Heart failure is the leading cause of death worldwide; therefore, developing effective therapies to treat heart failure has always been an urgent demand in cardiovascular medicine. Regenerative medicine holds great promise as a potentially better alternative therapy to drug treatment and donor/artificial heart transplantation. For cardiac regenerative modality, cardiomyocytes derived from stem cells may be the only cell source because adult cardiomyocytes lack proliferative ability. Herein, human induced pluripotent stem (hiPS) cells possess the obvious advantages of unlimited renewal capability and avoiding immune responses in recipients, attracting enormous interest in cardiac regenerative medicine (Takahashi et al., 2007). Although highly effective cardiomyocyte differentiation from hiPS cells by controlling the Wnt pathway has been realized (Lian et al., 2013) and the first clinical trial in the world using hiPS-derived cardiomyocytes (hiPS-CM) was conducted in Japan (Mallapaty, 2022; Miyagawa et al., 2022), differentiating maturely functional cardiomyocytes from hiPS cells is still a big unmet challenge (Karbassi et al., 2020).

Till now, a number of strategies to differentiate mature hiPS-CM have been reported (Tu et al., 2018), including physical stimulations (Ronaldson-Bouchard et al., 2018), gene editing (Kuppusamy et al., 2015), biochemical factors (Yang et al., 2014), functional culture substrates and three-dimensional culture (Ribeiro et al., 2015; Fong et al., 2016), and co-culturing with other cell types (Pasquier et al., 2017). Although a putative optimal methodology for the maturation may be a sophisticated integration of multiple effective agents rather than any single one, independent investigation for each effective agent is a prerequisite to realizing such integration. Currently, the metabolic feature of cardiomyocytes and the role of fatty acids in the maturation of hiPS-CM are attracting research attention (Slaats et al., 2020; Tani et al., 2022), and this has been encouraged by great success in developing a method to purify hiPS-CM from a differentiated cell pool by exploiting metabolic features of cardiomyocytes (Tohyama et al., 2013).

Human hearts shift their energetic substrates from lactate and glucose to fatty acids during the neonatal period (Isu et al., 2019; Horikoshi et al., 2019). However, the fatty acids supplied to cultured cells as the composition of fetal bovine serum (FBS) supplemented with culture media are quite limited. Noticing this deviation, Yang et al. (2019) treated hiPS-CM with media containing both glucose and a mixture of fatty acids designed to match content levels found in newborns and reported the induced cardiomyocyte hypertrophy and a significant increase in cardiomyocyte contractile force. Most recently, Yoshida et al. (2022) compared the types of fatty acids metabolized between cardiomyocytes in culture and heart cells in vivo, supplemented a mixture of fatty acids to the hiPS-CM culture medium, and found that the supplementation increased oxygen consumption and contractile force of the hiPS-CM. In another report, Lopez et al. (2021) showed that a more mature metabolic phenotype of hiPS-CM can be induced within a 3D culture by stimulating fatty acid oxidation.

In this regard, we found considerable shortages of ω–3 and ω–6 polyunsaturated fatty acids (PUFAs) in cultured rat fetal cardiomyocytes by comparing the fatty acid composition in the cultured cells and in the cells in neonate myocardial tissue (Karimata et al., 2013). In particular, the contents of docosahexaenoic acid (DHA) and arachidonic acid (AA) were significantly lower than those in the neonatal tissue. Then, we supplemented DHA and AA, respectively, to Dulbecco’s modified Eagle’s medium/F12 culture medium and evaluated the effects of the supplementations on the behavior of rat fetal cardiomyocytes (Yano et al., 2021). The supplementations were found to upregulate the expressions of mRNAs for cardiomyocyte differentiation, fatty acid metabolism, and cellular adhesion. The enhancement of cellular contractility was attributed to the improvement of intercellular connection rather than a direct enhancement of the contractile force.

This study is a succession to the work by Yano et al. (2021), and here we report the newly obtained results of the effects of DHA or AA supplementation on the behavior of hiPS-CM.

As shown in Fig. 2, hiPS-CM cultured with AA supplementation formed cellular clusters on the gels. DHA supplementation also stimulated cell aggregation to render higher cell density to places scattered on the gels compared to their neighbors; however, these places were not as big and obvious as those dark clusters with AA supplementation (Fig. 2b). The samples with different media did not have significantly different beat rates, though the beating rate under DHA or AA supplementation showed a lower tendency compared with that of the control (Table 2). Conversely, for the supplementations, the contractile ratios tended to be greater; in particular, the contractile ratio of the clusters induced by AA supplementation was significantly greater than that of the control (Table 2).

Fig. 4 shows the relative contractile force obtained in this study; the contractile force with DHA or AA supplementation tended to increase, although it was not significant among the samples. The maximum contractile force of around 5% strain for AA supplementation or around 10% for DHA supplementation was more obvious than the maximum in the control data. The existence of maximum contractile force vs. construct strain at a few percent may be regarded as the Frank-Starling type behavior and an assessment for cardiomyocyte maturation. However, the maxima in Fig. 4 were not remarkable enough to make a sound assessment.

The FEA results are presented in Fig. 5. Under the same contractile ratio, the uniformly distributed beating cells induced greater stress in the gel beneath the cells than the cellular cluster does (compare Figs. 5a with 5b). The contractile force of a whole gel was converted (Fig. 5c), and the gel with cell clusters consequently generated a smaller contractile force than the gel with uniformly distributed cells. This can explain why the AA supplementation with a significantly larger contractile ratio (shown in Table 2) did not give rise to a larger contractile force compared with DHA supplementation (Fig. 4).

Fig. 6 shows the mRNA expression of 17 genes in hiPS-CM cultured with the supplementations. The expressions of most of the genes did not significantly alter with the supplementations. Expression of transcriptional factor P300 was significantly upregulated with AA supplementation, which may correlate with the upregulation tendency in cTNT and MLC2a expressions with the supplementation. In contrast, DHA did not result in an obvious increase in the expression of genes associated with differentiation and maturation. Notably, MLC2v was downregulated with the supplementations; in particular, significantly downregulated with DHA supplementation.

Expression of CX43 with AA supplementation was upregulated, consistent with the phenomenon of cellular clustering with AA supplementation. However, in contrast, the important cellular adhesion protein CDH2 was significantly downregulated.

Genes coding for two critical factors with respect to fatty acid metabolism, the lipid transporter CD36 and the metabolic transcriptional factor ERRγ were upregulated (Fig. 6). Consistent with these upregulations, Fig. 7 shows that the precursor of fatty acid metabolism, BHB, was increased in the cytoplasm; meanwhile, the first intermediate of glucose metabolism, G6P, decreased. These alterations in the metabolites persisted with culture duration and the decrease in G6P on day 15 became significant.

The immunofluorescence images and the quantitative CTCF analysis are presented in Figs. 8a and 8b, respectively. MLC2v and cTNT were substantially expressed in the differentiated cells and the CTCF data may explain the enhanced tendency due to the DHA or AA supplementation.

However, the above-discussed data had certain inconsistencies. The expressions of cTNT and MLC2v assessed by RT-PCR (Fig. 6) disagreed with those by CTCF via immunostaining (Fig. 8), and the downregulated expression of CDH2 (Fig. 6) with AA supplementation did not coincide with the cell clustering (Fig. 2b). We speculate that these inconsistencies may be caused by the purification process of hiPS-CM samples for assessing mRNA expression as further discussed in the next section.

Fatty acids are essential components for our lives. Mammals can biosynthesize saturated and monounsaturated fatty acids in vivo (Iso et al., 2006; Matsuzaka et al., 2007), but cannot effectively synthesize PUFAs because they do not have the desaturation enzyme necessary for catalyzing a double bond at n–3 or n–6 positions. Therefore, PUFAs have to be supplied with food (Schmitz and Ecker, 2008). Considering the lack of DHA and AA during infant development, these two PUFAs have been supplemented in formulas. These supplementations exert positive effects on cognition, brain connectivity, and allergy in early childhood (Carlson and Colombo, 2016; Clandinin et al., 2005) as well as their cardioprotective effects (Shysh et al., 2016).

Research interests in the effects of fatty acids on the differentiation and growth of cardiomyocytes derived from stem cells are based on the fact that during the neonatal period, mammalian hearts shift their energetic substrates from lactate and glucose to fatty acids (Horikoshi et al., 2019; Isu et al., 2019), which is obviously disparate in the cell culture with glucose-based media. Taking the metabolic environment in vivo as a blueprint for differentiation and maturation of hiPS-CM (Slaats et al., 2020), Yang et al. (2019) treated hiPS-CM with media containing both glucose and a cocktail of fatty acids including palmitate, oleate, and linoleate and reported induced cardiomyocyte hypertrophy and a significant increase in cardiomyocyte contractile force. Yoshida et al. (2022) supplemented a mixture of fatty acids including oleic acid, stearic acid, myristic acid, linoleic acid, linolenic acid, palmitic acid, palmitoleic acid, and arachidonic acid to the hiPS-CM culture medium and found that the supplementation increased oxygen consumption and contractile force of the hiPS-CM. Ramachandra et al. (2018) reported that supplementing hiPS-CM with palmitate and oleate significantly enhanced mitochondrial remodeling, oxygen consumption rates, and ATP production.

Our previous study also paid attention to the metabolism feature of cardiomyocytes and found that the DHA and AA content in primary-cultured cardiomyocytes was significantly lower than in the neonatal tissue (Karimata et al., 2013) and that the supplementation of DHA or AA to the primary culture upregulated the expressions of mRNAs for cardiomyocyte differentiation (Nkx2.5), fatty acid metabolism (Cd36, peroxisome proliferator-activated receptors α (Pparα), and Pparδ), and cellular adhesion (Cdh2 and Cx43). The enhancement of cellular contractility was attributed to the improvement in intercellular connection rather than the cellular contractile force itself (Yano et al., 2021). Continuing the thread in this study, we further investigated the individual effects of these two PUFAs on the hiPS-CM culture rather than the integrated effects of a fatty acid mixture.

First, the uptake of DHA and AA into hiPS-CM was evidenced by the increase in CD36 expression (Fig. 6), the increase in BHB, and the decrease in G6P in the cytoplasm (Fig. 7). The alteration in the amount of the metabolites showed that substrates of the energy metabolism was replaced partially by the supplemented PUFAs. A putative activation of a metabolic pathway stimulated by AA supplementation in hiPS-CM, i.e., CD36_ERRγ_PGC1α_COX4 can be proposed (refer Fig. 6), which is different from our results of the rat cardiomyocyte primary culture where the activation of CD36_­PPARα/PPARδ was suggested (Yano et al., 2021). ERRγ and PPARs are nuclear receptors actively involved with cardiomyocyte metabolism (Huss et al., 2015; Montaigne et al., 2021). Estrogen-related receptors (ERR) largely function as a transcriptional activator in the heart to drive oxidative metabolism, usually interacting with PGC1α, which co-expresses in the heart and increases myocardial mitochondrial capacity assisting the metabolism shift from reliance on glycolysis to fatty acid oxidation (Huss et al., 2015). The upregulation tendency of nuclear receptor ERRγ in hiPS-CM instead of PPARs in the primary culture of rat fetal cardiomyocytes may be because the hiPS-CM in this study were at their earlier development state than the fetal cardiomyocytes in the primary culture, rather than due to the difference in species because the metabolism pathways in mammalian cardiomyocytes are quite conserved and stable (Lopaschuk and Jaswal, 2010; Chung et al., 2007).

As for the contractile behavior, the measured contractile force did not increase significantly although the relative force to control showed an increasing tendency. Similar to the primary culture, cellular aggregation and the increase in contractile ratio were observed. We expected that upregulation in CX43 and CDH2 mRNA could be detected; however, the supplementations led to a significant decrease in CDH2. We speculate that this was a result of the purification process in the mRNA experiment because culture on the gels did not present such a decrease in CDH2 (data not shown here). Although the vital transcriptional factor P300 (Ghosh, 2020) was upregulated with the supplementations, the genes related to the cardiomyocyte constitution and maturation were not consistently upmodulated (Fig. 6). The expression of cTNT and MLC2a instead of MLC2v increased with the AA supplementation, whereas their expressions decreased with the DHA supplementation. Immunofluorescence investigation in terms of CTCF for each cell (Fig. 8b) did not show the decrease accordingly. These results may also implicate the influence of the purification process and suggest positive effects of co-culture with other cell types.

In summary, we investigated the effects of individual supplementation of DHA or AA rather than their mixture on the hiPS-CM behavior and observed that the sole supplementation of albumin-conjugated DHA or AA can be taken up by hiPS-CM without other uptake-enhancing factors. The enhancement effect on the hiPS-CM contractile force was modest since the increase in the contractile force was not significant. Supplementation of AA was more effective than that of DHA because it significantly upregulated mRNA expressions of P300, CD36, and COX4. However, the FEA showed that the formation of clusters on a collagen gel attenuated the contractile force exerted from the gel to its surroundings.

This study is a continuation of our previous study on the primary culture of rat neonatal cardiomyocytes and intended to assess the effect of the supplementation of DHA or AA on the hiPS-CM. This is quite interesting as cardiac differentiation/maturation from hiPS cells is an intensively attractive research topic in cardiac regeneration. The maturation of cardiomyocytes derived from stem cells is a complicated process that surely involves multiple biophysical and biochemical factors acting in a properly temporal manner and any single factor may not possibly dominate the process. We report here that these two PUFAs, having been supplemented in infant formulas, have no direct and significant enhancement effect on the performance of the hiPS-CM when they were supplemented individually, although they were able to enter the cellular metabolic system. The AA supplementation may have some auxiliary effect on the maturation of hiPS-CM, as observed by the cluster formation and mRNA expression. We speculate that these putatively positive effects may be attributed to the alteration of cardiomyocytes membrane composition and the remodeling of some membrane functional molecules, such as cardiolipin (Khairallah et al., 2012; Lamaziere et al., 2015; El-Hafidi et al., 2020) rather than the uptake of AA into the energy metabolism. Based on the results of this study, further work may be focused on 1) the mechanism of the cluster formation and its potential for the promotion of cardiomyocyte maturation, and 2) the combination with other biochemical factors, such as Tafazzin and acyl-transferases, in the consideration of the membrane/functional molecule remodeling due to the AA supplementation.