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Prevention of liver cancer cachexia-induced cardiac wasting and heart failure

Jochen Springer, Anika Tschirner, Arash Haghikia, Stephan von Haehling, Hind Lal, Aleksandra Grzesiak, Elena Kaschina, Sandra Palus, Mareike Pötsch, Karoline von Websky, Berthold Hocher, Celine Latouche, Frederic Jaisser, Lars Morawietz, Andrew J.S. Coats, John Beadle, Josep M. Argiles, Thomas Thum, Gabor Földes, Wolfram Doehner, Denise Hilfiker-Kleiner, Thomas Force, Stefan D. Anker
DOI: http://dx.doi.org/10.1093/eurheartj/eht302 932-941 First published online: 29 August 2013

Abstract

Aims Symptoms of cancer cachexia (CC) include fatigue, shortness of breath, and impaired exercise capacity, which are also hallmark symptoms of heart failure (HF). Herein, we evaluate the effects of drugs commonly used to treat HF (bisoprolol, imidapril, spironolactone) on development of cardiac wasting, HF, and death in the rat hepatoma CC model (AH-130).

Methods and results Tumour-bearing rats showed a progressive loss of body weight and left-ventricular (LV) mass that was associated with a progressive deterioration in cardiac function. Strikingly, bisoprolol and spironolactone significantly reduced wasting of LV mass, attenuated cardiac dysfunction, and improved survival. In contrast, imidapril had no beneficial effect. Several key anabolic and catabolic pathways were dysregulated in the cachectic hearts and, in addition, we found enhanced fibrosis that was corrected by treatment with spironolactone. Finally, we found cardiac wasting and fibrotic remodelling in patients who died as a result of CC. In living cancer patients, with and without cachexia, serum levels of brain natriuretic peptide and aldosterone were elevated.

Conclusion Systemic effects of tumours lead not only to CC but also to cardiac wasting, associated with LV-dysfunction, fibrotic remodelling, and increased mortality. These adverse effects of the tumour on the heart and on survival can be mitigated by treatment with either the β-blocker bisoprolol or the aldosterone antagonist spironolactone. We suggest that clinical trials employing these agents be considered to attempt to limit this devastating complication of cancer.

  • Cancer cachexia
  • Heart failure
  • Cardiac wasting
  • Survival
  • Intervention

Introduction

Each year more than 12 million people worldwide are diagnosed with cancer, and that number is projected to rise to 27 million by 2050.1 Many of these patients also suffer from cachexia at some point in their course, which not only has a profound negative impact on quality of life,2 but also is estimated to directly account for >20% of cancer deaths.3,4 There are no established treatment regimes for cancer cachexia (CC) mainly because the underlying pathophysiological mechanisms leading to death in patients with CC are largely unknown. Hence, CC is a widespread, but poorly understood problem and represents a significant unmet medical need.5

Cachexia is clinically defined as weight loss of at least 5% that is not due to a reduction in oedema, occurs within a period of 12 months or less, and is due to an underlying disease including cancer.6 Although some studies demonstrated that CC is associated with loss of heart mass in rodent models,710 little is known of the consequences of that wasting on cardiac function, and whether it leads to heart failure or impaired survival. Three recent studies using the colon-26 mouse model showed an impaired cardiac function and suggested that nuclear factor κ B (NFκB) plays a crucial role.1113 However, Zhou et al.10 implicated myostatin and its receptor, the activin IIB receptor (ActRIIB), as the primary regulators of skeletal muscle wasting, acting via activation of the catabolic ubiquitin-proteasome system (UPS). It is not clear whether this mechanism is the key driver of cardiac muscle wasting, because Leinwand and co-workers14 have reported autophagy as the dominant pathway. In fact, it is not even clear if cardiac wasting is seen at all in humans with CC.

Herein, we evaluated the functional effects of CC on the heart employing the rat AH-130 hepatoma CC model and we identified drugs commonly used in heart failure reducing cardiac wasting and mortality.

Methods

For Methods, see Supplementary material online.

Results

The hepatoma tumour model displays cachexia, cardiac atrophy and dysfunction, and increased mortality

In our study, the AH-130 hepatoma model showed a tumour growth rate comparable to earlier reports (data not shown),15,16 and developed severe cachexia as indicated by a progressive loss of body weight, and loss of both fat and lean tissue (Figure 1A). This was associated with reduced food intake over time (Figure 1A). The loss of body weight is reflected in individual organs and tissues, including the heart, which showed clear-cut cardiac wasting as evidenced by a striking reduction in absolute heart weight (Figure 1A) and also of left-ventricular mass (LVmass) (Figure 1B). Cardiac contractility [as assessed by LV ejection fraction (LVEF) and LV fractional shortening (LVFS)] were normal in cancer animals until Day 9. Thereafter contractile function declined markedly (Figure 1B) and the heart showed an arrythmogenic impairment (see Supplementary material online, Figure S2). Of note, in untreated tumour-bearing animals, cardiac function on Day 11 was predictive of survival (see Supplementary material online, Figure S3). LV stroke volume (LVSV) and cardiac output (LVCO) at rest were reduced beginning on Day 7 (Figure 1B). Elevated tissue inhibitors of metalloproteinase-1 (TIMP-1) and monocyte chemoattractant protein-1 (MCP-1), as well as fibrinogen, suggests fibrotic remodelling (see Supplementary material online, Figure S4). These features were associated with a high mortality (87%, n = 73, Figure 2) within 16 days.

Figure 1

Alterations in weight, body composition, and cardiac function in the timeline experiment. (A) Loss of body weight, lean mass (=skeletal muscle), and fat mass as the cancer progressed. Tumour-bearing rats had a decreased food intake and the overall weight of the heart was progressively reduced. (B) The left-ventricular ejection fraction and fractional shortening were reduced starting Day 11 after tumour inoculation. A progressive loss of LV end-diastolic diameter (LVEDD) was seen and the stroke volume as well as the cardiac output was already reduced on Day 7, when the clinical definition of cachexia was met. LVmass was progressively lost compared with baseline, reaching its maximum of 58% loss at Day 13. LV end-systolic pressure (LVESP) was reduced on Days 11 and 13, while the end-diastolic pressure (LVEDP) was reduced starting Day 9 compared with sham. The contractility given as delta pressure/delta time (dP/dt) was affected starting Day 11. Time constant of relaxation (tau) was prolonged on Days 11 and 13, and heart rate was lower starting Day 7. *P < 0.05, **P < 0.01, ***P < 0.001 vs. sham.

Figure 2

Survival proportions. Only the best dose for each compound is shown (for complete survival analysis, see Supplementary material online, Figure S4). Rats were treated with 5 mg/kg/day (n = 23) of bisoprolol, 50 mg/kg/day (n = 16) of spironolactone, 0.4 mg/kg/day (n = 9) of Imidapril or placebo (n = 73). A high mortality was observed in the placebo group (87%), which was significantly reduced bisoprolol or spironolactone, see hazard ratios (HR) below the Kaplan–Meier curves.

Select agents used in heart failure improve survival in cancer cachexia

Based on the observed functional impairment of the heart in AH-130 rats (Figure 1B), we investigated the effect of drugs used as standard therapy to treat patients with more typical forms of heart failure, i.e. the β-1 selective antagonist bisoprolol (0.5, 2, 5, or 50 mg/kg/day), the aldosterone antagonist spironolactone (2, 5, and 50 mg/kg/day), and the angiotensin converting enzyme (ACE)-inhibitor imidapril (0.4, 1 or 10 mg/kg/day). We first performed dose–response experiments (Figure 2, Supplementary material online, Figure S5) and the optimal dose for each drug (5 mg/kg/day bisoprolol, 50 mg/kg/day spironolactone) was identified based on survival and cardiac function (Figure 2). Imidapril was ineffective in all doses tested, whereas bisoprolol and spironolactone not only preserved LV function but also markedly reduced mortality in AH-130 rats (Figure 2).

Spironolactone and bisoprolol, but not imidapril, reduce body wasting

Body weight and body composition were similar in all groups before tumour inoculation (Table 1). Treatment with bisoprolol or spironolactone markedly attenuated the loss of body weight, while imidapril was less effective (Table 1). Loss of fat mass was attenuated by bisoprolol, spironolactone, and imidapril (Table 1). Wasting of lean body mass was markedly attenuated by bisoprolol and spironolactone and to a lesser extent by imidapril (Table 1). Anorexia, decreased activity, and lower plasma albumin levels (Table 1, Supplementary material online, Figure S4), together with the weight loss, match the clinical definition of cachexia. Water consumption declined significantly (−24% vs. baseline) but was less reduced than food intake (−77% vs. baseline, P < 0.0001; see Supplementary material online, Table S3), suggesting the reduced food intake was primarily the result of secondary anorexia and not of an inability to access the food. Treatment with bisoprolol or spironolactone improved activity and food intake (Table 1). Imidapril had no significant effect on activity or food intake (Table 1).

View this table:
Table 1

Impact of treatment in tumour rats on weight and body composition

ShamPlaceboBisoprolol 5 mg/kg/daySpironolactone 50 mg/kg/dayImidapril 0.4 mg/kg/day
n1673231610
BW baseline, g203 ± 4206 ± 1204 ± 2200 ± 1204 ± 1
Δ BW, g60 ± 2***−53.7 ± 1.8−21.9 ± 10.5***−21.0 ± 11.0***−30.0 ± 14.1**
Fat baseline, g15.6 ± 0.618.8 ± 0.416.7 ± 0.615.4 ± 0.917.5 ± 0.7
Δ fat, g9.1 ± 0.9***−12.4 ± 0.4−5.9 ± 1.9***−6.7 ± 2.1***−8.9 ± 2.6*
Lean baseline, g158 ± 3158 ± 3 ± 1158 ± 2154 ± 3159 ± 2 
Δ lean, g41.7 ± 2.0***−39.8 ± 1.6−16.7 ± 7.7***−11.9 ± 8.7***−21.610.5**
Activity, (counts/24 h)67 192 ± 2847***29 509 ± 177543 755 ± 3741**44 817 ± 5286**29 379 ± 7205
Food intake (g/24 h)21.3 ± 0.8***4.30.510.9 ± 2.0***9.8 ± 1.7**7.0 ± 2.9
  • Body weight, activity, and food intake were assessed on Day 11.

  • BW, body weight.

  • *P < 0.05, **P < 0.01, ***P < 0.001 vs. sham or placebo treatment.

Spironolactone and bisoprolol, but not imidapril, attenuate tumour-induced cardiac dysfunction and wasting of cardiac muscle

Cardiac function was determined on Day 11 of the treatment protocol by echocardiography. The reduction in LVEF and LVFS was significantly attenuated in the spironolactone group and reduction tended to be attenuated in the bisoprolol group while imidapril had no beneficial effect (Table 2). Although LV end-diastolic volume (LVEDV) was higher in all treated groups compared with placebo, only bisoprolol- and spironolactone-treated groups had improved LVSV and LV end-diastolic diameter compared with placebo (Table 2). Consistent with cardiac dysfunction, AH-130 rats displayed a transient up-regulation of the cardiac stress marker brain natriuretic peptide (see Supplementary material online, Figure S4).

View this table:
Table 2

Impact of treatment in tumour rats on cardiac dimension and function

ShamPlaceboBisoprolol 5 mg/kg/daySpironolactone 50 mg/kg/dayImidapril 0.4 mg/kg/day
n1673231610
LVEF, %73 ± 2***52 ± 257 ± 466 ± 4**46 ± 6
LVFS, %52 ± 2***31 ± 235 ± 343 ± 3**30 ± 4
LVEDD, mm6.4 ± 0.1***5.7 ± 0.16.2 ± 0.3*6.3 ± 0.2*5.7 ± 0.3
LVESD, mm3.1 ± 0.1***3.9 ± 0.14.1 ± 0.23.6 ± 0.23.9 ± 0.2
LVEDV, µL269 ± 10***196 ± 10262 ± 31**268 ± 19**237 ± 24
LVSV, µL196 ± 10***105 ± 7160 ± 22**179 ± 18***99 ± 28
LVCO, mL/min81 ± 5***42 ± 346 ± 970 ± 8***44 ± 11
Δ LVmass, mg+110 ± 29***−101 ± 14−5 ± 31**+38 ± 12***−159 ± 57
Heart, mg770 ± 12506 ± 8573 ± 30**540 ± 23561 ± 54
HR, b.p.m.413 ± 7375 ± 13266 ± 22***384 ± 7332 ± 22
  • LVEF, left-ventricular ejection fraction; LVFS, left-ventricular fractional shortening; LVEDD, left-ventricular end-diastolic diameter; LVESD, left-ventricular end-systolic diameter; LVEDV, left-ventricular end-diastolic volume; LVSV, left-ventricular stroke volume; LVCO, left-ventricular cardiac output; LVmass, left-ventricular mass; HR, heart rate.

  • *P < 0.05, **P < 0.01, ***P < 0.001 vs. placebo.

Most importantly, the loss of LVmass was abrogated in rats treated with either bisoprolol or spironolactone, whereas imidapril had no significant effect on LV atrophy (Table 2). In the placebo-treated group, a loss of 21 ± 2% LVmass was observed on Day 11, while the LVmass was stabilized by bisoprolol (+2 ± 8%, P < 0.0001) and increased by spironolactone (+9 ± 3%, P < 0.0001). Imidapril had no effect (Table 2).

Coincident with the cardiac wasting, troponin-I and -T levels in plasma increased, suggesting ongoing cardiomyocyte necrosis (see Supplementary material online, Figure S4). Furthermore, apoptosis was increased as indicated by a higher activity of caspase-3 (see Supplementary material online, Figure S6). Bisoprolol and spironolactone reduced cachexia-induced activation of caspase-3 in cardiac tissue (Figure 3A). Enhanced activation of the UPS, as indicated by enzyme kinetic assessment in cardiac tissue of AH-130 rats, was observed on Day 11 (see Supplementary material online, Figure S6). Decreased trypsin-like activity of the UPS was seen in bisoprolol and spironolactone-treated animals (Figure 3B). In contrast, the peptidyl–glutamyl–protein-hydrolysing activity was increased by imidapril compared with placebo, and imidapril also increased chymotrypsin-like activity of the proteasome (Figure 3B). Imidapril significantly induced cardiac expression of FOXO3 (Figure 3C) in CC rats, while MuRF-1 was non-significantly induced (P = 0.07; Figure 3C), which may have contributed to the UPS effect. MAFbx was induced by CC and not regulated by treatment. In skeletal muscle, bisoprolol and spironolactone also reduced UPS activity, while imidapril only had a weak effect (see Supplementary material online, Figure S7). Although autophagy was recruited (see Supplementary material online, Figure S6), based on decreased expression of p62 and a change in the LC-3II to LC-3I (microtubule-associated proteins 1A/1B light chain 3A) ratio, autophagy was not differentially affected by any of the drug treatments (Figure 3C).

Figure 3

(A) Spironolactone reduced activity of caspase-3. (B) A lower trypsin-like activity of the proteasome was seen in groups treated with bisoprolol or spironolactone, while imidapril enhanced proteasome activity. (C) Imidapril-induced expression of FOXO3 and also its phosphorylation, possibly contributing to increased expression of MuRF-1. MAFbx is induced by cancer cachexia, and not regulated by bisoprolol or spironolactone. While there was no change in the reduction of p62 by treatment, an increase in LC-3 II /LC-3 I ratio was observed in the imidapril-treated group, suggesting increased autophagy. (D) Cancer cachexia caused loss of myosin heavy chain in myocardium, which was prevented by bisoprolol and spironolactone, while imidapril had no effect. Toponin-T was only reduced in the imidapril group and tropomyosin was not affected by cancer cachexia. ND, not done due to lack of material. *P < 0.05, **P < 0.01, ***P < 0.001 vs. placebo.

Bisoprolol and spironolactone preserve sarcomeric myosin heavy chain isoforms in the heart of cachectic rats

In skeletal muscle, cachexia alters the composition of the sarcomere by predominantly leading to loss of sarcomeric myosin heavy chain (MHC) protein while troponin and tropomyosin are less affected.17 Similarly, in the heart, we observed a predominant loss of MHC protein in tumour-bearing animals (Figure 3D) that was associated with a decrease in α-MHC and β-MHC mRNA levels (Figure 3D). Spironolactone and bisoprolol prevented the decrease in MHC protein and in α-MHC and β-MHC mRNA (Figure 3D). In contrast, imidapril failed to prevent the decrease in MHC protein. Similar to findings in skeletal muscle, there was no loss of tropomyosin and only a minor loss of troponin in the hearts of cachectic rats (Figure 3D).

Sprionolactone, bisoprolol, and imidapril differentially modulate anabolic and catabolic pathways in hearts of tumour-bearing rats

Tumour-bearing AH-130 rats demonstrated a marked reduction in anabolic signalling as indicated by a reduced expression of the insulin and insulin-like growth factor (IGF-1) receptors and reduced phosphorylation of key downstream targets, most importantly, protein kinase B (Akt). This, plus the resulting reduction in phosphorylation of glycogen synthase kinase 3α (GSK-3α) and GSK-3β, can be expected to lead to a marked reduction in activity of the critical regulator of protein synthesis, mammalian target of rapamycin complex 1 (mTORC1).18 Consistent with this, reduced phosphorylation of the direct mTORC1 target, eukaryotic initiation factor 4E binding protein-1 (4E-BP1), was observed (see Supplementary material online, Figure S8, Figure 4A). These data demonstrate a clear-cut reduction in the activity of this key anabolic pathway in the heart. Furthermore, when mTORC1 activity is reduced (as in the setting of energy stress) the degradative pathway of autophagy is typically recruited to attempt to restore homestasis. Remarkably, treatment with either bisoprolol or spironolactone restored phosphorylation of Akt, GSK-3α, and GSK-3β and at least spironolactone augmented mTORC1 activity as evidenced by increased 4E-BP1phosphorylation (Figure 4A). In contrast, imidapril up-regulated the expression of Akt protein without increasing the phosphorylation status of Akt and failed to restore mTORC1 activity (Figure 4A). Moreover, imidapril-treated rats displayed a marked up-regulation in expression of GSK-3α and, to a lesser extent, GSK-3β but with only a slight increase in phosphorylated (i.e. inhibited) GSK-3α (Figure 4A). This was associated with increased 5′ adenosine monophosphate-activated protein kinase (AMPK) (Figure 4A) which, together with increased expression and minimal phosphorylation of GSK-3s would lead to mTOR inhibition. In summary, signalling in CC is characterized by a profound catabolic profile involving multiple pathways that is partially rescued by bisoprolol and spironolactone.

Figure 4

Effect of treatment on signalling. (A) Anabolism in the hearts of tumour-bearing rats was increased by treatment with bisoprolol or spironolactone, which can be seen in the higher Akt phosphorylation, while imidapril failed to excerpt positive effects on anabolic signalling. (B) The negative growth regulator myostatin was induced in tumour-bearing animals and its levels were reduced to normal by bisoprolol or spironolactone treatment. *P < 0.05, **P < 0.01, ***P < 0.001 vs. placebo.

Finally, we found increased expression of the negative regulator of muscle growth, myostatin, in the myocardium of AH-130 rats (see Supplementary material online, Figure S8, Figure 4B). Myostatin was up-regulated 3.1-fold in AH-30 rats compared with sham (Figure 4B). Both bisoprolol and spironolactone reduced myostatin expression to sham levels (Figure 4B).

Effects of bisoprolol or spironolactone on GSK3α/β and α- and β-MHC regulation in cardiomyocytes in vitro

In order to determine whether the beneficial effects of the interventions on cardiac wasting were due to a direct or indirect effect on cardiomyocytes, we used isolated neonatal rat cardiomyocytes in culture (NRCM), which were serum-starved for 5 days prior to stimulation. Isoproterenol-induced hypertrophic growth of cardiomyocytes while at the same time reducing expression of α- and β-MHC mRNA (−83.0 ± 0.1 and −81.2 ± 0.1%, respectively, both P < 0.01), which was attenuated by bisoprolol and metoprolol (Figure 5A). In contrast, neither aldosterone nor spironolactone altered α- or β-MHC mRNA expression or GSK3α/β phosphorylation in cardiomyocytes under serum-starvation in vitro suggesting that it may be acting indirectly via effects on non-cardiomyocytes such as fibroblasts.19,20 Furthermore, bisoprolol-enhanced phosphorylation (=inhibition) of GSK3α/β in control and in isoproterenol stimulated NRCM (+29 ± 16%, vs. control, P = 0.03; Figure 5B). This suggests a direct role for the β-1 adrenoreceptor in the loss of sarcomeric proteins under cachectic conditions.

Figure 5

(A) Incubation of neonatal rat cardiomyocytes with isoproterenol (Iso) reduced mRNA expression of α- and β-MHC, which was rescued by bisoprolol (Biso), metoprolol (Meto), or the GSK3-inhibitor SB 425286. Aldosterone (Aldo) and spironolactone (Spiro) had no effect on α- and β-MHC-expression. (B) The ratio of phospho-GSK3 to GSK3 was somewhat shifted to pGSK by bisoprolol and to a lesser extent by metoprolol.

To address this issue, we first quantified aldosterone levels in blood taken from surviving cachectic animals at termination of the study and found them to be significantly elevated (Figure 6A). We also found progressive cardiac fibrosis (Figure 6B). In the intervention study, the increase in aldosterone levels in cachectic rats was even more pronounced, and was attenuated by bisoprolol and spironolactone (Figure 6C). Cortisol was increased in cachectic rats and was significantly reduced by spironolactone alone (Figure 6C). Interestingly, the weight of the adrenal glands was increased in tumour-bearing animals and significantly decreased by bisoprolol and spironolactone (Figure 6C). Renin plasma concentration was increased by CC, but no significant drug effect was observed (Figure 6C), suggesting that aldosterone is induced by both renin–angiotensin-system (RAS)-dependent and -independent mechanisms. Cancer cachexia also increased plasma noradrenalin levels, and these were reduced in the spironolactone group (Figure 6D). In the intervention study, fibrosis was quantified by measuring hydroxyproline (HP) levels in the tumour-bearing rats. We found that fibrosis was significantly increased in untreated rats but this was reduced by spironolactone treatment (Figure 6E). Thus CC-induced fibrosis is indeed driven, at least in part, by aldosterone.

Figure 6

(A) Rat aldosterone (aldo) plasma level in sham (open bars) and tumour-bearing animals (solid bars). (B) Cardiac fibrosis is occurring as early as 7 days after tumour inoculation. Scale bar: 100 µm. (C) Aldosterone and cortisol plasma levels were elevated in rats with cancer cachexia (reduced by spironolactone and to a lesser extent by bisoprolol), possible by both RAS-dependent and -independent mechanisms, as suggested by adrenal gland hypertrophy and evaluated renin levels. (D) Noradrenalin was induced by cancer cachexia, which was reduced by spironolactone. (E) Hydroxyproline (HP) as a marker of fibrosis, spironolactone reduced HP-expression compared with placebo. *P < 0.05, **P < 0.01, ***P < 0.001 vs. placebo cancer cachexia.

Cardiac wasting and the role of aldosterone in human cancer patients

We next asked how our findings in the rat model compared with findings in cancer patients. Patients who died as a result of CC manifested cardiac cachexia with reductions in heart weight of 25.6%, (Figure 7A) as well as reduced LV wall thickness (LVWT) (−12.1%) compared with controls who died of non-cancer-related illness. In LV-sections, we observed profound fibrosis in patients who died of cancer, irrespective of whether cachexia was present, consistent with extensive cardiac remodelling including fibrotic remodelling in both groups (Figure 7B). A second set of patients suffering from either non-small cell lung or colorectal cancer, with or without cachexia, also had significantly increased plasma levels of aldosterone (2.1-fold and 2.3-fold, respectively; Figure 7C). Plasma levels of cortisol were reduced in cancer patients but were at control levels in patients with CC. Renin was increased in cancer patients, with or without cachexia, (2.9-fold and 3.2-fold, respectively; Figure 7C). Furthermore, BNP was increased three-fold and 8.1-fold, respectively, (Figure 7D) compared with healthy controls (patient characteristics are given in Supplementary material online, Tables S2 and S3).

Figure 7

(A) Human cadaver heart weights and wall thickness (WT) of the posterior left (LV) and right ventricle (RV) wall of controls (open bar n = 11), cancer (grey bars, n = 12), and cancer cachexia (black bar, n = 14). (B) Representative sections of the LV posterior wall (top panels AZAN stain, lower panels Sirius Red stain). Fibrosis and perivascular fibrosis was seen in patients who died of cancer with or without cachexia. (C) Plasma aldosterone (aldo), cortisol, renin, and BNP levels in a second set of patients with cancer (n = 34), cancer cachexia (n = 20), and controls (n = 22). Scale bar 50 µm *P < 0.05, **P < 0.01 vs. control/sham, ##P < 0.01 vs. cancer.

Discussion

Here we show that LVmass is progressively lost and cardiac function becomes increasingly impaired as CC progresses in tumour-bearing rats. This type of cardiomyopathy is distinct from heart failure due to more typical aetiologies and from other forms of cardiac cachexia, in which an increase in LV diameter is a hallmark.21,22 Importantly, we found similarities between cardiac remodelling in our rat model and findings in cancer patients since those patients who died of CC had reduced LVmass and wall thickness, thereby validating our animal model. Fibrosis was seen in patients and animals which may, at least in part, be induced in response to increased aldosterone production,23 levels of which were higher in tumour-bearing rats as well as in cancer patients. The induction of aldosterone seems to be partly mediated via RAS, as renin plasma levels were increased in rats and humans, but may also be linked to RAS-independent mechanisms, as cortisol was also induced in rats and we observed hypertrophy of the adrenal glands in CC rats. Cortisol was not increased in human CC, but this may be due to therapy with glucocorticoids, which has been known to repress endogenous cortisol production.24 Increased plasma levels of fibrinogen, TIMP-1, and MCP-1 in the rat also indicate fibrotic remodelling and inflammation, augmenting the effects of aldosterone.25 Elevation of BNP, which was observed in the rat model,26,27 was also present in cancer patients, particularly in those with CC. Moreover, the increased noradrenalin levels indicate an activation of the sympathetic nervous system and increased noradrenalin levels have also been described in human CC.28

In contrast to Xu et al.11 who observed impaired cardiac function, but no cardiac wasting in CC mice, we observed a striking 58% loss of LVmass over time after tumour inoculation. Since this was higher than the maximal overall loss of lean mass, which was 35%, the heart appears to be more susceptible to catabolic stimuli/stress. The loss of LVmass was likely due to several mechanisms. In contrast to a prior report,14 we found increased UPS activity as well as increases in apoptosis (increased caspase-3 activity), necrosis (increased troponin levels), and autophagy (increased LC-3 II/I ratio and decreased p62). These mechanisms have been implicated in CC-induced skeletal muscle wasting.29 Recently, NFκB has been implicated in CC-induced cardiac wasting and functional impairments in mice and its inhibition reversed symptoms.12,13 However, in cancer, NFκB may be important for FAS-ligand-induced apoptosis,30 and elevated levels of the endogenous NFκB-inhibitor osteoprotegerin have been associated with increased mortality, particularly in cardiovascular disease.31 Therefore, we believe that using established cardiovascular drugs to treat CC-induced cardiomyopathy is more advantageous than direct NFκB-inhibition.

In addition to induced catabolism, we found a marked down-regulation of anabolic pathways in the heart, with reduced expression of insulin and IGF-1 receptors undoubtedly contributing to the reduced phospho-Akt. We also found elevated levels of myostatin in the myocardium of tumour-bearing rats, and this likely contributed to the loss of cardiac mass. Myostatin, which is a negative regulator of muscle growth, has been reported to inhibit Akt in cardiomyocytes.32 Enhanced cardiac expression of myostatin has recently been described in a mouse heart failure model that does not involve CC, suggesting it is a more generalized regulator of cachexia.33 In any case, all of the above-noted alterations would lead to inhibition of mTORC1,18 thereby inhibiting protein synthesis and increasing autophagy. Surprisingly, given the increase in UPS activity, we saw no up-regulation of forkhead box protein O (FOXO) or muscle ring finger protein-1 (MuRF-1), while muscle atrophy F-box protein (MAFbx) was induced by CC. This is distinctly different from findings in skeletal muscle, where a down-regulation of Akt signalling leads to an activation of FOXO with subsequent increases in MuRF-1 and MAFbx-levels.34,35

In our intervention study, a beneficial, dose-dependent effect on survival was found for the β-1 adrenoreceptor-specific antagonist, bisoprolol, and for the aldosterone antagonist, spironolactone, while the ACE-inhibitor, imidapril, failed to improve survival. Bisoprolol preserved body weight, and lean and fat mass, and improved the quality of life (locomotor activity and food intake) as well as cardiac function. Most importantly, the loss of LVmass was completely prevented by bisoprolol and spironolactone.

In our studies, imidapril reduced wasting of lean and fat mass. However, no effect was seen on cardiac wasting or cardiac function in our model suggesting that skeletal and cardiac muscle differ in their response to cancer-induced activation of RAS. In untreated tumour-bearing rats, cardiac function was predictive of outcomes, making cardiac function a clinically measureable predictor of survival. These findings also identify the heart as an important therapeutic target in CC. Notably, only spironolactone improved LV function, and this may reflect both the reduced cardiac fibrosis,36,37 as well as the preservation of LVmass.

The effect of the various drugs on cardiac function and outcome correlated with the signalling pathways regulating the anabolic/catabolic balance in the myocardium. Akt phosphorylation was normalized by bisoprolol and spironolactone, but not by imidapril. As for the role of aldosterone, spironolactone-induced Akt/mTORC1 signalling has been reported to inhibit myostatin signalling,38 and our findings are consistent with that.

Imidapril-induced key negative regulators of protein synthesis- total 4E-BP1, AMPK, and GSK-3, the latter profoundly so, while these negative regulators were not affected or were reduced by bisoprolol and spironolactone.

The intervention study is somewhat limited by a lack of histological analysis of the effect of drug treatment on the remodelling processes. However, biochemically we clearly showed distinct effects of each drug, which are in accordance with functional improvements of the heart and reduced mortality. Also, we were unable to measure blood pressure at the end of the intervention study, because the rats were terminally ill and hence deemed too sick to be catheterized under anaesthesia. Plasma levels of inflammatory cytokines were not assessed in our study and levels may have been altered by drug treatment. However, the focus of our study was on the effects of sympathetic activation and the RAAS—key players in heart failure—in the context of CC-induced cardiomyopathy.

In conclusion, CC drives wasting of the myocardium resulting in cardiac hypotrophy, distinguishing this form of heart failure from ‘classical’ heart failure with LV hypertrophy and dilatation. The heart is more susceptible to catabolic stimuli than skeletal muscle. Blockade of the mineralocorticoid receptor or sympathetic blockade can improve outcomes by enhancing anabolic signalling in the heart. In contrast ACE-inhibition is ineffective. Based on our findings, we suggest that clinical trials should be undertaken in this desperately ill patient population with very few viable treatment options.

Funding

J.M.A. was supported by grant SAF 26091-2011 from the Ministry of Science and Innovation (Spain), T.F. by HL091799 from NIH and the Scarperi family, F.J. by grant ANR-09-EBIO-024-01from the Agence Nationale pour la Recherche (France), D.H. by the Fondation Leducq and the HILF grant of Medical School Hannover, T.T. by grants of the German Ministry for Education and Research (IFB-Tx toTT, 01EO0802).

Conflict of interest: S.D.A., A.J.S.C., and J.S. received research grants from and are consultants for PsiOxus Therapeutics Ltd, S.D.A. and A.J.S.C. are shareholders of PsiOxus Therapeutics Ltd, A.J.S.C. receives honoraria from CSL Biotherapies, J.B. is a shareholder, employee, and Board Director of PsiOxus Therapeutics Ltd, T.F. received research support from Glaxo-Smith-Kline, B.H. is an employee of Immundiagnostik AG. All other authors declared no conflicts of interest.

References

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