*4.1. Rodents*

Rodent models are commonly used in cardiovascular research as they are easier to handle and house (leading to manageable costs), and have a relatively short life span, allowing the researcher to follow the natural history of the disease. Additionally, of great impact is the capacity to leverage mouse genetic manipulation for both gain/loss of function of specific genes. This includes capacity for temporal control of tissue-specific genetic constructs [115].

To evaluate the development, progression, and potential for regression of DCM, multiple genetically engineered rodent models have been developed. These models include constitutive and inducible transgenic overexpression and/or gene knockout that exhibit a DCM phenotype [116,117]. One of the first DCM mouse models to be described was the muscle Lin11, Isi1 & Mec-3 (LIM) protein (MLP)-null mouse. Deletion of MLP, an actin-associated cytoskeletal protein, leads to cardiac myocyte architectural disorganization through irregularities in the actin-based cytoskeletal structure. Mice deficient for MLP show many of the anatomical and physiological hallmarks of human DCM [92]. Desmin-deficient models are also commonly used, which exhibit severe loss of myocardial architecture

by degeneration and calcification [93]. Additionally, models with mutations in mitochondria can develop DCM with atrioventricular block due to deficient oxidative phosphorylation [91].

Several non-genetic methods, including drug and surgical techniques, are also used to induce the development of DCM in rodents. Surgical techniques include interruption of coronary arteries to produce myocardial infarction through permanent coronary ligation [94] or re-perfused infarction [95]. After an infarction, the DCM phenotype progressively develops in mice. Chronic doxorubicin [96,97] or isoproterenol [98,99] administration can lead to a dose-dependent dilated phenotype and overt heart failure over time owing to severe myocardial injury and cell death. Toxic drug-mediated cardiomyopathy is characterized by myocyte apoptosis and oxidative stress being highly specific forms of injury, which may also be useful in assessing cardiac responses to stress. It should be noted, however, that although these non-genetic models recapitulate many aspects of a DCM phenotype, DMD-associated DCM has noted differences in pathophysiology compared to these models.

*Mdx* mice that lack dystrophin are the most commonly used mouse model to study DMD. However, compared to patients with DMD, they exhibit a relatively minor cardiac phenotype [100,101]. Under baseline conditions, *mdx* mice do not demonstrate physiological indicators of heart failure early in life. However, disease can be readily unmasked by cardiac stressors. In efforts to make the baseline *mdx* cardiac phenotype more similar to that of patients, *mdx* mice have been crossed with utrophin knockout (KO) mice, which exhibit a more severe cardiomyopathy [102,103] and display the physiological indicators of end-stage heart failure, including a negative force-frequency relationship and a reduction in force development and impairment of relaxation [101]. Other symptomatic double knock-out strains have been generated by mutating genes involved in: (1) the DGC complex, including δ- sarcoglycan and dystrobrevin [118,119]; (2) muscle repair, such as dysferlin [120,121]; and (3) cytoskeleton-ECM interactions, including desmin and laminin [122,123]. A thorough characterization of the cardiomyopathy in these models will increase the usefulness of these animal models for research into treatments and diagnostics for DMD cardiomyopathy.
