## 2 In Vitro Models to Study CAC
### Cancer-Associated Cardiac Cachexia  
Preceding the cachexia diagnosis, cancer patients often report symptoms of fatigue and muscle weakness. However, those symptoms are aggravated by the decline in muscle mass all of which can impair the patient's quality of life [19; 60]. Cardiac abnormalities are often unaddressed pathological symptoms of CAC, they manifest as shortness of breath, pulmonary incapacity, and diminished exercise capacities, which are indicative of chronic heart failure [5]. These abnormalities are associated with enlarged oxygen consumption, thus prolonging of this occurrence increases energy expenditure. Consequently, this might result in energy inefficiency leading to a harmful energy balance and wasting, hence the designation of cardiac cachexia [11]. This occurrence is an independent predictor of increased morbidity and mortality in cancer patients [5; 27]. Cardiac wasting is mainly caused by loss of diaphragm and cardiac proteins, in part, as a result of patients' cancer treatment with cardiotoxic drugs to treat cancer or by tumour secretions that increase atrophy of cardiomyocytesocytes and affect cardiac contractility [5; 11].  
Overall, cardiac cachexia is promoted by the same mechanisms causing muscle wasting: cytokines and other pro-cache mediators secreted by the primary tumour, metastases and activated immune cells. In cardiac cachexia UPS and ALP (autophagy lysosomal pathway) are hyperactivated, but autophagy has a more preponderant role than in muscle wasting. This might justify the greater metabolic rate and protein turnover in the heart compared to skeletal muscle [5].  
Derived from embryonic BD1X rat ventricular tissue, the H9C2 cardiomyocytesoblasts cells have been extensively used as an in vitro model for skeletal and cardiac muscle [61]. The cell line can differentiate into cardiac myocytes when cultivated in a differentiation medium for 7 days supplemented with all-trans-retinoic acid (RA) increasing the expression of genes codifying proteins [62]. Their morphological features mirror immature embryonic cardiomyocytes' properties in electrophysiology, morphology (cell membrane), signalling (expression of g-signalling protein), and hormonal response [63; 64]. When stimulated with a hypertrophic agent these cells present nearly identical responses as those obtained in primary cardiomyocytes [65]. This discovery supports the relevance of H9C2 as a useful and reliable model to study cardiac hypertrophy in a molecular context and the development of the heart and diseases affecting the organ [65]. Still, the suitability of the murine cardiomyocytes model has been under scrutiny because the cell line is able to proliferate contrarily to primary cardiomyocytes. Additionally, H9C2 cells are unable to beat, unlike primary cardiac myocytes, for that limited function this line is not appropriate for contraction-based studies [65; 66]. The cells are rather easy to maintain in culture, however, since they are not immortalised, their lifespan is very limited and can only be cultured for a restricted number of passages [64]. The rise in passage number also increases the sensitivity to toxicity, and oxidative stress and the morphology of untreated cardiomyocytes also suffers alterations, meaning that cell parameters also vary [64]. It was found that the H9C2 cell line is reliable only in the first five passages. So, it is necessary to keep in mind that further passages of cardiomyocytes may not ensure the repeatability and reliability of the results because of the ageing culture [64]. The cell line is remarkably interesting in cytotoxic studies to analyse the cardiotoxicity safety of anticancer drugs namely, Doxorubicin [67], Mitoxantrone [68] and Pixantrone [69], focusing also on the mechanisms of myocyte damage and effect on cell apoptosis and necrosis.