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mRNA-1273 and BNT162b2 COVID-19 vaccines induce different cardiotoxic effects and functional disturbances in isolated cardiomyocytes

In this in vitro study, he authors from Germany and Hungary investigated the effect of mRNA-1273 (Moderna) and BNT162b2 (Pfizer/Biontech) COVID-19 vaccines on the function, structure, and viability of isolated rat left ventricular cardiomyocytes. The results showed, for the first time, that both mRNA-1273 and BNT162b2 induce cardiotoxic effects with disturbances of normal contractile function in rat cardiomyocytes. However, the effects of vaccines differed fundamentally in their pathophysiological mechanisms, which were very specific. 

In both vaccines (mRNA-1273 and BNT162b2), a messenger RNA (mRNA) sequence determines the structure and assembly of the immunogen, the SARS-CoV-2 spike (S) glycoprotein. The mRNA is protected from degradation by lipid nanoparticles (LNPs) and taken up by the cells as an LNP-mRNA complex through simple endocytosis. The authors noted that inadequate cardiac safety testing and exploration of the adverse event profile of mRNA vaccines led to adverse cardiac events, which have been classified as myocarditis and/or pericarditis. 

A common theory about the underlying pathophysiological mechanisms of myocardial injury is that there is a possible immunological cross-reaction. It has been suggested that antibodies directed against epitopes of the S protein, as a result of vaccination, may also react with epitopes of the α-myosin heavy chain (α-MHC). The authors, however, point out that α-MHC is a sarcomeric protein that is almost exclusively expressed in atria and cannot directly interact with circulating antibodies due to its intracellular localization.

 

 

About the study

The scientists studied the direct effect of mRNA-1273 and BNT162b2 on the function, structure, and viability of isolated rat cardiomyocytes over 72 hours. Since this model of isolated rat cardiomyocytes allows accurate quantification of myocyte contraction parameters up to 48 hours after isolation, the ‘functional state’ after 72 hours was assessed solely in a qualitative fashion.

The cellular and molecular effects directly attributed to the two mRNA vaccines were studied on the left ventricular cardiomyocytes isolated from the hearts of three-month-old male Wistar rats. At a stimulation frequency of 2 Hz, the function of cardiomyocytes was analyzed by determining the relative cell shortening, contraction velocity, and relaxation velocity. Confocal laser scanning microscopy for filamentous actin was used to image the sarcomere structure of isolated cardiomyocytes. AC16 human cardiomyocyte cell line of ventricular origin was used to investigate the uptake of LNP-mRNA complexes and the translation of the encoded S protein. 

 

Results

The effects of mRNA-1273 and BNT162b2 on the function of rat left ventricular myocytes 24 and 48 hours after application

After 24 h, no functional or morphological differences were detected between cells treated with mRNA-1273 or BNT162b2 and untreated control cells. The culture dishes incubated with mRNA-1273/BNT162b2 and untreated controls showed regularly contracting cells (percentages of 75% and 77%, respectively).

After 48 hours of incubation, the examination of the sarcomere structure revealed no irregularities in the structure of the parallel myofibrils in any of the treatment groups. 

 

The mRNA-1273 vaccine

48 h after the application of mRNA-1273, the number of contracting myocytes decreased and quantification of contraction parameters was no longer possible. The mRNA-1273 induced arrhythmic and completely irregular, partially ‘peristaltic’ contractions of myocytes, reducing myocardial cells that contract regularly to only 10%. The percentage of arrhythmic and irregularly beating cells was approximately 52%.

The analysis of the calcium transients in cardiomyocytes treated with mRNA-1273 showed arrhythmic, localized, and irregular transients.

The authors stated that arrhythmic and completely irregular contractions detected after incubation with mRNA-1273, together with the irregular and localized calcium transients, indicate significant dysfunction of ryanodine receptor 2 (RyR2) and direct impairment of sarcoplasmic reticulum-dependent calcium release. The RyR2 regulates the calcium release from the sarcoplasmic reticulum in cardiomyocytes and plays an integral role in excitation-contraction coupling. Numerous studies have identified that dysfunction of RyR2 causes arrhythmias, ventricular tachycardia, and sudden cardiac death.

Reduced dose of the mRNA-1273 to 1.0 μg/ml failed to improve these findings over time. A further reduction to 0.3 μg/ml increased the proportion of myocardial cells that regularly contract, but it was only transiently effective within 48 hours.

After 72 hours, the control dishes still had 41% contracting cardiomyocytes, whereas the cells incubated with mRNA-1273 almost completely ceased their function.

 

The mRNA BNT162b2 vaccine

After 24 or 48 hours, cells treated with BNT162b2 contracted rhythmically and uniformly. However, they exhibited an increase in relative cell shortening (+22.6%), contraction velocity (+31.9%), and relaxation velocity (+32.1%) compared to control myocytes.

The analysis of the calcium transients revealed a rhythmically and uniformly detectable systolic release and diastolic decrease of calcium in BNT162b2-treated cells and untreated control myocytes. The authors stated that the pattern of cell contraction that developed after the BNT162b2 application was largely consistent with the functional changes found in cardiomyocytes (or myocardium) after stimulation with the catecholamines.

As positive inotropic (enhanced contractility) and lusitropic (faster relaxation) effects are mediated mainly through the activation of protein kinase A (PKA), researchers evaluated myocyte PKA activity. After 48 h of incubation with BNT162b2, myocyte PKA activity increased to levels comparable to those seen in untreated cells that received β-adrenoceptor stimulation with isoprenaline, an agonist of β-adrenoceptors. The authors concluded that BNT162b2 induced a significant increase in PKA activity, increasing the intensity and dynamics of calcium transients in BNT162b2-treated myocytes and all contraction parameters described above. After 72 hours of incubation with BNT162b2, the proportion of beating myocytes decreased to 27%, compared with untreated controls.

In contrast, mRNA-1273 had no impact on PKA activity.

The authors noted that their findings of sustained PKA activation, induced by BNT162b2, could correspond histopathologically to catecholamine-induced cardiomyopathy. The permanent stimulation of β-adrenergic signaling mechanisms increases the energy demand of the myocardium, while the increase in heart rate simultaneously reduces the diastolic filling time of the coronary arteries, thereby also lowering the energy supply.

 

Illustration from the original articleOriginal figure from the article of Schreckenberg R, et al. Br J Pharmacol. 2024;181:345–361.

 

The uptake of mRNA and the translation of the vaccine-encoded S protein in rat and human cardiomyocytes

The authors then investigate the uptake of LNP-mRNA complexes and the translation of the encoded S protein in rat cardiac tissue. mRNA was detected in all sections of the left and right ventricular myocardium, the septum, and both atria. Cell-specific uptake was found in cardiomyocytes and non-myocytic cells, such as endothelial cells and fibroblasts. The cell fraction of non-myocytic cells consistently showed higher mRNA levels than myocyte fraction. After 48 h of incubation with mRNA-1273 or BNT162b2, positive test results were found exclusively for the intracellular fraction of treated cells, whereas the supernatant was always negative. 

The researchers also investigated the uptake of mRNA and the translation of the encoded S protein in the AC16 human cardiomyocyte cell line of ventricular origin. The results showed efficient uptake of LNP-mRNA complexes and the translation of the encoded S protein. In contrast to the isolated cardiomyocytes of adult rat hearts, the S protein was detected both in the cell fraction and the supernatant.

The authors suggested that the time kinetics in both cases indicate that S protein is a causal agent that must first be translated and enriched in the cytoplasmic compartment in a sufficient concentration. The utilized cell culture systems do not permit any definitive conclusions regarding the duration or extent of the S protein production, but all results for AC16 cells point to a highly efficient and potentially long-lasting translation.

 

Conclusion

Both, mRNA-1273 and BNT162b2 COVID-19 vaccines induced cardiotoxic effects and functional disturbances in isolated cardiomyocytes. mRNA-1273 vaccine induced arrhythmic and irregular contractions through extensive disruption of sarcoplasmic calcium release, whereas the BNT162b2 vaccine induced a pattern of cell contraction through chronic activation of PKA, consistent with catecholamine-induced cardiomyopathy. These cardiotoxic effects and functional disturbances are pathophysiologically consistent with cardiomyopathy, contrasting clinically diagnosed side effects, predominantly classified as myo-and/or pericarditis.

Impairment of RyR2 and persistent activation of PKA, both due to intracellular interactions of the S protein, are risk factors for sudden cardiac death, ventricular tachyarrhythmias, and contractile dysfunction. Both mechanisms also provide a possible explanation for persistent cardiac symptoms, observed in the context of long COVID syndrome.

According to the authors, it is imperative to investigate the effect of mRNA-1273 and BNT162b2 vaccines on the function of the cardiac conduction system. These cells are histologically different from working myocardial cells, but their heart rate and conduction velocity are also under the control of PKA. Furthermore, they recommended that the risk-benefit ratio of mRNA-based vaccines should be re-evaluated taking into account these experimental results and hidden cardiotoxic effects of the vaccines. These findings should be considered in future diagnosis and therapy of cardiac symptoms associated with mRNA-based COVID-19 vaccination.

 

This article was published in the British Journal of Pharmacology

 

 

Journal Reference

Schreckenberg R, Woitasky N, Itani N. et al. Cardiac side effects of RNA-based SARS-CoV-2 vaccines: Hidden cardiotoxic effects of mRNA-1273 and BNT162b2 on ventricular myocyte function and structure. Br J Pharmacol. 2024;181:345361.  https://doi.org/10.1111/bph.16262

 

 

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