The Genetic Mutation of ANO5 in Rabbits Recapitulates Human Cardiomyopathy

: The limb girdle muscular dystrophy type 2L (LGMD2L) is caused by mutations of the ANO5 gene in humans which encodes a 913 amino-acid integral membrane protein. Although cardiomyopathy has been reported in patients with an ANO5 mutation, the ANO5 mutant mice did not recapitulate this phenotype in previous studies. This study demonstrated that the ANO5 − / − rabbits recapitulated the typical signs of cardiomyopathy with decreased ejection fraction (EF) and fraction shortening (FS) with increased interstitial ﬁbrosis. This ANO5 − / − rabbit model would promote basic research to comprehend the pathogenesis and mechanism of ANO5 -related cardiomyopathy.

Ten anoctamin family members have been identified so far with important functions in various physiological process [8][9][10]. Due to the structural similarity between the ANO5 gene and other anoctamin proteins, it was proposed that ANO5 may have function of Ca 2+ -activated Cl − channels (CaCC). Recently, some anoctamins, including ANO1, ANO2, ANO8, and ANO9, have been linked to the regulation of CaCC activities; however, other anoctamins, such as ANO3 and ANO7, which are short on CaCC activities, have localization in cellular activities [6,11,12]. This suggests that the anoctamin family could possess different functional properties among different family members and also that the molecular and cellular mechanisms of the ANO5 gene still remains unclear.
Previous studies have reported that ANO5 knockout (KO) mice have different pathological manifestations. The ANO5 gene trapped mouse model shows signs of muscular dystrophy [13] without any obvious cardiomyopathy [14], reminiscent of the phenotype of LGMD2L patients. Currently, there are no ideal animal models to study the cardiomyopathy phenotype reported in ANO5-mutated patients, and also the cellular functional studies of the ANO5 function in skeletal muscle and myocardium still need to be further confirmed. Moreover, rabbits may be more popular animal models compared to mice in simulating some human diseases and have higher similarity with human beings in some respects, such as genetics, physiology, and anatomy, compared to mice [15]. Therefore, the phenotype of cardiomyopathy was studied using ANO5 −/− rabbits generated in our previous study [16]. Our data demonstrate that the typical phenotype of cardiomyopathy was identified in this ANO5 −/− rabbit model for the first time.

Animal and Ethics Statement
The ANO5 gene editing rabbits were generated by the CRISPR/Cas9 system in our laboratory [16] and kept at the Jilin University Laboratory Animal Centre.

Genotyping of Rabbits
Genotyping of ANO5 KO rabbits was performed according to the manufacturer's instructions as described previously [17,18]. Briefly, the exon of the ANO5 gene was amplified by PCR and Sanger sequenced for rabbits' mutations. The obtained sequences were compared with the corresponding reference sequence and analyzed using Snapgene (NC_013669.1). The same set of PCR primer was used as in our previous study [16].

Histology Analysis
The tissue sample of cardiac muscle was collected from ANO5 +/− , ANO5 −/− , and wild-type (WT) rabbits (15 months of age). Histology analysis was performed as per a previous study [16]. The 5 µm sections for HE, Masson's trichrome, and Van Gieson staining were performed as described previously [19,20]. The pictures of stained sections were captured by a Nikon TS100 microscope.

Echocardiography
Echocardiography was carried out as described previously [21,22]. The cardiac dimensions left ventricular end-diastolic diameter (LVDd), the percentage of fractional shortening (FS), and left ventricular ejection fraction (EF) were determined.

Statistical Analysis
The data were statistically analyzed by GraphPad prism 8.0.2 (t-test), and p < 0.05 was used as statistically significant, * p < 0.05.

The Breeding and Genotyping of ANO5 Gene-Edited Rabbits
The ANO5 KO rabbits were generated in our previous study [16]. To breed ANO5 gene-edited rabbits, the heterozygous male was mated with heterozygous female ANO5 gene-edited rabbit ( Figure S1A). The genotyping PCR result showed that 4 ANO5 +/− and 6 ANO5 −/− pups were generated in this study ( Figure S1B).

Pathological Changes of Cardiac in the ANO5 −/− Rabbits
To determine whether the disruption of ANO5 in rabbits induces the typical phenotype of cardiomyopathy, the histological and functional cardiac changes were evaluated and compared between gene-edited ANO5 (ANO5 +/− and ANO5 −/− ) and WT rabbits. The results showed that the increased interstitial fibrosis was determined in ANO5 −/− rabbits, but there was no significant difference in ANO5 +/− rabbits compared to WT control at the age of 15 months ( Figure 1A); 6 month old ANO5 −/− rabbits did not show significant differences compared to WT control ( Figure S2). In addition, the echocardiography result showed the significantly increased diastolic diameter of the left ventricle (LVDD), while EF and FS decreased in the ANO5 −/− rabbits, compared to the WT controls ( Figure 1B-E).
These data suggest that the typical phenotype of cardiomyopathy was identified in the ANO5 −/− rabbit models. Appl. Sci. 2020, 10

Pathological Changes of Cardiac in the ANO5 -/-Rabbits
To determine whether the disruption of ANO5 in rabbits induces the typical phenotype of cardiomyopathy, the histological and functional cardiac changes were evaluated and compared between gene-edited ANO5 (ANO5 +/-and ANO5 -/-) and WT rabbits. The results showed that the increased interstitial fibrosis was determined in ANO5 -/-rabbits, but there was no significant difference in ANO5 +/-rabbits compared to WT control at the age of 15 months ( Figure 1A); 6 month old ANO5 -/-rabbits did not show significant differences compared to WT control ( Figure S2). In addition, the echocardiography result showed the significantly increased diastolic diameter of the left ventricle (LVDD), while EF and FS decreased in the ANO5 -/-rabbits, compared to the WT controls ( Figure 1B-E). These data suggest that the typical phenotype of cardiomyopathy was identified in the ANO5 -/-rabbit models.

Discussion
The high expression of the ANO5 gene in human skeletal muscle, heart muscle, and bone [1][2][3]. Together with cardiomyopathy was reported in ANO5-deficient patients [7,23]. In our study, the ANO5 −/− 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 ANO5 −/− mouse [13,14]. The 15 month old ANO5 −/− rabbits showed cardiac changes close to ANO5-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 ANO5-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 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.