
Congenital heart disease
ICD-10 Code Q21-Q28
What is the clinical spectrum of Congenital Heart Disease?
The term congenital heart disease (CHD) covers all structural heart defects present at birth, singly or in combination.
Patients with CHD present with an abnormal structure of the cardiac chambers, valves, or great vessels that alters the normal pattern of blood flow.
Accounting for nearly one-third of all major congenital anomalies, CHD are the most prevalent human birth defects and the leading cause of infant mortality. The global prevalence of CHD is generally considered to be 9 per 1000 live births van der Linde et al., JACC, Nov 2011.
Congenital heart defects can have severe consequences on individuals' development, cognition, learning, behaviour, physical fitness, educational and employment opportunities Russell et al., JAHA, Mar 2018.
Individuals with CHD may develop cardiac complications such as endocarditis, supraventricular arrhythmias, ventricular arrhythmias, myocardial infarction, conduction disturbances, and pulmonary hypertension, even after surgical correction of the structural abnormalities.
The median age at death is variable depending on the severity of the heart defect, the number of complications, as well as age and gender. Nevertheless, the overall lifespan of individuals treated for CHD is reduced compared to the general population, particularly in the young. Death is commonly due to heart failure or sudden death Verheught et al., Europ. Heart J., May 2010.
While almost 90% of CHD patients are alive at age 20, for some severely complex CHD forms, such as truncus arteriosus and single ventricle, the survival rate is much poorer Tennant et al., Lancet, Feb 2010.
CHD has heterogenous presentations according to the varying simple, moderate and severe complexity of structural lesions Sabanayagam et al., Heart Fail. Clin., Aug 2018.
An example of moderate CHD is the ventricular septal defect (VSD), which accounts for about a fifth of all CHD. In VSD, a hole is present in the ventricular septum that normally separates the right ventricle from the left ventricle, enabling blood to flow between the left and the right ventricles, and causing left-to-right side abnormal pathways of blood flow called shunts. The severity of symptoms depends on the size of the hole. Most cases of small VSDs are asymptomatic, whereas those with larger holes can lead to symptoms of tachypnea, tachycardia, poor feeding and growth, and recurrent respiratory infections.
Long-lasting defects that cause left-to-right shunts like VSD, atrial septal defects (ASD), and patent ductus arteriosus (PDA), can lead to the Eisenmenger syndrome, classified as severely complex CHD. This is because, in recurrent left to right shunting, more blood flows into the pulmonary vasculature, leading to pulmonary hypertension, and ultimately to a reversal of shunt from right to left. The subsequent entry of deoxygenated blood into the systemic circulation can cause cyanosis, dyspnea on exertion, fatigue, syncope, and heart failure.
In the case of Tetralogy of Fallot (TOF), the shunt occurs the from right-to-left. This CHD has four main abnormalities: an outlet VSD, with a hole below the level of the aortic and pulmonary valves, a pulmonary stenosis or right ventricular outflow tract obstruction, a right ventricular hypertrophy, and an overriding aorta positioned directly over the VSD.
Typical clinical features of TOF are progressive cyanosis after birth, development of exertional dyspnea with episodes of severe cyanosis and hypoxia, known as "tet spells". If left untreated, this condition can lead to developmental delays due to reduced oxygen delivery to the body, pulmonary hypertension, arrhythmia, heart failure, and sudden cardiac death.
What do we know about the etiology of Congenital Heart Disease?
Cohort studies show that both genetic and environmental factors contribute to the development of congenital heart disease (CHD).
Owing to the complexity of genetic and environmental interactions, identifying causes of all CHD cases remains challenging.
Maternal chronic diseases, infections and medications are estimated to generate a 2% to 10% increase in risk of CHD Haxel et al., Pediatrics, Nov 2022.
The most frequent maternal diseases associated with CHD include pregestational diabetes, gestational diabetes diagnosed in the first or early second trimester, phenylketonuria, autoimmune disease such as Sjogren syndrome, and first trimester rubella infection.
Cohort study has shown that both T1DM and T2DM maternal pregestational diabetes was associated with a four-fold increase in risk of CHD in newborns. Oyen et al., Circulation, May 2016.
It is believed that hyperglycemia may have a teratogenic effect through the same signaling pathways that regulate insulin sensitivity and embryogenesis. In addition, it is possible that glucose may also affect gene expression in embryonic development via epigenetic changes such as histone acetylation.
Drugs that have been associated with an increased risk of cardiac malformations include retinoic acid, carbamazepine, paroxetine, angiotensin-converting enzyme inhibitors, and lithium Patorno et al., NEJM, Jun 2017.
Causes of growth failure and neurodevelopmental deficits in CHD are multi-factorial and involve patient‐specific genetic factors, maternal‐fetal environment abnormalities, cardiac developmental defects, and perioperative injuries Russell et al., JAHA, Mar 2018.
Likewise, causes leading to HF in CHD are diverse and include abnormal pressure of the right ventricle or left ventricle, myocardial ischemia, genetic predisposition, fibrosis and cardiac tissue damage during surgery Sabanayagam, Heart Fail Clin, Aug 2018.
Risk factors of genetic origin were shown to be variable, resulting in variable phenotypes Russell et al., JAHA, Mar 2018.
Importantly, as much as 70% of CHD cases do not have an identifiable genetic etiology.
Certain aneuploidies, such as trisomy 21, trisomy 18, trisomy 13, and monosomy X, account for 5 to 10% of patients with CHD.
Other cases of genetic contributions to CHD involve sub-chromosomal deletions and duplications, or monogenic mutations.
Copy number variants (CNVs) are often associated with congenital syndromes that affect multiple organs in addition to the heart, such as in DiGeorge syndrome (DGS), also known as 22q11.2 deletion syndrome, and Williams-Beuren syndrome with a deletion in 7p11.23 chromosomal region.
Rare monogenic pathogenic variants that cause syndromes associated with CHD include mutations in fibrillin-1 (Marfan syndrome), TXB5 (Holt-Oram syndrome), and PTPN11 (Noonan syndrome), that are typically inherited in an autosomal dominant manner. It is estimated that about 20 to 30% of syndromic CHD cases are due to monogenic pathogenic variants Maddhesiya & Mohapatra, Curr. Cardio. Rep., Feb 2024.
Monogenic mutations and CNVs also cause CHD that are non-syndromic - without any other associated abnormalities, although non-syndromic cases are less frequently linked to CNVs Pierpont et al., Circulation, Sep 2018.
In some instances genetic risk factors are common variants that occur frequently in the general population and that singly have a small effect on an individual's risk of developing CHD but that can contribute to CHD when combined with other common variants, epigenetic changes, and environmental factors.
Exome sequencing of probands and their unaffected parents has revealed that about 8-10% of both syndromic and non-syndromic CHD is caused by de novo coding variants. De novo genetic mutations can possibly occur as an error or under the influence of environmental factors during the embryonic development. In CHD, they are often related to genes that are essential for formation of a functioning heart anatomy, such as those coding for sarcomeres, Notch signaling pathways and primary cilia Jin et al., Nature Gen., Oct 2017.
Adding to the complexity of causal analysis are patient-specific pleiotropic, oligogenic and gene modifier effects that produce similar disease phenotypes for a variety of pathogenic variants as well as variable disease phenotypes even within the same family Pittman et al., BioRxiv, Apr 2022.
Moreover, genome-wide association studies have found that 90–95% of CHD-associated genetic variants reside in the noncoding genome.
Identification and analysis of CHD-associated single nucleotide polymorphisms (SNPs) that span noncoding regions of the genome indicates that these SNPs can potentially cause CHD by disrupting the gene regulation during heart development of the fetus Vashisht et al., BMC Gen., Jan 2025.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Face validity - How well does an animal model replicate the human disease phenotype?
No single animal model, whatever the animal species and experimental method employed, faithfully recapitulates the phenotypes, heterogeneity, complexity and severity of human congenital heart diseases (CHD).
Animal models of spontaneous CHD
Certain CHD types, such as DiGeorge syndrome (DGS) and Williams syndrome (WS), are unique to humans or have not been naturally observed in animals.
Other CHD were found to occur spontaneously in certain animal species, albeit at a varying frequency, indicating that susceptibility to specific CHD type is determined by species-specific factors.
In spite of their high degree of similarity to human CHD, clinical presentation in animal CHD may diverge.
For example, naturally occurring CHD in dogs, such as patent ductus arteriosus, pulmonary stenosis, and valvular aortic stenosis, show similarities to clinical features in humans Crofton et al., Cardiol Res., Oct 2023. Yet, presentations are not always identical, since human aortic stenosis typically occurs at the valve itself, while in dogs the fibrous tissue is commonly caudoventral to the valve.
The existing inter-species similarities in CHD phenotype have, unfortunately, not translated to a precise knowledge of disease mechanisms and to disease modifying treatments, neither for dogs nor for humans with valvular aortic stenosis.
In cats, it is the ventricular septal defect (VSD) that is the most common. In contrast to human VSD, defects are often small in cats with VSD and may not cause significant clinical symptoms Tidholm et al., J. Vet. Cardiol., Dec 2015.
Bicuspid aortic valve (BAV), in which there are two abnormal instead of three normal leaflets that regulate the blood flow into the aorta, is the most common CHD in humans. BAV is frequently associated with the development of thoracic aortic aneurysm (TAA) that can lead to internal bleeding and potentially life-threatening complications.
In humans, the most common type of BAV is Type A BAV, where the two commissures are located in an anteroposterior direction, resulting in left and right leaflets.
In contrast to the general human population, BAV occurs spontaneously only in certain animal species, only in certain anatomical/morphological types and only rarely Fernandez et al., Vet. Pathol., Feb 2020.
Hamsters that develop spontaneous BAV have BAV type A only, whereas genetically modified mouse strains have both type A and type B, with type B BAV being the predominant.
The Type A BAV in humans and hamsters have an anteroposterior orientation, while the Type B BAV in mice has a left-right orientation.
Between the tricuspid and the bicuspid condition, the aortic valve of hamsters shows a wide spectrum of intermediate morphologies, characterized by different degrees of fusion of the valve leaflets and presence of raphes of different size. The width of spectrum of intermediate BAV morphologies in hamster is more similar to BAV in humans than to BAV in mice.
Inter-species differences in BAV morphology are likely to affect susceptibility to disease symptoms. Even though both type 1 BAV in humans and type A BAV in animals are associated with complications like aortic stenosis, the extent, severity and progression of these complications are likely to differ based on species-specific physiological and genetic factors.
CHD in human mothers with chronic autoimmune diseases, such as Sjögren syndrome (SS) and systemic lupus erythematosus (SLE) are primarily linked to the presence of specific autoantibodies that can cross the placenta and cause inflammation, tissue damage and fibrosis in the fetal cardiac conduction system Moutasim et al., Int. Arch. Med., Jul 2009.
Spontaneous occurrence of SS in animals was not reported. Non-obese diabetic (NOD) mice are used as a model for SS, however, there is no evidence to suggest that NOD mice naturally develop CHD.
Although SLE can naturally occur in dogs, affecting various organs like joints and kidneys, it is not typically associated with CHD.
Genetically-engineered animal models of CHD
Experimental methods targeting genes that are involved in heart development and function, such as GATA4, NOTCH1, and TBX5, have succeeded in partially mimicking the phenotype of septal defects (ASD, VSD), outflow tract defects (TOF), and valve malformations (BAV) in mice McKellar et al., J. Thor. Cardiovasc. Surg., Aug 2007, Koening et al., JCI Ins., Nov 2017.
Mouse models of Williams syndrome (WS) engineered to contain heterozygous ELN deletion Osborne, Am. J. Med. Genet. C. Semin. Med. Genet., May 2010, presented with several hallmarks of WS, butt not the structural cardiac hypertrophy, conduction defects, and neurocognitive symptoms, to mention a few.
The syndromic features and coronary artery anomalies were not recapitulated in mouse models of DiGeorge syndrome either, despite numerous attempts, including by deleting a portion of the mouse chromosome 16B that is homologous to the human chromosome 22q11, by engineering null mutations of the Tbx1 gene, and by overexpressing several transgenes Baldini et al., Hum. Mol. Gen., Oct 2002.
Chemically-induced animal models of CHD
In rodent models of pregestational diabetes mellitus-induced CHD, streptozotocin (STZ) is administered in female mice and mouse embryos are cultured in high-glucose concentrations, to study the effects on formation of the left-right axis Hachisuga et al., PNAS, Sep 2015.
Animal models obtained in this manner frequently employ overtly aggressive diabetic conditions. The high dose of STZ to trigger pancreatic beta cells death and insulin deficiency, as well as concentrations of blood glucose in mice twice that of diabetic patients, lead to exaggerated cytotoxic phenotypes that are not relevant to the human clinical condition Singh, Front. Endocrinol., Feb 2024, Pandey et al., Biomed., Oct 2023.
It is also troubling that STZ does not induce insulin resistance, metabolic syndrome and insulin-secreting beta cells dysfunction, which are key features of T2DM. What STZ does induce in animals are symptoms that are not relevant for T2DM, such as decreased blood pressure and resting bradycardia. Certain CHDs, such as coarctation of the aorta and hypoplastic left heart syndrome do show decreased blood pressure but these are less commonly associated with CHD and can represent a hindrance for studying pregestational diabetes mellitus-induced CHD with high blood pressure.
Human thoracic aortic aneurysm (TAA) was modelled in animals by elastase, calcium chloride and angiotensin II induction Lindeman & Matsumura, Circ. Res., Feb 2019.
In the elastase method, porcine pancreatic elastase that degrades the key structural protein in the aortic wall, elastin, is infused into the aorta through a surgical incision. Calcium chloride can be administered into the perivascular space of the animal's aorta of rodents. Angiotensin II is infused into animals to induce hypertension and progression to heart failure.
While some of the pathophysiological features of aneurysm may be partially reflected, these chemically-induced TAA animal models do not fully reflect the human condition.
For example, the hallmarks of human TAA, aortic thrombus, atherosclerosis, and rupture, are absent in calcium chloride-induced TAA animal models.
The infusion of elastase alone was not sufficient to induce formation of aneurysm in rats, and it is believed that variation in phenotype of rodent models of aneurysm might be due to contaminants from porcine pancreatic tissue Carsten et al., J. Vasc. Surg., Jun 2001.
Surgically-induced animal models of CHD
Some animal models of CHD are developed by surgical inductions, such as by surgical creation of septal defects or by surgical altering of blood flow patterns.
For instance, aortic banding, where a band is placed around the aorta to create a partial obstruction, is commonly used to in animals to mimic hemodynamic changes seen in aortic stenosis.
Sternotomy is used to simulate surgical corrections of CHD, such as septal defects or valve abnormalities, and to mimic CHD-related complications.
Surgical endocarditis animal models are used to study congenital defects that predispose individuals to endocarditis, such as bicuspid aortic valves.
The response to surgical inductions are inconsistent across animal experiments, limiting the applicability of findings to humans Farag et al., Front. Vet. Sci. , Mar 2023.
Construct validity - How well do the mechanisms of disease induction in animals reflect the currently understood etiology of the human disease?
It was advanced that animal models were needed to better understand mechanisms of congenital heart disease (CHD), however, because of countless human-specific features covered in previous sections, insights obtained from animal studies have not translated to major benefits for CHD patients.
Animal models of spontaneous CHD
Since the underlying genetic, environmental, and epigenetic factors that lead to the formation of bicuspid aortic valve (BAV) differ between humans, hamsters, and mice, it is comprehensible that, to date, mechanisms that underly BAV remain unknown for most patients Fernandez et al., Vet. Pathol., Feb 2020.
It has been shown that rare sequence variants of GATA5 in humans are associated with the development of both BAV types A and B, whereas Gata5-defective mice develop only BAV type B, implicating intervention of additional factors in BAV pathogenesis, probably human-specific genetic modifiers.
Genetically-engineered animal models of CHD
In many cases, the human CHD gene defects were not correctly modeled in animals, since they were genetically engineered as homozygous gene knockouts instead of heterozygous point or truncating mutations.
Moreover the existing inter-species differences in the number of copies of a particular gene and the presence of redundant genes with similar or overlapping functions, are likely to have affected gene expression levels or or have provided resilience against loss of function in animal models. For example, in contrast to DiGeorge syndrome patients, Tbx1 haploinsufficiency is well tolerated in mice Pierpont et al., Circulation, Sep 2018.
Hypotheses on etiologies of human genetic disorders were often formed on the basis of similarities between the animal model phenotype and the human disease phenotype Baldini et al., Hum. Mol. Gen., Oct 2002. This approach is likely to have produce misleading hypothesis, since, depending on the animal species employed, an accurate disease mechanism can produce an unmatching phenotype, and an inaccurate disease mechanism can produce a matching phenotype.
For instance, transgenic mice with altered Hox A3 or Pax 3 expression exhibit some of the phenotypes that match DGS patients, such as cardiovascular defects and thymic hypoplasia, however, neither Pax 3 nor Hox A3 are implicated in the cause of human DGS. The existence of species-specific differences in genetic and molecular developmental pathways further shed doubt on construct validity of genetically engineered animal models of CHD.
Numerous mouse models have been generated over past decades in a bid to identify genetic changes that underly Williams syndrome (WS) Osborne, Am. J. Med. Genet. C. Semin. Med. Genet., May 2010. Yet, not all genes that are haploinsufficient in humans proved to be so in mice. In humans, WS is caused by deletion of a contiguous set of over 20 genes on chromosome 7. The size of the deletion can vary, and the specific genes deleted can influence the severity and range of symptoms. This human-specific complexity is not captured in mice, since they do not contain patient-specific genetic backgrounds, variations in deletion size, and combinatorial effects of multiple genes.
It seems equally unlikely that genetically engineered animal models of Sjögren syndrome (SS) and systemic lupus erythematosus (SLE) could yield human-relevant insights into mechanisms by which chronic autoimmune diseases induce CHD. In particular in the light of marked inter-species differences in innate and adaptive immunity Mestas & Hughes, J Immunol, Mar 2004, and in regulation of expression of immune system-related genes Yue et al., Nature, Nov 2014.
It was suggested that using animal models with a humanized immune system might improve translatability to humans, however, such approach would need to overcome too many unsurmountable hurdles: the equivalence to the human immune system was never demonstrated by objective measures, and the human-specific cross-talk between the human immune system and other human organ systems cannot be recapitulated in partially humanized animals.
Chemically-induced animal models of CHD
In STZ rodent models of pregestational diabetes-induced CHD, hyperglycemia is produced mainly through direct cytotoxic action on beta pancreatic cells rather than through a number of intermediate steps that lead to autoimmunity in TDM Singh, Front. Endocrinol., Feb 2024, Pandey et al., Biomed., Oct 2023. This method may therefore fail to capture the progressive cardiac remodeling in response to hyperglycemia. In addition, the administered chemicals could potentially be toxic for animal organs other than the pancreas, creating confounding adverse effects, higher morbidity, and high mortality.
Species-specific differences in glucose regulation, gene expression regulation, and immune system represent an additional barrier for understanding mechanisms of diabetes-induced human CHD.
Although thoracic aortic aneurysm (TAA) is recapitulated in animals via infusion of angiotensin II, it was also found to cause aortic dissections. Consequently, insights on disease mechanisms in animal models may diverge depending on whether one looks at aortic aneurysm or aortic dissection as cause of aortic rupture Carsten et al., J. Vasc. Surg., Jun 2001.
Moreover, the disease mechanisms of experimental induction in animals do not mimic the intricate interplay of genetic, physiological and environmental factors in human TAA. For instance, while the loss of elastin is a significant factor in the weakening of the aortic wall in human TAA, it is not the sole cause of aortic wall failure.
The use of inadequate animal models of aortic aneurysms have caused great damage to drug discovery Lindeman & Matsumura, Circ. Res., Feb 2019, and continue to do so, underscoring the need for an urgent shift to a human-based approach in biomedical research.
Surgically-induced animal models of CHD
Surgical induction of CHD does not accurately replicate the multifactorial basis of human CHD, and is therefore unlike to provide human-relevant insights on CHD disease mechanisms.
Moreover, the pain and psychological distress experienced by animals in surgical experiments profoundly affect the electrophysiology of the heart, producing misleading results. The use of anxiolytics, sedatives, analgesics, and anaesthesia during surgical procedures in animals can also produce electrophysiology-altering effects.
Predictive validity – How well do animal models predict safety and efficacy of therapies in human patients?
The predictive validity of animal experiments for cardiovascular diseases is poor and overall lower than the average for all indications BIO, New Clin. Dev. Succ. Rat., 2011-2020.
The likelihood for approval of drugs for heart conditions was only 4.8% over 2011-2020.
While efforts to optimize clinical practice and perioperative care have resulted in small incremental improvements, they have not led to major advances in clinical outcomes for CHD patients.
In addition, testing new treatments for infants with CHD is particularly challenging because of ethical complexities, risks for infants' development, rarity of CHD cases, and heterogeneity of CHD forms.
Patients with CHD benefit from drugs that manage cardiac hypertension, pulmonary hypertension, arrhythmia and HF Loss et al., Front. Pediatr., Nov 2021.
Many drugs used to treat CHD are also used for patients with acquired heart diseases as some of the mechanisms overlap. These treatments often include diuretics like furosemide and spironolactone to reduce the strain on the heart by removing excess fluid from the body, angiotensin-converting enzyme (ACE) inhibitors like captopril to relax blood vessels and improve blood flow, and negative inotrope beta-blockers like carvedilol and metoprolol to slow the heart rate.
Prostaglandin E1 helps keep the ductus arteriosus open in certain CHD, ensuring proper blood flow until surgery can be performed.
Vasodilators such endothelin receptor antagonist Bosentan and phosphodiesterase type 5 inhibitor Sildenafil are used for postoperative hypertension and treatment of CHD with pulmonary hypertension.
Positive inotropes such as dopamine and dobutamine are particularly used in cases where there is severe HF or myocardial infarction.
There is ongoing research for safety and effectiveness of newer drugs, such as angiotensin receptor-neprilysin inhibitor sacubitril/valsatran and sodium-glucose cotransporter-2 inhibitors, for treating children with CHD and HF.
Each of these treatments can be used alone or in combination, depending on the specific type of CHD, severity of patient's symptoms and patient's overall health.
Surgery is the mainstay of treatment for CHD. In many cases such as tetralogy of Fallot, transposition of the great arteries, and Ebstein's anomaly, surgical repair is the only definitive treatment.
Depending on the complexity and severity of the defect type, the result of surgery can be curative, reparative or palliative Geyer et al., Eu. J. Cardio. Prev. & Rehab., Feb 2007.
In contrast to patients with small PDA and ASD who rarely have long-term sequelae following surgery, patients with tetralogy of Fallot or anomalous pulmonary venous drainage can suffer from lifelong sequelae despite improvement after surgical correction.
Surgery in patients that suffer from severely complex CHD forms such as Eisenmenger syndrome, tricuspid atresia or single ventricle, does not fully correct the underlying defect, as consequence of which, treated patients may have significant lifelong impairment of function.
Some severe cases of CHD are treated in utero through fetal interventions, including balloon valvuloplasty, minimally invasive fetoscopic surgery and transplacental medications Keller et al., Medsc., Oct 2023, Haxel et al., Pediatrics, Nov 2022.
As the risk of fetal demise secondary to in-utero intervention is not negligible, it is carefully weighed against the risk of postnatal morbidity and mortality.
Numerous attempts of in-utero surgery in lambs, pigs and dogs have not translated to clear benefits for CHD patients. For example, fetal cardiac bypass in lambs have resulted in placental vascular resistance, cytokine activation, metabolic acidosis, hypoxia and death. Subsequently, open fetal cardiac surgery was largely abandoned.
Fetal pacemaker placement has been studied in a sheep model of congenital heart block, but its risk-benefit ratio for humans is currently unknown Keller et al., Medsc., Oct 2023.
Even though preclinical studies had shown promising results, there are concerns about the safety and efficacy of maternal therapy with intravenous immunoglobulin and fluorinated glucocorticoids to prevent maternal Sjögren syndrome/systemic lupus erythematosus-associated congenital heart block in fetus, particularly regarding the risk of neurotoxicity, stillbirth and growth restriction Phithakwatchara et al., J. Fet. Med., Jul 2017, Moutasim et al., Int. Arch. Med., Jul 2009.
Despite being necessary and lifesaving in many cases, surgical correction is not without risks for patients (mechanical injury during surgery, inadequate myocardial protection, ischemia-reperfusion injury) and can have long-term consequences.
Even when surgical treatment corrects the anomaly, patients may still have late complications, requiring careful monitoring and adapted treatments. For instance, atrial arrhythmias occurs in 15% of adults with CHD, which is nearly 3 times higher than in the general population Bouchardy et al., Circulation, Oct 2009.
Subvalvular aortic stenosis (SAS) is the most common CHD in dogs and is also prevalent in human children. It was expected that treatments for SAS developed in dogs would be efficient for humans, however, due to differences in anatomy, in physiology, in risks, and in potential complications, treatment of SAS in humans differs substantially from that in dogs.
Humans are generally treated surgically, whereas dogs are generally treated medically. In canine SAS patients, the median survival time with aortic balloon valvuloplasty is not significantly different than that with medical management with atenolol. In human medical practice, antihypertensive treatment with beta-blockers is uncommon in patients with severe aortic outflow tract obstructions, as it can often result in left ventricular dysfunction and hemodynamic compromise. By contrast, these results are not commonly reported after administration in canine patients, making atenolol a viable treatment option for this species Crofton et al., Cardiol Res., Oct 2023.
Further improvement in survival and quality of life of CHD patients will require a better understanding of pathogenicity of genetic variants of uncertain significance, of patient-specific underlying disease mechanisms and of risk factors Russell et al., JAHA, Mar 2018.
Ethical validity - How well do animal experiments align with human ethical principles?
Preclinical
Ethics is a human-specific philosophical concept. Animal experimentation is unethical in essence by human standards, since it involves physical constraint, psychological suffering and deprivation of freedom, social interactions, natural environment and life purpose.
In addition to this baseline, animal experiments inflict severe clinical harm in animals De Vleeschauwer et al., Animals, Aug 2023:
Table S4: Severity classification of clinical signs
Dyspnea, tachypnea and cyanosis: up to severe clinical signs
Cardiac function in zebrafish: a strongly reduced or erratic heart rate, frequently irregular heart rhythm: severe clinical signs
Table S5: Severity classification of chemical disease models
Hypertension: Rodent hypertension causing no more than moderate comorbidities
Table S13: Severity classification of genetically altered (GA) lines
Cardiomyopathy - Global heart failure with permanent respiratory distress and impairment of the general condition: severe clinical signs
Growth and body weight - severe growth retardation (runting): severe clinical signs
Mortality for rodent pups and rodents older than 2 weeks: up to severe
Table S8: Severity classification of other disease models
Hypoxia-induced pulmonary hypertension: moderate clinical signs
Table S3: Severity classification of surgery and surgical induction of disease
Surgery causing permanent or progressive heart failure (e.g., aortic banding, large myocardial infarction): severe clinical signs
Organ/cell transplantation or device testing where rejection/failure may lead to severe distress, death or impairment of the general condition of the animal (e.g., implantation of artificial heart or cardiac valve): severe clinical signs
Surgery intentionally causing severe infection or sepsis (e.g., cecal ligation and punction): severe clinical signs
Sternotomy: severe clinical signs
Major surgery without proper analgesia: severe clinical signs
Clinical
While there is no consensus on whether an unethical act can be justified by a pursuit of a hypothetically ethical outcome, it was suggested that animal research was necessary to advance efficient and safe treatments for human diseases.
However, statistics consistently show that clinical success rates of drugs developed and tested in animals is very low, raising the question of whether it is ethical to put the health of human patients at risk.
Phase I to II transition success rate, that primarily focuses on assessing the safety of drugs, was 50% for cardiovascular diseases over 2011-2020, below the average of all indications BIO, New Clin. Dev. Succ. Rat., 2011-2020.
With only 21% phase II to III transition success rate versus 28.9% average for all indications, cardiovascular diseases group is the 2nd lowest performing disease group in phase II to III transitions, underscoring the low effectiveness of drugs for cardiovascular diseases.
Intrinsic validity - How well do animal models capture the clinical heterogeneity of the human disease?
Animal models of congenital heart disease (CHD) do not recapitulate the human-specific inter-individual heterogeneity in clinical features and pathophysiology.
Extrinsic validity - How well does animal experimentation generate reliable and reproducible outcomes?
It is often argued that although animal models have severe limitations, experimenting on animals enables to gather insights that may be valuable. However, the basic precondition for a hypothetical benefit is not met since the majority of animal experiments is irreproducible Freedman et al., PLOS Biol., Jun 2015.
Contributing factors include flawed experimental design, variation in animal strains and experimental conditions, and lack of transparency on methodology and results of animal studies.
In a bid to improve the quality of reporting of animal experiments, the ARRIVE - Animal Research: Reporting of In Vivo Experiments - guidelines, were published in 2010 and updated to ARRIVE 2.0 in 2020 Arrive guidelines website.
Nonetheless, and in spite of significant investment in dissemination, various incentives and training of animal researchers, the Arrive guidelines remain poorly implemented Percie du Sert et al., BMC Vet. Res., Jul 2020, Bazoit, BioRxiv, Feb 2025.
As a result of weak relevance, rigor and reliability of animal studies, erroneous and misleading hypothesis are generated, animal and human lives are needlessly sacrificed and dozens of billions of dollars are annually wasted Yarborough et al., PLOS Biol., Jun 2018.
Beyond the problem of experimental design and reporting of animal studies, there are deep-seeded cultural reasons that are not likely to be addressed any time soon, such as the "publish or perish" culture that discourages from unbiased analysis and reporting of negative results and rewards exaggerated and overhyped claims Smaldino & McElreath, Roy. Soc. Op. Sci., Sep 2016.
In Summary
Congenital heart diseases (CHD) are characterized by structural heart defects present at birth. Depending on the complexity of structural lesions, CHD can have severe consequences on individuals' development, health, and lifespan.
Animal models of CHD do not faithfully recapitulate human-specific CHD phenotypes, heterogeneity, complexity and severity.
The multifactorial etiology of human CHD types is not captured in spontaneous, chemical, genetic and surgical animal models.
Inter-species differences in heart anatomy, development, electrophysiology, and gene expression represent a major barrier for understanding precise mechanisms that underpin individual CHD types.
While efforts to optimize clinical practice and perioperative care have resulted in small incremental improvements, they have not led to major advances in clinical outcomes for CHD patients.
At the same time, the severity of pain and distress in animals that are used for CHD modeling and testing treatments is extremely high, frequently raising ethical concerns.
A better understanding of human-relevant risk factors and patient-specific disease mechanisms can further improve prevention, survival and quality of life of individuals with CHD.
How is Human-Based In Vitro Testing the Answer to advance biomedical research into Congenital Heart Disease
*To model heterogenous presentations of CHD with varying complexity of structural defects, by using 3D bioprinting and microphysiological systems to fabricate heart models with specific CHD defects (hole, ischemia, tract obstruction).
*To study the effects of alterations in blood pressure, blood flow patterns, and other hemodynamic parameters, in CHD such as ventricular septal defect, on cardiac signaling pathways and electrophysiology, by simulating blood flow and pressure across a septal defect in a heart-chip.
*To study the consequences of CHD, such as reduced oxygen delivery to the body, on individuals' physical and cognitive capacities, by using multi-organ-chip with heart connected to muscle/brain.
*To identify new biomarkers of cardiac complications (arrhythmias, myocardial infarction, conduction disturbances) through multi-omics in cells derived from CHD patients with varying heart defects, complications, ethnicity, age and gender.
*To understand the impact of maternal chronic diseases on CHD development, severity and complications, by culturing the maternal iPSC-derived cardiac progenitor cells, that capture the donor's genetic and epigenetic background, under conditions that mimic pregestational diabetes, phenylketonuria or autoimmune diseases (glucose, ROS, phenylalanine, immune cells) Kostina et al., Stem Cell Rep., Mar 2024.
*To study individual and combined effects of genetic mutations on cardiogenesis and cardiac function, by using CHD patient-derived, gene silenced/knocked-out and healthy human isogenic cell lines Inacio et al., Cells, Feb 2023, Ang et al., Cell, Dec 2016, Liu et al., Gen. & Dis., Jul 2023.
*To identify new common and rare genetic variants that underpin CHD cases that do not have an identifiable genetic etiology, by correlating genetic data (sequencing, CRISPR gene editing/silencing) with phenotypic outcomes in vitro (myofilament structure, electrophysiology, contractility, elasticity, force-frequency relationship) Wu et al., Circ. Res., Aug 2019.
*To elucidate mechanisms by which combined genetic and environmental factors produce patient-specific specific CHD phenotypes, by complementing the cell culture of healthy human/CHD patient-derived tissues/heart organoids/heart-chip with epigenetic regulators, chemical agents, glucose, drugs, microbes.
*To explore epigenetic changes in chromatin accessibility and active transcription in CHD, in response to environmental factors, or during stages of human cardiogenesis, by conducting assay for transposase-accessible chromatin (ATAC-seq) and assay for histone H3 lysine 4 trimethylation (H3K4me3) in CHD patient iPSC-derived atrial/ventricular cardiomyocytes Liu et al., Gen. & Dis., Jul 2023.
*To study human-relevant phenotypic expression, kinetics, function and interaction of ion channels, ion exchangers, accessory proteins and regulatory auxiliary proteins, by qualitative and quantitative analysis (multi-omics, MEA, patch-clamp, genetically encoded indicators) in healthy human and CHD patient-derived cardiac tissues/cardiac organoids/heart-chip.
*To assess CHD type-specific functional properties of the heart, by real-time measurement of heart rate (MEA), cardiac contractile kinetics (force transducers, traction force microscopy, Ca imaging) and energetic efficiency (oxygen consumption, ATP production), in CHD patient-derived tissues/cardiac organoids/heart-chip.
*To study how abnormalities in neural crest cells contribute to CHD such as Tetralogy of Fallot, by modeling human cardiogenesis with endogenous neuronal integration in CHD patient iPSC-derived neuro-cardiac gastruloids, and selectively activating/silencing neural inputs through optogenetic stimulation Olmsted & Paluh, iScience, Jun 2022.
*To study temporal changes in human-specific expression patterns of genetic regulatory networks (transcription factors, lncRNA, miRNA) in cardiac cells differentiated from CHD patient-derived iPSC Ang et al., Cell, Dec 2016.
*To dissect individual and combined effects of pathogenic variants and external signals (growth factors, cytokines, hormones, toxins, teratogens, drugs, mechanical forces) on development of heart defects or fetal death, by exposing cardiac progenitor cells differentiated from hPSC/CHD patient-derived iPSC to external signals in a controlled cell culture environment Schmidt et al., Cell, Dec 2023, Volmert et al., Nature Comm., Dec 2023.
*To study the role of crosstalk between cardiac cell-types (cardiomyocytes, fibroblasts, neurons and endothelial cells) in cardiac development, fibrosis, angiogenesis, autonomic regulation, inflammation etc., by using donor-derived fetal heart samples or by engineering healthy/CHD patient-derived hiPSC co-culture systems.
*To identify new therapeutic leads and high-throughput screen drug candidates in CHD patient-derived tissues/cardiac organoids/heart-chip.
*To design and test gene and cell therapies for CHD, by examining how genetically-modified cells/normal cells interact with surrounding cells in hiPSC/ESC-derived embryoid bodies.
*To test devices and strategies of palliative surgery, by using computational fluid dynamics simulations and mock circulatory loops that recapitulate patient-specific circulatory systems and hemodynamics of surgical interventions Das et al., Fluids, Nov 2021.
*To evaluate the risk of developmental toxicity for new chemicals and drugs in human tissues/organoids/organ-on-chip.
*To stratify CHD patient populations according to disease mechanism types and/or response of patient-derived tissues to treatments.
*To test safety and efficacy of single and combination treatments in CHD patient-specific tissues/organoids/(multi)-organs-on-chip, in a precision medicine approach.
Although in vitro methods have inherent limitations, their relevance to human biology far exceeds that of animal research.
Animal model organisms were never comprehensively compared to humans and scientifically validated. Complementing in vitro methods with animal experiments is not effective for human patients, because species-specific differences prevent reliable translation of results to humans.
While animal research benefits from experimenting on a complete organism, model organisms fail to replicate the interplay of thousands of human-specific features, from molecular level to organism level, and are therefore not representative of the human organism.
To address the challenges of individual human-based in vitro models, they can be integrated with other human-based in vitro methods, AI-driven analysis, clinical data, and real-world patient data.
Have you leveraged in vitro methods in unique ways? We would love to hear how! Join the conversation to exchange ideas, collaborate and inspire new directions in human-based science!
