The term “cardiac cachexia” is often used to describe the unintended weight loss associated with heart disease, yet in the field of oncology, it can be used to describe cancer-induced cardiac atrophy, cardiac remodeling, and ventricular functional deficits. Although cancer-induced cardiac complications have garnered more attention in recent years, it remains an underdiagnosed component of disease progression. Animal models of cardiac cachexia report rapid loss of cardiac mass, accompanied by left ventricular (LV) wall thinning and chamber dilation (1). This is followed by systolic and diastolic functional deficits (1), increased ventricular fibrosis (2), and possibly heart failure (2,3). Tumor-bearing mice show reduced cardiac troponin I and α-myosin heavy chain (MHC) expression, as well as increased protein ubiquitination, brain natriuretic peptide expression, and peroxisome proliferator-activated receptor expression (2,3). Other reports indicate that cancer is capable of inducing dilated cardiomyopathy (4) and that this cardiac remodeling may be related to reductions in cardiomyocyte size and increased autophagy (5,6). This is supported by human studies demonstrating that cancer patients who died as a result of cancer cachexia showed a 25% reduction in heart weight when compared with cancer patients without cachexia, and by the fact that postmortem histology revealed extensive fibrosis in all cancer patients regardless of the presence of cachexia (6). Furthermore, a recent study of 16,500 Canadian patients who died of cancer identified that 7.5% of patients were also diagnosed with heart failure. Interestingly, heart failure appeared to be more prevalent in certain types of cancer: 14.4% in multiple myeloma, 10.4% in leukemia, 9.7% in lymphoma, 8.9% in male genital in urinary system cancers, 8.5% in lung cancer, and 7.6% in female breast cancer (7). Cardiac complications in cancer patients remain underdiagnosed and thus commonly unaddressed in the clinical setting.
Structured, aerobic exercise-based rehabilitation programs are becoming an important adjuvant therapy in the treatment of many cancers. Regular exercise in the cancer population significantly improves cardiopulmonary and musculoskeletal function, it can attenuate many common treatment-related side effects, and it can enhance quality of life (8). Chronic exercise is a powerful regulator of muscle function and metabolism, thereby providing a theoretical foundation for its use in treating cachexia-related conditions. However, no studies have investigated the influence of exercise-mediated autophagy on cancer-related cardiac atrophy and dysfunction. In addition, no studies have investigated the effects of exercise-mediated autophagy on tumor growth. Therefore, the purpose of this investigation was 1) to examine the effect of aerobic exercise training on cardiac function and cardiac autophagy protein expression and 2) to determine whether exercise-induced autophagy affects tumor growth.
Twelve-week-old female Fischer 344 rats (Harlan, Indianapolis, IN; n = 28) were housed in a temperature controlled facility with a 12-h light–12-h dark cycle. Animals were fed standard rat chow and distilled water ad libitum. Sedentary animals were housed in standard rat cages, whereas exercise-trained animals were housed in standard rat cages equipped with a running wheel. Spontaneous voluntary activity was recorded using a Vital View data acquisition system (MiniMitter, Bend, OR). Animals had access to the running wheel 24 h·d−1, 7 d·wk−1. All procedures were approved by the University of Northern Colorado’s Institutional Animal Care and Use Committee and complied with the Animal Welfare Act guidelines.
Rats were randomly assigned to one of four groups: 1) sedentary non-tumor bearing (SED, n = 7), 2) sedentary tumor bearing (SED + T, n = 7), 3) wheel run non-tumor bearing (WR, n = 7), or 4) wheel run tumor bearing (WR + T, n = 7). Animals remained either sedentary or voluntarily exercised for 6 wk. At week 4 of the protocol, animals in the tumor groups (SET + T and WR + T) were inoculated with MAT-B-III rat adenocarcinoma cells. All animals were sacrificed at the end of the 6-wk protocol. Tumor size was measured with calipers, and cardiac function was assessed using an isolated perfused working heart system. Heart and tumor tissue were collected and flash frozen in liquid nitrogen for subsequent biochemical analyses.
A rat mammary gland tumor cell line, 13762 MatBIII (American Type Culture Collection [ATCC], Manassas, VA), was used to grow a subcutaneous tumor in the flank of each animal. Cells were grown in McCoy’s 5a Modified Medium (ATCC) supplemented with 10% fetal bovine serum (ATCC), in an incubator set to 37°C and 5% CO2. On week 4 of the study, rats were inoculated subcutaneously in the left leg flank with 1 × 106 MatBIII cells (9,10). Rats were weighed, tumors were measured, and body condition (11) was assessed at the end of the each week of the 2-wk tumor growth period. Tumor length, width, and thickness were measured with calipers and recorded while the rat was sedated (40 mg·kg−1 ketamine, i.p.). These measurements were used to estimate tumor mass, relative tumor mass, and tumor volume. Tumor mass was estimated using the following formula: mass (g) = 0.79768 + (0.000456 × length × width × thickness of tumor in mm) (12). Relative tumor mass was calculated (in grams) as follows: estimated tumor mass/(total body mass − estimated tumor mass). Tumor volume was calculated using the following formula: volume (mm3) = (a × b 2)/2, where a was longest diameter and b was shortest diameter (13). Animals were to be euthanized if estimated tumor mass exceeded 25% of body mass, the percent loss of tumor free body mass exceeded 25% of starting mass, the tumor became ulcerated, or the animal received a body condition score ≤2 on two consecutive assessments. At the end of the 2-wk tumor-bearing period, tumors were excised from the left flank, and wet tumor masses were determined.
Isolated perfused working heart
Cardiac function was analyzed ex vivo using an isolated working heart model (AD Instruments, Colorado Springs, CO). Each animal was anesthetized with an intraperitoneal injection of heparinized (500 U) sodium pentobarbital (50 mg·kg−1), and the heart was quickly excised and placed into ice-cold Krebs–Henseleit buffer (120 mM NaCl, 5.9 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl, 25 mM NaHCO3, 17 mM glucose, and 0.5 mM EDTA) aerated with 95% O2–5% CO2. The ascending aorta was cannulated, and the heart was subjected to retrograde perfusion (Langendorff prep) while the heart was cleaned of all connective tissue and the coronary vasculature cleared of blood. The pulmonary vein was then cannulated, and blood flow was redirected from the aorta to the left atrium to initiate the working heart mode. Once stabilized, preload and afterload were standardized at 10 cm H2O and 100 cm H2O, respectively. A microtip catheter pressure transducer (Millar Inc., Houston, TX) was placed into the left ventricle via the apex of the heart for determination of LV developed pressure (LVDP), LV maximal rate of ventricular pressure development (+dP/dt), and LV rate of pressure decline (−dP/dt). Data collection began after a 10-min equilibration period. Hearts were paced at 240 bpm using electrodes attached to the cannulae. Data were collected and analyzed using a PowerLab data acquisition system (ADInstruments, Colorado Springs, CO). After isolated working heart experiments, the left ventricle was isolated, sectioned, flash frozen in liquid N2, and then stored at −80°C for subsequent biochemical analyses.
LV homogenates were analyzed for cardiac MHC protein expression (14,15). Approximately 100 mg of LV was homogenized via lysis buffer (1:10 w:v; 250 mM sucrose, 100 mM KCl, 5 mM EDTA, and 20 mM Tris–base, pH 6.8) with a Virtishear homogenizer (Virtis, Gardner, NJ), followed by sonication. Homogenates were centrifuged for 10 min at 1000g, 4°C, and the supernatant was discarded. The pellet was resuspended in ice-cold washing buffer (1:10 w:v; 175 mM KCl, 2 mM EDTA, and 20 mM Tris–base, pH 6.8, with 0.5% Triton X-100), and centrifuged for 10 min at 1000g, 4°C. Supernatant was discarded, and the pellet was again resuspended in washing buffer and centrifuged (same as before). The supernatant was again discarded, and the pellet was resuspended in final resuspension buffer (150 mM KCl and 20 mM Tris–base, pH 7.0) at a high concentration (1/12th previous volume). Protein concentration was determined by the Bradford method (16), and 10 μg protein was separated by SDS–PAGE on an 8% polyacrylamide gel with a 4% stacking gel at 100 V (Sure-Lock system; Life Technologies, Carlsbad, CA). Gels were stained with Coomassie blue and scanned, and protein bands were quantified by densitometry (ImageJ; National Institutes of Health, Bethesda, MD).
LV and tumor homogenates were analyzed for expression of the autophagy proteins LC3-II (lipid modified form of microtubule-associated proteins 1A/1B light chain 3B, Sigma L7543) and p62 (SQSTM1, AbCam Ab56416) by Western blot. Approximately 100 mg of tissue was homogenized in RIPA buffer (1:100 w:v; Sigma-Aldrich, St. Louis, MO) with a Virtishear homogenizer (Virtis). Homogenates were centrifuged for 10 min at 10,000g, 4°C, and the supernatant was collected for analysis. Protein concentration was determined by DC protein assay (Bio-Rad, Hercules, CA). Protein (30–40 μg) was loaded onto 4%–12% Bis–Tris gels and separated by SDS–PAGE. Proteins were transferred to PVDF membranes, membranes were blocked in 5% milk for 1 h at room temperature and then incubated with primary antibodies diluted in 5% nonfat milk (LC3-II, 1:500 dilution; p62, 1:5000) at 4°C overnight, and secondary antibodies were diluted in 5% nonfat milk (rabbit, 1:5000, Sigma A9169; mouse, 1:10,000, Sigma A2228) for 1 h at room temperature. Membranes were developed using ECL Select (GE Healthcare, RPN2235) and imaged using the MultiDoc-it Imaging System (UVP, LLC, Upland, CA). Membranes were stripped using 30% hydrogen peroxide and probed for GAPDH (loading control, 1:4000) in 5% nonfat milk, 4°C, overnight. Secondary antibody (mouse, 1:10,000) was diluted in 5% nonfat milk for 1 h at room temperature. Membranes were developed with ECL select as before. Protein concentrations were determined by densitometry (MultiDoc-it Imaging System, UVP) and normalized to GAPDH.
All data are presented as mean ± SEM and were analyzed using GraphPad Prism statistical software (La Jolla, CA). A one-way ANOVA was performed for each variable to determine differences between groups (SED, SED + T, WR, and WR + T). For selected variables, to determine differences between SED + T and WR + T groups, a Student’s t-test was performed. Also, to determine the differences in running distances between WR and WR + T groups, a Student’s t-test was performed for each time point. All analyses were two-tailed, and an alpha level of 0.05 was used to define statistical significance. For one-way ANOVA, if a significant difference (P < 0.05) was identified, Tukey’s post hoc testing was performed to identify where significant differences existed.
Effect of exercise and tumor burden on cardiac function
Rats were exercise trained in cages with running wheels or remained sedentary for 6 wk. During week 4 of the protocol, animals were inoculated in the left flank with adenocarcinoma cells to grow a localized tumor that allowed for convenient monitoring of tumor growth and morphology. At the end of the 6-wk protocol, hearts were excised and assessed for cardiac function via the isolated perfused working heart model. As summarized in Figure 1, sedentary tumor-bearing animals demonstrated impairment in both systolic and diastolic function. Specifically, SED + T animals had significantly (P < 0.05) lower LVDP and +dP/dt when compared with all other groups, and significantly (P < 0.05) lower –dP/dt when compared with SED and WR + T groups. Tumor load also appeared to result in cardiac atrophy, which was offset by exercise training. Wet heart mass of the SED + T group was significantly (P < 0.05) less compared with all other groups (Fig. 2), whereas there were no significant differences in body mass at the time of sacrifice (P > 0.05). These data indicate that tumor burden alone can negatively affect cardiac function, resulting in smaller hearts that function less effectively. Furthermore, these data also show that exercise is able to preserve cardiac mass and function from the deleterious cardiac effects of the cancer.
Effect of exercise and tumor burden on cardiac autophagy protein expression
To further investigate the cellular mechanisms of how tumor burden may negatively affect the myocardium, LV homogenates were analyzed for the expression of autophagy proteins LC3-II and p62. LC3-II is located on the inner and outer membrane of the autophagosome, whereas p62 is a docking and adapter protein that interacts with LC3-II and with autophagosome cargo proteins to assist in the incorporation and breakdown of cargo. Because muscle wasting due to tumor burden (cancer cachexia) has been linked to the upregulation of bulk protein degradation systems like the ubiquitin–proteasome system (17) and the autophagy–lysosome system (18), the present study attempted to determine whether the observed cardiac dysfunction might be attributed to enhanced autophagy. Animals from the SED + T group expressed significantly (P < 0.05) more LC3-II and significantly less p62 compared with all other groups (Fig. 3), indicating that autophagic flux was significantly upregulated in the hearts of sedentary tumor-bearing animals. This is likely one mechanism contributing to the severe cardiac dysfunction observed in tumor-bearing animals. Expression of LC3-II and p62 was not significantly different in the WR + T group when compared with the non-tumor-bearing groups, suggesting an exercise-induced cardioprotective effect.
Effect of exercise and tumor burden on cardiac myosin heavy chain protein expression
LV homogenates were also probed for protein expression of α- and β-MHC. Heart failure, hypertrophy, infarction, hormone deprivation, and even cancer can lead to an MHC isoform shift from α to β (4,19,20). Not surprisingly, animals in the SED + T group exhibited a significant (P < 0.05) shift from α-MHC to β-MHC, whereas SED and WR groups did not exhibit such a shift (Fig. 4). Furthermore, no significant differences were noted between WR + T and either the SED or the WR groups. These data indicate that tumor burden negatively affects cardiac function by shifting MHC expression toward the β isoform. This shift toward the β isoform, which has a higher economy of energy consumption but generates lower peak forces and slower velocities of shortening, translates into reductions in LVDP and +dP/dt. Although exercise appears to offer protection against this α- to β-MHC isoform shift, it did not eliminate the shift. Still, cardiac function in exercised tumor-bearing rats was not significantly different from SED or WR rats, indicating that exercise did preserve cardiac function in exercise-trained tumor-bearing rats.
Effect of exercise and tumor burden
Wheel running distances were monitored during the entirety of the study, and weekly running volumes were calculated. No significant differences in weekly wheel running volume (P > 0.05) were observed between WR and WR + T groups at any time point over the course of the 6-wk study (data not shown). However, animals in the WR + T group exhibited significantly smaller tumors compared with animals in the SED + T group (P < 0.05, Fig. 5). In line with this finding, tumors in WR + T animals had significantly less autophagy compared with SED + T animals (P < 0.05, Fig. 6). WR + T animals exhibited significantly less autophagy marker LC3-II protein expression compared with SED + T; however, no differences were observed between groups for protein expression of the autophagosome adapter protein p62.
Although no clinical evidence exists in support of any strategy to treat cardiac cachexia in cancer, several pharmacological strategies (e.g., angiotensin converting enzyme inhibitors, activin type II receptor antagonists, aldosterone antagonists, and NF-κB inhibitors) have been investigated in animal models with varying degrees of success. Likewise, we know little regarding the efficacy of exercise training in attenuating cardiac cachexia in cancer and even less in terms of mechanisms that may be protective. In the present study, tumor-bearing sedentary animals showed significant cardiac atrophy while tumor-bearing exercised animals had no cardiac atrophy. Clinically, cardiac atrophy can be the result of the cancer itself, cardiotoxic cancer therapies, or underlying heart disease unrelated to the cancer (1), yet here, the loss of heart mass is solely related to tumor burden because no cancer treatments were included in the experimental design. However, it does not appear as though this protective effect of exercise against cardiac atrophy is limited to the insult of cancer. We have previously shown that tumor-bearing rats undergoing chemotherapy with cardiotoxic cancer treatments such as anthracyclines show significant decreases in cardiac mass while exercised animals show no such reductions (21). Our data showing that cancer-induced cardiac atrophy was accompanied by cardiac dysfunction are supported by other studies (2,3,21). Exercise protected against cancer-induced cardiac dysfunction as evidenced by the fact that no significant differences were noted between sedentary controls and exercised tumor-bearing animals for LVDP, +dP/dt, or –dP/dt. The importance of cardiac atrophy and dysfunction in this context is highlighted by evidence suggesting that cardiorespiratory fitness is an independent predictor of cancer mortality even after a cancer diagnosis (22). Another point that should be emphasized is that exercise significantly attenuated tumor growth and the reduced tumor burden itself may partially explain the preservation of cardiac mass and function independently of the exercise intervention.
Hearts from sedentary tumor-bearing animals also showed enhanced autophagic flux evidenced by increased LC3-II and decreased p62 protein expression. The size of a muscle, such as the heart, is dictated by the balance of protein synthesis and protein degradation. The two major systems that govern protein degradation are the autophagy–lysosome system and the ubiquitin–proteasome system. Autophagy is a bulk degradation system capable of breaking down and recycling worn, damaged, or misfolded proteins, as well as larger protein aggregates and organelles that would otherwise be too big to be managed by the proteasome. The resulting carbohydrates, lipids, amino acids, and nucleic acids formed during degradation are released to support cellular metabolism and homeostasis (23,24). Basal levels of autophagy are necessary to support cellular homeostasis, and during times of cellular stress, an upregulation of autophagy is beneficial to the cell to provide adenosine triphosphate and remove harmful, toxic components such as reactive oxygen species (23,24). However, blocking autophagic flux in the heart has been reported to accelerate cell death and cause cardiac dysfunction (25). Although an upregulation of autophagy under very specific conditions of cellular stress can be beneficial for cardiac performance (e.g., during the ischemic phase of ischemia/reperfusion), if autophagy remains elevated for too long and becomes maladaptive, cardiac performance significantly diminishes (23). Recent reports, in line with the current study, have shown that cancer results in increased levels of the autophagy marker LC3-II in the heart which coincides with cardiac fibrosis and severe cardiac dysfunction (5,6). Maladaptive, elevated autophagy leads to excessive protein degradation which can needlessly catabolize myofibrillar contractile proteins and energetic organelles leading to a dysfunctional cell and ultimately a dysfunctional organ (26). Thus, an exercise-induced preservation of autophagic activity may be a possible mechanism explaining the preservation of cardiac mass in trained animals.
The present study also demonstrated that hearts from tumor-bearing animals have an α to β shift in MHC isoform expression. β-MHC is characterized by low adenosine triphosphatase activity and slower filament sliding velocity, yet it generates cross-bridge force with a greater economy of energy consumption when compared with the α isoform (27). Increased cardiac β-MHC expression is indicative of a stress response (19,28), which is likely an effort to preserve energy during stressful conditions. Although this is often viewed as a coping mechanism for the heart, others have shown that an α to β shift is disadvantageous under conditions of chronic stress and may actually exacerbate the severity of the dysfunction (27). Increased expression of β-MHC likely played a role in the cardiac dysfunction observed in tumor-bearing animals because increased cardiac β-MHC is associated with lower fractional shortening (27), reduced peak torsion, and reduced mechanical performance (29). Autophagy has a homeostatic role under normal baseline conditions but is activated as a result of reperfusion, starvation, aging, pressure overload, or other conditions associated with cell death and loss of cardiomyocytes (23,30). Other studies have shown that the upregulation of autophagy and an enhanced catabolic state can stimulate the upregulation of β-MHC (31,32). Thus, it appears as though cancer-induced MHC shifts are secondary to increases in cardiac autophagy.
Recent reports have identified exercise as the most effective treatment for excessive muscle atrophy due to disease (33,34). Exercise has been shown to improve metabolic function, promote protein anabolism, reduce inflammation, and support proper calcium handling in muscle to reduce disease-associated fatigue and improve quality of life and survival (33). Although the role of muscle autophagy in exercise is not well understood, preclinical studies show that exercise-mediated autophagy supports protein metabolism and anabolism to balance proteolysis and maintain a healthy muscle mass. This is achieved through the activation of AMPK and the upregulation of Glut4 on the muscle membrane surface (35). Moreover, exercise helps reduce inflammation through upregulation of IL-6, IL-10, and IL-1ra (34). Because chronic inflammatory diseases like Crohn’s disease have dysfunctional autophagy, it is possible that the exercise-mediated benefits of modulated autophagy observed in the hearts of animals in this study are due to reduced inflammation (36,37). Furthermore, the autophagy protein p62 is known to regulate participation in whole body metabolic regulation, and it is suggested that this is through p62’s regulation of muscle-derived cytokines (35,37). Therefore, the cardioprotection afforded by exercise in the present study may be due to the ability of exercise to modulate autophagy, keeping it in the beneficial zone: not too high as seen in the failing hearts of sedentary tumor-bearing animals, and not too low as can be seen in other cardiac pathologies. In doing so, the modulation of autophagy may lead to an improved metabolic profile, maintenance of muscle protein anabolism to catabolism ratios, which may preserve muscle size and function as well as reduce inflammation.
Another primary finding of this study is that the exercise-induced slowing of tumor growth was associated with a significant reduction in LC3-II, indicating that exercise downregulated tumor autophagy. Although preclinical studies have shown mixed results regarding the role of autophagy in cancer, several studies indicate that autophagy promotes tumor cell survival and tumor growth (38). In cancer cells, deficient autophagy through beclin1 haploinsufficiency promoted tumor growth, tumor necrosis, and inflammation, which was achieved through the activation of AKT signaling (38). Pancreatic tumor cell lines show an upregulation of autophagy and pharmacological inhibition of autophagy via chloroquine leads to robust regression of tumor growth (39). Zielinski et al. (40) reported that treadmill-trained mice implanted with EL-4 lymphoma cells had slowed tumor growth, and this was associated with reduced inflammation. As previously mentioned, chronic exercise training reduces inflammation, and autophagy has been implicated in supporting and regulating inflammatory pathways (36,37). Therefore, the exercise-mediated stunting of tumor growth observed in this study may be due to suppressed tumor autophagy and inflammation.
The current study provides novel evidence that chronic exercise can attenuate the atrophy and dysfunction associated with cancer-induced cardiac cachexia, which was associated with a reduction in cardiac autophagy and a subsequent shift in MHC isoform expression. In addition, this study showed that chronic exercise decreased tumor autophagy which may partially explain the exercise-induced stunting of tumor growth. As exercise-based cancer rehabilitation programs continue to grow in number, it is important to understand the benefits of exercise for cancer survivors and the underlying mechanism that explain those benefits. This study adds to the growing body of literature indicating that exercise not only attenuates the deleterious side effects of many standard cancer treatments (8,20,21), but it may potentially serve as an additional adjuvant anticancer therapy to alleviate the negative side effects of cancer itself.
Results of the present study are presented clearly, honestly, and without fabrication, falsification, or inappropriate data manipulation. This study was supported by the University of Northern Colorado.
The authors have no conflict of interest. Results of the present study do not constitute endorsement by the American College of Sports Medicine.
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Keywords:© 2018 American College of Sports Medicine
PHYSICAL ACTIVITY; DISEASE; HEART; MUSCLE; AUTOPHAGY