# The Genetic Mutation of _Ano5_ in Rabbits Recapitulates Human Cardiomyopathy
## 4 Discussion  
The high expression of the _ANOS_5 gene in human skeletal muscle, heart muscle, and bone [1; 2; 3]. Together with cardiomyopathy was reported in _ANOS_5-deficient patients [7; 23]. In our study, the _ANOS_5/- rabbits exhibited cardiomyopathy at 15 months as demonstrated by histological and functional cardiac analysis, while there is no obvious cardiomyopathy with normal thickness of the interventricular septum and cardiac functions in the _ANOS_5/- mouse [13; 14]. The 15 month old _ANOS_5/- rabbits showed cardiac changes close to _ANOS_5-deficiency patients (age 20-50) [24] and mice (age 6 months or later) [13] with the only symptoms muscular dystrophy typically appearing. Interestingly, the function of cardiac appeared abnormal by reduced left ventricular EF and FS. However, in some patients with cardiac arrhythmia [24], while the echocardiogram displayed LV dilatation and LV dysfunction as seen in other patients, but dilated cardiomyopathy may be a complication in muscles of _ANOS_5-deficient patients [7].  
There are some differences between mice and rabbits, which may trigger the performance of some aspects of the genetic disorder. The type of natural mutations is crucial for different consequences in animal models of different species. The rabbits, with a small indel in exon 12 were used to perform the  
Figure 1: **(A)** H&E staining, Masson’ trichrome, and Van Gieson staining of cardiac muscles from wild-type (WT), _ANOS_5/- and _ANOS_5/- rabbits. **(B)** Representative echocardiograms of _ANOS_5/- and WT control. The red arrows represent the position of left ventricular end-diastolic diameter (LVDD). **(C)** The left ventricular diastolic diameter increased in _ANOS_5/- rabbits. **(D)** The fractional shortening decreased in _ANOS_5/- rabbits. **(E)** The left ventricular ejection fraction (EF) decreased in _ANOS_5/- rabbits. The data is shown by means \(\pm\) SEM and was analyzed by \(t\)-tests. * \(p\) < 0.05. Normalized LVDD, The ratio of LV diastolic diameter to body weight.  
determination of cardiac pathology. The CRISPR-induced indels within the exon 12 or 13 of the _ANO5_ gene lead to the development of pathological alterations in various muscles and cardiac changes of the rabbit, resembling human patients with _ANO5_ mutations [25; 26]. In contrast, mice with exon 1 or exon 2 deletion did not develop muscle and cardiac phenotypes [14] which may illustrate the different consequences of cardiac pathology in _ANO5_ mutant models.  
To our knowledge, this novel animal model which recapitulates human cardiomyopathy will be beneficial for studying the potential impact of the disruption of _ANO5_ on cardiac changes and would also promote understanding of the pathogenesis mechanism of _ANO5_-related cardiomyopathy in the future.  
The following are available online at [[http://www.mdpi.com/2076-3417/10/14/4976/s1](http://www.mdpi.com/2076-3417/10/14/4976/s1)]([http://www.mdpi.com/2076-3417/10/14/4976/s1](http://www.mdpi.com/2076-3417/10/14/4976/s1)). Figure S1: The generation of F1 carrying ANO5 mutant rabbits; Figure S2: The histological of WT and _ANO5\({}^{-/-}\)_ at the age of 6 months.  
Conceptualization, S.T. and Z.L.; methodology, H.Y.; software, T.Z.; validation, J.L., H.Y. and L.L.; formal analysis, T.S.; investigation, T.Z.; resources, H.Y.; data curation, J.L.; writing-original draft preparation, T.S.; writing-review and editing, Z.L.; visualization, H.Y.; supervision, L.L.; project administration, Z.L.; funding acquisition, Z.L. All authors have read and agreed to the published version of the manuscript.  
This research was funded by the Changjiang Scholars and Innovative Research Team (no. IRT_16R32) and the China Postdoctoral Science Foundation (grant nos. 2019 TQ0113 and 2020M670859).  
We thank embryo manipulation technicians for the technical support.  
The authors declare no conflict of interest.