Introduction

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality worldwide, accounting for nearly 16 million deaths (~ 23% of global deaths) in 2021 [1, 2]. More people die each year from CVDs than from any other cause. With globalization, urbanization, and economic expansion, CVD is no longer a disease of just developed or westernized countries, with many nations adopting similar lifestyle changes promoting heart disease.

Animal models have provided great molecular, cellular, and physiological insights into disease and potential therapies [3,4,5], however there is no high-throughput, tractable primate model of CVD. Historically, use of non-human primates has remained limited due to their lack of experimental tractability, extraordinary cost, and ethical concerns [6,7,8,9]. Classical animal models of heart disease have traditionally involved studies in mice, rats, pigs, rabbits, and zebrafish [10, 11] – and with the invention of gene targeting in 1987 [12, 13] and the subsequent ascent of reverse genetics, widespread use of knock-out, knock-in, and transgenic animal models of arrhythmias [14], atherosclerosis [15], myocardial infarction [16], and heart failure [17] have engendered a deeper understanding of cardiac disease mechanisms at a genetic, biochemical and cellular level. With rapid advances in human genetics and genomics and accelerating progress in identifying cardiovascular disease genes in humans, mouse genetic models of CVD have become the supermodel for studying human biology due to the ease in manipulating their genes, coupled with their short generation time, large litter size, and comparatively cheap and easy husbandry [18]. However, with their expanding use has come a greater appreciation and concern that many aspects of human biology and pathology are poorly modeled in mice [19, 20], particularly involving major aspects of cardiovascular anatomy, physiology, and disease, limiting the utility of mice for cardiovascular research and pre-clinical testing [21, 22].

Cardiovascular Differences Between Humans and Mice

The major differences in cardiovascular structure, function, and biochemistry between humans and mice are summarized in Table 1. Mice differ greatly from humans in their conduction systems. Baseline mouse heart rates are 600–800 beats per minute [14], about five to ten times faster than humans. Their native pacemaker, the sinoatrial node is located in or nearer to the superior vena cava (SVC), rather than exclusively in the right atrium as in humans [21, 23]. Mice have two superior vena cava (SVC) veins [21, 23], while humans have one (the left SVC that persists in mice regresses in humans during embryonic development). Whereas humans have four pulmonary veins (a site of origin for atrial arrhythmias), mice have one [23]. The majority of mouse cardiomyocytes are multinucleated, whereas human cardiomyocytes are predominantly mononucleated; this fundamental difference contributes to the even more limited regenerative window of human myocytes [24]. Expression of different potassium-ion channels has also been observed, explaining why the repolarization phase of the cardiac action potential is more rapid in mice, severely limiting their use in evaluating anti-arrhythmic therapies [21, 25, 26]. Major differences in coronary anatomy also exist: human coronary arteries are extramural, whereas mouse coronaries are largely intramyocardial [27]. The main circulating cholesterol in mice is HDL, whereas in humans it is LDL [15, 28]. Mice develop few or no plaques in their coronary and carotid arteries, the main sites in humans [15, 28]. Spontaneous plaque rupture with overlying thrombosis causing downstream ischemia and infarction has never been found in mice [15, 28]. Opposite expression pattern of the myosin heavy chain (MHC) isoforms, the major component of cardiac sarcomeres has been found for mouse [29], accounting for the differences between mouse models engineered with human MHC cardiomyopathy-causing mutations [17] and human presentation of the disease. Mice have also not been found to spontaneously acquire many of the most common human CVDs such as coronary atherosclerosis, myocardial infarctions, heart failure, and arrhythmias [21, 25, 28, 30].

Table 1 Humans and mice differ greatly in cardiovascular anatomy, biology, physiology, and disease
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Search for a New Animal Model

These differences have led to a search for new model organisms that more closely resemble human physiology and disease but retain the genetic and practical advantages of mice. Pigs and rabbits had previously been considered, as they diverged earlier than mice from the primate lineage [31, 32], but other shortcomings preclude their widespread use, including the paucity of genetic approaches [33, 34]. Such limitations are magnified in non-human primates. Though sharing close ancestry to humans, due to high maintenance costs, low-throughput husbandry, and ethical concerns few genetically-engineered primate CVD models exist [6,7,8,9]. Current attempts have been made to use forward genetic strategies to screen primates, particularly baboon [35] and macaque [36], but these have been low throughput and focused on complex genetic traits.

The ideal model would have a relatively short generation time, be prolific, cheap, and easy to maintain, and have cardiovascular anatomy, physiology, and genetics that better mimic humans (Fig. 1a, b). Our lab has tested the mouse lemur (genus Microcebus) – a prolific lower (prosimian) primate indigenous to Madagascar, as a genetic model for study of primate-specific biology and disease [6, 37,38,39,40]. They are the smallest primates in the world (~ 60 gm), just twice the size of a mouse, and are genetically nearly twice as close to humans. They are also the fastest reproducing, with a generation time of 6–8 months and litters of 2–4 offspring [6], with an average lifespan of six years [41]. They have been studied by our collaborators at the French National Center for Scientific Research in Brunoy, France since the 1960 s, which has provided a basic understanding of their husbandry and some aspects of physiology, but little is known of their genetics, cardiac physiology, and pathology [6].

Fig. 1
figure 1

The mouse lemur is a primate model more closely related to humans than mice, with unique genetic advantages for cardiovascular research. (a) Phylogenetic tree showing that mouse lemurs are about half the genetic distance from humans than mice are. (Adapted from: Ezran C, et al. Genetics. 2017;206(2):651–64. https://doi.org/10.1534/genetics.116.199448, by permission from Oxford University Press) [6]. (b) Comparative radar plot between mouse and mouse lemur with values normalized on a scale from 0 to 1. Lemurs retain several practical advantages of mice (shorter generation time, small body size, multiple offspring, tractable and economical husbandry), while more closely resembling human biology (longer life span, slower heart rate, and ability to naturally acquire cardiovascular disease, CVD). Generation time represents the age of sexual maturity plus the gestation period. Cost per generation is based on laboratory maintenance rates from the National Institutes of Health [6]. Raw values for each parameter [6, 40] before normalization (mouse/mouse lemur): Divergence (90–110/60–75 million years); Weight (30/60 g); Generation time (2–3/6–8 months); Litter size (8–12/2–4 offspring); Life span (2/6 years); Cost per generation ($100/$500); Heart rate (600–800/300–389 beats per minute); CVD (absent/present). (c) The mouse lemur is a primate model well suited for the current -omics era, exemplifying tractable genetic tools, integration of high-resolution genomics and transcriptomics, and primate-specific phenotypes. Cartoons of the mouse lemur, chromosomes, and UMAP plot were created in BioRender. Chang, S. (2025) https://BioRender.com/oaqjzoi. Sample pedigree was drawn with QuickPed [50]

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Mouse Lemur, a New Tractable Primate Genetic Model Organism

In a series of three recent papers, we demonstrated the utility of the mouse lemur as a novel model organism for human physiology and disease (Fig. 1c) [38,39,40]. In one study, a rapid, point-of-care 3-lead ECG monitoring device was used to screen ~ 350 mouse lemurs, without need for sedation or anesthesia as the animals are quite docile [40]. More than 700 ECGs were performed over nine years, enabling longitudinal assessment in some individuals. The screen uncovered eight different naturally-occurring arrythmias and related pathologies, each of which mimics a significant human disease or condition – the first cases of heart disease in lemurs [40]. We constructed a pedigree and genetically mapped one of the familial lemur arrhythmias we identified, an autosomal recessive, episodic bradycardia resembling hereditary human sick sinus syndrome (SSS) that afflicted seven lemurs [40]. The most appealing candidate gene in the mapped interval was SLC41A2, a little studied magnesium transporter and novel disease gene [42, 43].

Mouse SLC41A2 knockouts do not show a cardiac pacemaker phenotype [44]. In contrast, our work revealed, in addition to the classic calcium transients that underlie pacemaker activity, the surprising presence of rhythmic magnesium transients in human iPSC-derived cardiac pacemaker cells (sinoatrial node cells, iSANCs). We found that SLC41A2 is expressed in iSANCs, localizes to the sarcoplasmic reticulum, and that CRISPR-mediated knockout of SLC41A2 alters magnesium dynamics and slows their calcium firing rate [40]. The results suggest SLC41A2 functions cell autonomously and primate-specifically in the cardiac pacemaker, and that intracellular magnesium dynamics have a crucial but previously unappreciated role in setting pacemaker rate. As such, mouse lemur is a valuable model for discovering new genes (SLC41A2), molecules (SLC41A2, magnesium), and mechanisms (autonomous magnesium spikes) of the primate pacemaker, and for identifying novel candidate genes and therapeutic targets for human arrhythmias. The approach can be used to elucidate other physiological and disease traits identified in this and other screens of this primate model organism.

To construct our mouse lemur cell atlas [38], we defined and biologically organized over 750 molecular cell types and their full expression profiles from 27 organs and tissues. This extensive atlas enabled global comparisons of cell type expression profiles, allowing us to define the molecular relationships of cell types across the body, and to explore primate cell type evolution by comparing lemur cell profiles to their counterparts in human and mouse. This revealed cell type specific patterns of primate cell specialization, as well as many cell types for which lemur is a better human model than mouse. For the lemur heart, we obtained transcriptomic profiles of over 4000 cardiac cells. This identified 15 heart cell types, including 5 major mammalian cardiac cell types: cardiomyocyte, endothelial cell, fibroblast, pericyte, and resident macrophage. Cardiomyocyte cells were further subdivided by chamber based on expression of mouse lemur orthologues of canonical chamber-specific marker genes. Additionally, small numbers of four rare heart cell types were found: cardiac conduction cell (sinoatrial or atrioventricular origin), Purkinje cell, epicardial cell, and cardiac neuron. However, many conduction cells were not identified, including sinoatrial P cell, transitional cell, atrioventricular P cell, atrioventricular bundle cell, and bundle branch cell.

The systematic analyses in our second atlas paper [39] make genetic, molecular, and physiological/disease studies tractable in lemur, and demonstrate that its genes, splice patterns, physiology and diseases also better model human than do mouse. The study identified hundreds of primate-specific genes, tens of thousands of splice variants, and key physiological and disease traits shared between humans and lemurs but absent in mice – highlighting high-priority targets for further research. We also introduced a reverse genetic approach in lemur, and applied it to several high priority genes (three primate genes missing in mice), describing the first null (nonsense) alleles in lemur and corroborating their transcriptional effects by showing substantial depletion of nonsense transcript reads. This has paved the way for a full genetic and phenotypic analysis of these key genes, and for identifying null mutations in many if not all other lemur genes.

Applying the methods and insights developed by the Telomere-to-Telomere (T2T) Consortium in assembling the complete human genome [45, 46], our team recently completed a near T2T assembly of both haplotypes of the mouse lemur genome [47, 48]. Using a trio-binning approach that integrated parental short-read sequencing data with PacBio High-Fidelity (HiFi), Oxford Nanopore Technologies (ONT) ultra-long, and HiC reads, we generated a chromosome-scale, phased diploid assembly with the principal (maternal) haplotype totaling 2.350 billion base pairs which includes gapless assemblies of numerous chromosomes. Compared to the current RefSeq (Mmur3.0), which consists of 7,677 scaffolds (contig N50: 211 kb), our assembly represents an improvement in contiguity, with 124 scaffolds (contig N50: 102 Mb) and a quality value (QV) of 61. Notably, some major cardiac genes are not annotated in the current RefSeq (Mmur3.0), including MYH6 and MYH7, two well-known cardiomyopathy-causing genes [49]. Our comprehensive lemur transcriptomic cell atlas [38] is being used to improve gene annotation of the new assembly. This high-quality, phased genome will facilitate more accurate genotype–phenotype mapping and be a valuable resource in evolutionary genomics and primate genetics.

Conclusions

Differences in cardiovascular anatomy, physiology, and biochemistry limit the utility of mice in modeling molecular and hemodynamic aspects of human cardiac disease. Species-specific differences in expression patterns of cardiac ion channels and sarcomeric proteins make it problematic to test potential therapies for arrhythmias and heart failure in mice. Moreover, mice do not develop clinically-significant atherosclerosis or plaque-induced myocardial infarction, the most common cause of human death. No high-throughput primate-specific model of CVD exists. We showed that systematic phenotypic screens and classical genetic mapping are possible in lemur, and use them to identify cardiac phenotypes (arrhythmias), a new disease gene (magnesium transporter), and novel physiological mechanism (magnesium in pacemaker automaticity), all conserved in lemur and human but missing or different in mice. The results establish the mouse lemur as a tractable model organism (Fig. 1c), providing a new paradigm for study of primate-specific cardiovascular genetics, biology, pathology, and therapy testing.

Key References

  • Chang S, Karanewsky CJ, Pendleton JL, Ren L, Anzeraey A, Froelicher V, et al. A primate model organism for cardiac arrhythmias identifies a magnesium transporter in pacemaker function. bioRxiv. 2025:2025.05.28.655959. https://doi.org/10.1101/2025.05.28.655959.

    • This study uses an ECG screen to identify naturally-occuring arrhythmias in mouse lemurs, and genetically maps a hereditary form of sinus bradycardia in this species to a new disease gene.

  • Ezran C, Liu S, Chang S, Ming J, Botvinnik O, Penland L, et al. A molecular cell atlas of mouse lemur, an emerging model primate. Nature. https://doi.org/10.1038/s41586-025-09113-9.

    • This work describers the construction and application of the mouse lemur cell atlas.

  • Ezran C, Liu S, Chang S, Ming J, Guethlein LA, Wang MFZ, et al. Mouse lemur cell atlas informs primate genes, physiology and disease. Nature. 2025;644(8075):185-96. https://doi.org/10.1038/s41586-025-09114-8.

    • This work uses the generated lemur cell atlas to uncover unidentified genes and novel splice isoforms, and introduces an experimental reverse genetics strategy to study primate genes absent in mouse.