Targeting Runx 1 protects against heart failure with preserved ejection fraction

Targeting Runx1 protects against heart failure with preserved ejection fraction | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var 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Yen Chin Koay , Dylan O’Toole , View ORCID Profile Pawel Herzyk , Colin Nixon , Karen Blyth , John F O’Sullivan , View ORCID Profile Ninian N Lang , View ORCID Profile Colin Berry , View ORCID Profile Thomas Braun , View ORCID Profile Gabriele G Schiattarella , View ORCID Profile Mauro Giacca , View ORCID Profile Martin W McBride , Stuart A Nicklin , Ewan R Cameron , View ORCID Profile Christopher M Loughrey , View ORCID Profile Eilidh A MacDonald doi: https://doi.org/10.1101/2025.01.24.634831 Ali Ali Mohamed Elbassioni 1 British Heart Foundation Glasgow Cardiovascular Research Centre, School of Cardiovascular & Metabolic Health, University of Glasgow , UK 2 Department of Cardiothoracic Surgery, Suez Canal University , Egypt Find this author on Google Scholar Find this author on PubMed Search for this author on this site Anmar A Raheem 1 British Heart Foundation Glasgow Cardiovascular Research Centre, School of Cardiovascular & Metabolic Health, University of Glasgow , UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jian Song 1 British Heart Foundation Glasgow Cardiovascular Research Centre, School of Cardiovascular & Metabolic Health, University of Glasgow , UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Alexander S Johnston 1 British Heart Foundation Glasgow Cardiovascular Research Centre, School of Cardiovascular & Metabolic Health, University of Glasgow , UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Cara Trivett 1 British Heart Foundation Glasgow Cardiovascular Research Centre, School of Cardiovascular & Metabolic Health, University of Glasgow , UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Hong Lin 1 British Heart Foundation Glasgow Cardiovascular Research Centre, School of Cardiovascular & Metabolic Health, University of Glasgow , UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Haobo Zhang 1 British Heart Foundation Glasgow Cardiovascular Research Centre, School of Cardiovascular & Metabolic Health, University of Glasgow , UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ashley Bradley 1 British Heart Foundation Glasgow Cardiovascular Research Centre, School of Cardiovascular & Metabolic Health, University of Glasgow , UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Erin Higgins 1 British Heart Foundation Glasgow Cardiovascular Research Centre, School of Cardiovascular & Metabolic Health, University of Glasgow , UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Leanne Mooney 1 British Heart Foundation Glasgow Cardiovascular Research Centre, School of Cardiovascular & Metabolic Health, University of Glasgow , UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Leanne Mooney Yen Chin Koay 3 University of Sydney , NSW Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Dylan O’Toole 1 British Heart Foundation Glasgow Cardiovascular Research Centre, School of Cardiovascular & Metabolic Health, University of Glasgow , UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Pawel Herzyk 4 Glasgow Polyomics, University of Glasgow , Garscube Campus, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Pawel Herzyk Colin Nixon 5 Cancer Research UK Scotland Institute , Switchback Road, Bearsden, Glasgow, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Karen Blyth 5 Cancer Research UK Scotland Institute , Switchback Road, Bearsden, Glasgow, UK 6 School of Cancer Sciences, University of Glasgow , Glasgow, G61 1QH Find this author on Google Scholar Find this author on PubMed Search for this author on this site John F O’Sullivan 3 University of Sydney , NSW Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ninian N Lang 1 British Heart Foundation Glasgow Cardiovascular Research Centre, School of Cardiovascular & Metabolic Health, University of Glasgow , UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ninian N Lang Colin Berry 1 British Heart Foundation Glasgow Cardiovascular Research Centre, School of Cardiovascular & Metabolic Health, University of Glasgow , UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Colin Berry Thomas Braun 7 Department of Cardiac Development and Remodelling, Max Planck Institute for Heart and Lung Research; Bad Nauheim , Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Thomas Braun Gabriele G Schiattarella 8 Deutsches Herzzentrum der Charité (DHZC), Charité -Universitätsmedizin Berlin , Berlin, Germany. 2DZHK (German Centre for Cardiovascular Research) , Partner Site Berlin, Berlin, Germany 9 Translational Approaches in Heart Failure and Cardiometabolic Disease, Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC) , Berlin, Germany 10 Division of Cardiology, Department of Advanced Biomedical Sciences, Federico II University , Naples, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Gabriele G Schiattarella Mauro Giacca 11 King’s College London British Heart Foundation Centre, School of Cardiovascular & Metabolic Medicine and Sciences , London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Mauro Giacca Martin W McBride 1 British Heart Foundation Glasgow Cardiovascular Research Centre, School of Cardiovascular & Metabolic Health, University of Glasgow , UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Martin W McBride Stuart A Nicklin 1 British Heart Foundation Glasgow Cardiovascular Research Centre, School of Cardiovascular & Metabolic Health, University of Glasgow , UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ewan R Cameron 12 School of Biodiversity One Health & Veterinary Medicine, University of Glasgow , Garscube Campus, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Christopher M Loughrey 1 British Heart Foundation Glasgow Cardiovascular Research Centre, School of Cardiovascular & Metabolic Health, University of Glasgow , UK 12 School of Biodiversity One Health & Veterinary Medicine, University of Glasgow , Garscube Campus, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Christopher M Loughrey Eilidh A MacDonald 1 British Heart Foundation Glasgow Cardiovascular Research Centre, School of Cardiovascular & Metabolic Health, University of Glasgow , UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Eilidh A MacDonald For correspondence: eilidh.macdonald.3{at}glasgow.ac.uk Abstract Full Text Info/History Metrics Preview PDF ABSTRACT Heart failure with preserved ejection fraction (HFpEF) is a public health problem and an elusive illness for which there are few treatment options. HFpEF is a systemic condition with a broad phenotype including diastolic dysfunction, pulmonary oedema, exercise intolerance, and left ventricular (LV) hypertrophy, collectively resulting in enhanced morbidity and mortality. Master-regulator transcription factor RUNX1 has recently been identified as a mediator of pathological changes in many cardiac diseases, however its role in HFpEF was unknown. Here we show that inhibition of Runx 1 limits adverse cardiac remodelling in a clinically relevant mouse model of HFpEF. Cardiomyocyte-specific tamoxifen-inducible Runx 1-deficient mice with HFpEF are protected, with preservation of diastolic function, and attenuation of pulmonary oedema, exercise intolerance, and hypertrophy. Furthermore, targeting Runx 1 in HFpEF by using gene transfer or small molecule inhibitors improves diastolic function, both in female and male mice. Overall, our research enhances our understanding of RUNX1 in cardiac disease and demonstrates a novel translational target for the treatment of HFpEF. Keywords: Heart failure with preserved ejection fraction, metabolic heart failure, diastolic dysfunction, hypertrophy, pulmonary oedema, exercise intolerance CLINICAL PERSPECTIVE Heart failure (HF) is a leading cause of death world-wide and traditionally divided into different subtypes according to cardiac ejection fraction (EF). In contrast to HF with reduced EF (HFrEF), there are limited treatment options for HF with preserved EF which is of considerable concern given that HFpEF is projected to become the dominant HF subtype in the future 1 . RUNX1 has been demonstrated to play an important role in the development of many cardiac and non-cardiac diseases. As a result, the potential for RUNX1 inhibitors as therapeutic agents across various conditions has become increasingly evident. In this study we established the therapeutic potential of targeting RUNX1 in the context of HFpEF. Targeting RUNX1 in cardiomyocytes markedly attenuates the development of the HFpEF phenotype and therefore this novel translational therapeutic target has great potential to address one of the biggest challenges in cardiac research. INTRODUCTION Heart failure (HF), a complex syndrome in which the heart is unable to meet the metabolic demands of the body, leads to considerable morbidity and mortality worldwide. It is classically categorised by the proportion of blood ejected from the left ventricle (LV) with each beat, the ejection fraction (EF). HF with reduced ejection fraction (HFrEF) has been heavily investigated for many years and there are several treatment options available that reduce mortality 2 . More elusive, however, is HF with preserved ejection fraction (HFpEF) which is increasing in prevalence and is poorly understood 3 . Despite multiple advances in the treatment of HFrEF, the classic HFrEF treatments are not convincingly effective for use in HFpEF, resulting in limited therapeutic options 4 . HFpEF is a multimorbidity syndrome, often developing alongside hypertension, metabolic stress, and diabetes, and results in diastolic dysfunction, pulmonary oedema, hypertrophy, and exercise intolerance 4 . HFpEF includes a wide range of clinical phenotypes and pathophysiological heterogeneity, and as such it is not clearly understood 5 . Therefore, elucidating molecular or cellular factors contributing to the development of HFpEF is essential and an important step toward identifying therapeutic targets. Master-regulator transcription factor, RUNX1, is minimally expressed in the adult heart but can be reactivated in the context of cardiac pathology. Using animal model systems, it has been shown to be a mediator and therapeutic target against adverse cardiac remodelling following myocardial infarction (MI), which is a major cause of HFrEF 6 – 8 , and in a transaortic constriction HFrEF model 9 . Targeting Runx 1 in the post-MI heart results in improved systolic function, calcium handling, and preservation of genes involved in oxidative phosphorylation 6 , 7 , 10 . Large scale analysis of RNAseq studies on human myocardium 11 – 17 demonstrates that Runx 1 expression is increased in several cardiac pathologies including myocardial infarction, hypertrophic cardiomyopathy, and dilated cardiomyopathy ( Supplemental Figure 1 , Supplemental Table 1 ). Further, serum samples from people admitted to hospital with decompensated HFpEF and HFrEF show that RUNX1 expression is higher in HFpEF than HFrEF (personal communication [Lang/Mooney]). Therefore, we hypothesised a potential role for RUNX1 in the pathophysiology of HFpEF, which is characterised by cardiac hypertrophy and stiffening 18 . The aim of this study was to use a preclinical model of HFpEF to interrogate the potential role of RUNX1 in the development of HFpEF and to identify its potential as a therapeutic target for the treatment of HFpEF. Download figure Open in new tab SUPPLEMENTAL FIGURE 1. View this table: View inline View popup Download powerpoint SUPPLEMENTAL TABLE 1. METHODS Detailed methods and statistical analysis are presented in the supplemental methods. We used a previously established 19 , two-hit model (2HM) that combines administration of a high-fat diet (HFD) and inhibition of nitric oxide synthase with N ω -nitro-L-arginine methyl ester (L-NAME) in drinking water to induce a HFpEF phenotype and compared changes to age-matched controls (CTRL) fed a regular chow diet and normal drinking water 19 . We utilised cardiomyocyte-specific tamoxifen-inducible Runx1 -deficient ( Runx1 Δ/Δ ) mice and floxed genetic-control mice ( Runx1 fl/fl ) generated as previously described 20 . RUNX1 was also targeted by either adeno-associated virus (AAV)-mediated delivery of shRNA or a small molecule inhibitor of RUNX1, Ro5-3335, as detailed in the supplemental methods. Bulk RNA sequencing and subsequent pathway analysis was performed on LV tissue samples from Runx1 fl/fl and Runx1 Δ/Δ mice at baseline and after the 2HM protocol. Using prior biological knowledge from the Ingenuity knowledge base, the cardiotoxicity networks and functional analyses were generated using QIAGEN Ingenuity Pathway Analysis (IPA) ( https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis/ ). The focus on cardiotoxicity processes considers the likely activation and inhibition of biological processes between the two strain comparisons (2HM- Runx1 fl/fl compared to CTRL- Runx1 fl/fl and 2HM- Runx1 Δ/Δ and CTRL- Runx1 Δ/Δ ) using a Z-score. Differences in Z-scores were identified. RESULTS Effect of cardiomyocyte-specific Runx1- deficiency on the development of HFpEF To evaluate the contribution of RUNX1 to the pathophysiology of HFpEF, we utilised male Runx1 Δ/Δ mice and floxed control mice ( Runx1 fl/fl ) on the 2HM and CTRL protocols compared to age-matched genetic controls (CTRL- Runx1 fl/fl : n = 19, CTRL- Runx1 Δ/Δ : n = 17, 2HM- Runx1 fl/fl : n = 21, 2HM- Runx1 Δ/Δ : n = 22, Figure 1a ). Each of the two-hits were observed in 2HM- Runx1 fl/fl and 2HM- Runx1 Δ/Δ male mice with an increase in body weight (8.0 ± 0.9g and 7.0 ± 0.9g, respectively both P<0.05; Figure 1b ) and systolic blood pressure (SBP, 33 ± 21mmHg and 30 ± 20mmHg respectively, both P<0.05; Figure 1c ) over the course of the protocol compared to respective CTRL groups. We confirmed that neither 2HM group had developed HFrEF by assessing whole heart contractile function as measured by fractional shortening via echocardiography ( Supplemental Figure 2 ). Download figure Open in new tab SUPPLEMENTAL FIGURE 2. Download figure Open in new tab Figure 1. Runx 1-deficient mice are protected against HFpEF phenotype. a) Schematic of two-hit protocol and experimental groups. b) Body weight over the experimental protocol in each of the groups. * p < 0.05 for 2HM- Runx 1 fl/fl (n = 21) compared to CTRL- Runx 1 fl/fl (n = 19), # p < 0.05 for 2HM- Runx 1 Δ/Δ (n = 22) compared to CTRL- Runx 1 Δ/Δ (n = 17), and † p < 0.05 for 2HM- Runx 1 Δ/Δ compared to 2HM- Runx 1 fl/fl by mixed-effects analysis. c) Systolic blood pressure (BP) over the experimental protocol. * p < 0.05 for 2HM- Runx 1 fl/fl compared to CTRL- Runx 1 fl/fl and # p < 0.05 for 2HM- Runx 1 Δ/Δ compared to CTRL- Runx 1 Δ/Δ by mixed-effects analysis. Characterisation of the HFpEF phenotype: d) Exercise intolerance was quantified by running distance. e) Pulmonary oedema quantified by wet to dry lung weight ratio. Hypertrophy was quantified by f) left ventricular (LV) weight normalised to tibial length (TL) and by g) cardiomyocyte cross sectional area assessed following Wheat Germ Agglutinin staining. Diastolic function quantified by h) E to A wave ratio from pulsed wave Doppler echocardiography and by the slope (β) of the end-diastolic pressure volume relationship derived from the exponential equation: (LVEDP= curve fitting constant × e [stiffness constant × LV end diastolic volume] ) i) representative curves (error lines denote 95% confidence interval; and j) data set. We then evaluated other key features of the HFpEF phenotype. At the end of the protocol, 2HM- Runx1 fl/fl mice had developed exercise intolerance, demonstrated by a reduction in running distance compared to CTRL- Runx1 fl/fl mice (128 ± 14m vs . 296 ± 18m respectively, P<0.05; Figure 1d ). 2HM- Runx1 Δ/Δ mice also had a reduction in running distance compared to their genetic control (2HM- Runx1 Δ/Δ : 192 ± 14m, CTRL- Runx1 Δ/Δ : 327 ± 22m, P<0.05; Figure 1d ) however, the exercise intolerance was attenuated because 2HM- Runx1 Δ/Δ mice ran greater distances than 2HM- Runx1 fl/fl mice (128 ± 14m vs . 192 ± 14m, P<0.05; Figure 1d ). Runx 1 deficiency also protected mice from developing pulmonary oedema as measured by the wet to dry lung weight. The wet to dry lung weight ratio was increased in 2HM- Runx1 fl/fl compared to in CTRL- Runx1 fl/fl mice (3.90 ± 0.2 vs . 2.84 ± 0.2, P<0.05; Figure 1e ). Conversely, there was no difference between 2HM- Runx1 Δ/Δ and CTRL- Runx1 Δ/Δ mice (3.14 ± 0.2 vs . 3.00 ± 0.1; Figure 1e ), and 2HM- Runx1 Δ/Δ mice had significantly lower wet to dry lung weight ratio than 2HM- Runx1 fl/fl mice (3.14 ± 0.2 vs . 3.90 ± 0.2, P<0.05; Figure 1e ). Runx 1 deficiency was also protective against development of hypertrophy as measured by LV weight normalised to tibial length (LV/TL). 2HM- Runx1 fl/fl had increased LV/TL compared to CTRL- Runx1 fl/fl mice (4.9 ± 2.3*10 - 3 vs . 3.5 ± 1.2*10 - 3 , P<0.05; Figure 1f ) and 2HM- Runx1 Δ/Δ mice had smaller LV/TL than 2HM- Runx1 fl/fl mice (4.2 ± 1.3*10 -3 , P<0.05; Figure 1f ) but no difference compared to CTRL- Runx1 Δ/Δ mice (3.7 ± 1.0 *10 -3 ; Figure 1f ). An additional indicator, relevant to concentric hypertrophy, is cardiomyocyte cross-sectional area. In contrast to 2HM- Runx1 fl/fl animals, 2HM- Runx1 Δ/Δ mice showed no significant increase in the cross-sectional area of cardiomyocytes compared to their relative control group (CTRL- Runx1 fl/fl : 352 ± 9.9 μm 2 , CTRL- Runx1 Δ/Δ : 323 ± 32.4 μm 2 , 2HM- Runx1 fl/fl : 482 ± 18.0 μm 2 , 2HM- Runx1 Δ/Δ : 289 ± 24.8 μm 2 ; Figure 1g , Supplemental Figure 2 ). Further, posterior and anterior wall thickness measured during systole with M-mode echocardiography was increased in the 2HM- Runx1 fl/fl mice but not in the 2HM- Runx1 Δ/Δ mice, compared to relevant controls ( Supplemental Figure 2 ). In addition to hypertrophy, there was a striking preservation of diastolic function in cardiomyocyte-specific Runx 1 knockdown mice, quantified by E to A wave ratio from pulsed-wave Doppler echocardiography. Compared to CTRL- Runx1 fl/fl and CTRL- Runx1 Δ/Δ , 2HM- Runx1 fl/fl had a higher E/A ratio whereas the E/A ratio in 2HM- Runx1 Δ/Δ mice was not different from either control group (CTRL- Runx1 fl/fl : 1.63 ± 0.10, CTRL- Runx1 Δ/Δ : 1.58 ± 0.09, 2HM- Runx1 fl/fl : 3.75 ± 0.29, 2HM- Runx1 Δ/Δ : 1.67 ± 0.09; Figure 1h , Supplemental Figure 2 ). An independent measure of LV chamber stiffness was calculated by fitting the slope of the load-independent end-diastolic pressure- volume relationship (EDPVR) measured using intracardiac pressure-volume catheters. 2HM- Runx1 fl/fl mice had a steeper EDPVR slope than 2HM- Runx1 Δ/Δ mice, indicating better diastolic function in the 2HM- Runx1 Δ/Δ mice (0.075 ± 0.008 vs . 0.018 ± 0.003, P<0.05; Figure 1i , 1j). Peripheral organs were also collected to investigate systemic effects of cardiomyocyte-specific Runx 1-deficiency. Liver, right kidney, and left kidney weights (all normalised to tibial length) were increased in 2HM- Runx1 fl/fl compared to CTRL- Runx1 fl/fl mice but were not different between Runx1 Δ/Δ mouse groups ( Supplemental Figure 2 ). Runx1 RNA interference using adeno-associated virus serotype 9 (AAV9) attenuates diastolic dysfunction in HFpEF Given the striking phenotypic differences observed in 2HM- Runx1 Δ/Δ mice compared to 2HM- Runx1 fl/fl , we then tested whether using translational approaches to target RUNX1 expression could prevent the development of HFpEF. To do this we utilised a viral vector-mediated gene delivery approach with AAV9- Runx 1-shRNA to knockdown Runx 1 in our 2HM of HFpEF. We injected 12-week-old C57BL/6N male mice via the tail vein with AAV9-scramble-shRNA (2HM-AAV9-scram, n = 10) or AAV9- Runx 1- shRNA (2HM-AAV9- Runx 1, n = 11) after which mice were placed on the 2HM protocol for 8 weeks for comparison to age-matched C57BL/6N mice on the 2HM (2HM-C57N, n = 18) or control (CTRL- C57N, n = 13) protocols ( Figure 2a ). Once again, we confirmed the efficacy of our 2HM by measuring changes in body weight ( Figure 2b ), SBP ( Figure 2c ), and preservation of fractional shortening from echocardiograpgy ( Supplemental Figure 3 ) over the duration of the protocol. Targeting Runx 1 with AAV9- Runx 1-shRNA was effective in preventing a number (but not all) of the key features of the HFpEF phenotype. Exercise intolerance was observed in all three 2HM groups compared to CTRL- C57N, but with no difference in running distance between 2HM groups (CTRL-C57N: 262 ± 12m, 2HM-C57N: 108 ± 3m, 2HM-AAV9-scram: 108 ± 23m, 2HM-AAV9- Runx 1: 163 ± 22m; Figure 2d ). Download figure Open in new tab SUPPLEMENTAL FIGURE 3. Download figure Open in new tab Figure 2. AAV9-mediated knockdown of Runx 1 protects against diastolic dysfunction. a) Schematic of two-hit protocol and experimental groups. b) Body weight over the experimental protocol i* p < 0.05 for 2HM-C57N (n = 18) compared to CTRL-C57N (n = 13), # p < 0.05 for 2HM-AAV9-scraM (n = 10) compared to CTRL-C57N, and † p < 0.05 for 2HM-AAV9- Runx 1 (n = 11) compared to CTRL-C57N by mixed-effects analysis. c) Systolic blood pressure (BP) over the experimental protocol. * p < 0.05 for 2HM-C57N compared to CTRL-C57N, # p < 0.05 for 2HM-AAV9-scram compared to CTRL-C57N, and † p < 0.05 for 2HM-AAV9- Runx 1 compared to CTRL-C57N by mixed-effects analysis. Characterisation of the HFpEF phenotype: d) Exercise intolerance was quantified by running distance. e) Pulmonary oedema quantified by wet to dry lung weight ratio. Hypertrophy was quantified by f) left ventricular (LV) weight normalised to tibial length (TL) and by g) cardiomyocyte cross sectional area assessed following Wheat Germ Agglutinin staining. Diastolic function quantified by E to A wave ratio from pulsed wave Doppler echocardiography on h) the final week and i) over the protocol; and by the slope (β) of the end-diastolic pressure volume relationship derived from the exponential equation: (LVEDP= curve fitting constant × e [stiffness constant × LV end diastolic volume] ) j) representative curves (error lines denote 95% confidence interval; and k) data set. AAV9- Runx 1 did, however, attenuate the development of pulmonary oedema compared to the other two 2HM groups, quantified by wet to dry lung weight ratio (CTRL-C57N: 3.18 ± 0.16, 2HM-C57N: 4.58 ± 0.07, 2HM-AAV9-scram: 4.36 ± 0.12, 2HM-AAV9- Runx 1: 3.68 ± 0.15; Figure 2e ). As with exercise testing, LV/TL was increased in 2HM groups compared to CTRL but was not different between 2HM groups (CTRL-C57N: 4.1 ± 0.2 *10 -3 , 2HM-C57N: 5.0 ± 0.2 *10 -3 , 2HM-AAV9-scram: 5.4 ± 0.2 *10 -3 , 2HM-AAV9- Runx 1: 4.9 ± 0.2 *10 -3 ; Figure 2f ). However, cardiomyocyte cross-sectional area of 2HM-AAV9-Runx1 was less than the 2HM-AAV9-scram group (2HM-AAV9-scram: 449 ± 10 μm 2 vs . 2HM-AAV9- Runx 1: 308 ± 23 μm 2 ; Figure 2g , Supplemental Figure 3 ). Most striking was the preservation of diastolic function by targeting Runx 1 with AAV9. E/A ratio was increased in both 2HM- C57N and 2HM-AAV9-scram groups compared to CTRL-C57N but was not increased in 2HM-AAV9- Runx 1 compared to CTRL-C57N (CTRL-C57N: 1.32 ± 0.10, 2HM-C57N: 2.55 ± 0.26, 2HM-AAV9-scram: 2.78 ± 0.33, 2HM-AAV9- Runx 1: 1.29 ± 0.12; Figure 2h , 2i; the latter figure demonstrating change over time, Supplemental Figure 3 ). This was also consistent with EDPVR, which was markedly lower in the 2HM-AAV9- Runx 1 group compared to 2HM-AAV9-scram (0.077 ± 0.009 vs . 0.019 ± 0.003, P<0.05; Figure 2j , 2i). Small molecule inhibition of RUNX1 remedies the HFpEF phenotype To take this one translational step further, we aimed to identify if inhibition of RUNX1 could ameliorate the HFpEF phenotype once it has already begun to develop using an established small molecule inhibitor of RUNX1 20 . 10-12 week-old C57BL/6N strain male mice were placed on the 2HM protocol for 10-12 weeks. Prior to drug treatment, in vivo parameters were utilised to ensure the HFpEF phenotype had developed and any mice that did not have HFpEF symptoms were excluded so that we were only attempting to treat mice with a phenotype to attenuate. Next, while mice remained on 2HM protocol, we injected small molecule inhibitors of RUNX1, either DMSO or Ro5-3335 every second day for two weeks prior to collecting end-point measurements and organometrics ( Figure 3a ). Although RUNX1 inhibition by Ro5-3335 injections did not change exercise tolerance (199.1 ± 50.2 vs . 192.5 ± 40.21, P>0.05; Figure 3b ), pulmonary oedema was reduced in 2HM-Ro5-3335 mice compared to 2HM-DMSO mice (4.26 ± 0.05 vs . 4.06 ± 0.07, P0.05, Figure 3d ). Diastolic dysfunction was attenuated in 2HM-Ro5-3335 mice compared to 2HM-DMSO. There was no difference in E/A wave ratio post-injection compared to pre-injection in the 2HM-DMSO mice (2.66 ± 0.35 vs . 2.82 ± 0.15, P>0.05; Figure 3e , left, Supplemental Figure 4 ) whereas the post-injection E/A wave ratio was reduced in the 2HM-Ro5-3335 mice compared to pre-injection, demonstrating diastolic dysfunction was attenuated (1.68 ± 0.14 vs . 2.51 ± 0.16, P<0.05; Figure 3e , right, Supplemental Figure 4 ). This was confirmed using PV loop assessment of diastolic function by EDPVR in 2HM-DMSO mice compared to the 2HM-Ro5-3335 group (0.048 ± 0.005 vs . 0.029 ± 0.005, P<0.05; Figure 3f ). Download figure Open in new tab SUPPLEMENTAL FIGURE 4. Download figure Open in new tab Figure 3. Runx 1 small molecule inhibitor Ro5-3335 remedies against diastolic dysfunction. a) Schematic of two-hit protocol and experimental groups, 2HM-DMSO (n = 12) and 2HM-Ro5-3335 (n = 14). Characterisation of the HFpEF phenotype: b) Exercise intolerance was quantified by running distance. c) Pulmonary oedema was quantified by wet to dry lung weight ratio. Hypertrophy was quantified by d) left ventricular (LV) weight normalised to tibial length (TL). Diastolic function quantified by e) E to A wave ratio from pulsed wave Doppler echocardiography and by the slope (β) of the end-diastolic pressure volume relationship derived from the exponential equation: (LVEDP= curve fitting constant × e [stiff- ness constant × LV end diastolic volume] ) f) representative curves (error lines denote 95% confidence interval; and g) data set. RNAseq predicts patterns of transcriptional changes consistent with a HFpEF phenotype To gain broader insight into the role of Runx 1 in HFpEF, we performed bulk RNAseq on analysis on LV tissue samples from Runx1 fl/fl and Runx1 Δ/Δ mice both at baseline (day 0, D0) and at the end of the 2HM study. There were not any significantly differentially expressed genes (DEG) between Runx1 fl/fl and Runx1 Δ/Δ at D0 and despite the large phenotypic differences, there were only 32 DEG between Runx1 fl/fl and Runx1 Δ/Δ mice at week 13 ( Supplemental Table 2 ). However, there were many differences when comparing each strain at week 13 compared to their respective baseline controls. Thus, because the transcriptomic snapshot at the end of the study does not depict the highly different phenotypes, we focussed on comparing the changes from D0 to the end time point within each strain. Using a false discovery rate (FDR) cut-off of ≤0.05 and log fold change (logFC) ±1, there were 1,866 DEG in 2HM- Runx 1 fl/fl mice at week 13 compared to D0 Runx 1 fl/fl mice ( Figure 4a and 4c ) and 3,691 DEG at week 13 in 2HM- Runx 1 Δ/Δ mice compared to the D0 ( Figure 4b and 4c ). The majority of DEG were shared between strains (1727 DEG: 92.6% of total DEG for Runx 1 fl/fl and 53.2% of total DEG for Runx 1 Δ/Δ ; Figure 4c ). Interestingly, the unique changes in the Runx 1 Δ/Δ mice across timepoints may account for the large functional differences observed because there were very few unique changes in the Runx1 fl/fl mice ( Figure 4c ). Using all significantly DEG in Runx1 fl/fl and Runx1 Δ/Δ mice at week 13 compared to baseline, we focused on cardiac toxicity functions defined by IPA software. We compared Z-scores (a statistical measure utilised to determine the significance and directionality of gene expression changes within a given pathway over that time course) from Runx1 fl/fl (week 13 of 2HM vs D0) and Runx1 Δ/Δ (week 13 of 2HM vs D0). We visualised the impact of Runx 1 deficiency by plotting the difference between Z-scores from Runx1 fl/fl (2HM vs D0) minus Runx1 Δ/Δ (2HM vs D0) mice (Z-diff; Figure 4d ). Whilst some predictive changes in cardiac toxicity functions demonstrated limited difference in Z-diff (yellow; Figure 4d ), the largest differences in Z-score were in congestive heart failure genes and cardiac damage genes. The genes included by IPA in these two cardiac toxicity functions were then plotted using a heat map ( Figure 4e and f ). View this table: View inline View popup Download powerpoint SUPPLEMENTAL TABLE 2. Download figure Open in new tab Figure 4. Cardiac differential gene expression (DEG) analysis of Runx 1 fl/fl and Runx 1 Δ/Δ mice between day 0 (D0) and 13 weeks of two-hit model (2HM) protocol. Volcano plots of all genes (orange-significantly downregulated, green-significantly upregulated and grey not changing) with the eight most regulated genes indicated in a) Runx 1 fl/fl and b) Runx 1 Δ/Δ mice. c) Venn diagram indicating unique changes and the large number of genes that are commonly differentially regulated between group comparison. d) Differences in functional predictions using Z-scores comparing Runx 1 fl/fl mice to Runx 1 Δ/Δ mice (red indicating an activation between Runx 1 fl/fl minus Runx 1 Δ/Δ , blue indicating an inhibition, and yellow indicating similar functional predictions in both groups). Heat map representing patterns of e) congestive heart failure and f) cardiac damage gene expression levels between Runx 1 fl/fl mice to Runx 1 Δ/Δ mice. Inhibition of Runx 1 in female mice: reversal of HFpEF phenotype To further increase the relevance and impact of our findings, we expanded our study in two ways: we used female mice to increase clinical relevance; and we waited to intervene with Runx 1 inhibition via RNA interference until HFpEF was already established in the mice, to test its utility as a therapy. It has been demonstrated that it is more difficult to induce a HFpEF phenotype via the 2HM in female mice compared to males in young mice 21 . Thus, in a cohort of C57-N strain females we waited until they were aged 14 weeks (∼40% older than previous data) before placing them on the 2HM protocol with a ramping dose of L-NAME ( Figure 5a ). Once again, we ensured efficacy of the two hits by measuring body weight and SBP in a female CTRL-C57N group (F-CTRL-C57N, n = 4) compared to a female 2HM-C57N group (F-2HM-C57N, n = 16; Figure 5b , 5c). We utilised our intermediary in vivo phenotypic measures exercise intolerance ( Figure 5d ) and diastolic dysfunction ( Figure 5e ) to confirm that at the 8-week time point the F-2HM-C57N group had established a HFpEF phenotype. Following this, we split the F-2HM-C57N group into two groups for AAV-mediated gene delivery such that they had consistent starting parameters ( Figure 5a ). One group was injected with AAV9-scramble-shRNA (F-2HM-AAV9-scram, n = 8) and a second injected with AAV9- Runx 1-shRNA to knockdown Runx 1 (F-2HM-AAV9- Runx 1, n = 8). Consistent with the male AAV study, there were no differences in running distance between groups 4 weeks following AAV injection (F-2HM-AAV9-scram: 188 ± 8m, F-2HM-AAV9- Runx 1: 182 ± 17m, p = 0.7560; Figure 5f ). We found pulmonary oedema was reduced in the F-2HM-AAV9- Runx 1 compared to F-2HM-AAV9-scram (3.97 ± 0.05 vs 4.29 ± 0.07, respectively, p = 0.0024; Figure 5h ) which was consistent with the male data ( Figure 2e ). Although hypertrophy (measure by LV weight normalised to TL) was not different between the 2HM-AAV-scram and 2HM-AAV- Runx 1 males ( Figure 2f ), it was reduced in F-2HM-AAV9- Runx 1 compared to F-2HM-AAV-scram (3.1 ± 0.1 *10 -3 vs 3.6 ± 0.2 *10 -3 , respectively, P<0.05; Figure 5i ). Finally, diastolic dysfunction was attenuated as measured both by E/A wave ratio from pulse wave Doppler echocardiography (F-2HM- AAV-scram: 1.99 ± 0.11 vs F-2HM-AAV9- Runx 1: 1.49 ± 0.05, p = 0.0007 Figure 5j , Supplemental Figure 5 ), and by the slope of the EDPVR (F-2HM-AAV-scram: 0.082 ± 0.0002 vs F-2HM-AAV9- Runx 1: 0.041 ± 0.0057, p = 0.0027; Figure 5k ). Download figure Open in new tab SUPPLEMENTAL FIGURE 5. Download figure Open in new tab Figure 5. AAV9-mediated knockdown of Runx 1 in female mice: partial reversal of HFpEF phenotype. a) Schematic of two-hit protocol and experimental groups, F-CTRL-C57N (n = 4), F-2HM-C57N (n = 16: F-2HM-AAV-scram, n = 8; F-2HM-AAV- Runx 1, n = 8). b) Body weight, c) systolic blood pressure (BP), d) exercise intolerance testing quantified by running distance, and diastolic function quantified by e) E to A wave ratio from pulsed wave Doppler echocardiography at week 8 in female control C57N-strain mice (F-CTRL-C57N) compared to female C57N-strain mice on the two hit model protocol (F-2HM-C57N). f) Exercise intolerance was quantified by running distance and g) pulmonary oedema was quantified by h) wet to dry lung weight ratio following removal of outlier identified by ROUT outlier test. Diastolic function quantified by i) E to A wave ratio from pulsed wave Doppler echocardiography; and by the slope (β) of the end-diastolic pressure volume relationship derived from the exponential equation: (LVEDP= curve fitting con- DISCUSSION This work identifies a critical role for RUNX1 in the development of HFpEF. Furthermore, we provide evidence that targeting Runx 1 in the context of HFpEF has clinical translational potential. In recent years, significant work has been done to establish a model with preserved EF which not only demonstrates increased hypertrophy but also phenotypes such as pulmonary oedema, exercise intolerance, and diastolic dysfunction and therefore is more representative of the multimorbidity, multi-system disorder of HFpEF in humans 19 , 22 . Runx 1 has been robustly demonstrated to play an important role in the context of cardiac disease, with a particular emphasis on its importance in adverse cardiac remodelling following MI 10 , 20 , 23 , 24 . Previous work has demonstrated the beneficial effects of targeting Runx 1 in the context of acute MI 20 and in the context of ischemic heart disease, however whether these benefits would be observed in a cardiac disease of a chronic progressive nature such as HFpEF was unknown. Therefore, we adapted a 2HM of HFpEF in our line of transgenic mice with cardiomyocyte Runx 1 deficiency, and then again with the C57-N strain mice using translational approaches to target Runx 1. Overall, this work has identified RUNX1 as a promising therapeutic target for treatment and prevention of HFpEF. Targeting Runx 1 with a cardiomyocyte-specific Runx 1-deficient mouse attenuates the development of a HFpEF phenotype. Runx 1-deficiency is highly protective against the development of HFpEF because despite the efficacy of the two-hits ( i.e. , mice in both 2HM groups gained weight and had increased SBP), the Runx 1-deficient mice did not develop all the signs of HFpEF whereas control mice had a classical HFpEF phenotype. Specifically, Runx 1-deficiency reduced the development of hypertrophy and exercise intolerance and completely protected against development of pulmonary oedema and diastolic dysfunction. Although in this study we have simply targeted a single gene ( Runx 1) in a single cell type (cardiomyocytes), the phenotypic outcome was evident systemically including effects on exercise intolerance, pulmonary oedema, and the mass of peripheral organs, reflecting the beneficial effects of targeting Runx 1 for both cardiac dysfunction and peripheral systems. We corroborated and translated these findings using RNAi therapy and small molecule inhibition of RUNX1. Interestingly, similar to the convincing protection of Runx 1-deficient mice, targeting Runx 1 with RNAi and small molecule inhibitors also attenuated diastolic dysfunction and pulmonary oedema. Not only were we able to prevent these two phenotypes with pre-treatment of AAV9 targeting Runx 1 but also by inhibiting Runx with the small molecule inhibitor Ro5-3335 after the establishment of HFpEF. We note that some parameters measured were less affected by these alternative approaches and may reflect the number of cardiomyocytes exposed to the therapy and/or, effects on non-cardiomyocytes or duration of exposure. Future work will aim to further understand the relative benefits of different approaches. To interrogate potential gene changes underlying the phenotypic differences observed when inhibiting Runx 1, we used RNAseq. This resulted in predictions using IPA software for changes in the regulation of diseases and functions when comparing the final time point (after 13 weeks of 2HM) tissue in both groups compared to day 0 (D0) heart tissue. In any chronic disease it is difficult to determine at which timepoint transcriptional changes might best be identified in order to discern differences between Runx1 fl/fl and Runx1 Δ/Δ mice because relevant changes in the transcriptome may precede phenotype differences. As such, it is perhaps unsurprising that the most DEG were shared between strains despite the stark phenotypic differences between the 2HM transgenic groups. However, IPA did predict an upregulation in congestive heart failure pathways in both Runx1 fl/fl and Runx1 Δ/Δ mice, with larger changes occurring in Runx1 fl/fl compared to Runx1 Δ/Δ mice despite more gene changes overall occurring in the Runx1 Δ/Δ mice. Interestingly, it was predicted that the upregulation of cardiac damage pathways would result in larger changes in Runx1 Δ/Δ mice compared to Runx1 fl/fl mice. Finally, we expanded the translational relevance of our work by performing a study in female mice. This enabled us to not only determine the effect of gene transfer in both sexes but also interrogate the translational potential of targeting Runx 1 with AAV9 after the HFpEF phenotype was fully established (in contrast to our male study where AAV was administered prior to mice being placed on the 2HM protocol). The AAV9 was injected following development of an evident HFpEF phenotype. Overall, inhibiting Runx 1 via RNAi was effective in reducing hypertrophy, pulmonary oedema, and diastolic dysfunction in the female 2HM, thus indicating a potential role for Runx 1 in the treatment of HFpEF in both females as well as males, and is capable of partially reversing the phenotype. Limitations to this study include the use of bulk RNAseq rather than a more targeted approach. It is possible that many of the DEG in our bulk tissue samples will be the result of transcriptional changes in non-cardiomyocyte cell types in the ventricle, potentially diluting cardiomyocyte-specific changes that are the result of the Runx 1-deficiency. Single-cell transcriptomic analysis, potentially at multiple time points, is part of the programme of future work. The aetiology of HFpEF and the associated changes in heart structure and diastolic function are complex and relatively poorly understood. The relative contributions of the metabolic changes at a cellular level and the chronic low-grade inflammation that accompanies metabolic stress and hypertension are not clear. It is remarkable that the relatively simple model developed by Schiattarella et al in 2019 and used again here can recapitulate many of the phenotypic changes associated with HFpEF given the subtleties of these physiological insults and their complex interplay. We acknowledge that RUNX1 is likely to modulate several aspects that mediate the pathogenesis of this syndrome. Interestingly, this is not at the level of the inducing factors, because weight gain and increased SBP are observed in both the Runx1 fl/fl and Runx1 Δ/Δ groups. Rather, it appears that RUNX1 is involved in the pathways that connect these factors (metabolic stress and increased SBP) to changes in the heart, leading to hypertrophy and diastolic dysfunction. It is intriguing that attenuation of Runx 1 function alleviates the deleterious effects observed both following MI and prevents and reverses key aspects of HFpEF, hinting at a more fundamental role in the response of heart tissue to damage and pathophysiological insult. Overall, this study clearly demonstrates that RUNX1 drives pathological changes in cardiomyocytes in the context of HFpEF. Inhibition of Runx 1 by gene transfer or the use of a small molecule inhibitor improves LV diastolic function and represents an exciting translational approach for the treatment of HFpEF. FUNDING This work was supported by a BHF programme grant RG/20/6/35095 to C.L., E.C, S.N. and C. B. C.B. and C.M.L were also supported by the British Heart Foundation (RE/18/6/34217). DISCLOSURE OF INTEREST Authors have nothing to disclose. DATA AVAILABILITY STATEMENT Data from this study are available upon request to the corresponding author. ACKNOWLEDGMENTS The authors thank Michael Dunne, Margaret Bell, and Catherine Hawksby and the Biological Services staff from the University of Glasgow Cardiovascular Research Unit for their surgical, animal, and technical assistance. We thank Douglas Strathdee, the Transgenic Technologies Lab, BSU and Histology lab at the Beatson Institute. Footnotes ↵ * AAME and AAR, as well as REFERENCES 1. ↵ Desai N , Olewinska E , Famulska A , Remuzat C , Francois C , Folkerts K . Heart failure with mildly reduced and preserved ejection fraction: A review of disease burden and remaining unmet medical needs within a new treatment landscape . Heart Fail Rev . 2024 ; 29 : 631 – 662 . OpenUrl CrossRef PubMed 2. ↵ Tsao CW , Lyass A , Enserro D , Larson MG , Ho JE , Kizer JR , Gottdiener JS , Psaty BM , Vasan RS . Temporal trends in the incidence of and mortality associated with heart failure with preserved and reduced ejection fraction . JACC Heart Fail . 2018 ; 6 : 678 – 685 . 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Share Targeting Runx 1 protects against heart failure with preserved ejection fraction Ali Ali Mohamed Elbassioni , Anmar A Raheem , Jian Song , Alexander S Johnston , Cara Trivett , Hong Lin , Haobo Zhang , Ashley Bradley , Erin Higgins , Leanne Mooney , Yen Chin Koay , Dylan O’Toole , Pawel Herzyk , Colin Nixon , Karen Blyth , John F O’Sullivan , Ninian N Lang , Colin Berry , Thomas Braun , Gabriele G Schiattarella , Mauro Giacca , Martin W McBride , Stuart A Nicklin , Ewan R Cameron , Christopher M Loughrey , Eilidh A MacDonald bioRxiv 2025.01.24.634831; doi: https://doi.org/10.1101/2025.01.24.634831 Share This Article: Copy Citation Tools Targeting Runx 1 protects against heart failure with preserved ejection fraction Ali Ali Mohamed Elbassioni , Anmar A Raheem , Jian Song , Alexander S Johnston , Cara Trivett , Hong Lin , Haobo Zhang , Ashley Bradley , Erin Higgins , Leanne Mooney , Yen Chin Koay , Dylan O’Toole , Pawel Herzyk , Colin Nixon , Karen Blyth , John F O’Sullivan , Ninian N Lang , Colin Berry , Thomas Braun , Gabriele G Schiattarella , Mauro Giacca , Martin W McBride , Stuart A Nicklin , Ewan R Cameron , Christopher M Loughrey , Eilidh A MacDonald bioRxiv 2025.01.24.634831; doi: https://doi.org/10.1101/2025.01.24.634831 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Physiology Subject Areas All Articles Animal Behavior and Cognition (7967) Biochemistry (18614) Bioengineering (14761) Bioinformatics (44129) Biophysics (22450) Cancer Biology (19580) Cell Biology (26733) Clinical Trials (138) Developmental Biology (13898) Ecology (20880) Epidemiology (2067) Evolutionary Biology (25312) Genetics (16096) Genomics (23390) Immunology (18595) Microbiology (42208) Molecular Biology (17938) Neuroscience (92853) Paleontology (693) Pathology (2969) Pharmacology and Toxicology (5063) Physiology (8060) Plant Biology (15899) Scientific Communication and Education (2091) Synthetic Biology (4538) Systems Biology (10182) Zoology (2375) window.__CF$cv$params={r:'a36d6d958f8693cc',t:'MTc4ODY5NzIyOQ==',u:'01a076a9c15178b3b23feab5fa29934b',ut:'XZQ3kuiKdIZY5cdXqVQOSrHAiG2apr7HSnOqm6ahFeE-1788697231-1.2.1.1-janKLFxsoGxTq1AzKO6csQYOXNVB1ucRTIkB6CpGniNpBmh36hpxN4Xe8gsu15z98AoEzPiPGXbmTPJyGzsSMRTses3Y73UYAhQ_cZmUO3Y',i:60};(function(){if(!document.body)return;var s=document.createElement('script');s.src='/cdn-cgi/challenge-platform/scripts/precursor/main.js';document.head.appendChild(s);})();