Harsha V. Renikunta,Katina Lazarow,Yiqi Gong,Praphulla Chandra Shukla,Vanasa Nageswaran,Hector Giral,Adelheid Kratzer,Lennart Opitz,Felix B. Engel,Arash Haghikia,Sarah Costantino,Francesco Paneni,Jens Peter von Kries,Katrin Streckfuss-Bömeke,Ulf Landmesser,Philipp Jakob
Large-scale microRNA functional high-throughput screening identifies miR-515-3p and miR-519e-3p as inducers of human cardiomyocyte proliferation
Cardiovascular medicine,Genetics,Molecular biology
Summary Ischemic cardiomyopathy, driven by loss of cardiomyocytes and inadequate proliferative response, persists to be a major global health problem. Using a functional high-throughput screening, we assessed differential proliferative potential of 2019 miRNAs after transient hypoxia by transfecting both miR-inhibitor and miR-mimic libraries in human iPSC-CM. Whereas miR-inhibitors failed to enhance EdU uptake, overexpression of 28 miRNAs substantially induced proliferative activity in hiPSC-CM, with an overrepresentation of miRNAs belonging to the primate-specific C19MC-cluster. Two of these miRNAs, miR-515-3p and miR-519e-3p, increased markers of early and late mitosis, indicative of cell division, and substantially alter signaling pathways relevant for cardiomyocyte proliferation in hiPSC-CM.
Large-scale microRNA functional high-throughput screening identifies miR-515-3p and miR-519e-3p as inducers of human cardiomyocyte proliferation

Ischemic cardiomyopathy, driven by loss of cardiomyocytes and inadequate proliferative response, persists to be a major global health problem. Using a functional high-throughput screening, we assessed differential proliferative potential of 2019 miRNAs after transient hypoxia by transfecting both miR-inhibitor and miR-mimic libraries in human iPSC-CM. Whereas miR-inhibitors failed to enhance EdU uptake, overexpression of 28 miRNAs substantially induced proliferative activity in hiPSC-CM, with an overrepresentation of miRNAs belonging to the primate-specific C19MC-cluster. Two of these miRNAs, miR-515-3p and miR-519e-3p, increased markers of early and late mitosis, indicative of cell division, and substantially alter signaling pathways relevant for cardiomyocyte proliferation in hiPSC-CM.

In adults, cardiac injury results in loss of cardiomyocytes and subsequent scar formation with heart failure symptoms. Despite progress made over the past decades, heart failure remains the most significant health burden worldwide. Current therapeutic approaches focus primarily on catheter-based or surgical re-perfusion strategies to minimize loss of contractile tissue as well as medical and adjunctive therapies to prevent adverse cardiac remodeling. However, potential therapies to remuscularize the heart do not exist. This lack is related to the very limited capacity of the adult heart to re-initiate cardiomyocytes turnover. Therefore, human hearts are prone to hypertrophic remodeling and fibrotic scarring, resulting in heart failure with potentially reduced left ventricular ejection fraction. Intriguingly, in recent years, the dogma of the heart as a post-mitotic organ has been challenged. Studies in postnatal murine hearts and humans at young age revealed cardiomyocytes proliferation,, albeit with an excessive decline in the aged population. Indeed, a small subset of cardiomyocytes retain the capacity to re-enter the cell cycle in adults that may reside preferentially in a hypoxic milieu., These findings reveal novel opportunities to explore therapeutic strategies for cardiomyocyte regeneration in the injured myocardium. Among these, microRNAs (miRNA), a class of small non-coding RNAs, have evolved as potent modulators of cell cycling in cardiomyocytes, some of which were identified from high-throughput screenings using overexpression of miRNAs.,,, Indeed, exogenous downregulation,,,, or overexpression,,,,, of miRNAs have both evolved as promising strategies to induce cardiomyocyte reentry into mitotic cell cycle in neonatal and adult mouse models. However, these observations were predominantly tested in non-human models and large functional screenings using both miRNA-inhibitors and miRNA-mimics in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) have not been investigated. Here, we tested overexpression and downregulation of 2019 miRNAs on proliferative activity in human cardiomyocytes, using hiPSC-CM. We show that individual downregulation of miRNAs is not efficient to induce cell cycle reentry. However, individual overexpression of 28 miRNAs yielded significant increase in proliferative activity in hiPSC-CM. Intriguingly, members of the human-specific C19MC cluster, miR-515-3p, and miR-519e-3p, enhance cytokinesis in human cardiomyocytes and further increase cardiomyocyte turnover after transient hypoxia. These findings are supported by our RNA-sequencing (RNA-seq) analysis that shows substantial transcriptomic regulation of structural sarcomeric genes and activation of signaling pathways involved in proliferation and cell division after overexpression of miR-515-3p and miR-519e-3p in hiPSC-CM.

We used human iPSC for differentiation into cardiomyocytes (hiPSC-CM) that were cultured for 50 to 60 days. HiPSC-CM displayed well-organized striated patterns visualized by cardiac troponin T (cTnT), and showed >90% cTnT+ CM (Figure 1A). We established a liposome-based transfection method to efficiently transfect miRNAs into hiPSC-CM. Fluorescent-labeled miRNAs (FAM-miR) indicated a high incorporation after transfection into hiPSC-CM (Figure 1B). In addition, fluorescence-activated cell sorting analysis showed a high transfection efficiency in FAM-miR-transfected hiPSC-CM (Figure 1C). Furthermore, a substantial induction of a cell-death phenotype with cell shrinking and detachment was observed after transfection with silencing RNAs targeting genes for cell survival, used as a positive control for transfection efficiency (Figure S1). We used overexpression of miR-1825, a miRNA previously reported to induce proliferation in murine cardiomyocytes, to test proliferative capacity in hiPSC-CM. Transfection with miR-1825-mimic increased DNA synthesis, as measured by incorporation of 5-ethynyl-2-deoxyuridin (EdU), in a dose-dependent manner (Figures 1D and 1E). Lactate dehydrogenase (LDH), a cytosolic enzyme that is released upon damage of the cell membrane and therefore quantifies cytotoxicity, was not increased after lipid-based transfection (Figure 1F). We also tested downregulation of endogenous miRNAs using anti-miRs that are known suppressors of cardiomyocyte turnover (i.e. anti-miR-99-5p, anti-miR-100-5p, and anti-miR-195-5p). Expression of endogenous miRNAs was substantially downregulated after anti-miR transfection, as shown for miR-195-5p (Figure 1G). Indeed, anti-miR-99-5p and anti-miR-195-5p significantly induced proliferative activity in hiPSC-CM as assessed by EdU incorporation (Figure 1H). In addition, Western blot analysis revealed downregulation of cyclin-dependent kinase inhibitor 1A (CDKN1A), an important cell cycle inhibitor, in hiPSC-CM treated with anti-miR-195 (Figure 1I). These results convincingly show that lipid-based transfection of miR-mimic and anti-miRs efficiently induce cell cycle reentry in hiPSC-CM. In order to explore miRNA modulation in a hypoxic milieu, that is present in patients with myocardial infarction, we exposed hiPSC-CM to transient hypoxia (0.5% O2). Interestingly, transient hypoxia further increased EdU incorporation in miR-1825-transfected hiPSC-CM that was not observed after miR-scrambled transfection (Figure 1J). These findings prompted us to use this model for investigating proliferative activity in hiPSC-CM by using a library of 2019 miR-mimics and anti-miRNAs.

We performed a functional high-throughput screening by using a miRNA library (Ambion; mirVana Human Library v19.0). Two thousand and nineteen miR-mimics were individually transferred to 384-wells pre-plated with hiPSC-CM. HiPSC-CM were transiently exposed to hypoxia for 2 h. Using a high-content imaging system, enhanced cell cycle activity of miRNA-transfected hiPSC-CM was assessed by EdU incorporation (Figure 2, Table S1, Figure S2). Z score, a normalizing method, was calculated for each candidate miRNA on the plate by subtracting all candidate wells in the plate and dividing the difference by median absolute deviation of all candidate wells in the plate. Transfection of miRNA-mimics identified 40 miRNAs that reached significant levels of cell cycle reentry in hiPSC-CM (Z score >3) (Figures 2A–2D). Twenty-eight miRNAs substantially increased number of EdU-positive hiPSC-CM in both replicates (Figure 2C, Table S2). We validated proliferative activity in a hit-picking screening, where these 28 miRNAs were assessed separately (Figure 2E). Interestingly, several miRNA-mimics identified in the high-throughput screening are members of miRNA-families, such as miR-148a-3p/miR-148b-3p (miRNA-148 family) and miR-212-3p/miR-132-3p (miRNA-212 family). In addition, five candidate miRNAs belong to the chromosome 19 miR-cluster (C19MC, hsa-miR-515-3p, hsa-miR-519e-3p, and hsa-miR-517c-3p) and adjacent miR-371-373 cluster (hsa-miR-371a-3p and hsa-miR-371b-3p), located on chromosome 19q13.41., This primate-specific imprinted cluster is not detected in the human heart muscle (Figure S3), but predominantly expressed in the placenta and in undifferentiated cells,, and experimental studies have investigated members of this cluster in the context of differentiation, invasion, and proliferation in different cell types of the placenta., As these biological features regulated by miRNAs of C19MC are highly relevant for cardiac regeneration, we further focused our study on miR-515-3p, miR-519e-3p, and miR-371a-3p that showed the highest proliferative activity within these clusters (Figures S4A, S4B, and Table S3).

We next assessed downregulation of 2019 miRNAs in hiPSC-CM on their proliferative effects (Figures 3A–3D and Table S4). We individually transfected 2019 anti-miRs into hiPSC-CM that were exposed to transient hypoxia for 2 h. Notably, two miRNA-inhibitors significantly induced proliferative activity (Z score >3 in both replicates) (Figures 3B–3D, Table S5, Figure S5). However, the increase of EdU-positive cardiomyocytes was modest (anti-miR-let-7c-5p: 6.69 ± 0.8%, anti-miR-365a-3p: 6.15 ± 0.6%) and validation of these candidates in a second screening, where anti-miR-let-7c-5p and anti-miR-365a-3p were assessed separately, did not show a substantial increase in EdU uptake as compared to mock-transfected hiPSC-CM (Figure 3E). Conclusively, as compared to the miR-mimic screening, induction of proliferative capacity after anti-miR transfection was not sufficient for cell cycle reentry in hiPSC-CM (Figure 3F; anti-miR screen from Figure 3C (blue circles) and miR-mimic screen from Figure 2C (gray and red circles) are shown for comparison). Therefore, we focused our study on candidate miRNAs derived from the miR-mimic screening (miR-mimic-515-3p, miR-mimic-519e-3p, and miR-mimic-371a-3p).

First, in order to define efficacy and toxicity, miR-mimic-515-3p, miR-mimic-519e-3p, and miR-mimic-371a-3p were transfected with increasing concentrations. Transfection with miR-mimic (60 nM) induced proliferative activity significantly above lower concentrations, whereas higher concentrations did not further increase cardiomyocyte turnover (Figures 4A and 4B). Importantly, miR-mimic (60 nM) transfection did not affect toxicity, as assessed by LDH assay (Figure 4C). Of note, transfection of hsa-miR-590-3p, previously reported to induce cardiomyocyte proliferation in murine cardiomyocytes,, did not result in enhanced EdU incorporation in hiPSC-CM (Figure 4A). We next tested if transient hypoxia, as observed for miR-mimic-1825, is critical for proliferative activity in hiPSC-CM. Interestingly, transfecting hiPSC-CM with miR-515-3p, miR-519e-3p not only substantially increased proliferation in normoxic conditions but also further increased EdU uptake in hiPSC-CM after transient hypoxia. Of note, this effect was not observed in miR-scrambled or miR-371a-3p-mimic-transfected hiPSC-CM (Figure 4D, 4E, S6A, and S6B). Furthermore, we analyzed phospho-histone H3 (H3P), a marker of late G2 mitosis (Figure 5). Transfection with miR-515-3p, miR-519e-3p, and miR-371a-3p-mimic increased number of H3P-positive hiPSC-CM as compared to miR-scrambled-treated hiPSC-CM. In addition, a transient period of hypoxia (2 h), as compared to normoxia, increased the number of H3P-positive hiPSC-CM in miR-519e-3p-mimic and miR-515-3p-mimic-transfected hiPSC-CM but not after transfection with miR-371a-3p-mimic (Figures 5A, 5B, S7A, and S7B). With respect to potential gender differences, we observed similar capacity of candidate miR-mimics to induce proliferation in hiPSC-CM derived from female and male donors (Figure S8). We also performed H3P staining over a period of five days to understand whether miRNA-induced increase in mitosis is persistent. Herein, we observed a peak at day three post transfection, suggesting a brief proliferative burst after lipid-based miRNA transfection (Figure S9). These results indicate that miR-515-3p, miR-519e-3p, and miR-371a-3p are robust inducers of cardiomyocyte proliferation. However, transient hypoxia did not result in an enhanced cardiomyocytes turnover after miR-371a-3p overexpression. As this may be relevant in a hypoxic milieu that is present in patients with myocardial infarction, we further focused our study on miR-515-3p and miR-519e-3p.

Cardiomyocyte proliferation involves regulation of genes that are necessary for cell cycle reentry. Therefore, we assessed gene expression of positive and negative cell cycle regulators after transfection of hiPSC-CM with miR-515-3p and miR-519e-3p (Figure 6). First, we confirmed, as already observed in HEK 293 cells, a substantial suppression of CDKN1A in hiPSC-CM both by RT-qPCR (Figure 6A) and immunofluorescence imaging (Figure 6B). Similarly, downregulation of CDKN1B was observed (Figure 6C), whereas CDKN1C was not significantly reduced (Figure 6D). Moreover, genes that restrain cell cycle progression within the Hippo-signaling pathway, LATS1 and MOB1B, were significantly downregulated after miR-515-3p and miR-519e-3p-mimic transfection (Figures 6E and 6F). Importantly, miR-515-3p and miR-519e-3p-transfected hiPSC-CM showed a substantial increase in cyclin A2 (CCNA2) expression, a key regulator of cell cycle progression through G1/S and G2/M phase., In addition, the cell cycle activator cyclin B1, was markedly upregulated, whereas cyclin D2, did not show a differential expression (Figures 6G–6I). In line, sarcomeric genes that are involved in structural maturation, such as myosin light chain-2, myosin light chain-7, and myomesin 3, were markedly downregulated (Figures 6J–6M). We also investigated members of the pocket protein family of tumor suppressors, namely RB transcriptional corepressor 1 (Rb1) and RB transcriptional corepressor like 2 (Rbl2) that are upregulated upon differentiation and suppress cell cycle activators and Rbl1 that is downregulated in adult cardiomyocytes.,, Herein, a marked downregulation of Rb1 and Rbl2 and upregulation of Rbl1 was detected after miR-mimic-515-3p transfection (Figures 6N–6P). Next, we validated our findings on protein level by conducting Western blots (Figure 6Q–6T). After transfection of miR-515-3p and miR-519e-3p in hiPSC-CM, a marked downregulation of CDKN1A and upregulation of CCNA2 on protein level was confirmed (Figures 6R and 6S). In addition, we observed a substantial increase of Aurora B-kinase, a midbody-associated protein marker needed for completion of cell division (Figure 6T). In conclusion, overexpression of miR-515-3p and miR-519e-3p substantially reduce expression of genes related to mitotic arrest and significantly upregulate genes involved in cell cycle reentry.

For identification of hiPSC-CM undergoing cytokinesis after miR-515-3p, miR-519e-3p, and miR-scrambled transfection, we visualized Aurora B-kinase. Herein, we were able to differentiate all stages of late mitosis, including movement of Aurora B-kinase to the midbody in cytokinesis (Figure 7A). In addition, miR-515-3p and miR-519e-3p-treated hiPSC-CM showed disorganization of sarcomere structures (Figure 7B). Importantly, the number of hiPSC-CM in prophase or stages of late mitosis was substantially increased after miR-515-3p and miR-519e-3p transfection, as indicated by Aurora B-kinase positive nuclei (Figure 7C). We also observed a substantially higher number of hiPSC-CM positive for localization of Aurora B-kinase in the midbodies, suggestive of cytokinesis after treatment with miR-515-3p and miR-519e-3p (Figures 7D and 7E). In conclusion, miR-515-3p and miR-519e-3p substantially induce early and late stages of mitosis and cell division.

To further understand pathways involved in the observed miRNA-induced regenerative potential, we performed RNA-seq after overexpression of miR-515-3p and miR-519e-3p in hiPSC-CM (Figure 8). The majority of genes modulated by miR-515-3p and miR-519e-3p are overlapping, as expected given the origin of the same miRNA family (Figures 8A and 8B). Further analysis identified two major gene clusters (Figure 8C). We used gene ontology (GO) enrichment analysis to investigate whether specific GO terms are associated with differentially expressed genes in the clusters (Figure 8D). Downregulated genes are strongly involved in structural and sarcomeric organization (Figure 8D, left). In contrary, upregulated enriched GO terms comprise processes involved in DNA replication, cell cycle, and cell division (Figure 8D, right). Notably, key signaling pathways regulating cardiomyocytes proliferation, such as the PI3K-Akt, the WNT/B-catenin, and TGF-B/SMAD (Figures 8E–8G), were enriched in miR-mimic-transfected hiPSC-CM. Moreover, we observed that Notch, Hippo/YAP, and NRG/ERBB signaling pathways showed a trend toward significant enrichment after miR-mimic overexpression (Figures 8H–8J). In addition, we analyzed transcription factors that are differentially regulated (Figure 8K). Interestingly, several cell cycle-related transcription factors from the E2F family (E2F1, E2F2, and E2F7), the MYB transcription factor family (MYBL1 and MYBL2), and FOXM1 were upregulated upon miR-mimic transfection (Figure 8K). As we observed an increase in proliferation after miR-mimic transfection and transient hypoxia as compared to normoxia, we also investigated enrichment of GO terms in the context of hypoxia. Indeed, genes relevant for response to hypoxia were significantly enriched in miR-mimic-treated hiPSC-CM (Figure 8L). Collectively, these findings suggest that the proliferative potential of miR-515-3p and miR-519e-3p is mediated by a broad panel of genes that de-repress inhibited proliferative pathways relevant for cardiomyocytes proliferation and cell division.

Our findings here provide evidence that overexpression of miRNAs is effective to induce proliferation in human iPSC-CM. We have tested overexpression and inhibition of more than two thousand miRNAs in hiPSC-CM using functional high-throughput screenings. Whereas downregulation of miRNAs did not robustly enhance cell cycle reentry, 28 out of 2019 miRNA-mimics significantly increased proliferative activity in hiPSC-CM. Derived from the high-throughput screening, we validated two miRNA members of the primate-specific C19MC cluster, miR-515-3p and miR-519e-3p. These miRNAs substantially increased markers of early and late mitosis in hiPSC-CM, indicative of cell division. Intriguingly, these miRNAs exert additive proliferative effects in hiPSC-CM upon transient hypoxia that was not observed with other candidate miRNAs, such as miR-371a-3p. These findings suggest that miR-515-3p and miR-519e-3p are potential attractive targets for induction of cardiomyocyte proliferation in a transient hypoxic milieu, such as present in myocardial infarction. Strategies to induce cardiomyocyte proliferation evolve as a novel therapeutic option in adults, as emerging evidence showed that cardiomyogenesis is not restricted to embryonic stages. Studies in mice and humans have shown that cardiomyocyte turnover persists in postnatal mice and humans in adolescence.,,,,, However, this reparative potential declines over the years with a very low number of cardiomyocytes undergoing cell cycle reentry in adults that are the most vulnerable to myocardial injury., In this regard, miRNA-targeted therapies have the potential to reinforce turnover of preexisting cardiomyocytes. Several experimental studies have shown that downregulation of endogenous miRNAs increase proliferation in vitro and in vivo in murine models.,,, Conversely, our high-throughput screening did not identify miRNA-inhibitors as inducers of cell cycle reentry in hiPSC-CM. Transfection of anti-let-7c that has been shown to increase turnover of murine cardiomyocytes in vivo, increased EdU uptake in the initial screening but failed in the validation screening. The absence of a significant proliferative response of the 2019 investigated miRNA-inhibitors can be related to inter-species differences or to the single lipid-based miRNA transfection used in our screening. If prolonged downregulation of endogenous miRNAs via adeno-associated virus may enhance proliferation in human-derived cardiomyocytes has to be determined. Intriguingly, overexpression of 28 out of 2019 miRNAs substantially enhanced cardiomyocyte turnover. Of note, whereas some of the miRNAs that were reported to induce proliferation in murine cardiomyocytes were confirmed in our screening, such as miR-1825 and miR-33b, others did not show a significant proliferative effect in hiPSC-CM after transient miRNA overexpression, such as miR-590, the miR-302/367 cluster, and the miR-17-92 family.,,, Interestingly, five (miR-515-3p, miR-519e-3p, miR-517c-3p, miR-371a-3p, and miR-371b-3p) out of 28 miRNA candidates from the screening belong to the primate-specific chromosome 19 miR-cluster (C19MC) and adjacent miR-371-373 family, that are expressed predominantly in placental tissue,,, and in human embryonic stem cells. As experimental and clinical studies indicate that miRNA members of the C19MC are involved in biological processes related to proliferation, migration, differentiation, extracellular matrix composition, and immunomodulation, we reasoned that these miRNAs are important for cardiomyocyte proliferation due to their involvement of biological processes that comprise key pathways for cardiac regeneration. Notably, the majority of members of the miR-515 and miR-371-373 family tested in the screening did not induce cardiomyocyte turnover, indicating that only a minority of the cluster harvest functional specialization to modulate proliferative pathways in cardiomyocytes. Indeed, we confirmed that miR-515-3p and miR-519e-3p substantially enhance expression of cell cycling progressors and suppress cell cycling inhibitors. In addition, Aurora B-kinase that is mandatory for cytokinesis was detected in a high number of miR-515-3p and miR-519e-3p-transfected hiPSC-CM and was substantially increased in the midbodies of cardiomyocytes, indicative of cell division. Importantly, transient hypoxia has an additive effect to the proliferative activity observed after miR-515-3p and miR-519e-3p transfection that was not evident in miR-scrambled and miR-371a-3p-treated hiPSC-CM. Interestingly, experimental studies suggest that a distinct population of adult cardiomyocytes maintain their proliferative capacity in a hypoxic environment,, and activation of cell cycling in cardiomyocytes is more prominent at the border zone of the infarcted myocardium., In this regard, miR-515-3p and miR-519e-3p may particularly enhance proliferation in these distinct cardiomyocyte populations. These findings are supported by our RNA-seq that observed enrichment of genes that respond to hypoxia in hiPSC-CM overexpressed with miR-515-3p and miR-519e-3p. Furthermore, our RNA-seq analysis in miR-515-3p and miR-519e-3p-treated hiPSC-CM reveals downregulation of genes involved in sarcomeric organization, most probably reflecting sarcomeric disassembly that is needed for cell division. In contrast, we observed a strong enrichment of genes that are involved in DNA replication, cell cycle, and cell division, supporting the substantial increase in Aurora B-kinase-positive hiPSC-CM as observed after miR-515-3p and miR-519e-3p-mimic transfection. Indeed, genes of core cardiomyocyte proliferative signaling pathways, such as PI3K-Akt, the WNT/B-catenin, and TGF-B/SMAD, were significantly enriched after miR-515-3p- and miR-519e-3p-mimic transfection. In addition, transcription factors (E2F family (E2F1, E2F2, and E2F7), FOXM1, MYBL1, and MYBL2 (alias B-Myb)) that are master regulators of genes regulating mitosis and cytokinesis,,, were substantially upregulated upon miR-mimic transfection. Interestingly, these transcription factors are downregulated in adult cardiomyocytes and after binucleation and fail to be reactivated in mature cardiomyocytes upon myocardial infarction,, suggesting that miRNA-mediated activation may re-initiate adult cardiomyocyte cell cycle reentry. Conclusively, these observations indicate that induction of proliferation is not dependent on single gene regulation, but rather on modulation of large gene networks that are cooperatively regulated by miR-515-3p and miR-519e-3p. In summary, our findings on molecular level, together with our functional data, strongly suggest that miR-515-3p and miR-519e-3p are effective for induction of cell cycle reentry and increase cell division in human-derived cardiomyocytes in a timely controlled manner.

This study provides a translational perspective to investigate miRNA-mimics derived from the high-throughput screening in pre-clinical models for assessment of miRNA-induced repair and regenerative potential. However, we are aware of the inherent limitations of hiPSC-CM technology used in this study. Notably, hiPSC-CM were cultured for 50 to 60 days to achieve a certain degree of maturation of hiPSC-CM. However, it has to be acknowledged that hiPSC-CM comprise an immature phenotype that share similarities with fetal rather than adult cardiomyocytes, which may also endorse a higher susceptibility to undergo proliferation after miRNA treatment. Therefore, whether application of these miRNAs in adult working cardiomyocytes in vivo results in cardiac repair processes will require further investigation. Secondarily, we did not investigate direct targets of miRNAs that contribute to the observed induction of cell division in hiPSC-CMs. Further studies are needed to evaluate specific molecular mechanisms involved in miRNA-induced activation of the cell cycling program in hiPSC-CM.

We explored proliferative potential of more than two thousand miRNAs upon overexpression and inhibition in hiPSC-CM after transient hypoxia. Importantly, efficient induction of cell cycling was restricted to overexpression of miRNAs. Primate-specific miR-515-3p and miR-519e-3p enhanced proliferation in hiPSC-CM through downregulation of pathways involved in sarcomere organization and upregulation of gene networks involved in cell division that is supported by substantial increase in proliferation using immune staining for early and late mitosis markers. Therefore, these miRNAs have the therapeutic potential for cardiac repair in patients with myocardial injury due to transient hypoxia, such as present in myocardial infarction.



Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Philipp Jakob, MD (Philipp.jakob@usz.ch).

This study did not generate new unique reagents.

Human induced pluripotent cell (hiPSC) derived Cardiomyocytes (hiPSC-CMs; WTD2 and Control 1) were obtained from University Medical Center Göttingen, Germany. Two Cell lines were generated with an episomal plasmid-based transduction. One female and one male hiPSC line was reprogrammed with the STEMCCA system, which is a humanized excisable lentiviral system containing all four reprogramming factors OCT4, SOX2, KLF4, and c-MYC in a single “stem cell cassette” (pHAGE2-EF1aFull-hOct4-F2A-hKlf4-IRES-hSox2-P2A-hcMyc-W-loxP). The pluripotent clones were expanded up to passage 8. HiPSCs were effectively differentiated and selected in vitro into >90% pure cardiomyocytes. Fifty to 60 or 110 to 120 days old hiPSC-CM were used for experiments.

As model system, human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) (hiPSC-CM) were used. HiPSCs were generated as previously described., Female and male donors were used to generate cardiomyocytes, with two different reprogramming-based approaches. Two female hiPSC lines were generated with an episomal plasmid-based transduction. One female and one male hiPSC line was reprogrammed with the STEMCCA system, which is a humanized excisable lentiviral system containing all four reprogramming factors OCT4, SOX2, KLF4, and c-MYC in a single “stem cell cassette” (pHAGE2-EF1aFull-hOct4-F2A-hKlf4-IRES-hSox2-P2A-hcMyc-W-loxP). The pluripotent clones were expanded up to passage 8. The established iPSCs were proven for their pluripotency by both in vitro studies - the expression of pluripotency markers and differentiation experiments - and in vivo teratoma formation assay. All protocols were approved by the Ethics Committee of the University Medical Center Göttingen (No. 10/9/15). Informed consent was obtained from all participants and all research was performed in accordance with relevant guidelines and regulations and in accordance with the Declaration of Helsinki.

Post differentiation, cardiac culture medium (RPME 1640 with Glutamax and HEPES, B27 supplement) was aspirated and replaced with cardio selection medium (RPMI 1640 without Glucose, Lactate/HEPES, hAlbumin, human recombinant L-Ascorbic Acid 2-Phosphate). The medium was exchanged every second day. Post selection, the medium was replaced with cardiac culture medium. HiPSCs were effectively differentiated and selected in vitro into >90% pure cardiomyocytes. Fifty to 60 or 110 to 120 days old hiPSC-CM were used for experiments. Cell detachment was performed using 0.25% trypsin, 0.02% EDTA and Stempro accutase, followed by collagenase diluted in RPME 1640 for cell dissociation. Thereafter, cells were seeded in cardio digestion medium (RPME 1640 with Glutamax and HEPES, B27 Supplement, 10% FBS, Thiazovivin (2 μM)). Medium was replaced to cardiac culture medium 48 h later. Cell culture plates were coated with Geltrex.

Lullaby stem (OZ Biosciences) was used for transfection of miRNAs using a standard forward transfection protocol. Medium was replaced with fresh cardiac culture medium 24 h post transfection. 48 h post transfection, hiPSC-CM were exposed to hypoxia (0.5% oxygen, 5% CO2) for 2 h. Thereafter, medium was exchanged to cardio culture medium containing EdU, or hiPSC-CM were fixed and stained with H3P or Aurora B-kinase and further proceeded for immunocytochemistry. Assessment of FAM-miR transfected hiPSC-CM was performed 24 h post transfection using fluorescent imaging or fluorescence-activated cell sorting (FACS). For FACS analysis, cells were briefly washed in PBS, trypsinized, and the detached cells were resuspended with freshly mixed staining solution (100 μL Annexin-biding buffer was added for each sample). The mix was carefully vortexed and incubated for a period of 20 min in dark at 4°C and samples were measured immediately at an Attune flow cytometer. All-stars Hs Cell Death Control siRNA (Qiagen) was used to observe a cell death phenotype in hiPSC-CM 48 h post transfection.

Staining was performed as previously described., HiPSC-CM were washed shortly with PBS and fixed with 3.7% formaldehyde (Sigma). Cells were washed three times with PBS and permeabilized with 0.5% Triton X-100 in PBS, and washed three times in PBS. HiPSC-CM were blocked for 1 h at RT with blocking buffer (10% Horse serum, 1% BSA, 0.3% Triton) in PBS and incubated with primary antibodies in dilution buffer (1x PBS, 1% BSA, 1% donkey or horse serum, 0, 3% Triton) overnight at 4°C. Subsequently, hiPSC-CM were washed three times with 0.1% Nonidet P40 in PBS, and incubated with corresponding secondary antibodies conjugated with Alexa Fluor 488 and Alexa Fluor 594 (1:500 Life technologies) for 1 h at RT. HiPSC-CM were washed three times with 0.1% BSA. EdU incorporation assay was performed by incubating cells with Click it reaction mix cocktail kit (Life Technologies, Invitrogen) for 30 min, according to manufacturer protocol. HiPSC-CM were washed three times with 0.1% BSA in PBS, followed by incubation with respective secondary antibodies for 1 h at RT. HiPSC-CM were later incubated with Hoechst (Nuclear stain) and washed three times with 1xPBS.

A high-throughput screening (HTS) by using a miRNA-library (Thermo Fisher Scientific/Ambion; mirVana Human Library v19.0; 2019 miR-mimics and miR-inhibitors) was performed in two individual screenings. Three thousand and five hundred hiPSC-CM/well were plated on Geltrex (Thermo Fisher Scientific) -coated 384-well plates (Corning) using an EL406 washer dispenser (BioTeK) in a laminar flow work cabinet to avoid contamination. For each miRNA, 400 nM dilution of the miRNA were complexed with Lullaby Stem (OZ Biosciences) according to the manufacturer protocol. The mixture was transferred to the 384-well assay plate (Corning) using a Freedom EVO liquid handling workstation (Tecan) resulting in a final concentration of 60 nM miRNA in a total volume of 50 μL per well with a slow dispense speed (1 μL/s). miRNA transfected hiPSC-CM were kept for 24 h at 37°C in 5% CO2. Medium exchange was performed by an EL406 washer dispenser (BioTeK) after 24 h 48 h post transfection, hiPSC-CM were exposed to transient hypoxia (0.5% oxygen, 5% CO2) for 2 h. Thereafter, cells were replaced with cardio culture medium containing EdU and incubated for 48 h. Thereafter, cells were fixed and processed for immunofluorescence analysis.

HiPSC-CM were fixed and stained. The immunofluorescence imaging was performed on an ArrayScan XTI High Content Screening Platform (ThermoScientific). 20× magnification objective was used, with a camera mode of 1104x1104 pixel. Three imaging channels were used, Channel 1 was represented as Hoechst (Nucleus marker/Thermo Fisher Scientific), Channel 2 as Troponin T (R&D systems), and Channel 3 as EdU (Thermo fisher scientific). Per well, 36 images from all three channels were acquired from a 384-well plate, covering the majority of the well area. Image analysis was performed with the integrated software, ThermoScientific HCS Studio, cell scan version 6.6.0 (Thermos Fisher Scientific). The first parameter was to identify the cell objects, where the primary object was selected as Channel 1 (Nuclei), later Channel 3 (EdU), as DNA synthesis localizes in the nuclei. Primary Object Identification Mask (Region of Interest = ROI) from Channel 1 (Nuclei) was carried over to Channel 3 without modification. Edu-positive nuclei/cells were selected based on the average intensity in the ROI being >2000. Object selection was based on Channel 1 and Channel 3, as hiPSC-CM were confluent enough for Channel 2. The two used criteria for image output was 1) number of valid objects from nuclei per well and 2) % of EdU-positive objects (nuclei) per well (related to the number of valid objects (nuclei)).

All data was processed using the KNIME analytics platform (KNIME). For plate-wise normalization, the Z score for each candidate miRNA on the plate was calculated. A Z score was calculated from the % of EdU-positives as follows: Z score = (x – median(x [subset]))/mad(x [subset]) Where x is the % of EdU-positives in the well, median(x [subset]) is the median from all candidate wells in the plate and mad(x [subset]) the median absolute deviation of all candidate wells in the plate. The mean for the results from the two replicates then was calculated using the following formula: (Z score R1+Z score R2)/2 and used for the final candidate ranking.

Total RNA was isolated from hiPSC-CM using the RNeasy Kit (Qiagen). Kits were used according to the manufacturer instructions. HiPSC-CM were seeded and transfected in 24- or 48-well cell culture plates. 48 h and 72 h post microRNA-transfection, cell culture medium was aspirated, and cells were washed three times with sterile DPBS. QIAzol lysis reagent was added onto cells and incubated for 5 min at RT. The resulting cell lysates were collected in 1.5 mL nuclease free reaction tubes (Eppendorf). MiRNeasy RNA extraction kit (Qiagen) was used to isolate the RNA according to the manufacturer instructions. The RNA concentration was measured using Nano drop at 260/280 nm. The isolated RNA was dissolved in 14 μL of RNAse-free water and stored at −80°C or continued for reverse transcription.

Gene expression quantification was performed according to standard protocols. The isolated RNA was dissolved in 14 μL of RNAse-free water and stored at −80°C or continued for reverse transcription. High-capacity reverse transcription kit (Thermo Fisher Scientific) for cDNA synthesis was used. SYBR green detection method was used for performing qPCR in 384-well plate and analyzed by Viia R real-time PCR system (Applied Bio systems). Fold changes in gene expression were analyzed by relative quantification by taking means of Ct values of gene of interest and a housekeeping gene (GAPDH) that served as a normalizing control. The fold change was calculated by 2-ΔΔCT formula. For expression of miRNA, RNA was isolated after transfection of hiPSC-CM with hsa-anti-miR-195-5p (Thermo Fisher Scientific) as mentioned above. TaqMan microRNA reverse transcription kit was used to perform reverse transcription and TaqMan microRNA fast advanced master mix and TaqMan microRNA primers (Thermo Fisher Scientific) were used. Expression of miR-195-5p was normalized with that of RNU48.

MiRNA-scrambled, hsa-miR-mimic-515-3p and hsa-miR-mimic-519e-3p were transfected into hiPSC-CM as described above. 72 h post transfections, cells were washed with DPBS three times. Thereafter QIAzol (Qiagen- MiRNeasy RNA extraction) was added. Resulting lysates were collected in 1.5 mL nuclease free reaction tubes (Eppendorf). RNA-Sequencing was performed by ArrayStar (Arraystar, Inc., USA). Total RNA was used to prepare the sequencing library using KAPA Stranded RNA-Seq Library Prep Kit (Illumina). Libraries were sequenced using HiSeq 4000 (Illumina). Image analysis and base calling was performed using the Solexa pipeline v1.8 (OffLine Base Caller software, v1.8). The raw reads were first cleaned by removing adapter sequences, trimming low quality ends, and filtering reads with low quality (phred quality <20) using Fastp (Version 0.20)). Sequence pseudo alignment of the resulting high-quality reads to the human reference genome (build GRCh38.p13, GENCODE release 32) and quantification of gene level expression was carried out using Kallisto (Version 0.46.1). To detect differentially expressed genes we applied a count based negative binomial model implemented in the software package EdgeR (R version: 4.0.3) using the GLM framework. Genes showing altered expression with adjusted (Benjamini and Hochberg method) p value <0.05 were considered differentially expressed.

HiPSC-CM were transfected with individual miRNAs. HiPSC-CM were lysed with freshly prepared RIPA lysis buffer 72 h post transfection. Cell lysates were collected and centrifuged at 13000 rpm for 20 min at 4°C. Supernatants from centrifugation were collected and the pellet was discarded. BCA protein assay kit (Pierce) was used to determine the protein concentration. Equal amount (40 μg) of proteins were resolved by 4–20% Pre cast protein gels (Bio-Rad) and blotted onto a polyvinylidene difluoride (PVDF) membrane. Membranes were blocked with 5% Milk or 5% BSA, incubated with primary antibodies over night at 4°C, followed by incubation with HRP-linked secondary antibodies. Chemiluminescent substrate (Supersignal westDura Extended) solution was added onto the membrane and the membrane was developed under chemiluminesence to detect antibody-labeled proteins (INTAS Detection System).

LDH assay was performed with the LDH assay kit (Pierce, Thermo Scientific) according to the manufacturer protocols. HiPSC-CM were plated in 96- or 384- well plates, and transfected with either scrambled-miRNA or miRNA-mimics using concentrations ranging from 20 nM to 100 nM to assess toxicity. Medium was carefully collected and placed in a 384-well plate. LDH start reagent was added onto the cells and incubated for a period of 30 min. Thereafter, LDH assay stop solution was added to stop the reaction and measured with a fluorescent plate reader (Tecan) at 490 nm and 690 nm absorbance.

Data are presented as means ± standard error of the mean (SEM). Bars represent average of a minimum of three to five independent experiments with an average of two replicates for each experiment. Data analysis was conducted using Prism 7 Software (Graphpad). We analyzed the data using un-paired Student’s t test for single comparisons. One-way or two-way ANOVA (using the post-hoc Tukey or Bonferroni’s test) were used for multiple comparisons. A value of p ≤ 0.05 was considered as statistically significant.