indicated antibodies. There is no significant difference in baseline levels of IGF-1 receptor (IGF-1R), phospho- or total Akt, and phospho- or total MAPK in transgenics. (B) Phospho-mTOR and phospho-p70S6K expression was diminished in muscles of angiotensin II–infused WT mice compared with pair-fed controls but was maintained in the angiotensin II–infused MLC/mIgf-1 mice. Additionally, expression of phospho-mTOR and phospho-p70S6K was increased at basal levels in transgenic mice compared with WT. (C) Phospho-Akt expression was maintained in angiotensin II–infused MLC/mIgf-1 mice, accompanied by diminished caspase-3 cleavage. (D) The accumulation of 14-kDa actin fragment was significantly reduced in the angiotensin II–infused MLC/mIgf-1 mice compared with WT mice.
In order to provide additional evidence of the role of the Akt/mTOR/p70S6K pathway in skeletal muscle wasting, we studied a rat ischemic cardiomyopathy model. Heart failure was induced by left anterior descending coronary artery (LAD) ligation, and muscles were harvested at 2 weeks. The rats with LAD ligation had reduced gastrocnemius (LAD-ligated, 1.5 ± 0.08 g; sham-operated 1.7 ± 0.1 g; P < 0.05) as well as soleus muscle mass (LAD-ligated, 122.8 ± 8.7 mg; sham, 152.4 ± 9.3 mg; P < 0.01). Western blotting showed that the levels of phospho-Akt and phospho-p70S6K were markedly reduced in gastrocnemius muscle from LAD-ligated rats compared with those from sham-operated rats. The levels of total Akt and total p70S6K were not different between LAD-ligated and sham-operated rats (Supplemental Figure 4).
Angiotensin II induces apoptosis of skeletal muscle, which is prevented by autocrine IGF-1. The activation of caspase-3 in our angiotensin II–infused animals prompted us to investigate whether apoptosis is involved in angiotensin II–induced muscle loss. Upstream components of the apoptosis cascade include Bad and cytochrome c. Akt has been shown to promote cell survival in part via its ability to phosphorylate Bad at Ser136 (33, 34). Phospho-Bad (Ser136) expression was reduced in the muscle of WT angiotensin II–infused mice compared with the pair-fed controls (Figure 6A). However, the phosphorylation of Bad was maintained in the MLC/mIgf-1 mice infused with angiotensin II. Furthermore, there was a significant increase in cytosolic cytochrome c release (Figure 6B) and DNA fragmentation (Figure 6C) in response to angiotensin II in WT mice compared with the pair-fed controls, whereas these increases were prevented in the MLC/mIgf-1 mice. TUNEL staining of gastrocnemius muscle sections showed that there was a significant increase in the number of TUNEL-positive nuclei, which was 21% ± 4% in angiotensin II–infused mice as opposed to 5% ± 2% in the pair-fed mice (P < 0.01) (Supplemental Figure 5).
Angiotensin II–induced muscle loss in WT mice is associated with (A) reduced levels of phospho-Bad in gastrocnemius muscle; (B) increased cytochrome c release into the cytosolic fraction (*P < 0.05, angiotensin II–infused vs. pair-fed WT); and (C) DNA fragmentation as detected by cell death ELISA (**P < 0.01, angiotensin II–infused vs. pair-fed WT). These changes were completely blunted in the transgenic mice (A–C).