Induction of cardiomyocyte proliferation to replace damaged heart tissue is a promising therapeutic approach. A recent drug screen revealed that cardiomyocytes require the mevalonate pathway for proliferation, although the specific mechanisms are unknown. In this study, we use human pluripotent stem cell-derived cardiomyocytes and cardiac organoids to further interrogate the role of the mevalonate pathway in cardiomyocyte proliferation. Chemical and genetic perturbations of the mevalonate pathway indicated that the post-translational modification, prenylation, regulates cardiomyocyte proliferation. We use prenyl probes and mass spectrometry to identify a catalogue of 40 prenylated proteins in human cardiac cells, including proteins where prenylated function had not yet been investigated. We show that multiple prenylated proteins control cardiomyocyte proliferation including RRAS2 and NAP1L4. We demonstrate that prenylation has differential effects on distinct proteins, with RRAS2 prenylation controlling membrane localization and NAP1L4 prenylation regulating cardiomyocyte mitosis and centrosome homeostasis. Together, these data show that protein prenylation is required for cardiomyocyte proliferation through multiple targets and these processes may need to be re-activated for cardiac regeneration. ### Competing Interest Statement E.R.P., R.J.M., and J.E.H. are co-inventors on patents relating to cardiac organoid maturation and cardiac therapeutics. J.E.H. is co-inventor on licensed patents for engineered heart muscle. E.R.P., R.J.M. and J.E.H. are co-founders, scientific advisors, and stockholders in Dynomics.

Myocardial injuries lead to cardiomyocyte loss and heart failure. Endogenous glucocorticoids, via the glucocorticoid receptor (GR), limit cardiomyocyte regeneration. Here we show that glucocorticoids suppress mammalian (murine) cardiomyocyte proliferative response to regenerative growth factors and cytokines. GR activation in neonatal cardiomyocytes upregulated MAPK-ERK inhibitors ERRFI1 and DUSP1. Using neuregulin 1 as a model, we demonstrated that glucocorticoids inhibit growth-factor-induced ERK activation, nuclear translocation and transcriptional output. Errfi1 and Dusp1 knockdown restored growth-factor-induced proliferation of glucocorticoid-exposed cardiomyocytes. Cardiac expression of DUSP1 and ERRFI1 increased postnatally, coinciding with regenerative capacity decline. In juvenile and adult cardiomyocytes, regenerative growth factors failed to induce the MAPK-ERK pathway and proliferation; however, DUSP1 inhibition restored these responses. GR antagonism enhanced growth-factor-induced cardiomyocyte protection, proliferation and cardiac function after adult myocardial injury. These findings reveal the emergence of a postnatal systemic brake on cardiomyocyte proliferative response to growth factors and support GR inhibition as a strategy to enhance growth-factor-based regenerative therapies.

Immunotherapy harnesses neoantigens encoded within the human genome, but their therapeutic potential is hampered by low expression, which may be controlled by the nonsense-mediated mRNA decay (NMD) pathway. This study investigates the impact of UPF1-knockdown on the expression of non-canonical/mutant proteins, employing proteogenomic to explore UPF1 role within the NMD pathway. Additionally, we conducted a comprehensive pan-cancer analysis of UPF1 expression and evaluated UPF1 expression in Triple-Negative Breast Cancer (TNBC) tissue in-vivo. Our findings reveal that UPF1-knockdown leads to increased translation of non-canonical/mutant proteins, particularly those originating from retained-introns, pseudogenes, long non-coding RNAs, and unannotated transcript biotypes. Moreover, our analysis demonstrates elevated UPF1 expression in various cancer types, with notably heightened protein levels in patient-derived TNBC tumors compared to adjacent tissues. This study elucidates UPF1 role in mitigating transcriptional noise by degrading transcripts encoding non-canonical/mutant proteins. Targeting this mechanism may reveal a new spectrum of neoantigens accessible to the antigen presentation pathway. Our novel findings provide a strong foundation for the development of therapeutic strategies aimed at targeting UPF1 or modulating the NMD pathway.

Cardiac injury, such as myocardial infarction (MI), results in permanent loss of cardiomyocytes and in many cases heart failure. Transgenic expression of the pro-proliferative transcription factor Myc and Cyclin T1 can drive substantial adult cardiomyocyte proliferation to replace lost cardiomyocytes. Herein, we show that Myc and Cyclin T1 induced cardiomyocyte proliferation leads to myocardial repair and functional (long-term) recovery post-MI in mice. To provide a more translational approach, we developed modified mRNA (modRNA) encoding Myc-Ccnt1 as a transient and non-integrating strategy for regeneration. One dose of Myc-Ccnt1 modRNA is sufficient to transiently drives cardiomyocyte proliferation in human pluripotent stem cell-derived cardiomyocytes and a mouse MI model, where it leads to better heart function. Using single nuclei sequencing and proteomics, we show this was functionally mediated by transcriptional activation of cell-cycle regulating genes, which ultimately results in mitosis and cytokinesis of cardiomyocytes. Collectively, these findings indicate that Myc-Ccnt1 modRNA has the potential to be an effective regenerative therapeutic.

Crosstalk between cardiac cells is critical for heart performance. Here we show that vascular cells within human cardiac organoids (hCOs) enhance their maturation, force of contraction, and utility in disease modeling. Herein we optimize our protocol to generate vascular populations in addition to epicardial, fibroblast, and cardiomyocyte cells that self-organize into in-vivo-like structures in hCOs. We identify mechanisms of communication between endothelial cells, pericytes, fibroblasts, and cardiomyocytes that ultimately contribute to cardiac organoid maturation. In particular, (1) endothelial-derived LAMA5 regulates expression of mature sarcomeric proteins and contractility, and (2) paracrine platelet-derived growth factor receptor β (PDGFRβ) signaling from vascular cells upregulates matrix deposition to augment hCO contractile force. Finally, we demonstrate that vascular cells determine the magnitude of diastolic dysfunction caused by inflammatory factors and identify a paracrine role of endothelin driving dysfunction. Together this study highlights the importance and role of vascular cells in organoid models.

Immunotherapy harnesses neoantigens encoded within the human genome, but their therapeutic potential is hampered by low expression, which may be controlled by the Nonsense-Mediated Decay (NMD) pathway. This study investigates the impact of UPF1-knockdown on the expression of non-canonical/mutant proteins, employing proteogenomic to explore UPF1 role within the NMD pathway. Additionally, we conducted a comprehensive pan-cancer analysis of UPF1 expression and evaluated UPF1 expression in Triple-Negative Breast Cancer (TNBC) tissue in-vivo. Our findings reveal that UPF1-knockdown leads to increased transcription of non-canonical/mutant proteins, particularly those originating from retained-introns, pseudogenes, long non-coding RNAs, and unannotated transcript biotypes. Moreover, our analysis demonstrates elevated UPF1 expression in various cancer types, with notably heightened protein levels in patient-derived TNBC tumours compared to adjacent tissues. This study elucidates UPF1 role in mitigating transcriptional noise by degrading transcripts encoding non-canonical/mutant proteins. Intriguingly, we observe an upregulation of the NMD pathway in cancer, potentially acting as a “neoantigen-masking” mechanism that suppresses non-canonical/mutant protein expression. Targeting this mechanism may reveal a new spectrum of neoantigens accessible to the antigen presentation pathway. Our novel findings provide a strong foundation for the development of therapeutic strategies aimed at targeting UPF1 or modulating the NMD pathway.

Immunotherapy interventions relies heavily on neoantigen availability. The human genome encodes non-canonical/mutant proteins that potentially contain neoantigenic peptides. Nevertheless, their typically low expression, potentially moderated by the Nonsense-Mediated Decay (NMD) pathway, restricts their therapeutic utility. In this study, we explored the NMD pathway influence on non-canonical/mutant protein expression, specifically focusing on UPF1 knockdown. We implemented proteogenomic approaches to ascertain if the encoding transcripts and their respective proteins were upregulated post-knockdown. Complementary to this, we conducted a comprehensive pan-cancer survey of UPF1 expression and an in vivo evaluation of UPF1 expression in Triple-Negative Breast Cancer (TNBC) tissue. Our empirical results delineated that UPF1 knockdown precipitates an increase in the transcription of non-canonical/mutant proteins, especially those originating from retained-introns, pseudogenes, long non-coding RNAs, and unannotated biotypes. Furthermore, the analysis revealed that UPF1 expression was conspicuously high across a range of neoplastic tissues, with protein levels notably amplified in patient derived TNBC tumours in comparison to adjacent tissues. Our study elucidates UPF1 functional role in attenuating transcriptional noise through the degradation of transcripts encoding non-canonical/mutant proteins. Interestingly, we observed an upregulation of the NMD pathway in cancer, potentially functioning as a “neoantigen masking” mechanism that subdues non-canonical/mutant protein expression. Suppressing this mechanism may unveil a new cadre of neoantigens accessible to the antigen presentation pathway. Our novel findings proffer a solid base for devising therapeutic strategies targeting UPF1 or the NMD pathway, given the pronounced presence of UPF1 in malignant cells, thus potentially augmenting immunotherapeutic responses in cancer.

The mammalian heart undergoes maturation in early neonatal life to meet the increased functional demands of postnatal life and coincidentally loses regenerative capacity. A relationship between mevalonate pathway shutdown and cell cycle arrest during maturation has recently been found. The mevalonate pathway generates end-products including coenzyme Q10 and cholesterol. However, metabolic intermediates farnesyl (FPP) and geranylgeranyl pyrophosphate (GGPP) are also important and are used in protein prenylation – a post-translational modification that alters subcellular localization, bimolecular interactions and protein processing.

PURPOSE OF REVIEW:This review summarizes the important role that metabolism plays in driving maturation of human pluripotent stem cell-derived cardiomyocytes.RECENT FINDINGS:Human pluripotent stem cell-derived cardiomyocytes provide a model system for human cardiac biology. However, these models have been unable to fully recapitulate the maturity observed in the adult heart. By simulating the glucose to fatty acid transition observed in neonatal mammals, human pluripotent stem cell-derived cardiomyocytes undergo structural and functional maturation also accompanied by transcriptional changes and cell cycle arrest. The role of metabolism in energy production, signaling, and epigenetic modifications illustrates that metabolism and cellular phenotype are intimately linked. Further understanding of key metabolic factors driving cardiac maturation will facilitate the generation of more mature human pluripotent stem cell-derived cardiomyocyte models. This will increase our understanding of cardiac biology and potentially lead to novel therapeutics to enhance heart function.