Diseases of the cardiovascular system are the most common cause of death not only in Germany (1.2), but also worldwide (3). Elderly people are particularly affected, but younger people can also suffer from high blood pressure, heart attacks, coronary artery disease (CAD), heart valve defects or cardiac arrhythmias and other symptoms.

Widespread disease with high costs

In 2021, 340,619 people died in Germany as a result of cardiovascular disease, which corresponds to just over a third of all deaths. In 2020, more than 1.5 million people were hospitalized due to cardiological problems (4). Around 20 million people in Germany suffer from high blood pressure (5). This also implicates that the costs for this disease profile are the highest compared to other diseases, summed up to a whopping €56.7 billion in 2020 (6).

This underlines the urgency to act on many levels to lower these gigantic numbers. This need results in a large number of research projects, especially in the field of cardiology, in which the underlying disease mechanisms are to be unfolded. Based on the knowledge of how these diseases develop, therapies and drugs can be developed for humans.

Drugs and therapies

It depends on the precise pathology and possible collateral illnesses whether the respective patient is only being treated with medication or whether (additionally) an operation is necessary. However, at some point in the disease course, a surgical intervention is often necessary, despite drug treatment.

Bypass surgery is often performed. A patient's own vein or artery (usually from the leg) is removed and used to bypass a narrowed or blocked vessel in the heart in order to re-supply the heart with oxygen. The replacement of heart valves is also a standard procedure in heart surgery today. If the heart is arrhythmic, a pacemaker can be implanted. It is often argued that these procedures could only be established thanks to previous animal experiments. But what is completely ignored are the numerous animal-tested methods that are not used because - in contrast to the outcome in animal experiments - they don’t work in humans. Furthermore, there is no evidence that the methods could not have been developed without agonizing experiments on animals. Above all, however, this is not an argument for future animal experiments. We may not be able to change the past but we can change the future.

Typical medications target the cardiovascular system (ACE inhibitors, beta blockers, digitalis), for example, by lowering blood pressure and heart rate, in some cases. Others influence blood clotting (acetylsalicylic acid, heparin, and coumarins) to prevent the clumping of blood platelets. Physicians can follow special guidelines (7) to prescribe the appropriate therapy for their patients.

What is striking when looking at the drugs: these classes have been on the market and, thus, in use for a very long time. There are also new approvals, but only about 6.6% of potential drug candidates make it from the preclinical phase to approval (8,9). Simply put: of 100 cardiovascular drugs that were effective and safe in the pre-clinical phase (i.e. primarily in animal experiments), fewer than 7 make it onto the market because they cannot prove their effectiveness and/or safety in human clinical trials. This is worse than the average number (all indications), which is a 92% failure rate (10). There is one strikingly evident explanation for this exorbitant failure rate: the obvious inability of animal experiments to predict the reaction(s) in humans.

Animal experiments in cardiology

There is not the one animal species that is singled out for us in animal experiments for cardiology. In the past, however, dogs were used particularly frequently for all research relating to the heart. The number has fallen but even nowadays, many dogs still suffer and die in the laboratory. The most common dog breed is the beagle.

Beagle dogs are often used for cardiology experiments.

Beagle dogs were used in experiments in Wuppertal, for example: During a first surgery, the animal's chest is cut open, a telemetry sensor is inserted into the main artery (aorta), and two sensor cables are attached directly to the heart. The chest is surgically closed. The sensor later serves to measure blood pressure and other blood values on the unanaesthetised dog. In a second surgery, the abdomen is cut open and the right kidney is wrapped in sterilized silk. The third surgery, in which the right main renal artery is blocked by inserting a vascular plug, takes place eight weeks after the kidney encasing. These interventions artificially produce high blood pressure. Eight weeks after the third surgery, all dogs display elevated blood pressure. Then a 50-week test phase begins, during which various active ingredients and combinations are tested. The dogs are observed and examined to assess the effects on blood pressure after once-daily dosing with a new drug, commonly used antihypertensive drugs, or combinations of both. What happens to the animals after the year-long study is not mentioned (11).

However, many small animals are also used for experiments. A heart attack is mimicked in mice by permanently constricting a heart artery. For this purpose, the chest is cut open on the left side under anesthesia, the heart is exposed, and an artery of the left heart is tied off with a thread. During the surgery, small pumps are also implanted under the skin of the animals, through which various test substances are administered to the mice after the operation. The animals are killed after 28 days at the latest (12).

In another experiment, 132 guinea pigs are used. The animals' chests are cut open under anesthesia and the exposed heart is touched with a metal rod cooled to minus 196°C, so the heart tissue dies off. This is how a heart attack is simulated. In the following week, 24 of the 132 guinea pigs die. Seven days after the freezing, small pieces of heart tissue grown from human cells are sewn onto the resulting scar. The pieces of tissue contain a different number of human heart cells or no heart cells at all. To ensure that the pieces of human tissue are not rejected by the guinea pig's immune system, the animals' immune system is suppressed with various drugs for 25 days from the 3rd day before the surgery. 33 guinea pigs die after the insertion of the human heart tissue. The remaining 75 animals are examined by heart ultrasound and killed in an undisclosed manner (13).

The reason why animal experiments in cardiology fail

The principle of the so-called animal model is used in cardiology as in (many) other fields of medical research: young, healthy animals are artificially manipulated in order to show symptoms similar to those of humans. Such a “model” is not just a foreign organism that has a different metabolism, different gene regulation, and a completely different lifestyle to that of humans. The diseases also do not correspond to the diseases of humans; merely individual symptoms are artificially caused. There is obviously a significant difference between a 75-year-old person suffering a heart attack due to a blocked artery, which may have developed gradually over decades as a result of an unhealthy lifestyle and unfavorable food choices and a young, healthy mouse having an artery constricted. Moreover, regarding blood clots, which therapy works best depends on their composition: whether it predominantly contains red or white blood cells or how high the proportion of fibrin fibers is influences the characteristics of the clot – and consequently the advised interventions (14). Not a single animal experiment is capable of reproducing this hugely important factor. Therefore it is not surprising that the success rate of potential new drugs is so catastrophically low. Another fatal point: Due to many misguidings, the search for therapy and drugs cannot be effective and cannot bring the optimally possible output.

Other models are constructed by genetically manipulating animals. While there are various genetic risk factors in humans, breeding them into a mouse or rat doesn't help much because these genetic expressions arise under a different metabolism and gene regulation, not to mention a completely different lifestyle. In addition, uniform, young, male animals are often used, which is even further from reality, since the risk in humans only increases from the age of 45 to 55 (15).

Lifestyle has the greatest influence, even when inheriting genetic dispositions are present. Countless and large, extensive studies show that a plant-based, low-salt diet, limiting or avoiding animal fats and proteins, avoiding obesity, avoiding cigarettes and alcohol, and regular exercise offer the best prevention for circulatory diseases (16–18). Particularly intriguing: switching to this (heart) healthy diet seems to reduce the risk of cardiovascular disease in people with a genetic predisposition to a level that is comparable to people who have no genetic predisposition (19). Even with children, parents should be careful since a lack of exercise can have a negative impact on the blood vessels in childhood and adolescence already (20).

That underlines the fact that pills or surgeries can only influence the symptoms and not eliminate the underlying causes. If patients do not change their lifestyle, the problems will either recur sooner or later or get worse which means more drugs will be prescribed. Always consider: no effect without side effects, no surgery without risk. Furthermore, if a clogged artery is widened again or a blood clot is removed, the immediate danger is averted – but if red meat continues to be washed down with wine, followed by smoking in an armchair with one hand conveniently placed in the bag of chips, then the next heart attack is almost pre-programmed.

Since the roots and progression of cardiovascular diseases in humans differ significantly from the highly artificial "animal models", it makes sense to change the research pattern for those diseases to relying on human-based, animal-free research and analysis methods.

The better choice: alternative methods

The great advantage of animal-free, human-based techniques is that research is carried out in the correct system resp. organism, i.e. with human cells and human tissue. Another advantage is that cells and tissues from healthy and (naturally) diseased people can be used - the comparison of this data can then provide conclusions about the development of the disease.

The range of alternative methods is already enormous. The methods presented here and many more can be looked up in the NAT database (link einsetzen: www.nat-database.org), the freely accessible database for research methods that do not use animal experiments. There are, for example, cell culture methods listed, with a special type being iPSCs, i.e. induced pluripotent stem cells. With this method, e.g. hair root cells can be obtained painlessly from patients. The cells are then reprogrammed into stem cells in the laboratory. These can be differentiated into different organ cells - for example heart muscle cells, which then can be used for research. However, these cells can be cultivated further and grow into millimeter-sized mini-organs, called organoids. If we take one step further, we get to the multi-organ chip: several organoids are connected to one another on a chip system via a circulatory system. In addition, 3D bio-printing is becoming increasingly important, with which even small hearts can be printed from human heart muscle cells.

Human-relevant results can already be obtained with classic cell cultures. In an in vitro model for atrial fibrillation, which many heart patients suffer from, atrial myocytes, i.e. muscle cells like those found in the heart atria, are produced using a special lab method. Cell lines are generated that display what is known as fibrillatory activity, i.e. disordered electrical impulses that also occur in atrial fibrillation in humans. If adding antiarrhythmic drugs (drugs used to treat atrial fibrillation) to these cell cultures, the uncontrolled impulses are suppressed. This is therefore an adequate research model, also for testing drugs (21).

If medically necessary interventions (biopsies, tissue removal) have to be carried out anyway, it makes sense to divert some material, which can then be used in the laboratory for tests. For example, a surgical intervention was performed on the aortic valve and the tissue was examined using immunocytochemistry in the laboratory. The pathogenesis of aortic valve calcification is still unclear, which is why the molecular mechanisms were evaluated. It was found that a specific signalling pathway could play a role in the development of the disease - and thus a potential target for new drugs has been identified (22).

Computational models have been predicting human reactions more reliably than animal experiments for a few years now, therefore there is great interest and potential in these In silico-technologies. Different methods are often combined. For example, human iPSCs can be differentiated into cardiomyocytes and the reaction of single cells and cell clusters to a specific drug is examined. A stimulus propagation via the heart muscle cells is measured with electrodes attached to the cells. Stimulus propagation results in heart contraction, so by stimulus propagation via cell (groups) it can be tested whether certain drugs could lead to cardiac arrhythmias. The data obtained is fed into a mathematical computer model that can predict possible side effects of new drugs before they are tested on test subjects. In this way, drug tests can be made faster, safer, and cheaper (23).

Virtual models are also gaining importance: researchers, industry, clinics, and authorities are collaborating on the Living Heart Project to create a digital heart model that can be used to plan drug tests, chemical tests, and operations (24).

Even a human body circuit can be simulated: heart-like pump systems that imitate heart chambers or heart valves can be constructed (25,26) in microfluidic circuit systems with iPSCs.

Organoids can be used to study congenital heart defects and their impact on the developing heart in early pregnancy. Embryonic cardiac organoids are grown from iPSCs. When exposed to different conditions, e.g., a sugar- and insulin-rich environment, simulating maternal diabetes, different biomarkers indicative of abnormal heart development can be observed. Depending on the culture medium, other maternal diseases, other congenital heart diseases and effects on the embryonic heart can also be examined. In addition, drug screening and toxicological studies can be carried out (27). The heart organoids even show a kind of heartbeat – they contract like a “real” heart does (28).

 
Mini hearts generated from human cells by Novoheart

A US study shows that organoids and multi-organ-chips are more reliable than animal experiments: 10 approved drugs were examined that had been shown to be harmless in animal experiments but had serious side effects on the heart or liver in humans and were therefore withdrawn from the market. The researchers analyzed the effects of these drugs in multi-organ chips with human heart and liver organoids and were able to unequivocally demonstrate the toxic effects found in humans for 7 out of 10 substances, compared to 0 out of 10 in animal studies (29).

3D printers are already in use in many private households, but they differ significantly from devices in laboratories used for 3D bioprinting. A special printer with various bio-inks prints a model calculated and created with a special computer programme. Instead of using different colors, the bioprinter prints a construction of different cells and gels. 3D printing can also be performed without animal components, which is referred to as “clean bioprinting” (30).

A complex heart organoid model can be printed using 3D bioprinting, for example. A hydrogel framework ensures three-dimensional stability, in which cardiomyocytes, fibroblasts, and endothelial cells are embedded. Heart-specific functionality can be detected via certain biomarkers. Interactions between the different cell types were also detected. This model can be used for mechanistic investigations regarding development and progression of heart diseases, as well as for testing drugs or chemicals, and their toxicity (31).

Another application is artificial elastic tissue replacement for pericardium, heart valves or blood vessels, which can be manufactured individually, i.e. patient-specifically. Here, a kind of hollow structure of a heart valve, for example, is produced using a spinning process with novel polymers, which is derived from the patient’s cells. The mending with the body's own tissue is facilitated by the porous structures. The waiver of animal materials (e.g. heart valves from pigs) also has the advantage of an immune-driven rejection being less likely and thus a more gentle healing phase. This could further shorten intensive care and hospital stays (32).

All methods can also be used for other research areas such as basic research for all types of cardiovascular diseases. Some of the highly efficient technologies have only existed for a few years and are already showing promising results. This great potential needs to be exploited further.

23 February 2023
Dipl. Biol. Julia Radzwill

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