Links between muscle phenotype and life history: differentiation of myosin heavy chain composition and muscle biochemistry in precocial and altricial pinniped pups.

In marine mammals, muscular development has been identified as a rate-limiting factor in achieving adult dive capacities. This study investigates the rate that myosin heavy chain (MHC) composition matures in a postural and locomotor skeletal muscle for four pinniped species with different lactation lengths: hooded seals, Cystophora cristata; harp seals, Pagophilus groenlandicus; northern fur seals, Callorhinus ursinus, and Steller sea lions, Eumetopias jubatus. The ontogeny of MHC isoform expression was compared with developmental rates of myoglobin concentrations, and aerobic (citrate synthase, ß-hydroxyacyl-CoA dehydrogenase) and anaerobic (lactate dehydrogenase) enzyme activities. Within taxonomic families, species with shorter lactation periods had more mature muscles biochemically at birth, and fiber types differentiated earlier during ontogeny (Phocidae: hooded > harp seals, Otariidae: northern fur seals > Steller sea lions). Northern fur seal neonates had the most phenotypically-mature muscles in this study, with no immature MHC isoforms. The relationship between muscle biochemistry and MHC composition became more pronounced with age, and developed to reflect swimming mode and activity levels. In adults, phocids had more slow-twitch oxidative protein in their primary locomotor muscle, the Longissimus dorsi (LD), than otariids which likely reflects oxygen-sparing strategies for the phocids' longer dives. Conversely, northern fur seal muscles had higher proportions of fast-twitch MHCs in the Pectoralis and LD, likely indicative of this species' smaller size and higher mass-specific metabolic rates. Thus, muscle phenotype is linked with species life history, and a mismatch between muscle biochemistry and MHC composition at weaning has important implications for the first year of independent foraging in pinniped pups.

Modeling Human Cancer-induced Cachexia.

Cachexia is a wasting syndrome characterized by pronounced skeletal muscle loss. In cancer, cachexia is associated with increased morbidity and mortality and decreased treatment tolerance. Although advances have been made in understanding the mechanisms of cachexia, translating these advances to the clinic has been challenging. One reason for this shortcoming may be the current animal models, which fail to fully recapitulate the etiology of human cancer-induced tissue wasting. Because pancreatic ductal adenocarcinoma (PDA) presents with a high incidence of cachexia, we engineered a mouse model of PDA that we named KPP. KPP mice, similar to PDA patients, progressively lose skeletal and adipose mass as a consequence of their tumors. In addition, KPP muscles exhibit a similar gene ontology as cachectic patients. We envision that the KPP model will be a useful resource for advancing our mechanistic understanding and ability to treat cancer cachexia.

Increased hypoxia-inducible factor-1α in striated muscle of tumor-bearing mice.

Cancer cachexia is a progressive wasting disease resulting in significant effects on the quality of life and high mortality. Most studies on cancer cachexia have focused on skeletal muscle; however, the heart is now recognized as a major site of cachexia-related effects. To elucidate possible mechanisms, a proteomic study was performed on the left ventricles of colon-26 (C26) adenocarcinoma tumor-bearing mice. The results revealed several changes in proteins involved in metabolism. An integrated pathway analysis of the results revealed a common mediator in hypoxia-inducible factor-1α (HIF-1α). Work by other laboratories has shown that extensive metabolic restructuring in the C26 mouse model causes changes in gene expression that may be affected directly by HIF-1α, such as glucose metabolic genes. M-mode echocardiography showed progressive decline in heart function by day 19, exhibited by significantly decreased ejection fraction and fractional shortening, along with posterior wall thickness. Using Western blot analysis, we confirmed that HIF-1α is significantly upregulated in the heart, whereas there were no changes in its regulatory proteins, prolyl hydroxylase domain-containing protein 2 (PHD2) and von Hippel-Lindau protein (VHL). PHD2 requires both oxygen and iron as cofactors for the hydroxylation of HIF-1α, marking it for ubiquination via VHL and subsequent destruction by the proteasome complex. We examined venous blood gas values in the tumor-bearing mice and found significantly lower oxygen concentration compared with control animals in the third week after tumor inoculation. We also examined select skeletal muscles to determine whether they are similarly affected. In the diaphragm, extensor digitorum longus, and soleus, we found significantly increased HIF-1α in tumor-bearing mice, indicating a hypoxic response, not only in the heart, but also in skeletal muscle. These results indicate that HIF-1α may contribute, in part, to the metabolic changes that occur during cancer cachexia.NEW & NOTEWORTHY We used proteomics and metadata analysis software to identify contributors to metabolic changes in striated muscle during cancer cachexia. We found increased expression of hypoxia-inducible factor-1α in the heart and skeletal muscle, suggesting a potential target for the therapeutic treatment of cancer cachexia.

Ibuprofen ameliorates fatigue- and depressive-like behavior in tumor-bearing mice.

AIMS: Cancer-related fatigue (CRF) is often accompanied by depressed mood, both of which reduce functional status and quality of life. Research suggests that increased expression of pro-inflammatory cytokines is associated with skeletal muscle wasting and depressive- and fatigue-like behaviors in rodents and cancer patients. We have previously shown that treatment with ibuprofen, a nonsteroidal anti-inflammatory drug, preserved muscle mass in tumor-bearing mice. Therefore, the purpose of the present study was to determine the behavioral effects of ibuprofen in a mouse model of CRF. MAIN METHODS: Mice were injected with colon-26 adenocarcinoma cells and treated with ibuprofen (10mg/kg) in the drinking water. Depressive-like behavior was determined using the forced swim test (FST). Fatigue-like behaviors were determined using voluntary wheel running activity (VWRA) and grip strength. The hippocampus, gastrocnemius muscle, and serum were collected for cytokine analysis. KEY FINDINGS: Tumor-bearing mice showed depressive-like behavior in the FST, which was not observed in mice treated with ibuprofen. VWRA and grip strength declined in tumor-bearing mice, and ibuprofen attenuated this decline. Tumor-bearing mice had decreased gastrocnemius muscle mass and increased expression of IL-6, MAFBx and MuRF mRNA, biomarkers of protein degradation, in the muscle. Expression of IL-1ß and IL-6 was also increased in the hippocampus. Treatment with ibuprofen improved muscle mass and reduced cytokine expression in both the muscle and hippocampus of tumor-bearing mice. SIGNIFICANCE: Ibuprofen treatment reduced skeletal muscle wasting, inflammation in the brain, and fatigue- and depressive-like behavior in tumor-bearing mice. Therefore, ibuprofen warrants evaluation as an adjuvant treatment for CRF.

Metalloproteinase expression is altered in cardiac and skeletal muscle in cancer cachexia.

Cardiac and skeletal muscle dysfunction is a recognized effect of cancer-induced cachexia, with alterations in heart function leading to heart failure and negatively impacting patient morbidity. Cachexia is a complex and multifaceted disease state with several potential contributors to cardiac and skeletal muscle dysfunction. Matrix metalloproteinases (MMPs) are a family of enzymes capable of degrading components of the extracellular matrix (ECM). Changes to the ECM cause disruption both in the connections between cells at the basement membrane and in cell-to-cell interactions. In the present study, we used a murine model of C26 adenocarcinoma-induced cancer cachexia to determine changes in MMP gene and protein expression in cardiac and skeletal muscle. We analyzed MMP-2, MMP-3, MMP-9, and MMP-14 as they have been shown to contribute to both cardiac and skeletal muscle ECM changes and, thereby, to pathology in models of heart failure and muscular dystrophy. In our model, cardiac and skeletal muscles showed a significant increase in RNA and protein levels of several MMPs and tissue inhibitors of metalloproteinases. Cardiac muscle showed significant protein increases in MMP-2, MMP-3, MMP-9, and MMP-14, whereas skeletal muscles showed increases in MMP-2, MMP-3, and MMP-14. Furthermore, collagen deposition was increased after C26 adenocarcinoma-induced cancer cachexia as indicated by an increased left ventricular picrosirius red-positive-stained area. Increases in serum hydroxyproline suggest increased collagen turnover, implicating skeletal muscle remodeling. Our findings demonstrate that cancer cachexia-associated matrix remodeling results in cardiac fibrosis and possible skeletal muscle remodeling. With these findings, MMPs represent a possible therapeutic target for the treatment of cancer-induced cachexia.

Losartan treatment attenuates tumor-induced myocardial dysfunction.

UNLABELLED: Fatigue and muscle wasting are common symptoms experienced by cancer patients. Data from animal models demonstrate that angiotensin is involved in tumor-induced muscle wasting, and that tumor growth can independently affect myocardial function, which could contribute to fatigue in cancer patients. In clinical studies, inhibitors of angiotensin converting enzyme (ACE) can prevent the development of chemotherapy-induced cardiovascular dysfunction, suggesting a mechanistic role for the renin-angiotensin-aldosterone system (RAAS). In the present study, we investigated whether an angiotensin (AT) 1-receptor antagonist could prevent the development of tumor-associated myocardial dysfunction. METHODS AND RESULTS: Colon26 adenocarcinoma (c26) cells were implanted into female CD2F1 mice at 8weeks of age. Simultaneously, mice were administered Losartan (10mg/kg) daily via their drinking water. In vivo echocardiography, blood pressure, in vitro cardiomyocyte function, cell proliferation assays, and measures of systemic inflammation and myocardial protein degradation were performed 19days following tumor cell injection. Losartan treatment prevented tumor-induced loss of muscle mass and in vitro c26 cell proliferation, decreased tumor weight, and attenuated myocardial expression of interleukin-6. Furthermore, Losartan treatment mitigated tumor-associated alterations in calcium signaling in cardiomyocytes, which was associated with improved myocyte contraction velocity, systolic function, and blood pressures in the hearts of tumor-bearing mice. CONCLUSIONS: These data suggest that Losartan may mitigate tumor-induced myocardial dysfunction and inflammation.

Fluoxetine prevents the development of depressive-like behavior in a mouse model of cancer related fatigue.

Cancer patients frequently suffer from fatigue, a complex syndrome associated with tiredness and depressed mood. Cancer-related fatigue (CRF) can be present at the time of diagnosis, escalates during treatment, and can persist for years after treatment. CRF negatively influences quality of life, limits functional independence, and is associated with decreased survival in patients with incurable disease. We have previously shown that increased pro-inflammatory cytokine expression in the brain contributes to depressive- and fatigue-like behaviors in a mouse model of CRF. Inflammatory cytokines increase the activity of indoleamine 2,3-dioxygenase (IDO) and kynurenine 3-monooxygenase (KMO), which competitively reduce serotonin synthesis. Reduced serotonin availability in the brain and increased production of alternative neuroactive metabolites of tryptophan are thought to contribute to the development of depression and fatigue. The purpose of this study was to determine the effects of fluoxetine, a selective serotonin reuptake inhibitor (SSRI), on brain cytokines and behavioral measures of fatigue and depression in tumor-bearing mice. Here we show that tumor growth increased brain expression of pro-inflammatory cytokines and KMO. Treatment with fluoxetine had no effect on tumor growth, muscle wasting, fatigue behavior, or cytokine expression in the brain. Fluoxetine, however, reduced depressive-like behaviors in tumor bearing mice. In conclusion, our data confirm that increased brain expression of pro-inflammatory cytokines is associated with tumor-induced fatigue- and depressive-like behaviors. However, it is possible to separate the effects of tumor growth on mood and fatigue-like behaviors using SSRIs such as fluoxetine.

Tumor growth increases neuroinflammation, fatigue and depressive-like behavior prior to alterations in muscle function.

Cancer patients frequently suffer from fatigue, a complex syndrome associated with loss of muscle mass, weakness, and depressed mood. Cancer-related fatigue (CRF) can be present at the time of diagnosis, during treatment, and persists for years after treatment. CRF negatively influences quality of life, limits functional independence, and is associated with decreased survival in patients with incurable disease. Currently there are no effective treatments to reduce CRF. The aim of this study was to use a mouse model of tumor growth and discriminate between two main components of fatigue: loss of muscle mass/function and altered mood/motivation. Here we show that tumor growth increased fatigue- and depressive-like behaviors, and reduced body and muscle mass. Decreased voluntary wheel running activity (VWRA) and increased depressive-like behavior in the forced swim and sucrose preference tests were evident in tumor-bearing mice within the first two weeks of tumor growth and preceded the loss of body and muscle mass. At three weeks, tumor-bearing mice had reduced grip strength but this was not associated with altered expression of myosin isoforms or impaired contractile properties of muscles. These increases in fatigue and depressive-like behaviors were paralleled by increased expression of IL-1ß mRNA in the cortex and hippocampus. Minocycline administration reduced tumor-induced expression of IL-1ß in the brain, reduced depressive-like behavior, and improved grip strength without altering muscle mass. Taken together, these results indicate that neuroinflammation and depressed mood, rather than muscle wasting, contribute to decreased voluntary activity and precede major changes in muscle contractile properties with tumor growth.

Patterns of tropomyosin and troponin-T isoform expression in jaw-closing muscles of mammals and reptiles that express masticatory myosin.

We recently reported that masticatory ('superfast') myosin is expressed in jaw-closing muscles of some rodent species. Most mammalian limb muscle fibers express tropomyosin-ß (Tm-ß), along with fast-type or slow-type tropomyosin-ß (Tm-ß), but jaw-closing muscle fibers in members of Carnivora express a unique isoform of Tm [Tm-masticatory (Tm-M)] and little or no Tm-ß. The goal of this study was to determine patterns of Tm and troponin-T (TnT) isoform expression in the jaw-closing muscles of rodents and other vertebrate species that express masticatory myosin, and compare the results to those from members of Carnivora. Comparisons of electrophoretic mobility, immunoblotting and mass spectrometry were used to probe the Tm and fast-type TnT isoform composition of jaw-closing and limb muscles of six species of Carnivora, eight species of Rodentia, five species of Marsupialia, big brown bat, long-tailed macaque and six species of Reptilia. Extensive heterogeneity exists in Tm and TnT isoform expression in jaw-closing muscles between phylogenetic groups, but there are fairly consistent patterns within each group. We propose that the differences in Tm and TnT isoform expression patterns between phylogenetic groups, which share the expression of masticatory myosin, may impart fundamental differences in thin-filament-mediated muscle activation to accommodate markedly different feeding styles that may require high force generation in some species (e.g. many members of Carnivora) and high speed in others (e.g. Rodentia).

Cardiac alterations in cancer-induced cachexia in mice.

Cachexia is a common syndrome in advanced cancer patients and causes up to 22% of cancer-related deaths. It remains elusive whether cancer cachexia causes heart failure. We investigated the effect of cancer cachexia on heart function and cardiac muscle structure in a mouse model. Male CD2F1 mice were inoculated with either colon-26 adenocarcinoma cells (Tumor group) or vehicle (PBS) (No Tumor group and Pair-fed group). Heart function as measured by fractional shortening in vivo using transthoracic echocardiography was performed on day 14 after tumor or PBS inoculation. At necropsy (day 17), hearts were collected for histology, transmission electron microscopy, RT-PCR and SDS-PAGE analysis. Mice from the Tumor group displayed a significantly reduced fractional shortening compared to mice in the No Tumor and Pair-fed groups. In hearts of the Tumor mice compared to the other groups, there was marked fibrosis and transmission electron microscopy revealed disrupted myocardial ultrastructure. Gene expression of troponin I, a regulator of cardiac muscle contraction, was reduced. Moreover, both mRNA and protein levels of myosin heavy chain (MHC) were altered whereby MHCalpha (adult isoform) was decreased and MHCbeta (fetal isoform) was increased indicating reactivation of the fetal gene expression pattern. In conclusion, heart function was diminished in mice with tumor-induced cachexia, and this impaired function was associated with increased fibrosis, disrupted myocardial structure and altered composition of contractile proteins of cardiac muscle.

Cancer cachexia is regulated by selective targeting of skeletal muscle gene products.

Cachexia is a syndrome characterized by wasting of skeletal muscle and contributes to nearly one-third of all cancer deaths. Cytokines and tumor factors mediate wasting by suppressing muscle gene products, but exactly which products are targeted by these cachectic factors is not well understood. Because of their functional relevance to muscle architecture, such targets are presumed to represent myofibrillar proteins, but whether these proteins are regulated in a general or a selective manner is also unclear. Here we demonstrate, using in vitro and in vivo models of muscle wasting, that cachectic factors are remarkably selective in targeting myosin heavy chain. In myotubes and mouse muscles, TNF-alpha plus IFN-gamma strongly reduced myosin expression through an RNA-dependent mechanism. Likewise, colon-26 tumors in mice caused the selective reduction of this myofibrillar protein, and this reduction correlated with wasting. Under these conditions, however, loss of myosin was associated with the ubiquitin-dependent proteasome pathway, which suggests that mechanisms used to regulate the expression of muscle proteins may be cachectic factor specific. These results shed new light on cancer cachexia by revealing that wasting does not result from a general downregulation of muscle proteins but rather is highly selective as to which proteins are targeted during the wasting state.

Degradation of rat cardiac troponin I during ischemia independent of reperfusion.

Cardiac troponin I (cTnI) degradation has been noted in the stunned myocardium of rodents after ischemia and reperfusion and is one proposed mechanism for the decreased left ventricular (LV) contractility in postischemic hearts. cTnI degradation has been best described after reperfusion of the ischemic myocardium. The effect of ischemia, independent of reperfusion, on cTnI breakdown has not been well characterized. We tested the hypothesis that progressive cTnI degradation occurs with increasing durations of ischemia and that this ischemia-based degradation is, in part, oxidant mediated. Isolated perfused rat hearts underwent global ischemia of 15, 20, or 25 min with and without reperfusion. A second series of hearts was treated with the antioxidants tiron (10 mM) and N-acetylcysteine (4 mM) before 20 min of global ischemia without reperfusion. cTnI degradation was measured using a cTnI-specific antibody and Western blot analyses. A progressive increase in cTnI degradation was seen with increasing duration of ischemia (no reperfusion), which correlated with the return of LV developed pressure during reperfusion. The extent of cTnI degradation was increased in hearts pretreated with antioxidants, although the qualitative degradation pattern was not altered. We conclude that a time-dependent cTnI breakdown occurs during global ischemia that is independent of reperfusion. cTnI breakdown during ischemia is further increased in the presence of antioxidants, suggesting ROS generated during ischemia may play a cTnI protective role.

Gender differences in myosin heavy chain-beta and phosphorylated phospholamban in diabetic rat hearts.

The objective of this study was to determine whether a gender difference exists in myosin heavy chain (MHC) isoform or sarcoplasmic reticulum protein levels in diabetic rat hearts. As is the case with normal rodent hearts, all four chambers of the control rat hearts expressed almost 100% MHC-alpha. In 6-wk diabetic rats, MHC-beta expression in ventricles of males was significantly greater (78 +/- 7%) than in females (50 +/- 5%). The cardiac sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a) protein level was decreased and the phospholamban (PLB) protein level was increased in the left ventricle of diabetic rats, but there was no difference between male and female diabetic rats. The phosphorylated PLB level was decreased more in male than in female diabetic rats. Insulin treatment completely normalized blood glucose level, cardiac SERCA2a and PLB protein levels, and the decrease in MHC-beta levels in both male and female diabetic rats. Insulin treatment completely normalized serum insulin and almost completely normalized phosphorylation of PLB at serine 16 in male diabetic rats. Although insulin treatment completely normalized serum insulin levels in male diabetic rats, in females it only partially normalized serum insulin levels. Also, insulin treatment almost completely normalized phosphorylation of PLB at threonine 17 in female diabetic rats; however, the increase was significantly greater than that identified for insulin-treated male diabetic rats. We conclude that higher levels of MHC-beta and dephosphorylated PLB may contribute to more contractile dysfunction in male than in female diabetic rat hearts, and that phosphorylation of PLB at threonine 17 is more responsive to insulin in female diabetic rat hearts.

Effects of peroxynitrite on isolated cardiac trabeculae: selective impact on myofibrillar energetic controllers.

Formation of peroxynitrite and cardiac protein nitration have been implicated in multiple cardiac disease states, but their contributions to disease initiation remain undefined. We have previously observed nitration of myofibrillar regions of cardiac myocytes in several experimental and clinical settings of cardiac myocyte dysfunction and postulated that oxidative insult to key components of the contractile apparatus may be initiating events. Here we tested the hypothesis that peroxynitrite alters myofibrillar contractile function, and investigated a mechanistic role for nitration in this process. Isolated rat ventricular trabeculae were exposed to physiologically relevant concentrations of peroxynitrite and ATP-dependent contractile responses were measured. Maximal trabecular force generation was significantly impaired following 300 nM peroxynitrite exposures. Several myofibrillar proteins demonstrated increased tyrosine nitration, the most significant increases occurred in the myosin heavy chain and the myofibrillar isoform of creatine kinase. Additional functional experiments were conducted using phosphocreatine (high energy phosphate substrate for myofibrillar creatine kinase) as the primary energy substrate. Myofibrillar creatine kinase-dependent force generation was impaired at peroxynitrite concentrations as low as 50 nM, suggesting potent inactivation of the enzyme. Extent of tyrosine nitration of myofibrillar creatine kinase was negatively correlated to myofibrillar creatine kinase-dependent force generation. These data demonstrate that the cardiac contractile apparatus is highly sensitive to peroxynitrite, and that MM-CK may be a uniquely vulnerable target.

Intracellular distribution of peroxynitrite during doxorubicin cardiomyopathy: evidence for selective impairment of myofibrillar creatine kinase.

Cardiac peroxynitrite and protein nitration are increased during doxorubicin cardiotoxicity, but the intracellular targets and functional consequences have not been defined. We investigated the intracellular distribution of protein nitration during doxorubicin cardiotoxicity in mice. Following in vivo cardiac function assessments by echocardiography, cardiac tissues were prepared for immunohistochemistry and electron microscopy 5 days after doxorubicin (20 mg kg(-1)) or vehicle control. Increased cardiac 3-nitrotyrosine was observed using light microscopy in doxorubicin treated animals. Immunogold electron microscopy (55,000x) revealed increased myofibrillar and mitochondrial 3-nitrotyrosine levels following doxorubicin, but cellular 3-nitrotyrosine density was 2 fold higher in myofibrils. We therefore investigated the actions of peroxynitrite on intact cardiac contractile apparatus. Skinned ventricular trabeculae were exposed to physiologically relevant peroxynitrite concentrations (50 or 300 nM) for 1 h, then Ca(2+) induced contractile responses were measured in the presence of ATP (4 mM) or phosphocreatine (12 mM) as high energy phosphate supplier. ATP maximal force generation was unaltered after 50 nM peroxynitrite, but phosphocreatine/ATP response was reduced (0.99+/-0.63 vs 1.59+/-0.11), suggesting selective inactivation of myofibrillar creatine kinase (MM-CK). Reduction of ATP maximal force was observed at 300 nM peroxynitrite and phosphocreatine/ATP response was further reduced (0.64+/-0.30). Western blotting showed concentration dependent nitration of MM-CK in treated trabeculae. Similarly, cardiac tissues from doxorubicin treated mice demonstrated increased nitration and inactivation of MM-CK compared to controls. These results demonstrate that peroxynitrite-related protein nitration are mechanistic events in doxorubicin cardiomyopathy and that the cardiac myofibril is an important oxidative target in this setting. Furthermore, MM-CK may be a uniquely vulnerable target to peroxynitrite in vivo.