Glia are present in all organisms with a central nervous system but considerably differ in their diversity, functions, and numbers. Coordinated efforts across many model systems have contributed to our understanding of glial-glial and neuron-glial interactions during nervous system development and disease, but human glia exhibit prominent species-specific attributes. Limited access to primary samples at critical developmental timepoints constrains our ability to assess glial contributions in human tissues. This challenge has been addressed throughout the past decade via advancements in human stem cell differentiation protocols that now offer the ability to model human astrocytes, oligodendrocytes, and microglia. Here, we review the use of novel 2D cell culture protocols, 3D organoid models, and bioengineered systems derived from human stem cells to study human glial development and the role of glia in neurodevelopmental disorders. Both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) can be differentiated into neurons and/or glia using various culturing techniques that are amenable to user-defined customizations , and studies from the past several decades have illuminated their immense importance to brain development in both health and disease . They noizations . Currentizations . Throughin vitro differentiation.There are two primary classes of CNS glia, each with unique developmental origins: macroglia and microglia. Macroglia refer to a class of neural cells within the CNS that share a common neuroectodermal origin with neurons . The twoDuring human fetal CNS development, neural stem cells  differentiate first into neurons and then astrocytes and OLs in a temporally restricted sequence. Neurogenesis begins early, around 6-8 gestation weeks, in the human fetus . Around in vitro astrocyte and OL differentiation from human stem cells.Although it is not fully known what drives the gliogenic switch, a combination of extrinsic, intrinsic, and epigenetic signals have been implicated from studies across multiple model systems. Early rodent studies identified activators of the Janus kinase/signal transducer and activator of transcription (JAK-STAT) pathway, including ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), and cardiotrophin 1, as cytokines involved in the initiation of astrogenesis . Bone moEmbryonic yolk sac progenitors are the precursors to all tissue-resident macrophages, including microglia. Prior to the gliogenic switch, microglia have already invaded the CNS, making them one of the first CNS glial residents of the nervous system. Primitive ameboid-like microglia have been observed in the human brain as early as 5 gestational weeks, thus developing side-by-side with neurons prior to the presence of astrocytes and OLs . MicroglSall1 has been identified as a microglia-specific signature gene that is not expressed in other mononuclear phagocytes or CNS cells  , and neuin vitro system that recapitulates in vivo features. The ability to study glia using human stem cell models is critical to gaining insights into their essential operations within the brain.An alternative solution to studying human CNS glia is to utilize stem cells from human patients. Stem cells can be differentiated into the various cell types of the brain, including radial glia, neurons, astrocytes, OLs, and microglia. Recent advancements to stem cell technologies, outlined in detail in the following sections, enable functional modeling of the human brain using an in vitro. With this pretext, we walk through current 2D, 3D, and bioengineered approaches for generating astrocytes, OLs, and microglia and studying their contributions to NDDs.In the sections below, we will focus on stem cell models of human glia. We will start by discussing various sources of stem cells and their advantages for disease modeling. Next, we will highlight common metrics used to measure “success” in generating glia in vitro fertilization, so there is a limited number of existing and new hESC lines. Alternatively, hiPSCs are reprogrammed from adult fibroblast cells using some combination of the Yamanaka TFs: Oct3/4, Sox2, c-Myc, and Klf4  vs. White (0.77%) individuals highlights a combination of underlying genetic susceptibilities and socioeconomic access to treatment . HoweverOne of the major obstacles within the stem cell and glial fields is how we determine “success” of differentiations. Adding to this challenge, there is no current consensus on the appropriate methods or signatures required to define each of the major glial cell types. Here, we will briefly provide perspective on the most common readouts used to measure success of glial differentiation protocols.in vivo /immunocytochemistry (ICC) or with fluorescence-activated cell sorting (FACS) is one of the most frequently used methods to test glial induction. This method is quite simple: detection of a cell type-specific protein within a cell delineates its identity. However, many glial markers lack specificity and/or sensitivity. For example, GFAP is commonly used to detect astrocytes, but the protein is also present in radial glia, is a marker for astrocyte reactivity, and exists in only a subset of differentiated astrocytes in vivo . In the in vivo , but howfeatures  and becain vitro glia acquire the same phenotypes as seen in vivo. Functional studies can also be performed via in vitro cultures (in vitro models or with in vivo observations. This is particularly relevant for cell types (microglia and astrocytes) where in vitro phenotypes can diverge from the CNS environment (RNA-sequencing (RNA-seq) is a widely used technique for assessing differentiation success. Transcriptomics analyses allow users to unbiasedly compare expression data of stem cell-derived glia with primary glia  and can cultures  and/or xcultures . Some maironment .Altogether, combining the use of glial markers, transcriptomic data, and functional studies can help ensure the success of producing glial cells from human stem cells. Metrics of “successful differentiation” remain ambiguous, but it is clear that the combination of each of the above readouts should be a requisite benchmark for claims of successful glial differentiation.The use of human stem cells to study glia and their contributions to NDDs has flourished in the last two decades. The generation of glia from human stem cells has unveiled new areas of research into their development, maturation, and involvement in neurological disorders. 2D monolayer culture protocols of glial differentiation from human stem cells remain a steadfast way to obtain astrocytes, OLs, and microglia. These cultures all begin with pluripotent stem cell colonies, sometimes maintained on top of stromal feeders, such as mouse embryonic fibroblasts (MEFs). Stromal feeders support stem cell health but can also result in the transfer of animal pathogens, elicit immune responses, and introduce cell contamination . As an a+ cells but may simultaneously induce aspects of astrocyte reactivity (+ days). To obtain aquaporin-4 (AQP4)+ and S100β+ cells, markers associated with more mature astrocytes, CNTF and/or brain-derived neurotrophic factor (BDNF) are frequently added to the culture media  to further confirm their functionality in vivo. Subsequent studies tested additional supplements, including SHH, Noggin, and/or PDGF-AA, to also successfully obtain OPCs and mature OLs from hESCs + OLs. Likewise, SCZ hiPSC-macroglial progenitor cells implanted into Shiverer mice resulted in reduced myelination, delayed astrogenesis, and behavioral abnormalities (The role of OLs in the primary pathogenesis of schizophrenia (SCZ) has been long discussed, primarily due to the identification of abnormalities in and differential expression of myelination-related genes in patients with chronic SCZ . With recontrols , suggestotential . When cumalities . Altogetin vivo. After about a month, these embryoid bodies (EBs) were immunoreactive for markers of early yolk sac progenitors. Upon selection and monolayer culturing in supplemented NGD media, the progenitors formed microglia-like precursors, which differentiated into MGLs after an additional month in culture. Comparisons of hiPSC-derived MGLs to fetal human microglia demonstrated evidence of similar, yet less extensive, genetic signatures and marker expression. Additionally, MGLs functionally resembled fetal human microglia in their ability to phagocytose, react to cytokines and chemokines, and respond to cell damage.Generating microglia from human stem cells is more challenging due to their mesodermal developmental origin, which means they do not follow the “default” neuroectodermal differentiation pathway that benefits other CNS lineage glia in traditional NPC cultures. Instead, microglia originate from erythromyeloid progenitor cells of the yolk sac that propagate during hematopoiesis. These progenitors further develop into primitive macrophages that invade the developing neural tube where they transition into microglial progenitors . In the + hematopoietic progenitors  and adapt a quiescent microglial phenotype. Another protocol published by Douvaras and colleagues used a similar approach but instead cultured hiPSCs or hESCs in normoxic conditions  organoids from human stem cells . Like neobserved .when neurons and astrocytes are combined, which enables precision over the timing of initiating neuron-glial interactions.An alternative, but less common, method to study astrocytes is to grow both neurons and astrocytes separately in 2D cultures and then combine them into 3D spheres . In thisTREX1 or hESCs with patient-specific TREX1 mutations induced using CRISPR/Cas9. AGS-organoids were significantly smaller than controls, recapitulating the microcephaly observed in patients with AGS. AGS-organoids also experienced a high degree of neuronal cell death, which was linked to increased neurotoxic type 1 interferon release by TREX1-deficient astrocytes. Additionally, when healthy control organoids and neurons were cultured in conditioned media from AGS-astrocytes, they experienced increased cell death and organoid size reduction. The authors tested Lamivudine (3TC) and Stavudine (d4T), two FDA-approved HIV antiviral drugs, on TREX1-deficient organoids and observed partial rescue of the neurotoxicity, neuronal death, and size reduction phenotypes. Another recent study utilized cortical organoids with CRISPR/Cas9-induced mutations in hiPSCs to model tuberous sclerosis (TSC) , a rare genetic encephalopathy that results in severe intellectual disability in infants . Organoiis (TSC) . TSC is The ability to study the functions of OLs, particularly axonal wrapping and subsequent myelination, in 2D cultures is limited. This has incentivized the use of 3D cultures as a reproducible system to study OL development and function. Initial protocols to produce brain organoids typically lacked OLs, which was surprising given that OLs are derived from the same radial glia population as astrocytes and neurons. When profiling their organoids using single-cell RNA-seq, several groups observed a small number of endogenous OPCs , citing +/OLIG2+ double-positive OPCs and PDGFRα+ OPCs could be detected within hOLSs, and these cells continued to mature over the next 100 days into functional myelinating OLs. RNA-seq data revealed similar gene expression between hOLS-derived OL lineage cells and primary human OPCs and OLs isolated via immunopanning, establishing a robust and reproducible protocol that can be used to study OL development in a manner that mimics the in vivo cell environment. TF approaches have also been used to generate OLs in brain region-specific organoids. Using modifications to their own organoid differentiation protocol, the Paşca lab also produced human oligodendrocyte spheroids (hOLSs) . In contPLP1 gene caused significant reductions to the number of OLs present within the oligocortical spheroids. While the mechanistic outcomes behind PLP1 disruptions have yet to be uncovered, this validated approach provides hope toward future disease modeling using OL-containing organoids to explore patient-specific OL pathogenesis.Despite an abundance of evidence implicating OLs in numerous NDDs, including schizophrenia , AlexandStudying the roles of microglia and their development within the brain using 3D neuroectodermal cultures is challenging due to their distinct mesodermal lineage. Groups have largely pursued two approaches to tackle this obstacle: (1) add primary or human stem cell-derived microglia to brain organoids to form multilineage structures or (2) minimally pattern organoids that contain progenitor cells from multiple germ layers. Using the first approach, The second approach for introducing MGLs into organoids has focused on innately growing microglia within the organoid structure itself. One study identified the presence of a population of mesodermal precursors within cerebral organoids using single-cell RNA-seq , which wAPOE3 patients were induced with homozygous APOE4 alleles using CRISPR/Cas9 and subsequently differentiated into MGLs (APOE3-MGLs (controls) or APOE4-MGLs (AD-risk) were embedded into 2-month-old APP duplication organoids and cultured for a month. Organoids with APOE4-MGLs exhibited increased accumulation of Aβ aggregates, suggesting that the presence of APOE4 negatively impacts MGL ability to clear extracellular Aβ. This phenotype was reversible when the APOE4 mutation was converted to APOE3. A simultaneously published study also formed brain organoids from hiPSCs derived from familial and sporadic AD patients and used a dual chamber microfluidics device to study their cellular interactions  has been used to create microchannels on a glass coverslip coated with the cell-adhesive molecule poly-D-lysine . hiPSC-din vivo cues driving development and maturation of each of these cell types. Additionally, more robust markers to confirm output quality are needed.Despite great progress in using human stem cells to model the developing brain, there are still several limitations that 2D, 3D, and bio-engineered models have yet to overcome. One primary challenge with these current methods is difficulty in mass scale-up and standardization. There is considerable variability across different hiPSC and hESC lines and many protocols require several months to produce mature, functional glia. Currently, the field lacks consensus on differentiation protocols due to the fact that we still do not fully understand the in vitro cell cultures are unable to fully recapitulate complex cell-cell interactions and do not thoroughly represent the adult glial cell heterogeneity observed in vivo. Additionally, due to the limited progenitor pool heterogeneity within 2D and 3D cultures and the limited size expansion of organoids, it can be difficult to reproducibly study whole brain interactions. The incomplete and random nature of the intrinsic cytoarchitecture also serves as a restriction to studying neural circuit formation and aberrant circuitry in disease models. Moreover, in vitro studies are limited in their ability to study the influence of systemic changes on CNS physiology. As organoid platforms become more sophisticated, many of these limitations may dissipate. Additionally, there is potential to integrate organoid cultures with other systems to interrogate the influences of external factors, such as the gut microbiome, circulating hormones, and environmental exposures, on gliogenesis and maturation.Although organoids have aided in advancing cellular complexity and long-term maturation studies, the diversity in cell types and ability to produce fully mature cells still does not match the complexity of the human brain . At presIn the meantime, one of the biggest unmet needs in the field is reliable glial markers. Many current glial markers are co-expressed by multiple cell types, e.g., S100β is expressed in both astrocytes and OPCs , and/or We are at an exciting stage where our human glial toolset is expanding faster than our answers to fundamental biological questions about glial development and their contribution to NDDs. As we look to the next decade, we expect that a comprehensive understanding of how glia develop, function, and mature will allow us to better determine the roles of human glia in health and disease.SL composed the manuscript. Both authors developed the sections to be included in the review and edited and finalized the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The Joint Program Executive Office for Chemical, Biological, Radiological, and Nuclear Defense (JPEO-CBRND) began development of a broad-spectrum antiviral countermeasure against deliberate use of high-consequence viral hemorrhagic fevers (VHFs) in 2016. The effort featured comprehensive preclinical research, including laboratory testing and rapid advancement of lead molecules into nonhuman primate (NHP) models of Ebola virus disease (EVD). Remdesivir  was the first small molecule therapeutic to successfully emerge from this effort. Remdesivir is an inhibitor of RNA-dependent RNA polymerase, a viral enzyme that is essential for viral replication. Its robust potency and broad-spectrum antiviral activity against certain RNA viruses including Ebola virus and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) led to its clinical evaluation in randomized, controlled trials (RCTs) in human patients during the 2018 EVD outbreak in the Democratic Republic of the Congo (DRC) and the ongoing Coronavirus Disease 2019 (COVID-19) pandemic today. Remdesivir was recently approved by the US Food and Drug Administration (FDA) for the treatment of COVID-19 requiring hospitalization. Substantial gaps remain in improving the outcomes of acute viral infections for patients afflicted with both EVD and COVID-19, including how to increase therapeutic breadth and strategies for the prevention and treatment of severe disease. Combination therapy that joins therapeutics with complimentary mechanisms of action appear promising, both preclinically and in RCTs. Importantly, significant programmatic challenges endure pertaining to a clear drug and biological product development pathway for therapeutics targeting biodefense and emerging pathogens when human efficacy studies are not ethical or feasible. For example, remdesivir’s clinical development was facilitated by outbreaks of Ebola and SARS-CoV-2; as such, the development pathway employed for remdesivir is likely to be the exception rather than the rule.The current regulatory licensure pathway for therapeutics targeting rare, weaponizable VHF agents is likely to require use of FDA’s established Animal Rule . The FDA may grant marketing approval based on adequate and well-controlled animal efficacy studies when the results of those studies establish that the drug is safe and likely to produce clinical benefit in humans. In practical terms, this is anticipated to include a series of rigorous, well-documented, animal challenge studies, to include aerosol challenge, combined with human safety data. While small clinical studies against naturally occurring, high-consequence pathogens are typically performed where possible, approval for the therapeutics currently under development against biodefense pathogens will likely require the Animal Rule pathway utilizing studies in NHPs. We review the development of remdesivir as illustrative of the effort that will be needed to field future therapeutics against highly lethal, infectious agents. Development of medical countermeasures (MCMs) to treat and prevent viral infections with highly lethal filoviruses, such as Ebola virus (EBOV) and Marburg virus (MARV) is very difficult. Recently, US Food and Drug Administration (FDA)-approved vaccines and therapeutics for disease caused by EBOV and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infections were obtained using the standard regulatory framework to address the new products’ safety and efficacy profile with randomized clinical trials. Clinical trials were feasible as sufficient numbers of patients were available due to the large scale outbreaks with both Ebola and SARS-CoV-2. However, the opportunity to conduct human studies against emerging viral pathogens is relatively rare, and effectively impossible for high-consequence pathogens likely to be used in biowarfare and bioterrorism, such as the filoviruses. The alternative regulatory pathway for development of new drugs targeting biodefense pathogens that cannot be conducted when human efficacy studies are not ethical and field trials are not feasible is through the FDA’s Animal Rule. In this review, we highlight how remdesivir’s preclinical development path, based upon the Animal Rule for an EVD indication, was leveraged to support its ultimate regulatory approval as an antiviral agent for the treatment of Coronavirus Disease 2019 (COVID-19). The preclinical and clinical development of remdesivir for both SARS-CoV-2 and filovirus treatment including evidence from Animal Rule and clinical studies is also reviewed. The challenges associated with employing the Animal Rule for development of multiple biodefense therapeutics against numerous and distinct viral biothreats within Biosafety Level 4 are discussed. The issues of developing pharmaceuticals for weaponized viruses are also examined, along with a proposed pathway for drug study and approval to meet FDA requirements. Human filovirus infections are characterized by high morbidity and mortality in patients and exhibit case fatality rates ranging from 25% to 90%. The majority of infections have been associated with outbreaks of Ebola Zaire (ZEBOV), and minor outbreaks with other species of Ebola or the closely related Marburg virus (MARV) have also been noted . While ZFilovirus infections are validated threats to the warfighter, and the development of medical countermeasures (MCMs) for prevention and treatment is a priority requirement . The chaWe review the development of the antiviral remdesivir (Veklury) here to illustrate the challenges of fielding broad-spectrum biowarfare countermeasures, particularly those associated with development under the US Food and Drug Administration’s (FDA) Animal Rule . RemdesiRemdesivir is active against a host of other viral threats, most notably Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), where it recently obtained FDA licensure after demonstrated reduction in time to recovery in hospitalized Coronavirus Disease 2019 (COVID-19) patients [The Joint Project Manager for Chemical, Biological, Radiological, and Nuclear Medical , a subordinate element of the Joint Program Executive Office for Chemical, Biological, Radiological, and Nuclear Defense (JPEO-CBRND), funded preclinical research of a broad-spectrum antiviral therapeutic, remdesivir (GS-5734), with Gilead Sciences (Gilead). JPM CBRN Medical agreed to provide funding to support remdesivir preclinical development to assess its antiviral activity, which included laboratory tests to define its activity spectrum against biodefense viral pathogens of interest, including EBOV and MARV filoviruses. Later studies also included animal testing, conducted under Biosafety Level 4 conditions to evaluate remdesivir safety and efficacy and demonstrate positive proof-of-concept against filovirus infections. A significant portion of the preclinical testing was carried out at the US Army Medical Research Institute of Infectious Diseases (USAMRIID) in collaboration with Gilead. USAMRIID research demonstrated remdesivir’ s activity against Ebola in primates and generated data that could support further investigation of remdesivir as an Ebola treatment for humans . The outRemdesivir is an antiviral drug invented and manufactured by Gilead Sciences, a US pharmaceutical company with expertise in antiviral drug development. Remdesivir is a nucleotide prodrug inhibitor of viral RNA-dependent RNA polymerase (RdRp), a viral enzyme that is essential for viral replication. The chemical name for remdesivir is 2-ethylbutyl N-{(S)-[2-C- modification that facilitates passive prodrug diffusion into target cells. Delivery of the monophosphate nucleoside moiety thereby bypasses the initial rate-limiting phosphorylation step in nucleotide metabolism. Remdesivir distributes into cells where it is metabolized to the nucleoside monophosphate intermediate catalyzed by carboxyesterase 1 and/or cathepsin A, depending upon the cell type, and a phosphoramidase. Nucleoside analogs require further phosphorylation by intracellular nucleotide and nucleoside diphosphate kinases to create the triphosphate active form of the drug. Remdesivir triphosphate acts as an analog of adenosine triphosphate (ATP) and competes against the endogenous natural ATP pool with high selectivity (3.65-fold) over the natural ATP substrate for incorporation into nascent RNA chains . The proNucleoside/nucleotide analogs (NAs) have different mechanisms of action on viral DNA-dependent and RNA-dependent RNA polymerases by inducing chain termination, directly inhibiting the viral polymerase, or by acting as mutagens in the viral genome . There aExploratory animal studies were initiated with remdesivir at the USAMRIID to determine an effective dose against EBOV infection and to establish the treatment window against EBOV using rhesus macaques. A recent natural history study of EVD in rhesus monkeys demonstrates how intramuscular (IM) exposure to 1,000 plaque-forming units (PFUs) of EBOV manifests consistently across infected animals and culminates with mortality events occurring 7 to 10 days after exposure . Therape6 PFU equivalents are detected on days 4 to 6 postexposure. The characteristics of EVD in aerosol exposure (AE) studies in NHPs are nearly identical to those in IM exposure studies with regard to survival, time to death, viremia peak, and infectious virus profile [Transmission of EBOV by small-particle aerosols has been documented in experimental settings using NHPs . Rhesus  profile . A rhesu profile . Signifi profile . This feThe findings in the aerosolized Ebola model raise important questions regarding the optimal route of administration based on desired sites of therapeutic effect in the body. Vigorous humoral and cellular responses in lung tissue in particular are likely to be a critical first line of defense. Intranasal IgA mAbs may have advantages over conventional IgG monoclonals administered intravenously. The same may hold true for vaccines. In the case of thereutics, strategies to target small molecules to the lung should be considered. In a similar vein, the study raises important questions regarding the comparability of parenteral routes of inoculation to aerosolization of agents in animal models. While Ebola behaved similarly between IM and inhalational routes in the NHP model, other biowarfare agents cannot be expected a priori to do the same.The safety and pharmacokinetics of remdesivir were studied in 131 subjects in 4 healthy volunteer studies . In geneRemdesivir has been administered to tens of thousands of patients as treatment for COVID-19, at hundreds of sites in regulated clinical trials or compassionate use administration. Diarrhea, hepatic and renal abnormalities, rash, and hypotension, the latter as a precursor to SAEs including septic shock and multiorgan failure, have been the most common reported outcomes. In uncontrolled compassionate use settings, 23% had SAEs, many likely related to the underlying disease, with 8% discontinuing therapy due to side effects . AdverseThe Pamoja Tulinde Maisha  study was a randomized controlled trial (RCT) in the DRC comparing 4 EBOV therapeutics to include the ZMapp mAb cocktail, the mAb mAb114 (Ebanga), the Regeneron mAb cocktail REGN-EB3 (Inmazeb), and remdesivir . The resThe 2 antibody therapeutics, REGN-EB3 and mAb114, were found to provide a statistically significant mortality benefit against acute EVD compared to ZMapp and remdesivir. The difference in the results for the early and late diagnosis cohorts supports the argument that treatment with any antiviral therapy for filovirus disease must start early to have the highest probability of success. While this remains to be proven clinically, poor outcomes in late presenting patients in PALM support the limitations of delayed antiviral monotherapy later in the disease course as immunologic dysfunction begins to predominate . CombinaRemdesivir inhibits viral replication by the virus’ RdRp. Remdesivir likely reduces viral load but not before or within enough time to prevent clinical decline in treated individuals in the PALM study. The study outcomes in the PALM trial raise several questions regarding direct-acting antiviral (DAA) treatment, including the need to understand the relationship between the required proportional drop in infectious virus to realize a sustained response with clinical improvement. Madelain and colleagues modeled viral kinetics in NHPs during therapy with DAAs and indicated that current DAAs do not achieve the viral load reduction required at day 5 (mimicking severe disease in humans) to provide 100% survival . MadelaiNew England Journal of Medicine documented a transmission chain from a survivor of Ebola infection treated with mAb114. The patient suffered a recrudescent infection 149 days after initial discharge, transmitted his infection to his spouse , and ultimately, this chain of transmission led to the infections of 91 cases in 6 health zones over a period of 4 months [The difference in the results for the early and late diagnosis cohorts supports the argument that early treatment with any antiviral therapy for filovirus disease must start early to have the highest probability of success. Combining an antibody-based therapy with an antiviral is likely to be additive for improving efficacy for both early and late stage disease. Only remdesivir has broad-spectrum activity against other filoviruses in animal models. Additionally, the ability to enter key immune sanctuary sites such as the brain and testes may have benefit against disease sequelae in survivors. A small placebo-controlled study of EBOV eradication from the semen among 38 survivors in Liberia and Guinea was recently published. Remdesivir was shown to significantly reduce persistent EBOV infection in the Partnership for Research on Ebola Virus in Liberia (PREVAIL) IV study indicating bioavailability in the testes . A recen4 months . TreatmeFurthermore, combination or sequential therapy may prevent post-EVD sequelae and development of poorly understood persistent infections by eradicating EBOV localized in immunologically privileged sites. A combination therapy study in patients has not been conducted to date, and the Ebola outbreak has since subsided, suggesting that an NHP bridging study might be useful. Interestingly, Cross and colleagues recently reported that combination therapy consisting of remdesivir and a human antibody targeting MARV achieved 80% protection against advanced disease in a MARV challenge model using rhesus macaques while remdesivir only or mAb only treatment yielded no survivors . While tOther considerations for use of remdesivir as a filovirus DAA include the following: (1) detection and treatment for the warfighter likely will be initiated earlier and result in greater efficacy; (2) EBOV mAb treatments still have an overall mortality rate of 30% at the affected population level (which may be decreased for warfighters promptly diagnosed and treated); (3) EBOV mAb monotherapy likely will not prevent all sequelae, including persistence; (4) remdesivir treatment may benefit EBOV survivors with persistent virus replication; and (5) combination or sequential therapy may be additive and improve overall outcomes including preventing persistent infection.Nature Cell Research has shown that both remdesivir and chloroquine effectively inhibit the SARS-CoV-2 virus in vitro  [Specific studies against coronavirus infections in vitro demonstrated activity against coronaviruses isolated from bats and circulating human coronaviruses in primary lung cells. In vivo rodent studies of SARS-CoV-2 pathogenesis have shown prophylactic and early therapeutic administration of remdesivir significantly reduced lung viral load, improved clinical signs, and enhanced respiratory function . Subsequ in man) . Taking A number of clinical studies of remdesivir against COVID-19 have been conducted at various sites around the world using remdesivir alone or in combination with other drugs. One of the largest studies completed thus far is the Adaptive COVID-19 Treatment Trial (ACTT-1) study sponsored by the National Institutes for Allergy and Infectious Disease (NIAID). ACTT-1 compared remdesivir treatment to placebo in hospitalized COVID-19 patients with lower respiratory tract involvement. There were 1,063 patients randomized (541 remdesivir versus 522 placebo), with a median recovery time of 11 days for remdesivir versus 15 days with placebo. Kaplan–Meier estimates of mortality by 14 days were 7.1% with remdesivir and 11.9% with placebo . Gilead Antiviral drugs require early treatment initiation for maximum efficacy . DAA treCombined therapy of remdesivir with Janus kinase (JAK) inhibitor baricitinib (ACTIV) recently reported a reduction in average hospital stay from 8 to 7 days for the combination, and 18 to 10 days for those on high-flow oxygen or receiving noninvasive ventilation . RemdesiThe results of the ACTIV-3 trial investigating treatment of COVID patients with mAbs have found that SARS-CoV-2 mAb products in development may be harmful in more advanced disease and use is not recommended in hospitalized patients by the NIH or Infectious Disease Society of America –63. BamlAntibody-dependent enhancement (ADE) of filovirus infection has been demonstrated in mAbs derived from human survivors of Ebola infection . All of In contrast to these published reports, Bournazos and colleagues suggested that the observed ADE published by others is an in vitro phenomenon not replicated in vivo . The rouThe PALM trial underscored the need for very early recognition and initiation of specific therapy as mortality increased with time following onset of therapy . In addiEBOV- and MARV-induced diseases, in both humans and NHPs, are similar in presentation, pathology, and mortality  adapted IM inoculation produces consistent infection in NHPs.IM injection allows for delivery of a consistent quantity of virus among animals and between studies.IM inoculation produces key disease manifestations that closely resemble those occurring in fatal human cases.The FDA has indicated a preference for IM studies in the critical pathway for licensure of filovirus MCMs because of the above considerations.To date, no Good Laboratory Practice (GLP) studies utilizing an inhalational aerosol challenge have been conducted in a Biosafety Level 4 setting.Consequently, the IM model may be an acceptable route of exposure for determination of filovirus MCM efficacy supporting the Animal Rule licensure pathway by the FDA. It is important to note that this may not be the case with other biothreat agents and aerosol challenge represent a more physiologically relevant challenge route for biodefense-related product development.Although not required for expensive, complex, and regulated pivotal studies under the Animal Rule, we argue here that products being developed for a biodefense indication should include one or more aerosol challenge studies as part of the development package. An aerosol study will help elucidate lung or potentially other respiratory-related differences in pathogenesis. Studies should be conducted preferably in NHPs likely to have the greatest similarly to human response based on prior studies . The antAdvantages of the Animal Rule include optimal modeling of target exposure including dose, route (aerosol), and timing of interventions over the course of one or more regulated GLP studies. Naïve, lab-reared animals lacking comorbidities also help to reduce confounding variables that complicate study interpretation. Study of AE is generally not possible during natural outbreaks. Less virulent filovirus outbreaks tend to be short-lived and relatively easily to contain when local healthcare teams are trained and equipped to respond. A prepositioned or rapidly deployable clinical team may be needed in the outbreak area to be able to conduct regulated trials serendipitously in most cases. Disadvantages of Animal Rule studies include the current shortage and increased cost of NHPs for research use due to export limitations in China and competing primate studies for COVID-19, relevance to naturally occurring disease where patients may often present later in the course of illness, rendering countermeasures requiring immediate application ineffective. Treatment windows may vary between patients and NHPs, and the large number of animals required in some cases may increase costs to the level of clinical trials. Viral kinetic modeling may address some of these limitations. Animal Rule studies also represent an opportunity to study disease processes in much greater depth with the use of routine necropsy in contrast to rare autopsies performed in VHF patients due to infection control considerations. They may help to inform the appropriate timing and/or trigger for intervention with the complementary combination therapies such as immunomodulators.The advantages of developing a product with an existing commercial indication include a less expensive developmental pathway, a large safety database, existing GMP drug manufacturing, and supply chain for the existing commercial indication, which results in drug availability. Products developed solely for a biowarfare indication are developed for a small population and are thus likely to be expensive, may be in limited supply, and may require the USG to assume lead responsibility for bench-to-bedside product development in some cases. The massive increase in human safety and efficacy data and clinical experience with remdesivir coupled with greater manufacturing capacity will positively impact further development of remdesivir for filovirus infection. This will ensure broad availability of remdesivir for supplemental indications such as treatment MARV infection and may expedite the pathway to licensure.Development of MCMs for biowarfare and rare disease indications are more expensive, time consuming, and riskier than starting development with a product already approved for a commercial indication. Developing an MCM without a commercial indication is a more difficult path than repurposing a drug that already has or will soon have an approved indication. With static or declining development budgets for biowarfare or rare disease indications in DOD, repurposing drugs with a commercial indication is an attractive method of delivering safe, approved MCMs for the warfighter. To fortify the emerging arsenal of therapeutic agents that might be employed in a deliberate bioterrorism attack, a broader range of viral threats could be evaluated and developed in NHPs under the Animal Rule. High-priority viral pathogens currently in need of safe and efficacious therapeutics include Marburg, Sudan-strain Ebola, Crimean-Congo, Lassa, Hanta, and other hemorrhagic fever viruses.Animal models should be standardized where possible for comparability, allowing meaningful direct comparison of experimental outcomes. Investigators should drive for improved translational relevance by refining and benchmarking future studies with respect to previously published work. While limited studies—carefully planned and prepositioned—may be possible in outbreak settings, use of the animal rule is far more likely to be required given the epidemiology and relative rarity of sustained outbreaks of these diseases. These studies should also be planned with a view to establishing comparability and expanding on known clinical data to date.Given the outcome of the PALM study, the clinical benefit of remdesivir relative to other therapeutics  was not sufficient to warrant continued development for treatment of EVD as monotherapy. The use of remdesivir in combination with mAb-based treatments has not yet been assessed in a clinical study, and combination therapy may well offer a clinical benefit particularly for patients who are diagnosed late in the course of their disease. Given poor efficacy in the late stage treatment cohort with mAB114 and REGN-EB3, there is clearly room for improvement to provide the best possible benefit for these patients.Due to their high specificity, these recently developed monoclonal ZEBOV therapeutics are capable of being used to treat other Ebola species to include Sudan, Tai Forrest, and Bundibugyo. Of the remaining filoviruses without a clinical treatment, MARV is a logical choice for continued development given the severity of disease encountered in nature. MARV infections in nature have been shown to be highly lethal with case fatality rates up to 90%, and there are no MCMs available for treatment . MonotheCombination therapy between these agents (and remdesivir) had already been demonstrated in NHPs. A recent study in primates assessed the efficacy of a combination of remdesivir and a candidate Marburg mAb, and the combination of the two was more efficacious in treating late stage disease than either drug alone . The benThe FDA created the Animal Rule as a pathway to develop medical countermeasures for indications that cannot ethically or reasonably be studied in humans. The intent of the Animal Rule is to provide a means to develop drugs and biological products to reduce or prevent life-threatening conditions caused by exposure to lethal diseases. Product efficacy under the Animal Rule is established based on adequate and well-controlled studies in animal models of the human disease, and safety is evaluated under the preexisting requirements for drugs and biological products . The FDARequired studies under the Animal Rule include the following: (1) a Natural History study demonstrating pathology closely mimicking human disease; (2) PK studies to estimate the human-equivalent dose (HED); (3) a dose ranging study estimating the efficacious HED in animals; (4) at least 2 pivotal GLP animal studies at the estimated HED; and (5) often one or more postmarketing studies. To date, only 2 antiviral agents have been developed and approved under the Animal Rule—TPOXX and TEMBEXA—both for the treatment of smallpox. Both of these products used multiple animal species in well-developed models of poxvirus disease to demonstrate efficacy ,82.A number of outstanding questions remain moving forward in the development of anti-filoviral countermeasures. The recent development efforts related to remdesivir are illustrative. Timing is key, and it appears that earlier remdesivir administration leads to better outcomes. Patients enrolled in the PALM trial were treated an average of 6 days after the onset of symptoms, compared with 4 days after known experimental infection in the NHP models .Finally, combination therapies should be considered in patients with moderate–severe Ebola and COVID-19. It is clear that remdesivir as monotherapy provided modest improvements. However, in patients with either disease in the advanced state, viral replication rapidly transitions to immune dysregulation. In that regard, late antiviral therapy in moderate–severe patients is likely to have less impact. Combination therapies with other therapeutic classes should also be considered to include both monoclonal therapies primarily targeting viral replication and immunomodulating agents. In the latter case, the recent ACTT-2 trial comparing remdesivir with and without baricitinib, a JAK1/2 immunomodulator, have shown modest additional reductions in mortality in hospitalized patients with severe COVID-19 not yet on mechanical ventilation . HoweverGiven a significant mortality reduction with mAbs in the PALM trial against Ebola, a combination trial with remdesivir should be considered. The advantages may include further mortality reduction, but perhaps more importantly, reduction in sequelae often observed in survivors. Viral persistence in immunologically privileged sites is thought to account for these phenomena. These can include persistent neurologic symptoms of headache, cognitive impairment, and fatigue, as well as persistence in semen with possible ongoing sexual transmission. The PREVAIL IV trial (NCT02818582) conducted in Guinea and Liberia was the first placebo-controlled study to evaluate clearance from immune-privileged sites with an antiviral. This relatively small study demonstrated reductions in the carrier state among male Ebola survivors . Future The antiviral remdesivir is illustrative of the challenges and potential to develop broad-spectrum biowarfare countermeasures.Remdesivir was originally developed as an antiviral countermeasure against filovirus infection (Ebola). Remdesivir compared to monoclonal antibodies met the DOD’s requirement for efficacy but did not outperform 2 monoclonal antibody therapeutics in a recent head-to-head trial during a ZEBOV outbreak in the DRC.The US military has supported animal studies demonstrating the efficacy of remdesivir to treat MARV (a distinct filovirus species) infection and aerosolized EBOV infection.Remdesivir has since been found to have potential application against a host of other viral threats, most notably COVID-19, where it was recently licensed after demonstrated reductions in mortality and duration of hospitalization.While both the Ebola epidemic and COVID-19 pandemic allowed clinical testing of remdesivir, this is expected to be the exception, rather than the rule, for obtaining new product regulatory approval. Biodefense countermeasures will need to continue to rely on the FDA’s Animal Rule for future development. The Animal Rule is often the only path forward for FDA approval of medical countermeasures for biothreat agents where clinical trials cannot be conducted. Supportive clinical data should be sought where possible, considering ex vivo methods. Examples include in vitro antimicrobial inhibitory activity of human-dosed plasma or passive transfer of vaccine antibodies.https://www.accessdata.fda.gov/drugsatfda_docs/label/2020/214787Orig1s000lbl.pdfU.S. Food and Drug Administration. FDA label for remdesivir. 2020. Available from: 10.1056/NEJMoa1910993. Epub 2019 Nov 27. PMID: 31774950.Mulangu S, Dodd L, Davey R Jr, Tshiani Mbaya O, Proschan M., Mukadi D, and the PALM Consortium Study Team. A Randomized, Controlled Trial of Ebola Virus Disease Therapeutics. N Engl J Med. 2019 Dec 12;381(24):2293–2303. doi: 10.1016/S1473-3099(20)30282-6. Epub 2020 Jun 18. PMID: 32563280; PMCID: PMC7302789.Iversen P, Kane C, Zeng X, Panchal R, Warren T, Radoshitzky S, et al. Recent successes in therapeutics for Ebola virus disease: no time for complacency. Lancet Infect Dis. 2020 Sep;20(9):e231–e237. doi: 10.1038/s41467-018-06215-z PubMed PMID: 30275474; PubMed Central PMCID: PMC6167368.Madelain V, Baize S, Jacquot F, Reynard S, Fizet A, Barron S, et al. Ebola viral dynamics in nonhuman primates provides insights into virus immuno-pathogenesis and antiviral strategies. Nat Commun. 2018 Oct 1;9(1):4013. doi: 10.1038/nature17180. Epub 2016 Mar 2. Erratum in: ACS Chem Biol. 2016 May 20;11(5):1463. PMID: 26934220; PMCID: PMC5551389.Warren T, Jordan R, Lo M, Ray A, Mackman R, Soloveva V, et al. Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys. Nature. 2016 Mar 17;531(7594):381–5. doi:


Inertial sensors are increasingly used in rodent research, in particular for estimating head orientation relative to gravity, or head tilt. Despite this growing interest, the accuracy of tilt estimates computed from rodent head inertial data has never been assessed. Using readily available inertial measurement units mounted onto the head of freely moving rats, we benchmarked a set of tilt estimation methods against concurrent 3D optical motion capture. We show that, while low-pass filtered head acceleration signals only provided reliable tilt estimates in static conditions, sensor calibration combined with an appropriate choice of orientation filter and parameters could yield average tilt estimation errors below  Microelectromechanical inertial sensors are the core components of a multitude of wearable devices used to measure the orientation and kinematics of body parts. These devices have found key applications in healthcare  ,2,3 as wMeasuring head kinematics in rodents is particularly relevant as these animals heavily rely on head movements for exploring their environment. Head mobility contributes to efficient acquisition of olfactory and tactile (whisker-based) information in rats and mice ,21,22, bHead orientation relative to gravity  is among the key parameters which can be computed using 6-axis IMUs . As in humans, abnormal head tilt in rodents is most often associated with a central or peripheral vestibular dysfunction. Experimentally-induced lesions of the vestibular system in rats and mice are a classical approach for studying the associated neuronal compensatory mechanisms  and provIn the context of experimentally-induced vestibular lesions in rodents, head tilt has traditionally been rated on a discrete scale by an observer, offering a qualitative estimation rather than a quantitative measurement ,33,34,35Here, we address the question of the appropriate methodology to obtain reliable head tilt estimates in freely moving rats using readily available IMUs. We first discuss the problem of dealing with sensor offsets and then assess the level of precision of different IMU-based head tilt estimation methods using concurrent 3D video motion capture. Our results show that popular IMU filters offer satisfactory results when implemented with the right set of parameters in conjunction with sensor calibration, with average tilt estimation errors below Animals were adult Long Evans rats . Wired IMUs consisted of a small printed circuit board . Injectp); offsets  controlled by an Arduino Uno. To ensure an homogeneous sampling of tilt orientations, rotation angles were programmed to align gravity with the points of a spherical Fibonacci lattice in the IMU reference frame . Each oroptimize  package A rat implanted with a rigid 3D-printed support containing a wired IMU and a set of four infrared reflective markers , enabling just-in-time code compilation. The output of these filters are a unit quaternion for each sample representing the rotation aligning the z-axis of the sensor reference frame with the gravitational acceleration vector while preserving sensor azimuth. Time series of the gravitational acceleration vector’s coordinates were then obtained by rotating Head attitude  was computed from IMU data using an extended Kalman filter (EKF) and two classical complementary filters (Madgwick and Mahony) ,44. Thesn signal , we compBefore calculating IMU-based tilt estimates and comparing them with motion capture data, an initial step was to correct for the small misalignment between IMU axes and the head-bound reference frame calculated using reflective markers see . This waThe time-series of unit-vectors representing synchronized IMU .To compute head tilt maps, a spherical Fibonacci lattice of 5000 points was generated and used as vertices for Delaunay triangulation. The number of IMU samples during which the estimated gravitational acceleration vector fell within each triangle was computed to obtain a spherical density map. These spherical head tilt maps were converted to 2D maps using the Lambert azimuthal equal-area projection of the cartopy toolbox  was lower than 12° sribution . After az-axis of the sensor reference frame with the gravitational acceleration vector  is classically calculated from 6-axis IMU data using Kalman filters or a family of less computationally demanding algorithms designated as complementary filters. Because their performance likely depends on movement statistics, we wished to compare these different types of filters in our actual recording conditions, i.e., using head inertial data collected from freely moving rats. To assess the level of precision of an extended Kalman filter (EKF) and of two complementary filters (Madgwick and Mahony) ,44, we usholding . Head tisholding . The parsholding .−1 and the use of an EKF is advisable for accurate tilt measurements at higher angular speed values.In the case of the Madgwick filter, correcting for sensor offsets reduced the optimal average head tilt estimation error by 27% and 45% during movement and immobility, respectively d. Our moHz  9]. Dur. DurHz  or the vestibular nerve ,31,34. THead tilt estimation in rodents still largely relies on observational methods ,30,31,32z vs. x and y were indeed more consistent when considering them in a reference frame aligned with gravity , which peaked 24 te group a. Notablte group , probable lesion  but was e lesion . A secone lesion b,d. The on . Our measurements are however consistent with the typical time course of this global rating in kainate- and arsanilate-induced UVL: an effect peaking in the first few hours after injection followed by an almost complete recovery within days for kainate ,29,30 an−1 (−1), using a Madgwick filter seems like a most reasonable solution, while the EKF is probably a safer choice for higher angular speed applications.Using optical motion capture as a ground truth measurement of 3D orientation, we benchmarked IMU-based methods for computing head tilt estimates in freely moving rats. Our results show that all methods achieved the same level of precision  and some data pre-processing dedicated to isolating tracking errors. Optical motion capture nevertheless represents a powerful tool for obtaining ground truth estimates of three-dimensional body movements, an approach adopted in the present work but also successfully used for automatically annotating anatomical landmarks on videos of rodents ,39. It nDespite a lower accuracy compared to optical motion capture systems, IMUs have a number of advantages which make them more tractable and versatile, besides of course offering a considerably cheaper solution. They require a minimal calibration procedure compared to a multiple-camera setup, generate small data files compared to markerless 3D video and do not bear the inherent difficulties of video recordings, such as occlusions, parasite reflections and a necessary compromise between resolution and the size of the field of view. Finally, the need for specifically preparing the animal for inertial measurements will often not be an issue as a growing number of headborne devices such as electrophysiology headstages already incorporate an IMU. For all these reasons, an IMU-based head tilt estimation solution is significantly easier to deploy than most optical approaches, with a minimal compromise in terms of precision. A direct comparison would however be interesting to carry out between IMUs and affordable lighthouse-based 3D localization systems used for virtual reality applications .Head tilt and circling are among the main behavioral manifestations of an asymmetrical vestibular impairment in rodents together with nystagmus. Head tilt is of primary interest in the context of preclinical research as it mirrors an equivalent symptom in patients with unilateral inner ear injuries. Contrary to eye movements, the assessment of head tilt in rodents has traditionally suffered from a lack of objective and quantitative measurements. In the case of UVL studies, a battery of static and dynamic features is typically rated on a discrete scale by an observer ,30,31,32Here, we show that IMUs offer an attractive solution for a quantitative full scale analysis of head kinematics, including both head tilt and circling but also complementary features such as the degree of head mobility and the animal’s overall level of activity . As illuIMU-based head tilt estimation also offers a route toward a more ethological assessment of vestibulo-postural functions in rodents, such as the ones ensuring head stabilization in space . These fBeyond vestibular research, headborne IMUs offer new opportunities for quantitative approaches in rodent behavioral studies. A wide range of different behavioral activities indeed have a distinct signature in IMU data, such as locomotion, rearing, grooming or sniffing ,9,21,24.


Cardiac function highly relies on sufficient energy supply. Perturbations in myocardial energy metabolism play a causative role in cardiac pathogenesis. Accumulating evidence has suggested that modifications of cardiac metabolism are also an essential part of the adaptive responses to various physiological conditions in the heart to meet specific energy needs. The review highlighted some new studies on basic myocardial energy substrate metabolism and updated recent findings regarding cardiac metabolic remodeling and their associated mechanisms under physiological conditions, including exercise and cardiac development. Studying basic metabolic profiles in the heart in these conditions can contribute to understanding the significance of metabolic regulation in the heart during physiological adaption and gaining further insights into the maladaptive metabolic changes associated with cardiac pathogenesis, thus opening up new avenues to exploring novel therapeutic strategies in cardiac diseases. The heart demands high energy production and turnover to maintain its proper functioning. Cardiomyocytes navigate their metabolic activities to meet specific energy demands with each contraction cycle. Multifaceted interactions and coordinations between metabolic pathways and networks underlie the integrated metabolic homeostasis of the heart regarding ATP production, biosynthesis, signaling regulation, and control of redox state. Perturbations in myocardial energy metabolism have been considered causative for cardiac pathogenesis, eventually progressing to ventricular dysfunction, arrhythmias, and heart failure, contributing to poor prognosis and high cardiovascular mortality rates. Therefore, it is of great significance to elucidate the critical role of metabolic regulation in the heart.Metabolic remodeling, in general concept, refers to the reconstruction of the metabolic landscape or metabolomics profiling that results in a shift toward a greater reliance on alternative fuel metabolism , which iIn the review, we summarized continuing research progress on myocardial energy substrate utilization and also discussed a portion of recent findings regarding cardiac metabolic remodeling and the associated mechanisms under different physiological conditions, including exercise and cardiac development. Reviewing and discussing the current progress on cardiac metabolism in these conditions can contribute to better understanding the significance of metabolic regulations in the heart during physiological adaption and receiving fresh perspectives on the maladaptive metabolic changes underlying cardiac pathogenesis.. Acetate, which accounted for the highest percentage of short-chain FAs produced by gut bacteria, was avidly extracted by the heart, accounting for 2% of myocardial carbon uptake. Meanwhile, the heart secreted most of the essential amino acids, particularly those with greater nitrogen content. Among them, glutamine and alanine were the two most abundant amino acids secreted, with the release of the former nearly equal to glutamate uptake. In addition, histidine was identified to be the most highly secreted essential amino acid, and considering that myoglobin, a heme protein abundantly present in cardiac tissues, contained a greater histidine content than other cardiac proteins, they could be serving as a potential carbon reservoir , for the posttranslational O-linked glycosylation (O-GlcNAcylation), via increasing activating transcription factor 4 and its direct transcriptional target, fructose-6-phosphate aminotransferase 1. While Nox4 TG and WT mice exhibited similar cardiac energetics and function at baseline, the former showed a significantly preserved energetic state when challenged with acute isoproterenol compared with WT control. Together, these results suggested that Nox4-redirected glucose utilization and subsequently increased fatty acid oxidation might be a beneficial response of the myocardium adapting to stress.Unlike most tissues, the heart mainly utilizes long-chain fatty acids, accounting for 60–70% of its fuel metabolism. Distinctively, glucose oxidation is less favored in the heart because the heart has very little glycogen available. It gets depleted very quickly under hypoxic conditions, plus little gluconeogenesis occurs in the heart. Metabolic remodeling involving glucose and lipid metabolism has been intensely characterized in the heart with preexisting dysfunctions, such as heart failure, myocardial infarction, and diabetic cardiomyopathy. In mature animal models of heart failure that is most commonly elicited by either pressure-overload or ischemia, the energy-deficient heart switches from fatty acid oxidation to more oxygen-efficient glucose oxidation accompanied by markedly upregulated cardiac glucose uptake . Such meex vivo model of perfused, isolated hearts. In recent years, the rapid development and application of isotopic labeling and metabolomics have greatly encouraged and facilitated the assessment of fuel availability and fate in the heart in vivo. For example, in vivo [U-13C] glucose labeling combined with isotopomer analysis with nuclear magnetic resonance spectroscopy (NMR spectroscopy). Male adult mice received 30-min continuous intravenous infusion of [U-13C] glucose before their hearts were excised for the ex vivo analysis. In pressure overloaded TAC hearts that exhibited significant concentric hypertrophy and reduced EF, 13C was significantly enriched in lactate, succinate, glutamate, glutamine, and aspartate in the heart compared with the sham group but were overall less pronounced in the plasma. There were no significant changes of 13C enrichment between volume overloaded shunt-treated hearts that showed prominent eccentric hypertrophy with unchanged LVEF and the sham group. However, increased plasma 13C enrichment of succinate was detected in the shunt group while the abundance of aspartate was too low in the plasma to trace its 13C enrichment. These findings indicated that their experimental approach primarily captured the cardiac-driven metabolic flux of glucose instead of systemic effects. The enrichment of  lactate metabolized from  pyruvate following glycolysis was significantly increased in TAC hearts together with markedly upregulated expressions of glucose transporter 1 (GLUT1), hexokinase-1, and lactate dehydrogenase A (LDHA) compared with the sham group. Both the enrichment of  glutamate as a readout for the activity of pyruvate dehydrogenase (PDH) and  glutamate indicative of anaplerotic reactions in the TCA cycle was significantly increased in TAC hearts compared with sham together with increased gene expression of PDH E1 component subunit α (PDHαE1), which suggested augmented pyruvate flux directly into the TCA cycle and greater anaplerosis. Such changes were not observed in the shunt group. However, the gene expression of pyruvate dehydrogenase kinase 4 (PDK4) was markedly decreased compared with the sham group. In addition, other than in shunt, TAC hearts exhibited a significant increase in the enrichment of  glutamine and the total levels of glutamine measured by NMR as well as the protein levels of glutamine synthetase and glutaminase compared with sham. The in vivo13C-labeling strategy employed in this study held unique advantages of directly tracing the metabolic pathway activities over conventional studies in which pathway analysis relied heavily on detecting changes in gene, mRNA, and protein expression levels that indirectly reflected pathway activities. The results gained also corroborated with the previously observed general pattern of increased glucose metabolism in pressure overload-induced heart failure.Previously, most studies investigating cardiac fatty acid and glucose metabolism were performed in an 13C6-glucose administered through a standardized 13C6-containing liquid diet. By overexpressing cardiac phosphatase-deficient or kinase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2/PFKFB), the GlycoLo and GlycoHi hearts were excised and subjected to NMR and ion exchange chromatography-mass spectrometry (IC-MS) to qualify metabolite abundance and 13C enrichment. In this study, GlycoLo mice hearts with low PFK-1 activity exhibited increased glycogen synthetic metabolites such as glucose-6-phosphate (G6P) and fructose-6-phosphate, and glucose-1-phosphate together with increased 13C-labeled glycogen (13C-labeled 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) and phosphoribosyl pyrophosphate were markedly increased in the GlycoLo hearts. Further investigation revealed that the metabolic routing of glucose-derived 13C carbons to AICAR induced by low PFK-1 activity was associated with chaperones-containing large multimeric complexes and metabolic enzymes, such as phosphoribosylaminoimidazole carboxylase. This study focused on the relatively slower biosynthetic pathways of glucose metabolism compared with the majority of in vivo13C labeling approaches previously reported  activities that catalyze ketone bodies back to acetyl coenzyme A (acetyl-CoA)  production and cardiomyocyte apoptosis while enhancing the mRNA expressions of antioxidant enzymes such as metallothionein 2, catalase, and superoxide dismutases in H9c2 cells that were exposed to 5-mmol/L 3-OHB. Besides, the magnitude of 3-OHB-induced increase in histone hyperacetylation was greater in Bdh1 TG mice than WT control treated with the same infusion. The novel Bdh1 TG model provided further evidence of intervening myocardial ketone body metabolism as a potential therapeutic strategy for treating heart failure.A recent study demonstrated that ketone bodies could serve as a primary fuel source for cardiac energy production instead of a “thrifty-fuel” as high 3-OHB concentrations in aerobically perfused hearts extracted from 12-week-old male mice could markedly boost ketone oxidation and efficiently produce ATP without suppressing physiological glucose and palmitate metabolism rates . This fityl-CoA) . Permanetyl-CoA) . Therefotyl-CoA) . UchihasThe myocardium has been considered a net lactate consumer that uptakes lactate directly proportional to its circulating concentrations over the past few decades . During 2O2-induced oxidative stress. H2O2-treated cardiomyocytes exhibited enhanced glutaminolysis and a subsequent increase in ATP production through glutamine anaplerotic reactions to compensate for compromised substrate oxidation under immense oxidative stress, thus conferring cardioprotective effects in the stressed heart.Amino acids play an obligatory role in maintaining the metabolic milieu of the heart, thus gaining more and more attention for exerting a marked influence on cardiac pathophysiology. Amino acids, such as glutamine and glutamate, can replenish a small amount of ATP in the stressed heart via non-oxidative metabolism, thus free from contributing to acidification and free radical formation . GlutamiIn recent years, there has also been some rapid progress on branched-chain amino acids (BCAAs) metabolism involved in cardiac metabolic remodeling, and it is receiving more attention nowadays than ever before. BCAAs, referring to leucine, isoleucine, and valine, serves as a major nitrogen donor for glutamine synthesis in muscles where BCAA catabolism primarily occurs due to robust BCAA transaminase (BCAT) activity that converts BCAAs and α-KG to α-ketoacids (BCKA) and glutamate. Defective BCAA catabolism and subsequent tissue accumulation of toxic byproducts such as medium- and long-chain acylcarnitines (AC), valine catabolite, 3-hydroxyisobutyrate (3HIB) as well as BCAA themselves have been extensively reported to impair fasting glucose and stimulate mammalian target of rapamycin complex 1(mTORC1) overactivation-induced insulin resistance (IR) .ad libitum manner exhibited significant cardiac hypertrophic responses compared with littermate control, whereas oscillatory fluctuations in mTORC1 activities observed in control littermates were completely abolished in CBK mice, indicating that clock-defective mice chronically developed increased sensitivity to a BCAA diet. Given that BCAA-induced hypertrophic growth in the healthy mouse hearts did not establish an accumulative effect, mice were subjected to TAC surgery to induce cardiac pressure overload and were fed with either the LHB diet or EHB diet. LHB mice exhibited significant hypertrophy and fibrosis and declined function compared with EHB mice, implying that repetitive BCAA consumption at the end of the active phase could build up a damaging effect on the pressure-overloaded heart. Overall, this study was the first to probe how circadian rhythms converged with BCAA metabolism to modulate cardiac remodeling, which recapitulated the importance of the often-neglected temporal regulation of cardiac metabolites in both the normal and stressed heart.Dietary BCAAs have been repeatedly observed to have a damaging effect on the stressed heart . HoweverAs a prevalent yet understudied BCAA intermediate, the metabolic fate of BCKA has been surrounded with inquisitive eyes and was recently traced to be primarily reanimated back to valine and subsequent catabolite, 3-hydroxyisobutyrate in a BCAT-dependent manner in the heart of recruited male rats . FurtherDespite these exciting discoveries, it is notable that BCAAs accounted for less than 5% of total carbon combustion in the heart; thus, defective BCAA catabolism observed in heart failure is likely to be mediated by alternative mechanisms beyond the control of energy production.In addition to the above experiment studies using animal models primarily focused on characterizing each substrate utilization and the underlying molecular mechanisms in the heart individually, offering only a partial insight into the whole picture of dynamic adaptation of nutrient utilization in the heart, many recent studies focus on the comprehensive regulation among different metabolic substrates and the dynamic adaptation of nutrient utilization in the heart.Hi mice) that expressed sustained glycolysis through the increased activity of phosphofructokinase-2 (PFK-2). Both transgenic mice and their WT littermates were fed with either a standard low-fat chow diet (LFD) or challenged with a 7-day high-fat diet (HFD). As a result, both the genotype and the diet manipulation significantly affected global cardiac metabolism due to increased depletion of early glycolytic intermediates by upregulated glycolysis. Subsequent global proteomic analysis revealed an increased abundance of proteins involved in both glucose and lipid metabolism in HFD-treated GlycoHi hearts, whereas HFD-treated WT mice had consistently greater abundance in lipid metabolism proteins but declined abundance in proteins involved in glucose metabolism, suggesting that GlycoHi mouse hearts were able to sustain elevated expression of glycolytic proteins against short-term HFD challenge. There was also a striking increase of BCAAs in HFD-treated GlycoHi mice, accompanied by a diet-independent decrease in BCKD E1α subunit. Such an increase in BCAAs was considered a unique effect as other amino acids had a decreased abundance in GlycoHi hearts compared to WT. This study depicted both genotype and diet-dependent impact of sustained glycolysis on global cardiac metabolism, in which PFK-2 induced increase in the content of BCAAs and PDK4 was identified as novel compensatory mechanisms of the heart to manage nutrient stress. This study also provides clues to potential outcomes of metabolic remodeling that could occur if glucose metabolism takes up a larger proportion of cardiac energy metabolism in a healthy heart.–/– male mice exhibited decreased BCAA oxidation, increased individual BCAAs, and decreased BCKAs in the heart, with circulating BCAA levels remaining unchanged compared with their control counterparts. These results indicated that increased accumulation of BCAAs instead of BCKAs should be held responsible for triggering the mTOR-mediated hypertrophic responses in the heart, and BCKAs could exert an inhibitory effect on insulin-promoted cardiac glucose oxidation potentially through downregulating AKT and PDH activities.O-GlcNAcylation. Although there were no evident pathological changes at baseline, KO mice subjected to myocardial IR insults exhibited worsened I/R injury compared with WT littermates when administered with a high level of BCAA supplements. Such negative changes could be rescued by either 3,6-dichlorobenzo[b]thiophene-2-carboxylic acid (BT2) treatment or GLUT1 overexpression in double transgenic PP2CmKO/GLUT1TG mice. Li’s study revealed a novel regulatory role of BCAA in cardiac glucose metabolism and an increased vulnerability of the PP2CmKO heart to IR injury following defective BCAA catabolism.Additionally, Reciprocally, cardiac glucose metabolism was found to negatively regulate BCAA breakdown through the actions of the above-described transcriptional regulator, Kruppel like factor 15 (KLF15) . RNA micAltogether, these studies have advanced our understanding of how fatty acid and glucose metabolism regulates BCAA and contributes to the normal physiology of the heart as well as how alterations in BCAA catabolism impact the well-characterized derangements in glucose and lipid metabolism intensifies maladaptive metabolic remodeling in the stressed heart. This emerging new paradigm implicating defective metabolic regulation in the pathogenesis of major adverse cardiac events could provide essential knowledge for developing efficacious therapeutic strategies such as pharmacologically inhibiting BCKD activity with BT2 treatment to restore BCAA catabolic flux in pressure-overload induced heart failure and myocardial IR injury .However, the fundamental structure supporting cardiac metabolic plasticity and flexibility is, no doubt, the mitochondria. Whether it is lipids, polysaccharides, or proteins, they are all degraded into acetyl-CoA and then enter the TCA cycle coupled with oxidative phosphorylation apparatus inside the mitochondria to generate ATP. Therefore, the mitochondria serve as a converging hub for energy substrate crossovers and play a central role in metabolic homeostasis. Dysfunctional mitochondria are directly responsible for remodeled metabolic patterns and disruption of energy homeostasis in the heart, which underlie the pathological outcomes of many heart diseases. For example, deficient mitochondrial pyruvate carrier 1, which transports pyruvate from glycolysis into the mitochondria, could lead to ventricular hypertrophy, heart failure, and premature death in mice hearts upon a series of metabolic remodeling, predominantly glucose overreliance and diminished TCA activity . MeanwhiAlthough metabolic remodeling predominantly occurs in the cardiac pathological condition in response to mechanical stress, myocardial metabolism can also adapt to the energy demand under the different physiological conditions. While the cardiac metabolism remodeling in pathological conditions has been well-reviewed , much feExercises have been reported to induce cardiomyocyte hypertrophy, promote physiological cardiac growth, and improve cardiovascular health. The metabolic responses ensuing different types of exercises vary, shaping the heart muscle broadly into either eccentric or concentric hypertrophy with individual heterogeneity . EnduranSo far, how exercise-induced adaptations in cardiac metabolism promote cardiac remodeling has not yet been fully investigated. First of all, metabolic flexibility of the mitochondria as an efficient adaptation to energy resources plays a pivotal role in balancing periodic shifts between primary fuel utilization such as glucose and FA oxidation as well as employing alternative fuels upon dynamic energy demand and supply in the heart. How endurance exercises promote cardiac mitochondrial structural and functional quality has been extensively reported in cardiac disease models, particularly in diabetic and failing hearts . For exa–/–) mice compared to sedentary (eNOS–/–) mice. In contrast, trained WT mice exhibited significantly increased cardiac GLUT1 expression and upregulated insulin sensitivity that markedly promoted cardiac glucose uptake, all of which remained unchanged in trained eNOS–/– mice. These findings revealed novel eNOS-dependent mitochondrial biogenesis induced by endurance exercise, which substantially orchestrated metabolic adaptations of the heart to exercise through promoting basal and insulin-stimulated cardiac glucose uptake.In non-stressed hearts of 8-week-old male mice, endurance exercise was reported to induce mitochondrial biogenesis through the actions of nitric oxide, a key mediator of cardiovascular responses to exercises . CompareBesides, Lo) and phosphatase-deficient (GlycoHi) TG mice were developed to constitutively manipulate myocardial PFK-1 activity, which resulted in mild cardiac hypertrophy in both GlycoLo and GlycoHi mice accompanied by irreversible mitochondrial inflexibility. Only GlycoLo hearts exhibited seemingly beneficial cardiac remodeling patterns similar to but not exceeding that of exercise-adapted WT mice and without evident upstream AKT activation. Additionally, Hi hearts that expressed sustained glycolysis through increased PFK-2 activity. Both GlycoHi mice and their WT littermates at 30–36 weeks of age were fed with either a standard LFD or challenged with a 7-day HFD. As a result, both the genotype and the diet manipulation significantly affected global cardiac metabolism. Pathway analysis uncovered a marked increase of PDK4 in HFD-treated WT hearts, and such increase was even more pronounced in HFD-treated GlycoHi mouse hearts reflected through PDH activity. Besides, fasted GlycoHi mouse hearts exhibited significantly higher PDK4 levels than fasted WT hearts, and fed GlycoHi mouse hearts also had greater PDK4 levels than fed WT mouse hearts, suggesting that enhanced glycolysis could elicit an increase in PDK4 to dial down glucose oxidation. The above two studies together provided unique evidence for the pervasive effects of sustained glycolysis on cardiac metabolic remodeling at the mechanistic level in response to exercises and nutrient availability, respectively, which further contributed to an improved understanding of dynamic interactions between diet, exercises, and genotypes, the three major components necessary to determine individual cardiac health.Lo mice. Meanwhile, myocardial expression of Cited4, whose cardiac overexpression has been shown to induce physiological hypertrophy with normal systolic function that mimics the effects of endurance exercises , whose reduction has previously been reported to serve as a central signal controlling exercise-induced physiological hypertrophy , was obsxercises , was fouiSA) developed a concentric LV hypertrophy accompanied with increased systolic function at 28-week old compared to littermate controls, which resembled adaptive cardiac hypertrophy. Retrogradely perfused mTORC1iSA heart at 12-week old exhibited markedly decreased glucose uptake and oxidation while the downstream product of hexokinase 2, G6P, was significantly increased, suggesting a considerably decreased glucose influx to the glycolytic pathway. Further investigation discovered a persistent decrease in protein expression and enzymatic activity of glucose 6-phosphate isomerase (GPI) from as early as 12-week old all the way to 28-week old. WT mice subjected to 6-day wheel running showed increased mTOR phosphorylation at Ser 2448 accompanied by decreased GPI protein expression but increased enzymatic activities compared with sedentary WT mice, whereas oral administration of rapamycin for the last 8 weeks of the project prevented the increase in GPI activity at 12-week old and hypertrophic alterations seen at 28-week old in both mTORC iSA mice and trained WT mice. WT mice subjected to a 6-week wheel running protocol developed significantly increased cardiac hypertrophic changes together with increased GPI activity, which were again prevented with the rapamycin diet, suggesting a consistent rapamycin inhibition on mTORC1-induced glucose changes during both short-term and long-term voluntary exercises. The mTORC1 signaling has been considered as a metabolic rheostat that integrates inputs from nutrient sensing with cell growth -1 has been a primary focus during the fetal metabolic transition whose deficiency has been reported to markedly attenuate cardiomyogenesis of embryonic stem cells in mice . Highly In addition, A transcriptome analysis conducted by in vitro human embryonic stem cell-derived cardiomyocytes (hESC-CMs) with an immature metabolic phenotype have been extensively used to study human fetal cardiomyocyte metabolic maturation and potentially provide alternative cell-based approaches to cardiac regeneration and repair (in vivo study revealed drastically downregulated fetal cardiac glucose uptake albeit relatively stable uterine glucose supply during late gestation or the early postnatal period. Additionally, fetal and neonatal hearts from diabetic pregnancy of Akita heterozygous mice showed elevated mitotic activity and increased biventricular wall thickness in company with decreased cardiomyocyte size compared with control littermates, suggesting that hyperglycemia could impede fetal cardiac maturation and potentially contribute to cardiomyopathy in infants of diabetic mothers (So far, various animal species have been recruited to study fetal heart growth, such as the murine and ovine fetal hearts described above. However, considering distinct interspecies differences in embryo development and limited samples of human fetal heart, d repair . Nakano  mothers . This stin vitro hESC-CMs metabolic maturation well corroborated with in vivo discoveries regarding fetal cardiac metabolic changes during terminal maturation, leading to a more comprehensive understanding of fetal cardiac metabolic remodeling.13C isotope labeling revealed that glucose flux branching into the PPP pathway was significantly lowered at week 12. Besides, although there was no significant difference in overall protein O-GlcNAcylation, both UDP-GlcNAc abundance and glutamine-fructose-6-phosphate aminotransferase 2 expression were significantly reduced at week 12, suggesting reduced flux to the HBP pathway. These findings indicated that the metabolic flux that passed through the glycolytic pathway was preferentially directed toward the entry of the TCA cycle. hiPSC-CM at week 12 also exhibited a more mature mitochondrial network and a tendency to shift to a more oxidative metabolic profile over time as fatty acid oxidation increasingly contributed to total oxygen consumption rate at the cost of glucose oxidation.in vivo and in vitro. In addition, studying the progression of fetal cardiac remodeling conferred substantial value on deciphering the pathological metabolic alterations in the ischemic and failing hearts, thus encouraging the development of minimally invasive and regenerative therapeutics for a series of heart diseases. The main findings from these new studies have been summarized in Overall speaking, the fetal metabolic transition is achieved through coordinated control and regulation of substrate partitioning under the combinatorial effects of the metabolic and signaling network, especially the repeatedly highlighted HIF1α signaling both This review primarily focused on presenting the current knowledge of myocardial substrate metabolism and the latest progress regarding cardiac metabolic remodeling under physiological conditions such as during exercise and cardiac development. Especially, many novel signaling pathways have been linked to the metabolic remodeling in the heart under physiological conditions of endurance exercises and cardiac development, which remarkably increases our knowledge of the heart’s potential in response to the states with diverse energy requirements under the physiological condition. We also dabbled into the correlation of metabolic patterns between physiological and pathological cardiac metabolism, which all together strived to gain a deeper understanding of the impact of metabolic remodeling in the heart under physiological conditions. Despite the significant impacts of these advances in this field, many aspects remain unknown and require future investigations:It is no doubt that every substrate matters, and each substrate serves not only as a fuel source but also as an indispensable part of the metabolic signaling pathways that together induce structural and functional remodeling in the heart. The current studies bring new insights into the substrate utilization of the heart in physiological conditions. First, although glucose and fatty acid metabolism remain a research hotspot, cardiac lactate and ketone metabolism have gained growing attention, especially the ketone body, whose increase serves as a general feature of maladaptive metabolism in the aging heart and the failing heart. As a result, a large body of research has been devoted to investigating their pervasive cardioprotective effects by applying a ketogenic diet or administering exogenous ketogenic agents to induce therapeutic ketosis. Secondly, many emerging roles of BCAA metabolism in the heart have been brought to light, such as the relationship between cardiac BCAA oxidation and insulin resistance; transcriptional regulation of BACC catabolism by upstream regulators such as KFL15, BCAT2, BCKDH, and PP2Cm; third, most importantly, as previous studies primarily focused on characterizing each substrate utilization and the underlying molecular mechanisms in the heart individually, many recent studies focus on the interaction among different metabolic substrates and the dynamic adaptation of nutrient utilization in the heart, by using a combination of the various animal models, such as the newly discovered metabolic interplay and reciprocal regulation between glucose and BCAA metabolism. These studies highlighted the comprehensive regulations in cardiac metabolism.Despite the exciting findings from the recent studies, it remains largely unknown regarding the complex regulatory mechanisms governing cardiac energy substrate metabolism under physiological conditions, not to mention numerous unidentified metabolic remodeling processes under pathological stimuli. For example, although physiological and pathological myocardial hypertrophy has long been considered as two separate entities and sustained, mTORC1 activation was heavily incriminated in producing divergent hypertrophic outcomes in them, the recently reported intermediate non-pathological-hypertrophic phenotype in the presence of persistent mTORC1 activation indicative of a continuum of influence of this key regulator on cardiac growth. Additionally, glycolytic activities were observed to exhibit a periodic pattern in exercise-adapted hearts, and genetically downregulated cardiac glycolysis could maximize physiological myocardial hypertrophy. The more newly discovered metabolic pathways aside from the most characterized IGF-1 and mTOR signalings involved in exercise-promoted cardioprotection, such as the eNOS and the SNARK signaling, greatly contributed to identifying novel promising molecular targets. Furthermore, the differential role of BCAAs and BCKAs on mediating cardiac IR has been recently clarified, and recruiting BCAA oxidation as a promising treatment target to improve insulin sensitivity in heart diseases associated with IR has become a new focus. So far, only a few genes and transcriptional factors involved in BCAA metabolism were identified and analyzed as shown above, which says that very little is known about the underpinning pathways and molecular regulation of BCAA metabolism in response to various physiological and pathological stimuli as well as its potential interactions with other metabolic processes in the heart, which require further investigations in the future. It is beyond doubt that cardiac research on metabolism is heading toward the path of multidisciplinary analysis and quantification with a plethora of powerful and promising molecular methods that can more accurately and comprehensively characterize cardiac metabolism.ex vivo and in vitro models and experimentation exerted more uncertainty in interpreting the gained results to the in vivo system. On top of these traditional techniques, the application of novel strategies to enable deep metabolic network tracing or studies employing advanced analytical technologies and informatics to unravel the dynamic and complex metabolic nature of cardiomyocytes. Systemic delivery of metabolic substrates, such as C16:1n7, could exert a localized effect on exercise-induced cardiac growth, which served as a fresh reminder that metabolic remodeling under any circumstance is an outcome of both local and systemic metabolic responses. The advancement of metabolomics and isotope tracing as well as their individual and combined applications in studying heart metabolism ex vivo and in vivo has provided novel and alternative approaches for characterizing cardiac metabolism. So far, it remains challenging to investigate cardiac metabolic changes in response to exercise training due to its dynamic nature of systemic involvement, thus causing larger magnitudes of more complex discrepancies. Therefore, the application of metabolomics, isotope tracing, and other future technological innovations will substantially provide more accuracy and reduce the difficulties in delineating numerous exercise-promoted cardiac metabolic signatures. In addition, alternative experimental approaches such as in vitro hESC-CMs and hiPSC-CMs testing revealed more knowledge of the metabolic changes during fetal cardiomyocyte proliferation and maturation. Studying metabolic remodeling in the developing heart also brought promising therapeutic targets as fetal metabolic gene reprogramming has been repeatedly recognized in the adult heart under pathological conditions.The conventional Recent studies also provide evidence for developing new therapeutic strategies. For example, most recent studies have indicated that dietary BCAAs could be used as a potentially efficacious intervention for heart failure in some reports. However, in others, they have been shown to increase the susceptibility of the heart to pathological stimuli. The conflict on this topic remains unsolved as the specific experimental models and diet protocols employed in each study varied, and the hemostasis of BCAA metabolism in the heart is always the result of multifactorial regulation, such as the often understudied temporal regulations. There are a significant number of targets for potential therapeutic interventions waiting to be discovered within the metabolic realm, in which pharmacological targeting of energy metabolic signaling and pathways has just emerged. Therefore, our quest has just begun to uncover the entire blueprint of cardiac metabolism.XS performed the literature searching and drafted the manuscript. HQ revised the manuscript. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.