Inhibition of the renin–angiotensin system improves physiological outcomes in mice with mild or severe cancer cachexia

Authors


Correspondence to: Kate T. Murphy, Department of Physiology, Basic and Clinical Myology Laboratory, The University of Melbourne, Victoria 3010 Australia, Tel.: +61 3 8344 0065, Fax: +61 3 8344 5818, E-mail: ktmurphy@unimelb.edu.au

Abstract

Cancer cachexia describes the progressive skeletal muscle wasting and weakness associated with many cancers. Cachexia reduces mobility and quality of life and accounts for 20–30% of all cancer-related deaths. Activation of the renin–angiotensin system causes skeletal muscle wasting and weakness. We tested the hypothesis that treatment with the angiotensin converting enzyme (ACE) inhibitor, perindopril, would enhance whole body and skeletal muscle function in cachectic mice bearing Colon-26 (C-26) tumors. CD2F1 mice received a subcutaneous injection of phosphate buffered saline or C-26 tumor cells inducing either a mild or severe cachexia. The following day, one cohort of C-26 mice began receiving perindopril in their drinking water (4 mg kg−1 day−1) for 21 days. In mild and severe cachexia, perindopril increased measures of whole body function (grip strength and rotarod) and reduced fatigue in isolated contracting diaphragm muscle strips (p < 0.05). In severely cachectic mice, perindopril reduced tumor growth, improved locomotor activity and reduced fatigue of tibialis anterior muscles in situ (p < 0.05), which was associated with increased oxidative enzyme capacity (succinate deyhydrogenase, p < 0.05). Perindopril attenuated the increase in MuRF-1 and IL-6 mRNA expression and enhanced Akt phosphorylation in severely cachectic mice but neither body nor muscle mass was increased. These findings support the therapeutic potential of ACE inhibition for enhancing whole body function and reducing fatigue of respiratory muscles in early and late stage cancer cachexia and should be confirmed in future clinical trials. Since ACE inhibition alone did not enhance body or muscle mass, co-treatment with an anabolic agent may be required to address these aspects of cancer cachexia.

Abbreviations
ACE

Angiotensin converting enzyme

Ang

angiotensin

AT1A

angiotensin type 1A receptor

C-26

Colon-26

CHO

carbohydrate

CSA

cross-sectional area

EDL

extensor digitorum longus

NSCLC

nonsmall cell lung cancer

PBS

phosphate buffered saline

Perind

perindopril

RAS

renin–angiotensin system

RER

respiratory exchange ratio

SDH

succinate dehydrogenase

TA

tibialis anterior

math formula

carbon dioxide production

math formula

oxygen consumption

Cancer cachexia is a complex multifactorial syndrome characterized by a progressive loss of skeletal muscle mass with or without loss of fat mass and is associated with significant functional impairments.[1] Cachexia is present in up to 80% of patients with advanced cancer and in 60–80% of those diagnosed with gastrointestinal, pancreatic and lung cancer.[2] It can develop in stages from pre-cachexia to cachexia to refractory cachexia.[1] Cachexia causes severe fatigue and reduces mobility, leading to a loss of functional independence and a reduction in overall quality of life.[3] It can also impair the response to chemotherapy, and the eventual failure of respiratory and cardiac muscle function is responsible for 20–30% of all cancer-related deaths.[2] Treatments are therefore needed to improve patient quality of life and reduce mortality.

The renin–angiotensin system (RAS) is typically associated with the regulation of blood pressure and water balance and has well-characterized effects including vasoconstriction. RAS inhibition is used widely for treating hypertension. Local RAS exist in multiple tissues and have diverse effects.[4] Angiotensin peptides such as angiotensin I (Ang I) and Ang II are produced within skeletal muscle[5, 6] and stimulation of these peptides may contribute to muscle breakdown, either directly or indirectly by numerous mechanisms including enhanced protein degradation,[7] reduced protein synthesis,[8] inflammation,[9] and apoptosis.[10] RAS expression is up-regulated in several conditions associated with muscle wasting and weakness, including sarcopenia,[11] muscular dystrophies,[12] chronic heart failure[13] and chronic renal failure[14]; and may contribute to their pathophysiology. RAS upregulation occurs in patients with colorectal liver metastases[15] and laryngeal cancer[16] which are both associated with severe cachexia.[17] RAS inhibition therefore represents a potential therapeutic strategy for reducing cancer cachexia. Supporting this contention is our recent finding that mice lacking the angiotensin type 1A receptor (AT1A−/−) have increased whole body and skeletal muscle function compared with wild type mice.[18]

AT1 antagonism and inhibition of the angiotensin converting enzyme (ACE), which mediates conversion of Ang I to Ang II, have been shown to improve skeletal muscle pathophysiology in conditions associated with muscle wasting including laceration injury,[19] sarcopenia[20] and the muscular dystrophies.[21] Only one preclinical study has investigated the therapeutic potential of RAS inhibition for attenuating cancer cachexia and found that treatment with a high dose (30 mg kg−1) of the ACE inhibitor, imidapril, attenuated both the loss of body mass and tumor growth in a murine model of cancer cachexia.[7] However, this study did not test whether imidapril also improved skeletal muscle function, which is an important unresolved question since this is the main outcome affecting the quality of life and mortality of patients with cancer cachexia.[3] Although there are no listed clinical studies (on PubMed), a nonpeer-reviewed report of a Phase III clinical trial by Ark Therapeutics found that imidapril significantly attenuated the loss of body mass and reduction in grip strength in cachectic patients with nonsmall cell lung cancer (NSCLC) and colorectal cancer, but not pancreatic cancer.[22] However, when the results for all patients were combined, the improvements were not statistically significant. Taken together, the limited data available support the therapeutic potential of RAS inhibition for cancer cachexia,[23] but there is an unmet need to more comprehensively investigate the efficacy of RAS inhibition for attenuating functional deficits in cancer cachexia. We examined the efficacy of the long-acting ACE inhibitor, perindopril, for enhancing whole body and skeletal muscle function in mildly cachectic and severely cachectic mice bearing Colon-26 (C-26) tumors. As treatments are initiated at different stages of the cachexia spectrum, it is important to test the efficacy of potential therapies in models with varying degrees of cachexia to maximize translation of preclinical findings. We therefore tested the hypothesis that perindopril would increase whole body and skeletal muscle function in both mildly cachectic and severely cachectic C-26 tumor-bearing mice.

Material and Methods

All experiments were approved by the Animal Ethics Committee of The University of Melbourne and conducted in accordance with the Australian code of practice for the care and use of animals for scientific purposes as stipulated by the National Health and Medical Research Council (Australia).

C-26 cell line and inoculation

Frozen C-26 cells inducing mild cachexia were kindly donated by Dr. Paul Gregorevic (Baker IDI, Melbourne, Australia) via the NCI Frederick Cancer DCT Tumor Repository (Frederick, MD), and frozen C-26 cells inducing severe cachexia were kindly donated by Prof. Martha Belury (The Ohio State University, Columbus, OH). We have previously described the procedures used to thaw and count the cells and have characterized the functional and metabolic impairments in mildly cachectic and severely cachectic C-26 tumor-bearing mice in detail elsewhere.[24] On the day of inoculation (day 1), all mice were anesthetized via an intraperitoneal (i.p.) injection of a mixture of ketamine (100 mg kg−1) and xylazine (10 mg kg−1; VM Supplies, Chelsea Heights, VIC, Australia), such that they were unresponsive to tactile stimuli. Mice were shaved on the dorsal side and given a subcutaneous injection of either 5 × 105 C-26 cells suspended in 100 µl of sterilized phosphate buffered saline (PBS), or 100 µl of sterilized PBS only (control). Mice recovered from anesthesia on a heat pad and were given a subcutaneous injection of atipamezole (Antisedan; 1 mg kg−1; VM Supplies) to partially reverse the effects of xylazine and promote faster recovery from sedation. Body mass and tumor size were monitored daily after inoculation.

Experimental animals

Mildly cachectic mice

To assess the effects of perindopril in mildly cachectic C-26 tumor-bearing mice, 12-week-old male CD2F1 mice were allocated randomly into one of three experimental groups: a mildly cachectic C-26 tumor-bearing group fed ad libitum (C-26 mild, n = 8); a control group injected with PBS alone and pair-fed to the mildly cachectic mice (PBS, n = 8); and a mildly cachectic C-26 tumor-bearing group given perindopril in their drinking water and pair-fed to the mildly cachectic mice (C-26 mild + Perind, n = 8).

Severely cachectic mice

To assess the effects of perindopril in severely cachectic C-26 tumor-bearing mice, 12-week-old male CD2F1 mice were allocated randomly into one of three experimental groups: a severely cachectic C-26 tumor-bearing group fed ad libitum (C-26 severe, n = 12); a control group injected with PBS alone and pair-fed to the severely cachectic mice (PBS, n = 8); and a severely cachectic C-26 tumor-bearing group given perindopril in their drinking water and pair-fed to the severely cachectic mice (C-26 severe + Perind, n = 8). All mice were obtained from the Animal Resources Centre (Canning Vale, Western Australia) and housed in the Biological Research Facility at The University of Melbourne under a 12:12-hr light-dark cycle. Water was available ad libitum and both water and standard laboratory chow was provided, changed and monitored daily. The amount of water consumed per mouse per day was determined and expressed as cumulative water intake.

Perindopril treatment

One day after C-26/PBS injection some mice began receiving perindopril in their drinking water (25.5 µg ml−1, Sigma-Aldrich, Castle Hill, NSW, Australia). The concentration of perindopril used was designed to provide a dose of 4 mg kg−1, which has been shown previously to significantly reduce serum ACE and Ang II levels in rodents.[25] Body mass and water consumption was determined daily to allow for calculation of the actual intake of perindopril (mg) per kg body mass.

Grip strength and rotarod test

As described in detail previously,[24] whole body strength and whole body mobility and co-ordination were assessed on day 20 by means of a grip strength meter (Columbus Instruments, Columbus, OH) and rotarod performance test (Rotamex-5, Columbus Instruments).

Metabolism and locomotor activity

On day 20 (19 days after tumor inoculation), whole body metabolism and locomotor activity were measured using an Open Field Metabolic Chamber (Columbus Instruments) in severely cachectic mice since we have shown previously that they have alterations in these parameters (see Supporting Information Methods for detailed protocols).[24]

Assessment of contractile properties

On day 21, mice were anesthetized with sodium pentobarbitone (Nembutal; 60 mg/kg; Sigm-Aldrich) via i.p. injection. The methods for assessment of the contractile properties of the mouse tibialis anterior (TA) muscle in situ have been described in detail previously.[26] At the conclusion of the contractile measurements in situ, the TA, extensor digitorum longus (EDL), soleus, plantaris, gastrocnemius and quadriceps muscles as well as the epididymal fat were carefully excised, blotted on filter paper and weighed on an analytical balance. The entire diaphragm and rib cage were then surgically excised and costal diaphragm muscle strips composed of longitudinally arranged full-length muscle fibers, were isolated and prepared for functional assessment in vitro, as described in detail elsewhere.[26] Upon completion of the functional analyses, the diaphragm muscle strip was trimmed of tendon and any adhering nonmuscle tissue, blotted once on filter paper and weighed on an analytical balance. Mice were killed as a consequence of diaphragm and heart excision while still anesthetized deeply.

Skeletal muscle histology

Serial sections (5 µm) were cut transversely through the TA muscle using a refrigerated (−20°C) cryostat (CTI Cryostat; IEC, Needham Heights, MA). Sections were reacted with laminin, succinate deyhydrogenase (SDH) and N2.261 for determination of myofiber cross-sectional area (CSA), activity of oxidative enzymes and the percentage of myosin IIa isoforms, respectively (see Supporting Information Methods for detailed protocols). Digital images were obtained using an upright microscope with camera (Axio Imager D1, Carl Zeiss, Wrek, Göttingen, Germany), controlled and quantified using AxioVision AC software (AxioVision AC Rel. 4.7.1, Carl Zeiss).

Real-Time RT-PCR analyses

As stimulation of Ang I and Ang II contributes to skeletal muscle breakdown by numerous mechanisms including enhanced protein degradation[7] and inflammation,[9] we examined the effect of perindopril on the expression of genes involved in these pathways. Gene expression of MuRF-1 and atrogin-1 was examined since protein degradation involves their transcriptional activation. Gene expression of inflammatory genes (IL-6, TNF-α) was assessed since we have previously reported differences in mRNA expression of these genes in cachectic C-26 tumor-bearing mice and have had difficulties obtaining reliable antibodies for these proteins.[24] AT1 and ACE mRNA expression was examined due to a lack of specificity of available antibodies precluding assessment of protein expression.[27] The description of procedures for RNA extraction, cDNA synthesis and PCR analysis are described in Supporting Information Methods.

Western blot analyses

Western blotting was performed as described previously.[28] Twenty-six well gels were employed and all 24 samples (n = 8/group) ran on one gel to obviate gel-to-gel variation as a potential concern in these analyses. Membranes were incubated overnight at 4°C with the following antibodies (all 1:1,000 in blocking buffer): p-Akt (Ser473; #8271, Cell Signaling, Danvers, MA); Akt (#9272, Cell Signaling); and caspase-3 (#9662, Cell Signaling). The signal was imaged using ChemiDoc XRS machine (Bio-Rad) and blots were quantified using Image Lab software (Bio-Rad). A total protein stain (BLOT-FastStain™, G-Biosciences, St Louis, MO) was performed by incubating membranes in Fixer for 3 min at RT, and then in Developer for 1 min at RT followed by 30 min at 4°C. The signal was imaged using ChemiDoc XRS machine and blots were quantified using Image Lab software.

Statistical analyses

All values are expressed as mean ± SEM unless stated otherwise. Groups were compared using a Student's t-test, a one-way ANOVA or a two-way ANOVA, where appropriate. Fiber type proportions are presented as 95% confidence intervals of the mean. Differences were considered significant when no overlap existed between the 95% confidence intervals. Bonferonni's post hoc test was used to determine significant differences between individual groups. The level of significance was set at p < 0.05 for all comparisons.

Results

Perindopril prevented the decrease in body mass in mice with mild cachexia

Untreated mildly cachectic mice drank more than PBS controls (p < 0.01), but perindopril had no effect on water intake (Fig. 1a). Actual perindopril intake of C-26 mild + Perind mice (3.8 mg kg−1) was close to the target intake (4 mg kg−1, Fig. 1b). There were no significant differences between groups in body mass expressed as a percentage of initial (Fig. 1c) or tumor size during the experimental period (Fig. 1e). However, when body mass expressed as a percentage of initial was compared at the end of the experimental period only, there was a significant reduction in the untreated tumor-bearing mice compared with controls (p < 0.05), which was prevented with perindopril (PBS, 103.9 ± 0.9; C-26 mild, 98.3 ± 0.7; C-26 + Perind, 102.0 ± 1.7). Calculation of tumor-free body mass also revealed that perindopril prevented the small but significant 2.5% decrease in tumor-free body mass in the untreated mildly cachectic mice (Fig. 1d). Tumor mass normalized to body mass was not different between the untreated and perindopril-treated tumor-bearing mice (Fig. 1f).

Figure 1.

Perindopril prevents weight loss in mildly cachectic C-26 tumor-bearing mice. Measured daily water intake was expressed as cumulative water intake (a) and used to calculate actual average perindopril intake for the C-26 mild + Perind mice (b). Daily body mass (c) and tumor area (e) were also recorded and at the end of the experimental period the tumor was surgically excised and weighed, allowing for calculation of the percentage change in tumor-free body mass (d) and for tumor mass to be normalized to body mass (f). Mass of selected hindlimb muscles (g) the heart and epididymal fat (h) were also assessed. EDL, extensor digitorum longus; Plant, plantaris; TA, tibialis anterior; Gastroc, gastrocnemius; Quad, quadriceps. Data are mean ± SEM; n = 7 for PBS; 6 for C-26 mild and 7 for C-26 mild + Perind * p < 0.05 vs. PBS; † p < 0.05 vs. C-26 mild; ap < 0.01 PBS vs. C-26 mild group main effect. (Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.)

There were no significant differences in muscle or fat mass between groups (Figs. 1g and 1h), but perindopril reduced heart mass by 18 and 16% compared to PBS controls and untreated mice, respectively (Fig. 1h). When normalized for initial body mass there were no differences between controls and untreated mildly cachectic mice in the mass of any of the muscles examined as well as the heart and epididymal fat (Supporting Information Table S1). However, perindopril-treated mice had a significantly lower normalized heart mass compared with PBS controls and untreated tumor-bearing mice (Supporting Information Table S1).

Perindopril improved whole body function and diaphragm muscle fatigue resistance in mice with mild cachexia

Grip strength and latency-to-fall during a rotarod test were not different between PBS controls and untreated mildly cachectic mice (Figs. 2a and 2b). However, perindopril increased grip strength and latency-to-fall compared with untreated tumor-bearing mice by 64 and 52%, respectively (p < 0.05).

Figure 2.

Perindopril enhances whole body strength and mobility and delays muscle fatigue in mildly cachectic C-26 tumor-bearing mice. Whole body strength was assessed using a grip strength meter (a) and whole body mobility was assessed via latency-to-fall during a rotarod test (b). Functional properties of TA muscles included peak tetanic force (c), specific (normalized) force production (d), frequency-force relationship (e) and force production during and after 4 min of (fatiguing) intermittent stimulation (f). Functional properties of diaphragm muscle strips included specific (normalized) force production (g) and specific force production during and after 4 min of (fatiguing) intermittent stimulation (h). Data are means ± SEM; n = 7 for PBS; 10 for C-26 mild and 6 for C-26 mild + Perind. †p < 0.05 vs. C-26 mild; ap < 0.05 PBS vs. C-26 mild group main effect; cp < 0.05 C-26 mild vs. C-26 mild + Perind group main effect.

There were no statistically significant differences between groups in peak twitch force, time-to-peak twitch tension, one-half relaxation time or maximum rate of force development during a twitch of TA muscles (data not shown). Similarly, no differences between groups were found in peak tetanic force, specific (normalized) force, force production over a range of stimulation frequencies, or force production during and after a 4 min intermittent stimulation (fatiguing) protocol (Fig. 2).

In diaphragm muscle strips there were no significant differences between groups in peak twitch force (data not shown), twitch characteristics (time-to-peak twitch tension, one-half relaxation time, maximum rate of force development, data not shown), peak tetanic force (data not shown) and specific force production over a range of stimulation frequencies (Fig. 2g). However, specific force production during and following a 4 min (fatiguing) stimulation protocol was lower in untreated mildly cachectic tumor-bearing mice compared with controls (p < 0.001) and this was prevented completely with perindopril (Fig. 2h).

Perindopril enhanced oxidative enzyme activity in TA muscles of mice with mild cachexia

TA muscle cross sections were reacted for myosin IIa (N2.261, green), laminin (red) and SDH activity (blue) to identify type IIa fibers, visualize all fibers, and indicate activity of oxidative enzymes (SDH), respectively (Fig. 3a). There were no differences between groups in the proportion of type IIa and type IIx/b fibers nor differences in the CSA of type IIa and type IIx/b fibers (Figs. 3b and 3c). SDH reaction intensity was not different between PBS controls and untreated tumor-bearing mice, but perindopril-treated tumor-bearing mice had higher average SDH and higher SDH intensity in type IIa fibers compared with PBS controls (p < 0.05, Fig. 3d).

Figure 3.

Perindopril enhances fiber oxidative enzyme activity in TA muscles from mildly cachectic C-26 tumor-bearing mice. Representative images of muscle cross-sections reacted for myosin IIa (N2.261, green), laminin (red) and SDH (blue) to indicate type IIa fibers, muscle fiber CSA, and oxidative enzyme activity, respectively (a). Quantification of N2.261, laminin and SDH based on reaction intensity facilitated determination of the proportion of type IIa and type IIx/b fibers (non-N2.261 reacting fibers, b); the area of the type IIa and type IIx/b fibers (c); and the SDH activity based on reaction intensity of the type IIa and type IIx/b fibers (d). Data are means ± SEM (c and d) or means ± 95% CI (b); n = 7 for PBS; 10 for C-26 mild and 6 for C-26 mild + Perind. *p < 0.05 vs. PBS. Scale bars = 100 µm.

Perindopril reduced tumor size but not body mass in mice with severe cachexia

Severely cachectic tumor-bearing mice drank less water than PBS controls which was exacerbated by perindopril (p < 0.05, Fig. 4a). This was attributed to their smaller body mass since water intake normalized to body mass showed that untreated tumor-bearing mice actually drank more than controls (p < 0.01) and there was no difference in water intake between perindopril-treated mice and controls (p < 0.27, data not shown). Actual perindopril intake in the severely cachectic mice (3.4 mg kg−1) was slightly less than the target intake (4 mg kg−1, Fig. 4b). Severely cachectic tumor-bearing mice had a progressive reduction in body mass from day 10 which was exacerbated with perindopril (p < 0.05, Fig. 4c). Over the 21 day period, pair-fed PBS controls lost ∼6% body mass whereas severely cachectic untreated mice lost ∼22% tumor-free body mass (p < 0.001), and perindopril-treated mice had an ∼28% loss of tumor-free body mass (p < 0.001, Fig. 4d). Perindopril slowed the progressive increase in tumor size (p < 0.05) and at the end of the experimental period, reduced tumor mass by 34% (p < 0.001, Figs. 4e and 4f).

Figure 4.

Perindopril reduces tumor size in severely cachectic C-26 tumor-bearing mice. Cumulative water intake (a) and actual average perindopril intake for the C-26 severe + Perind mice (b). Daily body mass (c) and tumor area (e), the percentage change in tumor-free body mass (d) and tumor mass normalized to body mass (f). Mass of selected hindlimb muscles (g) the heart and epididymal fat (h) were also assessed. EDL, extensor digitorum longus; Plant, plantaris; TA, tibialis anterior; Gastroc, gastrocnemius; Quad, quadriceps. Data are mean ± SEM; n = 15 for PBS; 14 for C-26 severe and 18 for C-26 severe + Perind. *p < 0.05 vs. PBS; †p < 0.05 vs. C-26 severe. ap < 0.05 PBS vs. C-26 severe group main effect; bp < 0.05 PBS vs.C-26 severe + Perind group main effect; cp < 0.05 C-26 severe vs. C-26 severe + Perind group main effect. (Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.)

In untreated severely cachectic mice absolute mass of the EDL (−19% vs. PBS), TA (−18%) and gastrocnemius muscles (−16%) as well as the heart (−7%) and epididymal fat (−92%) were reduced compared with PBS controls (p < 0.01, Figs. 4g and 4h). Perindopril caused a larger decrease in heart mass (−26% vs. PBS, p < 0.001). When normalized for initial body mass, mass of the EDL (−20% vs. PBS), TA (−19%), gastrocnemius (−17%), heart (−12%) and epididymal fat (−92%) were reduced in severely cachectic untreated tumor-bearing mice compared with PBS controls (Supporting Information Table S2).

Perindopril improved whole body and skeletal muscle function in mice with severe cachexia

Untreated severely cachectic tumor-bearing mice had reduced grip strength (−22%) and latency-to-fall during a rotarod test (−52%) compared with PBS controls (p < 0.05), but these impairments were prevented completely with perindopril (p < 0.05, Figs. 5a and 5b).

Figure 5.

Perindopril prevents the reduction in whole body strength and mobility and delays muscle fatigue in severely cachectic C-26 tumor-bearing mice. Whole body strength (a) and mobility (b). Functional properties of TA muscles included peak tetanic force (c), specific (normalized) force production (d), frequency-force relationship (e) and absolute force production during and after 4 min of (fatiguing) intermittent stimulation (f). Functional properties of diaphragm muscle strips included frequency-force relationship (g) and specific force production during and following 4 min of (fatiguing) intermittent stimulation (h). Data are means ± SEM; n = 7 for PBS; 10 for C-26 severe and 6 for C-26 severe + Perind. * p < 0.05 vs. PBS; †p < 0.05 vs. C-26 severe; ap < 0.05 PBS vs. C-26 severe group main effect; bp < 0.05 PBS vs. C-26 severe + Perind group main effect; cp < 0.05 C-26 severe vs. C-26 severe + Perind group main effect.

Peak twitch force of TA muscles was 26% lower in untreated tumor-bearing mice compared with controls (p < 0.01) and this was prevented completely with perindopril (data not shown). Twitch characteristics (time-to-peak twitch tension, one-half relaxation time, maximum rate of force development) were not significantly different between groups (data not shown). TA muscles from untreated tumor-bearing mice produced lower peak tetanic force than controls (−31%, p < 0.001) which was unaltered with perindopril (Fig. 5c). Since specific force was not different between controls, the lower peak tetanic force was attributed to the smaller CSA of muscles from severely cachectic mice (Fig. 5d). Examination of the frequency-force relationship revealed that TA muscles from untreated tumor-bearing mice produced lower forces than controls at stimulation frequencies ≥ 30 Hz (p < 0.01, Fig. 5e). Perindopril induced a small but significant increase in force production in severely cachectic tumor-bearing mice, with the greatest improvement occurring at a stimulation frequency of 40 Hz (p < 0.05, Fig. 5e). Force production during and after a 4 min intermittent stimulation protocol was lower in untreated tumor-bearing mice compared with controls (p < 0.001), but fatigue resistance was improved with perindopril (p < 0.001, Fig. 5f).

In diaphragm muscle strips there were no significant differences between groups in peak twitch force and twitch characteristics (data not shown). The frequency-force relationship revealed that diaphragm muscle strips from untreated severely cachectic mice produced lower specific forces than controls (p < 0.02) and this was not altered with perindopril (Fig. 5g). Specific force during and after a 4 min fatigue protocol was lower in untreated severely cachectic mice compared with controls (p < 0.02) but this was prevented completely with perindopril (p < 0.001, Fig. 5h).

Perindopril enhanced oxidative enzyme activity in mice with severe cachexia

Examination of TA muscle cross sections reacted for myosin IIa, laminin and SDH activity (Fig. 6a) revealed no differences between groups in the proportion of type IIa and type IIx/b fibers (Fig. 6b). Average fiber CSA and area of the type IIx/b fibers was smaller in untreated severely cachectic mice compared with controls (p < 0.05) and was not altered with perindopril (Fig. 6c). SDH intensity was not different between PBS controls and untreated severely cachectic mice, but perindopril increased average fiber SDH and SDH intensity in type IIx/b fibers compared with both PBS controls and untreated tumor-bearing mice (p < 0.05, Fig. 6d).

Figure 6.

Perindopril enhances fiber oxidative enzyme activity in TA muscles from severely cachectic C-26 tumor-bearing mice. Representative images of muscle sections reacted for myosin IIa (N2.261, green), laminin (red) and SDH (blue) (a) and group data including: the proportion of type IIa and type IIx/b fibers (b); the area of the type IIa and type IIx/b fibers (c); and the SDH activity of the type IIa and type IIx/b fibers (d). Data are means ± SEM (c and d) or means ± 95% CI (b); n = 7 for PBS; 8 for C-26 severe and 8 for C-26 severe + Perind. *p < 0.05 vs. PBS; †p < 0.05 vs. C-26 severe. Scale bars = 100 µm.

Perindopril improved locomotor activity in mice with severe cachexia

Severely cachectic tumor-bearing mice moved less than controls during the dark cycle and when the light and dark cycles were combined (p < 0.05), and this was not altered with perindopril (Supporting Information Table S3). Movement duration during the dark cycle was also lower in the untreated severely cachectic mice compared with controls (p < 0.05), but there was no significant difference in movement duration between perindopril-treated tumor-bearing mice and PBS controls (Supporting Information Table S3). Untreated severely cachectic mice moved more slowly and rested for longer compared with controls (p < 0.05) but this was prevented with perindopril (Supporting Information Table S3).

Perindopril prevented the metabolic impairments in mice with severe cachexia

Average oxygen consumption ( math formula), carbon dioxide production ( math formula) and respiratory exchange ratio (RER) were lower during all cycles in untreated severely cachectic mice compared with controls (Supporting Information Table S4). However, these effects were prevented with perindopril (Supporting Information Table S4). Perindopril also prevented the reduction in CHO oxidation and the increase in fat oxidation in severely cachectic tumor-bearing mice (Supporting Information Table S4).

Perindopril reduced MuRF-1 and IL-6 mRNA expression and enhanced Akt phosphorylation in mice with severe cachexia

The effect of perindopril on mRNA expression of ubiquitin ligases MuRF-1 and atrogin-1 was examined (Supporting Information Figs. S1a and S1b). Perindopril prevented the twofold increase in MuRF-1 mRNA and attenuated the threefold increase in atrogin-1 mRNA in severely cachectic mice (p < 0.05).

Mediators of inflammation were investigated by assessing the mRNA expression of the inflammatory genes IL-6 and TNF-α (Supporting Information Figs. S1c and S1e). Perindopril prevented the fivefold increase in IL-6 mRNA in severely cachectic tumor-bearing mice (p < 0.01), but there were no differences between groups in TNF-α mRNA (p < 0.41).

Protein expression of full-length (35 kDa) caspase3, a mediator of apoptosis, was not significantly different between groups, but perindopril attenuated the 69% increase in expression of cleaved, active caspase3 (19, 17 kDa) in severely cachectic mice (p < 0.05, Supporting Information Figs. S2a and S2c).

To examine changes in the protein synthesis pathway, the expression of phosphorylated and total Akt was assessed (Supporting Information Figs. S2a and S2b). Severely cachectic mice had a 39% lower expression of phosphorylated Akt normalized to total Akt compared with controls, which was attenuated with perindopril (p < 0.05).

To determine the efficacy of perindopril to inhibit ACE, ACE mRNA was assessed (Supporting Information Fig. S1d) and it was also of interest to examine whether ACE inhibition reduced AT1 mRNA expression (Supporting Information Fig. S1f). Perindopril prevented the increase in ACE mRNA in severely cachectic tumor-bearing mice (p < 0.05), but there were no differences between groups in AT1 mRNA (p < 0.90).

Cardiac α-MHC mRNA expression in severely cachectic mice was examined since cardiac dysfunction has been associated with reduced α-MHC expression[29, 30] and perindopril has been shown to increase cardiac α-MHC expression.[31] Untreated tumor-bearing mice had a 60% decrease in α-MHC mRNA compared with controls (p < 0.05), but there was no significant difference between controls and perindopril-treated tumor-bearing mice (Supporting Information Fig. S1g).

Discussion

The most important finding of clinical relevance from this study was the demonstrated efficacy of the long-acting ACE inhibitor, perindopril, to enhance whole body and skeletal muscle function in tumor-bearing mice at different stages of the cachexia spectrum. In both mildly and severely cachectic mice, perindopril improved whole body function and reduced fatigue of diaphragm muscle strips. In severely cachectic mice, perindopril also improved locomotor activity and attenuated the deleterious metabolic aberrations in untreated cachectic mice. Taken together, these findings highlight the therapeutic potential of ACE inhibition for enhancing whole body function and reducing fatigue of respiratory muscles in early and late stage cancer cachexia and should be confirmed in future clinical studies.

Patients with cancer cachexia have a 25% reduction in grip strength[32] which affects their ability to perform everyday tasks such as rising from a chair or bed, performing home duties and maintaining personal hygiene. The reduction in grip strength in cachectic patients is also correlated strongly with postoperative complications.[32] Perindopril prevented completely the 22% decrease in grip strength in severely cachectic mice and increased grip strength in mildly cachectic mice. These findings are consistent with a Phase III clinical trial showing improved grip strength with imidapril in cachectic NSCLC patients.[22] They also indicate that ACE inhibition could improve the ability of affected patients to perform even the simplest tasks and may reduce their risk of postoperative complications.

Cachectic patients have impaired mobility and lower levels of physical activity that reduces their functional independence.[33] Perindopril prevented the decrease in mobility as assessed by rotarod performance and attenuated the reduction in locomotor activity in severely cachectic mice, and enhanced mobility in mildly cachectic mice. The reduction in physical activity levels in cachectic patients is due to a combination of pain, fatigue and depression, and a vicious cycle ensues whereby reduced physical activity levels compound the depression. Physical activity levels in our severely cachectic mice were monitored over 24 hr, but it would be of interest to examine whether perindopril increased the performance of voluntary exercise in tumor-bearing mice. The maintenance of physical activity levels might not only attenuate cachexia, but also reduce depression.

Respiratory failure is one of the major causes of death in cancer cachexia.[34] Remarkably, perindopril was able to reduce fatigue of isolated diaphragm muscle strips from mice with mild and severe cachexia. These findings support those of a recent study reporting diaphragm muscle wasting after Ang II infusion in mice,[35] and are consistent with perindopril attenuating the reduction in math formula and math formula in severely cachectic mice. The improved fatigue resistance with perindopril is clinically significant since respiratory muscles work continuously during life and cachectic patients often die from respiratory failure. By reducing fatigue of respiratory muscles, ACE inhibition could potentially prolong survival of cachectic patients.

Limb muscle strength is impaired in cancer cachexia,[36] causing affected patients to reduce their functional independence.[3] Perindopril attenuated the reduction in submaximal force and reduced fatigue in TA muscles of severely cachectic mice. Given that the force required of muscles for normal daily activities is never close to maximal,[37] the improved submaximal forces with perindopril would be beneficial for performing normal daily tasks. The fatigue resistance with perindopril was associated with increased fiber oxidative enzyme capacity (SDH) in both the fast oxidative type IIa fibers and the fast glycolytic type IIx/b fibers. As severe fatigue is a devastating consequence of cachexia that affects nearly all cancer patients,[38] attenuating fatigue would dramatically improve the quality of life of affected patients. The improvements in whole body and skeletal muscle function with perindopril occurred without affecting muscle mass or fiber size; findings consistent with our previous observation that AT1A−/− mice have increased whole body and skeletal muscle function despite having smaller muscles.[18] The improvements in these mice were attributed to a fiber-type shift toward a greater proportion of fast glycolytic type IIx/b fibers and a smaller proportion of fast oxidative type IIa fibers.[18] However, a similar fiber-type shift was not found in the current study so the mechanisms contributing to the improvement in muscle function with perindopril remain uncertain and should be investigated in future studies. Although some previous studies have found no effect of acute RAS inhibition on muscle mass in dystrophic mdx mice,[39] other studies have reported increased muscle mass.[21] Real-Time RT-PCR analyses confirmed that perindopril prevented the increase in ACE mRNA in severely cachectic mice. Perindopril also attenuated expression of genes involved in protein degradation (MuRF-1, atrogin-1) and inflammation (IL-6), and proteins involved in apoptosis (caspase3), as well as increased expression of proteins involved in protein synthesis (phosphorylated Akt). These findings are consistent with previous reports showing that stimulation of Ang I and Ang II induce skeletal muscle breakdown by enhancing protein degradation,[7] inflammation[9] and muscle apoptosis,[10] and by reducing protein synthesis.[8]

Metabolic abnormalities including increased fat oxidation and reduced CHO oxidation are prevalent in patients with cancer cachexia and are thought to contribute to the pathogenesis.[40] Remarkably, perindopril prevented the metabolic alterations in severely cachectic tumor-bearing mice, returning fat and CHO oxidation and RER to levels similar to those of PBS controls. The increase in fat oxidation and the reduction in CHO oxidation in cachectic patients has been linked to insulin resistance[41] and correction of these abnormalities is consistent with the well characterized improvement in whole body sensitivity with ACE inhibition.[42]

Experimental studies in rodents have reported that ACE inhibition reduced the size of various tumors including colorectal liver tumors[43] and MAC16 colon tumors.[7] Retrospective studies have also shown that ACE inhibition is associated with attenuation of tumor growth of the prostate, lung, colon and breast.[44] To our knowledge, this is the first study showing that ACE inhibition reduced the size of C-26 tumors from severely cachectic mice. Interestingly, perindopril had no effect on C-26 tumor size in mildly cachectic mice. ACE inhibition has been proposed to inhibit tumor angiogenesis and growth, and to induce apoptosis.[45] Although beyond the scope of this study, future studies should compare the effects of perindopril on the angiogenesis, growth and apoptosis of tumors induced with the two different C-26 cell lines. Importantly, the differing effects of perindopril on C-26 tumor size indicate that the improvements in whole body and skeletal muscle function in the severely cachectic mice was not simply due to a smaller tumor burden.

Cardiac atrophy leading to cardiac failure is one of the major causes of death in cancer cachexia and is the cause of >7% of all cancer-related deaths.[46] Anti-cancer therapies can cause cardiotoxicity and exacerbate the cardiac dysfunction in cancer patients.[47] Consistent with our previous study, cardiac atrophy was apparent in mice with severe but not mild cachexia.[24] Heart failure has been linked to reduced expression of α-MHC due to slowed rates of rise of force and reduced ejection time[29] and functional cardiac impairments in C-26 tumor-bearing mice have been associated with reduced α-MHC expression.[30] Although assessment of cardiac function was beyond the scope of the current study, the mRNA expression of α-MHC was reduced in C-26 tumor-bearing mice indicating impaired cardiac function. ACE inhibitors have well known cardioprotective effects and can prevent cardiotoxicity and improve cardiac function in patients receiving chemotherapy.[48] ACE inhibitors also reduce heart mass by lowering blood pressure, decreasing cardiac load[49] and increasing cardiac α-MHC expression.[31] Consistent with these effects, perindopril reduced heart mass and attenuated the decrease in α-MHC mRNA in tumor-bearing mice, indicating functional improvement with perindopril and that the reduction in heart mass was physiological rather than pathological.

Conclusions

Our preclinical findings support the therapeutic potential of RAS inhibition for enhancing whole body and skeletal muscle function at the early and late stages of the cachexia spectrum. The improvements could enhance the capacity of affected patients to perform tasks of everyday living, improve quality of life and reduce the risk of postoperative complications. Attenuating diaphragm muscle fatigue could also reduce mortality due to respiratory failure. As ACE inhibition did not enhance body or muscle mass, co-treatment with an anabolic agent may prove even more efficacious for cancer cachexia. Future clinical trials in patients with cancer cachexia should be conducted to confirm our experimental findings.

Acknowledgements

The authors thank Prof. Martha Belury (Department of Human Nutrition, The Ohio State University) for kindly donating the C-26 cells and Prof. Donna McCarthy (College of Nursing, The Ohio State University) for arranging the shipment of these cells. They thank Assoc. Prof. Graham R. Robertson (ANZAC Research Institute, University of Sydney) for providing access to the animal colony.

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