Fibroblasts Drive Metabolic Reprogramming in Pacemaker Cardiomyocytes
Abstract
Background:
The sino atrial node (SAN) is characterized by the microenvironment of pacemaker cardiomyocytes (PCs) encased with fibroblasts. An altered microenvironment leads to rhythm failure. Operable cell or tissue models are either generally lacking or difficult to handle. The biological process behind the milieu of SANs to evoke pacemaker rhythm is unknown. We explored how fibroblasts interact with PCs and regulate metabolic reprogramming and rhythmic activity in the SAN.
Methods:
Tbx18 (T-box transcription factor 18)-induced PCs and fibroblasts were used for cocultures and engineered tissues, which were used as the in vitro models to explore how fibroblasts regulate the functional integrity of SANs. RNA-sequencing, metabolomics, and cellular and molecular techniques were applied to characterize the molecular signals underlying metabolic reprogramming and identify its critical regulators. These pathways were further validated in vivo in rodents and induced human pluripotent stem cell-derived cardiomyocytes.
Results:
We observed that rhythmicity in Tbx18-induced PCs was regulated by aerobic glycolysis. Fibroblasts critically activated metabolic reprogramming and aerobic glycolysis within PCs, and, therefore, regulated pacemaker activity in PCs. The metabolic reprogramming was attributed to the exclusive induction of Aldoc (aldolase c) within PCs after fibroblast-PC integration. Fibroblasts activated the integrin-dependent mitogen-activated protein kinase-E2F1 signal through cell-cell contact and turned on Aldoc expression in PCs. Interruption of fibroblast-PC interaction or Aldoc knockdown nullified electrical activity. Engineered Tbx18-PC tissue sheets were generated to recapitulate the microenvironment within SANs. Aldoc-driven rhythmic machinery could be replicated within tissue sheets. Similar machinery was faithfully validated in de novo PCs of adult mice and rats, and in human PCs derived from induced pluripotent stem cells.
Conclusions:
Fibroblasts drive Aldoc-mediated metabolic reprogramming and rhythmic regulation in SANs. This work details the cellular machinery behind the complex milieu of vertebrate SANs and opens a new direction for future therapy.
Graphical Abstract
Novelty and Significance
What Is Known?
•
The sinoatrial node (SAN) initiates electric impulses for every heartbeat, and its dysfunction causes a slow heart rate or cardiac arrest.
•
SAN is characterized by the microenvironment of pacemaker cardiomyocytes (PCs) encased with fibroblasts.
•
The disintegration of PCs with fibroblasts results in SAN dysfunction.
What New Information Does This Article Contribute?
•
Fibroblasts induce metabolic reprogramming and activate the PC-specific expression of Aldoc (aldolase c) within SANs through integrin-dependent cell contact.
•
The activation of aldolase c critically maintains intrinsic aerobic glycolysis in PCs and regulates pacemaker activities.
•
The engineered tissue sheets are established as an in vitro model of the SAN microenvironment.
From the induced rat PCs and engineered tissue sheet models to human PCs and in vivo animal experiments, we observed fibroblasts induce aerobic glycolysis and regulate rhythmicity by upregulating Aldoc in PCs. In addition to explaining the mechanisms of SAN failure, Aldoc-driven energy replenishment may also be used as a future device-free therapy to restore SAN dysfunction. Establishment of engineered tissue sheets can be used as an in vitro experimental model for the study of SAN diseases and preclinical tests.
In This Issue, see p 1
Meet the First Author, see p 2
Editorial, see p 21
The sinoatrial node (SAN) initiates electric impulses for every heartbeat to maintain life. Its dysfunction causes a slow heart rate, insufficient blood supply, and detrimental consequences such as cardiac arrest.1 In contrast to atrial or ventricular cardiac tissue, the SAN consists of a network of pacemaker cardiomyocytes (PCs) encased with abundant fibroblasts and a heterogeneous connective tissue microenvironment.2–5 This unique structure of SANs is well conserved across vertebrate species.5–7 The microenvironment, through the integration of PCs, mesenchymal lineages (including fibroblasts), and extracellular matrix (ECM) organization, is required for the rhythmic activity of SANs during embryogenesis.4 Failure of ECM organization and likely fibroblast integration results in electrical dysfunction of SANs.4 At the other extreme, extensive fibrosis of the SAN also leads to pacemaker failure.8,9 Alteration of the microenvironment underlies the pathogenesis of SAN disorders. Although the molecular mechanisms underlying the ability of individual PCs to generate rhythmic electrical impulses have been well studied,10,11 the biological process behind the microenvironmental niche in SANs, especially fibroblast-PC interactions, remains poorly understood.
The SAN is tiny with a paucity of PCs.3,12 Operable cell or tissue models are either generally lacking or difficult to handle.10 To study functional interactions within the microenvironment, recapitulation of the complex structure of the PCs and fibroblasts within the SAN is necessary, but such models have yet to be developed.13 This presents a barrier to studying the biological function of this critical tissue and obtaining models of SAN diseases.10 Recently, induced PCs, generated by Tbx18 (T-box transcription factor 18) transduction or biomaterial, recapitulated not only electrical and morphological phenotypes, but also the metabolic properties of native SAN cardiomyocytes.14–16 It is possible that Tbx18-induced PCs (Tbx18-PCs), considered a replacement for native PCs, might be used to establish engineered models to study unknown SAN biological processes, especially intercellular functional interactions within the microenvironment.17
In this study, we used Tbx18-PCs and engineered tissue to explore how fibroblasts regulate the functional integrity of SANs. Fibroblasts drove PC-specific expression of Aldoc (aldolase c, an enzyme involved in glycolysis metabolism) through integrin-dependent cell contact. This machinery critically maintained intrinsic aerobic glycolysis in PCs and regulated pacemaker activities. Aldoc-mediated rhythmic activity was faithfully validated in an in vitro engineered model, in vivo in mice, and in human-induced pluripotent stem cell-derived cardiomyocytes. These findings highlight the importance of the SAN microenvironment in determining its energy metabolism and rhythmicity. Moreover, tissue engineered with Tbx18-PCs could be a feasible in vitro platform to study SAN physiology.
Methods
A detailed, expanded methods section is provided in the Supplemental Material. The Institutional Animal Care and Use Committee at Taipei Veterans General Hospital approved all animal experiments.
Data Availability
All data supporting the findings of this study are available within the article and its Supplemental Material. The whole transcriptome analysis data set (Figure 1A and Figure 3A) is provided in the Sequence Read Archive data at NCBI (PRJNA743181 and PRJNA743409).
Statistical Analysis
Statistical analysis was performed with GraphPad Prism version 8.3.0 (GraphPad Software, CA). Data are expressed as the mean±SD. The Shapiro-Wilks normality test was used to determine the use of Student t test, 1-way ANOVA, or Mann Whitney test as appropriate. Repeated measures ANOVA with the least significant difference (LSD) post hoc test was used when repeated measures were necessary. Multiple comparison correction was performed using the false discovery rate control approach and the Benjamini–Hochberg method when multiple comparisons of different variants were needed.18 A P value <0.05 was considered statistically significant.
Results
Glycolysis Metabolism Regulated Rhythmicity in Tbx18-PCs
We hypothesized that a cell-specific biological process of PCs, which is different from that of quiescent ventricular cardiomyocytes (VMs), would underlie the tailored integration of fibroblasts and PCs for functional integrity.4,19 Therefore, Tbx18-PCs were selected as the cell model.14,20 The differential biological processes between PCs and VMs were explored. Whole-transcriptome expression was compared between Tbx18-PCs and control-VMs (Figure 1A, Supplemental Data S1).14,20 Glucose metabolism and glycolysis accounted for the top canonical pathways (Figure 1B, Table S1, and Supplemental Data S2) and were predominant in gene ontology analysis (Table S2 and Supplemental Data S3). Glucose is metabolized to pyruvate via a complex enzyme network (Figure 1C). The metabolic genes involved in glycolysis were mostly increased in Tbx18-PCs. Aldoc was an exception, as its transcripts significantly decreased (Figure 1C). The genes in other metabolic pathways, including the tricarboxylic acid cycle cycle, pentose phosphate pathway, pyruvate oxidation, and fatty acid metabolism were either not different or slightly increased between Tbx18-PCs and control-VMs (Figure S1). The differential change in Aldoc transcripts between Tbx18-PCs and control-VMs was confirmed via real-time PCR (Figure 1D).
To correlate functional changes in transcriptome expression, glycolysis and mitochondrial function in Tbx18-PCs were further analyzed using Seahorse functional assays. Glycolysis activity, including basal and compensatory glycolysis, and proton efflux rate in Tbx18-PCs were lower than in control-VMs (Figure 1E). Mitochondrial function, including basal respiration, proton leak, and ATP production, did not differ between the 2 groups (Figure S2). The downregulation of glycolysis was correlated with the reduced levels of Aldoc and suggested that the increased expression of metabolic genes other than Aldoc was likely compensatory. To confirm this idea, we performed metabolomics analysis via liquid chromatography–mass spectrometry to comprehensively delineate the metabolite levels of the aforementioned pathways in Tbx18-PCs and control-VMs.
The levels of tricarboxylic acid cycle cycle metabolites, energy molecules (eg, ATP and NADH), pyruvate conversion metabolites, and pentose phosphate pathway metabolites were mostly not statistically different between Tbx18-PCs and control-VMs (Figure S3). Only within the glycolysis process, almost all metabolites decreased. A maximal decrease was observed in the levels of glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP, Figure 1F), which reached the nadir of all glycolysis metabolites (Figure 1G). Aldoc catalyses reversible aldolase cleavage of fructose 1,6-bisphosphate to DHAP and G3P (Figure 1C).21 The critical reduction in DHAP and G3P levels was consistent with the reduction in Aldoc expression. The reduction in lactate levels (end-product of glycolysis) was further confirmed by a colorimetric assay (Figure 1H). These results reflected low glycolysis activity in the single culture of PCs and demonstrated the key role of reduced Aldoc expression in determining glycolysis status.
Whether glycolysis activity in PCs has functional implications must be determined. Supplementation with sodium pyruvate to replenish the energy supply from glycolysis in Tbx18-PCs increased the beating rates of PCs and potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4 (Hcn4) expression (distinct ion channels that initiate electrical impulses in PCs, Figure 1I). Treatment with 2-deoxy-D-glucose to inhibit glycolysis decreased both beating rates and Hcn4 expression in Tbx18-PCs (Figure 1I). Considered a critical hub in the glycolysis process, adenoviral vector-mediated overexpression of Aldoc was performed to reverse Aldoc expression in Tbx18-PCs (Figure 1J). The increased expression of Aldoc improved beating rates and Hcn4 expression (Figure 1K). These results indicate that reduced Aldoc levels and glycolysis status dysregulated PC rhythmicity.
Fibroblasts Drive Glycolysis Adaptation Through Aldoc in Tbx18-PCs
Next, we determined whether fibroblasts regulate pacemaker rhythm through intrinsic glycolysis within Tbx18-PCs. Coculture with fibroblasts improved global glycolysis function, including basal and compensatory glycolysis, as well as the proton efflux rate, compared with Tbx18-PCs alone (Figure 2A). Mitochondrial function (oxidative phosphorylation), including basal respiration, spare respiratory capacity, proton leak, and ATP production did not differ between the 2 groups (Figure 2B). Based on metabolomics analysis, compared with the drastic decrease in G3P and DHAP in the single culture of Tbx18-PCs, coculture with fibroblasts significantly increased G3P and DHAP, as well as fructose 1,6-bisphosphate, through the reverse reaction of aldolase from DHAP (Figure 2C).22 This led to the upregulation of downstream metabolites, such as 2-phosphoglyceric acid and phosphoenolpyruvate. The levels of tricarboxylic acid cycle cycle metabolites, energy molecules (eg, ATP and NADH), pyruvate conversion metabolites, and pentose phosphate pathway metabolites were mostly marginally increased or not statistically different between Tbx18-PCs and cocultures (Figure S4). The increased levels of DHAP were validated by an ELISA (Figure 2D).
Aldoc expression in the cocultures was higher than that in the single PC cultures (Figure 2E), thus supporting the notion that Aldoc expression underlies the increase in G3P and DHAP levels. In addition, coculture with fibroblasts was associated with better pacemaker phenotypes such as beating rate (Figure 2F), as well as with the expression of PC-specific genes (Hcn4 and Cx45 [connexin 45], Figure 2G). Spontaneous local Ca2+ release events are a hallmark of automaticity in PCs.23,24 Spontaneous local Ca2+ release events could be observed in Tbx18-PC cocultures. The inhibition of Aldoc by the Aldoc siRNAs decreased both spontaneous local Ca2+ release events and oscillating calcium transients (Figure S5). The spontaneous local Ca2+ release event period, as observed in the Tbx18-PC cocultures, was linearly correlated with the cycle length of oscillating calcium transients (Figure S5). Overall, these findings suggest that Aldoc regulates both membrane and calcium clocks within PCs.
The improvement in glycolysis was related to the intrinsic regulation of PCs but not to contamination of fibroblasts. First, lactate levels increased in contact cocultures, which supported the improvement of glycolysis function after coculture with fibroblasts (Figure 2H). However, if we performed a separate coculture (coculture of fibroblasts and PCs, but PCs and fibroblasts were separated by porous membranes), the levels of lactate did not increase. Lactate levels in the single culture of fibroblasts were also low. These findings indicate that the microenvironment contributed to the improvement in glycolytic activity through contact between fibroblasts and PCs. Moreover, we did not observe any Aldoc protein expression in fibroblasts (Figure 2I and Figure S6). These results indicate that the improvement in glycolysis was due to intrinsic regulation of Aldoc within PCs.
The regulatory enzymes of glycolysis in Tbx18-PC cocultures were different from those in cocultures of control-VMs and fibroblasts (Figure S7). In contrast to increased Aldoc expressions within Tbx18-PCs, fibroblasts increased the transcripts of aldolase a in control-VMs after coculture. Therefore, we observed that glycolysis function and DHAP levels improved in both Tbx18-PCs and control-VMs after coculture with fibroblasts (Figure S7).
Fibroblasts Switched on Aldoc Expression in PCs Through Integrin-Dependent Signals
The mechanisms by which fibroblasts regulate Aldoc expression in PCs were further explored. After coculture with fibroblasts, PCs were isolated via cell sorting (Figure S8). The whole transcriptome expression in isolated PCs from PC-fibroblast cocultures was compared with that from PCs in single PC cultures (Figure 3A, Supplemental Data S4). The analysis of glycolysis-related genes revealed that the highest transcriptional changes (4.3-fold increment) were observed at Aldoc levels compared with the other glycolysis enzymes (Figure 3B). Other metabolic genes related to pyruvate oxidation, tricarboxylic acid cycle, the pentose phosphate pathway, and fatty acid metabolism were either minimally or not statistically different (Figure S9). Again, Aldoc was the key enzyme critically regulated by fibroblasts. The relevant pathways related to metabolic/energetic regulation of automaticity, mainly those within the calcium clock, were analyzed (Figure S10).24,25 The spontaneous electrical activity of PCs, especially calcium clock, is driven by a cAMP-mediated phosphorylation, which critically relies on the balance between the cAMP production by adenylyl cyclases and degradation by cyclic nucleotide Pde (phosphodiesterases).24,25 The coculture with fibroblasts decreased the expression of Pde4a (phosphodiesterase) and might regulate cAMP-mediated protein phosphorylation and the calcium clock in PCs.26
We performed Ingenuity Pathway Analysis to explore the pathways to potentially regulate Aldoc expression, including canonical pathways and gene ontology (Figure 3C, Tables S3 and S4, Supplemental Data S5 and S6). Within canonical pathways (Table S3), those related to ECM (eg, collagen and laminin), relevant surface receptors (integrins) and their downstream signals (PI3K [phosphoinositide 3-kinases]/Akt and MAPK [mitogen-activated protein kinase]) were repeatedly observed. These results were compatible with the finding that direct contact between fibroblast-PCs and the ECM is critical to drive metabolic reprogramming in PCs. Therefore, we comprehensively analyzed integrins, which are the predominant ECM-binding receptors in cardiomyocytes (Figure 3D).27 The Itgb1 (integrin subunit β1) transcripts were the most abundant and increased significantly. We further treated Tbx18-PC coculture with an Itgb1 inhibitory antibody, which decreased integrin activation, Aldoc expression (Figure 3E), and the beating rate (control versus Itgb-1 antibody: 140.4±92.0 bpm, n=23 versus 20.6±55.7 bpm, n=16, P=1.2×10−5). This indicates that fibroblasts activate Aldoc and glycolysis activity through integrin-dependent signals.
The common downstream signals of integrins within cardiomyocytes include PI3K/Akt and MAPK (p38 and ERK).27,28 The expression of phosphorylated and total Akt did not change between cocultures and single cultures of PCs (Figure 3F and Figure S11A and S12). Treatment with a PI3K inhibitor (wortmannin) in PC cocultures increased Aldoc expression (Figure 3G). These results indicate that PI3K/Akt activity did not activate Aldoc expressions after coculture. Considering MAPK pathways, ERK activation was suppressed after coculture as p-ERK (phosphorylated ERK) was lower in cocultures than in the single culture of PCs (Figure 3H and Figure S11B and S13). Instead, phosphorylated and total p38-MAPK increased after coculture (Figure 3H and Figure S11C and S14). Treatment of cocultures with a p38-MAPK inhibitor (SB203580) decreased Aldoc expression, suggesting that p38-MAPK activation is a downstream signal of integrins (Figure 3I) to increase Aldoc expression. The p38-MAPK activation induces the dissociation of Rb and E2F1, and increases E2F1-driven transcriptional activity.29 Increased expression of Rb, phosphorylated Rb, and E2F1 was observed in cocultures (Figure 3J, Figure S11D and S11E, S15, and S16). There are multiple E2F1 binding sites on the promotor region of Aldoc, suggesting that E2F1 is a transcriptional regulator of Aldoc (Figure 3K). Inhibition of p38-MAPK, Rb, and E2F1 via siRNA decreased Aldoc expression (Figure 3L and Figure S17). We further performed an in vivo experiment to clarify the link between Aldoc expression and integrin-dependent signaling. Mice that received an intraperitoneal injection of the Itgb1 inhibitory antibody (a functional blocking antibody) had lower Aldoc expression in SANs than control mice (Figure 3M). Overall, these results suggest that fibroblasts induce Aldoc expression in PCs through β1-integrin activation and downstream p38-MAPK/E2F1 signaling.
Engineered Tbx18-PC Tissue Sheets Recapitulated Aldoc-Driven Rhythmic Machinery
The microenvironment with fibroblasts is essential for the functional integrity of pacemaker tissue. We further constructed an in vitro Tbx18-PC tissue sheet to mimic the 3-dimensional microenvironment of in vivo SANs to study the regulatory role of Aldoc in pacemaker rhythmicity. The PC tissue sheet was induced by re-expression of Tbx18 in an engineered tissue, which was constructed by mixed culture of VMs and fibroblasts with Matrigel (Figure 4A). Compared with control tissue sheets (GFP expression), re-expression of Tbx18 induced spontaneous electrical firing that was recorded with a microelectrode array (Figure 4B). A sympathomimetic drug (epinephrine, α- and β-receptor sympathetic agonist) increased the beating rate of Tbx18-PC tissue sheets (Figure 4C). Through immunofluorescence staining, PCs with distinct pacemaker ion channels (HCN4, Figure 4D and Figure S18) and Cx45 (Figure S19) were observed in Tbx18-PC tissue sheets but not in controls. PC tissue sheets also had higher expression of PC-specific genes (Hcn4 and Cx45) than control tissue sheets (Figure 4E). These findings suggest that engineered Tbx18-PC tissue sheets recapitulated the phenotypes of a native SAN. Aldoc-driven rhythmic machinery within a 3-dimensional microenvironment was further explored. Aldoc expression in PC tissue sheets was higher than controls (Figure 4F). Treatment of PC tissue sheets with Aldoc siRNAs reduced Aldoc expression (Figure S20) and spontaneous electrical activity (Figure 4G). Engineered Tbx18-PC tissue sheets recapitulated the microenvironment of de novo SAN tissues and suggested that the Aldoc-driven glycolysis machinery regulates PC rhythmicity in a 3-dimensional microenvironment.
The Regulation of In Vivo Pacemaker Rhythms by Aldoc in Vertebrates
The regional distribution of Aldoc and its physiological function in the hearts of vertebrates were further explored. In rats and mice, through immunofluorescence staining, Aldoc expression was observed exclusively within SANs but not in the atrium or ventricle (Figure 5A). This finding was consistent with the abundance of Aldoc transcripts in rat SANs, while Aldoc expression was almost undetectable in the atria and ventricles (Figure 5B).30 We further performed in vivo knockdown of Aldoc in mouse SANs to determine whether Aldoc regulates the pacemaker activity of SANs. The efficiency of AAV9 (adeno-associated virus 9) Aldoc siRNAs in reducing Aldoc expression was first confirmed via in vitro transduction of mouse cardiomyocytes (Figure S21). Then, AAV9 Aldoc siRNAs were delivered into the pericardial recess around mouse SANs through a mini-thoracotomy and reached ≈80% transduction efficiency (Figure S22). The in vivo Aldoc expression in the mouse SAN was successfully reduced (Figure 5C and 5D). Mice that received Aldoc siRNAs had a lower spontaneous heart rate than those that received the scrambled controls (Figure 5E and 5F). In addition, the responses to epinephrine were nullified in mice that received Aldoc siRNAs (Figure 5F). Accordingly, the expression of Aldoc within the SAN might not only drive glycolysis machinery but also regulate in vivo PC rhythmicity.
Aldoc Regulated Pacemaker Activity in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes
Induced pluripotent stem cell-derived cardiomyocytes (IPS-CMs) were used as a human cell model to study whether Aldoc regulates pacemaker activity in human PCs. HCN4 (+) PCs accounted for ≈10% of IPS-CMs31,32 and were responsible for the rhythmic beating of IPS-CMs.33 Aldoc expression was predominantly observed in human HCN4 (+) PCs (Figure 6A and 6B). Overexpression of Aldoc in IPS-CMs via adenoviral vectors significantly increased Aldoc levels in IPS-CMs (Figure 6C) and the electrical firing rate of IPS-CMs (Figure 6D and 6E). Human PCs share similar machinery in which Aldoc drives metabolic adaptation to regulate pacemaker rhythm, highlighting their translational potential for the study of human SAN physiology or disease prevention.
Discussion
Years after observing the clinical linkage of the microenvironmental niche in SANs with pacemaker failure, the exploration of its biological process remains a rocky terrain.11,34 From the induced rat PCs and engineered tissue sheet models to human PCs and in vivo animal experiments, we found that fibroblasts induce metabolic reprogramming by upregulating Aldoc in PCs through integrin-dependent MAPK-E2F1 signals. This enhanced aerobic glycolysis and regulated the rhythmicity of SANs. Interruption of the fibroblast-PC Aldoc interaction stagnated SAN electrical activity. Aldoc-driven energy replenishment may restore SAN dysfunction and might be a future device-free therapeutic target. Establishment of engineered tissue sheets can hopefully be used as an in vitro experimental model for the study of SAN diseases and preclinical tests.
Fibroblast and PC interactions in SANs are rarely addressed. Only a mathematical model suggests that fibroblasts, as stretch sensors during atrial diastole, raise the spontaneous depolarization rate of PCs.35 Our findings uncovered functional implications of the cellular architecture of SANs showing that fibroblasts drive PC-specific Aldoc expression to modulate cardiac rhythm. Constitutive Aldoc expression has mainly been observed in cancer cells or neuronal cells in the brain and causes cancer progression and neurodegeneration.21,36,37 The physiological roles of Aldoc in the heart are far from clear. The constitutive expression of Aldoc in the atria and ventricles is seen in amphibians but not in mammals.36,38 Aldoc seems inducible only in quiescent cardiomyocytes during hypoxia, which activates glycolysis (anaerobic glycolysis) to compensate for energy deficiency from impaired mitochondrial function.39–41
In SANs, the presence of fibroblast-PC interactions activated integrin-dependent pathways and maintained constitutive Aldoc expression within PCs. Even while the mitochondrial oxidative phosphorylation function remained activated and unchanged (as shown in the Seahorse, whole transcriptome, and metabolomics analyses), Aldoc in PCs activated glycolysis (aerobic glycolysis, Warburg effect)42 and regulated beating rates in SANs. This phenomenon was observed either at rest or after treatment with sympathomimetic drugs to mimic exercise. Aerobic glycolysis is an important energy machinery for proliferative cells, including cancer cells, stem cells, and endothelial cells in the heart.42 The present results extend the physiological implications of aerobic glycolysis from cell proliferation to relentless electrical firing in PCs. Most intriguingly, this machinery needs to be maintained by the microenvironment between fibroblasts and PCs.
The upregulation of Itga5 and Itgb1 after the coculture with fibroblasts in the present work suggests that the integrin heterodimer (α5β1) is a critical receptor that drives fibroblast-induced glycolysis signals. The α5β1 is predominantly a fibronectin receptor, which corresponds to the abundant expression of fibronectin in the sinus node ECM.30 Fibroblast-driven Aldoc-mediated PC regulation is similarly observed across different vertebral PC cells, including rats, mice, and human cells, suggesting its physiological importance in SAN function. The critical role of α5β1 in the fibroblast-SAN interaction, ECM, and microenvironment is worth further investigation. The paucity of connexin expression at fibroblast-PC contacts, including Cx45 and Cx43 (data not shown), might be associated with low electrical conductivity across heterocellular junctions. It remains unclear whether the low conductivity between fibroblasts and PCs might be related to the initiation or organization of rhythmic electrical activity.
Aldoc expression are confined to the central nervous system and is not found in the peripheral nervous system (which innervates the heart or SAN). Aldoc is only detected in Purkinje cells, a specific type of cerebellar neuronal cell within the cerebellum.43,44 In other areas of the central nervous system, Aldoc is not detected within neuronal cells and is only observed in nonneuronal cells (astrocytes, some cells of the pia mater) within the hippocampus and thalamus.43,44 Additionally, in our study, Aldoc was found to be expressed exclusively within the cytoplasm of cardiomyocytes in SANs by immunofluorescence staining. These findings suggest that Aldoc is activated within PCs but not via SAN innervation by nerve terminals.
We could never successfully explore the machinery behind PC and fibroblast interactions without the engineered Tbx18-PC and tissue sheet models. Tbx18-PCs and tissue sheets recapitulate the phenotypes of de novo SANs including distinct genes related to pacemaker functions (Hcn4, Cx45), spontaneous electrical firing, the calcium clock, and responses to autonomic stimulation.14 In addition, metabolic machinery including Aldoc-driven glycolysis and PC rhythmicity was similarly replicated in engineered Tbx18-PCs, native SANs in animals, and PCs among IPS-CMs. This similarity underlies the successful translation of fibroblast-driven glycolysis pathways in induced Tbx18-PCs and their rhythmicity to de novo PCs. The engineered Tbx18-PCs and tissue sheets may represent an in vitro platform to study SAN diseases, at least those related to energy metabolism. The SAN is a complex 3-dimensional structure comprising different structures, such as pacemaker cells, transitional cells, fibroblasts, and autonomic nerve terminals. The present engineered model only partially recapitulated SAN phenotypes. Additionally, it should be noted that not all VMs were transduced with Tbx18 and successfully reprogrammed. The Tbx18 transduction rate was 87.3±10.9% (n=3). The reprogramming rate (ie, the ratio of HCN4 [+] cells within Tbx18-transduced cardiomyocytes) was 25.8±14.7%.
Clinical reports addressing Aldoc in SAN pathologies are not well established. This is probably not a limitation but rather offers new hope to understand the mechanisms of SAN diseases and develop future therapy. For example, age-dependent degeneration is a common cause of SAN dysfunction,11 and a reduction in blood Aldoc expression is observed with aging.45 Exploring the potential role of reduced Aldoc expression in the aging SAN might give birth to a target strategy involving Aldoc replenishment and restoration of SAN failure. This could not only be considered a future device-free therapy but also part of preventive medicine. Regulation of glucose-related cardiac metabolism by diet, lifestyle, exercise, or medication for the primary prevention of cardiac disease is not new.46
Article Information
Author Contributions
P.-C. Chou, C.-H. Weng, and J.-D. Liu performed immunostaining, PCR, in vivo and in vitro cell experiments, and animal experiments; K.-C. Yang performed whole transcriptome analysis. M.-L. Cheng performed metabolomics analysis; R.-B. Yang and Y.-C. Lin performed the integrin and mechanism experiments. B.-C. Shyu assisted in microelectrode array experiments. M. Hsiao and S.-K. Shyue assisted in the gene interference experiments. S.-P. Chen reviewed the results. P.-C. Chou, C.-H. Weng, and C.-M. Liu wrote the article. Y.-F. Hu designed the project and revised the article.
Supplemental Materials
Expanded Methods and Materials
Data Set 1–6
Acknowledgments
We thank Wei-Chi Wang for bioinformatics analysis and I-Chien Wu for statistical analysis consultation.
Footnote
Nonstandard Abbreviations and Acronyms
- Aldoc
- aldolase c
- Cx45
- connexin 45
- DHAP
- dihydroxyacetone phosphate
- ECM
- extracellular matrix
- G3P
- glyceraldehyde 3-phosphate
- Hcn4
- potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4
- IPS-CMs
- induced pluripotent stem cell-derived cardiomyocytes
- Itgb1
- integrin subunit β1
- MAPK
- mitogen-activated protein kinase
- PCs
- pacemaker cardiomyocytes
- Pde
- phosphodiesterase
- Pde4a
- phosphodiesterase
- p-ERK
- phosphorylated ERK
- PI3K
- phosphoinositide 3-kinase
- SAN
- sinoatrial node
- Tbx18
- T-box transcription factor 18
- Tbx18-PC
- Tbx18-induced PC
- VMs
- ventricular cardiomyocytes
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Received: 28 November 2021
Revision received: 28 April 2022
Accepted: 3 May 2022
Published online: 25 May 2022
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This work was supported by Taipei Veterans General Hospital (V110C-039, V110B-043, V111C-047, VGHUST111-G6-3-2, VTA111-A-1-2), the Ministry of Science and Technology (110-2628-B-075-015, 110-2314-B-075-063-MY3), National Health Research Institutes (NHRI-109BCC0-MF-202014-02), and Academia Sinica (AS-TM-110-01-01).
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