Glucose inhibits cardiac muscle maturation through nucleotide biosynthesis
Congenital heart disease ( CHD ) is the most common type of birth defect affecting 0 . 8% of human live births ( Fahed et al . , 2013 ) . Although genetic factors play a significant role in the development of CHD , current genomic technologies , including exome sequencing and SNP arrays , have provided a genetic diagnosis for only 11% of the probands ( Gelb et al . , 2013 ) , highlighting the crucial role of non-genetic contributors . Among the non-genetic factors that influence the fetal heart , maternal hyperglycemia is the most common medical condition , associated with a 2–5-fold increase in CHD independent of genetic contributors ( Centers for Disease Control , 1990; Simeone et al . , 2015; Yogev and Visser , 2009 ) . Diabetic pregnancy is often accompanied by maternal complications including vasculopathy , neuropathy , and insulin resistance , which potentially affect fetal cardiac formation indirectly . These systemic complications are often subclinical , hindering the dissection of the pathomechanism of CHD in diabetic pregnancy . Thus , despite the established association between maternal hyperglycemia and malformation of the fetal heart , little is known about how glucose levels impact cardiomyocyte development and how hyperglycemia affects heart formation in diabetic pregnancy ( Gaspar et al . , 2014 ) . The metabolic environment is one potential non-genetic determinant of cell proliferation and differentiation . Cells display distinct metabolic characteristics depending on their differentiation stage ( Carey et al . , 2015; Tohyama et al . , 2016; Wang et al . , 2009 ) , and the fuel type used by the cells serves not merely as a source of energy but also as a critical regulator of self-renewal and differentiation of stem or progenitor cells ( Harris et al . , 2013; Oburoglu et al . , 2014; Shiraki et al . , 2014; Shyh-Chang et al . , 2013 ) . However , little is known about the mechanism . Cardiomyocytes shift their energy substrate during late embryonic and neonatal stages ( Makinde et al . , 1998 ) . Glucose is the major energy source during the early developmental stages . Oxidative phosphorylation is low until E10 . 5 of developing rodent hearts and rapidly increases between E10 . 5 and E14 . 5 ( Cox and Gunberg , 1972 ) . This coincides with the rapid maturation of the mitochondrial structure in embryonic cardiomyocytes ( Mackler et al . , 1971 ) . Shortly after birth , fatty acid oxidation becomes the predominant source of ATP production to meet the high energy demand of the maturing heart ( Warshaw and Terry , 1970 ) . These metabolic changes occur as a consequence of changes in the expression of metabolic enzymes and transporters . However , it remains unclear whether and how these metabolic changes , in turn , regulate the cardiac differentiation program . Here , we describe the use of in vitro human embryonic stem cell-derived cardiomyocytes ( hESC-CMs ) and an in vivo murine diabetic model to show that glucose not only induces cardiomyocyte proliferation but also inhibits cardiomyocyte maturation . Glucose can be metabolized in multiple catabolic and anabolic pathways , including glycolysis , oxidative phosphorylation , the pentose phosphate pathway ( PPP ) and the hexosamine biosynthesis pathway . Our chemical screening revealed that the pro-mitotic and anti-maturation effect of high glucose is regulated by glucose-derived deoxynucleotide biosynthesis through PPP . In vivo measurement of 18F-FDG accumulation revealed that glucose uptake is drastically suppressed during the late gestational and early postnatal stages . Exposure to high blood glucose in a murine model of diabetic pregnancy resulted in higher mitosis and delayed maturation of fetal cardiomyocytes in vivo . Together , our data uncover how the dynamics of glucose metabolism impact late embryonic cardiogenesis .
hESC-CMs were differentiated in monolayer in a chemically defined condition , reproducibly yielding ~90% of MF20+ cardiomyocytes at day 14 with multiple cell lines including WA09 ( H9 ) and UCLA4 hESCs ( Figure 1A–C ) ( Arshi et al . , 2013; Minami et al . , 2012 ) . hESC-CMs start to beat synchronously at around day 6–7 in our system . To characterize the differentiation stages , mRNA expression profiles from H9 hESC-CMs were serially examined by RNA-seq at five distinct stages ( GSE84815 ) : undifferentiated hESC ( day 0 ) ; mesodermal precursor stage ( hMP , day 2 ) ; cardiac progenitor stage ( hCP , day 5 ) ; immature cardiomyocyte ( hCM14 ) ; and hESC-CMS differentiated for 14 additional days ( hCM28 ) . The expression data were analyzed using signatures collected from MSigDB , a body atlas and primary cell atlas ( Mabbott et al . , 2013; Su et al . , 2004; Subramanian et al . , 2005 ) . As expected , the stem cell signature decreases during these five stages , while signatures associated with heart and smooth muscle increase , further suggesting that our protocol leads to a high degree of enrichment for cardiomyocytes ( Figure 1D and E ) . This differentiation course is comparable to that reported in previous publications ( Paige et al . , 2012; Wamstad et al . , 2012 ) . To examine the impact of glucose levels on cardiac differentiation , hESC-CMs were cultured in media containing various concentration of glucose , starting at the hCM14 stage when cells are already differentiated to immature cardiomyocytes ( Figure 2A , Video 1 and 2 ) . The basal differentiation medium contains 25 mM glucose , 0 . 9 mM pyruvate , essential and nonessential amino acids , and human albumin ( G25 medium ) . Interestingly , glucose dose-dependently suppressed the expression of TNNT2 ( a cardiac marker ) , NKX2-5 ( a cardiac marker ) , and PPARGC1A ( a mitochondrial marker ) ( Figure 2B ) . Gene expression profiling by RNA-seq revealed that genes that are related to cardiac muscle and function are enriched in hESC-CMs in low glucose medium , and that genes that are associated with mitosis and cell cycle are enriched in the high-glucose group genome-wide ( Figure 2C , Figure 2—figure supplement 1B; GSE84814 ) . These data suggest that low glucose after day 14 induces the differentiation and suppresses the cell cycle of hESC-CMs . To validate these results , hESC-CM proliferation was analyzed by pH3 staining and EdU flow cytometry analysis . Low glucose decreased mitotic activity at day 28 without affecting the viability of hESC-CMs ( Figure 2D , E ) . In addition , hESC-CMs in low glucose medium showed more robust staining of α-actinin , although the sarcomere length did not significantly change ( Figure 2F , Figure 2—figure supplement 1B ) . MitoTracker staining and flow cytometry analyses revealed that hESC-CMs cultured in low glucose media have increased mitochondrial contents and inter-myofibrillar distribution of mitochondria , characteristic of differentiated cardiomyocytes ( Figure 2F and G ) . Addition of 2-DG ( 2-deoxy-D-glucose ) , a competitive inhibitor of glucose phosphorylation , induced higher levels of MitoTracker and MF20 expression even in the presence of 5 mM or 25 mM glucose ( Figure 2—figure supplement 1C ) , suggesting that the effect is specific to glucose and not to changes in osmotic pressure . Consistently , flow cytometry showed a significant increase in cell size under glucose-restricted conditions ( Figure 2I ) . Together , these results demonstrate that glucose dose-dependently suppresses the maturation of cardiomyocyte cellular architecture and the upregulation of cardiac genes in hESC-CMs . We next compared the metabolic and functional maturity of hESC-CMs cultured in the presence and absence of glucose by six methods . First , hESC-CMs were stained with JC-1 , a green fluorescent dye that generates red fluorescence upon formation of aggregates in active mitochondria . The level of red fluorescence is often used as an indicator of mitochondrial membrane potential and , therefore , mitochondrial activity . Immunofluorescent staining revealed that mitochondria in glucose-reduced hESC-CMs are more elongated ( Figure 3A ) , and flow cytometry analysis revealed that the level of JC-1 aggregation is significantly higher in glucose-reduced hESC-CMs cultured in both regular 0 . 9 mM and 10 mM pyruvate conditions . ( Figure 3B ) . These data suggest that high glucose inhibits the functional maturation of mitochondria . Second , intracellular lactate levels of hESC-CMs were measured using Laconic , a fluorescence resonance energy transfer ( FRET ) -based lactate nanosensor ( San Martín et al . , 2013 ) . After glucose deprivation , the Laconic construct was introduced into hESC-CMs via adenovirus as described previously ( John et al . , 2008 ) . In hESC-CMs differentiated in standard 25 mM glucose , bath-applied lactate ( 4 mM ) caused a rapid increase in intracellular lactate ( measured as a decrease FRET ratio ) , demonstrating the efficacy of the probe . Subsequent addition of pyruvate evoked a similar but smaller elevation of intracellular lactate . Under these conditions , inhibition of mitochondrial respiration with sodium cyanide ( NaCN ) had only a minor effect on the intracellular lactate level . This result suggests that hESC-CMs that are differentiated in standard 25 mM glucose do not actively metabolize pyruvate ( Figure 3C , upper panel ) . By contrast , addition of 4 mM pyruvate to glucose-reduced hESC-CMs did not cause intracellular lactate accumulation , and addition of NaCN in the presence of pyruvate resulted in a substantial increase in lactate level . This result is consistent with pyruvate utilization by mitochondria ( Figure 3C , lower panel ) . Together , these data suggest that mitochondria metabolize pyruvate in hESC-CMs that are cultured in glucose-reduced conditions , but not in the presence of glucose . Third , we assessed cellular respiration of hESC-CM using the XF24 Extracellular Flux Analyzer ( Seahorse Bioscience ) , in which oxygen consumption rate ( OCR ) was measured in real time in a basal state and in response to oligomycin ( an ATP synthase inhibitor ) , FCCP ( carbonilcyanide p-triflouromethoxyphenylhydrazone , a mitochondrial uncoupler ) , and rotenone or myxothiazol ( complex I or III inhibitors , respectively ) ( Figure 3D ) . Although base-line mitochondrial respiration was not changed ( Figure 3E ) , ATP-linked respiration was elevated in the no glucose condition ( Figure 3F and G ) . Glucose-reduced hESC-CMs also demonstrated substantially larger maximum respiration capacity , as indicated by the response to FCCP ( Figure 3H ) . These results corroborate the increased capacity of cellular respiration in hESC-CMs cultured under low glucose conditions . Fourth , the Ca2+ kinetics of hESC-CMs were assessed using a Ca2+ transient assay ( Shimizu et al . , 2015 ) . Although the peak amplitude of the transient ( ΔF/F0 ) did not show a significant difference , the maximum upstroke ( Vmax ) was significantly faster and the time to 50% decay was significantly shorter in the glucose-reduced group ( Figure 3I ) . This pattern is consistent with the previous report demonstrating the role of thyroid hormone on hiPSC-CM maturation ( Yang et al . , 2014 ) , and suggestive of an inhibitory role of high glucose on hESC-CM maturation . Fifth , taking advantage of our monolayer culture system , we examined electrophysiological properties , using a multi-electrode array ( MEA ) culture plate as reported previously ( Zhu et al . , 2017 ) . Maximum upstroke velocity ( dV/dtmax ) of field potential is a reliable parameter for approximating the electrophysiological maturity of cardiomyocytes derived from pluripotent stem cells ( Haase et al . , 2009; Ma et al . , 2011; Zhang et al . , 2009 ) . Compared with hESC-CMs cultured in 25 mM glucose , those hESC-CMs cultured in the absence of glucose displayed a significant increase in dV/dtmax ( Figure 3—figure supplement 1A–C ) . Finally , the monolayer culture method allowed us to measure cell contractility by digital image correlation using the MotionGUI program ( Huebsch et al . , 2015 ) . Both average maximum contraction speed and average maximum relaxation speed were higher in those hESC-CMs cultured in 0 mM glucose ( Figure 3J ) . Together , these data suggest that glucose reduction promotes the functional maturation of hESC-CMs at the metabolic , electrophysiological , and biomechanical levels . Having determined that glucose reduction induces cardiac maturation at morphological , genetic metabolic , and functional levels , we next sought to analyze the mechanism by which glucose blocks cardiac maturation . Glucose is metabolized by multiple pathways involving both catabolic reactions ( anaerobic glycolysis and the aerobic TCA cycle ) and anabolic reactions ( PPP , the hexosamine pathway , etc . ) . We first examined the impact of glucose reduction on the global metabolomics signature . Mass spectrometry revealed that glucose deprivation resulted in a significant decrease in the levels of the metabolites in purine metabolism , pyrimidine metabolism , the PPP , the hexosamine pathway , and glycolysis , whereas lipid precursors , amino acids , glutamine , and glutamate , as well as urea cycle metabolites , were not significantly affected ( Figure 4A and Figure 4—figure supplement 1 ) . ATP levels were not significantly different under glucose-restricted conditions ( Figure 4B ) , neither were any specific stress pathways significantly increased when examined by RNA-seq , suggesting that the cells were not energy-starved in the absence of glucose in the cell culture media . To identify the metabolic pathway responsible for the improved cardiac maturation by glucose reduction , we conducted a systematic screening using chemical inhibitors for the various glucose metabolic pathways in the monolayer 384-well format ( Figure 4C ) . Consistent with flow cytometry for MitoTracker and MF20 ( Figure 2—figure supplement 1C ) , 2-DG dose-dependently abolished the glucose-dependent inhibition of TNNT2 and NKX2-5 mRNA levels in hESC-CM ( Figure 4D and Figure 4—figure supplement 4A ) . 3PO ( 3-[3-pyridinyl]-1-[4-pyridinyl]-2-propen-1-one ) , an inhibitor of the phosphofructokinase PFKFB3 ( which is a regulator of PFK1 ) , did not affect the level of hESC-CM maturity at any concentration of glucose ( Figure 4E and Figure 4—figure supplement 4B ) . The failure of 3PO to recapitulate the effect of glucose deprivation suggests that glucose metabolites downstream of PFK1 are not essential for the glucose-dependent inhibition of cardiac maturation . Consistently , sodium oxamate ( a lactate dehydrogenase [LDH] inhibitor ) did not block the inhibitory effect of glucose ( Figure 4F and Figure 4—figure supplement 4C ) . Interestingly , however , 6AN ( 6-[cyclohexa-2 , 5-dien-1-ylideneamino] naphthalene-2-sulfonate ) and DHEA ( didehydroepiandrosterone ) , both inhibitors of glucose-6-phosphate dehydrogenase ( G6PD ) in the oxidative arm of the PPP , recapitulated glucose reduction ( Figure 4G and H , Figure 4—figure supplement 4D–E ) . As summarized in Figure 4—figure supplement 3 , our chemical inhibitor screening suggests that the PPP plays a critical role in the inhibition of cardiac maturation and that blocking this pathway by either use of chemical inhibitors or glucose deprivation induces cardiac maturation . Although mitochondria are a major source of reactive oxygen species ( ROS ) and physiological levels of ROS promote cellular differentiation ( Crespo et al . , 2010 ) , the level of ROS measured by DCFDA ( dichlorodihydrofluorescein diacetate ) did not increase in the absence of glucose and neither did ROS inhibition had a significant impact on TNNT2 expression level ( Figure 4—figure supplement 3F ) , suggesting that the increase in ROS is not responsible for the induction of cardiac maturation . The oxidative arm of the PPP generates two major products: reducing power in the form of NADPH and 5-carbon sugars that supply the backbone for nucleotide biosynthesis . To test whether glucose level impacts cardiac maturation by affecting nucleotide biosynthesis , we rescued nucleotide synthesis by adding uridine to hESC-CMs cultured in low glucose media . Under glucose starvation , supplementation of uridine is known to rescue the growth of bacteria , yeast and malignant cells ( Linker et al . , 1985 ) . In our hESC-CM culture system , uridine restored cell proliferation even in low-glucose conditions ( Figure 5A and B ) . Interestingly , uridine dose-dependently reduced the level of TNNT2 even in glucose-deprived conditions ( Figure 5C and Figure 5—figure supplement 1A ) , suggesting that glucose-mediated inhibition of cardiac maturation is dependent on the supply of nucleotides and not of NADPH . In order to test whether nucleotides are necessary for the glucose-dependent inhibition of cardiac maturation , nucleotide biosynthesis was blocked by multiple methods . Addition of an excess amount of thymidine ( unlike uridine ) blocks the synthesis of DNA by inhibiting the formation of deoxycytidine ( i . e . , the thymidine block method ) , which is commonly used to synchronize the cell cycle ( Reichard et al . , 1960; Xeros , 1962 ) . When excess thymidine was added to hESC-CMs , the expression of TNNT2 and NKX2-5 were increased ( Figure 5D and Figure 5—figure supplement 1B ) . To further confirm this effect , we blocked deoxynucleotide synthesis by hydroxyurea ( HU ) , an inhibitor of ribonucleotide reductase ( RNR ) that catalyzes the formation of deoxyribo-nucleotides . Consistent with the results from thymidine block , HU dose-dependently induced the expression of TNNT2 and NKX2-5 ( Figure 5E and Figure 5—figure supplement 1C ) . RNAi-based knockdown of RRM2B , a key subunit of RNR , also resulted in a significant increase in TNNT2 expression level , even in the presence of 25 mM glucose ( Figure 4—figure supplement 2 ) . Together , these gain- and loss-of-function data suggest that nucleotide biosynthesis is a key regulatory pathway of the pro-mitotic–anti-maturation effect of glucose . Nucleotide synthesis is a key step in DNA replication and thus cell cycling . Therefore , it is not clear whether the maturation of hESC-CMs that results from deprivation of glucose is due to the cell-cycle block in general or to the effects of nucleotides themselves . To examine whether cell-cycle arrest in general is an essential trigger for cardiac maturation , we blocked the mitotic activity of hESC-CMs by a CDK4/6 inhibitor and paclitaxel ( Taxol; an inhibitor of microtubule breakdown ) , both of which block the cell cycle without directly inhibiting the nucleotide kinetics . Interestingly , neither CDK4/6 inhibitor nor paclitaxel induced cardiac maturation ( Figure 5—figure supplement 2A–B ) . Together , these data suggest that cell-cycle arrest by itself is not crucial for the promotion of cardiac maturation . Rather , nucleotide deprivation is a key mechanism for cardiac maturation . These in vitro data suggest that glucose reduction promotes cardiac maturation while inhibiting cardiomyocyte proliferation . An intriguing possibility is that the same mechanism underlies cardiac maturation in the in vivo natural counterpart . During normal embryogenesis , however , the blood glucose level is primarily regulated by maternal metabolism and stays relatively stable in utero , leading us to hypothesize that cellular glucose uptake becomes restricted during late fetal stages . To test this possibility , we measured the glucose uptake in the fetal hearts using 18F-labeled 2-deoxy-2-fluoroglucose ( FDG ) . 18F-labeled FDG was injected intravenously via the maternal tail vein at E10 . 5 , E12 . 5 , and E15 . 5 or intraperitoneally into P1 and P7 pups . After 2 hr , the mice were imaged by PET/CT , the hearts were dissected , and cardiac accumulation was measured quantitatively . Interestingly , the normalized cardiac accumulation progressively and rapidly decreased from E10 . 5 to P7 , with 0 . 11% and 0 . 05% 18F-FDG uptake in P1 and P7 hearts , respectively , compared to E10 . 5 hearts ( Figure 6 ) . These data suggest that cardiac glucose uptake becomes significantly restricted during late gestational and early postnatal stages , creating an intracellular glucose deprivation condition during natural in vivo development . We next tested whether hyperglycemia promotes the proliferation and inhibits the maturation of cardiomyocytes in vivo using fetuses and neonates from diabetic pregnancy . Akita heterozygous mice carry a single amino-acid substitution in the Ins2 gene and exhibit multiple disorders associated with maturity-onset diabetes of the young ( MODY ) ( Barber et al . , 2005; Fujita et al . , 2001; Wang et al . , 1999; Yaguchi et al . , 2003; Yoshioka et al . , 1997 ) . By crossing an Akita female with a wild-type male , we created a diabetic pregnancy condition in which wild-type fetuses ( half of the pups in the litters ) and their littermates are exposed to hyperglycemia ( Figure 7A ) . In the C57BL/6 background , the average blood-glucose levels of the Akita mothers that we used were significantly higher than those of sex-matched control littermates ( 215 ± 84 vs 71 ± 12 mg/dl , respectively; p<0 . 0005 ) . Fetal and neonatal Tnnt2+ cardiomyocytes from wild-type hearts from wild-type mothers and wild-type hearts from Akita mothers were examined for mitotic activity in an in vivo EdU incorporation assay at E16 . 5 and P0 stages , when cardiomyocytes are not yet multinucleated or multiploidic . As shown in Figure 7B and C , the number of cardiomyocytes in S phase was significantly higher at both E16 . 5 and P0 in the diabetes group . Histological analyses showed that the number of phosphorylated histone H3-positive cardiomyocytes ( pH3+/Tnnt2+ ) is higher at E16 . 5 in the embryos from diabetic pregnancies ( Figure 7D and E ) . These data suggest that fetal cardiomyocytes are more mitotic when exposed to maternal hyperglycemia . To examine whether hyperglycemia inhibits the maturation of fetal cardiomyocytes in vivo , we analyzed the fetal and neonatal hearts from diabetic pregnancies . The level of Tnnt2 expression was significantly lower in the hearts from the diabetic pregnancies ( Figure 7F ) . A hallmark of the congenital heart disease associated with diabetic pregnancy is asymmetric cardiac hypertrophy . Although heart weight/body weight ratio did not show a difference in our mouse model , the thickness of left and right ventricular free walls was significantly increased in the hearts from diabetic pregnancies at P1 ( Figure 7G ) . Consistently , the cardiomyocyte size measured by flow cytometry was significantly smaller in the diabetic pregnancy group ( Figure 7H ) . These data suggest that overproliferation and/or delayed maturation underlie the pathological mechanism of cardiomyopathy associated with diabetic pregnancy .
Glucose is the most fundamental and commonly available nutrient for the cells . Hence , the activity of the glucose metabolic pathways is tightly regulated in cells . Glucose is broken down to extract energy through the glycolysis pathway and also shunts to supply 5-carbon sugars and NADPH through the PPP . In our study , chemical inhibition of glucose metabolic pathways in hESC-CMs revealed that it is not the catabolic breakdown of glucose to extract energy but rather the anabolic use of glucose to build nucleotides that is responsible for the glucose-dependent inhibition of cardiac maturation . Most of the proliferating cells synthesize nucleotides de novo from glucose , glutamine , and CO2 . In our hESC-CM experiments , blocking the PPP and nucleotide biosynthesis inhibited the glucose-mediated induction of mitosis and suppression of maturation , and supplementation of nucleotides was sufficient to recapitulate the effect of glucose ( Figures 4 and 5 ) . These data suggest that nucleotide biosynthesis via the PPP is the key regulator of the pro-mitotic/anti-maturation effect of glucose . Interestingly , cardiomyocyte maturation was not fully induced by blocking of the cell cycle with a CDK4/6 inhibitor or paclitaxel , neither of which directly impact the nucleotide kinetics . Therefore , it is not the cell cycle in general but the nucleotides themselves that blocks the maturation ( Figure 5—figure supplement 2 ) . It is well-documented that there is generally an inverse correlation between cell proliferation and differentiation during developmental stages ( Ruijtenberg and van den Heuvel , 2016 ) . Our data raise an intriguing possibility that nucleotide biosynthesis serves as a nodal point balancing cell proliferation and differentiation during development . Clinically , maternal diabetes can accompany multiple complications including neuropathy , microvasculopathy , nephropathy , and insulin resistance . Although meta-analysis predicts that hyperglycemia itself is a major teratogen during diabetic pregnancy ( Reece et al . , 1996 ) , it is often difficult to dissect the impacts of maternal complications on CHD as they are often subclinical . To our knowledge , our in vitro study is the first to demonstrate that environmental glucose itself , if excessive , directly impacts cardiac differentiation . The formation of the heart is regulated by both genetic and non-genetic factors , with the latter playing important roles particularly during late-stage cardiogenesis . An interesting aspect of the interaction between genetic and non-genetic mechanisms is that they seem to reinforce each other mutually . Our data suggest that the glucose metabolic environment is , on the one hand , a consequence of changes in cardiac genetic program , and on the other hand , a cause of the changes in cardiac gene expression . Understanding the metabolic signature of hESC-CMs will potentially open new methods for purifying these cells ( Tohyama et al . , 2016; Tohyama et al . , 2013 ) or inducing their maturation ( Drawnel et al . , 2014 ) . Considering that the inhibitors of the PPP and nucleotide biosynthesis have entered clinical trials for cancer treatment ( Tennant et al . , 2010; Vander Heiden , 2011 ) , our data raise the possibility that manipulating this pathway may allow us to control the proliferation and maturation of cardiomyocytes for regenerative medicine . With the advances in fetal diagnosis and surgical techniques , the number of CHD patients who survive childhood ( and so have adult CHD ) is growing rapidly by nearly 5% per year ( Brickner et al . , 2000 ) . Maternal hyperglycemia is a common medical condition associated with 2–5-fold increase in CHD ( Centers for Disease Control , 1990; Simeone et al . , 2015; Yogev and Visser , 2009 ) . Currently , 60 million women of reproductive age ( 18–44 years old ) worldwide and approximately 3 million in the U . S . have diabetes mellitus . This number is predicted to double by 2030 , posing a huge medical and economic burden ( Gabbay-Benziv et al . , 2015 ) . Our findings will lay a foundation for understanding how the glucose environment regulates cardiogenesis and how disturbance of non-genetic factors affects the genetic program during the pathological development of the heart .
Wild-type and Akita mice were maintained on the C57BL/6 background according to the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health ( NIH Publication No . 85–23 , revised 1996 ) . Housing and experiments were performed according to the Institutional Approval for Appropriate Care and Use of Laboratory Animals by the UCLA Institutional Animal Care and Use Committee ( Protocol #2008-127-07 ) . H9 ( WA09 ) and UCLA4 ( UCLA stem cell core ) hESC lines were maintained as described before ( Arshi et al . , 2013 ) . Authentication of hESCs was achieved by confirming the expression of pluripotency genes and protein markers . hESCs were routinely verified as mycoplasma-free using a PCR-based assay . hESCs were grown and differentiated in a chemically defined condition ( Minami et al . , 2012; Young et al . , 2016; Zhu et al . , 2017 ) . Usage of all the human embryonic stem cell lines is approved by the UCLA Embryonic Stem Cell Research Oversight ( ESCRO ) Committee and the Institutional Review Boards ( IRB ) ( approval #2009-006-04 ) . For the RNA-seq analyses shown in Figures 1D , E and 2C , RNA was extracted from hESC , hMP , hCP , hCM14 , hCM28 , hCM28 ( in 25 mM glucose ) and hCM28 ( in 0 mM glucose ) using TRIZOL ( TheroFisher ) and RNeasy kit ( QIAGEN ) . 500 ng of DNaseI-treated RNA was used as input material for library preparation using the Illumina TruSeq mRNA kit ( Illumina , RS-122–2001 ) , according to manufacturer’s instructions . Final libraries were sequenced as single-end 50 bp on the Illumina HiSeq2000 platform ( GSE84814 ) . Libraries for RNA-Seq shown in Figures 1D , E and 2C were prepared with the KAPA Stranded RNA-Seq Kit . The workflow consists of mRNA enrichment , cDNA generation , end repair , A-tailing , adaptor ligation , strand selection and PCR amplification . Different adaptors were used for multiplexing samples in one lane . Sequencing was performed on an Illumina HiSeq 3000 for a paired end 2 × 150 run ( GSE84815 ) . A data quality check was done on an Illumina SAV . De-multiplexing was performed with the Illumina Bcl2fastq2 v 2 . 17 program . Short read sequences generated from an Illumina Sequencer were aligned to the UCSC human reference genome hg19 downloaded from support . Illumina . com ( http://support . illumina . com/sequencing/sequencing_software/igenome . html ) using TopHat from the Tuxedo Tools . The average overall read mapping rate reached over 82 percent ( average = 82 . 43 percent ) . The output was in the form of BAM ( Binary Sequence Alignment/Map format ) files . These outputs contain information for assigning location and quantifying the short-read alignments obtained from the RNA-seq samples . This is necessary for downstream analyses such as annotation , transcript abundance comparison and polymorphism detection . Counts or gene expression matrices were generated using HTSeq , which quantified the reads per transcript . The expression matrices were log transformed and normalized using the rlog function in the Deseq2 package . The normalized gene expression matrices were used as input for SaVanT ( Signature Visualization Tools ) , which allowed for the visualization of molecular signatures directly related to heart development as seen in Figures 1D , E and 2C . hESC-CMs and mouse embryonic hearts were washed three times with PBS and incubated at 37°C in a dissociation enzyme solution with occasional pipetting to a single-cell suspension . The enzyme solution contained 1% penicillin/streptomycin ( ThermoFisher , 15140–122 ) , 10% fetal bovine serum ( Hyclone ) , collagenase 2 mg/ml ( Worthington , CLS-2 ) , dispase 0 . 25 mg/ml ( Gibco , 17105–041 ) , and DNAase I ( ThermoFisher ) in PBS . The cells were analyzed with the following antibodies: MF20 ( mouse , 1:100 , Hybridoma Bank ) , Tnnt2 ( rabbit , 1:250 , Sigma-Aldrich ) , MitoTracker Orange CMTMRos ( ThermoFisher ) , JC-1 ( Abcam ) , and EdU ( 100 μl of 10 mM EdU solution per 10 g of mouse , ThermoFisher ) . For MF20 and Tnnt2 , FITC-conjugated anti-mouse IgG secondary antibody ( BD Biosciences ) was used . Stained cells were analyzed by a flow cytometer ( LSRII , BD Biosciences ) . Data analysis was performed using FACSDiva ( BD Biosciences ) . Cells were fixed with 4% paraformaldehyde , blocked for an hour with 5% normal goat serum , and incubated with mouse alpha actinin antibody ( Sigma ) followed by Alexa fluor 488-conjugated secondary antibody ( ThermoFisher ) . Images were taken with Zeiss LSM780 confocal microscopy . Sarcomere lengths were analyzed using Zeiss Zen software . Total DNA including mtDNA was extracted from cells using the PureLink DNA kit ( ThermoFisher ) , and DNA purity and quantity were determined using a spectrophotometer . To determine the ratio between mitochondrial and nuclear DNA , qRT-PCR was performed on a Roche Lightcycler 480 using SYBR Green dye . Mitochondrial gene expression was corrected for nuclear gene expression values , and normalized to the value of the control group for each experiment as described before . Forward and reverse primer sequences are as follows: UUR forward , CAC CCA AGA ACA GGG TTT GT; UUR reverse , TGG CCA TGG GTA TGT TGT TA for mt DNA; B2-microglobulin forward , TGC TGT CTC CAT GTT TGA TGT ATC T; and B2-microglobulin reverse , TCT CTG CTC CCC ACC TCT AAG T . Ca2+ transient was measured as described ( Shimizu et al . , 2015 ) . Briefly , hESC-CMs cultured in the presence or absence of glucose were loaded with 5 µM fluo-4 AM and imaged in Tyrode buffer containing 138 . 2 mM NaCl , 4 . 6 mM KCl , 1 . 2 mM MgCl , 15 mM glucose and 20 mM HEPES according to the manufacturer’s instruction . Images were recorded on a Zeiss LSM 780 confocal microscope . Data analysis was carried out using the Zeiss Zen and ImageJ . Contractility assessments were performed by utilizing a video-based technique with the UCSF Gladstone-developed Matlab program MotionGUI ( Huebsch et al . , 2015 ) . The videos were converted from . mts to . avi format at native resolution using commercially available software and loaded into the MotionGUI program . The conversion between pixels and real distance was performed within the MotionGUI program using a reference image with unit divisions of 100 um , taken under the same objective and video zoom settings as the cell videos , to yield a pixel size of 0 . 681125 . This pixel size was used for all contractility assessments . Motion vectors were calculated and the data were evaluated upon completion . All samples were subjected to the same post-processing procedures in order to ensure consistency during comparative analysis . Each video sample was post-processed using neighbor-based cleaning with the vector-based cleaning criterion within the program . The threshold for this post-processing method was set to two for all samples and was adequate for improving the signal-to-noise ratio enough to identify peaks clearly corresponding to beating events in most samples . A small number of videos suffering from significant noise issues were separately subjected to fast Fourier transform ( FFT ) frequency domain cleaning with a cut-off frequency of 1 Hz . Only one post-processing method was applied to one video at one time . All other parameters of the MotionGUI program not outlined here were set to their respective default values . The lactate biosensor Laconic was a gift from Dr . Barros ( San Martín et al . , 2013 ) . Overexpression of Laconic in hESC-CM was achieved using engineered adenoviruses encoding the construct . Expression of the construct was sufficiently high after 36–48 hr for FRET experiments or microscopy imaging . All cells were imaged live without fixation . Images ( 16 bit ) were acquired using a microscope ( Eclipse TE300; Nikon ) fitted with a 60× ( 1 . 4 NA ) oil immersion lens ( Nikon ) and equipped with a filter cube comprising a CFP bandpass excitation filter , 436/20b , together with a longpass dichroic mirror ( 455DCLP; Chroma Technology Corp ) . Two LEDs ( Philips Lumileds ) , one emitting at 455 ± 20 nm ( royal blue ) and the other emitting at 505 ± 15 nm ( cyan ) were used as light sources . Ratiometric FRET measurements were obtained from the YFP and CFP images acquired simultaneously using a Dual View image splitter ( Optical Insights ) equipped with a 505 nm longpass dichroic filter to separate the CFP and YFP signals , a CFP emission filter ( 480/30 ) , and a YFP emission filter ( 535/40 ) ( John et al . , 2008 ) . Images were captured with a Cascade 512B digital camera ( Photometrics ) . Reagents indicated in Figure 3C were added and followed by washing . hESC-CMs were seeded onto a matrigel-coated XF24 Cell Culture Microplate ( Seahorse Bioscience ) at 2–7 . 5 104 cells/well with or without glucose ( 25 mM glucose of cardiac differentiation media ) . Oxygen consumption rate ( OCR ) was measured using an XF24 Extracellular Flux Analyser ( Seahorse Bioscience ) in unbuffered DMEM assay medium supplemented with 1 mM pyruvate , 2 mM glutamine and with or without 25 mM glucose . OCR was measured before and after the sequential addition of 0 . 75 µM oligomycin , 0 . 5 µM FCCP and 0 . 75 µM of rotenone/myxothiazol . OCR was normalized to protein concentration using a Bradford assay ( Bio-Rad ) . Mitochondrial respiration was calculated as the difference between total and rotenone/myxothiazol rates . Maximal respiration was the response to FCCP . ATP-linked respiration was represented by the oligomycin-sensitive respiration rate , whereas uncoupled respiration was represented by the difference between oligomycin and rotenone/myxothiazol rates . hESC-CMs at the stage of hCM14 were plated on microelectrode arrays ( MEAs ) containing 120 integrated TiN electrodes ( 30 µm diameter , 200 µm interelectrode spacing ) . The MEAs were placed in an incubator with a temperature of 37°C and 5% CO2 . Two days were given to allow the cardiomyocytes to well attach the MEAs before recording started . Local field potentials at each electrode were collected over a period of 5 min every day in total with a sampling rate of 1 KHz using the MEA2100-HS120 system ( Multichannel systems , Reutlingen , Germany ) . Data analysis was carried out using the MC_DataTool ( Multichannel Systems ) , Origin ( OriginLab Corporation ) and Matlab ( MathWorks ) . Data shown are based on three independent hESC-CM preparations . The experiments were performed as described ( Krall et al . , 2016 ) . Briefly , cells were seeded in 6-well plates , so that the final cell count at the time of metabolite extraction was about 7*105 and this was even across all cell lines . To extract intracellular metabolites , cells were briefly rinsed with cold 150 mM ammonium acetate ( pH 7 . 3 ) , followed by the addition of 1 ml cold 80% MeOH on dry ice . Cell scrapers were used to detach cells , and the cell suspension was transferred into Eppendorf tubes . Extracted metabolites were transferred into glass vials and dried down under vacuum . For the LC-MS-based analysis , the samples were resuspended in 70% acetonitrile and 50 μl were injected onto a Luna NH2 column ( 150 mm x 2 mm , Phenomenex ) . Separation was achieved using A ) 5 mM NH4AcO ( pH 9 . 9 ) and B ) ACN . The gradient started with 15% A ) going to 90% A ) over 18 min , followed by an isocratic step for 9 min and reversal to the initial 15% A ) for 7 min . Metabolites were quantified with TraceFinder 3 . 3 using accurate mass measurements ( ≤3 ppm ) and retention times of pure standards . Data analysis was performed using the statistical language R . RNA was extracted from the tissue or the cells cultured with a specific concentration of glucose together with or without titrated metabolic pathway inhibitors using the Direct-zol RNA mini prep kit ( Zymo research ) . RNA was reverse-transcribed into complementary DNA using the qScript cDNA synthesis kit ( Quanta Biosciences ) . Quantitative reverse-transcriptase PCR was performed using Viia7 ( Applied Biosystems/ThermoFisher ) . In Figures 1B , 2B and 4C–E , Figure 4—figure supplement 3A–E , Figure 5—figure supplement 1A–C , and Figure 5—figure supplement 2A–B , each bar represents the average of biological duplicates with at least three independent wells , each of which is triplicated for qPCR reaction . The relative mRNA level is normalized to the expression level of 25 mM glucose without any chemicals . Forward and reverse primer sequences are as follows: GAPDH forward , TTGAGGTCAATGAAGGGGTC; GAPDH reverse , GAAGGTGAAGGTCGGAGTCA; TNNT2 forward , CAGAGCGGAAAAGTGGGAAGA; TNNT2 reverse , TCGTTGATCCTGTTTCGGAGA; NKX2-5 forward , GTTGTCCGCCTCTGTCTTCT;NKX2-5 reverse , TCTATCCACGTGCCTACAGC; PPARGC1A forward , GGTGCCTTCAGTTCACTCTCA; and PPARGC1A reverse , AACCAGAGCAGCACACTCGAT . 18F-FDG was obtained from the UCLA Department of Nuclear Medicine . Warmed pregnant mice or pups were injected intravenously or intraperitoneally , respectively , with ~90 microCi ( ~3 . 33 MBq ) of 18F-FDG . After 2 hr , the mice were sacrificed . Preliminary experiments suggested that 2 hr was sufficiently long for 18F-FDG to reach maximum accumulation in each organ and embryo . Fetal or neonatal hearts were separated from the other tissue ( carcass ) , and the mass and radioactivity in both the hearts and the carcasses were measured using a standard balance and a Wizard 3’ automatic gamma counter ( Perkin Elmer ) , respectively . The radioactivity levels in the pup carcasses were higher than the detection limit of the gamma counter , so the expected gamma counter values for the pup carcasses were calculated on the basis of the decay-corrected injected dose of 18F-FDG and known conversion values between microCi and CPM on the gamma counter . To calculate the ‘Normalized FDG accumulation’ , radioactive accumulation in each heart was divided by heart weight and then further divided by the total radioactivity in each embryo or pup . This last normalization is to account for differences in 18F-FDG injected dose and accessibility to the embryos and pups . Averages and standard errors of the mean ( SEM ) were calculated , and the values were normalized such that E10 . 5 embryo FDG accumulation was set to 100 . Mouse embryos were isolated in cold PBS and fixed in 4% PFA for 1~2 hr , followed by equilibration in 30% sucrose in PBS solution overnight . The tissues were placed in 1:1 30% sucrose/OCT ( Tissue-Tek , Electron Microscopy Sciences ) solution for 1–2 hr , then in 100% OCT compound for 1 hr at 4°C , and embedded in 100% OCT compound , carefully oriented in Cryomolds ( Ted Pella ) . The blocks were immediately frozen on dry ice with isopropanol and stored at −80°C . The sections were cut 5 μm with a Leica CM3050 S cryostat . The following primary and secondary antibodies were used: α-actinin ( mouse , 1:200 , Sigma-Aldrich ) , Phospho-Histone 3 ( rabbit , 1:250 , Millipore ) , as well as Alexa Fluor 488 ( green ) - and Alexa Fluor 594 ( red ) -conjugated secondary antibodies specific to the appropriate species , which were used ( 1:500; ThermoFisher ) for fluorescent staining . Sections were mounted with antifade mounting medium with DAPI ( ThermoFisher ) , and analyzed by using AxioImager D1 ( Carl Zeiss Microimaging , Inc ) . ANOVA and Student’s t-test were used to determine whether any statistically significant difference exists among independent groups .
The heart switches its energy substrate from glucose to fatty acids at birth , and maternal hyperglycemia is associated with congenital heart disease . However , little is known about how blood glucose impacts heart formation . Using a chemically defined human pluripotent stem-cell-derived cardiomyocyte differentiation system , we found that high glucose inhibits the maturation of cardiomyocytes at genetic , structural , metabolic , electrophysiological , and biomechanical levels by promoting nucleotide biosynthesis through the pentose phosphate pathway . Blood glucose level in embryos is stable in utero during normal pregnancy , but glucose uptake by fetal cardiac tissue is drastically reduced in late gestational stages . In a murine model of diabetic pregnancy , fetal hearts showed cardiomyopathy with increased mitotic activity and decreased maturity . These data suggest that high glucose suppresses cardiac maturation , providing a possible mechanistic basis for congenital heart disease in diabetic pregnancy .
Congenital heart disease is the most common type of birth defect , affecting nearly 1 in 100 children born . It can involve a weak heart , narrowed arteries , narrowed heart valves , or the main arteries of the heart switching places . These conditions can be fatal if untreated and often need surgery to correct . The mother’s blood sugar levels during pregnancy can have a large effect on how likely the baby is to have congenital heart disease . If a pregnant woman has poorly controlled diabetes with rapidly fluctuating sugar levels , she may be at a higher risk of having a child with the condition . High sugar levels in the mother’s blood make the baby up to five times more likely to have congenital heart disease . It has been difficult to find out exactly how sugar levels interfere with heart development because diabetes can affect the fetus in many ways . Nakano et al . used stem cells and experiments in pregnant mice with diabetes to hone in on how high sugar levels affect the fetus’s heart development . First , heart cells were grown from human stem cells , and exposed to high levels of glucose in a dish . This revealed a new mechanism for how high sugar levels affect heart formation: the cells created too many nucleotides , the building blocks of molecules such as DNA . It turns out that high glucose levels boosted a chemical process in the cell known as the pentose phosphate pathway . Some of the products of this pathway are nucleotides . This made the cells divide rapidly , but did not allow them to mature well compared with cells exposed to normal levels of sugar . In another experiment , Nakano et al . found similar results in pregnant diabetic mice . The heart cells in mouse fetuses also divided quickly but matured slowly when exposed to high sugar levels . An estimated 60 million women at an age to have children have diabetes . These new findings help us to understand why and how these women are more likely to have children with congenital heart disease , and further study will hopefully lead to a better way to prevent this condition .