A novel glycopeptide from mountain-cultivated ginseng residue protects type 2 diabetic symptoms-induced heart failure.

ETHNOPHARMACOLOGICAL RELEVANCE: Mountain-cultivated Panax ginseng C.A.Mey. (MCG) with high market price and various properties was valuable special local product in Northeast of Asia. MCG has been historically used to mitigate heart failure (HF) for thousand years, HF is a clinical manifestation of deficiency of "heart-qi" in traditional Chinese medicine. However, there was little report focus on the activities of extracted residue of MCG. AIM OF THE STUDY: A novel glycopeptide (APMCG-1) was isolated from step ethanol precipitations of alkaline protease-assisted extract from MCG residue. MATERIALS AND METHODS: The molecular weight and subunit structure of APMCG-1 were determined by FT-IR, HPLC and GPC technologies, as well as the H9c2 cells, Tg (kdrl:EGFP) zebrafish were performed to evaluated the protective effect of APMCG-1. RESULTS: APMCG-1 was identified as a glycopeptide containing seven monosaccharides and seven amino acids via O-lined bonds. Further, in vitro, APMCG-1 significantly decreased reactive oxygen species production and lactate dehydrogenase contents in palmitic acid (PA)-induced H9c2 cells. APMCG-1 also attenuated endoplasmic reticulum stress and mitochondria-mediated apoptosis in H9c2 cells via the PI3K/AKT signaling pathway. More importantly, APMCG-1 reduced the blood glucose, lipid contents, the levels of heart injury, oxidative stress and inflammation of 5 days post fertilization Tg (kdrl:EGFP) zebrafish with type 2 diabetic symptoms in vivo. CONCLUSIONS: APMCG-1 protects PA-induced H9c2 cells while reducing cardiac dysfunction in zebrafish with type 2 diabetic symptoms. The present study provides a new insight into the development of natural glycopeptides as heart-related drug therapies.

The Multifaceted Roles of Hippo-YAP in Cardiovascular Diseases.

The Hippo-yes-associated protein (YAP) signaling pathway plays a crucial role in cell proliferation, differentiation, and death. It is known to have impact on the progression and development of cardiovascular diseases (CVDs) as well as in the regeneration of cardiomyocytes (CMs). However, further research is needed to understand the molecular mechanisms by which the Hippo-YAP pathway affects the pathological processes of CVDs in order to evaluate its potential clinical applications. In this review, we have summarized the recent findings on the role of the Hippo-YAP pathway in CVDs such as myocardial infarction, heart failure, and cardiomyopathy, as well as its in CM development. This review calls attention to the potential roles of the Hippo-YAP pathway as a relevant target for the future treatment of CVDs.

Cell cycle visualization tools to study cardiomyocyte proliferation in real-time.

Cardiomyocytes in the adult human heart are quiescent and those lost following heart injury are not replaced by proliferating survivors. Considerable effort has been made to understand the mechanisms underlying cardiomyocyte cell cycle exit and re-entry, with view to discovering therapeutics that could stimulate cardiomyocyte proliferation and heart regeneration. The advent of large compound libraries and robotic liquid handling platforms has enabled the screening of thousands of conditions in a single experiment but success of these screens depends on the appropriateness and quality of the model used. Quantification of (human) cardiomyocyte proliferation in high throughput has remained problematic because conventional antibody-based staining is costly, technically challenging and does not discriminate between cardiomyocyte division and failure in karyokinesis or cytokinesis. Live cell imaging has provided alternatives that facilitate high-throughput screening but these have other limitations. Here, we (i) review the cell cycle features of cardiomyocytes, (ii) discuss various cell cycle fluorescent reporter systems, and (iii) speculate on what could improve their predictive value in the context of cardiomyocyte proliferation. Finally, we consider how these new methods can be used in combination with state-of-the-art three-dimensional human cardiac organoid platforms to identify pro-proliferative signalling pathways that could stimulate regeneration of the human heart.

Native mechano-regulative matrix properties stabilize alternans dynamics and reduce spiral wave stabilization in cardiac tissue.

The stability of wave conduction in the heart is strongly related to the proper interplay between the electrophysiological activation and mechanical contraction of myocytes and extracellular matrix (ECM) properties. In this study, we statistically compare bioengineered cardiac tissues cultured on soft hydrogels ( E ≃ 12 kPa) and rigid glass substrates by focusing on the critical threshold of alternans, network-physiological tissue properties, and the formation of stable spiral waves that manifest after wave breakups. For the classification of wave dynamics, we use an improved signal oversampling technique and introduce simple probability maps to identify and visualize spatially concordant and discordant alternans as V- and X-shaped probability distributions. We found that cardiac tissues cultured on ECM-mimicking soft hydrogels show a lower variability of the calcium transient durations among cells in the tissue. This lowers the likelihood of forming stable spiral waves because of the larger dynamical range that tissues can be stably entrained with to form alternans and larger spatial spiral tip movement that increases the chance of self-termination on the tissue boundary. Conclusively, we show that a dysfunction in the excitation-contraction coupling dynamics facilitates life-threatening arrhythmic states such as spiral waves and, thus, highlights the importance of the network-physiological interplay between contractile myocytes and the ECM.

Zebrafish as a Model System for Brugada Syndrome.

Brugada syndrome (BrS) is an inheritable cardiac arrhythmogenic disease, associated with an increased risk of sudden cardiac death. It is most common in males around the age of 40 and the prevalence is higher in Asia than in Europe and the United States. The pathophysiology underlying BrS is not completely understood, but several hypotheses have been proposed. So far, the best effective treatment is the implantation of an implantable cardioverter-defibrillator (ICD), but device-related complications are not uncommon. Therefore, there is an urgent need to improve diagnosis and risk stratification and to find new treatment options. To this end, research should further elucidate the genetic basis and pathophysiological mechanisms of BrS. Several experimental models are being used to gain insight into these aspects. The zebrafish (Danio rerio) is a widely used animal model for the study of cardiac arrhythmias, as its cardiac electrophysiology shows interesting similarities to humans. However, zebrafish have only been used in a limited number of studies on BrS, and the potential role of zebrafish in studying the mechanisms of BrS has not been reviewed. Therefore, the present review aims to evaluate zebrafish as an animal model for BrS. We conclude that zebrafish can be considered as a valuable experimental model for BrS research, not only for gene editing technologies, but also for screening potential BrS drugs.

Diabetes induces modifications in costameric proteins and increases cardiomyocyte stiffness.

Several studies have demonstrated that Diabetes mellitus can increase the risk of cardiovascular disease and remains the principal cause of death in these patients. Costameres connect the sarcolemma with the cytoskeleton and extracellular matrix, facilitating the transmission of mechanical forces and cell signaling. They are related to cardiac physiology because individual cardiac cells are connected by intercalated discs that synchronize muscle contraction. Diabetes impacts the nano-mechanical properties of cardiomyocytes, resulting in increased cellular and left ventricular stiffness, as evidenced in clinical studies of these patients. The question of whether costameric proteins are affected by diabetes in the heart has not been studied. This work analyzes whether T1DM modifies the costameric proteins and coincidentally changes the cellular mechanics in the same cardiomyocytes. The samples were analyzed by immunotechniques using laser confocal microscopy. Significant statistical differences were found in the spatial arrangement of the costameric proteins. However, these differences are not due to their expression. Atomic force microscopy was used to compare intrinsic cellular stiffness between diabetic and normal cardiomyocytes and obtain the first elasticity map sections of diabetic living cardiomyocytes. Data obtained demonstrated that diabetic cardiomyocytes had higher stiffness than control. The present work shows experimental evidence that intracellular changes related to cell-cell and cell-extracellular matrix communication occur, which could be related to cardiac pathogenic mechanisms. These changes could contribute to alterations in the mechanical and electrical properties of cardiomyocytes and consequently, to diabetic cardiomyopathy.

Environmental Cues Facilitate Maturation and Patterning of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

BACKGROUND/AIMS: Advances in induced pluripotent stem cell (iPSC) technology allow for reprogramming of adult somatic cells into stem cells from which patient- and disease-specific cardiomyocytes (CMs) can be derived. Yet, the potential of iPSC technology to revolutionize cardiovascular research is limited, in part, by the embryonic nature of these cells. Here, we test the hypothesis that decellularized porcine left ventricular extracellular cardiac matrix (ECM) provides environmental cues that promote transcriptional maturation and patterning of iPSC-CMs in culture. METHODS: Cardiac progenitor cells were plated on ECM or standard tissue plates (2D monolayer) for 30 days, after which CM orientation and single cell transcriptomics were evaluated using confocal imaging and singe cell RNA-sequencing, respectively. RESULTS: Cardiac progenitors differentiated on left ventricular ECM formed longitudinal fibers that differed quantitatively from progenitors differentiated in standard 2D conditions. Unsupervised clustering of single cell transcriptomics identified a CM cluster expressing a higher level of genes related to CM maturation. CMs differentiated on ECM were overrepresented in this cluster, indicating a bias toward CM maturation, compared to cells differentiated in standard 2D monolayer conditions. CONCLUSION: Our data suggest that environmental cues related to the left ventricular ECM may promote differentiation to a more mature CM state compared to cells differentiated on a standard 2D monolayer, while facilitating organization into longitudinal micro-fibers. Our study highlights the utility of ECM as a differentiation substrate to promote CM maturation and fiber orientation in vitro .

Multidirectional Filamented Light Biofabrication Creates Aligned and Contractile Cardiac Tissues.

Biofabricating 3D cardiac tissues that mimic the native myocardial tissue is a pivotal challenge in tissue engineering. In this study, we fabricate 3D cardiac tissues with controlled, multidirectional cellular alignment and directed or twisting contractility. We show that multidirectional filamented light can be used to biofabricate high-density (up to 60 × 106 cells mL-1) tissues, with directed uniaxial contractility (3.8x) and improved cell-to-cell connectivity (1.6x gap junction expression). Furthermore, by using multidirectional light projection, we can partially overcome cell-induced light attenuation, and fabricate larger tissues with multidirectional cellular alignment. For example, we fabricate a tri-layered myocardium-like tissue and a bi-layered tissue with torsional contractility. The approach provides a new strategy to rapidly fabricate aligned cardiac tissues relevant to regenerative medicine and biohybrid robotics.

Alteration of N-glycosylation of CDON promotes H2O2-induced DNA damage in H9c2 cardiomyocytes.

Protein glycosylation is involved in DNA damage. Recently, DNA damage has been connected with the pathogenesis of heart failure. Cell adhesion associated, oncogene regulated (CDON), considered as an N-linked glycoprotein, is a transmembrane receptor for modulating cardiac function. But the role of CDON and its glycosylation in DNA damage remains unknown. In this study, we found that the knockdown of CDON caused DNA double-strand breaks as indicated by an increase in phosphorylated histone H2AX (γH2AX) protein level, immunofluorescent intensity of γH2AX and tail DNA moment in H9c2 cardiomyocytes. Conversely, overexpression of CDON leads to decreasing DNA damage induced by hydrogen peroxide (H2O2) and upregulating the expression of genes related to DNA repair pathways-homologous recombination (HR) and non-homologous end joining (NHEJ). Moreover, we expressed nine predicted N-glycosylation site mutants in H9c2 cells prior to treatment with H2O2. The results showed that mutation of N-glycosylation sites (N99Q, N179Q, and N870Q) increased the accumulation of DNA damage and downregulated the expression of HR-related genes, demonstrating that CDON N-glycosylation on DNA damage is site-specific and these specific N-glycan sites may regulate HR repair-related transcript abundance of genes. Our data highlight that N-glycosylation of CDON is critical to cardiomyocyte DNA lesion. It may uncover the potential strategies targeting DNA damage pathway in heart disease.

Maturation of pluripotent stem cell-derived cardiomyocytes: limitations and challenges from metabolic aspects.

Acute coronary syndromes, such as myocardial infarction (MI), lack effective therapies beyond heart transplantation, which is often hindered by donor scarcity and postoperative complications. Human induced pluripotent stem cells (hiPSCs) offer the possibility of myocardial regeneration by differentiating into cardiomyocytes. However, hiPSC-derived cardiomyocytes (hiPSC-cardiomyocytes) exhibit fetal-like calcium flux and energy metabolism, which inhibits their engraftment. Several strategies have been explored to improve the therapeutic efficacy of hiPSC-cardiomyocytes, such as selectively enhancing energy substrate utilization and improving the transplantation environment. In this review, we have discussed the impact of altered mitochondrial biogenesis and metabolic switching on the maturation of hiPSC-cardiomyocytes. Additionally, we have discussed the limitations inherent in current methodologies for assessing metabolism in hiPSC-cardiomyocytes, and the challenges in achieving sufficient metabolic flexibility akin to that in the healthy adult heart.

Promotion of maturation in CDM3-induced embryonic stem cell-derived cardiomyocytes by palmitic acid.

BACKGROUND: Myocardial infarction leads to myocardial necrosis, and cardiomyocytes are non-renewable. Fatty acid-containing cardiomyocyte maturation medium promotes maturation of stem cell-derived cardiomyocytes. OBJECTIVE: To study the effect palmitic acid on maturation of cardiomyocytes derived from human embryonic stem cells (hESCs) to optimize differentiation for potential treatment of myocardial infarction by hESCs. METHODS: hESCs were differentiated into cardiomyocytes using standard chemically defined medium 3 (CDM3). Up to day 20 of differentiation, 200 Mm palmitic acid were added, and then the culture was continued for another 8 days to mimic the environment in which human cardiomyocytes mainly use fatty acids as the main energy source. Light microscopy, transmission electron microscopy, immunofluorescence, reverse transcription-polymerase chain reaction, and cellular ATP assays, were carried out to analyze the expression of relevant cardiomyocyte-related genes, cell morphology, metabolism levels, and other indicators cardiomyocyte maturity. RESULTS: Cardiomyocytes derived from hESCs under exogenous palmitic acid had an elongated pike shape and a more regular arrangement. Sarcomere stripes were clear, and the cells color was clearly visible. The cell perimeter and elongation rate were also increased. Myogenic fibers were abundant, myofibrillar z-lines were regularly, the numbers of mitochondria and mitochondrial cristae were higher, more myofilaments were observed, and the structure of round-like discs was occasionally seen. Expression of mature cardiomyocyte-associated genes TNNT2, MYL2 and MYH6, and cardiomyocyte-associated genes KCNJ4, RYR2,and PPARα, was upregulated (p < 0.05). Expression of MYH7, MYL7, KCND2, KCND3, GJA1 and TNNI1 genes was unaffected (p > 0.05). Expression of mature cardiomyocyte-associated sarcomere protein MYL2 was significantly increased (p < 0.05), MYH7 protein expression was unaffected (p > 0.05). hESC-derived cardiomyocytes exposed to exogenous palmitic acid produced more ATP per unit time (p < 0.05). CONCLUSION: Exogenous palmitic acid induced more mature hESC-CMs in terms of the cellular architecture, expression of cardiomyocyte maturation genes adnprotein, and metabolism.

Colchicine Prevents Cardiac Rupture in Mice with Myocardial Infarction by Inhibiting P53-Dependent Apoptosis.

Cardiac rupture is a fatal complication following myocardial infarction (MI) and there are currently no effective pharmacological strategies for preventing this condition. In this study, we investigated the effect of colchicine on post-infarct cardiac rupture in mice and its underlying mechanisms.We induced MI in mice by permanently ligating the left anterior descending artery. Oral colchicine or vehicle was administered at a dose of 0.1 mg/kg/day from day 1 to day 7 after MI. Cultured neonatal cardiomyocytes and fibroblasts were exposed to normoxia or anoxia and treated with colchicine.Colchicine significantly improved the survival rate (colchicine, n = 46: 82.6% versus vehicle, n = 42: 61.9%, P < 0.05) at 1 week after MI. Histological analysis revealed colchicine significantly reduced the infarct size and the number of macrophages around the infarct area. Colchicine decreased apoptosis in the myocardium of the border zone and cultured cardiomyocytes and fibroblasts as assessed by TUNEL assay. Colchicine also attenuated the activation of p53 and decreased the expression of cleaved-caspase 3 and bax, as assessed by Western blotting.Colchicine prevents cardiac rupture via inhibition of apoptosis, which is attributable to the downregulation of p53 activity. Our findings suggest that colchicine may be a prospective preventive medicine for cardiac rupture, however, large clinical trials are required.

RNA-Seq analysis of human heart tissue reveals SARS-CoV-2 infection and inappropriate activation of the TNF-NF-κB pathway in cardiomyocytes.

The negative impact of SARS-CoV-2 virus infection on cardiovascular disease (CVD) patients is well established. This research article explores the cellular pathways involved in underlying heart diseases after infection. The systemic inflammatory response to SARS-CoV-2 infection likely exacerbates this increased cardiovascular risk; however, whether the virus directly infects cardiomyocytes remains unknown due to limited multi-omics data. While public transcriptome data exists for COVID-19 infection in different cell types (including cardiomyocytes), infection times vary between studies. We used available RNA-seq data from human heart tissue to delineate SARS-CoV-2 infection and heart failure aetiology specific gene expression signatures. A total of fifty-four samples from four studies were analysed. Our aim was to investigate specific transcriptome changes occurring in cardiac tissue with SARS-CoV-2 infection compared to non-infected controls. Our data establish that SARS-CoV-2 infects cardiomyocytes by the TNF-NF-κB pathway, potentially triggering acute cardiovascular complications and increasing the long-term cardiovascular risk in COVID-19 patients.

In Vivo and In Vitro Approaches to Modeling Hypoplastic Left Heart Syndrome.

PURPOSE OF REVIEW: Hypoplastic left heart syndrome (HLHS) is a critical congenital heart defect characterized by the underdevelopment of left-sided heart structures, leading to significant circulatory challenges, and necessitating multiple surgeries for survival. Despite advancements in surgical interventions, long-term outcomes often involve heart failure, highlighting the need for a deeper understanding of HLHS pathogenesis. Current in vivo and in vitro models aim to recapitulate HLHS anatomy and physiology, yet they face limitations in accuracy and complexity. RECENT FINDINGS: In vivo models, including those in chick, lamb, and mouse, provide insights into hemodynamic and genetic factors influencing HLHS. In vitro models using human induced pluripotent stem cells offer valuable platforms for studying genetic mutations and cellular mechanisms. This review evaluates these models' utility and limitations, and proposes future directions for developing more sophisticated models to enhance our understanding and treatment of HLHS.

Cardiomyocyte proliferation and regeneration in congenital heart disease.

Despite advances in prenatal screening and a notable decrease in mortality rates, congenital heart disease (CHD) remains the most prevalent congenital disorder in newborns globally. Current therapeutic surgical approaches face challenges due to the significant rise in complications and disabilities. Emerging cardiac regenerative therapies offer promising adjuncts for CHD treatment. One novel avenue involves investigating methods to stimulate cardiomyocyte proliferation. However, the mechanism of altered cardiomyocyte proliferation in CHD is not fully understood, and there are few feasible approaches to stimulate cardiomyocyte cell cycling for optimal healing in CHD patients. In this review, we explore recent progress in understanding genetic and epigenetic mechanisms underlying defective cardiomyocyte proliferation in CHD from development through birth. Targeting cell cycle pathways shows promise for enhancing cardiomyocyte cytokinesis, division, and regeneration to repair heart defects. Advancements in human disease modeling techniques, CRISPR-based genome and epigenome editing, and next-generation sequencing technologies will expedite the exploration of abnormal machinery governing cardiomyocyte differentiation, proliferation, and maturation across diverse genetic backgrounds of CHD. Ongoing studies on screening drugs that regulate cell cycling are poised to translate this nascent technology of enhancing cardiomyocyte proliferation into a new therapeutic paradigm for CHD surgical interventions.

Cellular calcium handling and electrophysiology are modulated by chronic physiological pacing in human induced pluripotent stem cell-derived cardiomyocytes.

Electric pacing of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) has been increasingly used to simulate cardiac arrhythmias in vitro and to enhance cardiomyocyte maturity. However, the impact of electric pacing on cellular electrophysiology and Ca2+-handling in differentiated hiPSC-CM is less characterized. Here we studied the effects of electric pacing for 24h or 7d at a physiological rate of 60 bpm on cellular electrophysiology and Ca2+-cycling in late-stage, differentiated hiPSC-CM (>90% troponin+, >60d post differentiation). Electric culture pacing for 7d did not influence cardiomyocyte cell size, apoptosis or generation of reactive oxygen species in differentiated hiPSC-CM compared to 24h pacing. However, epifluorescence measurements revealed that electric pacing for 7d improved systolic Ca2+-transient amplitude and Ca2+-transient upstroke, which could be explained by elevated sarcoplasmic reticulum Ca2+-load and SERCA activity. Diastolic Ca2+-leak was not changed in line-scanning confocal microscopy suggesting that the improvement in systolic Ca2+-release was not associated with a higher open probability of RyR2 during diastole. While bulk cytosolic Na+-concentration and NCX activity were not changed, patch-clamp studies revealed that chronic pacing caused a slight abbreviation of the action potential duration (APD) in hiPSC-CM. We found in whole-cell voltage-clamp measurements that chronic pacing for 7d led to a decrease in late Na+-current, which might explain the changes in APD. In conclusion, our results show that chronic pacing improves systolic Ca2+-handling and modulates the electrophysiology of late-stage, differentiated iPSC-CM. This study might help to understand the effects of electric pacing and its numerous applications in stem cell research including arrhythmia simulation.

SIRT1-FOXOs signaling pathway: A potential target for attenuating cardiomyopathy.

Cardiomyopathy constitutes a global health burden. It refers to myocardial injury that causes alterations in cardiac structure and function, ultimately leading to heart failure. Currently, there is no definitive treatment for cardiomyopathy. This is because existing treatments primarily focus on drug interventions to attenuate symptoms rather than addressing the underlying causes of the disease. Notably, the cardiomyocyte loss is one of the key risk factors for cardiomyopathy. This loss can occur through various mechanisms such as metabolic disturbances, cardiac stress (e.g., oxidative stress), apoptosis as well as cell death resulting from disorders in autophagic flux, etc. Sirtuins (SIRTs) are categorized as class III histone deacetylases, with their enzyme activity primarily reliant on the substrate nicotinamide adenine dinucleotide (NAD (+)). Among them, Sirtuin 1 (SIRT1) is the most intensively studied in the cardiovascular system. Forkhead O transcription factors (FOXOs) are the downstream effectors of SIRT1. Several reports have shown that SIRT1 can form a signaling pathway with FOXOs in myocardial tissue, and this pathway plays a key regulatory role in cell loss. Thus, this review describes the basic mechanism of SIRT1-FOXOs in inhibiting cardiomyocyte loss and its favorable role in cardiomyopathy. Additionally, we summarized the SIRT1-FOXOs related regulation factor and prospects the SIRT1-FOXOs potential clinical application, which provide reference for the development of cardiomyopathy treatment.

Melatonin alleviates high glucose-induced cardiomyocyte injury through suppressing mitochondrial FUNDC1-DRP1 axis.

OBJECTIVES: To use H9c2 cardiomyocytes to establish a diabetic cardiomyopathic model by exposing these cells to high glucose (HG), followed by treating them with melatonin (MEL) or plasmid vectors overexpressing FUN14 Domain Containing 1 (FUNDC1). METHODS: We employed quantitative real-time PCR, mitochondrial staining, and biochemical assays to measure the activity of various antioxidant and mitochondrial complex functions under various treatment conditions. KEY FINDINGS: Our results showed that HG induced the expression of FUNDC1 and increased mitochondrial oxidative stress and fragmentation, while MEL treatment reversed most of these pathological effects. Moreover, HG exposure activated dynamin-related protein 1 expression and its translocation to mitochondria. Modulation of AMP-activated protein kinase level was found to be another pathological hallmark. In silico molecular docking, analysis revealed that MEL could directly bind the catalytic groove of FUNDC1 through Van der Waal's force and hydrogen bonding. Finally, MEL ameliorated diabetic cardiomyopathy-induced mitochondrial injury through FUNDC1 in vivo. CONCLUSIONS: Hyperglycemia induced mitochondrial fragmentation and altered electron transport chain complex functions, which could be ameliorated by MEL treatment, suggesting its potential as a cardiovascular therapeutic.

Mitogen-activated protein kinase signalling in rat hearts during postnatal development: MAPKs, MAP3Ks, MAP4Ks and DUSPs.

Mammalian cardiomyocytes become terminally-differentiated during the perinatal period. In rodents, cytokinesis ceases after a final division cycle immediately after birth. Nuclear division continues and most cardiomyocytes become binucleated by ∼11 days. Subsequent growth results from an increase in cardiomyocyte size. The mechanisms involved remain under investigation. Mitogen-activated protein kinases (MAPKs) regulate cell growth/death: extracellular signal-regulated kinases 1/2 (ERK1/2) promote proliferation, whilst c-Jun N-terminal kinases (JNKs) and p38-MAPKs respond to cellular stresses. We assessed their regulation in rat hearts during postnatal development (2, 7, 14, and 28 days, 12 weeks) during which time there was rapid, substantial downregulation of mitosis/cytokinesis genes (Cenpa/e/f, Aurkb, Anln, Cdca8, Orc6) with lesser downregulation of DNA replication genes (Orcs1-5, Mcms2-7). MAPK activation was assessed by immunoblotting for total and phosphorylated (activated) kinases. Total ERK1/2 was downregulated, but not JNKs or p38-MAPKs, whilst phosphorylation of all MAPKs increased relative to total protein albeit transiently for JNKs. These profiles differed from activation of Akt (also involved in cardiomyocyte growth). Dual-specificity phosphatases, upstream MAPK kinase kinases (MAP3Ks), and MAP3K kinases (MAP4Ks) identified in neonatal rat cardiomyocytes by RNASeq were differentially regulated during postnatal cardiac development. The MAP3Ks that we could assess by immunoblotting (RAF kinases and Map3k3) showed greater downregulation of the protein than mRNA. MAP3K2/MAP3K3/MAP4K5 were upregulated in human failing heart samples and may be part of the "foetal gene programme" of re-expressed genes in disease. Thus, MAPKs, along with kinases and phosphatases that regulate them, potentially play a significant role in postnatal remodelling of the heart.

Differentiation of Sinoatrial-like Cardiomyocytes as a Biological Pacemaker Model.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are widely used for disease modeling and pharmacological screening. However, their application has mainly focused on inherited cardiopathies affecting ventricular cardiomyocytes, leading to extensive knowledge on generating ventricular-like hiPSC-CMs. Electronic pacemakers, despite their utility, have significant disadvantages, including lack of hormonal responsiveness, infection risk, limited battery life, and inability to adapt to changes in heart size. Therefore, developing an in vitro multiscale model of the human sinoatrial node (SAN) pacemaker using hiPSC-CM and SAN-like cardiomyocyte differentiation protocols is essential. This would enhance the understanding of SAN-related pathologies and support targeted therapies. Generating SAN-like cardiomyocytes offers the potential for biological pacemakers and specialized conduction tissues, promising significant benefits for patients with conduction system defects. This review focuses on arrythmias related to pacemaker dysfunction, examining protocols' advantages and drawbacks for generating SAN-like cardiomyocytes from hESCs/hiPSCs, and discussing therapeutic approaches involving their engraftment in animal models.