Cardiomyocyte crosstalk with endothelium modulates cardiac structure, function, and ischemia-reperfusion injury susceptibility through erythropoietin

Erythropoietin (EPO) exerts non-canonical roles beyond erythropoiesis that are developmentally, structurally, and physiologically relevant for the heart as a paracrine factor. The role for paracrine EPO signalling and cellular crosstalk in the adult is uncertain. Here, we provided novel evidence showing cardiomyocyte restricted loss of function in Epo in adult mice induced hyper-compensatory increases in Epo expression by adjacent cardiac endothelial cells via HIF-2α independent mechanisms. These hearts showed concentric cellular hypertrophy, elevated contractility and relaxation, and greater resistance to ischemia-reperfusion injury. Voluntary exercise capacity compared to control hearts was improved independent of any changes to whole-body metabolism or blood O2 content or delivery (i.e., hematocrit). Our findings suggest cardiac EPO had a localized effect within the normoxic heart, which was regulated by cell-specific EPO-reciprocity between cardiomyocytes and endothelium. Within the heart, hyper-compensated endothelial Epo expression was accompanied by elevated Vegfr1 and Vegfb RNA, that upon pharmacological pan-inhibition of VEGF-VEGFR signaling, resulted in a paradoxical upregulation in whole-heart Epo. Thus, we provide the first evidence that a novel EPO-EPOR/VEGF-VEGFR axis exists to carefully mediate cardiac homeostasis via cardiomyocyte-endothelial EPO crosstalk.

We previously generated a constitutive, cardiomyocyte specific Epo knockout mouse driven by the Mlc2v promoter (Allwood et al., 2024).When cardiomyocyte Epo is abolished during embryogenesis, cardiac cellular proliferation is reduced, leading to irreversible changes to overall morphology, function, and response to ischemic injury in the adult heart (Allwood et al., 2024).However, these mice also show a surprising transcriptional increase in endothelial cell derived Epo in compensation to loss of cardiomyocyte gene expression.This prohibits the distinction between developmental adaptations from physiological effects in the adult.To resolve whether cardiomyocyte-derived EPO signalling was physiologically relevant following a normal course of cardiogenic development, we used alpha-myosin heavy chain tamoxifen-induced Cre-LoxP loss of function in adult mice.
Herein we show that following the loss of cardiomyocyte derived Epo in the adult heart, compensatory hyperexpression persists by endothelial cells in a HIF2α-independent manner.This response was associated with concentric cellular hypertrophy, elevated contractile function, and a greater resistance to ischemia-reperfusion injury.Functionally, this phenotype translated to better voluntary exercise capacity, which was unrelated to changes in whole-body metabolism nor any change in hematocrit.Our findings suggest the overexpression of endogenous cardiac EPO acted locally, not systemically.In the absence of cardiomyocyte Epo, we observed concomitant upregulation of Epo, Vegfr1, and Vegfb RNA in the whole heart.When VEGF-VEGFR signaling was inhibited, a further increase in cardiac Epo could be observed.Collectively, our findings provide the first evidence for a paracrine cardioendothelial feedback loop by the EPO-EPOR/VEGF-VEGFR axis for maintaining cardiac homeostasis in the adult mouse.
2 Materials and methods

Ethical approval
Adult mice (C57Bl6 background) were bred and aged to 16 weeks of maturity for experiments and housed at 23 °C-24 °C with 45% humidity and maintained on a 12 h light/dark cycle with food and water provided ad libitum.This study was approved by the Animal Care Committee at the University of Guelph and all experiments were carried out in accordance with the guidelines from the Canadian Council on Animal Care.

Generation of EPO Δ/Δ knockout mice
Inducible CreLox transgenic mice expressing Epo LoxP (Zeigler et al., 2010) and Cre recombinase under the control of the cardiomyocyte-specific promoter, alpha-myosin heavy chain (αMHC-MerCreMer), were used in this study (Sohal et al., 2001) (Jackson Laboratory Strain # 005657, Supplementary Figure S1).Briefly, the 5′loxP site was inserted into intron 1 of the Epo gene (located 94 base pairs upstream from exon), and the 3' loxP site was inserted into intron 4 (located 86 base pairs downstream of the exon 4), with the NEO cassette flanked by both loxP sites.

Voluntary wheel running
EPO fl/fl and EPO Δ/Δ mice were subjected to a 3-day voluntary wheel running protocol.Mice were housed individually and allowed to run freely on an in-cage wheel (12 cm in diameter).Rotations were transmitted to a cycling computer (VDO M2.1 WR Cycling Computer) and distance, time, and pace were recorded.Data were presented as the averages from all 3 days of voluntary wheel running following a 24-h acclimation period.

CLAMS: metabolic analyses
The Comprehensive Laboratory Animal Monitoring System (CLAMS) metabolic caging apparatus (Columbus Instruments Oxymax) is a sealed indirect calorimeter used for the simultaneous measurement of multiple parameters, including oxygen consumption (VO 2 ), carbon dioxide production (VCO 2 ), and calculation of respiratory exchange ratio (RER) across 24-h.EPO fl/fl and EPO Δ/Δ mice were weighed and individually placed into the CLAMS caging for a 24-h acclimatization period followed by a subsequent 24-h data collection period.Mice were maintained on a 12-h light/dark cycle and provided food and water ad libitum.Data was recorded every 15 min for metabolic readings (VO 2 , VCO 2 ) and total energy expenditure.The RER was calculated as the quotient of VCO 2 /VO 2 .

Hematocrit and hemoglobin
For the determination of hematocrit, blood was collected from the left ventricle and saphenous vein of EPO fl/fl and EPO Δ/Δ mice and centrifuged in heparinized microcapillary tubes (5000rpm at 23 °C for 10 min).To calculate hematocrit, the length of red blood cells was divided by the length of total blood volume and expressed as a percentage.To measure hemoglobin (g/L), saphenous vein blood was collected by microcuvettes and measured by a HemoCue ® Hb 201 (cat# 111716 Life Supply).
Echocardiography was performed using the Vevo2100 system (VisualSonics Inc., Toronto, ON, Canada) with the 40 MHz MS550D ultrasound transducer.Mice were maintained at 37.5 °C throughout data collection, as confirmed with a TH-5 rectal probe thermometer (Physiotemp Instruments LLC, Clifton, NJ, United States).B-Mode images were captured from the parasternal long axis mid-papillary region to measure the left ventricle endocardial length (i.e., LV chamber length) from the aortic annulus to the apex during in diastole, as previously described (Vinhas et al., 2013).M-Mode images were collected from the parasternal long axis mid-papillary region and analyzed using the left ventricle trace function from the cardiac package (VisualSonics Inc., Toronto, ON, Canada) as previously described (Platt et al., 2017).Measurements represent the average data collected over three consecutive cardiac cycles.Data collection and analyses were performed in a blinded manner.

Invasive hemodynamics
Cardiac function was investigated in vivo by invasive hemodynamics.Mice were anaesthetized using an isoflurane/ oxygen mix (2%:100%).Animals were maintained at 37.5 °C throughout data collection using a heated water pad.A 1.2F catheter (FTS-1211B-0018; Transonic Scisense Inc.) was inserted into the right carotid artery and advanced into the left ventricle to collect hemodynamics measurements.Hemodynamics were collected for 15 min.All hemodynamic signals were digitized at a sampling rate of 2000 Hz and recorded by computer using iWorx ® analytic software (Labscribe2, Dover, NH, USA).Data collection and analyses were performed in a blinded manner.Tissues were collected, weighed, and randomly assigned to either histology, qPCR, or western blotting.

Langendorff preparation
The ex vivo Langendorff preparation provides the direct assessment of systolic and diastolic cardiac function (and its susceptibility to ischemia-reperfusion injury), independent of preload, afterload, and heart rate, without the influence of neurohormonal effects or humoral factors in the blood.Accounting for these variables allows for conclusions to be made about purely intrinsic cardiac function.Mice were heparinised (200 IU/kg of body weight) for ex vivo cardiac assessment.After 20 minutes, mice were anesthetized using isoflurane, followed by a midline thoracic incision made to rapidly excise the heart.The heart was rinsed in ice-cold phosphate buffered saline and the aorta was cannulated onto a 21-gauge needle to allow for retrograde perfusion.Hearts were perfused with carbogenated (95% O2: 5% CO2) Krebs Henseleit buffer (pH of 7.4) at 70-75 mmHg.The Krebs Henseleit buffer contained the following compounds (in mM/L): 118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 0.5 mM C3H3NaO3, 0.05 mM EDTA, 11 mM glucose, and 2 mM CaCl2.The left atrium was removed to allow the insertion of a deflated balloon attached to a pressure catheter into the left ventricle.The balloon was inflated to achieve an end diastolic pressure of 5-8 mmHg.Hearts were paced using a Grass SD9 Stimulator at a frequency of 7 Hz.A stabilization period of 20 min was followed by recording of baseline measurements, followed by 25 min of global no-flow ischemia.Afterwards, the perfusate line was reopened and hearts were re-perfused for 45 min.To assess cytoprotective function ex vivo, percent recovery of left ventricular pressure, and rate of change in pressure (dP/dt max -index of contractility/inotropy, and dP/ dt min -index of relaxation) were calculated after 45 min of reperfusion.Data collection and analyses were performed in a blinded manner.

Serum EPO ELISA
Blood was collected from the left ventricle via cardiac puncture and allowed to clot for 2 h on ice, then centrifuged in 1.5 mL Eppendorf tubes at (4,000 rpm at 4 °C for 20 min).The supernatant was removed, snap frozen in liquid nitrogen, and stored at −80 °C for quantification of serum EPO levels.EPO protein concentration was quantified from serum using a Quantikine Mouse EPO ELISA (MEP00B, R&D Systems) according to the manufacturer's instructions.

qPCR molecular analyses
Tissues selected for qPCR were snap frozen in liquid nitrogen and kept at −80 °C (n = 8 per group for EPO fl/fl and EPO Δ/Δ , n = 7 EPO fl/-Cre +/− ).Total RNA was isolated from the free left ventricle and kidneys using TRIzol (Invitrogen, Burlington, ON, Canada) with the Qiagen RNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.RNA samples were treated with DNase (Qiagen), according to manufacturer's instructions.Prior to cDNA synthesis, RNA concentrations were quantified (NanoDrop, ND1000; Thermo Fisher Scientific, Waltham, Massachusetts, USA).Protein contamination was assessed by measuring absorbance at 280 nm.Generation of cDNA was completed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems by Thermo Fisher Scientific, Waltham, Massachusetts, USA) according to the manufacturer's instructions, using 2000 ng of RNA per sample.Target gene RNA was quantified using the Platinum SYBR Green qPCR SuperMix-UDG with ROX (Invitrogen, Burlington, ON, Canada) using the primers listed in Table 1.Primers were designed to span an exon-exon region to eliminate any possibility of priming genomic contamination.qPCR was performed using 7,500 Real Time PCR detection system (Applied Biosystems, Foster City, CA, USA) with the following protocol: 1 cycle at 50 °C for 2 min, 1 cycle at 95 °C for 5 min, then 40 cycles at 95 °C for 15 s, 1 min at 60 °C for all genes (excluding Epo, where the annealing temperature was 58 °C), followed by a dissociation curve to assess specificity of the reaction (Supplementary Figure S2).Samples were run in duplicate 25 uL reactions.Undetectable Epo RNA was assigned the value of the limit of detection of the assay (CT = 40).Results were analyzed according to the delta-delta CT method using reference genes (Table 1) and normalized to the EPO fl/fl group.

RNA fluorescent in situ hybridization
The hearts were excised and block fixed in 10% buffered formalin for 24 h, followed by transfer into 70% ethanol for storage before processing and embedding in paraffin wax.Tissue microarrays were created using the tissue micro array (TMA) Master II instrument from 3D Histech Ltd.Regions of interest from the left ventricle were selected, cored, and added into a host paraffin block to create an array of 84 tissue cores, each measuring 2.0 mm in diameter (biological replicates n = 3).

Histology
Mice were exsanguinated and 10 mL of 1x PBS, 10 mL of 0.5 mol/L KCl, and 10 mL of 10% buffered formalin (VWR, Mississauga, ON, Canada) were perfused through the right carotid artery to fix cardiomyocytes in diastole.Tissues were harvested, stored in 10% buffered formalin for 24 h, and then transferred into 70% ethanol.Hearts were processed and embedded in paraffin wax.Cross sections of the heart (5 μm) were mounted onto charged 1.2 mm Superfrost slides (Fisher Scientific).Paraffin embedded sections were then stained with either hematoxylin and eosin or Picrosirius Red for the determination of cardiomyocyte cross-sectional area (CSA) or percent fibrosis, respectively, within the left ventricle.Bright field images were acquired using an Olympus FSX 100 light microscope and analyzed blinded using ImageJ (for CSA) and cellSens (for interstitial fibrosis).

Immunoblotting
For probing of HIF-2α, left ventricular samples were homogenized in a buffer with a phosphatase (PhosSTOP, cat.# 4906845001, Sigma) and protease inhibitor cocktail (cat.#P8340, Sigma), and separation of nuclear from cytoplasmic extracts was performed using the NE-PER kit as per the manufacturer's instructions (cat.# 78833, Life Technologies).Nuclear protein extract concentrations were measured by bicinchoninic acid assay (cat.# 23277, Fisher Scientific).Samples were equally loaded (20ug/ well) and separated by 10% SDS-PAGE, followed by immunoblotting.Nitrocellulose membranes were rinsed in ddH2O and then incubated in reversible Ponceau Stain for 7 min to confirm equal protein transfer.The stain was stripped using 200uM NaOH for 1 min and rinsed in ddH2O for 5 min x 3. Membranes were blocked (5% skim milk in 1x TBST (0.1% tween)) and incubated in a primary HIF-2α antibody (Sun et al., 2015) (1: 1000; cat.# NB100-122SS, Bio-Techne; 5% bovine serum albumin in 1x TBST) overnight at 4 °C.Membranes were washed for 5 min x 3 in TBST and then incubated with a goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (1:1000, cat.# HAF008, Bio-Techne; 1% skim milk for 1 h at 22 °C).Membranes were washed for 5 min x 3 in 1x TBST.Signal was detected and quantified via enhanced chemiluminescence (cat.# 1705060, Bio-Rad) using a FluorChem HD imaging system (Alpha Innotech, Santa Clara, CA, USA).Values were obtained by measuring the target band (normalized to Ponceau) relative to the EPO fl/fl group.

Treatment with VEGF receptor tyrosine kinase inhibitor, axitinib
Axitinib is an FDA-approved selective inhibitor of cellular phosphorylation of VEGF receptor tyrosine kinases (VEGFR-1, VEGFR-2, VEGFR-3) for the treatment of advanced renal cell carcinoma (Tyler and FCSHP, 2012;INLYTA, 2023).Axitinib (cat.#S1005, Selleck Chemicals) was prepared as previously described (Ma and Waxman, 2009).Briefly, axitinib was suspended at 5 mg/mL in polyethylene glycol 400 (cat.# PX1286B-2, Sigma) and sonicated at room temperature for 30 min until dissolved.Using 0.1N HCl, the pH was adjusted to 2.5, followed by a second round of 10-min sonication.To achieve a final ratio of 3:7 (v/v) of polyethylene glycol 400 to water, acidified water (pH 2.5) was added.The solution was prepared fresh and stored in the dark at 4 °C.Axitinib was administered once a day through i. p. injection at a dose of 25 mg/kg body weight in a volume of 5 μL/g body weight (Ma and Waxman, 2009) for 4 days x 2 cycles (with 2 days rest).Data (invasive hemodynamics, saphenous vein hemoglobin levels, and tissues for qPCR) were collected 24 h after the last injection.

Cell apoptotic assay
A one-step TUNEL in situ apoptosis kit (cat.# E-CK-A320, Elabscience Biotechnology Co.) was used to quantify differences in cell apoptosis between EPO fl/fl and EPO Δ/Δ hearts according to the manufacturer's instructions.Fluorescent images were acquired using an Olympus FSX 100 microscope and the amount of green fluorescence of TUNEL-positive cells was analyzed blinded using ImageJ.

Statistical analyses
Graphing and statistical analyses of the data presented was performed using Prism version 9 software developed by (GraphPad Inc., La Jolla, CA, USA).Power calculations were used to determine the number of mice needed to detect a significant effect.Results were reported as mean ± SD (morphometrics, serum, hematocrit, qPCR, western blotting) or mean ± SEM (CLAMS, echocardiography, histology, invasive hemodynamics, Langendorff).To confirm whether data was normally distributed, a Shapiro-Wilk test was used.If the data was normally distributed, either a one-way ANOVA followed by Dunnett's or Tukey's post-hoc test or an unpaired Student's t-test was performed.If the data was not normally distributed, a Kruskal-Wallis test with Dunn's post-hoc test or Mann-Whitney U test was performed.Simple linear regression correlation analyses were run comparing Epo RNA expression to dP/dt max , dP/dt min , and dP/dt@LVP40 values and goodness-of-fit values were provided.p-value <0.05 was considered significant.

Cardiomyocyte-specific deletion of Epo induced compensatory overexpression of Epo in the heart by endothelial cells
In the developing fetus, mice null for Epo and the Epor die by E13.5 due to impaired cardiogenesis and anemia (Wu et al., 1999;Kertesz et al., 2004).Our previous work using the Mlc2v promoter established cardiomyocyte specific Epo deletion during embryogenesis in mice induces a phenotype with less cardiomyocyte hypoplasia, compensatory cellular hypertrophy, and upregulation of Epo in the heart by the endothelial cell persisting into adulthood (Allwood et al., 2024).Therefore, the presence of cardiac EPO is critical for proper development of the heart, yet endogenous cardiac Epo expression, regulation, and physiological significance in the adult heart remains unknown.For this reason, in our follow up study, we hypothesized that the demand for compensatory cardiac EPO would be reduced in a fully, and normally, developed adult heart.Upon induced deletion of adult cardiomyocyte Epo in mice after an ordinary course of cardiogenesis, we expected whole-heart morphology to be normal and cardiac Epo production to be low.To verify this, mice were subjected to morphometric and quantitative tissue PCR analysis.In line with our previous reports (Allwood et al., 2024), the morphological measurements revealed no gross differences in body weight, heart weight, left ventricular weight, or heart weight/tibial length (HW/TL) ratio between groups (Supplementary Figure S3C; Table 2).Surprisingly, after tamoxifen induced MerCreMer deletion of Epo expression, there was a significant upregulation in cardiac Epo in EPO Δ/Δ mice compared to the EPO fl/fl group by qPCR (Figure 1A).This data was confirmed by a mildly graded response in mice with only one floxed EPO allele (i.e., EPO fl/-Cre +/− ) (Figure 1A), suggesting this phenomenon does not exist in an all-or-nothing manner.Further, we sought to confirm our data was not specific to male mice.Indeed, female EPO Δ/Δ mice also showed a significant increase in wholeheart Epo upon targeted cardiomyocyte Epo deletion (Figure 1B) with no apparent sex effect (Supplementary Figure S4).To identify the specific cell type(s) responsible for Epo expression under normoxic conditions in EPO fl/fl and EPO Δ/Δ mice, we used RNA fluorescent in situ hybridization.As expected, we observed low basal Epo mRNA production by the cardiomyocyte (Figure 1Ci) and endothelial cell (Figure 1Cii) in EPO fl/fl mice.By contrast, in the EPO Δ/Δ mice, an over-abundance of Epo signal (shown using two regions of interest, Figure 1Ciii, iv) was co-localized with the endothelial cells, indicating that upon successful cardiomyocyte-Epo knockout, the endothelium compensated for the loss by increasing its own Epo expression.Therefore, not only have we verified this phenomenon in the adult mouse using a second independent CreLox line to support our earlier work (Allwood et al., 2024), but we also showed hyper-compensated cardiac Epo occurs regardless of floxed allele homozygosity (EPO Δ/Δ ) or heterozygosity (EPO fl/-: Cre +/− ), and in both sexes.Importantly, despite marked Epo overexpression in the adult EPO Δ/Δ mouse, on a whole-body and organ level, there were no visual abnormalities (Supplementary Figure S3A).There was also no change in renal Epo expression (Figure 2A), serum EPO (Figure 2B), or hematocrit (Figure 2C), suggesting that there would be no altered physiological consequences from compensatory cardiac endothelial overexpression.
3.2 EPO Δ/Δ mice had greater voluntary wheel running capacity independent of changes in hematocrit and whole-body metabolism Exposing mice to either a physiological or pathological challenge could reveal additional insight to the innate compensatory mechanisms.We therefore subjected mice to an exercise test, and considering there was no change in hematocrit, we hypothesized no difference would be observed between groups.Yet, voluntary wheel running across three consecutive days revealed EPO Δ/Δ mice had increased running performance compared to control animals (Figure 2D).Since the difference in exercise capacity was not the result of erythropoiesis, we determined it could be linked to reported effects of EPO as a regulator of energy homeostasis as it increases metabolic activity, cellular respiratory capacity, and oxygen utilization shown previously in transgenic mice (Wang et al., 2013).However, using whole-body indirect calorimetry (CLAMS setup), our data showed there were no differences in VO 2 (Figures 2E, F), VCO 2 (Figures 2G, H), RER (Figures 2I, J), or total energy expenditure between groups (Figure 2K, L).We cannot exclude the possibility for differences in myocardial oxygen consumption at the level of the skeletal muscle by these data alone.It is plausible that EPO Δ/Δ mice were more efficient at extracting oxygen for mitochondrial cellular respiration (i.e., ATP production).Indeed, positive hypertrophic cardiac remodeling might account for the heightened exercise tolerance due to increased cardiac output.

EPO Δ/Δ mice demonstrated concentric cellular hypertrophy
Prior work showed that constitutive deletion of cardiomyocytespecific EPO during embryogenesis causes early hypoplasia, and eventually, cardiac Epo over-expression led to hypertrophy, with no change in overall cardiac mass in adult hearts (Allwood et al., 2024).We sought to explore the physiological significance of cardiomyocyte deleted Epo in adult mice, independent of cardiogenesis.By echocardiography, there was a significant decrease in end systolic dimension (ESD) and a trending reduction in end diastolic dimension in EPO Δ/Δ mice (EDD, Figures 3A, B; Table 3).The left ventricle chamber length was decreased (Figures 3A, C) and posterior wall thickness was increased (Figures 3A, D).While heart rate (Figure 3E) and cardiac output (Figure 3F) were unchanged, EPO Δ/Δ mice had greater ejection fractions (Figure 3G), suggesting systolic function was being augmented.Together, these parameters indicated a concentric hypertrophy phenotype in the EPO Δ/Δ mice.To confirm these findings, we evaluated histologically cardiomyocytes in perfusionfixed hearts (Figure 3H), identifying a significant increase in EPO Δ/Δ cross-sectional area compared to control (Figure 3I).These data combined suggest that as cells of the left ventricle wall widened, the length of the hearts shortened, resulting in no difference in global heart mass.
Both pathological (e.g., hypertension) and physiological (e.g., exercise) conditions stimulate cellular and gross ventricular hypertrophy with inverse relationships to long-term health (Schoenfeld et al., 1998).However, a hallmark feature of pathological remodeling is the re-expression of certain fetal genes (Schoenfeld et al., 1998).Therefore, we quantified relative RNA levels of Anp, Bnp, α-Mhc, ß-Mhc, and the ratio of ß-Mhc/α-Mhc between groups by qPCR.No differences amongst Anp, Bnp, α-Mhc expression, or the ratio of ß-Mhc/α-Mhc (Figure 3J) were seen though ß-Mhc RNA was increased in EPO Δ/Δ mice.An incipient cause of ß-Mhc re-expression during cardiac hypertrophy and normal aging is linked to elevated fibrosis (Pandya et al., 2006).Accordingly, we quantified interstitial fibrosis of the left ventricle between groups (Figures 3Ki,ii), finding no difference.EPO Δ/Δ mice had compensatory cardiac function (i.e., increased ejection fraction) and the EPO Δ/Δ phenotype is inconsistent with pathological remodeling.Nonetheless, we reconciled this line of inquiry with a comprehensive assessment of cardiac function by invasive hemodynamics.

EPO Δ/Δ mice have heightened cardiac function in vivo
To gain a deeper understanding of the functional role of cardiac Epo overexpression and concentric cellular hypertrophy on cardiac function in our model, invasive hemodynamic analyses were performed (Figure 4A; Table 3).There were no differences in heart rate (Figure 4B).In terms of systolic function, EPO Δ/Δ mice demonstrated elevated left ventricular systolic pressures (LVP, Figure 4C) and an increase in cardiac contractility (dP/dt max , dP/dt@LVP40, Figures 4D, E).To assess diastolic function, a multi-parameter approach was used (Ogilvie et al., 2020).For this reason, we reported indices of both relaxation and compliance: dP/dt min (active relaxation phase), Tau Logistic (active relaxation phase), and end diastolic pressure (EDP, passive filling phase).dP/dt min was significantly improved (Figure 4D), though this was not reflected by differences in EDP (Figure 4F) or Tau Logistic (Figure 4G).Taken together, both systolic and diastolic function were superior in the EPO Δ/Δ mice, suggesting endogenous cardiac EPO production was associated with better cardiac function.Supraphysiological levels of exogenous rhEPO confer inotropic and lusitropic effects ex vivo, therefore we were interested in the correlation between the levels of upregulated endogenous Epo RNA and these key hemodynamic parameters recorded in vivo.Simple linear regression analyses demonstrated a strong positive relationship between Epo RNA expression and dP/dt max , dP/dt min , and dP/dt@LVP40, with goodness-of-fits of R 2 = 0.49, 0.48, and 0.67, respectively (Figures 4H-J).Therefore, our results indicate that in mice with higher endogenous cardiac Epo expression, inotropic and lusitropic function are greater.

Cardiac-derived EPO has inotropic, lusitropic, chronotropic, and cardioprotective benefits in an ex vivo isolated heart preparation
The in vivo assessment of cardiac function carries limitations-the data collected are dependent on preload, afterload, and heart rate.Conversely, the Langendorff preparation (i.e., ex vivo) allows for the isolated evaluation of systolic and diastolic cardiac function at a healthy baseline or after ischemic insult under controlled conditions without the influence of systemic neurohormonal or immunological factors.Since exogenous rhEPO has shown cardioprotective effects in vivo, investigating the physiological autocrine and/or paracrine effects of endogenous cardiac EPO on the heart was of particular interest.The EPO Δ/Δ group demonstrated a significant increase in baseline LVP (Figures 5A, B (top left)), developed pressure (Figure 5C), and dP/dt max and dP/dt min (Figure 5A (bottom left), D).While there were no significant differences in EDP or paced heart rate as they were manually set and controlled, intrinsic heart rate prior to pacing was elevated in EPO Δ/Δ mice (Table 4).After baseline recordings, mice were subjected to 25 min of global, no-flow ischemia, followed by 45 min of reperfusion (Figure 5A (right)).EPO Δ/Δ mice displayed better recovery post-ischemia: 71% of LVP (Figure 5E), 76% of dP/ dt max , (Figure 5F), and 61% of dP/dt min (Figure 5G), whereas the EPO fl/fl mice recovered only 27% of LVP (Figure 5E), 28% of dP/ dt max (Figure 5F), and 25% of dP/dt min (Figure 5G).Therefore, independent of heart rate, afterload, preload, and whole-body neurohormonal influence, hearts overexpressing endogenous cardiac EPO also displayed positive inotropic, lusitropic, chronotropic, and cardioprotective qualities.Having established the physiological impact of hyper-compensated cardiac Epo, we aimed to clarify some of the factors involved in regulating this phenomenon.

Marked overexpression of left ventricular
Epo in EPO Δ/Δ mice was HIF2α-independent HIF-1α and HIF-2α are both primary transcription factors for regulating the hypoxic response.However, in terms of the transactivation of the Epo promoter, HIF-2α, and not HIF-1α, is the primary regulator in the adult kidney, liver, and brain (Warnecke et al., 2004;Gruber et al., 2007;Rankin et al., 2007;Yamashita et al., 2008;Yeo et al., 2008;Wang et al., 2015;Urrutia et al., 2016;Ghosh et al., 2021).Although there was no overt indication of hypoxia in this model (i.e., no differences in hematocrit or renal EPO expression), we aimed to rule out the canonical PHD2/VHL/ HIF2α axis as the mechanism for mediating cardiac overexpression of Epo in EPO Δ/Δ mice.By immunoblotting, we showed no difference in the HIF-2α protein levels in left ventricular nuclear extracts of EPO fl/fl and EPO Δ/Δ mice (Figures 6A-C).This finding confirmed the overexpression of left ventricular Epo in EPO Δ/Δ mice was HIF2α-independent.Considering the unexpected nature of our findings, we wanted to conclusively exclude hypoxia as a stimulus for cardiac overexpression of Epo by investigating HIF1α-specific downstream target genes.

Overexpression of cardiac
Epo in EPO Δ/Δ mice was accompanied by elevated Vegfb and Vegfr1 gene expression Under hypoxic conditions, HIF-1α escapes oxygen-dependent degradation, translocates to the nucleus, and complexes with HIF-1β (Wang et al., 1995).Working in concert with hepatocyte nuclear factor 4 (HNF-4) (Galson et al., 1995;Huang et al., 1997;Jelkmann, 2007) and the transcriptional co-activators, p300 and cAMP response element (CREB)-binding protein (Firth et al., 1995;Bunn et al., 1998), the HIF-1 complex binds the hypoxia response element to initiate the transcription Significance was considered when p < 0.05 compared to EPO fl/fl (determined by an unpaired, two-tailed t-test and shown using bolded values and "*").The pink box "(A)" is used to show cardiomyocyte-Epo colocalization in EPO fl/fl , purple box "(B)" is used to show endothelial-Epo colocalization in EPO fl/fl , purple boxes "ROI 1" and "ROI 2" show upregulated EPO signal from endothelial cells.A nuclei marker was used (Dapi, blue).Scale bar represents 50 µm.A one-way ANOVA followed by a Dunnett's post-hoc test was used to detect differences in left ventricular expression between EPO fl/fl , EPO fl/-Cre+/−+TAM , and EPO Δ/Δ mice.An unpaired, two-tailed t-test was used to detect a difference between EPO fl/fl-female and EPO Δ/Δ-female .Data are expressed as mean ± SD and were considered significant when p < 0.05.
Frontiers in Physiology frontiersin.org09 of >500 downstream target genes (Mole et al., 2009), including Vegfa (Forsythe et al., 1996), Glut1 (Iyer et al., 1998), Ho-1 (Lee et al., 1997), Pgk-1 (Semenzas et al., 1994;Li et al., 1996a), Cas 8 (Zhao et al., 2018).Therefore, to rule out hypoxia and the involvement of HIF-1α in regulating cardiac Epo overexpression, we examined the expression levels of select HIF1α-specific target genes (e.g., Vegfa, Glut1, Hmox-1, Pgk-1, Cas 8) by qPCR.We measured no differences in their gene expression, and even downregulation of Cas 8 in EPO Δ/Δ mice (Figure 7).This indicated the upstream regulation of Epo in the EPO Δ/Δ mice by hypoxia and HIF-1α was unlikely.Epo is also regulated by HIF-independent mechanisms in a tissue-specific manner (Yasuda et al., 1998;Tam et al., 2006).One study demonstrates potent inhibition of VEGF induces hepatic synthesis of Epo and subsequent erythropoiesis through a HIF1α-independent mechanism (Tam et al., 2006).Therefore, we investigated the hypoxiaindependent VEGF isoform, Vegfb, and its receptor, Vegfr1, by qPCR analyses.Interestingly, both genes were significantly upregulated in EPO Δ/Δ mice (Figure 7).Co-upregulation of endothelial-derived EPO and whole-heart Vegfb and Vegfr1 suggested a complex cellular interaction was governing this novel physiological concentric hypertrophy and cardioprotective phenotype.To identify a relationship between cardiac EPO and VEGF, we used the FDA-approved VEGF-specific tyrosine kinase inhibitor, axitinib, to interrupt VEGF signaling and observe the corresponding impact on Epo RNA levels in the heart.Greater voluntary wheel running capacity in EPO Δ/Δ compared to EPO fl/fl mice occurred independent from differences in hematocrit and wholebody metabolism.(A) Kidney Epo RNA expression (normalized to β-Actin) in EPO fl/fl and EPO Δ/Δ mice, (B) serum EPO, (C) hematocrit (%) collected from the saphenous vein of EPO fl/fl and EPO Δ/Δ mice, and (D) average voluntary wheel running distance (km) across 3 days.Comprehensive Laboratory Animal Monitoring System (CLAMS) measured (E, F) VO 2 (mL/kg/min), (G, H) VCO 2 (mL/kg/min), (I, J) RER (VCO 2 /VO 2 ), and (K-L) total energy expenditure (TEE, kcal/hour) in EPO fl/fl and EPO Δ/Δ mice during their sleep and wake phases.An unpaired, two-tailed t-test was used to detect differences.Data are expressed as mean ± SD (for qPCR, serum EPO levels, and hematocrit) or mean ± SEM (for average running distance and whole-body metabolic readings).Data were considered significant when p < 0.05.

Axitinib disrupted the VEGF-VEGFRdependent crosstalk between endothelial cells and cardiomyocytes, which further upregulated EPO expression in mice
Several independent labs show active systemic crosstalk between EPO-EPOR and VEGF-VEGFR signal transduction pathways.rhEPO increases Vegf expression (Ribatti et al., 1999;Westenbrink et al., 2008;Lu et al., 2012;Oztas et al., 2020) resulting in angiogenesis and attenuated interstitial fibrosis, while VEGF-VEGFR blockade induces non-renal Epo expression (Tam et al., 2006) and erythropoiesis (Tam et al., 2006;Johnson et al., 2017).Whether these pathways interact similarly in the heart by endogenous forms of EPO and VEGF, is unknown.Therefore, to improve our understanding of the interplay between VEGF-VEGFR and EPO-EPOR in the heart, we interrupted VEGF-VEGFR signaling by inhibition using axitinib (Ma and Waxman, 2009) in EPO fl/fl mice.Preclinically, in a mouse model of prostate cancer, Perfusion fixed hearts (representative hematoxylin and eosin histological images from (i) EPO fl/fl and (ii) EPO Δ/Δ hearts-scale bars represent 50µm and 1000 µm) were used to measure differences in (I) cardiomyocyte cross-sectional area (CSA).(J) Expression of fetal genes (atrial natriuretic peptide (Anp), brain natriuretic peptide (Bnp), alpha myosin heavy chain (αMhc), beta myosin heavy chain (βMhc), and βMhc/αMhc ratio) was quantified.(K) Quantification of interstitial fibrosis (%) from (i) EPO fl/fl and (ii) EPO Δ/Δ hearts stained with Picrosirius Red.Scale bar represent 216 µm.An unpaired, twotailed t-test was used to detect differences.Data are expressed as mean ± SEM (for echocardiography, CSA, and interstitial fibrosis) or mean ± SD (qPCR).Data were considered significant when p < 0.05.supressing tumour patency (i.e., blood perfusion) without disrupting blood perfusion to normal tissues (e.g., heart, kidney, liver, lung, muscle) (Ma and Waxman, 2009).In our study, axitinib was welltolerated, and mice did not display any overt signs of distress during, or after, treatment.Short-term axitinib treatment did not elicit changes in erythropoiesis, as determined by no differences detected in hemoglobin levels measured between EPO fl/fl and EPO fl/fl+AXI groups (Figure 8A).When VEGF signaling was inhibited, cardiac expression of Epo RNA was significantly elevated by qPCR (Figure 8B).However, by invasive hemodynamics, we observed blunted heart rates and reduced left ventricle relaxation in axitinib-treated mice (Supplementary Table S2).These data were expected since tyrosine kinase inhibitors affect the cardiac conduction system, causing bradycardia and QTc prolongation (Kloth et al., 2015;Shopp et al., 2022), and are known to have cardiotoxic effects (Force and Kolaja, 2011;Dobbin et al., 2021).Our findings indicated cardiac Epo upregulation following VEGF inhibition does not rescue hearts from suspected axitinib-related bradycardia and cardiotoxicity.Importantly, however, we revealed the existence of a previously unrecognized link between VEGF and EPO in the heart.Future work is needed to fully define how the cardiac EPO-EPOR and VEGF-VEGFR axes are coordinated.

Discussion
Here we show that targeted deletion of the Epo gene in the adult cardiomyocyte results in compensatory overexpression by cardiac endothelial cells by HIF2α-independent conditions.The presence of excess Epo in the heart translated to notable changes in cardiac Significance was considered when p < 0.05 compared to EPO fl/fl (determined by an unpaired, two-tailed t-test and shown using bolded values and "*").
Frontiers in Physiology frontiersin.orgstructure, and improvements in function and cytoprotection.Hyperproduction of Epo by the heart did not affect systemic erythropoiesis, suggesting EPO only acted locally via paracrine signaling between cardiomyocytes and endothelial cells.Pharmacological interruption of VEGF-VEGFR using the tyrosine kinase inhibitor, axitinib, allowed us to study how cardiac Epo expression could be modulated in the absence of VEGF signaling.Indeed, cardiac Epo RNA levels were upregulated in axitinib-treated mice.These data suggested a dynamic and interactive crosstalk between cardiomyocytes and endothelial cells involving the VEGF-VEGFR pathway that regulate the upstream production and subsequent physiological effects of cardiac EPO under hypoxiaindependent conditions.Taken together, in the adult mouse heart, endothelial EPO is an important regulator of cell infrastructure, cardiac contractility, and ischemia-reperfusion susceptibility, such that excess Epo is associated with superior cardiac function directly independent of systemic red blood cell production.
Recent studies establish EPO as a pleiotropic cytokine.However, interpretation of these data does not always consider the form of EPO in question-exogenous vs. endogenous.rhEPO is differentially glycosylated compared to endogenous EPO (Rush et al., 1995;Skibeli et al., 2001;Lasne et al., 2002) and the oligo branching patterns and sialic acid content modify the pharmacodynamics and biological activity in vivo (i.e., stability and receptor binding) (Weikert et al., 1999;Skibeli et al., 2001).Therefore, high dose rhEPO may facilitate non-specific receptor binding, signal transduction, and physiological outcomes that do not apply to endogenous EPO.According to three ex vivo studies (Kaygisiz et al., 2006;Piuhola et al., 2008;Hefer et al., 2012), exogenous rhEPO is reportedly inotropic.The changes in contractility and relaxation are attributed to cAMP (Kaygisiz et al., 2006), increased calcium transients (rate and magnitude) and myofilament function via PI3-K and PKCε (Hefer et al., 2012), and endothelin-1 signaling (Piuhola et al., 2008).In another ex vivo study, hypoxia induces high endogenous plasma EPO levels, which correlate to increased atrial contraction (Sterin-Borda et al., 2003).Collectively, these studies suggest a positive primary effect of EPO on myocardial inotropic function.
Until now, the interpretation and application of these findings to an in vivo endogenous EPO system was limited.Therefore, using both ex vivo (i.e., Langendorff isolated hearts) and in vivo (i.e., invasive hemodynamics) preparations, our data revealed the improved cardiac functional effects conferred by endogenous EPO in EPO Δ/Δ mice for the first time.It is plausible endogenous EPO improved calcium handling by increasing cAMP-dependent protein kinase A (PKA) phosphorylation of phospholamban (PLB) (Simmerman and Jones, 1998), which stimulated subsequent sarcoplasmic reticulum calcium uptake (Kranias et al., 1985) and release by the ryanodine receptors (RyR) (Valdivia et al., 1995).Indeed, calcium transient assays and western blotting of key proteins (i.e., PLB, RyR) may clarify this line of inquiry.Alternatively, greater cardiac function in EPO overexpressing mice could be attributed to cardiomyocyte hypertrophy (i.e., CSA ∝ force output) (An et al., 1991).Investigating additional biochemical parameters (e.g., nitric oxide, endothelial or neuronal nitric oxide synthase, TGF-β, cyclic GMP) could clarify the mechanism(s) underlying this work, albeit An unpaired two-tailed t-test was used to detect differences.Data are expressed as mean ± SEM and were considered significant when p < 0.05.
Frontiers in Physiology frontiersin.org13 they remain unclear.Since both cardiomyocytes (Wright et al., 2004) and endothelial cells (Anagnostou et al., 1994;Beleslin-Cokic et al., 2004;Yang et al., 2014) express the Epor, the EPO-mediated contractile effects could be the result of total activation of one, or both, cell receptors (Sterin-Borda et al., 2003).Thus, studies relying on transgenic knockout mice, radiolabeled EPO-receptor binding assays, immunoprecipitation, and western blotting, will ultimately improve our knowledge of the non-canonical EPO receptor activation mechanisms that govern EPO-mediated improvements on cardiac function.
We are the first to report that targeted deletion of Epo from the cardiomyocyte, both in the embryonic (Allwood et al., 2024) and  Significance was considered when p < 0.05 compared to EPO fl/fl (determined by an unpaired, two-tailed t-test and shown using bolded values and "*").
Frontiers in Physiology frontiersin.org14 now adult hearts (data presented here), results in a hyper-compensated response by the cardiac endothelial cell.This phenomenon suggests a critical physiological role for cardiac EPO, which must be conserved.The findings presented here were previously unrecognized.Using the same EPO fl/fl mice, Zeigler et al. characterized a conditional EPO-deficient model for chronic kidney disease (CKD) by tamoxifen-induced wholebody Epo knockout in the adult mouse (Zeigler et al., 2010).Considering 80%-95% efficiency of Cre recombination (Sohal et al., 2001;Hsieh et al., 2007;Bersell et al., 2013), their model achieves near ubiquitous Epo knockout from all cell types within the heart (Zeigler et al., 2010).Since the focus of their study was to establish the CKD model and prove EPOdeficient mice still participate in stress-induced erythropoiesis, no further physiological investigations were performed on the heart.Therefore, our study partially resolves this limitation and confirms a critical role for cardioendothelial derived Epo in modulating cardiac morphology, function, and susceptibility to ischemia.However, the complex cellular interplay we observed may not be limited to the cardiomyocytes and endothelial cells.For instance, decades of research indicate renal EPO synthesis is the sum of multiple EPOproducing cells (e.g., cells of the interstitial cortex and outer medulla (Maxwell et al., 1993), proximal convoluted tubule cells (Loya et al., 1994;Haidar et al., 1997), and peritubular fibroblasts (Koury et al., 1988;Lacombe et al., 1988))-therefore, it is plausible the novel regulatory network presented here extends to and includes other cardiac cells (e.g., smooth muscle cells, cardiac progenitors, monocytes/macrophages).While cardiac fibroblast cell isolation revealed no detectable Epo mRNA signal from either group (data not shown), other resident cardiac cells capable of Epo production in the heart could be identified using single cell RNA sequencing, fluorescent in situ hybridization, or Epo reporter mice should be used (Kragesteen et al., 2023).These findings would greatly inform the relevance of non-erythropoietic non-renal EPO, and open new avenues for EPO regulation to be explored.
Cardiac structure and function are governed by complex cellular interplay, that when disturbed, can cause pathology (Yin et al., 2017).A one-way ANOVA followed by a Tukey's post-hoc test was used to detect differences amongst the three groups.Data are expressed as mean ± SD and were considered significant when p < 0.05.

FIGURE 7
Vegfb and Vegfr1 RNA expression were upregulated in the left ventricle of EPO Δ/Δ mice.An unpaired two-tailed t-test was used to detect differences between EPO fl/fl and EPO Δ/Δ for individual genes of interest using qPCR.Data are expressed as mean ± SD and were considered significant when p < 0.05.
Frontiers in Physiology frontiersin.org15 Given their proximity, non-myocytes (e.g., fibroblasts, endothelial cells) regulate cardiomyocyte growth and development either through direct cell-to-cell contact or by the release of paracrine factors (Long et al., 1991;Gray et al., 1998;Yin et al., 2017).Cardiomyocytes, the force-producing cells of the heart, are unique from non-myocytes in that they very rarely self-replicate (Long et al., 1991;Li et al., 1996b;Soonpaa et al., 1996;Poolman and Brooks, 1998).Therefore, should cardiomyocyte-derived EPO be critical for global heart function, it is reasonable to speculate there is a feedforward mechanism involving other cell type(s) to compensate for any loss (proposed mechanism presented in Figure 9).Mice null for either Epo or the Epor die due to vascular abnormalities and anemia (Wu et al., 1999;Kertesz et al., 2004;Allwood et al., 2024).The data presented here emphasize that cardiac Epo production is also imperative for homeostatic function in the adult heart and is thus regulated by complex paracrine mechanisms that allow for redundancy to ensure adequate paracrine EPO is ever-present.Interestingly, this is not the case for VEGF-A.Upon cardiomyocyte or endothelial cell specific Vegfa deletion, no other VEGF isoforms or neighbouring cell types will increase their expression to compensate (Giordano et al., 2001).In these models, a lack of paracrine support leads to profound detriments in vascular homeostasis and cardiac function.The presence of redundancy for one system (i.e., EPO) and not the other (i.e., VEGF), is perhaps surprising considering the parallels between these growth factors and their upstream regulation by the HIFs, but could be explained, at least in part, by the isoform and/or transcription factors involved.
Hypoxia induced Epo synthesis in the kidney, liver, and brain is modulated by HIF-2α (Warnecke et al., 2004;Gruber et al., 2007;Rankin et al., 2007;Yamashita et al., 2008;Wang et al., 2015;Urrutia et al., 2016).However, the transcription factor responsible for cardiac Epo expression under hypoxic conditions (Chu et al., 2007;El Hasnaoui-Saadani et al., 2013;Deji et al., 2015) has received less attention.In one study, acute Vhl inactivation induces cardiac Epo production in a HIF1α-dependent manner.However, HIF-2α was not investigated under these conditions, therefore we cannot rule out its involvement in mediating the cardiac EPO response.Here, we provide evidence of the hypoxia mimetic, CoCl 2 , stabilized HIF-2α in the heart (Figure 6), which induced downstream local Epo production (data not shown).Yasuda et al., ) induced Epo production in the uterus is not regulated by hypoxia (Yasuda et al., 1998).In another study, potent VEGF inhibition increases hepatic Epo synthesis and modulates erythropoiesis in a HIF1α-independent manner (Tam et al., 2006).Our data show cardiac Epo overexpression in the EPO Δ/Δ heart was independent of HIF-2α stabilization (Figure 6) and investigations on HIF-1α target genes further suggest that Epo regulation was not related to a hypoxic stimulus (Figure 7).To our knowledge, this is the first report of cardiac Epo overexpression that occurs independent of HIF-1α and HIF-2α stabilization under normoxic conditions.The identity of the transcription factor(s)/repressors responsible for initiating this phenomenon remain to be solved.Using siRNA knock-down, chromatin immunoprecipitation assays that reveal DNA-protein interactions at the Epo enhancer binding sites, or transgenic knockout models, the mechanisms regulating hypoxia-independent cardiac Epo production may become apparent.
Considering cross-sectional area of a muscle is proportional to its force output, this seems reasonable.To maintain the same organ weight and size, a cardiomyocyte with increased cross-sectional area would either need to 1) shorten lengthwise or 2) undergo apoptosis.To address the former, in our previous study, we confirmed cardiogenesis is modulated by the presence of cardiac Epo-therefore, how long, and wide a cardiomyocyte grows is dependent upon endothelial cell (Colliva et al., 2020) and EPO signaling (Colliva et al., 2020;Allwood et al., 2024).Second, apoptosis is a hallmark feature of the transition from compensatory hypertrophy to heart failure (for review (Van Empel et al., 2005)).Accordingly, reduced contractile function of the heart and increased caspase 8 expression (Kruidering and Evan, 2000) would accompany cardiomyocyte apoptosis, which was not the case for the EPO Δ/Δ mice as confirmed by no difference in the amount of apoptotic cell death by TUNEL assay (Supplementary Figure S5A).Next, while we did not reconcile the transcription factor(s) responsible for regulating hypoxia-independent Epo overexpression, we established it was not via the canonical axis involving HIF-1 or HIF-2.Using next-generation chromatin immunoprecipitation assays/sequencing, the protein-DNA interactions that mediate this response might be uncovered.Further, by modifying the Epo gene construct, LacZ-tagged Epo reporter mice could reveal novel enhancer/promoter regions responsible for cardiac Epo expression.(i.e., 5′ kidney inducibility element, 3' liver inducibility element).

Conclusion
We have uncovered a novel paradigm wherein adult cardiomyocyte Epo deletion induced endothelial cell derived Epo and subsequent Vegfb expression, which together appeared to stimulate cardiomyocyte hypertrophy in a feedforward manner.Along with more efficient cardiac force generation, EPO Δ/Δ mice demonstrated superior resistance to ischemic-reperfusion injury.Accordingly, there was a complex cellular interplay involving the EPO-EPOR and VEGF-VEGFR transduction pathways, which ultimately modulated cardiac structure and function, though future work is required to fully elucidate the mechanisms involved.Together in the heart, these pathways act in concert.The author(s) declared that they were an editorial board member of Frontiers, at the time of submission.This had no impact on the peer review process and the final decision.

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FIGURE 1 Epo
FIGURE 1Epo RNA expression was significantly upregulated in male and female EPO Δ/Δ mice by qPCR.(A) Left ventricular expression of Epo RNA (normalized to Eef1e1) of EPO fl/fl , EPO fl/-Cre+/−+TAM , and EPO Δ/Δ mice.(B) Left ventricular expression of Epo RNA (normalized to Rpl32) of female EPO fl/fl and EPO Δ/Δ mice.Panels (Ci-iv): Cardiomyocytes and endothelial cells contributed to basal Epo expression in EPO fl/fl , which upon cardiomyocyte specific Epo deletion (EPO Δ/Δ ), became overcompensated by the endothelial cells, leading to hyper-expression.(i) A cardiomyocyte (Myh6, TRITC) and (ii) endothelial cell (Kdr, Cy5) show co-localization with EPO (FITC, green) in EPO fl/fl .However, in EPO Δ/Δ mice, the cardiomyocyte does not appear to be a contributing source, confirming successful knockout of EPO.Rather, (iii, iv) show two regions of interests (RIO) to highlight intense endothelial-derived EPO signal.The pink box "(A)" is used to show cardiomyocyte-Epo colocalization in EPO fl/fl , purple box "(B)" is used to show endothelial-Epo colocalization in EPO fl/fl , purple boxes "ROI 1" and "ROI 2" show upregulated EPO signal from endothelial cells.A nuclei marker was used (Dapi, blue).Scale bar represents 50 µm.A one-way ANOVA followed by a Dunnett's post-hoc test was used to detect differences in left ventricular expression between EPO fl/fl , EPO fl/-Cre+/−+TAM , and EPO Δ/Δ mice.An unpaired, two-tailed t-test was used to detect a difference between EPO fl/fl-female and EPO Δ/Δ-female .Data are expressed as mean ± SD and were considered significant when p < 0.05.

FIGURE 4
FIGURE 4 Improved inotropic and lusitropic cardiac function was observed in vivo by invasive hemodynamics in EPO Δ/Δ compared to EPO fl/fl mice.(A) Representative left ventricle pressure (LVP, top) and dP/dt max and dP/dt min (bottom) tracings from EPO fl/fl and EPO Δ/Δ mice.(B) Heart rate (bpm), (C) left ventricular systolic pressure (mmHg), (D) dP/dt max and dP/dt min (mmHg/s), (E) dP/dt@LVP40 (mmHg/s), (F) end diastolic pressure (EDP), and (G) Tau logistic (ms).Simple linear regression correlation analyses were run comparing Epo RNA expression to (H) dP/dt max , (I) dP/dt@LVP40, and (J) dP/ dt min values and detected a positive correlation (p = 0.0014 for each) with moderate to strong goodness-of-fit values (R 2 = 0.49, 0.67, 0.48, respectively).An unpaired two-tailed t-test was used to detect differences.Data are expressed as mean ± SEM and were considered significant when p < 0.05.

FIGURE 5 EPO
FIGURE 5EPO Δ/Δ mice demonstrated greater inotropic and lusitropic cardiac function by the ex vivo Langendorff preparation, accompanied by superior cytoprotective abilities post-ischemia/reperfusion. (A) Representative isolated heart (Langendorff) function tracings at baseline (red and green) and postischemia reperfusion (pink and purple) for EPO fl/fl and EPO Δ/Δ mice.Baseline measurements included (B) left ventricle systolic pressure (mmHg), (C) developed pressure (mmHg), (D) dP/dt max and min (mmHg/s).Baseline was followed by global no-flow ischemia and subsequent reperfusion, generating data for (E) % left ventricular pressure (LVP) recovery, (F) % dP/dt max recovery, and (G) % dP/dt min recovery.An unpaired two-tailed t-test was used to detect differences.Data are expressed as mean ± SEM and were considered significant when p < 0.05.

FIGURE 6
FIGURE 6Marked overexpression of left ventricular Epo in EPO Δ/Δ mice was HIF2α-independent.(A) Western blot permitted the quantification of HIF-2α protein from nuclear extracts of left ventricle tissue of EPO fl/fl , EPO Δ/Δ , and EPO fl/fl mice treated with cobalt chloride (CoCl 2 ) at 3 timepoints-3h postinjection, 1.5h post-injection, and 45min post-injection.(B) Representative western blot of HIF-2α and (C) ponceau stain, which demonstrates equal loading (20 µg per well).A one-way ANOVA followed by a Tukey's post-hoc test was used to detect differences amongst the three groups.Data are expressed as mean ± SD and were considered significant when p < 0.05.

FIGURE 8
FIGURE 8Pan-VEGFR inhibition via 8 days of axitinib treatment (i.p.) significantly upregulated left ventricular Epo RNA expression in EPO fl/fl mice independent of erythropoiesis.(A) Saphenous hemoglobin levels and (B) left ventricle Epo RNA expression by qPCR in EPO fl/fl compared to EPO fl/fl+AXI mice.Data are expressed as mean ± SD and were considered significant when p < 0.05 as determined by unpaired, two-tailed t-test.

TABLE 2
Morphometrics.HW, heart weight; TL, tibial length; LV, left ventricle peak pressure.Data is presented as mean ± SD.

TABLE 3
In vivo cardiac assessment using echocardiography and invasive hemodynamics.LVP, left ventricle peak pressure; LV EDP, left ventricle end diastolic pressure.Data is presented as mean ± SEM.

TABLE 4
Ex vivo cardiac assessment using Langendorff isolated heart preparation.LVP, left ventricle systolic pressure; LV EDP, left ventricle end diastolic pressure.Data is presented as mean ± SEM.