Graphical abstract graphic file with name fx1.jpg [69]Open in a new tab Highlights * • ENPP1 is known to initiate an aberrant metabolic cascade after myocardial infarction * • A humanized monoclonal antibody targeting human ENPP1 is engineered (hENPP1mAb) * • Systemic administration of hENPP1mAb rescues post-infarct metabolic defects * • In humanized mice, a single dose of hENPP1mAb rescues post-infarct heart function __________________________________________________________________ After myocardial infarction, the ectonucleotidase ENPP1 initiates an aberrant metabolic cascade that disrupts nucleotide biosynthesis and worsens heart repair. Li et al. engineer a humanized monoclonal antibody targeting ENPP1 (hENPP1mAb) and demonstrate that a single dose of hENPP1mAb in humanized animals rescues metabolic defects and enhances post-infarct heart function. Introduction The adult mammalian heart has a limited ability to regenerate and heals via a fibrotic repair response after acute myocardial infarction (MI).[70]^1 MI contributes to 40%–70% of all cases of incident heart failure, and the degree of cardiac fibrosis in failing hearts has been shown to be an independent prognostic indicator of survival and adverse cardiovascular events.[71]^2^,[72]^3 Modulation of cardiac wound healing to redirect the cardiac injury response from a fibrotic to a reparative remains a broad goal of cardiovascular therapeutics. Following MI, the cardiac microenvironment significantly changes with the recruitment of a diverse population of cells in a precise spatiotemporal manner,[73]^4 enabling cellular crosstalk between myocytes and non-myocytes that is thought to affect various phases of wound healing.[74]^5 Cell-cell crosstalk has been regarded as an attractive target to enhance cardiac repair as crosstalk affects multiple repair processes such as fibrosis, inflammation, angiogenesis, and cell metabolism.[75]^5 Yet, there are no drugs available that enhance cardiac wound healing after acute MI. We have recently described a metabolic crosstalk between myocytes and non-myocytes in the infarcted heart that affected cardiac repair.[76]^6 Following ischemic cardiac injury, the extracellular ATP concentration increases from extravasation of intracellular ATP from injured myocytes.[77]^7^,[78]^8 We showed that an ectonucleotidase ENPP1 (ectonucleotide phosphodiesterase/pyrophosphatase 1) was significantly upregulated in the infarcted heart.[79]^6 ENPP1 is a type II transmembrane protein that hydrolyzes extracellular ATP into AMP.[80]^6^,[81]^9 We showed that AMP generated by ENPP1 initiated a deleterious metabolic crosstalk between myocytes and non-myocytes that led to the formation of adenine and purine nucleosides and disrupted pyrimidine biosynthesis in cycling and non-cycling cells. A metabolic catastrophe caused by defects in pyrimidine biosynthesis resulted in functional defects in both myocytes and non-myocytes and worsened cardiac repair.[82]^6 These observations suggest that ENPP1 could serve as a potential molecular target for augmenting cardiac repair. However, the efficacy of targeting human ENPP1 for cardiac repair remains unknown. Here, we engineer a humanized monoclonal antibody targeting human ENPP1, create humanized ENPP1 mice that express the human instead of the murine ortholog, and demonstrate that administration of hENPP1mAb leads to transcriptional and metabolic rewiring of the infarcted heart and leads to superior post-infarct heart function. In animals genetically engineered to express antibody clearance kinetics similar to that in humans, we demonstrate that a single dose of humanized hENPP1mAb is sufficient to enhance post-infarct cardiac repair with significantly better preservation of post-infarct heart function. Results Generation of a humanized monoclonal antibody against human ENPP1 that is potent and highly specific We initially generated a monoclonal antibody targeting the extracellular catalytic domain of human ENPP1. For this purpose, a peptide of 840 amino acids that represented the catalytic domain of human ENPP1, and synthesized in a mammalian cell line, was injected into wild-type BALB/c mice followed by harvesting of splenic cells, generation of hybridomas, screening of hybridoma supernatants, and selection of the appropriate clone ([83]Figure S1). The selected monoclonal was subsequently humanized for development as a clinical candidate, where the complementarity-determining regions (CDRs) of the antibody were grafted onto a human antibody scaffold,[84]^10 and the degree of “humanness” of the humanized antibody was scored with proprietary software. The final humanized ENPP1 monoclonal antibody (hENPP1mAb) was thereafter synthesized in a recombinant manner in a stable mammalian cell line ([85]Figure S1). We overexpressed human ENPP1 in human embryonal kidney (HEK) cells and using flow cytometry observed the dissociation constant (K[D], that provides a measure of avidity) of hENPP1mAb to be 0.08 nM ([86]Figure 1A). A luciferase-based cell-free assay to determine the potency of hENPP1mAb to inhibit human ENPP1 catalytic activity demonstrated an IC[50] of 1.3 nM ([87]Figure 1B). As the crystal structure of ENPP1 has been described,[88]^11^,[89]^12 we next performed homology modeling and protein-protein docking interactions using computational tools, and hENPP1mAb was predicted to bind near the active site of human ENPP1 ([90]Figure 1C). All the best-scoring models on protein-protein docking demonstrated that hENPP1mAb significantly blocked access to the enzyme site, and portions of the CDR L-2 and CDR H-3 were predicted to insert into the active catalytic site of human ENPP1 ([91]Figure 1D). We next examined the binding of hENPP1mAb to a variety of related human ectonucleotidases and other human proteins. As members of the ENPP family such as ENPP3, ENPP4, and ENPP5 have been shown to possess ectonucleotidase activity,[92]^13 we cloned and overexpressed these genes in HEK cells, but hENPP1mAb did not bind to other members of the human ENPP family on flow cytometry, demonstrating binding specificity only to human ENPP1 ([93]Figure 1E). hENPP1mAb also did not bind to other ectonucleotidases active in the heart such as CD39 or CD73, but ENPP3, CD39, and CD73 antigens were recognized by antibodies specific to those antigens ([94]Figure S2A). As there are no antigen-specific antibodies for ENPP4/5 for flow cytometry, to demonstrate membrane expression, we created ENPP-GFP fusion constructs and demonstrate successful expression of hENPP5 on the cell surface but lack of binding to hENPP1mAb on flow cytometry ([95]Figures S2B and S2C). As a positive control, with flow cytometry, hENPP1mAb recognized hENPP1-GFP fusion constructs expressed on the cell surface demonstrating the specificity of hENPP1mAb for hENPP1 ([96]Figures S2B and S2C) To determine off-target binding to any human protein, we utilized a membrane array system where more than 6,000 secreted and membrane-bound human proteins are expressed on the cell surface of HEK cells.[97]^14^,[98]^15 An immunoassay is then performed and scored to determine the binding of hENPP1mAb to each of the greater than 6,000 human proteins on the array. Using this assay, with appropriate positive and negative controls, we observed that hENPP1mAb only binds to the secreted and membrane-bound forms of hENPP1 and does not demonstrate specific binding to any other human protein ([99]Figures 1F–1H). Figure 1. [100]Figure 1 [101]Open in a new tab Avidity, potency, homology modeling, and off-target binding assays for hENPP1mAb (A) Dissociation constant of hENPP1mAb calculated with flow cytometry to determine concentration-dependent binding of hENPP1mAb to HEK cells overexpressing human ENPP1. Calculated K[D] value (measure of avidity) shown (n = 3). (B) Potency curve of hENPP1mAb in inhibiting human ENPP1 catalytic activity in a concentration-dependent manner. Calculated IC50 shown (n = 3). (C and D) Homology modeling and protein-protein docking interactions show the (C) docked model of hENPP1mAb and human ENPP1. The antibody chains (CDR of light and heavy chains) occlude the enzyme active site (dark gray surface) (human ENPP1 antigen in blue, Fab segment of hENPP1mAb in green, CDRs color coded as shown). (D) Magnified image demonstrating several CDRs of hENPP1mAb inserting into human ENPP1 catalytic domain. (E) Flow cytometry to determine binding of hENPP1mAb to other members of human ENPP family and to other phosphatases (n = 5/group). (F–H) Retrogenix membrane array screening by immunoblotting to determine binding of hENPP1mAb to 6,101 human plasma membrane proteins and 396 human heterodimers expressed on HEK cells. (F) hENPP1mAb shows a significant specific interaction with human ENPP1 isoforms (red) (plasma membrane isoforms upper and middle, and tethered secreted form, lower). Note hENPP1mAb also shows binding to IGHG that may serve as IgG receptors. (G) Rituximab is used as a positive control to determine any non-specific binding of a mAb and demonstrates no binding to human ENPP1 and binding to IGHG proteins. (H) PBS is used as a negative control for the entire assay and demonstrates signal against the IGHG proteins as well. Immunoblotting demonstrates representative images of n = 3. Data are represented as mean ± SEM. Generation of a humanized ENPP1 animal for determining in vivo efficacy of hENPP1mAb As the ENPP1mAb was initially isolated from mice following injection of the human ENPP1 peptide fragment, we next determined species reactivity of hENPP1mAb against ENPP1 orthologs of different species. For this purpose, we cloned the ENPP1cDNA of mouse, rat, pig, monkey, and human, overexpressed them in HEK cell lines, and determined binding of the hENPP1mAb to ENPP1 orthologs of different species by flow cytometry ([102]Figure 2A). We observed that hENPP1mAb exhibited binding to human and monkey ENPP1 but not to ENPP1 orthologs of other species tested ([103]Figures 2A and [104]S2C). Figure 2. [105]Figure 2 [106]Open in a new tab Species reactivity of hENPP1mAb and development of humanized ENPP1 mice (A) Flow cytometry to determine binding of hENPP1mAb against mouse, rat, pig, monkey, and human ENPP1 overexpressed in HEK cell line. HEK cells expressing eGFP used as a negative control (n = 3 for eGFP and monkey, n = 5 in mouse, rat, pig, and human). (B) Schematic representation of generation of the humanized ENPP1 mouse. Using CRISPR-Cas9, human ENPP1CDS with a PolyA signal at the 3′ end is inserted to replace the 1st exon of murine ENPP1 gene. (C) Agarose gel electrophoresis of RT-PCR products of heart tissue from humanized ENPP1 mice or wild-type C57BL/6J mice (n = 3 animals/group). Discriminatory PCR primers are used to distinguish murine and human ENPP1 expression. (D) qPCR demonstrating ENPP1 gene expression in the injured region of the heart compared with uninjured region at 7 days after MI (n = 3 animals/group). (E) qPCR on infarcted heart of humanized ENPP1 mouse at day 7 post MI demonstrating the absence of murine ENPP1 and expression of human ENPP1 in the infarcted region (n = 3 animals/group). (F) Immunostaining for ENPP1 (green, arrowheads) and cardiac troponin I (red) in the injured regions at day 7 after MI. Magnified images demonstrate cells in the infarcted region of the inset expressing human ENPP1 (arrowheads). Note that ENPP1 expression is present in troponin-negative regions. Data are represented as mean ± SEM. ∗p < 0.05, Statistical significance was determined using Student’s t test, 2 tailed. As the hENPP1mAb did not exhibit binding to ENPP1 orthologs of lower species, and to test the efficacy of the hENPP1mAb in vivo, we genetically engineered a mouse expressing human ENPP1 and not the mouse ortholog (humanized ENPP1 mouse). To generate the humanized ENPP1 mouse, we used CRISPR-Cas9 genome editing to knock in the hENPP1CDS into the murine ENPP1 gene loci (first exon). A polyadenylation signal was included in the knockin cassette (downstream of hENPP1 CDS) to prevent murine ENPP1 gene transcription ([107]Figure 2B). Using discriminatory primers, we observed that hearts of humanized ENPP1 animals expressed human but not murine ENPP1 ([108]Figure 2C). Next, we subjected the humanized ENPP1 animal to MI via ligation of the left anterior descending coronary artery and observed by qPCR that the expression of ENPP1 gene increased significantly in the infarcted area ([109]Figure 2D) similar to what we had observed in post-MI wild-type animals. The infarcted heart of the humanized ENPP1 animals demonstrated increased gene expression of human but not mouse ENPP1 ([110]Figure 2E) suggesting that regulatory elements driving post-injury ENPP1 expression were not disrupted in the humanized ENPP1 mouse. Immunostaining of the infarcted region demonstrated upregulated ENPP1 protein expression predominantly in the region of the scar ([111]Figure 2F). The humanized ENPP1 mouse thus recapitulates ENPP1 expression in the wild-type mouse after MI and provides a platform to test the efficacy of hENPP1mAb for MI. hENPP1mAb protects animals against MI-induced cardiac dysfunction We subjected the humanized ENPP1 mouse to MI. The hENPP1mAb has an IgG1 backbone and, in contrast to humans, the half-life of an IgG antibody is in the order of a few days in mice and thus necessitates repeated administration.[112]^16 We injected the hENPP1mAb, 10 mg/kg, i.p. (intra-peritoneally) twice weekly (every 3 days) and administered the first dose 3 days prior to injury followed by repeated i.p. administration every 3 days for 12 days post injury ([113]Figure 3A). Control animals were injected with human IgG in an identical manner and concentration ([114]Figure 3A). Western blotting on whole heart lysates demonstrated that ENPP1 protein expression in the heart increases robustly within 7 days of MI ([115]Figures 3B and 3C). To confirm that the hENPP1mAb inhibits ENPP1 nucleotidase activity in vivo, we first isolated infarcted hearts of humanized ENPP1 animals at 7 days post MI. We measured ATP hydrolytic activity and observed that in IgG-injected animals, injury induced an increase in ATP hydrolytic activity consistent with increased ENPP1 activity, but in hENPP1mAb-injected animals, there was no increase in ectonucleotidase activity after injury ([116]Figure 3D). Animals were subjected to weekly B and M mode echocardiography, and we observed that by day 7 post MI, ejection fraction (EF) and ractional shortening (FS) in hENPP1mAb-injected animals were almost double that of IgG-injected animals (EF 47.34% ± 2.86% in hENPP1mAb versus 24.95% ± 2.95% in IgG group, and FS 23.70% ± 1.55% in hENPP1mAb versus 11.66% ± 1.48% in IgG groups, p < 0.01) ([117]Figures 3E and 3F). The end systolic dimension of the ventricle (LVIDs) was also significantly decreased in the hENPP1mAb-injected animals compared to IgG-injected controls, demonstrating decreased post-infarct ventricular dilatation in hENPP1mAb-injected animals ([118]Figures 3E and 3F). The functional benefits persisted at 4 weeks approximately 2 weeks after completion of the last dose of hENPP1mAb administration ([119]Figure 3F). To determine the effects of the hENPP1mAb on post-infarct heart failure, we stratified the post-infarct EF (week 4) as severe heart failure (EF<20%), moderate heart failure (EF: 20%–40%), and mild heart failure (EF>40%). The fraction of animals that developed severe heart failure at 4 weeks in the IgG-injected group was 52% but only 5% of the animals in the hENPP1mAb-injected group developed severe heart failure demonstrating an order of magnitude improvement in the severity of post-infarct heart failure with hENPP1mAb therapy ([120]Figure 3G; [121]Table S1). Analysis of survival curves also did not show any significant difference between the IgG and the hENPP1mAb-injected animals ([122]Figure S3). To examine the scar size and cardiac remodeling in greater detail, we next performed contrast-enhanced gated cardiac computed tomography (CT) scans of live animals at 14 days following hENPP1mAb or IgG injection. Transverse cuts on gated cardiac CT demonstrated a far more dilated left ventricle in the IgG-injected animals, and coronal slices demonstrated a smaller anterior wall scar with thicker ventricular walls in the hENPP1mAb-injected animals ([123]Figure 3H). Consistent with echocardiography, EF estimated by gated cardiac CT imaging was also significantly higher in the hENPP1mAb-injected animals ([124]Figure 3I), and robust vigorous contraction of the left ventricular walls was noted on gated cardiac CT imaging in the hENPP1mAb-injected animals ([125]Videos S1 and [126]S2). We next performed strain imaging to determine the contractile forces (wall strain) generated by various segments of the infarcted mouse heart in hENPP1mAb versus IgG-injected animals.[127]^17 Strain imaging simply represents the deformation of the cardiac muscle in systole and can be represented as ΔL/L[0],[128]^18 where ΔL represents the change of the muscle fiber/segment in length at the end of systole and L[0] is the initial muscle fiber length prior to onset of systole (as the length is shortening during systole, strain numbers are usually represented as negative in value). The segments of the heart were divided from base to apex and posterior to anterior ([129]Figure 3J). We observed that the wall strain (contractile force) was significantly greater (deeper color on heatmap) across all segments of hearts of animals that received hENPP1mAb ([130]Figure 3J). Quantification of strain forces demonstrated several fold higher strain forces generated in the myocardial segments of animals that received hENPP1mAb compared to IgG ([131]Figure 3K). Figure 3. [132]Figure 3 [133]Open in a new tab hENPP1mAb attenuates post-infarct cardiac dysfunction in humanized ENPP1 animals (A) Strategy for hENPP1mAb administration in humanized ENPP1 animals subjected to MI. (B) Western blotting for ENPP1 in wild-type mice hearts at 3, 7, and 14 days following MI. (C) Quantitative densitometry of ENPP1 level (n = 3). (D) Extracellular ATP hydrolytic activity in injured and uninjured hearts of animals treated with IgG or hENPP1mAb (n = 4 animals/group). (E) B (top) and M-mode (below) echocardiogram demonstrating superior contractile function in hENPP1mAb-treated animals. Diastolic (green line) and systolic internal dimensions (yellow line) in hearts of hENPP1mAb/IgG-treated animals. (F) Ejection fraction, fractional shortening, and left ventricular (LV) chamber size in systole (LVIDs) and diastole (LVIDd) in IgG or hENPP1mAb-treated animals at 1, 2, and 4 weeks following MI (n = 21/IgG and n = 19/hENPP1mAb). (G) Pie chart illustrating the fraction of animals with mild, moderate, and severe reduction in EF at 4 weeks after injury following IgG or hENPP1mAb administration. (H) 4D gated cardiac CT showing transverse and coronal views of the heart of IgG or hENPP1mAb-injected animals at day 14 post MI (arrowheads point to the thin wall post infarct scar that is decreased in hENPP1mAb-injected groups). (I) Ejection fraction measurement by gated cardiac CT (n = 7 animals/group). (J) Myocardial strain analysis of cardiac segments in longitudinal axis at day 7 post MI in IgG versus hENPP1mAb-treated animals. Heatmap demonstrating wall strain generated with deeper color corresponding to greater contractile force. (K) Myocardial deformation measurements to demonstrate strain forces generated at various cardiac segments between IgG and hENPP1mAb-treated animals. GLS, global longitudinal strain); Post, posterior base; Post. Mid; Post. Apex; Ant., Anterior apex; Ant. Mid and Ant. base. (n = 9 animals/group). Data are expressed as mean ± SEM. ∗∗p < 0.01, ∗p < 0.05, ns: not significant. Statistical significance was determined using ordinary one-way ANOVA with Tukey’s multiple comparison test (C and D), unpaired multiple t test (F), or Student’s t test, 2 tailed (I and K). Video S1. CT scans of the heart coronal sections of live animals at 14 days post myocardial infarction following IgG or hENPP1mAb treatment, related to Figure 3 [134]Download video file^ (3.2MB, mp4) Video S2. CT scans of the heart transverses section of live animals at 14 days post myocardial infarction following IgG or hENPP1mAb treatment, related to Figure 3 [135]Download video file^ (2.1MB, mp4) hENPP1mAb administration is associated with histologic evidence of superior post-infarct repair We next examined histological correlates of superior post-infarct heart function. The degree of post-MI fibrosis is known to be an independent prognostic factor regulating cardiovascular outcomes after MI with higher degree of fibrosis associated with significantly worse outcomes.[136]^3 We performed Masson trichrome staining to determine the degree of cardiac fibrosis at 4 weeks after injury and observed significantly decreased fibrosis in hearts of hENPP1mAb-injected animals compared to IgG-injected controls ([137]Figures 4A and 4B). We stratified the severity of fibrosis at 4 weeks post MI according to the ratio of fibrotic area to the left ventricular surface area (severe fibrosis: >40%, moderate fibrosis: 20%–40%, and mild fibrosis: <20%). In IgG-injected animals, 60% of the animals exhibited severe fibrosis at 4 weeks post MI but only 14% of the animals that received hENPP1mAb developed severe fibrosis ([138]Figure 4C; [139]Table S2). In contrast to thin scarred walls in IgG-injected animals, hematoxylin-eosin staining demonstrated thicker walls in the hearts of hENPP1mAb-injected animals suggestive of superior cardiac remodeling ([140]Figure 4D). Peri-infarct hypertrophy is an adverse prognostic sign after MI,[141]^19 and we measured the effects of hENPP1mAb on post-infarct cardiac hypertrophy. At 4 weeks after MI, the heart weight and the heart weight/body weight ratios were significantly decreased in animals that received hENPP1mAb compared to IgG with no change in body weight alone ([142]Figure 4E). Immunofluorescent staining with myocyte markers demonstrated significantly decreased myocyte size (surface area) in the infarcted hearts of hENPP1mAb-injected animals at 4 weeks after injury consistent with decreased post-infarct hypertrophy ([143]Figure 4F). Immunostaining for endothelial capillaries demonstrated a significant increase in CD31-expressing capillaries suggestive of a superior cardiac repair process ([144]Figure 4G). Figure 4. [145]Figure 4 [146]Open in a new tab Humanized ENPP1 animals treated with hENPP1mAb after MI exhibit histologic evidence of superior cardiac repair (A) Masson trichrome staining to demonstrate scar size as a fraction of LV surface area measured 4 weeks after injury at the apex and mid ventricle in IgG or hENPP1mAb-injected humanized ENPP1 animals. (B) Quantitation of scar surface area (n = 15/IgG and 22/hENPP1mAb) and (C) pie chart illustrating the fraction of animals with mild, moderate, and severe fibrosis following IgG or hENPP1mAb administration. (D) Hematoxylin/eosin staining to demonstrate the thickness of infarcted wall at 4 weeks after MI in IgG or hENPP1mAb-injected animals with quantification of wall thickness (n = 11/IgG and n = 12/hENPP1mAb). (E) Heart weight (HW), body weight (BW), and HW/BW ratio in IgG versus hENPP1mAb-treated animals (n = 21/IgG and n = 19/hENPP1mAb). (F) Immunostaining for cardiac troponin and wheat germ agglutinin to determine myocyte surface area and quantification (surrogate for cardiac muscle hypertrophy) 4 weeks after MI in IgG or hENPP1mAb-injected animals (n = 10/IgG and n = 9/hENPP1mAb). (G) Staining for endothelial cells (CD31) to determine capillary formation (arrowheads) 4 weeks after MI in IgG or hENPP1mAb-treated animals and quantification of capillary formation (n = 13/IgG and n = 16/hENPP1mAb). Data are represented as mean ± SEM. ∗∗p < 0.01, ∗p < 0.05, ns: not significant. Statistical significance was determined using Student’s t test, 2 tailed. hENPP1mAb alters the transcriptional repair response in myocytes and non-myocytes after MI We next investigated the mechanisms of benefit of hENPP1mAb and performed single-nuclear transcriptomics to determine how the transcriptional repair response is altered by hENPP1mAb in a cell-specific manner. For this purpose, we harvested the hearts of hENPP1mAb or IgG-treated animals at 7 days following MI and harvested nuclei to perform single-nuclear transcriptomics using the 10× Genomics platform. Uniform manifold approximation and projection (UMAP) analysis demonstrated expected population of cells in the infarcted region including myocytes, macrophages, endothelial cells, and fibroblasts ([147]Figures 5A and [148]S4). Distribution of cells across the IgG and hENPP1mAb populations demonstrated increased myocytes and endothelial cells and decreased fibroblasts and macrophages in the hENPP1mAb group compared to the IgG group ([149]Figures 5B and 5C). We have previously demonstrated that ENPP1 is expressed by the non-myocyte population and predominantly by cardiac fibroblasts.[150]^6 We also validated our observation by examining published single-cell transcriptomic datasets[151]^20 from infarcted human hearts and observed the expression of ENPP1 in the non-myocyte population including human cardiac fibroblasts ([152]Figure S5). Our findings are also consistent with another recently published single-nuclear transcriptomic dataset in individuals with cardiomyopathy that demonstrated ENPP1 expression in myofibroblasts in end-stage cardiomyopathic hearts.[153]^21 We first examined gene expression changes in the entire fibroblast population, and gene ontogeny (GO) analysis demonstrated that cardiac fibroblasts in the hENPP1mAb-treated group exhibited the downregulation of genes related to cytoskeletal organization, extracellular matrix (ECM), cell migration, and cell substrate adhesion ([154]Figure 5D). As this suggested that genes associated with or identifying activated fibroblasts or myofibroblasts were downregulated in the hENPP1mAb group, we performed subcluster analysis of fibroblasts to determine how the fibroblast populations were altered in the hENPP1mAb-treated group. UMAP analysis based on transcriptional signatures was used to subcluster the fibroblasts into 3 populations across both the hENPP1mAb and IgG-treated groups ([155]Figure 5E; [156]Table S6). Fibroblasts of the IgG and the hENPP1mAb-injected hearts were asymmetrically distributed throughout these fibroblast subclusters with fibroblasts from IgG-treated animals predominantly contributing to cluster 0 and fibroblasts from hENPP1mAb-treated animals predominantly contributing to cluster 1 ([157]Figures 5F and 5G). Subcluster 0 represented the myofibroblast population with abundant expression of matrix and myofibroblast genes including Col1a1, Postn, and Acta 2 ([158]Figure 5H) and thus the number of cells contributing to myofibroblasts (cluster 0) in the hENPP1mAb group was significantly lower compared to the IgG group ([159]Figures 5G and 5H). In contrast, subcluster 1 of the fibroblast population predominantly comprised fibroblasts from the hENPP1mAb-injected animals ([160]Figure 5G). Cluster 1 population of fibroblasts represented a population that exhibited decreased expression of ECM genes such as collagens and myofibroblast genes compared to cluster 0 ([161]Figure 5I). In contrast to Col1 and Col3-encoding genes that were enriched in cluster 0 fibroblasts, cluster 1 fibroblasts expressed collagen genes coding for COL4 or COL5 ([162]Figure 5I) that typically support vasculature and maintain ECM structure.[163]^22^,[164]^23 Other ECM genes such as laminin, decorin, and spondin that are not typically enriched in scar tissue but thought to play a role in matrix organization, cardiac muscle support,[165]^24 and matricellular signaling[166]^25 were abundantly expressed in cluster 1 compared to cluster 0 ([167]Figure 5I; [168]Table S6). A dot-blot analysis clearly demonstrated that ECM and myofibroblasts signatures were significantly downregulated overall in fibroblasts in the hearts of hENPP1mAb-injected animals ([169]Figure 5J). We confirmed these observations and performed qPCR on the hearts of IgG and hENPP1mAb-treated animals at day 3 and day 7 following MI and observed significant downregulation of ECM and myofibroblast genes in the hearts of hENPP1mAb-injected animals at day 7 post MI ([170]Figure S6A). We also compared the expression of ECM and myofibroblast genes in the hearts of IgG and hENPP1mAb injected animals (day 7 MI) to sham-injured animals and observed that hENPP1mAb significantly altered the transcriptional response with ECM gene expression in the hearts of hENPP1mAb animals closer to sham-injured hearts than that in IgG-injected animals ([171]Figure S6B). The administration of hENPP1mAb thus alters the transcriptional repair response in cardiac fibroblasts and results in an altered fibroblast population with muted myofibroblast and ECM signatures. Figure 5. [172]Figure 5 [173]Open in a new tab Single-nuclei RNA sequencing of hearts of humanized ENPP1 animals treated with IgG or hENPP1mAb and harvested at 7 days following MI (A) Uniform manifold approximation and projection (UMAP) demonstrating different phenotypes of cell clusters in the infarcted heart and (B) distribution of cells from IgG and hENPP1mAb-treated animals across these clusters (n = 3 animals/group). (C) Fraction of different cell populations in IgG versus hENPP1mAb-injected animals. (D) Gene ontology analysis of main pathways differentially downregulated in cardiac fibroblasts in hENPP1mAb-treated animals versus IgG control animals. (E) UMAP demonstrating subclustering of fibroblast population across IgG and hENPP1mAb groups and (F) distribution of fibroblasts of IgG versus hENPP1mAb groups across these fibroblast subclusters. (G) Fraction of fibroblasts in IgG or hENPP1mAb-treated groups contributing to the fibroblast subclusters with cluster 0 contributed by IgG-injected group and cluster 1 by the hENPP1mAb group. (p < 0.05 in cluster 0, p < 0.01 in cluster 1, and no significance in cluster 2). (H) Expression of ECM and myofibroblast genes (Col1a1, Postn, and Acta2) across these fibroblast subclusters with abundant expression of ECM genes and myofibroblast marker Postn in subcluster 0 compared to subcluster 1. (I) Dot plot demonstrating distribution of abundantly expressed genes representing the fibroblast subclusters (Note: myofibroblast and ECM genes are abundant in subcluster 0 compared to subcluster 1). (J) Dot blot representing expression of myofibroblast and ECM genes in the entire cardiac fibroblast population of IgG versus hENPP1mAb animals. Data are expressed as mean ± SEM. ∗∗p < 0.01. Statistical significance was determined using Student’s t test, 2 tailed. As cardiac contractile function and wall tension were increased in the hearts of hENPP1mAb-injected animals, we next examined transcriptional changes in the myocyte population. A GO analysis of genes differentially upregulated in myocytes of hearts of hENPP1mAb versus IgG-injected animals demonstrated genes belonging to metabolic pathways affecting glycolysis, glycogen metabolism, and cardiac contractile process ([174]Figure S7A). Genes regulating glycolysis directly like phosphofructokinase (Pfkm) or Enolase (Eno) or affecting regulation of key glycolytic enzymes were differentially upregulated in the myocytes of hENPP1mAb-injected animals ([175]Figure S7B). Expression of genes encoding for contractile proteins such as (Tnnt2, Tnni3, Myh6, and Ttn) was also significantly upregulated in the myocytes of animals treated with hENPP1mAb ([176]Figure S7C). The facilitation of glycolysis and increased expression of contractile genes provide an underlying mechanism of enhanced post-infarct heart function in hENPP1mAb-injected animals. Increased post-infarct inflammation is associated with adverse infarct outcomes in humans, and we examined the expression of inflammatory gene signatures in macrophages and observed that the expression of inflammatory genes was significantly suppressed in hENPP1mAb-injected animals ([177]Figure S7D). We have shown that an ENPP1-mediated metabolic cascade increases cell death in the infarcted heart, and we looked at pro-apoptotic signatures across the entire cell population in the infarcted heart and observed that hENPP1mAb decreased cell death signatures ([178]Figure S7E). hENPP1mAb rescues metabolic defects in the infarcted heart and enhances aerobic cellular respiration of the infarcted heart ENPP1 is an ectonucleotidase and we have shown previously that ENPP1-mediated hydrolysis initiates a metabolic cascade that disrupts pyrimidine biosynthesis.[179]^6 To determine the effects of hENPP1mAb on the cardiac metabolome, we harvested hearts of IgG versus hENPP1mAb-injected animals at 7 days following MI, extracted metabolites, and performed metabolomic analysis using liquid chromatography and mass spectrometry. We observed that pyrimidines or intermediary metabolites connected with pyrimidine biosynthesis were significantly upregulated in the hearts of hENPP1mAb-injected animals ([180]Figure 6A). Pyrimidines or pyrimidine bases such as uridine, uracil, ornithine, cytidine, and deoxycytidine were increased in the infarcted hearts of hENPP1mAb-injected animals along with increased ribose and metabolites of the pentose phosphate pathway required for ribose synthesis ([181]Figure 6A). A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis pointed to pyrimidine metabolism as the most significantly differentially expressed metabolic pathway in the hearts of hENPP1mAb versus IgG-injected animals ([182]Figure 6B). Pyrimidine biosynthesis is connected to NAD biosynthesis as PRPP (phosphoribosyl pyrophosphate is required for both), and we had shown previously that ENPP1 metabolic cascade disrupts pyrimidine biosynthesis at the PRPP incorporation step.[183]^6 We examined NAD+/NADH levels and observed the NAD, NADH, and nicotinamide levels to be significantly higher in the infarcted hearts of hENPP1mAb-injected animals ([184]Figure 6C). These findings are overall consistent with increased expression of glycolytic genes in myocytes with hENPP1mAb noted earlier. To determine whether increased NAD levels is associated with increased cellular respiration, we performed Seahorse cellular respiration assays to determine the oxygen consumption rate (OCR) in homogenates of infarcted hearts of hENPP1mAb versus IgG-injected animals. We observed that the OCR measured at various mitochondrial respiratory complexes (complex I and II) was significantly greater in the hearts of hENPP1mAb versus IgG-injected animals ([185]Figure 6D). Taken together, these observations demonstrate the ability of hENPP1mAb to augment or rescue pyrimidine biosynthesis in infarcted hearts and lead to higher NAD levels with significantly higher aerobic cellular respiration. Figure 6. [186]Figure 6 [187]Open in a new tab hENPP1mAb administration in humanized mice leads to the rescue of metabolic pathways and augmented cellular respiration in the infarcted heart (A) Metabolomic analysis of hearts of animals injected with IgG or hENPP1mAb at day 7 post MI demonstrating metabolites that are significantly upregulated (p < 0.05) in hearts of hENPP1mAb-injected animals (arrows point to pyrimidines or metabolites in pentose phosphate pathway, demonstrating rescue of pyrimidines in hENPP1mAb-injected animals compared to IgG-injected animals, n = 5/IgG and 4/hENPP1mAb). (B) KEGG analysis of metabolic pathways that are significantly upregulated in hearts of hENPP1mAb injected animals demonstrating that pyrimidine biosynthetic pathway is the most significant metabolic pathway. (C) Quantification of NAD, NADH, and nicotinamide levels in hearts of animals injected with hENPP1mAb versus IgG as early as 3 days post infarction (n = 6/group). (D) Seahorse cellular respiration on heart homogenates (day 3 post MI) demonstrating oxygen consumption rate (OCR) at mitochondrial electron transport complexes I and II normalized to MitoTracker Deep Red (MTDR) fluorescence (n = 10/group). Data are expressed as mean ± SEM. ∗∗p < 0.01. Statistics was determined using Student’s t test, 2 tailed. hENPP1mAb is safe and well tolerated We next investigated the safety profile of hENPP1mAb and first assessed the biodistribution of hENPP1mAb. For this purpose, we performed pITC-desferrioxamine conjugation to label hENPP1mAb with the radioisotope Zr89, which decays with positron (β+) emission, and intravenously injected a single dose of the radiolabeled hENPP1mAb or similarly labeled IgG. The animals were imaged in a microPET/CT scanner and images acquired serially from 0.5 h after injection to 240 h ([188]Figure S8A). We observed that 24 h after initial administration and distribution, signal intensity from hENPP1mAb was mainly localized to the heart (9.10 ± 0.18 %ID/cc), liver (8.40 ± 0.11 %ID/cc), and bones (4.63 ± 0.28 %ID/cc) ([189]Figure S8A). At 144 h after initial administration, the signal intensity of hENPP1mAb compared to that from IgG was significantly higher in multiple organs including heart (5.83 ± 0.30 vs. 1.90 ± 0.09 %ID/cc), liver (5.81 ± 0.21 vs. 5.01 ± 0.19 %ID/cc), kidneys (3.82 ± 0.10 vs. 2.09 ± 0.28 %ID/cc), lungs (3.25 ± 0.14 vs. 1.40 ± 0.20 %ID/cc), and bones (8.60 ± 0.61 vs. 2.53 ± 0.38 %ID/cc) confirming target-specific organ-wide distribution of hENPP1mAb ([190]Figure S8B). In the heart, we observed a significant drop in signal intensity from 24 to 72 h that likely corresponds to the clearance of a humanized IgG from mice ([191]Figure S8C). To determine any potential toxicity associated with the hENPP1mAb, we administered the antibody to humanized ENPP1 animals every 3 days for 2 weeks as performed earlier for therapeutic intervention and harvested the organs at 4 weeks after the first dose was administered. Histology did not show any evidence of toxicity in major organs such as the lung, heart, liver, kidney, and spleen ([192]Figure S8D). As osteoblasts in bone express ENPP1[193]^26 and hENPP1mAb was observed to bind to bone tissue in the biodistribution study, we administered hENPP1mAb for 2 weeks as aforementioned and then performed CT scans at 4 weeks after initial dose to determine changes in bone densitometry, but we observed that hENPP1mAb did not have any effects on bone density ([194]Figure S9A). Loss-of-function mutations in ENPP1 have been associated with a rare genetic disease that causes ectopic calcification in neonatal life in humans.[195]^27^,[196]^28 We harvested the aorta, heart, kidney, and heart valves at 4 weeks following the initial administration of hENPP1mAb as aforementioned, but Von Kossa staining did not demonstrate any calcification of tissues ([197]Figure S9B). Serum chemistry including calcium and phosphate levels following 2 weeks of hENPP1mAb treatment did not demonstrate any abnormalities and no differences were observed compared to IgG-injected animals ([198]Table S3). Complete and differential blood counts also did not demonstrate any abnormality following 2 weeks of hENPP1mAb administration ([199]Table S3). There was also no evidence of acute toxicity associated with hENPP1mAb administration. At day 3 following hENPP1mAb administration, serum chemistry and blood counts were within the normal range and no different from that of IgG-injected animals ([200]Table S4). There was no evidence of weight loss compared to baseline at day 3 or 1 and 2 weeks following hENPP1mAb administration ([201]Table S4; [202]Figure S9C). As ENPP1 has been shown to affect B cell function,[203]^29 we also examined B cell subsets from the peripheral circulation and the spleen by flow cytometry following injection of hENPP1mAb, but did not observe any difference in B cell subsets compared to IgG-injected animals within 7 days of hENPP1mAb/IgG injection ([204]Figure S10). These data suggest that hENPP1mAb is not associated with any obvious signs of toxicity in the organs examined. A single “shot” of hENPP1mAb after cardiac injury is sufficient to enhance repair and rescue post-infarct heart function We had administered the hENPP1mAb prior to infarction with repeated administration after MI to account for the time needed for the hENPP1mAb to be systemically absorbed via i.p. route and the shorter half-life of human IgG in mice. However, administration of a drug prior to a clinical event is not a clinically viable strategy. To overcome issues of pharmacokinetics of human IgG in mice, we adopted to use the Tg32 mice (FcRN−/− hFcRn (32)T). These animals have the murine Fcgrt gene (Fc receptor, IgG alpha chain transporter) knocked out and carry a transgene expressing the human FCGRT gene under its native promoter (Tg32).[205]^30 These animals thus demonstrate antibody clearance similar to humans and are extremely useful for determining the half-life of humanized monoclonal antibodies in mice, and pharmacokinetic data obtained from these animals correspond closely with phase 1 data in human trials.[206]^16^,[207]^31 We first used the Tg32 animal to determine the half-life of the hENPP1mAb. For this purpose, we injected a single dose (10 mg/kg) of the antibody intravenously and sampled peripheral blood at various time points following administration to determine the concentration of hENPP1mAb (by measuring human IgG) in peripheral blood of mice. Control animals received polyclonal human IgG and using this system, we determined an approximate half-life of 14 days in Tg32 animals with the clearance of the hENPP1mAb closely mirroring that of human IgG ([208]Table S5). It is to be noted that as our pK measurements are being carried out in Tg32 animals, that do not express human ENPP1, it does not take into account target-mediated drug disposition as a potential factor regulating the clearance of the drug. We have shown previously that ENPP1 expression peaks at 7 days after MI and the injured region undergoes maximal transcriptional changes within the first 2 weeks after MI.[209]^6^,[210]^22 As the estimated hENPP1mAb half-life was 14 days, we hypothesized that a single dose of the hENPP1mAb administered after injury should be sufficient to maintain the inhibition of hENPP1 and lead to superior cardiac repair. To address this hypothesis, we crossed humanized ENPP1 animals with Tg32 animals to create progeny humanized ENPP1/Tg32 animals that were homozygous for all alleles ([211]Figure 7A). We subjected these animals to MI and injected a single dose of hENPP1mAb (100 mg/kg) or control IgG intravenously on the day of injury within 2–4 h of MI ([212]Figure 7B). We adopted to administer a higher dose to serve as a loading dose that provides advantages of rapidly achieving high plasma levels and drives an immediate clinical response. Echocardiography was performed at weekly intervals and B and M mode echocardiography demonstrated significantly superior cardiac contractile performance with the EF and FS almost double that of IgG-injected animals (EF at 7 days: 42.73% ± 5.07% in hENPP1mAb versus 23.96% ± 4.73% in IgG groups; FS at 7 days: 21.56% ± 2.95% in hENPP1mAb-injected animals versus 11.37% ± 2.51% in IgG-injected controls, p < 0.05) ([213]Figures 7C and 7D). The dilatation of the ventricle, measured by end systolic ventricular dimension (LVIDs), was also significantly less in hENPP1mAb-injected animals compared to IgG-injected controls ([214]Figures 7C and 7D). Measurement of adverse post-infarct hypertrophy by determining heart weight and body weight ratios at 4 weeks following injury demonstrated significantly decreased post-infarct hypertrophy in hENPP1mAb-injected animals without any change in body weight ([215]Figure 7E). Finally, histological analysis with Masson trichome staining at 4 weeks post MI demonstrated significantly decreased fibrosis in the hearts of animals injected with a single dose of hENPP1mAb ([216]Figure 7F). These observations demonstrate that a single dose of hENPP1mAb is sufficient to rescue post-MI heart function. Figure 7. [217]Figure 7 [218]Open in a new tab A single dose of hENPP1mAb administered in humanized ENPP1/Tg32 animals after MI is sufficient to significantly rescue post-infarct cardiac function (A) Genetic strategy of generating humanized ENPP1/Tg32 animals and (B) determining the effects of a single dose of hENPP1mAb administered after MI. (C) B mode (top) and M mode (below) echocardiogram demonstrating cardiac contractile function and chamber dilatation in IgG versus hENPP1mAb-injected animals. Green line points to cardiac dimensions in diastole and yellow lines point to dimensions in systole. (D) Ejection fraction, fractional shortening, and LV dimensions in systole (LVIds) and diastole (LVIDd) at 1, 2, and 4 weeks after MI following a single shot of hENPP1mAb or IgG after MI (n = 11/IgG and n = 12/hENPP1mAb). (E) Heart weight, body weight, and heart weight/body weight ratios of hearts harvested at 4 weeks in animals receiving a single dose of hENPP1mAb or IgG after MI (n = 11/IgG and n = 12/hENPP1mAb). (F) Masson trichrome staining to demonstrate fibrosis at 4 weeks post MI in animals receiving a single dose of hENPP1mAb or IgG and quantification of fibrosis (n = 9/IgG and n = 11/hENPP1mAb). Data represented as mean ± SEM, ∗∗p < 0.01, ∗p < 0.05, ns: not significant. Statistical significance was determined using unpaired multiple t test (D) or Student’s t test, 2 tailed (E and F). Discussion Cardiac repair comprises a complex and highly regulated sequence of spatiotemporal events driven by the recruitment of a diverse population of cells in the infarcted region. Crosstalk between cells regulates critical repair processes such as fibrosis, inflammation, cell death, and post-infarct cardiac remodeling. The field of cardiac regenerative therapies has evolved from a myocyte-centric approach to an understanding that cellular crosstalk between different population of cells in the injured heart holds immense therapeutic potential.[219]^5^,[220]^32 This body of work targets a myocyte-non-myocyte crosstalk that regulates extracellular nucleotide metabolism in the infarcted heart, and demonstrates that inhibiting a metabolic cascade that is initiated by ENPP1 rescues metabolic defects, induces beneficial transcriptional repair response across myocyte and non-myocyte populations, and leads to superior post-infarct heart function. Of all the mammalian ectonucleotidases expressed in the infarcted heart, genetic loss-of-function studies have demonstrated that ENPP1 is the principal ectonucleotidase mediating extracellular ATP hydrolysis.[221]^6 Our strategy thus builds on this rational premise that if deleterious metabolic cascades initiated by the principal cardiac ectonucleotidase ENPP1 can be attenuated, then an adverse metabolic catastrophe in cycling and non-cycling cells can be avoided with beneficial effects on post-infarct cardiac function. Rather than affecting a specific phenotype of cell or cellular process, hENPP1mAb-mediated inhibition of ENPP1 by rescuing defects in pyrimidine and NAD metabolism affected a diverse population of cells including myocytes, fibroblasts, endothelial cells, and inflammatory cells. Such pleiotropic beneficial effects on a diverse population of cells result in decreased fibroblast activation, decreased expression of ECM genes, increased glycolysis, and improved myocyte energetics with increased contractility. It is to be noted that even though hENPP1mAb decreased fibrosis and ECM gene expression, there was no occurrence of cardiac rupture. This is likely because of timing of peak ENPP1 expression. Cardiac rupture typically occurs within the first few days of MI, but peak ENPP1 expression occurs in the heart at 7 days after injury and thus beyond the stage when the infarcted heart is most vulnerable to rupture. As such, targeting ENPP1 decreased ECM gene expression without increasing rupture risk. The beneficial metabolic effects on myocytes also likely resulted in superior myocyte function, which decreases the chance of post-infarct rupture. Thus, hENPP1mAb by exerting beneficial effects on different cardiac cell populations in cardiac repair, whether direct or secondary, stands to have an advantage over therapeutic strategies aimed at targeting focused processes such as modulation of fibrosis or angiogenesis. Our study has limitations as well. Modulation of metabolism in the human heart remains an untested therapeutic strategy for MI or prevention of heart failure, though metabolic abnormalities are thought to contribute toward worsening heart function. In this regard, although our study demonstrates the therapeutic efficacy of the hENPP1mAb injected after the occurrence of MI, the transcriptomic, phenotypic, and metabolic data reflect the delivery of the therapeutic prior to MI. However, the functional benefits were similar when the hENPP1mAb was administered after MI, suggesting that mechanisms of benefit of hENPP1mAb including effects on pyrimidine and NAD biosynthesis likely underlie mechanisms of benefit when the therapeutic agent is injected within a few hours of MI. The therapeutic window after MI during which hENPP1mAb would continue to exert beneficial results is also not clear from our study as the hENPP1mAb was administered within a few hours of infarction. However, as wound healing events occur rapidly after cardiac injury, early administration of hENPP1mAb would likely offer the maximal clinical benefit. Our study also did not examine in great detail potential safety concerns related to antibody-dependent cytotoxicity, but we did not see any occurrence of cardiac rupture with no difference in post-MI mortality, thus suggesting that antibody-dependent cellular cytotoxicity, even if present, was not of sufficient magnitude to affect clinical outcomes. Mutations in ENPP1 are known to cause generalized ectopic calcification in neonatal life and hypophosphatemic rickets in adulthood,[222]^33 so our therapeutic strategy should be accompanied by careful monitoring for ectopic calcification and calcium and phosphate balance for a few months following administration of hENPP1mAb. Although we did not see any calcification in the short term in the animals that received hENPP1mAb, the degree and pattern of expression of ENPP1 in the humanized knockin mice that we have used was not extensively evaluated in all tissues, emphasizing the need for thorough monitoring of ectopic calcification and other potential adverse effects on bone mass and calcium/phosphate balance. Notwithstanding the aforementioned limitations, prevention of coronary thrombosis and attenuating the sympatho-adrenergic and renin-angiotensin system form the mainstay of therapies for MI. There are no drugs currently available that augment tissue repair in the heart or other organs. In this regard, this body of work identifies a monoclonal antibody, we have engineered to affect cardiac repair, by beneficially modulating post-infarct metabolism in both myocytes and non-myocytes. Transcriptional changes after MI occur rapidly and the infarcted region is known to become transcriptionally mature within 2 weeks of injury.[223]^22 As the half-life of the hENPP1mAb is anticipated to be close to 14 days in humans, a single dose of the hENPP1mAb administered after MI would potentially be sufficient to enhance heart repair and attenuate post-infarct decline in heart function. Limitations of the study Cardiac metabolism has not been therapeutically targeted for MI or heart failure in humans and thus this remains an untested strategy. ENPP1 mutations can potentially lead to calcium and phosphate imbalance, and future human phase 1 studies would be needed to determine the safety of the monoclonal antibody. Our study also uses a single-dose-based strategy for MI based on the half-life of the antibody as well as on the native repair response of the injured heart. Our study does not address multi-dose safety particularly if the therapeutic is envisioned to treat chronic heart failure. Resource availability Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Arjun Deb (adeb@mednet.ucla.edu). Materials availability There are certain restrictions to the availability of the hENPP1mAb. The humanized antibody is manufactured by a CRO in batches and manufacturing is expensive and thus may limit the amounts we are able to provide to investigators. Requests for the hENPP1mAb or other unique reagents generated in this study should be directed to and will be fulfilled by the [224]lead contact, Arjun Deb (adeb@mednet.ucla.edu), with a Materials Transfer Agreement. Data and code availability Original/source data of RNA sequencing (accession number: GEO: [225]GSE225826 ) have been deposited at [226]https://www.ncbi.nlm.nih.gov/geo/ and are publicly available as of the date of publication. This study did not report new original code. Any additional information required to reanalyze the data reported in this paper is available from the [227]lead contact upon request. Acknowledgments