Congenital heart disease

How similar are human and animal cardiovascular systems?

 

This is not an exhaustive list of species-specific differences, nor can one be made given their unknown full extent, but rather an example of how these differences impact the face, construct, predictive, and intrinsic validity of animal models.

 

Not all species-specific differences can be accounted for in animal models, as there are hundreds of them, their relevance to congenital heart disease is unclear, and their interaction with other organ systems in the animal model makes it difficult to predict how they would have behaved within the human system.

 

Cardiovascular systems of human and non-human animals differ at every level, from molecular to organism level, contributing to poor translation of animal studies to humans.

 

Species-specific Differences in Cardiomyocyte Electrophysiology

Divergencies in cardiac electrophysiology across species, including in cellular and tissue-level function of depolarizing currents, repolarizing currents, early afterdepolarizations, and delayed afterdepolarizations, have implications for understanding human-specific cardiac function, for identifying vulnerabilities to human cardiac anomalies, for dissecting the impact of congenital heart disease (CHD) on cardiac function, for assessing the risk of drug-induced cardiotoxicity, and for predicting efficacy of drugs.

 

At the heart of the cardiac function is the action potential (AP) - a temporary change in the electrical membrane potential of a myocyte, allowing it to transmit a signal.

During the cardiac cycle, the AP goes through phases that are generally consistent across all animal species: depolarisation (phase 0), initial repolarisation (phase 1), plateau (phase 2), repolarisation (phase 3) and resting potential (phase 4).

However, electrical currents that initiate and propagate the AP - inward sodium current (INa), inward calcium current (ICa), transient outward potassium current (Ito), delayed rectifier potassium (ultrarapid Ikur, rapid IKr and slow Iks) currents, intermediate conductance calcium-activated potassium (IKCa) current and inward rectifier potassium current (IK1) - have species-specific expression, regulation, kinetics (activation, inactivation, recovery, conductance), function and interaction Edwards & Louch, Clin. Med. Ins. Cardio., Feb 2017.

 

Molecular and functional differences in major depolarizing currents

During depolarisation, both Ca2+ entering through L-type Ca2+ channels (LTCCs) in the sarcolemma, and Ca2+ released from the sarcoplasmic reticulum (SR) through type 2 ryanodine receptors (RyR), participate to contraction.

During repolarisation, a portion approximately equal to L-type Ca2+ current (ICaL) influx is extruded from the cell by the Na+-Ca2+ exchanger, NCX. That exchanger activity differs substantially across species, reflecting the degree to which sarcolemmal Ca2+ fluxes support contractile requirements.

In the mouse and rat, the influx via ICaL amounts to only 7% of the total Ca2+ cycled during each beat, with the vast remainder being released from and pumped back into the SR at steady state. In non-diseased larger mammals, including rabbits and humans, the NCX contribution to calcium clearance is roughly 4-fold higher.

This reduced contribution for sarcolemmal Ca2+ fluxes in rats results from up to 4-fold higher SR calcium ATPase (SERCA2) activity, and fundamentally reduced NCX activity in the rat.

These differences may be explained by higher intracellular [Na+] in rats and mice, differential regulation and expression of NCX1 splice variants or other poorly understood species-specific variables.

Peak ICaL is slightly reduced in rats compared with larger mammals, in part due to lower LTCC expression. Also, the voltage at which ICaL begins to activate is more negative in rats than in rabbits and guinea pigs, which can affect the overall cardiac AP, potentially influencing how each species responds to certain drugs and interventions.

 

There is little data on inter-species functional differences in Ina. In contrast, the molecular basis of INa, mediated by Nav1.5 channel, has been studied in a number of different species.

Although Nav1.5 is the alpha subunit core component in all cases, significant and variable expression of other isoforms has been observed across species. 

In addition, a wide variety of auxiliary beta subunits, adhesion proteins, modulatory proteins, and signaling enzymes, may contribute to species-dependent effects on INa function and response to drugs by altering the kinetics, gating responses, cellular localisation and pharmacology of Na channels Edwards & Louch, Clin. Med. Ins. Cardio., Feb 2017.

 

Molecular and functional differences in repolarizing currents

Rapidly developing repolarizing currents

Varying function and kinetics of the different K+ currents is the primary explanation for major species differences in cardiac electrophysiology Edwards & Louch, Clin. Med. Ins. Cardio., Feb 2017.

In contrast to humans who have longer AP, repolarization in mice and rats’ myocytes proceeds so rapidly that channel types which conduct K+ currents that develop slowly will rarely be recruited.

Indeed, in mice and rats, ultrarapid IKur and rapid IKr currents are generally much larger than in humans, shaping the triangular AP waveforms and shortening AP durations.

 

Even among mammals with longer AP, for reasons that are not well understood, repolarizing currents exhibit remarkable variability.

Although Ito is believed to play a part in early repolarisation in several species, there are species-specific differences in phenotypic expression (density, distribution, composition, accessory proteins and regulatory subunits) and in kinetics of fast Ito (Ito,f) and slow Ito (Ito,s) channels that affect AP duration and the heart's response to stress or pharmacological agents. Sala et al., EP Europace, Sep 2018.

The faster kinetics of Ito,f channel in rodents compared to humans also contributes to shorter AP durations and much higher heart rates than in humans.

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In comparison to Ito in humans, Ito in dogs exhibit slower reduction in current and faster recovery from inactivation. In addition, high expression of Ito,f in the epicardium relative to endocardium underlies a large portion the transmural Ito gradient in most species, and in the dog, this gradient is very large due to region-specific expression of Ito,f.

Intermediate conductance calcium-activated potassium (IKCa) channels, such as KCa3.1, contribute to the repolarization process during phase 3 by allowing K⁺ ions to exit the cell. Guinea pigs have lower expression levels of KCa3.1 channel than humans, which may explain why they are not responsive to drugs such as Clotrimazole and Senicapoc Hamlin & Keene, Curr. Op. Tox., Oct 2020.

 

Slowly developing repolarizing currents

Species-specific differences in delayed rectifiers LKur, LKr and LKs has particular importance for susceptibility to ventricular long QT (LQT)–associated arrhythmias such as torsades de pointes. As such, LKr is recognized as a critical target for understanding the potential for cardiotoxicity of drugs Edwards & Louch, Clin. Med. Ins. Cardio., Feb 2017.

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In myocytes of animal species with brief AP, LKr make a small contribution to repolarization when hERG recover from their conformational inactivation.

In contrast to LKr, activation kinetics of LKs are too slow to contribute to repolarization in species that have shorter AP and exhibits only very slight inactivation.

In larger mammals with longer AP, which includes humans, the susceptibility to AP prolongation and LQT-associated arrhythmia depends on the balance between LKr and LKs.

Notably, in animal species with robust IKs, such as guinea pig and dog, inhibition of IKr causes voltage-dependent recruitment of IKs, thereby resisting further AP and QT prolongation. However, species with less pronounced IKs, including humans, and to a lesser extent rabbits, but not rats, show higher sensitivity to IKr blockade Arpadffy-Lovas et al., Can. J. Physiol. Pharmac., Sep 2022.

This characteristic is therefore believed to be crucial for human susceptibility to ventricular QT prolongation and torsades de pointes, particularly during hERG blockade that occurs in drug-induced cardiotoxicity and congenital LQT syndrome.

 

As a consequence of these inter-species differences, antiarrhythmic drugs that work by blocking the Ikr, such as Ibutilide and Dofetilide, do not have an effect in mice and rats Hamlin & Keene, Curr. Op. Tox., Oct 2020.

 

The phenotypic expression of IK1 current, that helps maintain the resting membrane potential by allowing K⁺ ions to flow into myocytes more easily than out, thanks to the blocking of the channels by intracellular magnesium ions (Mg2+), also diverges across species, which modifies both the trajectory of late repolarization and the stability of resting potential Edwards & Louch, Clin. Med. Ins. Cardio., Feb 2017.

In addition, IK1 conductance is positively regulated by extracellular K+ in the physiologic range, meaning that changes in extracellular K+ influence resting potential and terminal repolarization in a nonlinear way that depends on a balance between this change in IK1 conductance and an opposing change in Nernst equilibrium for K+.

Because baseline IK1 conductance and plasma K+ vary among species, tissues, and pathologic states, these changes are clinically relevant.

For instance, inhibition of the IK1 current significantly prolonged rat ventricular repolarization, but only slightly prolonged it in dogs, and did not affect it in humans Arpadffy-Lovas et al., Can. J. Physiol. Pharmac., Sep 2022.

Guinea pig IK1 is relatively large and may also exhibit greater contribution of Mg2+-dependent rectification compared to cats and rabbits. Both rabbits and dog exhibit larger ventricular IK1 expression than humans. In humans, IK1 expression is lower in the atria than in the ventricles, while the murine IK1 is at least as large in the atria compared with ventricles.

 

These characteristics may confer a larger susceptibility of human myocytes to spontaneous calcium release events (SCRE) from the SR during diastole, compared to other large mammals. Depending on the cellular environment and context, this SCRE can potentially lead to the generation of delayed afterdepolarizations (DAD), increase in the firing rate and arrhythmia.

 

Species-specific Mechanisms of Early Afterdepolarizations

Mechanisms that underly early afterdepolarizations (EAD) are fundamentally different across animal species Edwards & Louch, Clin. Med. Ins. Cardio., Feb 2017.

These EAD, that can occur either during the plateau phase of the AP (phase 2) or during late repolarization (phase 3), are significant because they can trigger abnormal AP and lead to arrhythmias.

In humans, dogs, sheep, guinea pigs, and rabbits, EAD are generally initiated late in the plateau and are driven by reactivation of the LTCC pool combined with reduction of K+ channel conductance.

By contrast, in mice, in which rapidly repolarizing potassium currents Ito and IKur are highly expressed, it is INa that is the greatest contributor to EAD.

Computational analyses involving a human atrial model suggest that, as in the mouse, INa reactivation is the initiating current in these EADs, however, atria of humans do not repolarize on the same timescale as in mice.

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The fact that the mouse if frequently used to model and test therapies for arrhythmia, despite its species-specific features of EAD, contributes to poor translation of preclinical studies to humans.

 

Inter-species Differences in Delayed Afterdepolarizations

Delayed Afterdepolarizations (DAD) occur after the completion of repolarization (Phase 4), during the resting membrane potential Edwards & Louch, Clin. Med. Ins. Cardio., Feb 2017.

Although the basic mechanism of DAD, that involves intracellular Ca2+ overload that triggers spontaneous release of Ca2+ from SR followed by depolarizing influx of Na+ via NCX, are shared across species, susceptibility to DAD events is variable depending on species-specific parameters such as RyR sensitivity, cellular Ca2+ flux balance, cellular ultrastructure (T-tubules, dyads) and heart rate.

For example, rabbit myocytes exhibit rapid loss of SR Ca2+ due to prominent Ca2+ extrusion by NCX. In rat myocytes, however, SR Ca2+ reuptake by SERCA dominates over NCX function, and SR Ca2+ content is maintained.

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The full extent and mechanisms of these inter-species differences in DAD events are not well understood, and yet, they are likely to affect both the cardiac function and response to treatments. Variations in RyR sensitivity can affect the timing and amount of Ca2+ released, which in turn influences the heart's electrical activity and contractility. Variation in T-tubule density and orientation of dyads can impair Ca2+ handling. Imbalances in Ca2+ flux can disrupt the excitation-contraction coupling process, leading to arrhythmias and heart failure.

 

Inter-species Differences at the Tissue-Level

Inter-species differences at tissue and organ levels can be both structural or functional, affecting susceptibility to arrhythmia through an impact on arrhythmogenic tissue substrate or through interactions between tissues substrates and triggers of arrhythmia Edwards & Louch, Clin. Med. Ins. Cardio., Feb 2017.

 

Species-specific differences in structural and physiological characteristics such as tissue size, conduction system structure, gap junction conductance, expression of ion channels, current expression, diastolic interval and restitution properties, play a part in inter-species variations to susceptibility to re-entry, ectopy and dispersion of repolarization.

 

Re-entry is a common mechanism in many types of arrhythmias, such as atrial flutter, atrial fibrillation, and ventricular tachycardia, in which a propagating impulse continues to circulate and repeatedly stimulate the heart muscle, leading to rapid and irregular heartbeats.

In ectopy on the other hand, the triggering of irregular heartbeats originates from outside of the sinoatrial node that, if frequently repeated, can result in arrhythmias.

Dispersion of repolarization presents with differences in repolarization times across different heart regions, predisposing the heart to arrhythmias.

 

In humans, dispersion of repolarization is an important precursor of congenital and acquired arrhythmias. By contrast, the smaller heart size, low transmural thickness and different ion channel expression in rodents is likely to result in less pronounced dispersion of repolarization, a lower likelihood of re-entry, and a higher likelihood of ectopy, compared to humans.

In humans, the largest regional differences in AP duration occur transmurally and AP dispersion across the left ventricle (LV) wall is believed to be one of the most important sources of arrhythmogenic substrate.

As for larger animal models such as dogs, pigs and sheep, since the underlying mechanisms of transmural variation in humans remain unclear, there is insufficient evidence to suggest they would be human-relevant.

 

In humans, APD alternans, in which the AP duration alternates from beat to beat in cardiomyocytes, is considered as a precursor to arrhythmias such as ventricular fibrillation.

Given the above species-specific differences, it is very likely that rodents exhibit differing mechanisms of cellular alternans and differing susceptibility to alternans-dependent arrhythmia than humans.

Even in case of use of larger animal models, such as pigs, sheep and dogs, these various species-specific features are likely to present a hindrance for successful translation of preclinical studies to a clinical setting.

 

 

Species specific-differences in heart anatomy and bioenergetics

Including in cellular, tissue and organ-level heart morphology, function and metabolic needs, have implications for understanding change in human-specific cardiac contractile kinetics in response to electrical, mechanical and chemical stimuli, for dissecting the causes and the effects of human heart diseases, for elucidating the mechanisms of CHD, for understanding human-specific heart morphogenesis, and for discovering human-relevant treatments.

 

Although the heart anatomy, physiology, and bioenergetics of smaller mammals (rodents) and bigger mammals (humans, pigs, sheep) share several similarities, there are also massive inter-species differences.

 

Inter-species differences in heart size, shape, and metabolic demands lead to variations in cardiac electrical conduction structure, function, speed and efficiency that reflect evolutionary adaptations to species' physiological needs Hamlin et al., Am. Heart. Assoc., Jan 2011.

 

The mouse has at least 5-fold higher heart rate compared to the human. Pigs and sheep have lower metabolic rates compared to dogs.

The energy per beat for the human heart is almost 3 times greater than for the mouse heart. The rate of synthesis of ATP by creatin kinase, that ensures that myocardial energy levels remain sufficient to support continuous and rhythmic contractions is similar between the two species, pointing to differences in energy transfer efficiency.

 

In humans, the heart is trapezoidal in shape with a blunt apex. Pigs and dogs also have a blunt apex, but the heart shape in pigs is more rounded while in dogs the heart is ovoid. By contrast, the heart of sheep is conical in shape with a pronounced apex.

These inter-species difference in heart shape can be explained by anatomical context, body structure and posture, activity level, and metabolic needs.

In dogs, the coronary arteries have extensive collateralization with many arterial anastomoses. In case of blockage or narrowing in one of the main coronary arteries, these vessels can act as natural bypasses by providing an alternative route for blood to reach heart tissues. In contrast, humans, pigs, and sheep have less developed collateral networks. The highly protective role of collateralization in dogs is likely to produce inter-species differences in phenotypes of coronary artery diseases.

 

Owing to its transparency and rapid development, the zebrafish is commonly used to study embryogenesis, however, it has a 2-chambered heart with a single atrium and a single ventricle, which makes it unsuitable to mimic human heart morphogenesis Pierpont et al., Circulation, Sep 2018.

 

Amphibians, such as frogs and salamanders, present a three-chambered heart, with two atria that receive oxygenated and deoxygenated blood separately and one ventricle where the blood mixes before being pumped out to the rest of the body.

 

The cardiovascular system of each animal has evolved differently in order to meet the demands of that particular species. The heart of a small animal such as mouse can beat up to 800 times per minute, which is 10 times faster than in humans, while an elephant has a heart rate of only 35 beats per minute Milani-Nejad & Janssen, Pharma. & Therap., Mar 2014.

 

Due to inter-species differences in conduction system properties, the P–R interval, that reflects the time it takes for electrical impulses to travel from the atria to the ventricles, can vary significantly.

Generally smaller mammals like mice and rats have faster heart rates and shorter P–R intervals, while larger animals like humans and pigs have slower heart rates and longer P–R intervals.

For example, in mice, the P–R interval is typically around 40 to 60 milliseconds while in humans it ranges from 120 to 200 milliseconds. The P-R interval in sheep is similar to humans, in dogs it is about 60 to 100 milliseconds and in pigs 100 to 150 milliseconds Milani-Nejad & Janssen, Pharma. & Therap., Mar 2014.

 

Comparison of gross anatomy shows that mice and human both have 4-chambered hearts, that both hearts are about 0.5% of body weight, and that both share the same right atrial and mean systemic arterial pressures, to mention a few examples. There are, nevertheless, enormous differences between mouse and human hearts that cannot be explained by simple allometric scaling.

Although animal species of similar body weight and heart weight tend to share more comparable heart rates, the size and weight are by far not the only varying factors.

 

Major inter-species differences exist in cardiac contractile kinetics (speed, force, rate, synchronisation, energetic efficiency) of cardiac processes, including excitation-contraction coupling of electric induction to mechanical action, contraction (systole) and relaxation (diastole) of the myocardium.

These differences are believed to be driven by numerous species-specific features, including in calcium handling, sarcomere length, composition of myofilaments, and adrenergic stimulation.

 

In addition, mice modulate their heart rate only minimally. Even during strenuous exercise, the mouse heart rate increases from the resting rate by 50% at most, whereas in humans, the heart rate can increase by 300%. As a result, exercise-induced increases in heart rate, combined with stroke volume per contraction, can be up to 10-fold in the human, whereas in the mouse it is only up to 2-fold Janssen et al., Circ. Res., Jul 2016.

 

Dimension-wise, the fundamental units of muscle structure and function, sarcomere and myocyte are nearly identical in both species. In both species, the contractile process results from a complex interaction between calcium ions, troponin and tropomyosin, followed by cross-bridge cycling between myosin heads and actin filaments that, driven by ATP hydrolysis, generates the force needed for muscle contraction.

Despite that, these components are tuned to operate in an entirely different matter to produce entirely different cardiac contractile kinetics Janssen et al., Circ. Res., Jul 2016.

Notably, there are critical inter-species differences in myosin heavy chain (MyHC) isoforms alpha (V1), alpha-beta (V2), and beta (V3) that affect cardiac contractile kinetics and energetic properties.

In rodents, the heart predominantly expresses the alpha-MyHC isoform, which is associated with higher ATPase activity, high cross-bridge turnover rates, high resting heart rates and faster contractile velocity.

In contrast, the human heart primarily expresses the beta-MyHC isoform, which has lower ATPase activity, slower contractile velocity and lower resting heart rates Hamlin & Altschuld, Circulation, Jan 2011,  Miyata et al., Circ. Res., Mar 2000.

Pigs and sheep myocardium is more similar to humans than to rodents in that regard.

Rodents can increase the expression of alpha-MyHC isoform in response to physiological stimuli such as stress hormone elevation and exercise. Conversely, the human heart's response to stress involves a decrease in alpha-MyHC expression with a shift towards beta-MyHC, contributing to impaired cardiac function.

Moreover, in humans, mutations in the beta-MyHC gene (MYH7) are commonly associated with familial hypertrophic cardiomyopathy (FHC), a condition characterized by asymmetric left ventricular hypertrophy and myofibrillar disarray. Transgenic mouse models that carry mutations in the beta-MyHC gene can recapitulate a similar phenotype as in humans, however, the predominant expression of alpha-MyHC in rodents can influence the severity and progression of modeled cardiac conditions.

 

In the human heart, the ratio of two major isoforms of the protein titin in myocardium - N2BA (more compliant) : N2B (stiffer) - is typically around 30:70 in normal conditions. This ratio can shift towards a higher proportion of N2BA in conditions like dilated cardiomyopathy Milani-Nejad & Janssen, Pharma. & Therap., Mar 2014.

By contrast, mice and rats, overwhelmingly express the N2B isoform, with a ratio of 80:20, resulting in a higher passive stiffness in rodent hearts. In spite of that, unlike humans, rodents do not naturally develop conditions like heart failure with preserved ejection fraction, probably owing to species-specific compensatory mechanisms. Pigs also predominantly express the N2B isoform with a N2BA : N2B ratio of 60:40.

 

Another significant species-specific difference lies in reliance on sarcolemma Ca2+ versus sarcoplasmic reticulum (SR) Ca2+. In mice and rats, the sarcolemma Ca2+ contribution to calcium transient is about 10-fold lower than in humans. As a result, when the SR is rendered not functional, the sarcolemma Ca2+ can provide sufficient calcium for muscle contraction in humans but not in mice and rats Bers, Ann. Rev. Physiol., Nov 2007.

In humans, pharmacological inhibition of SR can shift the force-frequency relationship from positive to negative, meaning that as the heart rate increases the force of contraction decreases. This outcome could possibly be caused by an excessive accumulation of Ca2+ in the SR, leading to SR dysfunction, dysregulation of sarcolemma Ca2+-induced SR Ca2+ release, desensitization of troponin or excessive Ca2+-induced ROS.

In rabbits, however, the force–frequency relationship remains positive in this situation and the force of contraction increases as the heart rate increases, likely due to a species-specific reliance on compensatory mechanisms.

 

The heart cycle length, which is the time between successive heartbeats (also known as the R-R interval), and the heart rate, are much faster in mice than in humans. namely, in mice, the heart cycle length is roughly 100 ms at 600 bpm (rest) and 80 ms at 750 bpm (peak exercise), whereas in humans these processes take place in 1000 ms at 60 bmp (rest) and 350 ms at 170 bmp (peak exercise).

In contrast to humans, mice barely need to elevate their heart rate during strenuous exercise. In other terms, the mouse does not need large kinetics reserves - increases in contraction and relaxation speed to support their metabolic needs.

Humans, on the other hand, rely heavily on the ability of the heart to increase cardiac output when demand for oxygen increases. Contrarily to mice, humans have a lower tolerance for increased heart rates during exercise, and are therefore more prone to negative effects such as cardiac strain or severe symptoms seen in humans with heart diseases Desai & Bernstein, DICM, Jan 2011. In effect, the inability of the heart to sufficiently accelerate kinetics on an increase in heart rate can lead to inappropriate ventricular filling, insufficient cardiac output, and ultimately death.

 

In light of these major inter-species differences in heart function, it appears clear that the mouse is not an adequate model for studying mechanisms that lead to human heart failure.

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In humans and dogs, aortic valves are both anatomically connected to their respective mitral valves via an intervalvar septum. By contrast, the aortic valves of pigs and sheep, do not have the same degree of anatomical similarity to human aortic valves, and sheep do not have an intervalvar septum at all Crofton et al., Cardiol Res., Oct 2023.

 

Even though non-human primates (NHP) and humans are evolutionary closer than other commonly used animal species, their hearts present numerous differences, including in rate, cardiac output, and left ventricular morphology.

Furthermore, NHP do not have the same susceptibility to certain cardiovascular disease, such as atherosclerosis and myocardial fibrosis. Chimpanzees, for example, tend to suffer from fibrotic heart disease while humans are more prone to ischemic heart disease. This divergence in susceptibility can be explained by differences in evolutionary adaptation, genetics, diet, lifestyle, and environmental factors Ward & Gilad, eLife, Apr 2019. 

NHP often engage in short bursts of resistance physical activity, such as climbing, which create pressure stress on the cardiovascular system. By contrast, the human cardiac system has evolved to adapt to a predominantly moderate-intensity endurance physical activity with possibility to increase the cardiac output in case of necessity. This evolutionary adaptation in humans is believed to have hindered humans' ability to handle pressure stress, making them more susceptible than NHP to conditions like hypertensive heart disease Shave et al., PNAS, Sep 2019.

 

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Species-specific differences in metabolism

Divergences in metabolism across species, including in organism-level, organ-level, tissue-level, and cellular-level physiology and metabolic rates, have implications for understanding the impact of human-specific diet, genetic, environmental, and lifestyle factors on human cardiac function, for predicting human-relevant ADME properties of drugs, and for testing safety and efficacy of therapies.

 

Metabolic rates of mammals are adapted to their body sizes, physiology, diet, behaviour, environment and activity levels Pettersen et al., J. Exper. Biol., Jan 2018.

For instance, in comparison to humans, mice, rats, and rabbits have higher metabolic rates, while pigs and sheep have lower metabolic rates. Ectothermic animals, like amphibians, who have evolved to be more energy-efficient, have lower metabolic rates than mammals, regardless of their size.

It follows that the cardiac function is evolutionary adapted so that enough oxygenated blood can meet the metabolic demand. These species-specific evolutionary adaptations are likely to have played a role in species-specific differences in heart function, susceptibility to heart disease, and heart disease phenotype. 

 

In contrast to humans, mice, hamsters, and several other animal species, have the ability to enter torpor in situations of caloric restriction and sub-thermoneutral ambient temperatures Hamlin & Altschuld, Circulation, Jan 2011. During the state of torpor, mice experience a sharp decrease in heart rate and blood pressure.

 

Human metabolism also differs significantly from that of other primates. In spite of their higher physical activity levels, NHP have lower metabolic rates, presumably due to higher energy demands associated with the human brain Yegian et al., PNAS, Nov 2024.

 

Inter-specific differences in metabolic rates also affect pharmacokinetics of drugs. Animals with higher metabolic rates may metabolize drugs more quickly, leading to faster clearance Toutain et al., Comp. Vet. Pharmac., Jan 2010.

For example, adenosine, that is used as anti-arrhythmic agent in treatment of certain types of supraventricular tachycardias, is less effective in dogs than in humans, presumably due to species-specific differences in levels of cellular uptake and adenosine deaminase catabolic enzyme Hamlin & Keene, Curr. Op. Tox., Oct 2020.

Atropine, an antimuscarinic drugs commonly used to treat bradycardia, is less effective in rabbits than in humans, likely because rabbits possess a unique form of carboxylesterase, known as atropinesterase, that efficiently hydrolyzes atropine, and that is lacking in humans Gradinaru, Rab. Gen., Jul 2021.

 

 

Species-specific differences in embryogenesis

Including in molecular, cellular, tissue, organ and organism-level timing, regulatory networks and signaling pathways, have implications for understanding human-specific embryogenesis, for dissecting the impact of genetic, epigenetic, and environmental factors on the development of the human heart, for identifying factors that contribute to CHD and to adult heart disease, and for assessing the risk of developmental toxicity for new chemicals and drugs.

 

The heart is the first organ formed during mammalian embryogenesis Zubrzycki et al., Int. J. Mol. Sci., Jun 2024. The development of the heart is a very sensitive process and any perturbation in developmental processes are susceptible to lead to congenital heart defects. 

 

Mice are commonly used to study the impact of genetic, epigenetic, and environmental factors on heart development, in spite of the fact that the full extent of inter-species differences in heart development is currently unknown.

Crucially, human-based studies of embryogenesis have allowed to reveal a long list of inter-species differences in expression patterns of genes that are critical for the development of a fully functional human heart Gao et al., PLOS Bio., May 2021.

 

Human-specific features of embryogenesis, including in the disc-like shape of the human embryo, spatial relationship to extraembryonic tissues, implantation, and gestational timing, all play a part in human-specific electrophysiology and predisposition to cardiac diseases.

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At a gross level of comparison, the process of heart development presents with several similarities across mammalian species.

In all mammalians, the heart is composed of four chambers with distinct structure, biochemistry, and electrophysiology: the left atrium (LA), the right atrium (RA), the left ventricle (LV), and the right ventricle (RV). The early development of all mammalian species follows a similar sequence of stages: zygote, morula, and blastocyst that implants into the uterine wall.

Although the pre-implantation development may look overtly similar across species, there are major inter-species differences in the timing and process of maternal genome inactivation, in the genes and transposons activated at zygotic genome activation (ZGA), in the timing and molecular pathways of blastocyst lineage specification, and in the presence or absence of a mechanism to promote embryo diapause Rossant et al., Cell, Jan 2022.

 

Since the developmental processes and the need for maternal contributions vary among species, species with longer gestation periods tend to have later ZGA compared to those with shorter gestation periods.

For instance, in human embryos, ZGA takes place at a later stage than in the mouse, meaning that the human embryo becomes transcriptionally active and starts producing its own proteins necessary for further development later than in mice.

Indeed, in humans, who typically have a gestation period of around 280 days, the ZGA occurs around the 4-cell to 8-cell stage, while in rodents, who have a gestation period of around 19 to 23 days, the ZGA occurs at the 2-cell stage, leading to rapid heart development.

Whole-embryo and single-cell transcriptome analysis have shown that only 40% of human ZGA genes are shared with those of mouse ZGA, and many of these genes are of unknown function Rossant & Tam, Cell, Jan 2017.

 

In human embryogenesis, implantation of the blastocyte to the uterine wall typically starts around 5 to 6 days after fertilization. Although the ZGA in pigs, dogs and sheep occurs at the same time as in humans, the heart formation process is 2-fold shorter. The formation of all 4 chambers is completed before the 9th week of gestation in humans, 4th week in pigs, dogs and sheep, and 2nd week in mice and rats.

In contrast to humans, several animal species, including mice and rats, have developed mechanisms of embryonic diapause which allows them to synchronize the birth of their offspring with more favorable environmental conditions and which influence the timing of heart development.

 

The stages of blastocyst lineage specification are broadly similar across all mammals. The initial stages of lineage specification, such as differentiation into trophoblast (TE), that contributes to the placenta, and the inner cell mass (ICM), start around the time of ZGA. Further differentiation within the ICM into the epiblast (EPI), that gives rise to the fetus, and the primitive endoderm (PE), which forms the yolk sac, occurs after ZGA. Following differentiation of the ICM, gastrulation occurs, in which EPI undergoes further differentiation to form the three germ layers (ectoderm, mesoderm, and endoderm).

The anterior primitive streak that forms in the EPI gives rise to the mesodermal embryonic tissue, which further differentiates into cardiogenic mesoderm.

 

In spite of these basic similarities, the arrangement of cardiac progenitor cells in the cardiogenic mesoderm that give rise to the early heart tube diverges across species Ivanovitch et al., J. Cardiovasc. Dev. Dis., Nov 2017.

Namely, in humans, the cardiogenic mesoderm further forms two separate cardiac primordia bilaterally that fuse at the midline to create a primitive heart tube. By contrast, in mice, the cardiogenic mesoderm forms a single crescent-shaped primordium.

This species-specific divergence has repercussions for the study of CHD pathogenesis. For instance, the single crescent-shaped primordium in mice allows for a more continuous and integrated formation of the heart tube. Conversely, in humans, the heart tube formation requires a more complex process of fusion that involves precise coordination, which can lead to a higher susceptibility to CHD if the fusion process is disrupted.

In both species, the first heart field (FHF) forms the left ventricle and parts of the atria. The primitive heart tube then undergoes looping to establish the shape and orientation of the heart. The second heart field (SHF) contributes to the developing heart tube, forming the right ventricle, parts of the atria, and the outflow tract.

 

In mammals, the core program for heart developmental process is driven by a complex interaction of thousands of genes working into genetic regulatory networks (GRN).

However, the composition and the expression patterns of these GRN, which involve transcription factors, signaling pathways, and noncoding RNA, differ between humans and other species.

 

For instance, the specific sequences and regulatory elements of OCT4 have been found to differ between humans and mice, producing variations in how this gene crucial for maintaining pluripotency is regulated. Indeed, humans have distinct binding sites for transcription factors and human-specific distal enhancers for OCT4 that are not present in mice.

Only about 5% of Oct4 and Nanog transcription factors binding sites are shared across the genomes of mouse and human embryonic stem cells (ESC), suggesting that the regulatory networks governing pluripotency and gene expression in ESC are highly species-specific.

Additionally, human OCT4 levels are more sensitive to external signals such as growth factors, cytokines, and ROS, which can induce changes in heart development and potentially lead to CHD Zhang & Wu, CHD: Br. Heart, Jun 2024.

Species-specific transposon sequences make up a significant proportion of the remaining species-specific binding sites. For example, owing to the evolutionary history of endogenous retroviruses that were integrated into the human genome and co-opted for regulatory functions in human development, the HERV-H (Human Endogenous Retrovirus type H) transposon is specifically expressed in the human ICM, where it shapes human-specific stem cell gene expression.

While the transcription factor estrogen-related receptor beta (ESRRB) is expressed in certain human tissues, its role in maintaining pluripotency in human ESC is less prominent compared to its role in mouse ESC.

These numerous species-specific differences in transcriptional frameworks established from ZGA to blastocyst, and in the time frames of lineage formation, together indicate that understanding human pluripotency cannot simply be extrapolated from mouse studies and will require an in-depth analysis of the progression to pluripotency in the human embryo itself Rossant & Tam, Cell, Jan 2017.

 

Equally, in both humans and mice, the FGF/ERK signaling pathway influences the expression of transcription factors NANOG (for EPI) and GATA6 (for PE), that are critical for the segregation of the ICM into the EPI and PE lineages. However, the regulation, timing, duration and cross talk between FGF/ERK and other signaling pathways, such as Wnt, BMP and Hippo, that ensure precise control of cell proliferation, differentiation and cell fate decisions during organogenesis, differ across species Mossahebi-Mohammadi et al., Front. Cell Dev. Biol., Feb 2020.

 

Transcription factors such as NKX2-5, GATA4, FOXC1, MYH6 and TBX5 are expressed during heart tube formation, heart looping, chamber formation, valve formation, maturation and remodeling.

Some, like NKX2-5 and GATA4 are expressed by most cardiac precursors in the FHF and SHF, while others, like TBX5 are preferentially expressed in the FHF Ivanovitch et al., J. Cardiovasc. Dev. Dis., Nov 2017.

Pathogenic variants of these key transcription factors are associated with various CHD, including VSD and ASD. Their expression is conserved across species, nonetheless, species-specific differences exist in the timing, synergy, and regulatory mechanisms of expressions Al-Maqtari et al., PLOS One, Mar 2017.

 

MicroRNAs (miRNA) affect the final protein output through inhibition of translation and/or mRNA degradation. These miRNA act as fine-tuning modulators across numerous GRN that govern the species-specific heart anatomy, physiology and susceptibility to congenital and adult heart diseases.

For instance, the 22q11.2 deletion DiGeorge syndrome involves haploinsufficiency of the DGCR8 gene, which is essential for miRNA processing, leading to impaired miRNA production.

In combination with a background of haploinsufficiency for genes implicated in cardiac structure and function, miRNA can decrease the expression of genes that are haploinsufficient, exacerbating the phenotypic effects, or, on the contrary, balance the reduced gene dosage by targeting other genes and pathways.

Comparative analysis of miRNA expressed in the heart of 20 species representative of all major vertebrate groups, including humans, macaques, dogs, and mice, has allowed to identify dozens of miRNA with expression profiles that are specific to a single species or a group of closely related species Nachtigall et al., BMC Gen., Mar 2021.

 

These findings are consistent with GWAS that show that up to 95% of CHD-associated genetic variants reside in the noncoding genome that plays a role in the regulation of gene expression during heart development of the human fetus Vashisht et al., BMC Gen., Jan 2025. 

 

 

Species-specific differences in gene expression

Species-specific differences in genetic, epigenetic, transcriptional and post-transcriptional regulation have implications for understanding human-specific heart development, for elucidating mechanisms of congenital heart diseases, for identifying human-relevant therapeutic targets, and for developing safe and effective therapies.

 

Understanding how human genes are expressed and translated during organogenesis and adult function is crucial for understanding mechanisms of congenital heart diseases. Unfortunately, since the majority of gene expression studies are conducted in animal models, our knowledge of drivers of human heart pathologies remains poor. Recent advances in sequencing, in vitro and AI tools have enabled to bring to light numerous human-specific features of gene expression regulation.

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Species-specific epigenetic, transcriptional, and post-transcriptional regulation​

Cross-species comparison of DNA methylation, chromatin accessibility, and transcriptome of the adult human and mouse hearts, has revealed human-specific and mouse-specific differentially expressed genes (DEG) in the atria and the ventricles Gao et al., PLOS Bio., May 2021.

In total, 199 human-specific DEG were identified and found to be enriched in the gene ontology (GO) terms such as synaptic signaling and neurogenesis, while 150 mouse-specific DEG were revealed and shown to be related to ion transport. 

For instance, STAT4, of the signal transducer and activator of transcription (STAT) gene family, is a human-specific DEG that has been implicated in myocardial ischemic heart disease.

In the ventricles, human-specific and mouse-specific DEG with higher expression were localized in cardiomyocytes. In the atria, human-specific and mouse-specific DEG with higher expression were found in fibroblasts, neurons, endothelial cells, and cardiomyocytes.

In addition, comparison of binding motifs of transcription factors in human and mouse hearts has identified binding motifs enriched only in the human heart or only in the mouse heart, indicating that these transcription factors play species-specific roles in heart development, responses to environmental signals, epigenetic regulation and/or heart disease pathogenesis.

 

What is more, genes associated with several heart diseases were identified among 19 and 5 gene pairs that showed cis-regulatory relationships between long noncoding RNA (lncRNA) and protein-coding genes in the atria and the ventricles, respectively. For example, mutations in NKX2-6 have been linked to familial atrial fibrillation, mutation in KCNJ3 to hereditary bradyarrhythmias, and mutations in MYL2 to hypertrophic cardiomyopathy Gao et al., PLOS Bio., May 2021.

 

LncRNA have recently emerged as one of the key regulators of the heart development during embryogenesis and adult heart function.

The various lncRNA have been found to act through a variety of regulatory mechanisms, including epigenetic (by recruiting epigenetic modifiers to specific genomic regions), post-transcriptional (by influencing the stability, translation, or degradation of mRNA) and through other noncoding RNA regulators (by sequestering miRNA and preventing them from binding to their target mRNA).

In spite of presence of conserved regions, sequences of lncRNA were found to vary significantly between species. In organs of different species, including in the heart, species-specific lncRNAs were shown to have distinct expression profiles Sarropoulos et al., Nature, Jun 2019.

 

Humans are more genetically similar to NHP than to other species commonly used to model cardiovascular diseases. And yet, there are crucial differences between humans and other primates in gene regulation of cardiac development and function.

NHP do not have the same susceptibility to atherosclerosis and myocardial fibrosis. For example, chimpanzees are less prone to ischemic heart disease than humans. Comparison of transcriptional responses to hypoxia in humans and in NHP has revealed over hundred genes with species-specific expression and post-transcriptional modification in hypoxic condition that are likely to contribute to human-specific  vulnerability to heart diseases Ward & Gilad, eLife, Apr 2019.

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Species-specific gene dosage and gene redundancy​

The existence of inter-species differences in the number of copies of a particular gene and the presence of redundant genes with similar or overlapping functions can produce misleading findings.

For example, while Nkx2-5 is essential for cardiac development in both mice and humans, Nkx2-6 provides additional redundancy in mice, which is not observed in humans Srivastava & Olson, Nature, Sep 2000.

In contrast to humans, zebrafish often have two different genetic loci encoding slightly different versions of the same gene, including those that play a role in cardiac development Pierpont et al., Circulation, Sep 2018. As a result, these duplicate genes, known as paralogs, can have redundant, complementary, or even distinct functions. In zebrafish, for instance, genes coding for transcription factors nkx2.5 and tbx5, which are crucial for formation of the heart tube, atria, ventricles, and septa, have paralogs that can compensate for each other.

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Human-specific cardiac genomic innovation​

Characterization of the cardiac transcriptional and translational profiles of humans, chimpanzees, gorillas, rhesus macaques, mice and rats, using RNA sequencing and Ribo-seq, has brought to light hundreds of species-specific and lineage-specific genomic innovations that emerged during primate evolution Ruiz-Orera et al., Nature Cardiovasc. Res., Sep 2024.

In essence, the three classes of human cardiac genomic innovations are young small open reading frames (sORF), young genes and genes with human-specific cardiac-enriched expression.

 

These include 551 genes and 504 small open reading frames (sORF) microproteins that are uniquely expressed and translated in human myocytes.

Moreover, 76 evolutionarily conserved genes were found to display recently evolved human-specific cardiac-enriched expression.

Of particular relevance for susceptibility of humans to heart diseases, the overwhelming majority of human young genes, young sORFs and genes with recently acquired cardiac-enriched expression were downregulated in tissues derived from patients with dilated cardiomyopathy.

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Human-specific up/down-regulation of cardiac genes ​

When translational efficiencies (TE) ratios of normalized Ribo-seq and RNA-seq counts per coding sequences were compared across mammalian hearts, mitochondrial oxidative phosphorylation (OXPHOS) complexes IV (cytochrome c oxidase) and V (ATP synthase) displayed the highest TE variation, suggesting species-specific cardiac adaptations to metabolic needs.

Comparison of upregulated or downregulated cardiac genes across primate species only, has shown that cardiac-specific sarcomere proteins MYH6, MYL2 and MYOZ2 were significantly higher in humans than in other primates. The ratios of MYH7 (beta-myosin heavy chain) : MYH6 (alpha-myosin heavy chain) and MYL2 (myosin light chain 2) : MYL7 (myosin light chain 2) play significant roles in heart contraction, particularly in the context of adaptation to physiological and pathological conditions. An increased MYH7:MYH6 ratio is often observed in heart failure while an increased MYL2:MYL7 ratio has been associated with improved contractility and maturation of cardiac tissues Ruiz-Orera et al., Nature Cardiovasc. Res., Sep 2024.

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Recent evolutionary acquisition of human cardiac-enriched genes​

Among evolutionary conserved genes, 76 were found to have recently acquired cardiac-enriched expression in humans, where they exert a function in the heart, are associated with genetic predisposition with cardiac diseases and serve as potential cardiac biomarkers or therapeutic targets Ruiz-Orera et al., Nature Cardiovasc. Res., Sep 2024.

In the same manner, there are human cardiac-enriched genes that are enriched in non-cardiac tissues of other species, suggesting a switch in patterns of tissue expression across evolution.

 

For example, in humans, the gene SGLT1 is expressed in the heart, but in NHP, rats, and mice, it is expressed only in the kidneys. The beneficial effect of dual inhibitors of SGLT1 and SGLT2 in treating heart failure was discovered serendipitously, in course of treatment of T2DM patients with kidney disorder. Had the efficacy of this treatment been tested in rodents and NHP, a promising therapeutic lead would have been discarded as false negative.

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