The ability of mitochondria to coordinate this myriad of functions is achieved through the exquisite regulation of fundamental dynamic properties, including remodeling of the mitochondrial network via fission and fusion, motility and mitophagy. In this Review, we summarize the current understanding of the mechanisms by which these dynamic properties of the mitochondria support mitochondrial function, review their impact on human cortical development and highlight areas in need of further research.Mitochondria are signaling hubs responsible for the generation of energy through oxidative phosphorylation, the production of key metabolites that serve the bioenergetic and biosynthetic needs of the cell, calcium (Ca Summary: This Review highlights the mechanisms by which the dynamic properties of the mitochondria impact human cortical development. Modeling the processes of cortical development is essential for understanding the specific molecular mechanisms involved in human brain development and for elucidating the etiology of many neurological diseases. While the importance of the mitochondria during brain development has long been known, an emerging view emphasizes that mitochondrial homeostasis is tightly regulated during cortical development, and more importantly that mitochondrial morphology and function underlie many of the physiological processes that take place during this fundamental time in human growth.2+) buffering and apoptosis. Mitochondrial homeostasis is maintained through the concerted execution of mitochondrial dynamics (fusion and fission), cristae dynamics, motility and mitophagy , metabolite synthesis, calcium  and human embryonic stem cells (hESCs) to model the earliest stages of development and disease may be a powerful tool with which to gain insight into these complex pathways . In thisThe cerebral cortex is the center responsible for crucial human abilities such as perception, thought, language, attention, episodic memory and voluntary movement . Each ofIn humans, cortical neurogenesis starts at gestational week (GW) 5 and ends around GW20 . The rem2 molecule. NADH and FADH2 can then feed the ETC complexes for ATP generation. The TCA cycle intermediates provide building blocks for macromolecules such as fatty acids, nucleotides, hemes and porphyrins. Thus, mitochondrial homeostasis influences not only energy production, but also the biosynthesis of these essential intermediates that are essential for cell survival. In general, a more fused mitochondrial network supports OXPHOS metabolism and a more fragmented mitochondrial network shifts the energetic state toward glycolysis, as seen during reprogramming. Many stem cells rely on glycolysis and have a fragmented mitochondrial network that fuses during neuronal differentiation and supports a transition to OXPHOS. Mutations in various genes involved in mitochondrial respiration have been described. These genetic dysregulations have been associated with severe neurodevelopmental disorders  that convert NADH molecules into ATP. This process, termed oxidative phosphorylation (OXPHOS), leads to the bulk of cellular ATP production and is essential for meeting the bioenergetic needs of the cell . In the isorders .The reduction of oxygen by the electron transport chain leads to reactive oxygen species (ROS) superoxide byproducts, the accumulation of which can be damaging to cellular processes. During homeostatic conditions, ROS can also modulate metabolism by redox signaling and post-translational modifications, such as acetylation and thiol oxidation.Around GW7, vRG cells that divide asymmetrically produce progeny that adopt neuronal fates directly, or re-enter the cell cycle as IPCs . IPCs ar2 that are not transferred to complex IV and by oxygen  and globally to initiate switches in metabolism. Calcium homeostasis is maintained through the coordinated interaction of the endoplasmic reticulum (ER) and the mitochondria . DespiteThe cortical plate develops into the layered cerebral cortex, in which each layer contains a characteristic group of neuronal subtypes. This variety of pyramidal neuron subtypes is determined mainly by the laminar position of their cell bodies, molecular and electrophysiological properties, somatic and dendritic morphology, and axonal connectivity . Despitey oxygen . These eectivity .Box 2. After the neurogenic stages described above are complete, the radial glia detach from the apical surface and become gliogenic, generating astrocytes and oligodendrocytes, or transform into ependymal cells . In addiMitochondria are major signaling organelles that undergo constant dynamic morphological changes, in flux between fragmented and fused states, in response to the energy demands of the cell  and 3. TMitochondrial fusion is necessary for mitochondrial DNA (mtDNA) homogenization and tissue maturation, as well as for the formation and assembly of the electron transport chain (ETC)  Chan, 2. Unlike Fusion of the inner membrane begins through the action of OPA1. OPA1 exists as two proteolytically cleaved proteins, designated as long OPA1 (OPA1-L) and short OPA1 (OPA1-S) . OPA1-L MFN2 and OPA1, respectively  executes fission upon activation by several post-translational modifications, including phosphorylation, ubiquitylation, sumoylation and O-GlcNAcylation. The enzymes that perform this activation of dynamin-related proteins have not been completely identified, but extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2) and cyclin dependent kinase 1 (CDK1) phosphorylate DRP1 at serine 616. Additional phosphorylation sites at serines 600, 637, 693 and 656 by calmodulin-dependent protein kinase 1α (CaM kinase Iα), protein kinase A (PKA), glycogen synthase kinase 3β (GSK3β) and A-kinase anchor protein 1 (AKAP1) have been identified – among others – providing insight into specific pathway that promote increased mitochondrial fragmentation . Once ac2+ signaling   . The clior DRP1) . Individor DRP1) . With gror DRP1)  (Bartsakor DRP1) . Underst2+ dynamics  that encodes QIL1, a mitochondrial protein and component of the MICOS complex, induces severe mitochondrial encephalopathy, hepatopathy and lactate acidosis Fig. 4). gulation  and 2  may have DRP1-independent approaches to removing damaged parts of the mitochondria without sacrificing the whole organelle , mitochondrial dynamics were impaired, axonal trafficking disturbed and neuronal development inhibited (Mitophagy is vital for proper neurodevelopment, as dysregulated mitophagy has been found to be pathogenic in multiple syndromes and may lead to increased levels of neuroinflammation. In roblasts . Similarnhibited . Pink1/pnhibited . During nhibited . In casenhibited , which mnhibited .Within the human cortex there is extraordinary diversity of cell types, all of which maintain pools of mitochondria that have adapted unique motility behaviors to travel within the cell.Astrocytes are glial cells that serve to support the function of synapses through regulating neurotransmitter uptake and recycling, as well as nutrient uptake from nearby blood vessels making them essential to neuronal survival . AstrocyAlthough mitochondrial dynamics are much less well studied in oligodendrocyte and microglial populations, there is growing interest in their function in neurodevelopment and the contribution of mitochondrial dynamics to neuronal homeostasis. Mitochondria are required for oligodendrocyte differentiation and myelination; increased Opa1 and Fis1 transcription during differentiation suggest changes in mitochondrial morphology may be important to these processes . OligodeAs resident immune cells of the central nervous system, microglia are central in regulating inflammatory responses in the brain , but a cAs described in this Review, the exact regulation of the dynamic properties of the mitochondria in the context of the human cortex remains largely unexplored. A substantial barrier to understanding the spatiotemporal mechanisms of early human neural differentiation and maturation has been the lack of a model that faithfully recapitulates human embryonic cortical development. Furthermore, the practical and ethical considerations of accessing post-mortem human embryonic tissue further adds to the challenges of studying early developmental disorders. Human iPSC-derived systems coupled with gene editing, classical biochemistry, high resolution microscopy and proteomics are rapidly evolving as a relatively high-throughput means of modeling these earliest stages of development . The abiThe field of organoid biology is growing at incredible speed. Although some studies caution on the activation of cellular stress pathways that may impair cell-type specification , extensiLandmark studies have shown the fundamental differences in mitochondrial morphology and function within the soma, dendrites and axons of mature cortical neurons . It has


With the high morbidity and mortality rates, cardiovascular diseases have become one of the most concerning diseases worldwide. The heart of adult mammals can hardly regenerate naturally after injury because adult cardiomyocytes have already exited the cell cycle, which subseqently triggers cardiac remodeling and heart failure. Although a series of pharmacological treatments and surgical methods have been utilized to improve heart functions, they cannot replenish the massive loss of beating cardiomyocytes after injury. Here, we summarize the latest research progress in cardiac regeneration and heart repair through altering cardiomyocyte fate plasticity, which is emerging as an effective strategy to compensate for the loss of functional cardiomyocytes and improve the impaired heart functions. First, residual cardiomyocytes in damaged hearts re-enter the cell cycle to acquire the proliferative capacity by the modifications of cell cycle-related genes or regulation of growth-related signals. Additionally, non-cardiomyocytes such as cardiac fibroblasts, were shown to be reprogrammed into cardiomyocytes and thus favor the repair of damaged hearts. Moreover, pluripotent stem cells have been shown to transform into cardiomyocytes to promote heart healing after myocardial infarction (MI). Furthermore, in vitro and in vivo studies demonstrated that environmental oxygen, energy metabolism, extracellular factors, nerves, non-coding RNAs, etc. play the key regulatory functions in cardiac regeneration. These findings provide the theoretical basis of targeting cellular fate plasticity to induce cardiomyocyte proliferation or formation, and also provide the clues for stimulating heart repair after injury. So, finding an effective way to replenish the loss of cardiomyocytes during heart attack has been a hot topic of interest.In the past few decades, the prevalence of cardiovascular diseases (CVDs) have increased significantly and become one of the leading causes of mortality worldwide.Cardiomyocyte plasticity plays a critical role in cardiac adaptive responses such as myocardial remodeling and heart repair. In response to various stimuli, the heart will gradually gain appropriate renewal potential to replace necrotic or apoptotic cardiomyocytes after injury, bringing hope to patients with MI. Recently, increasing evidence has suggested that targeting the plasticity of cell fate is one new potential approach for cardiac regeneration, which can be mainly achieved by reprogramming non-cardiomyocytes into cardiomyocytes, the differentiation of pluripotent stem cells into cardiomyocytes, and the proliferation of pre-existing cardiomyocytes.6 and facilitate the blood pumping into the circulatory system by coordinating contraction and diastole. Mammalian cardiomyocytes have been thought as terminally differentiated cells that have little ability to proliferate. Recently, some groundbreaking studies has debunked this concept and provided the compelling evidence. Fetal and neonatal cardiomyocytes have been shown to proliferate and then repair the damaged tissues.7 Nevertheless, adult mammalian hearts hardly regenerate functional myocardium after injury due to inadequate cardiomyocyte renewal.9 However, recent studies have revealed that damaged hearts in MI are able to acquire regenerative potential by targeting some key signal pathways that induce cardiomyocytes to re-enter the cell cycle.Cardiomyocytes account for 75% of left ventricular volume in healthy adults,10 It was lately indicated that cardiac fibroblasts can be directly reprogrammed into induced cardiac-like myocytes (iCLM) by introducing cardiogenic transcription factors, Gata4, Mef2c, and Tbx5 (GMT), thereby providing a potential source of cells for heart repair.12 It has also been demonstrated that regulation of some critical microRNAs (miRNAs) and epigenetic modification improve the efficiency of reprogramming non-cardiomyocytes into cardiomyocytes.16 Thus, reprogramming non-cardiomyocytes into cardiomyocytes in situ would represent a powerful and attractive alternative strategy for myocardial regeneration.Non-cardiomyocytes are also important components of adult hearts, most of which are cardiac fibroblasts.18 Pluripotent stem cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can differentiate into almost any somatic cells including cardiac myocytes.19 Transplantation of pluripotent stem cell-derived cardiomyocytes combined with stem cells can not only significantly increase the retention of transplanted cardiomyocytes and improve the survival, but also promote neovascularization and anti-inflammatory through paracrine pathway, thereby repairing the damaged heart and improving cardiac functions.20Pluripotent stem cell-based therapy has been suggested as a promising treatment for cardiovascular diseases.26 Besides, hypoxia, energy metabolism, extracellular matrix, nerve, epicardial factors, and inflammation and other factors, are also involved in the regulation of cardiomyocyte proliferation and heart repair. Targeting these factors in infarcted hearts become the new therapeutic strategy for cardiac regeneration. In this review, we summarize these progress on regulating the cell fate plasticity for cardiac regeneration and how these pathways could be targeted for therapeutic benefit.Up to now, a series of cell cycle regulators, signal pathways, non-coding RNAs, and other molecules have been shown to be involved in post-injury cardiomyocyte proliferation and heart repair, which may be used to develop innovative cardiac regenerative drugs for clinical application Fig. .21–26 Be27 Then, it was reported that after infected with transcription factors Oct3/4, Sox2, c-Myc, and Klf4 combined with retroviral transduction, fibroblasts can be reprogrammed into iPSCs.28 Interestingly, later studies found that in vitro fibroblasts also can be directly reprogrammed into iCLM after the integration of the transcription factors GMT, which provide a potential source of cells for heart repair.11 Previously, just a small portion of these cells were shown to be beating cardiomyocytes, so it has attracted much attention to improve the reprogramming efficiency.Generally, cardiac fibroblasts are activated after MI and recruited to the injured site to form scar tissue to replace the injured heart muscle. Therefore, reprogramming these cells into functional cardiomyocytes would be an ideal strategy for heart repair in response to ischemic injury. It was initially found that the transcription factor encoded by the myogenic regulator MYOD1 induces many types of cells to differentiate into skeletal muscles.31 GMT or GMT plus Mesp1 and Myocd combined with miR-133 are capable to regulate the gene expression of mouse embryonic fibroblasts (MEF), adult mouse cardiac fibroblasts (MCF) and human cardiac fibroblasts (HCF) from fibroblast phenotype to cardiomyocyte-like phenotype, and in turn accelerate cardiomyocytes reprogramming.32 Additionally, reprogramming efficiency of cardiomyocytes induced by GMT in vitro and in vivo was enhanced by transforming growth factors and Wnt inhibitors.33 Interestingly, epigenetic factors play the promoting or inhibitory roles in cardiac reprogramming.34 For example, inhibition of H3K4 methyltransferase Mll1 increased the efficiency of embryonic and cardiac fibroblasts transforming into functional iCLM.35 These findings provide new insights into molecular mechanisms of fibroblast-cardiomyocyte transformation. Single-cell transcriptomics have also been applied to reconstruct reprogramming trajectories. It provides a more comprehensive understanding of cellular reprogramming by identifying intermediate cell populations, signal pathways and potential regulators involved in iCLM reprogramming.36 Notably, direct reprogramming in vivo appears to improve post-MI heart function in mice more efficiently than in vitro, suggesting that the reprogramming effectiveness depends in part on functional cardiomyocytes, and other factors are also committed to heart repair together.37 Thus, although it has been revealed that targeting signal pathways, growth factors, miRNAs and small molecules further facilitate iCLM reprogramming, the more in-depth exploration of its regulatory mechanism is required before it is applied to the clinic.Currently, endogenous mouse cardiac fibroblasts have been found converted into iCLM in vivo upon GMT or GHMT (GMT plus Hand2) transduction.38 Because of its simplicity, versatility, specificity and high efficiency, it has been widely used in the biomedical field.40 The CRISPR/Cas9 system can directly activate the expression of downstream target genes with high precision.41 CRISPR interference was used to repress the expression of EGF-like domain 7 (Egfl7) in zebrafish to investigate its role in angiogenesis.42 Moreover, several studies showed that genetically engineered stem cells by CRISPR/Cas9 protected the damaged heart and improved cardiac functions.44 Currently, CRISPR/Cas9 can reprogram the human fibroblast lineage into induced cardiac progenitor cells, which then differentiate into cardiomyocytes, smooth muscle cells, and endothelial cells in vitro.45 The finding provides a new source of cells for disease modeling, drug screening, and rational and personalized cardiac cell therapy. Due to the off-target effect and unstable editing efficiency of gene editing, more work needs to be done in establishing effective and achievable delivery methods and identifying therapeutic targets.Clustered, regularly interspaced short palindromic repeat (CRISPR)-Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements.19 Previous studies suggested that hPSC-derived cardiomyocytes (hPSC-CMs) can be produced by simulating some developmental signaling cues.46 Indeed, hESCs are able to differentiate into functional cardiomyocytes following a mature cardiac differentiation protocol,49 and transplantation of hESCs-derived cardiomyocytes (hESC-CMs) and hiPSCs-derived cardiomyocytes (hiPSC-CMs) in vivo can promote the regeneration of the damaged heart and improve cardiac functions.50 Thus, hPSC-CMs have broad prospects in cardiac regeneration after injury. It has been reported that ESC-derived cardiomyocytes (ESC-CMs) express genes that play critical roles in cardiac development, and the electrophysiological and contractional phenotypes of the heart.52 Moreover, ESC-CMs transplanted into the infarcted heart can survive and improve cardiac functions.53 Nevertheless, transplantation of ESCs resulted in severe teratomas in the implanted area, which are attributed to the immune response and the accumulation of undifferentiated ESCs.56 IPSCs have unlimited proliferation capacity and can differentiate into cardiomyocytes.57 Cardiac systolic function in MI hearts was significantly improved at 4 and 12 weeks after transplantation of iPSC-derived cardiomyocytes (iPSC-CMs).58 However, as expected, the immaturity of iPSC-CMS in transplanted animals often leads to ventricular tachycardia. At the same time, iPSC-CMs trigger an immune response mediated by natural killer cells (NKCs), resulting in a low engrafting.59 Currently, attempts have been made to improve the implantation of pluripotent stem cell-based therapy and inhibit the occurrence of tumors and arrhythmias by obtaining high purity and mature iPSC-CMs (Table 61. Enrichment of cardiomyocytes by vascular cell adhesion molecule 1 (VCAMI)-coupled magnetic dynabeads62 and hydrogel63 or cell sheet holder64 have been shown to improve therapeutic effects. However, more investigations are required to determine the safety and effectiveness of pluripotent stem cell therapies, especially in clinical stage.Human pluripotent stem cells (hPSCs) are mainly consisted of human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs), and have the capability to differentiate into almost any proliferative somatic cells.65 and exogenous implanted CSCs improved cardiac functions of damaged hearts.66 However, by utilizing advanced molecular and genetic techniques, lately reports uncovered that CSCs eventually do not transform into functional cardiomyocytes. The benefit of infarcted mice after intramyocardial injection of CSCs was shown to be associated with an acute inflammatory-based wound-healing response.68 It suggests that CSCs do not produce new cardiomyocytes and its effects on hearts after MI are complicated.It had been argued that cardiac stem cells (CSCs) could differentiate into cardiomyocytes, smooth muscle cells and endothelial cells,69 Nevertheless, adult amphibians and zebrafish have a strong cardiac regeneration ability.74 Similarly, fetal and newborn mouse hearts can still regenerate myocardium through cell division after injury.75 Recently, increasing evidence showed that targeting the cell cycle of adult cardiomyocytes is able to induce pre-existing cardiomyocyte to proliferate, which has become one new strategy for cardiac regeneration after injury. The activation or inactivation of cardiomyocyte proliferation is linked to many pathophysiological factors and regulatory genes. Cardiac microenvironment, extracellular matrix, neuromodulation, etc have been found involved in cardiac regeneration by impacting cell proliferation   to oxidative metabolism. The change leads to the production and accumulation of large amounts of reactive oxygen species (ROS), further destroying the genome.78 With the change of energy supply pattern before and after birth, the increase of oxidative stress plays a critical role in cell cycle arrest.91 Meanwhile, the inhibition of glycolysis resulted in impaired regeneration ability of neonatal mouse cardiomyocytes.92 Recent studies have revealed that pyruvate dehydrogenase kinase (PDK) regulates glycolysis and pyruvate metabolism of cardiomyocytes of the border area in zebrafish after injury.93 Moreover, the loss of PDK4 can increase the proliferation of cardiomyocytes and improve left ventricular function after MI and reduce remodeling.94 These findings suggest that energy metabolism plays a critical role in cardiomyocyte proliferation after injury. Additionally, mice overexpressing the nuclear receptor PPARβ/δ can increase the expression of type 4 glucose transporter (Glut4) in the myocardium to promote glucose utilization, and significantly reduce myocardial damage caused by I/R injury.95 Thus, the regulatory mechanism of energy metabolism beneficial to the repair and regeneration of the heart after injury is complicated and influenced by many factors.As known, the energy supply of adult cardiomyocytes mainly comes from the oxidative phosphorylation of mitochondria.23. Increasing evidence suggest that ECM is a highly dynamic regulatory network in cardiac regeneration and plays a vital role under stress or in pathological conditions.96The integrity of the ECM microenvironment plays critical roles in regulating cardiac systolic and diastolic function. Cardiac ECM not only provide mechanical support but also act as a key factor to regulate cell survival, growth and development. Under stress conditions, ECM macromolecules are involved in the pathogenesis of ventricular dysfunction and heart failure by driving a variety of cellular biological responses. It was reported that the ECM protein Agrin has a positive effect on the mitosis of cardiomyocytes derived from mouse and human-induced pluripotent stem cells.97 It has been shown that the nerves at the bottom of the limbs of amphibians cannot regenerate after being cut off before or after amputation.98 For instance, the development of arm morphology and the regeneration of other organisms, such as chicken embryos, suggesting that cholinergic neurons play a vital role in the reformation of biological structures.100 Newborn mice can regenerate the heart within one week of birth, while adult heart almost lose the ability to regenerate.75 The evidence showed that pharmacological interventions on cholinergic signaling transduction (sympathetic) are able to inhibit the development of the cell cycle after AR and MI in zebrafish and newborn mice, thus affecting cardiac regeneration.101 It provides important insights into the role of neuromodulation in cardiac regeneration.A large number of studies have shown that nerve signal plays critical roles in regulating cardiac regeneration.102 In mammalian heart repair, cardiomyocyte survival and blood vessel formation were supported by epicardial cells.103 Revascularization, which is critical to supporting tissue repair and heart functions after MI, is also regulated by epicardial and endocardial signalings.104 In addition, the increase of the expression and activity of follistatin-like 1 (Fstl1) in the epicardium can stimulate the re-entry of cell cycle, and ultimately improve the impaired cardiac functions and survival.25 Therefore, The epicardium also plays a crucial role in response to cardiac regeneration and angiogenesis after injury.The epicardium is an important source of progenitor cells and provides nutrition and structural support for adult heart. Many studies have confirmed that the thin mesothelial cell layer covering the chamber-the epicardium is a key factor in the repair and regeneration of a damaged heart. The epicardium of the zebrafish heart is activated when it is damaged and is committed to cardiac regeneration by producing factors that maintain heart function.105 the subsets of macrophages with pro-inflammatory properties derived from Ly6Chigh monocytes, which are recruited through chemokine dependent pathway, infiltrate into the infarcted area and maintained the pro-inflammatory environment.107 Studies have shown that macrophages are essential for the regeneration of the newborn heart.110 Macrophages infiltration induced by monocyte chemoattractant protein-1 (MCP-1) can improve cardiac functions in mice after MI.111 Therefore, an in-depth understanding of the behavior of immune cells involved in heart repair will help develop new therapies for MI.It has been known for decades that the immune system coordinates immune cells to regulate tissue repair, angiogenesis, and fibrosis. In the early stage of heart infarction, white blood cells (including neutrophils and monocytes) rapidly infiltrate the infarcted area, activating the production of myofibroblasts and vascular cells for cardiac repair.112 It has been shown that cyclin mutant kinase 9 (CDK9) is involved in an important signal pathway for cardiac hypertrophy.113 CDK9 also plays a key role in responding to myocardial injury by directly binding to GATA-binding protein 4 (GATA4).114 This association provides theoretical support for CDK9 to paly a regulatory role in cardiomyocyte proliferation.115 It has been shown that CDK9 ablation inhibit the proliferation of zebrafish cardiomyocytes and has a negative effect on the repair of laser-damaged hearts.116 Later, it was found that endogenous CDK inhibitors trigger adult cardiomyocytes to re-enter the cell cycle and proliferate actively.117 These studies indicate that CDKs play a key role in regulating cardiomyocyte proliferation.The intervention of cell cycle regulators, such as cyclin or its dependent kinases (CDKs) enables the remaining cardiomyocytes to re-enter the cell cycle and maintain the proliferation signaling.7 Moreover, the synergistic effect of MEIS1 and its cofactor homeobox B13 (Hoxb13) can jointly regulate the proliferation window of cardiomyocytes after birth and the repair and regeneration of the adult heart.118 On the contrary, Tbx20, a member of the Tbx1 subfamily of T-box (Tbx) genes, directly repressed the expression of cell cycle inhibitors p21, MEIS1 and Btg2. Overexpression of Tbx20 in adult mouse cardiomyocytes can promote cell proliferation and significantly improve heart repair after MI.121 Besides, Tbx20 participated in the signaling cascade of Tbx20-PROK2-PROKR1 and regulated angiogenesis.122 The above findings provide further mechanistic insights into the link between cardiomyocyte proliferation and cardiac regeneration.The myeloid ecotropic viral integration site 1 homolog (MEIS1), is a member of the TALE homeobox gene family, is a regulator of cell cycle arrest in post-natal cardiomyocytes. It was uncovered that loss of MEIS1 in adult mice induced cardiomyocytes to re-enter the cell cycle.125 Hippo pathway signaling cascade is highly conserved in mammals. Its core components are MST1 or MST2, LATS1 or LATS2 kinases and two accessory molecules SAV1 and MOB kinase activator 1 (MOB1).126 Activation of upstream components of the Hippo pathway, including Mst1 and Lats2, phosphorylates Yes-associated protein (Yap), which is a transcription factor that shuttle between the cytoplasm and nucleus to participates in cell survival and proliferation.127 The decrease of Yap binding to TEAD1, a transcription enhancer in the nucleus, causes a decrease in the expression of genes involved in cell growth and survival.128 Conversely, the inactivation of the Hippo pathway or activation of its downstream effector, the Yap transcription co-activator, promotes cardiac regeneration and improves heart function.132 Overexpression of activated Yap is sufficient to promote mitosis of cardiomyocytes. Additionally, some evidence suggests that Hippo signaling can regulate the cell cycle re-entry of adult cardiomyocytes. The genetic depletion interferes with the Hippo pathway to stimulate cardiomyocytes through the S phase, mitosis, and cytokinesis, exhibiting greater re-entry of the cardiomyocyte cell cycle.133 Thus, targeting Hippo pathway to regulate cardiomyocytes fate may be an attractive approach for heart repair after MI. However, the inhibited Hippo pathway can also activate a positive feedback mechanism called Yap-TEAD1-Oncostatin M (OSM), which eventually worsens cardiac dysfunction caused by pressure overload.131Recent studies have suggested that Hippo pathway plays a pivotal role in regulating cardiomyocytes proliferation and can affect the heart size through regulation of apoptosis, proliferation, and cellular fate.134 Notch signal was found to be activated after heart injury in zebrafish, with the increased expression of various Notch receptors and ligands.135 The overall inhibition of the Notch signaling hindered cardiomyocyte proliferation and resulted in scarring.137 Inhibition of Notch signal in neonatal cardiomyocytes blocked cell proliferation and induced cell apoptosis, suggesting that Notch signal may be manipulated to affect the division of postnatal cardiomyocytes.85 In the damaged adult hearts, only the endocardial and epicardial cells near the infracted region expressed the Notch-related genes, suggesting that activation of the Notch pathway occurs in the endocardium and the effect of Notch signaling activation on myocardium may be indirect.138 Additionally, ectopic activation of Notch signaling in myocardium limits the pathological remodeling process of the heart after MI.139 Moreover, the regulation of cardiac development is regulated by a complex network of multiple signal pathways, such as Notch signal, Hippo pathway and so on.The repair and regeneration of organs usually involves response to cellular signal pathways ralated to its development. Many studies in mice and zebrafish have confirmed that Notch signal plays a vital role in regulating heart repair and cardiac regeneration.141 The myocardium of germline mice deficient in the NRG1, ErbB2, or ErbB4 genes thinned. These genes are necessary during the second trimester to produce fetal cardiomyocytes.144 In zebrafish heart, blocking NRG1 signal inhibited injury-induced cardiomyocyte proliferation. In the absence of heart damage, the reactivation of NRG1 may stimulate many signs of cardiac regeneration.81 These results suggest that NRG1 is the key node between myocardial injury and physiological cardiac regeneration. Additionally, activation of NRG1/ErbB4 signaling pathways induced the proliferation of differentiated cardiomyocytes, thereby enhancing cardiac regeneration after myocardial injury and improving heart functions. Importantly, NRG1-induced myocardial regeneration may attenuate the hypertrophic phenotype after MI, thus improving ventricular remodeling.146 This study presents one possible way to repair the heart by delivering recombinant growth factors. Additionally, on the basis of the fact that NRG1 stimulated cardiomyocyte proliferation in young adult mice,148 studies have found that NRG1 can also induce cardiomyocyte proliferation in infants under 6 months of age with heart disease.149 It suggests that NRG1 signal pathway is conservative and may serve as an effective therapeutic strategy for children with heart diseases.The NRG1 gene is a member of the epidermal growth factor (EGF) gene family and exerts biological functions through the ErbB family of tyrosine kinase receptors, including ErbB1, 2, 3, and 4.150 are functional RNA molecules that almost do not encode proteins.151 Recently, protective functions of some miRNAs and lncRNAs in cardiac regeneration and heart repair have been verified.153 In addition, circular RNA (circRNA) forms a covalently closed loop, which help to maintain its stability and enhance its miRNA/protein binding ability.154 There is evidence that certain types of circRNAs may play a significant role in controlling cell proliferation, development and even tissue regeneration  and long non-coding RNAs (lncRNAs),157 MiRNAs regulate the growth and development of the heart.159 Recent studies have demonstrated that the exogenous administration of miRNAs such as hsa-miR-590 and hsa-miR-199a stimulated cell cycle re-ertry of isolated cardiomyocytes, and promoted the regeneration of mouse hearts after MI.162 Some miRNAs also showed the capability to negatively regulate cardiomyocytes proliferation and cell cycle re-entry.164 For instance, MI can induce the expression of pro-apoptotic miR-34 family members. The inhibited expression of miR-34 family members or by increasing the expression of target genes  can effectively improve the impaired cardiac functions.166 It has been found that miRNAs can regulate a variety of signaling pathways involved in cardiac regeneration,168 including the MAPK signal, Hippo signal,169 Wnt signal, PI3K-Akt signal, and pluripotency pathways. Interestingly, miRNAs have also been shown to regulate the differentiation of stem cells into cardiomyocytes.170MiRNAs play a critical role in gene expression by binding to the 3’UTR of target mRNA. MiRNA binding can induce translational blocking of mRNA or direct mRNA cleavage.173 Regulation of cell cycle genes is a feasible way to induce cardiomyocyte cycle re-entry.7 Studies have shown that lncRNAs can play a crucial function in cardiac regeneration by promoting the proliferation of pre-existing cardiomyocytes. Although lncRNAs do not translate into proteins, they have been shown to regulate the cell cycle re-entry of cardiomyocytes.175 Mechanistic analysis revealed that lncRNAs are also involved in multiple signal and metabolic pathways related to cardiac regeneration, such as Hippo pathway.176 Further, lncRNA can interact with the 3’UTR of mRNA to form molecular complexes to improve the stability of the mRNA and ultimately produce beneficial biological functions in heart failure.177 Therefore, it is necessary to further explore the regulatory mechanisms of some key lncRNAs on cardiomyocyte proliferation.A large body of evidence has showed that lncRNA plays a unique role in cardiovascular diseases.178 CircRNA microarray analysis uncovered that there were significant differences in the expression of some circRNAs in newborn and adult mouse hearts, suggesting that circRNAs may be involved in heart development. Generally, circRNAs can interact with RNA-binding proteins to form complexes that promote the expression of key downstream targets to exert their biological effects. For example, circFndc3b can interact with RNA-binding proteins to promote the expression of VEGF, thereby promoting the reduction of cell apoptosis and improving cardiac regeneration.179 Besides, circRNA, as a miRNA sponge, regulate the expression of its downstream molecules, ultimately activating or inactivating relevant signaling pathways and generating corresponding biological functions.180 It was found that loss of circNfix promoted cardiomyocyte proliferation. Mechanically, On the one hand, circNfix as a sponge of miR-214 promoted GSK-3 expression and further inhibited the activity of catenin. Besides, circNfix-related super-enhancers regulated the expression of circNfix by recruiting the transcription factor MEIS1 and eventually served as an effective therapeutic target for improving heart function and preventing heart failure after MI.181In recent years, circRNAs have been recognized as a new type of non-coding RNAs with regulatory functions. It has a typical covalently closed structure, highly stable expression, and resistance to nucleases.Non-coding RNAs are used as a potential target for the treatment of cardiovascular diseases, but needs further evaluation in pre-clinical studies and clinical trials. The exploration of the potential regulatory mechanisms of non-coding RNAs in various pathological processes will favor the deeper understanding of cardiac homeostasis and the development of cardiovascular diseases. In the future, non-coding RNAs will become an important part of the emerging field of cardiac regenerative medicine.Collectively, targeting the cell fate plasticity is emerging as a promising and essential strategy for repairing damaged hearts. Some important approaches for cardiac regeneration have been summarized and described here: Promoting cell cycle re-entry and mitosis of pre-existing cardiomyocytes in damaged hearts; Reprogramming non-cardiomyocytes such as cardiac fibroblasts into cardiomyocytes; Inducing pluripotent stem cells such as ESCs and iPSCs differentiate into cardiomyocytes. Cardiacogenesis and cardiomyocyte proliferation are regulated by cell cycle regulators, non-coding RNAs, etc, which are also associated with multiple signaling pathways, actually forming a highly coordinated and orderly network. All the findings provide new insights into underlying mechanisms of cardiac development and regeneration, and also confer novel therapeutic approaches for heart damage. In the future, multidisciplinary cooperation and in-depth understanding of cardiomyocyte fate plasticity are of great significance for guiding the treatment of cardiovascular diseases. Of course, the approaches to induce cardiac regeneration are beyond the three strategies we mentioned above. We need continue to focus on the treatment theories and technologies in this field.182 But its potential as a clinical therapeutic drug remains to be evaluated in a large number of clinical trials. The proliferative ability of cardiomyocytes also involves different pathophysiological microenvironment, including inflammation, ROS, extracellular matrix, neuromodulation, etc. These pathophysiological microenvironments enrich our understanding about cardiac regeneration events. However, their mechanisms in heart repair after MI needs to be further explored in a more comprehensive way. The reprogramming of fibroblasts into iCLM is able to repair infarcted myocardium and improve cardiac functions. In the future, more work should be committed to exploring the key regulators and enhancing the efficiency of non-cardiomyocyte reprogramming.Cardiomyocyte differentiation of pluripotent stem cells is an important way to induce cardiac regeneration, but some moral disputes may arise due to the particularity of its source. At present, their clinical safety and efficacy have yet to be verified in clinical trials. Additionally, the occurrence of arrhythmias and severe immune response after transplantation in vivo also indicated that cardiomyocytes derived from pluripotent stem cells are immature and lack the function of adult cardiomyocytes. In recent years, some progress has been made in the study of signaling pathways regulating the proliferation of cardiomyocytes, but the effects of these pathways still need to be further verified in animal models closer to humans, so as to evaluate their possible therapeutic effects and side effects in a more detailed and comprehensive manner. In addition, non-coding RNAs have been identified as a vital new regulator of cardiovascular risk, and can be used as a diagnostic biomarker for CVDs.


How is palliative care being delivered to patients with noncancer illness , and is it being delivered differently than for patients with cancer?In this cohort study, among 145 709 adults who died of cancer or terminal noncancer illness and received palliative care in their last year of life, palliative care was more likely to be initiated earlier, initiated in the hospital setting, and delivered across multiple care settings in patients with cancer than in those with organ failure or dementia. Patients with cancer were more likely to receive care using a consultative or specialist model instead of a generalist model and received palliative care more often from general practitioners and physicians with subspecialty training compared with patients with chronic organ failure or dementia.Differences in the delivery of palliative care across distinct types of serious illness have important implications for the organization and scaled implementation of palliative care programs, including enhancement of practitioner education and training and improvements in equitable access to care across all settings. Palliative care improves health outcomes, but studies of the differences in the delivery of palliative care to patients with different types of serious illness are lacking.To examine the delivery of palliative care among adults in their last year of life who died of terminal noncancer illness compared with those who died of cancer.This population-based cohort study used linked health administrative data of adults who received palliative care in their last year of life and died between January 1, 2010, and December 31, 2017, in Ontario, Canada.Cause of death .Components of palliative care delivery, including timing and location of initiation, model of care, physician mix, care settings, and location of death.A total of 145 709 adults received palliative care ; 21 054 died of chronic organ failure , 14 033 died of dementia, and 110 622 died of cancer. Palliative care was initiated earlier (>90 days before death) in patients with cancer (32 010 [28.9%]) than in those with organ failure  or dementia . A lower proportion of patients with cancer had palliative care initiated in the home (16 088 [14.5%]) compared with patients with chronic organ failure  or dementia . Patients with cancer received palliative care across multiple care settings (92 107 [83.3%]) more often than patients with chronic organ failure  or dementia . Palliative care was more often delivered to patients with cancer (80 615 [72.9%]) using a consultative or specialist instead of a generalist model compared with patients with chronic organ failure  or dementia . Patients with cancer (42 718 [38.6%]) received shared palliative care more often from general practitioners and physicians with subspecialty training, compared with patients with chronic organ failure  or dementia .In this cohort study, there were substantial patient- and practitioner-level differences in the delivery of palliative care across distinct types of serious illness. These patient- and practitioner-level differences have important implications for the organization and scaled implementation of palliative care programs, including enhancement of practitioner education and training and improvements in equitable access to care across all settings. This cohort study examines the delivery of palliative care among adults in their last year of life who died of terminal noncancer illness compared with those who died of cancer. It focuses on the prevention, early identification, assessment, and management of physical issues, including pain and other distressing symptoms, psychological distress, spiritual distress, and social needs.7 However, studies11 contrasting palliative care for patients with cancer and those with terminal noncancer illness report disparities in access and differences in the magnitude of its associated benefits.Palliative care improves quality of life, reduces symptom burden and unwanted health care use, and helps patients die in their preferred location.9 of 235 159 adults who died in Ontario, Canada, found that patients dying of cancer were more likely to receive palliative care than patients dying of noncancer illness, which included chronic organ failure and dementia. Relative to their death, patients with cancer were more likely to receive palliative care earlier than patients with noncancer illness. Prior population-based research12 demonstrated associated benefits of certain components of palliative care among 74 986 patients dying with heart failure (HF). Patients who received palliative care in the home from specialist palliative care practitioners had increased odds of a home death, a place where most patients prefer to die.A population-based study13 has evaluated differences in certain components of palliative care delivery for specific conditions , there is limited evidence investigating how the type of serious illness a patient has is associated with gaps in the delivery of multiple key components of palliative care. The current study is novel because it describes how multiple key elements of palliative care are being delivered across distinct types of serious illness at a population level with an aim to identify these gaps. The objective of this study was to describe the delivery of palliative care among adults in their last year of life who died of terminal noncancer illness  compared with those who died of cancer.The focus of practitioners, decision makers, and health services researchers is shifting from an examination of the efficacy of palliative care across distinct types of serious illness to an examination of the successful design of palliative care programs to scale and implement them. Although prior research16 Ethics approval was obtained from Sinai Health System’s Research Ethics Board. This study followed the Reporting of Studies Conducted Using Observational Routinely-Collected Health Data (RECORD) reporting guidelines.17We conducted a population-based cohort study in Ontario, Canada, using linked clinical and health administrative databases. Ontario is Canada’s most populous province, with more than 10 million adults. All residents of Ontario have access to hospital care and physicians’ services, and those 65 years or older are provided universal prescription drug insurance coverage. The administrative data sets used in this study were linked using encoded identifiers at the patient level at ICES   who died of cancer or common terminal noncancer causes and received newly physician-delivered palliative care in their last year of life between January 1, 2010, and December 31, 2017. We identified the delivery of palliative care based on a set of unique physician claims fee codes , and dementia because these conditions are recognized as having unique trajectories of functional decline at the end of life that may influence a person’s palliative care needs.23 Cause of death was determined according to the International Statisitcal Classification of Diseases and Related Health Problems—Tenth Revision, Canada (ICD-10-CA) disease code on the patient’s death certificate.The study index date was 365 days before the patient’s death. The primary exposure was the patient’s type of serious illness, categorized according to the cause of death as cancer or noncancer illness. We defined terminal noncancer illness as HF, chronic obstructive pulmonary disease (COPD), end-stage kidney disease (ESKD), cirrhosis, stroke, and dementia because these diseases represent the most common terminal noncancer conditions.31 Currently, there are no established standards on when to initiate palliative care.After the identification of differences in the outcome of timing of palliative care initiation according to illness type, we conducted a prespecified, exploratory, hypothesis-generating analysis using the timing of palliative care relative to death as the exposure to measure the potential association of timing with location of death. The timing of palliative care was categorized according to commonly used periods in the published literature: 30 days or less, 31 to 90 days, and 91 days or more before death.32 and hospital frailty risk score,33 using a 5-year look-back period. We also measured year of death, use of acute health care services in the 1 year before the study index date, and the timing of first palliative care consultation relative to death.We measured demographic and clinical variables, including age, sex, socioeconomic status, rural location of residence, comorbidities and chronic conditions,31 The secondary outcomes were (1) location of initiation , (2) model of care , (3) physician mix , (4) care setting , (5) total number of palliative care visits before death, and (6) location of death, which included home (including nursing homes and hospice), hospital (including intensive care unit), and other .19 Formal palliative care in Ontario is predominantly provided by physicians, nurse practitioners, registered nurses, social workers and personal support workers in hospitals, outpatient clinics, and at home. Patients generally require a referral from a physician to access specialized palliative care services but such services may be provided by nonpalliative care specialists without a referral.Most palliative care is delivered by general practitioners in Ontario. We considered physicians to be palliative care specialists if their annual billing was comprised of more than 10% of palliative care fee codes, which is based on a previously validated method with a sensitivity of 76.0% and a specificity of 97.8%.34 These models are derived using the proportion of palliative care fee codes claimed by physicians, which classifies them as a palliative care specialist or palliative care generalist as described above. The 4 models of palliative care were (1) no physician-based palliative care , (2) generalist palliative care , (3) consultative palliative care , and (4) specialist palliative care .We measured the delivery of 4 models of physician palliative care delivery.The setting of palliative care was determined using physician claims, which included the locations in which care was delivered. There were 5 possible settings where palliative services could be delivered: (1) clinic, (2) home, (3) inpatient setting, (4) multiple locations, and (5) third-party case management settings.Descriptive statistics were used to measure patient characteristics, the delivery of palliative care, and locations of death. The association between type of serious illness (using cancer as the comparator) and the timing of palliative care relative to death (using ≤30 days as the reference) and the timing of palliative care (using ≤30 days as the comparator) and the location of death (using home as the reference) were estimated using a multinomial logistic generalized estimating equation approach. After the identification of differences in the outcome of timing of palliative care initiation according to illness type, we conducted a prespecified, exploratory, hypothesis-generating analysis using the timing of palliative care relative to death as the exposure to measure the potential association of the timing with the location of death. We stratified location of death outcomes by type of serious illness because we did not intend to directly compare the magnitude of the associations given that they are confounded by indication. Models were adjusted for age, sex, comorbidities, rurality, neighborhood income, hospital frailty risk score, and total number of hospitalizations in the 1 year before the study index date. We did not account for clustering by physician or facility because most people receive end-of-life care from many physicians in multiple care settings.35 All analyses were performed using SAS statistical software, version 9.4 (SAS Institute Inc).We used standardized differences to identify important differences for all secondary outcomes. Standardized differences  of 0.2, 0.5, and 0.8 can be used to represent small, medium, and large effect sizes, respectively. An effect size index of 0.1 or less is considered to indicate good balance among variables.A total of 145 709 adults  received palliative care . The medPalliative care was initiated earlier (>90 days before death) in patients with cancer (32 010 [28.9%]) than in those with organ failure  or dementia  . After aThe models of generalist and specialist palliative care along with the physician mix also differed between patients with distinct types of serious illness and are presented in A higher proportion of patients with cancer (69 208 [62.6%]) died at home compared with patients with chronic organ failure (11 232 [53.3%]), but the proportion was lower than for patients with dementia (10 520 [75.0%]) . Differe42This cohort study of 145 709 adults who died of cancer or noncancer illness and received palliative care in their last year of life found important differences in the delivery of palliative care across different types of serious illness for patients with cancer compared with those with chronic organ failure or dementia. In general, patients dying of cancer were younger, had a lower burden of medical comorbidity, and had lower prior health care use compared with patients dying of terminal noncancer illness. Palliative care was also initiated earlier and in the hospital or clinic setting for patients with cancer. For patients with cancer, palliative care was more often delivered by palliative care specialists across multiple locations. More patients with cancer also received palliative care from both general practitioners and physicians with subspecialty training compared with patients with chronic organ failure and dementia. These findings identify patient-level disparities in access to palliative care, which may be associated with the presence of established palliative care programs in cancer centers to provide multidimensional care for patients with cancer. These findings also suggest practitioner-level deficiencies in palliative care for patients with terminal noncancer illness that may be associated with differences in specialist self-rated knowledge of end-of-life care or recognition of their patient’s palliative needs and subsequent referral during their illness.43 Still, the fact that more patients with terminal noncancer illness died in the hospital despite having had palliative care initiated at home raises interesting questions that require further exploration because home palliative care is strongly associated with a home death.15 The fact that a much larger proportion of patients with cancer received palliative care in multiple settings (which includes home) may simply reflect differences in where patients are identified as having palliative care needs. Alternatively, this finding may be influenced by the availability and needs of family caregivers, especially for persons with dementia.The association between timing and location of death should be considered as exploratory, hypothesis-generating findings and interpreted with caution because of the risk of confounding by indication. A higher proportion of patients with chronic organ failure and dementia had palliative care initiated 30 days or less before death. This late initiation of palliative care in a population of patients with high medical complexity may limit sufficient time to build a trusting relationship with their palliative care practitioners and clarify their goals of care and preferences for location of death.11 demonstrating differences in the associated benefits of palliative care across types of serious illness. A population-based cohort study11 of 230 921 adults who died in Ontario found differences in the magnitude of association between the early initiation of palliative care (≥60 days before death) and health care use at the end of life across types of serious illness compared with late initiation (<60 days before death). In that study,11 early palliative care was associated with more than 2-fold higher odds of acute health care use at the end of life in patients dying of chronic organ failure and 1.5-fold odds in patients dying of dementia compared with acute health care use in patients dying of cancer. Prior research5 also examined differences in the associated benefits of different elements of palliative care delivery among 74 986 patients dying of HF. A subgroup analysis of the 35 292 patients who received palliative care found a strong association between palliative care initiated in the home, palliative care delivered across multiple settings, and models of care that involve specialist palliative care practitioners and a home death.5 The current study identified disparities among these components of delivery across different types of serious illness. Future work is required to measure the association of these differences with important outcomes and to identify differences in preferences for specific types of treatments between patients with different types of serious illness. Given that there are twice as many patients with noncancer illness and palliative care needs than there are with cancer, it is clear that there are not enough palliative care specialists to meet current or future demand.44 Health care systems should begin to focus on building capacity to integrate palliative care across all health care settings and levels (primary to tertiary care) throughout an illness, according to the patient’s needs. This work will require a commitment to delivering palliative care by all health care professionals with basic palliative care training, reserving specialist palliative care using multidisciplinary teams for referral of complex cases.7Our findings are supported by prior research47 In addition, patients with specific terminal noncancer illnesses, such as HF, ESKD, and cirrhosis, likely have differences in other care needs, such as ongoing dialysis, that occur during an exacerbation of their underlying disease. Second, this study described multiple differences in the components of palliative care as delivered by both generalist and specialist palliative care physicians. A stratified analysis of care delivery across illness trajectories comparing delivery by generalist and specialist palliative care practitioners would help identify further gaps in access to specialized palliative care and its associated benefits.48 However, a description of the real-world delivery of palliative care is important to identify gaps in care and likely strengthens the generalizability of the study to care provided in other similar health care systems. Third, the study did not measure delivery of palliative care by health care practitioners other than physicians, which may include nurse practitioners or social workers.50 Still, capturing the delivery of palliative care using physician fee codes in administrative data is a strength of the study given that care classification has been less successful in health systems without universal coverage.51 The prescription of medication  may be an effective alternative or complementary approach to measuring the delivery of palliative care, including the timing of its initiation, beyond physician claims. Further work is required to address challenges associated with identifying the indication, dispensing, and continuous use of medications used with palliative intent. Fourth, the possibility that physicians were delivering palliative care and not using specific palliative care physician fee codes would serve to underestimate the magnitude of our findings. In Ontario, physicians are directed to use these codes for seriously ill patients who wish to forgo aggressive treatment of their underlying disease, with a focus on maintaining comfort. In practice, many physicians use the consensus definition from the International Association for Hospice and Palliative Care to determine if they are delivering palliative care.7 Fifth, the study intentionally used information on a patient’s death certificate to define the cohort to maximize specificity because of the concern that other approaches may introduce too much heterogeneity and other sources of bias.4This study has limitations. First, individual palliative care needs or specific palliative therapies were not measured among patients in the cohort. Patients with terminal noncancer illness frequently have a higher burden of palliative care needs associated with higher health care use, worse functional impairments, and higher levels of anxiety and depression compared with patients with cancer.There are substantial patient- and practitioner-level differences in the delivery of palliative care across distinct types of serious illness. These patient- and practitioner-level differences have important implications for the organization and scaled implementation of palliative care programs, including enhancement of practitioner education and training and improvements in equitable access to care across all settings.


The bone-derived hormone fibroblast growth factor 23 (FGF23) acts in concert with parathyroid hormone (PTH) and the active vitamin D metabolite calcitriol in the regulation of calcium (Ca) and phosphate (P) homeostasis. More factors are being identified to regulate FGF23 levels and the endocrine loops between the three hormones. The present review summarizes the complex regulation of FGF23 and the disturbed FGF23/Klotho system in chronic kidney disease (CKD). In addition to the reduced ability of the injured kidney to regulate plasma levels of FGF23, several CKD-related factors have been shown to stimulate FGF23 production. The high circulating FGF23 levels have detrimental effects on erythropoiesis, the cardio-vascular system and the immune system, all contributing to the disturbed system biology in CKD. Moreover, new factors secreted by the injured kidney and the uremic calcified vasculature play a role in the mineral and bone disorder in CKD and create a vicious pathological crosstalk. The discovery of the bone-derived hormone fibroblast growth factor 23 (FGF23) in 2000 led to a new understanding of the mineral homeostasis . T. T12]. TThe circadian rhythms in hormonal levels have a systemic impact on all aspects of physiology. Results from our laboratory showed the existence of a circadian rhythm of plasma FGF23 levels A 31,32],3231,32]In mammals the master pacemaker of circadian rhythmicity is in the hypothalamic suprachiasmatic nucleus (SCN). The SCN receives light cues about day and night and it is accordingly coordinating the peripheral and central clocks via neuronal, hormonal and metabolic signaling pathways . In addiBone remodeling is a complex process by which old bone is removed and replaced by new bone, requiring interaction between different bone cells, a process which to a large extent is coordinated by osteocytes . The diuFurthermore, plasma phosphate and PTH have circadian rhythms that depend on the feeding pattern . This phIn the kidney, FGF23 binds to FGFR. The obligate co-receptor Klotho is expressed in the distal tubules and to less extend in the proximal renal tubules ,41. InhiIt is probably the transmembrane Klotho that is expressed in small quantities in the proximal renal tubules, which is the co-receptor responsive for FGF23–FGFR interaction in the kidney. It has also been proposed that a circulating soluble Klotho fragment acts as a hormone on the proximal tubule, causing endocytosis of the NaPi co-transporters ,45. KlotCYP27B1), is opposite to that of PTH. The 1α-hydroxylase is expressed in the proximal renal tubule and is regulated by both FGF23, PTH and calcitriol itself. FGF23 suppresses the 1α-hydroxylase expression and activity [In the distal convoluted tubule FGF23 increases calcium reabsorption. FGF23 is acting via the FGFR–Klotho complex and activation of ERK1/2, SGK1 and WNK4 upregulates expression of the calcium-selective channel protein TRPV5 at the luminal membrane and hereby reduces renal calcium excretion . The phoactivity ,50. A practivity ,49. In aactivity .2+/PTH relationship during normocalcemia and acute hypocalcemia in the rat and demonstrated that FGF23 has an inhibitory tonus on PTH secretion, when plasma Ca2+ is within the normal range. However, if plasma Ca2+ is low and increased PTH secretion is needed in order to restore Ca2+ levels, this inhibitory effect of FGF23 on PTH secretion is abolished. Furthermore, the same experimental in vivo study showed that the suppressive tonus of FGF23 in normocalcemia was mediated through the FGFR [The direct interplay of FGF23 and PTH takes place at the level of bone and in the parathyroid glands. In bone, PTH stimulates biosynthesis of FGF23 via activation of the orphan nuclear receptor Nurr1 . The parthe FGFR .+/K+-ATPase activity via interaction with the α-1-subunit and hereby causing an increased abundance of Na+/K+-ATPase in the plasma membrane [+/K+-ATPase by ouabain did not affect the PTH secretory response to hypocalcemia [+/K+-ATPase activity. Furthermore, it has since been demonstrated that parathyroid specific deletion of Klotho did not alter the PTH acute response to changes in extracellular calcium or FGF23. Additionally, in the absence of parathyroid Klotho, the calcineurin-NFAT pathway was found to mediate the suppression of PTH secretion by FGF23 [The role of parathyroid Klotho remains controversial. Klotho has been proposed to modulate the parathyroid Namembrane . This recalcemia . Our resby FGF23 . The mecby FGF23 .A central complication in CKD is the mineral and bone disorder (CKD-MBD), a complex systemic disorder characterized by disturbances in calcium and phosphate balance, plasma levels of PTH and calcitriol along with changes in bone morphology, bone density and remodeling activity. Development of calcification in soft tissue especially the arteries and heart valves are also part of this complex syndrome . DiscovePatients with CKD have severely increased plasma levels of FGF23 and deficiency of Klotho. Epidemiological studies have repeatedly reported that FGF23 is an important factor associated with increased risk of progression of kidney disease, loss of kidney graft function, cardiovascular disease and mortality ,64,65,66The mechanisms behind this early increase in FGF23 are not completely understood. It has been proposed to be an early physiological mechanism in order to increase phosphate excretion in the functional nephrons . WhetherTherefore, we investigated the role of the kidney in the regulation of FGF23 in experimental models in the rat ,19. RemoIn a model of chronic kidney disease, the 5/6 nephrectomized rat, we have repeatedly measured severely increased plasma levels of FGF23 ,77,78,79FGF23 plasma levels increase early in CKD progression . BesidesRegulation through the bone FGFR maintains to be an important regulator of FGF23 in kidney insufficiency ,18,19. A2+ is sensed by the CaSR which is expressed in bone. However, the molecular mechanism behind the rapid influx/efflux of Ca2+ in bone is not fully understood [2+ and the Ca2+ set point on bone surface. Inducing acute hypocalcemia by short lasting EGTA infusion in normal rats lowered plasma Ca2+ significantly. Then the EGTA infusion was stopped and subsequently a rapid increase in plasma Ca2+ took place (within 10 min) with further recovery to basal levels. EGTA infusion in anephric rats caused lower calcium levels and recovery to a lower levels of plasma Ca2+  and highest in the inactive phase (afternoon) in normal rats. As rats are nocturnal animals, the corresponding value in human would be opposite to the time point. This diurnal cycle was maintained but shifted in time in all CKD rats. The circadian rhythm of plasma FGF23 was disturbed including a shifted acrophase in CKD rats fed a high phosphate diet or standard diet. FGF23 circadian rhythmicity was completely abolished in CKD rats fed a low phosphate diet, which also remarkedly lowered circulating FGF23 in these rats. Plasma levels of Klotho were similar during day and night both in normal and uremic rats. Circulating PTH levels changed significantly during the 24 h clock with lowest levels in the active phase (early morning) and in the highest levels inactive phase (midday). This diurnal variation was completely abolished in CKD rats fed a high phosphate diet. Low phosphate diet preserved significant circadian rhythm although acrophases were shifted [We examined the circadian rhythm of mineral parameters in normal rats and 5/6 nephrectomized (CKD) rats fed a low, normal or high phosphate diet . Plasma  shifted . Disturb shifted . A distu shifted ,102.We further examined the circadian clock operating the parathyroid gland in normal and in CKD rats suffering from secondary hyperparathyroidism. It was shown that an internal molecular circadian clock operates in the parathyroids and that a circadian rhythmicity of genes, regulating cell cycle was demonstrated . In the Early in kidney injury the expression of Klotho is downregulated. The precise mechanisms have not been completely clarified. A rapid unilateral decline in kidney Klotho expression was demonstrated in a model of unilateral obstructive nephropathy (UUO), despite the condition is not a state of uremia . Its dowWe and others have demonstrated that induction of FGF23 expression in acute and chronic injured kidney tissue regardless of animal model ,107,108.Due to the very high concentration of FGF23 in plasma measured in CKD, it is thought to activate FGFRs independently of Klotho and several off-target effects of FGF23 have been reported. It has been shown in experimental CKD models that FGF23 activates FGFR4 and the downstream calcineurin-NFAT pathway in cardiomyocytes and induces left ventricular hypertrophy ,113—a coCKD patients suffering from severe kidney insufficiency have concomitant very high plasma levels of both hormones FGF23 and PTH. As such, the inhibitory effect of FGF23 on PTH secretion is abolished. In addition to the downregulation of CaSR, VDR and FGFR in the dysplastic parathyroid gland, it has been proposed to be explained by the downregulation of Klotho as shown by some groups ,122,123.2+ concentration in FGF23′s regulation of PTH, may illustrate an additional mechanism behind the resistance of the parathyroid gland to FGF23′s suppressive effect, as the concurrent low Ca2+ levels overrule this inhibition [2+ within the normal range, as pharmacological inhibition of the FGFR resulted in increased PTH secretion [Still, hypocalcemia is a common complication in kidney failure. Therefore, our findings of a superior role of the extracellular Cahibition . As prevecretion . TherefoWhereas FGF23 impose a minute-to-minute regulation of PTH secretion, PTH stimulation of FGF23 expression is a slower stimulus. In uremia circulating PTH is necessary for generating the increase in FGF23 expression in bone and the high plasma levels of FGF23, as shown in experimental studies by us and others. Parathyroidectomy prevented the FGF23 increase in a CKD model. Additionally, it reduced FGF23 after CKD was inflicted in the rat ,125. In In recent years, new factors have been proposed to be implicated in the disturbed mineral and bone disorder in CKD . Kidney Recently, we have examined such a new paradigm and its potential impact on CKD-MBD. We used the unilateral nephrectomy model (UUO), which is ideal to study the rapid development of unilateral renal fibrosis  in a non-uremic milieu as the contralateral kidney maintains a normal glomerular filtration . ActivinIn a study on atherosclerotic diabetic CKD mice, treatment with Dkk1 antibodies improved the renal osteodystrophy and uremic vasculopathy . TreatmeA pathological tissue crosstalk is a feature of CKD-MBD, and the disturbed system biology in CKD goes beyond impaired kidney function and kidney-derived factors. We proposed that vascular calcification in CKD could affect bone homeostasis, based on the findings of large upregulation of Wnt inhibitors and TGFβ family in the uremic vasculature. Among the upregulated signal molecules, we found potential circulating factors such as sclerostin, SFRP4 and activin A . Recentl2+ affects the feed-back loop between FGF23 and PTH. How FGF23 is regulated by extracellular phosphate has not yet been completely clarified, but improvements have been made in understanding extracellular phosphate sensing. Thus, CaSR has binding sites for phosphate, where binding turns the receptor into an inactive state. Yet, how the two ligands Ca2+ and phosphate compete in CaSR activation/inactivation is not yet understood. In bone, Pit2 and FGFR1 has been proposed to be involved in phosphate sensing (Recent research has improved our understanding of the complex regulation of the hormones involved in mineral homeostasis. More local and circulating factors have been demonstrated to regulate FGF23 expression. Additionally, the extracellular concentration of Ca sensing . A circadian rhythm of FGF23 has been demonstrated, yet the mechanism leading to the diurnal variation in plasma levels is not fully understood, although they are related to feeding. A molecular circadian clock is active in the parathyroid gland but gets disturbed in CKD. Additionally, the plasma levels of activin A and phosphate changed their diurnal pattern in CKD. These findings are important when interpreting biochemistry results in CKD patients. Still, the clinical importance of a disturbed circadian rhythmicity in CKD-MBD has yet to be finally established, so has its implications in the development of parathyroid hyperplasia. 2+ and phosphate reabsorption in the kidney and the plasma levels of calcitriol. In addition, the key role of the kidney in regulating the plasma levels of intact FGF23 has been identified by a high extraction ratio of 40% between the renal artery and vein. This important degradation of the hormone diminishes in CKD, resulting in increased plasma levels. Expression of FGF23 is induced in injured kidney and probably has a role in the inflammation and fibrosis processes via autocrine/paracrine signaling. Kidney-derived FGF23 does not contribute to the increased plasma levels of FGF23 in CKD. The kidney has long been known for its important role in the balance of calcium and phosphate by regulating CaRecently, new factors related to Wnt or TGF-β signaling have been shown to be secreted from the injured kidney and the uremic calcified vasculature, and they may have an important role in CKD-MBD. Future studies may further provide understanding of each of these factors and their potential as therapeutic targets in CKD and CKD-MBD.