Evaluating spiny mice (Acomys) as a model for cardiac research

Complex tissue regeneration is extremely rare among adult mammals. An exception, however, is the superior tissue healing of multiple organs in spiny mice (Acomys). While Acomys species exhibit the remarkable ability to heal complex tissue with minimal scarring, little is known about their cardiac structure and function. In this study, we characterized cardiac structure, anatomy, and in vivo function, as well as cardiomyocyte characteristics in Acomys compared to the most commonly used cardiac mouse model, the C57BL6 mouse strain (Mus). Our results demonstrate comparable cardiac anatomy, structure and function between the two rodent species, but reveal significant differences in their cardiomyocyte characteristics. These findings establish Acomys as a new mammalian model for cardiac research.


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Significant clinical advances in revascularization and medical therapies have 32 improved the mortality rate after myocardial infarction (MI). 1 Unfortunately, there are no 33 therapies to limit myocardial damage during MI and millions of patients progress to develop 34 heart failure (HF). This is because the normal healing response to tissue injury in adult 35 mammals is fibrotic repair; an effective short-term strategy, but one that leads to loss of tissue 36 function. Following injury to the heart, a limited ability to replace functional cardiomyocytes 37 results in irreversible damage, scar formation, and compromised heart function. These events 38 will ultimately lead to HF. 2 Although robust cardiac regeneration occurs in some adult 39 vertebrates (e.g., zebrafish, salamanders, newts, etc.) and neonate mammals, 3-6 adult 40 mammalian hearts exhibit poor regenerative capacity. 7-9 The lack of adult mammalian models 41 demonstrating cardiac regeneration has contributed to the current knowledge gap in the field. 42 The ability to regenerate injured organs is widespread in the animal kingdom. Fishes, 43 newts, and salamanders have extensive regenerative ability, and can functionally replace most 44 tissues and organs after amputation. Specifically, zebrafish have become a well-characterized 45 model for adult cardiac regeneration with the documented ability to recover from a plethora of 46 heart injuries including apical resection, 6 cryo-injury, 10 and coronary artery ligation. In contrast, 47 most adult mammals generally exhibit poor regenerative capacity, especially as it pertains to 48 recovering from heart damage. Spiny mice (Acomys spp.) are murid rodents found throughout 49 Africa, the Middle East and Western Asia. These rodents exhibit a number of special traits, 11 50 tantamount among them is the ability to regenerate skin and complex tissue. 12-16 Moreover, a 51 recent study examining acute and chronic kidney injury documented enhanced cytoprotection 52 in Acomys cahirinus compared to two laboratory mouse strains. 17 At present, the extent to 53 which spiny mice respond to heart injury remains unknown. In order to characterize the heart 54 injury response in a comparative framework, it is imperative to accurately determine Acomys 55 heart structure and function. Here, we report detailed cardiac characterization of A. cahirinus 56 in comparison to the commonly used C57BL6 mouse strain. The results of this study 57 demonstrate comparable cardiac anatomy, structure and function between Acomys and Mus 58 hearts but differences in cardiomyocyte phenotype between species.   sterile PBS) to prevent coagulation. Ten minutes later, under isoflurane anesthesia, hearts were 96 excised and cannulated with a blunt tip 23g needle and clamp. 10 ml of 1x PBS was perfused 97 intra-aortically, followed by 1.5ml Batson's 17 polymer mixture (2.5ml base monomer + 600ul 98 Catalyst + 3 drops Promoter; Polysciences). Polymer was mixed and ten minutes later was 99 injected over five minutes and then left to cure on ice for 3 hours. Once cured, hearts were 100 briefly washed with ddH2O and then placed in potassium hydroxide (maceration solution) for 101 one hour at room temp. After maceration incubation, hearts were laid with the anterior side up 102 and the left anterior descending artery was imaged using a Nikon Camera (DS-12) after 103 application of several drops of ddH2O to clear overlying tissue.

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Tissue from all species were collected and weighed immediately at sacrifice. The dry 107 lung weight was collected after 3 days of 65° C incubation. Hearts were perfused with PBS 108 (VWR International) followed by 4% buffered formalin (VWR International) fixation via 109 cannulation of the ascending aorta. Hearts were post-fixed overnight at 4 0 C. Hearts were then 110 sectioned into 3-mm cross-sectional slices followed by paraffin embedding. Tissue was 111 transferred to 70% ethanol until sectioning into 4-μm sections starting at the level of papillary 112 muscle.

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Immunohistochemical assessments were carried out on deparaffinized and rehydrated 116 sections as previously described. 18 After deparaffinization, washing, TRIS heat induced epitope 117 retrieval, and protein and streptavidin/biotin blocking, slides were incubated with primary   Values are expressed as mean ± standard error of mean (SEM). We used unpaired 178 Student t test to estimate differences, as appropriate. Throughout the analyses, a P value < 0.05 179 was considered statistically significant. All statistical analyses were performed using the Prism

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Heart structure and coronary tree anatomy are similar between Acomys and Mus 184 To establish a basis for accurate comparison across species, we first set out to 185 determine whether baseline differences in cardiac gravimetrics exist between Acomys and Mus. 186 First, we assessed heart weight (HW) and normalized it to body size (body weight [BW] and 187 tibia length [TL]) to account for overall size differences between species. Although Acomys 188 demonstrated a higher mean HW than Mus ( Figure 1B), normalized to BW, Acomys exhibited 189 a slighly lower HW than Mus ( Figure 1C). There were no differences between species when 190 HW was normalized to TL ( Figure 1D). Acomys demonstrated heavier wet and dry lung weight   Figure 6C). ICa,T is present in 67% of Acomys, compared to <5% of Mus 248 ventricular cardiomyocytes ( Figure 6D). Cell capacitance was significantly lower in Acomys 249 compared to Mus ( Figure 6E), consistent with morphometric analysis (above, Figure 4B),.

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Additionally, maximal conductance density trended greater in Mus than Acomys (p=0.06, 251 Figure 6F). Voltage-dependent activation and inactivation of ICa,L was significantly shifted 252 positive for Acomys compared to Mus ( Figure 6G and H). Taken together these data are 253 consistent with a 'young' adult heart physiological phenotype in the mature Acomys heart.  Figure 2). Interestingly, Acomys hearts were similar structurally and anatomically to Mus. 299 Additionally, there were no significant differences for in vivo cardiac functional parameters 300 between the two groups ( Figure 3). These findings are crucial in planning future myocardial 301 ischemia studies as they indicate that similar techniques can be used to induce comparable 302 injury across species.

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The adult mammalian heart loses its regenerative capability after the first week of life