Despite cardiovascular diseases represent the leading cause of death in western countries, knowledge of the underlying molecular (patho)mechanisms is still incomplete, which hinders the development of novel therapies. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide the basis to study the physiology and pathophysiology of heart diseases in a human model system. Nevertheless, the use of iPSC-CMs is limited by their immature phenotype compared to adult ventricular CMs. We recently designed a maturation medium (MM), which induced the metabolic and functional maturation of iPSC-CMs in 2D cultures. Whether this medium is also suitable to enhance the maturation of engineered heart muscles (EHMs) consisting of iPSC-CMs and cardiac fibroblasts has not been investigated so far. Therefore, we investigated the effect of MM for the cultivation of EHMs in comparison to the commonly used standard medium which is supplemented with VEGF, bFGF and IGF-1 (SM). 

To better compare the results, we also supplied MM with the same growth factors (GF) as present in SM (MM+GF). After EHM formation (70:30 ratio of hiPSC-CMs and human ventricular fibroblasts, HVFs), the tissues were randomized and cultivated in SM or MM+GF for 21 days. Afterwards, we characterized the contractility, structure and gene expression in the EHMs. The measurements of the contractile function revealed an established force-length relationship (Frank Starling mechanism) and calcium concentration dependent force of the EHMs. Interestingly, EHMs that were cultured in MM+GF had higher active force development as well as an enhanced inotropic response to isoprenaline, compared to EHMs cultured in SM.Measurement of mRNA expression levels using qPCR revealed increased ratios of MYL2/MYL7 and MYH7/MY6, demonstrating the structural maturation of the EHMs in MM+GF. Expression levels of genes involved in cell metabolism and mitochondrial development, such as OPA1, CPT1B and CD36 showed a trend towards an increased expression under the influence of MM+GF. Notably, we observed an increased beating rate of EHMs in MM+GF in comparison to SM, which is contradictory to a reduced spontaneous beating rate commonly observed during iPSC-CM maturation in MM. We hypothesized that accumulation of intracellular lipids and lipotoxicity may cause the increased beating frequency through cellular stress. Indeed, cultivation of iPSC-CMs in MM+GF (2D culture) led to a strong accumulation of intracellular lipids, which could be prevented by using MM without GF. Furthermore, EHMs cultured in MM without GF showed a reduction in spontaneous beating frequency during the 3-week maturation period in comparison to MM+GF, while the contractile performance and mRNA expression of different maturation markers such as MYL2/MYL7 or MYH7/MYH6 were maintained.

In summary, our results demonstrate that cultivation of EHMs in MM+GF strongly enhances the contractile function, structure and expression of maturation markers in comparison to SM. However, EHMs in MM+GF revealed an increased beating spontaneous rate, which may be caused by lipotoxicity specifically induced by supplementation with GF. Current studies aim to establish a model system for the modelling of metabolic diseases (for example, diabetic cardiomyopathy) by culturing EHMs in MM with modifications mimicking the conditions in diabetic patients.