Effect of dapagliflozin on rat and human cardiac fibroblasts physiology cultured in diabetic media conditions

Effect of dapagliflozin on rat and human cardiac fibroblasts physiology cultured in diabetic media conditions | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effect of dapagliflozin on rat and human cardiac fibroblasts physiology cultured in diabetic media conditions Obaalologhi Wilfred This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5461630/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Heart failure (HF) is a disease caused by defects in myocardial structure and function which results to impaired ventricular filling of the ejection of blood, and mostly due to reduced myocardial function of the left ventricle, impairment of the myocardium, endocardium, great vessels, pericardium, heart valves, either in combination or alone, presenting symptoms which include fatigue, oedema, shortness of breath, abdominal distention and right hypochondrial pain. HF, which is classed into HF with reduced ejection fraction (HFrEF) and HF with preserved ejection fraction (HFpEF) has been linked to elevated glucose levels seen in diabetic patients, which is also linked to increased fibrosis which impacts on myocardial function. Unfortunately, no effective drug is yet developed for the treatment HFpEF. In the current study, I investigated the effect of dapagliflozin (dapa) on cardiac fibroblasts (CFs) physiology in diabetic media conditions using rat cardiac fibroblasts (RcFbs) and human cardiac fibroblasts (HcFbs). Experiments were performed to study the effects of elevated glucose with/without dapa on gene expressions of human collagen-1 (col1a2), calcium/calmodulin-dependent protein kinase II delta (CAMK2D) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using quantitative polymerase chain reaction (qPCR), Ca 2+ release and migration and proliferation assays of CFs were assessed using confocal microscopy and countess counter. Our study demonstrates that dapa has a potential in wound healing, and has been shown to increase Ca 2+ mobilization in diabetic cultured cells. Although dapa impaired col1a2 expression in diabetic condition, it showed no significant change on CAMK2D expression in diabetic conditions. The implication of these findings suggests that dapa could be a potentially therapeutic agent in treating HF as it has been shown to improve wound healing (via the scratch assay), increased proliferation of cells and increased col1a2 expression. Cardiac & Cardiovascular Systems Cardiothoracic Surgery Clinical Pharmacology Heart failure diabetic dapagliflozin cardiac fibroblasts left ventricle Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Heart failure (HF) is a disease caused by defects in myocardial structure and function which results to impaired ventricular filling of the ejection of blood. While HF is mostly due to reduced myocardial function of the left ventricle, impairment of the myocardium, endocardium, great vessels, pericardium and heart valves, either in combination or alone is also linked with HF (Inamdar & Inamdar, 2016), and presents as chamber remodeling, interstitial fibrosis and decreased ventricular compliance (Travers et al., 2016) with symptoms including fatigue, edema, shortness of breath, abdominal distention and right hypochondrial pain (Yancy et al., 2013; Watson et al., 2000). The prevalence of HF, according to Bui et al, (2011) and Ponikowski et al, (2014) is estimated to be over 23 million, and this is expected to rise as global population increases (Conrad et al, 2018). Notwithstanding the advancement in HF treatment over the last decades, mortality and morbidity continue to rise (Lesyuk et al, 2018). Clinically, HF can be classified into: (i) HF with reduced ejection fraction (HFrEF), a form of HF where there is impairment in the systolic force generation of the heart, thereby causing reduction in the volume of expelled blood with each contraction (ejection fraction); and (ii) HF with preserved ejection fraction (HFpEF) where standard parameters of systolic function are mostly maintained, however, there is still impairment of diastolic filling and relaxation (Ponikowski et al, 2016). Although HF can be predominantly left, right or bi-ventricular based on the location of deficit in the heart, depending on the onset, it can further be classed either as acute or chronic (Inamdar & Inamdar, 2016). Although HFpEF is more dominant in females and older adults, in all patients with HFpEF, the EF is normally over 50%, with normal left-ventricular (LV) chamber volume accompanied by thick and stiff LV wall, thereby increasing the LV mass:end-diastolic volume ratio (Ohtani et al., 2012). Whereas, in patients with HFrEF, the LV is usually dilated and LV mass/end-diastolic volume ratio can be reduced or normal, however, the volume of myofibrils and cardiomyocyte diameter are elevated in HFpEF compared to HFrEF (Dassanayaka and Jones, 2015). While HFrEF patients respond favourably to treatment regimen with better prognosis, this is not the case for HFpEF patients as they do not respond to standard pharmacological interventions (except for nitrates), thus prognosis is poor during the decompensated phase of HF (Othani et al., 2012; Zamani et al., 2015; Glean et al., 2015). Recent studies have implicated cardiac fibrosis in the aetiology of almost both types of HF, especially in the pathophysiology of HFpEF (Moreo et al, 2009; Gonza´lez et al, 2018) where cardiac fibroblasts (CFs) deposits excess extracellular matrix (ECM) proteins which leads to myocardial remodelling and eventually resulting to decrease in tissue compliance and acceleration of HF progression (Travers et al., 2016). This event consequently leads to systolic and diastolic dysfunction observed in many pathophysiological dysfunction (Kong et al., 2013) which is observed in diabetic and ischemic heart disease patients, and accompanied with morbidity and mortality (Tian et al., 2017). CFs are important cell type, mainly of embryonic epicardial and endothelial roots (Moore-Morris et al., 2014; Ali et al., 2014), and in healthy hearts, CFs produce and secrete fibrillar collagens (Zhang et al., 2022). Collagen I and III fibers, for instance, functions as scaffold for the myocardium. Hence imbalances in the production and degradation of collagen in the ECM leading to collagens expansion causes myocardial fibrosis. Imbalances such as this occur as a result of cardiomyocytes death, or stimuli that induces increase in the synthesis of collagen (Rathod et al., 2016). Hence, under physiological conditions, CFs maintain homeostasis of ECM – distributing mechanical forces through the cardiac tissue, therefore giving the structural scaffold for cardiomyocytes and initiating electric conduction in the heart (Souders et al., 2009; Camelliti et al., 2005; Porter et al., 2009). An indicator for HFpEF is hypertrophic growth of cardiac myocytes, and ECM accumulation caused mainly by fibrillar collagen (Zile et al., 2004). In a previous study (Zile et al., 2015), the increase in left ventricular cavity and myocardial diastolic stiffness observed in HFpEF patients is due to abnormality in myocyte structural proteins and elevated myocardial collagen synthesis. Again, resident fibroblasts also appear to be the primary cells involved in fibrillar collagens production in response to left ventricular pressure-overload (LVPO) in murine models of clinically relevant heart disease (Moore-Morris et al., 2014). Thus, different mechanisms of myocardial fibrosis exist, however, high accumulation of ECM which impedes cardiac function is the main cause of fibrotic heart disease (Tian et al., 2017). Although CFs appear to be flat, spindle shaped with multiple processes, the cardiac milieu varies greatly with respect to the species when examined between healthy and injured myocardium (Banerjee et al., 2007). Whereas previous studies have shown that CFs accounts for the vast majority cells found in the human and adult rodent myocardium (Banerjee et al., 2007; Camelliti et al., 2005; Zak, 1974), in adult murine hearts, CFs make up of less than 20% of the cell population, significantly lower than previously reported (Ali et al., 2014; Pinto et al., 2016). Although, Gittenberger et al., (1998) and Muñoz-Chápuli et al., (2001) have previously reported that the bulk of resident fibroblasts come from the embryonic epicardium, there is still much to learn about how different resident and invading cells contribute to the active cardiac myofibroblasts, including the development of more precise molecular markers for CFs. Also, the regenerative ability of the heart is limited, with the process of repairing involving removing necrotic cardiomyocytes and then the replacement of fibrotic scar tissue to preserve the structure and function of myocardial integrity. In connective tissues, CFs help in performing these functions by converting to myofibroblasts, their active form by secreting high levels of ECM proteins, thereby encouraging a profibrotic condition. Thus, cardiac fibrosis initiates pathological events that promotes cardiomyocyte hypertrophy and apoptosis, chamber dilatation which eventually leads to the development of congestive heart failure (CHF) (Baudino et al., 2006; Cohn et al., 2000). In spite of the role of fibrosis in cardiovascular diseases (CVD), cardiac fibrosis remains poorly understood; with current clinical interventions effectively targeting CFs and its pathological contributions to disease progression limited (Travers et al., 2016). Elevated glucose levels observed in diabetes have been linked to increased ECM deposition or fibrosis, and has been reported to have an impact on myocardial function (Weber et al. 1987, 1988). Studies have revealed the profibrotic effects of increased glucose levels in isolated fibroblasts and models of diabetic disease (Tang et al. 2007; Asbun et al. 2005; Zaman et al. 2004). For instance, diabetes, a metabolic disease, is mainly characterized by high levels of blood glucose. Impairment of glucose metabolism, proteins and lipids generates alterations both micro and macrovascular circulation, paving way for the risk of numerous complications in diabetic patients especially, cardiovascular complications (Rask-Madsen and King, 2013) and delayed wound healing (Brem and Tomic-Canic, 2007) amongst others. A key abnormality in diabetes, hyperglycaemia, has been shown to play a role in the development of inflammation in diabetic complications, as an earlier study shows that increase in blood sugar levels enhances inflammation and impedes wound healing by altering angiogenesis (Braiman-Wiksman et al., 2007). In vitro studies reveal that hyperglycaemia reduces migration (Lamers et al., 2011 and Kido et al., 2017), proliferation (Stolzing et al., 2006) and collagen synthesis (Willershausen-Zonnchen et al., 1991) as well as increase apoptosis (Baumgartner-Parzer et al., 1995; Deveci et al., 2005) in cells of various types. During wound healing, fibroblasts, keratinocytes, macrophages, endothelial cells and platelets coordinate in a complex manner, with successful healing process involving cell proliferation and migration, deposition and remodeling of collagen, and angiogenesis and wound contraction. Fibroblasts, being the most abundant cell types found in connective tissues are primarily involved in the production and remodelling of the ECM (Willershausen-Zonnchen et al., 1991). Currently, therapies such as sodium-glucose co-transporter 2 (SGLT2) inhibitors and beta-blockers which targets the renin–angiotensin–aldosterone system (RAAS) exists for the treatment of HFrEF, however, no effective drug is yet to be developed for the treatment HFpEF even though multiple randomized clinical trials have been conducted (Pfeffer et al, 2019; Seferovic et al, 2019; Ponikowski et al, 2016), posing a serious concern as HFpEF is now common and responsible for over 50% of all HF cases globally (Vasan et al, 2018), which could be linked to its association with comorbidities including diabetes, hypertension and ageing (Streng et al, 2018; Dunlay et al, 2017; Bekfani et al, 2016). Although, biochemical mediators such as angiotensin II (AngII) have been shown to regulate the fibroblasts – ECM interactions (Watson et al. 1998; Burgess et al. 1994), the effects of dapagliflozin (dapa) in response to elevated glucose in CFs requires further investigation. In the current study, I investigated the effect of dapagliflozin on rat and human cardiac fibroblasts physiology cultured in diabetic media conditions. Experiments were performed to study the effects of glucose in the presence of dapa on gene expressions of human collagen-1 (col1a2), human calcium/calmodulin-dependent protein kinase II delta (CAMK2D) and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using quantitative polymerase chain reaction (qPCR). I have also assessed the effects of dapa on Ca 2+ release in these cells following stimulation with AngII using confocal imaging technique. Additional experiments were then carried out to assay the migration and proliferation abilities and phenotypes of CFs in response to drug in glucose media conditions. MATERIALS AND METHODS Materials Human cardiac fibroblasts (HcFbs), Fibroblast Growth Medium 3 (Fb medium), Dulbecco’s Modified Eagle’s Medium (DMEM), Basic fibroblast growth factor (recombinant human), insulin (recombinant human), fetal bovine serum (FBS), fetal calf serum (FCS) and penicillin/streptomycin were all purchased from Promo Cell GmbH (Heidelberg, Germany). Rat cardiac fibroblasts (RcFbs) was a gift from Professor Suzan Currie from the University of Strathclyde. D-Glucose anhydrous, Palmitate, ReddyMix PCR Master Mix were bought from Thermo Fisher Scientific (Loughborough, UK). Angiotensin II (AngII) and dapagliflozin were supplied by Sigma-Aldrich (St. Louis, MO), Cal-520® was purchased from AAT Bioquest (California, USA). Integrated DNA Technologies (Leuven, Belgium) supplied both the forward and reverse primers for col1a2, CAMK2D and GAPDH, and EvaGreen® qPCR Supermix, 5x was purchased from Solis BioDyne (Tartu, Estonia). Ethical consideration The was approved by the ethical committee, and the study conforms to the United Kingdom Animal procedures act 1986, and with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication, 8th Edition, 2011). Cell culture HcFbs (1 mL vial stored in liquid nitrogen storage) and RcFbs (seeded in 2x 96-well plates) were maintained according to manufacturer’s instructions (Gao et al., 2020; Tao et al., 2021). All cell culture experiments were carried out in the cell culture room aseptically, in the Kendro KS12 safety hood (Kendro Ltd., Germany). Briefly, prior to thawing the HcFbs, hFibroblast culture media containing 89% Fb media, 1% penicillin/streptomycin and 10% FBS was made, and cells were removed from the liquid nitrogen storage and quickly thawed by placing vial in a 37 o C water bath for ~ 1 min. Cells were monitored during thawing and were removed from the bath just prior to complete thawing, and were quickly transferred into a 15 mL conical tube with 9 mL room temperature hFibroblast media (previously prepared) and centrifuged at 1,200 rpm for 3 mins and supernatant was aspirated, leaving the cell pellet undisturbed. To achieve a single cell suspension, cell pellet was re-suspended in 5 mL of hFibroblast media and mixed properly by pipetting up and down. The cell suspension was transferred to a T25 cell culture flask and incubated in humidified temperature-controlled Galaxy 170 S incubator (Eppendorf/New Brunswick Scientific, Stevenage, UK) (5% O 2 , 5% CO 2 and balance N 2 ) overnight, just as hFibroblast media was replaced every 2 days. At a confluency of about 80–90%, CFs created a swirling pattern, and confluent cells were split from the T25 cell culture flask into a T75 cell culture. This was done by retrieving the T25 flask from the incubator and aspirating hFibroblast media, and washing the cells twice with 5 mL Phosphate-buffered saline (PBS). Then, 1 mL of 0.25% trypsin was added to the cells and incubated at 37 o C for 5 mins to ensure the cells dissociation from the culture flask. The cells were then checked under the Olympus Optical microscope CK2-TR (Olympus Optical Co., Ltd., Japan) to confirm cells have completely detached from the culture flask (flask was tapped on the bench to induce mechanistic force to dissociate cells where necessary). The cells were further re-suspended in 5 mL of trypsin inhibitor and pipetted up and down, and used to washed down the flask again as cells were collected as a single cell suspension, transferred into a 15 mL conical tube and centrifuged at 1,200 rpm for 3 mins. Supernatant was aspirated, leaving the cell pellet undisturbed and was then re-suspended in 7 mL of fresh hFibroblast media. The cell suspension was pipetted up and down 2–3 times and a single cell suspension was achieved and transferred to a T75 cell culture flask and incubated at 37 o C and 5% CO 2 overnight. hFibroblast media was replaced every 2–3 days as required. The cells in the T75 cell culture flask were confirmed to have attained 80–90% confluency using the microscope, and was used to produce enough cells to freeze 3 x 1 mL aliquots. Briefly, 1.5 mL of freezing media (90% FBS + 10% DMSO) and 1.5 mL of hFibroblast (99% FCS + 1% penicillin/streptomycin) were prepared and set aside. The hFibroblast media was aspirated from the T75 culture flask, cells were washed twice with 5 mL PBS and aspirated, and then 4 mL of 0.25% trypsin was added to the cells and then incubated at 37 o C for 5 mins cells to dissociate completely from the culture flask. The flask was retrieved and observed under the microscope to confirm cells detachment, and where necessary, the bottom of the flask was gently tapped on the bench to dislodge cells. The cells were then re-suspended in 6 mL of trypsin inhibitor and pipetted up to wash the flask again and a single cell suspension collected. The cell suspension was then transferred into a 50 mL conical tube and centrifuged at 1200 rpm for 2 mins. Thereafter, the supernatant was carefully removed leaving the cell pellet intact. The cell pellet was then re-suspended in 1.5 mL of hFibroblast media, and then, 1.5 mL of freezing media was added to cell suspension, bringing the total volume to 3 mL which was aliquoted into 3x 1 mL aliquots across three cryovials, wrapped in 5 layers of tissue paper and immediately placed cells in -80 o C freezer for 72 h, before finally transferring cells to -150 o C freezer. Whereas, Fb media was used as media for the HcFbs, I have used DMEM for the RcFbs. Cells were passaged at about 80–90% confluency, and cells in the passages 9 (HcFbs) and 1 (RcFbs) respectively were used for experiments in the current study. Drug Treatment RcFbs and HcFbs were grown to about 70–80% confluence in their respective growth medium (as mentioned above) in different media conditions. For experiments on RcFbs, media conditions were low glucose (LG; 17.7 mM), high glucose (HG; 30 mM), palmitate (P; 2.5 mM) and HG + P (30 mM + 2.5 mM). For HcFbs experiments, the conditions where were LG (7.7 mM), HG (30 mM) and HG + P (30 mM + 2.5 mM). Media conditions were set up in triplicates, and were treated with and without dapa (1µM) for 24 h in their respective growth medium containing 1% and 10% serum respectively after serum starving for 24 h. For RcFbs, stock concentrations of drugs were diluted in DMEM with supplements (1% penicillin/streptomycin and 1% L-glutamine), and in HcFbs experiments, stock drug concentration was diluted in hFibroblast media (1% penicillin/streptomycin) before adding to the cells. Drug concentrations were based on relevant concentrations in previous studies (Tian et al., 2021; Arow et al., 2020; McMullen et al., 2020). Cell imaging Cell growth and phenotype were monitored with the aid of Olympus Optical CK2-TR inverted microscope (Olympus Optical Co., Ltd., Japan) and a Digital Bio JULI™ Smart fluorescent cell analyser (Nanoentek Inc., Seoul, Korea) at 10× magnification (Fig. 1 ). Cell proliferation assays Proliferation assay was performed on RcFbs upon reaching a confluency of 60–80%. Briefly, from each well, DMEM media was aspirated and the cells were serum-starved for 24 h and consequently treated with dapa in the different media conditions as previously mentioned. After 24 h, plate was retrieved from the incubator, media was aspirated and 200µl PBS used to wash the cells, and replaced with 100µl trypsin and incubated for 3 mins. Then, 200µl of FBS-supplemented DMEM was added to each well, and mixed properly by pipetting up and down, and then transferred into Eppendorf tubes which were centrifuged for 10 mins at 2600 rpm. Cell pellet was extracted for analysis as supernatant was discarded. Then, 10µl of serum free media (SFM) and 10µl of Trypan blue were then added to the tubes, from which 10µl of the mixture was loaded to the cell counter chamber slide, and inserted into the Countess II Automated Cell Counter (Invitrogen, Paisley, Renfrewshire, UK) to determine cell concentration per ml (Fig. 2 ). Intracellular Calcium Measurement 200 µL cell suspension of RcFbs were seeded into clear bottomed, dark sided 96-well plate (which was later confirmed to be at a density of 3.0 × 10 4 cells/well), and incubated for 24 h before treatment with dapa in different media conditions. The culture medium was aspirated. 2 mL of 1x Fibroblasts Krebs/Tyrode solution (136.9mM NaCl, 5.4mM KCl, 1.3mM CaCl 2 , 0.4mM MgSO 4 ·7H 2 O, 0.5mM MgCl 2 .6H 2 O, 0.3mM Na 2 HPO 4 .2H 2 O, 0.4mM KH 2 PO 4 , 5.6mM glucose, 4.2mM NaHCO 3 ; pH = 7.4) was added to Cal-520 AM calcium dye stock (1 µM), and 50 µl from the solution was transferred into each well and allowed to incubate on the bench at room temperature for 20 mins. The solution was then aspirated and then washed with 50 µl 1x Fibroblasts Krebs/Tyrode solution once. The 96-well plate was carefully positioned, and cells were imaged to record baseline fluorescence for 1 min. Then 0.3 µM AngII (1.5 mL of 1x Fibroblasts Krebs/Tyrode solution + 4.5 µl of AngII) was added to initiate Ca 2+ release from the endoplasmic reticulum (ER). Fluorescence measurements were taken using a spinning disk confocal microscopy system using a 488 nM laser with emission collected using an EM-CCD camera (Prime, Photometrics, Tucson USA). Analysis of data was then performed with ImageJ version IJ 1.46r. Background measurement from each well was taken with no cells, and using the formula F = F raw -F background , background was subtracted from all measurements. From the F image, a ‘z-project’ was created and time the last 20 images were created when the AngII addition was added was determined, and random cells were circled out and input on the ROI manager, representing the F 0 values. Then, the formula, F normalized =F/F 0 was used to determine Ca 2+ transient amplitude as the area under the curve on the ImageJ software. Ca 2+ excitation was then obtained by subtracting 1 from the acquired peak values (Fig. 3 ). Cell migration assays Cell migration assay was then performed on HcFbs seeded into 6-wells plate previously assigned reference scratches on the bottom. The cells were left to grow to grow in hFibroblasts media until 100% confluent. At confluency, media was aspirated from each well, and cells serum-starved for 24 h. Following this, the plate was retrieved and an initial scratch was made inside each well with a 200 uL pipette tip, as much as close to the reference line. SFM was aspirated to wash of the wounds, and 3 mL fresh SFM added to each well before the plate was photographed using Digital Bio JULI™ Smart fluorescent cell analyser (Nanoentek Inc., Seoul, Korea) at 10× magnification. The cells in the presence of 10% FCS-supplemented media were then treated with dapa accordingly, and incubated for 24 h. The plate was thereafter retrieved and the cells photographed again for comparison. Assay was analysed by measuring the difference in scratch width and converting this into a percentage migration (Fig. 4 ). Gene expression analysis Sample preparation 2 mL HcFbs cell suspension was seeded into 3x 6-wells plate and left to grow until 100% confluent. At confluency, media was aspirated from each well, and cells serum-starved for 24 h. Then, the cells in the presence of 10% FCS-supplemented media were treated with dapa in different media conditions, and incubated for 24 h. After this, the media was aspirated, and the cells lysed by adding 175 µL Lysis Buffer RA1 and 1.75 µL β-mercaptoethanol (β-ME) in each well. Scrappers where then used to scrub the bottom of each well to collect cells, and were transferred into sterile 1.5 mL Eppendorf tubes and vortexed vigorously. Total RNA purification from the cultured cells was then carried out using NucleoSpin® RNA Mini Kit for RNA purification (Macherey-Nagel™ GmbH & Co. KG, Duren, Germany) according to manufacturer’s instructions. Total RNA was then quantified using the Epoch BioTek® (Fisher Scientific, Loughborough, UK) before being stored in the − 80 o C freezer till the next day. Reverse – Transcription : briefly, the total RNA was subjected to reverse transcription using the commercially available High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Lithuania) according to manufacturer’s instructions. First, 10 µL of a master mix containing 2.0µL of 10 x RT buffer, 0.8 µL of 25 x dNTP, 0.8 µL of 10 x random primers, 1.0 µL of Multiscribe and 4.2 µL of Nuclease-free H 2 O was added into PCR tubes with 10 µL RNA, and then placed in the Applied biosystems 2720 Thermal Cycler® (Life Technologies Holdings Pte Ltd., Singapore) with the following conditions: 25 o C for 10 mins to maximise primer RNA template binding, 37 o C for 120 mins for reverse transcription, and 85 o C for 5 mins to deactivate reverse transcription. cDNA was then stored in the − 20 o C fridge for later use. Quantitative real-time PCR (qPCR) : qPCR was performed to measure the expression level of the mRNA (Fig. 5 ) using EvaGreen® qPCR Supermix 5x master mix and fluorescently tagged primers. The primers for the genes of interest used are shown on Table 1 . qPCR reactions to determine the expression of mRNA of the genes of interest were set up in a 96-well plates in triplicate, and the results were normalized against those of GAPDH, and run using the SYBR method in the ViiA7TM real-time PCR System (Life Technologies, Paisley, UK) in the following conditions: 50 o C for 2 min, 95 o C for 10 min and 40 cycles of 95 o C for 15 secs, 60 o C for 1 min. The data (Table 2 ) were analysed using the 2 ΔΔ CT method. Statistical Analysis Data were analysed using GraphPad Prism 9 (GraphPad Software, San Diego, CA), and presented as mean values ± S.E.M of n observations (n, representing the number of samples). Comparisons were made by one – or two-way analysis of variance (ANOVA) followed by either Tukey’s or Benferroni’s multiple comparison tests as appropriate. In this study, a value of p < 0.05 indicated statistical significance. RESULTS Effect of dapagliflozin on cardiac fibroblast phenotype Healthy CFs were only established using bright field imaging. In assessing the CFs phenotype in the absence and presence of dapa on different media conditions the cells were subjected to, cells were treated with either the same concentration of drug (1 µM) or without drug for 24 h and then imaged (Fig. 1 ; LG = low glucose; HG = high glucose; P = palmitate; HG + P = high glucose with palmitate). In RcFbs (Fig. 1 A), there was no observed changes in cell phenotype in LG and HG media conditions, and no obvious cell loss. There was no visible shape in cells in P media conditions, but associated cell loss with the formation of vacuole-like structures. While there seem not to be any phenotypic change, there was an obvious cell loss in HG + P media conditions. However, in media conditions treated with dapa, the shape of cells remained intact with no changes, and there was insignificant cell loss in LG and HG media conditions. While P media conditions exhibited obvious cell death with the formation of vacuole-like structures, HG + P media conditions showed decreased cell death and less vacuole formed. In HcFbs (Fig. 1 B), there was no significant alterations in cell phenotype across all media conditions, with or without dapa treatment, CFs appeared flat, spindle shaped with multiple processes. Effect of dapagliflozin on RcFbs proliferation Next, proliferation of RcFbs in different media conditions, in the absence (Fig. 2 A) and presence of 1µM dapa (Fig. 2 B) was investigated. Proliferation assay revealed HG and HG + P in the absence of dapa significantly influenced cell proliferation, with LG showing lowest proliferation of cells. Thus, there was a significant increase in cell proliferation in HG and HG + P media conditions when compared to LG media condition (Fig. 2 A; p*= 0.03, p*** = 0.0001). Dapa however showed no influence on cell proliferation across all media conditions investigated. But, when compared using 2-way ANOVA, there was a significant drop in cell proliferation of cells in HG media condition treated with dapa compared to those without dapa treatment (Fig. 2 C; p* = 0.01). There also seem to be a trend of increasing proliferation in LG without dapa compared to dapa treated LG condition, but this was not significant (Fig. 2 C). Effect of dapagliflozin on AngII-induced Ca 2+ mobilization in RcFbs To elucidate the effect of dapa on cellular calcium, the influence of dapa on Ca 2+ mobilization in RcFbs in the different media conditions were examined. AngII (0.3µM) was used to induce intracellular Ca 2+ release. Figure 3 shows Ca 2+ mobilization from random cells (n = 6) from across different media conditions. In the group not receiving dapa treatment, there was an increase in Ca 2+ release in LG cultured cells compared to those cultured in other media conditions (Fig. 3 A; p***= 0.0007, p**** ˂0.0001). In media conditions treated with dapa (1µM), a significant increase in Ca 2+ mobilization was also observed in HG media compared to all other media conditions (Fig. 3 B; p****˂0.0001). When a group comparison test was performed, result indicated that there was a significant increase in Ca 2+ mobilization observed only in cells cultured in HG media condition subjected to dapa compared to their untreated counterparts (Fig. 3 C; p**= 0.004). Effect of dapagliflozin on HcFbs migration The effect of dapa on HcFbs migration cultured on different media conditions was examined using a wound healing assay after 24 h. In media conditions without dapa treatment, LG showed significant migration compared to other groups (figure 4A, 4C; p** = 0.0068, p*** = 0.0009). Though there seem to be an increase in migration in HG media cultured cells, this was not statistically significant when compared with other media conditions treated with dapa (figure 4B, 4D). The result of the multiple comparison test shows that cells cultured in HG media treated with dapa displayed significant migration rate, compared to their untreated counterpart (figure 4E; p** = 0.0106) after 24 h. Effect of dapagliflozin on gene expressions of human collagen-1 (col1a2), human calcium/calmodulin-dependent protein kinase II delta (CAMK2D) in HcFbs. Table 1 qPCR primers design of primers Genes Sequences Melting temperature T m / o C Molecular weight /MW Col1a2 F: 5’-GAG CGG TAA CAA GGG TGA GC -3’ R: 5’-CTT CCC TAG GGC CTC TC -3’ 58.3 57.1 6,256.1 5,979.9 CAMK2D F: 5’- AGT CAG AAG AGA CTC GTG TGT − 3’ R: 5’- TGA TGG GTA CTG TTG GTG ACC − 3’ 55.2 56.8 6,510.3 6,508.3 GAPDH F: 5’- ACA ACT TTG GTA TCG TGG AAG G -3’ R: 5’- GCC ATC ACG CCA CAG TTT C -3’ 55.1 57.1 6,814.5 5,708.8 The effect of dapa on gene expressions of Col1a2 and CAMK2D in HcFbs in the different media conditions were studied. For Col1a2 analysis, when compared with the control group in media conditions not receiving dapa treatment, Col1a2 expression was significantly reduced in HG, but increased in HG + P (Fig. 5 A; p*** = 0.006, p**** <0.0001) respectively. However, there was a significant decrease in its expression in HG + P and LG media treated with dapa when compared to controls (Fig. 5 B; p* = 0.03, p** = 0.0084). When a 2-way ANOVA was performed, cells cultured in HG media which received dapa exhibited a significant increase in Col1a2 gene expression compared to their untreated counterpart (Fig. 5 C; p** = 0.006). Similarly, in dapa-free media conditions, CAMK2D showed a significant reduction in HG, but increased expression in HG + P cultured cells (Fig. 5 D; p** = 0.0083, p***<0.0001) respectively. Dapa however showed no significant effect on CAMK2D expression across all media conditions. Table 2 CT values of genes of interest obtained from ViiA7TM real-time PCR System. Groups Col 1a2 Col 1a2 Col 1a2 GAPDH GAPDH GAPDH CAMK 2D CAMK 2D CAMK 2D GAPDH GAPDH GAPDH LG +dapa 33.868 33.184 33.837 29.572 31.161 30.162 30.698 33.107 31.066 34.574 33.132 36.835 LG (C) -dapa 31.746 31.920 31.528 29.523 31.312 31.289 28.733 30.399 28.743 31.069 30.418 31.287 HG + dapa 32.502 34.845 34.330 33.121 Not determined 33.145 37.137 32.830 35.266 Not determined 32.349 31.920 HG -dapa 33.763 33.532 32.830 31.115 30.414 30.986 30.404 30.638 29.650 30.390 30.724 30.073 HG + P + dapa 33.268 33.884 32.962 30.566 31.182 31.285 29.990 30.187 29.658 31.704 29.955 29.972 HG + P -dapa 30.851 30.851 30.851 31.659 31.659 31.659 30.478 30.478 30.478 31.659 31.659 31.659 DISCUSSION Cardiac fibrosis is the hallmark of diabetic cardiomyopathy which may be evident even in the absence of any obvious cardiomyocyte damage, indicating a role for cardiac fibroblast in the pathological process, although, mechanism underlying this is unknown (Ling-Yu Zhang et al., 2021). In the present study, the effects of diabetic media conditions in the presence of dapagliflozin on rat and human CFs have been evaluated to aid a better understanding of the profiles of glucose and dapa in CFs physiology and function. Here, the effects of glucose and dapa on CFs cell phenotype, proliferation, wound healing, Ca 2+ mobilization, and gene expressions of col1a2 and CAMK2D were assessed and documented. Palmitate has been used as part of the media conditions in the present study owing to its ability to inhibit and/or retard oxidation reactions by preventing damaging effects of oxidation in animal cells and tissues (NCBI, 2022). The current study demonstrates that the relatively low concentrations of dapa (1µM) and glucose used in the treatment of CFs in the presence of serum was sufficient in evaluating their potential to affect cell phenotype and function as other studies have used similar concentrations in achieving promising results (Tian et al., 2021; Arow et al., 2020; McMullen et al., 2020). Although, a more realistic result would have been obtained if a concentration-response curve of the drug was produced to have a broader idea of the activity of the drug on these cells. Dapa is an SGLT2 inhibitor indicated for the management of type 2 diabetes mellitus (Obermeier et al., 2010) and functions by inhibiting the SGLT2 receptor, thereby preventing the reabsorption of glucose in the kidney, thus reducing circulating blood glucose, and increasing excreted glucose (Albarrán and Ampudia-Blasco, 2013). In the group not receiving treated with dapa, RcFbs did not display any alterations in cell phenotype across the media conditions investigated, however, formation of vacuole-like structures and cell viability were evident in the palmitate media conditions, with obvious cell loss in HG + P media condition. Following treatment of cells with dapa, cell phenotype remained unchanged, even though P media conditions exhibited formation of vacuole-like structures. In HcFbs, there were no obvious cell death and no alterations in phenotype as CFs maintained flat, spindle shaped with multiple processes. This therefore suggest that neither dapa nor glucose, nor the combination of both at their respective concentrations induced any phenotypic alterations on these cells. Even though CFs form the bulk of matrix producing cells in the heart and are responsible for cardiac remodeling and fibrosis (Porter and Turner, 2009), previous studies have concentrated on the effect of hyperglycemia on cardiac myocytes, with few studying CFs. Diabetes, being an independent risk factor for CVD, with diabetic patients predisposed to CV complications including fibrosis, it is pertinent to evaluate the influence of high glucose on fibroblast activation to enhance identification of specific targets that could be beneficial in delaying disease progression. I therefore sought to establish the role of high glucose on proliferation of CFs. Here it was established that in the absence of dapa, HG significantly increased CF proliferation. This observation is in line with other studies where isolated CFs from diabetic myocardium showed more proliferation than those isolated from healthy control myocardium (Shamhart et al., 2009), with high glucose found to be stimulating CFs proliferation in vitro (Asbun and Villarreal, 2006; Neumann et al., 2002). Although, Zhang et al., (2007) had previously reported that CFs proliferation was decreased in high glucose containing medium relative to low glucose medium, this may be attributed to the fact the cells studied were cultured in cover slip coated with collagen. However, in the presence of dapa, HG-induced CFs proliferation was significantly reduced. This suggests that dapa has a role in decreasing CFs proliferation. The effects of glucose on HcFbs migration was also investigated with/without dapa, and it was found that CFs cultured in dapa-free HG media migrated slowly compared to those cultured in LG conditions, which is in agreement with an earlier study which established impediment of wound healing in condition of increased blood sugar levels in diabetic patients (Kido et al., 2017; Lamers et al., 2011; Braiman-Wiksman et al., 2007) as well as a similar observation found in human gingivial fibroblast (Buranasin et al., 2018). However, this observation contradicts Shamhart et al., (2014) which reported that adult CFs stimulated with high glucose migrated faster than fibroblasts in low glucose. However, in the presence of dapa, CFs migration was increased in HG condition, implying that dapa enhances CF wound healing. Diabetes and hyperglycaemia induce the mobilization of intracellular Na + and Ca 2+ (Baartscheer et al., 2017). Thus, rise in Na + and Ca 2+ promotes events leading to dysregulation of mitochondrial homeostasis, such as increased ROS production, cardiac hypertrophy, and remodelling (Uthman et al., 2018) and eventual cell death (Odagiri et al., 2009). Ang II plays a significant role in maintaining cardiac performance via the RAAS II system (Urata et al., 1990), and directly binds to G-protein-coupled AngII receptor type 1 (AT 1 ), therefore stimulating Ca 2+ /calmodulin-dependent phosphorylation of L-type calcium channels, resulting in an increase in influx of Ca 2+ (Correa et al., 2015; Goette and Lendeckel, 2008). In this current study, I have sought to evaluate Ca 2+ mobilization in the ER of RcFbs with/without dapa in diabetic media conditions. Interestingly, at 1µM concentration, dapa sufficiently enhanced AngII-mediated intracellular Ca 2+ mobilization in HG cultured cells compared to other groups, especially exerting a decreasing effect in intracellular Ca 2+ mobilization in LG cultured cells. To the best of our knowledge, no study has looked into this using CFs, hence, the data suggests that dapa promotes Ca 2+ mobilization in diabetic CFs. However, in a recent study, cardiomyocytes displayed a reduction in amplitude of Ca 2+ transients (Arow et al., 2020). Since no higher concentrations of the drug were used, I are unsure of any exacerbating effect it may have on Ca 2+ -induced CF responses. Although relevant studies have documented the effect of excess Ca 2+ release on CFs apoptosis and necrosis (Li et al., 2010; Pinton et al., 2008). Considering the role CFs play in the disposition of ECM and cardiomyocyte function, effects of high glucose on CFs could cause a direct impact on cardiac contraction which could result to impaired diastolic function. Since cardiac fibrosis is a serious occurrence in diabetic cardiomyopathy and pathological remodelling of the heart, and as collagen and CAMKII are well established important molecules in cardiac physiology (Villarreal et al., 2009; Bers, 2008; Beckendorf et al., 2018), and implicated in CF dysfunction (Horn and Trafford 2016; Martin et al., 2014), the effect of dapa on gene expressions of col1a2 and CAMK2D were measured in HcFbs. It was found that HG decreased col1a2 expression but influenced col1a2 expression in HG + P condition not treated with dapa, which is similar to a previous study that saw an increase in col1 expression in hyperglycemic conditions (Shamhart et al., 2014; Asbun et al., 2005; Tang et al., 2007). However, in the presence of dapa, there was decreased expression of col1a2 in HG + P. Interestingly, dapa influenced the expression col1a2 in HG, suggesting that dapa plays a role in CFs collagen expression, especially in diabetic states. The discrepancy in expression between HG and HGP could be due to the presence of palmitate in the HG + P media. Furthermore, in the absence of dapa, there was a significant increase in CAMK2D expression in HG + P (but decrease in HG) conditions, however, no significant improvement was observed when treated with dapa, suggesting that dapa did not influence the CAMK2D expression. To the best of our knowledge, this is the first study that would explore the influence of dapa on CAMK2D gene expression in CFs in diabetic media conditions. STUDY LIMITATIONS In performing the experiments, some limitations were encountered. First, contamination of the media (which resulted in the death of most cells) and repression of cell growth (for the ones that eventually survived when media was supplemented with growth factor and insulin) was an issue encountered. For this reason, there were not sufficient cells with which to perform all the experiments, especially with the same cell line. Thus, by filter-sterilizing the media using a syringe and 0.2um filter and optimizing the growth factor and insulin supplemented in the media, this could be overcome. Again, CFs from both rat humans were subjected to different experimental conditions, and this hampered on statistical inferences. Secondly, there was no enough time, and samples to check the efficiency of the primers. Even though qPCR worked, it is necessary to first check the efficiency of primers and optimize where necessary, since it’s the first time these primers would be used in the lab. Contamination and degradation of RNA may have impacted the results of the gene analysis, as during the extraction process, samples were not kept on ice. Lastly, as HcFbs were used at high passage number (P9), this could have affected the outcome of the result as high passages affect cell properties. CONCLUSSION Despite the limitations encountered, our study has demonstrated that the concentration of glucose and dapa used in the in vitro experiment was able to potentially affect CFs function, but not phenotype as neither glucose nor the drug brought about any phenotypic alterations of the CFs studied. Without dapa treatment, CFs proliferated more, but migrated slowly in diabetic media conditions, and these effects were reversed following treatment with dapa. Thus, dapa has a potential in wound healing. Also, dapa has been shown to sufficiently enhance Ca 2+ mobilization in the ER of HG cultured cells, as lower mobilization could be detrimental to the cells, however, its role in CFs is not fully understood. While diabetic media condition increased col1a2 gene expression, this expression was impaired in the presence of dapa. The study further concludes that dapa showed no effect on CAMK2D expression in high glucose media. The implication of these findings suggests that dapa could be a potentially therapeutic agent in treating HF as it has shown to improve wound healing (via the scratch assay) and proliferation of cells. FUTURE EXPERIMENT Since CFs were not verified in vivo, further investigation into the effect of diabetic media conditions on CFs should be performed using in vivo study, as this will establish strong evidence of the impact of dapa on diabetic CFs. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5461630","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":378661025,"identity":"d3bf7294-e412-438d-be20-d1df5def9fb8","order_by":0,"name":"Obaalologhi Wilfred","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIiWNgGAWjYFACxsYDFUCyH0nIgJCWhgNngOTMBoRqQloYGMBaNhwgVgu/RHLDgYN7bGQ3Hz9j/Jrnzx95BvbmbRL4tEjOSGw4cOBZmvG2Mzlm1rxtBoYNPMfK8GoxuJ3YcPjDgcOJ2w7kmBnzNhgwNkjkmBHUcuDAgf+Jm/vfmBnz/DGwb5B/Q5SWA4kbJHKMH/OwGSQ2SPDg1yI5/yFIS7LxjBvPyhjnthknt/GkFVvg08LPc/zhgwMH7GT7+5M3f3jzR862n/3wxhv4tCADNrB72IhVDgLMH0hRPQpGwSgYBSMHAAAPolUVhnPrKQAAAABJRU5ErkJggg==","orcid":"","institution":"Augusta University","correspondingAuthor":true,"prefix":"","firstName":"Obaalologhi","middleName":"","lastName":"Wilfred","suffix":""}],"badges":[],"createdAt":"2024-11-15 15:41:55","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-5461630/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5461630/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":69251437,"identity":"312bbf6e-84f1-40be-9cde-ef9f6fc71020","added_by":"auto","created_at":"2024-11-18 11:47:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1529415,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of dapa on CFs phenotype after 24 h in different media conditions. \u003cstrong\u003e(A)\u003c/strong\u003e RcFbs in the absence and presence of dapa(1µM) treatment in 1% serum. \u003cstrong\u003e(B)\u003c/strong\u003e HcFbs in the absence and presence of dapa (1µM) treatment in 10% serum. Magnification x10. Images are from single experiment, representative of three others. Dapa: dapagliflozin.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5461630/v1/2551648c443fdd5da6ddd342.png"},{"id":69251438,"identity":"f92fbbf4-b164-4d11-a286-eb7662fe6a7e","added_by":"auto","created_at":"2024-11-18 11:47:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":110850,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of dapa on RcFbs proliferation after 24 h in different media conditions. RcFbs proliferation without \u003cstrong\u003e(A)\u003c/strong\u003e and with dapa \u003cstrong\u003e(B)\u003c/strong\u003e in the presence of 1% serum. Dapa: dapagliflozin, 1 µM. data = means ± SD (n=4; p*= 0.03, p***= 0.0001, p* = 0.01). Analysis was performed by one-way ANOVA (A, B) and two-way ANOVA (C) with a Turkey’s (2A) and Benferroni’s (2C) multiple comparison tests.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5461630/v1/c67b378a37ee76464783c0cd.png"},{"id":69251436,"identity":"25743cfe-7f9b-4f64-bf54-17410bdd9827","added_by":"auto","created_at":"2024-11-18 11:47:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":110834,"visible":true,"origin":"","legend":"\u003cp\u003eAngII - induced Ca\u003csup\u003e2+\u003c/sup\u003e mobilization in RcFbs after 24 h in different media conditions. Ca\u003csup\u003e2+\u003c/sup\u003e mobilization without \u003cstrong\u003e(A)\u003c/strong\u003e and with dapa \u003cstrong\u003e(B)\u003c/strong\u003e in the presence of 1% serum. Dapa: dapagliflozin, 1 µM; AngII - angiotensin II, 0.3µM. data = means ± SD (n=18; p** = 0.004, p*** = 0.0007, p**** ˂0.0001). Analysis was performed by one-way ANOVA (A, B) and two-way ANOVA (C) with a Turkey’s multiple comparison test.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5461630/v1/87edf4fd79e0dbb14ed7f965.png"},{"id":69251439,"identity":"78e6cccb-b0da-4a22-bfac-a9bd1ca8b238","added_by":"auto","created_at":"2024-11-18 11:47:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":806588,"visible":true,"origin":"","legend":"\u003cp\u003eHcFbs migration after 24 h in different media conditions in the without \u003cstrong\u003e(A\u003c/strong\u003e\u0026amp;\u003cstrong\u003eC)\u003c/strong\u003e and with dapa \u003cstrong\u003e(B\u003c/strong\u003e\u0026amp;\u003cstrong\u003eD)\u003c/strong\u003e in the presence of 10% serum. (A\u0026amp;B) shows images from the scratch assay at 10x magnification, with red parallel lines indicating edges where growth of cells terminated, and width measurements taken. Data represents four assays. Dapa: dapagliflozin, 1 µM. data = means ± SD (n=4; p** = 0.0068, p*** = 0.0009, p** = 0.0106). Analysis was performed by one-way ANOVA (A\u0026amp;B) and two-way ANOVA (C) with a Turkey’s (4C) and Benferroni’s (4E) multiple comparison tests.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5461630/v1/7eab17b2405b290bf98ac728.png"},{"id":69251441,"identity":"d9617984-bd8e-4926-8ada-5af49416abf0","added_by":"auto","created_at":"2024-11-18 11:47:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":184160,"visible":true,"origin":"","legend":"\u003cp\u003eqPCR analysis of fibrotic markers (col1a2: \u003cstrong\u003eA-C\u003c/strong\u003e; and CAMK2D: \u003cstrong\u003eD-F\u003c/strong\u003e) in HcFbs, and normalised for GAPDH. Dapa: dapagliflozin, 1 µM; col1a2 - collagen-1; calcium/calmodulin-dependent protein kinase II delta – CAMK2D; human glyceraldehyde-3-phosphate dehydrogenase - GAPDH. data = means ± SD; (n = 3; HG +dapa: n = 2; p**** \u0026lt;0.0001, p***=0.006, p** = 0.0084, p*=0.03, p** = 0.006, p** = 0.006) in each group.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5461630/v1/f7d664e969766b75b4ac5169.png"},{"id":69252743,"identity":"f504ea30-f1d4-4e9a-bd43-375024eca09b","added_by":"auto","created_at":"2024-11-18 11:55:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3576098,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5461630/v1/904c02cb-5dc6-493e-b96e-efc29452d6ba.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eEffect of dapagliflozin on rat and human cardiac fibroblasts physiology cultured in diabetic media conditions\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eHeart failure (HF) is a disease caused by defects in myocardial structure and function which results to impaired ventricular filling of the ejection of blood. While HF is mostly due to reduced myocardial function of the left ventricle, impairment of the myocardium, endocardium, great vessels, pericardium and heart valves, either in combination or alone is also linked with HF (Inamdar \u0026amp; Inamdar, 2016), and presents as chamber remodeling, interstitial fibrosis and decreased ventricular compliance (Travers et al., 2016) with symptoms including fatigue, edema, shortness of breath, abdominal distention and right hypochondrial pain (Yancy et al., 2013; Watson et al., 2000). The prevalence of HF, according to Bui et al, (2011) and Ponikowski et al, (2014) is estimated to be over 23\u0026nbsp;million, and this is expected to rise as global population increases (Conrad et al, 2018). Notwithstanding the advancement in HF treatment over the last decades, mortality and morbidity continue to rise (Lesyuk et al, 2018). Clinically, HF can be classified into: (i) HF with reduced ejection fraction (HFrEF), a form of HF where there is impairment in the systolic force generation of the heart, thereby causing reduction in the volume of expelled blood with each contraction (ejection fraction); and (ii) HF with preserved ejection fraction (HFpEF) where standard parameters of systolic function are mostly maintained, however, there is still impairment of diastolic filling and relaxation (Ponikowski et al, 2016). Although HF can be predominantly left, right or bi-ventricular based on the location of deficit in the heart, depending on the onset, it can further be classed either as acute or chronic (Inamdar \u0026amp; Inamdar, 2016). Although HFpEF is more dominant in females and older adults, in all patients with HFpEF, the EF is normally over 50%, with normal left-ventricular (LV) chamber volume accompanied by thick and stiff LV wall, thereby increasing the LV mass:end-diastolic volume ratio (Ohtani et al., 2012). Whereas, in patients with HFrEF, the LV is usually dilated and LV mass/end-diastolic volume ratio can be reduced or normal, however, the volume of myofibrils and cardiomyocyte diameter are elevated in HFpEF compared to HFrEF (Dassanayaka and Jones, 2015). While HFrEF patients respond favourably to treatment regimen with better prognosis, this is not the case for HFpEF patients as they do not respond to standard pharmacological interventions (except for nitrates), thus prognosis is poor during the decompensated phase of HF (Othani et al., 2012; Zamani et al., 2015; Glean et al., 2015).\u003c/p\u003e \u003cp\u003eRecent studies have implicated cardiac fibrosis in the aetiology of almost both types of HF, especially in the pathophysiology of HFpEF (Moreo et al, 2009; Gonza\u0026acute;lez et al, 2018) where cardiac fibroblasts (CFs) deposits excess extracellular matrix (ECM) proteins which leads to myocardial remodelling and eventually resulting to decrease in tissue compliance and acceleration of HF progression (Travers et al., 2016). This event consequently leads to systolic and diastolic dysfunction observed in many pathophysiological dysfunction (Kong et al., 2013) which is observed in diabetic and ischemic heart disease patients, and accompanied with morbidity and mortality (Tian et al., 2017). CFs are important cell type, mainly of embryonic epicardial and endothelial roots (Moore-Morris et al., 2014; Ali et al., 2014), and in healthy hearts, CFs produce and secrete fibrillar collagens (Zhang et al., 2022). Collagen I and III fibers, for instance, functions as scaffold for the myocardium. Hence imbalances in the production and degradation of collagen in the ECM leading to collagens expansion causes myocardial fibrosis. Imbalances such as this occur as a result of cardiomyocytes death, or stimuli that induces increase in the synthesis of collagen (Rathod et al., 2016). Hence, under physiological conditions, CFs maintain homeostasis of ECM \u0026ndash; distributing mechanical forces through the cardiac tissue, therefore giving the structural scaffold for cardiomyocytes and initiating electric conduction in the heart (Souders et al., 2009; Camelliti et al., 2005; Porter et al., 2009). An indicator for HFpEF is hypertrophic growth of cardiac myocytes, and ECM accumulation caused mainly by fibrillar collagen (Zile et al., 2004). In a previous study (Zile et al., 2015), the increase in left ventricular cavity and myocardial diastolic stiffness observed in HFpEF patients is due to abnormality in myocyte structural proteins and elevated myocardial collagen synthesis. Again, resident fibroblasts also appear to be the primary cells involved in fibrillar collagens production in response to left ventricular pressure-overload (LVPO) in murine models of clinically relevant heart disease (Moore-Morris et al., 2014). Thus, different mechanisms of myocardial fibrosis exist, however, high accumulation of ECM which impedes cardiac function is the main cause of fibrotic heart disease (Tian et al., 2017).\u003c/p\u003e \u003cp\u003eAlthough CFs appear to be flat, spindle shaped with multiple processes, the cardiac milieu varies greatly with respect to the species when examined between healthy and injured myocardium (Banerjee et al., 2007). Whereas previous studies have shown that CFs accounts for the vast majority cells found in the human and adult rodent myocardium (Banerjee et al., 2007; Camelliti et al., 2005; Zak, 1974), in adult murine hearts, CFs make up of less than 20% of the cell population, significantly lower than previously reported (Ali et al., 2014; Pinto et al., 2016). Although, Gittenberger et al., (1998) and Mu\u0026ntilde;oz-Ch\u0026aacute;puli et al., (2001) have previously reported that the bulk of resident fibroblasts come from the embryonic epicardium, there is still much to learn about how different resident and invading cells contribute to the active cardiac myofibroblasts, including the development of more precise molecular markers for CFs. Also, the regenerative ability of the heart is limited, with the process of repairing involving removing necrotic cardiomyocytes and then the replacement of fibrotic scar tissue to preserve the structure and function of myocardial integrity. In connective tissues, CFs help in performing these functions by converting to myofibroblasts, their active form by secreting high levels of ECM proteins, thereby encouraging a profibrotic condition. Thus, cardiac fibrosis initiates pathological events that promotes cardiomyocyte hypertrophy and apoptosis, chamber dilatation which eventually leads to the development of congestive heart failure (CHF) (Baudino et al., 2006; Cohn et al., 2000). In spite of the role of fibrosis in cardiovascular diseases (CVD), cardiac fibrosis remains poorly understood; with current clinical interventions effectively targeting CFs and its pathological contributions to disease progression limited (Travers et al., 2016).\u003c/p\u003e \u003cp\u003eElevated glucose levels observed in diabetes have been linked to increased ECM deposition or fibrosis, and has been reported to have an impact on myocardial function (Weber et al. 1987, 1988). Studies have revealed the profibrotic effects of increased glucose levels in isolated fibroblasts and models of diabetic disease (Tang et al. 2007; Asbun et al. 2005; Zaman et al. 2004). For instance, diabetes, a metabolic disease, is mainly characterized by high levels of blood glucose. Impairment of glucose metabolism, proteins and lipids generates alterations both micro and macrovascular circulation, paving way for the risk of numerous complications in diabetic patients especially, cardiovascular complications (Rask-Madsen and King, 2013) and delayed wound healing (Brem and Tomic-Canic, 2007) amongst others. A key abnormality in diabetes, hyperglycaemia, has been shown to play a role in the development of inflammation in diabetic complications, as an earlier study shows that increase in blood sugar levels enhances inflammation and impedes wound healing by altering angiogenesis (Braiman-Wiksman et al., 2007). \u003cem\u003eIn vitro\u003c/em\u003e studies reveal that hyperglycaemia reduces migration (Lamers et al., 2011 and Kido et al., 2017), proliferation (Stolzing et al., 2006) and collagen synthesis (Willershausen-Zonnchen et al., 1991) as well as increase apoptosis (Baumgartner-Parzer et al., 1995; Deveci et al., 2005) in cells of various types. During wound healing, fibroblasts, keratinocytes, macrophages, endothelial cells and platelets coordinate in a complex manner, with successful healing process involving cell proliferation and migration, deposition and remodeling of collagen, and angiogenesis and wound contraction. Fibroblasts, being the most abundant cell types found in connective tissues are primarily involved in the production and remodelling of the ECM (Willershausen-Zonnchen et al., 1991).\u003c/p\u003e \u003cp\u003eCurrently, therapies such as sodium-glucose co-transporter 2 (SGLT2) inhibitors and beta-blockers which targets the renin\u0026ndash;angiotensin\u0026ndash;aldosterone system (RAAS) exists for the treatment of HFrEF, however, no effective drug is yet to be developed for the treatment HFpEF even though multiple randomized clinical trials have been conducted (Pfeffer et al, 2019; Seferovic et al, 2019; Ponikowski et al, 2016), posing a serious concern as HFpEF is now common and responsible for over 50% of all HF cases globally (Vasan et al, 2018), which could be linked to its association with comorbidities including diabetes, hypertension and ageing (Streng et al, 2018; Dunlay et al, 2017; Bekfani et al, 2016). Although, biochemical mediators such as angiotensin II (AngII) have been shown to regulate the fibroblasts \u0026ndash; ECM interactions (Watson et al. 1998; Burgess et al. 1994), the effects of dapagliflozin (dapa) in response to elevated glucose in CFs requires further investigation. In the current study, I investigated the effect of dapagliflozin on rat and human cardiac fibroblasts physiology cultured in diabetic media conditions. Experiments were performed to study the effects of glucose in the presence of dapa on gene expressions of human collagen-1 (col1a2), human calcium/calmodulin-dependent protein kinase II delta (CAMK2D) and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using quantitative polymerase chain reaction (qPCR). I have also assessed the effects of dapa on Ca\u003csup\u003e2+\u003c/sup\u003e release in these cells following stimulation with AngII using confocal imaging technique. Additional experiments were then carried out to assay the migration and proliferation abilities and phenotypes of CFs in response to drug in glucose media conditions.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eHuman cardiac fibroblasts (HcFbs), Fibroblast Growth Medium 3 (Fb medium), Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM), Basic fibroblast growth factor (recombinant human), insulin (recombinant human), fetal bovine serum (FBS), fetal calf serum (FCS) and penicillin/streptomycin were all purchased from Promo Cell GmbH (Heidelberg, Germany). Rat cardiac fibroblasts (RcFbs) was a gift from Professor Suzan Currie from the University of Strathclyde. D-Glucose anhydrous, Palmitate, ReddyMix PCR Master Mix were bought from Thermo Fisher Scientific (Loughborough, UK). Angiotensin II (AngII) and dapagliflozin were supplied by Sigma-Aldrich (St. Louis, MO), Cal-520\u0026reg; was purchased from AAT Bioquest (California, USA). Integrated DNA Technologies (Leuven, Belgium) supplied both the forward and reverse primers for col1a2, CAMK2D and GAPDH, and EvaGreen\u0026reg; qPCR Supermix, 5x was purchased from Solis BioDyne (Tartu, Estonia).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEthical consideration\u003c/h3\u003e\n\u003cp\u003eThe was approved by the ethical committee, and the study conforms to the United Kingdom Animal procedures act 1986, and with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication, 8th Edition, 2011).\u003c/p\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eHcFbs (1 mL vial stored in liquid nitrogen storage) and RcFbs (seeded in 2x 96-well plates) were maintained according to manufacturer\u0026rsquo;s instructions (Gao et al., 2020; Tao et al., 2021). All cell culture experiments were carried out in the cell culture room aseptically, in the Kendro KS12 safety hood (Kendro Ltd., Germany). Briefly, prior to thawing the HcFbs, hFibroblast culture media containing 89% Fb media, 1% penicillin/streptomycin and 10% FBS was made, and cells were removed from the liquid nitrogen storage and quickly thawed by placing vial in a 37\u003csup\u003eo\u003c/sup\u003eC water bath for ~\u0026thinsp;1 min. Cells were monitored during thawing and were removed from the bath just prior to complete thawing, and were quickly transferred into a 15 mL conical tube with 9 mL room temperature hFibroblast media (previously prepared) and centrifuged at 1,200 rpm for 3 mins and supernatant was aspirated, leaving the cell pellet undisturbed. To achieve a single cell suspension, cell pellet was re-suspended in 5 mL of hFibroblast media and mixed properly by pipetting up and down. The cell suspension was transferred to a T25 cell culture flask and incubated in humidified temperature-controlled Galaxy 170 S incubator (Eppendorf/New Brunswick Scientific, Stevenage, UK) (5% O\u003csub\u003e2\u003c/sub\u003e, 5% CO\u003csub\u003e2\u003c/sub\u003e and balance N\u003csub\u003e2\u003c/sub\u003e) overnight, just as hFibroblast media was replaced every 2 days.\u003c/p\u003e \u003cp\u003eAt a confluency of about 80\u0026ndash;90%, CFs created a swirling pattern, and confluent cells were split from the T25 cell culture flask into a T75 cell culture. This was done by retrieving the T25 flask from the incubator and aspirating hFibroblast media, and washing the cells twice with 5 mL Phosphate-buffered saline (PBS). Then, 1 mL of 0.25% trypsin was added to the cells and incubated at 37\u003csup\u003eo\u003c/sup\u003eC for 5 mins to ensure the cells dissociation from the culture flask. The cells were then checked under the Olympus Optical microscope CK2-TR (Olympus Optical Co., Ltd., Japan) to confirm cells have completely detached from the culture flask (flask was tapped on the bench to induce mechanistic force to dissociate cells where necessary). The cells were further re-suspended in 5 mL of trypsin inhibitor and pipetted up and down, and used to washed down the flask again as cells were collected as a single cell suspension, transferred into a 15 mL conical tube and centrifuged at 1,200 rpm for 3 mins. Supernatant was aspirated, leaving the cell pellet undisturbed and was then re-suspended in 7 mL of fresh hFibroblast media. The cell suspension was pipetted up and down 2\u0026ndash;3 times and a single cell suspension was achieved and transferred to a T75 cell culture flask and incubated at 37\u003csup\u003eo\u003c/sup\u003eC and 5% CO\u003csub\u003e2\u003c/sub\u003e overnight. hFibroblast media was replaced every 2\u0026ndash;3 days as required.\u003c/p\u003e \u003cp\u003eThe cells in the T75 cell culture flask were confirmed to have attained 80\u0026ndash;90% confluency using the microscope, and was used to produce enough cells to freeze 3 x 1 mL aliquots. Briefly, 1.5 mL of freezing media (90% FBS\u0026thinsp;+\u0026thinsp;10% DMSO) and 1.5 mL of hFibroblast (99% FCS\u0026thinsp;+\u0026thinsp;1% penicillin/streptomycin) were prepared and set aside. The hFibroblast media was aspirated from the T75 culture flask, cells were washed twice with 5 mL PBS and aspirated, and then 4 mL of 0.25% trypsin was added to the cells and then incubated at 37 \u003csup\u003eo\u003c/sup\u003eC for 5 mins cells to dissociate completely from the culture flask. The flask was retrieved and observed under the microscope to confirm cells detachment, and where necessary, the bottom of the flask was gently tapped on the bench to dislodge cells. The cells were then re-suspended in 6 mL of trypsin inhibitor and pipetted up to wash the flask again and a single cell suspension collected. The cell suspension was then transferred into a 50 mL conical tube and centrifuged at 1200 rpm for 2 mins. Thereafter, the supernatant was carefully removed leaving the cell pellet intact. The cell pellet was then re-suspended in 1.5 mL of hFibroblast media, and then, 1.5 mL of freezing media was added to cell suspension, bringing the total volume to 3 mL which was aliquoted into 3x 1 mL aliquots across three cryovials, wrapped in 5 layers of tissue paper and immediately placed cells in -80 \u003csup\u003eo\u003c/sup\u003eC freezer for 72 h, before finally transferring cells to -150 \u003csup\u003eo\u003c/sup\u003eC freezer.\u003c/p\u003e \u003cp\u003eWhereas, Fb media was used as media for the HcFbs, I have used DMEM for the RcFbs. Cells were passaged at about 80\u0026ndash;90% confluency, and cells in the passages 9 (HcFbs) and 1 (RcFbs) respectively were used for experiments in the current study.\u003c/p\u003e\n\u003ch3\u003eDrug Treatment\u003c/h3\u003e\n\u003cp\u003eRcFbs and HcFbs were grown to about 70\u0026ndash;80% confluence in their respective growth medium (as mentioned above) in different media conditions. For experiments on RcFbs, media conditions were low glucose (LG; 17.7 mM), high glucose (HG; 30 mM), palmitate (P; 2.5 mM) and HG\u0026thinsp;+\u0026thinsp;P (30 mM\u0026thinsp;+\u0026thinsp;2.5 mM). For HcFbs experiments, the conditions where were LG (7.7 mM), HG (30 mM) and HG\u0026thinsp;+\u0026thinsp;P (30 mM\u0026thinsp;+\u0026thinsp;2.5 mM). Media conditions were set up in triplicates, and were treated with and without dapa (1\u0026micro;M) for 24 h in their respective growth medium containing 1% and 10% serum respectively after serum starving for 24 h. For RcFbs, stock concentrations of drugs were diluted in DMEM with supplements (1% penicillin/streptomycin and 1% L-glutamine), and in HcFbs experiments, stock drug concentration was diluted in hFibroblast media (1% penicillin/streptomycin) before adding to the cells. Drug concentrations were based on relevant concentrations in previous studies (Tian et al., 2021; Arow et al., 2020; McMullen et al., 2020).\u003c/p\u003e\n\u003ch3\u003eCell imaging\u003c/h3\u003e\n\u003cp\u003eCell growth and phenotype were monitored with the aid of Olympus Optical CK2-TR inverted microscope (Olympus Optical Co., Ltd., Japan) and a Digital Bio JULI\u0026trade; Smart fluorescent cell analyser (Nanoentek Inc., Seoul, Korea) at 10\u0026times; magnification (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell proliferation assays\u003c/h2\u003e \u003cp\u003eProliferation assay was performed on RcFbs upon reaching a confluency of 60\u0026ndash;80%. Briefly, from each well, DMEM media was aspirated and the cells were serum-starved for 24 h and consequently treated with dapa in the different media conditions as previously mentioned. After 24 h, plate was retrieved from the incubator, media was aspirated and 200\u0026micro;l PBS used to wash the cells, and replaced with 100\u0026micro;l trypsin and incubated for 3 mins. Then, 200\u0026micro;l of FBS-supplemented DMEM was added to each well, and mixed properly by pipetting up and down, and then transferred into Eppendorf tubes which were centrifuged for 10 mins at 2600 rpm. Cell pellet was extracted for analysis as supernatant was discarded. Then, 10\u0026micro;l of serum free media (SFM) and 10\u0026micro;l of Trypan blue were then added to the tubes, from which 10\u0026micro;l of the mixture was loaded to the cell counter chamber slide, and inserted into the Countess II Automated Cell Counter (Invitrogen, Paisley, Renfrewshire, UK) to determine cell concentration per ml (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIntracellular Calcium Measurement\u003c/h3\u003e\n\u003cp\u003e200 \u0026micro;L cell suspension of RcFbs were seeded into clear bottomed, dark sided 96-well plate (which was later confirmed to be at a density of 3.0 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well), and incubated for 24 h before treatment with dapa in different media conditions. The culture medium was aspirated. 2 mL of 1x Fibroblasts Krebs/Tyrode solution (136.9mM NaCl, 5.4mM KCl, 1.3mM CaCl\u003csub\u003e2\u003c/sub\u003e, 0.4mM MgSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, 0.5mM MgCl\u003csub\u003e2\u003c/sub\u003e.6H\u003csub\u003e2\u003c/sub\u003eO, 0.3mM Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e.2H\u003csub\u003e2\u003c/sub\u003eO, 0.4mM KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 5.6mM glucose, 4.2mM NaHCO\u003csub\u003e3\u003c/sub\u003e; pH\u0026thinsp;=\u0026thinsp;7.4) was added to Cal-520 AM calcium dye stock (1 \u0026micro;M), and 50 \u0026micro;l from the solution was transferred into each well and allowed to incubate on the bench at room temperature for 20 mins. The solution was then aspirated and then washed with 50 \u0026micro;l 1x Fibroblasts Krebs/Tyrode solution once. The 96-well plate was carefully positioned, and cells were imaged to record baseline fluorescence for 1 min. Then 0.3 \u0026micro;M AngII (1.5 mL of 1x Fibroblasts Krebs/Tyrode solution\u0026thinsp;+\u0026thinsp;4.5 \u0026micro;l of AngII) was added to initiate Ca\u003csup\u003e2+\u003c/sup\u003e release from the endoplasmic reticulum (ER). Fluorescence measurements were taken using a spinning disk confocal microscopy system using a 488 nM laser with emission collected using an EM-CCD camera (Prime, Photometrics, Tucson USA). Analysis of data was then performed with ImageJ version IJ 1.46r. Background measurement from each well was taken with no cells, and using the formula F\u0026thinsp;=\u0026thinsp;F\u003csub\u003eraw\u003c/sub\u003e-F\u003csub\u003ebackground\u003c/sub\u003e, background was subtracted from all measurements. From the F image, a \u0026lsquo;z-project\u0026rsquo; was created and time the last 20 images were created when the AngII addition was added was determined, and random cells were circled out and input on the ROI manager, representing the F\u003csub\u003e0\u003c/sub\u003e values. Then, the formula, F\u003csub\u003enormalized\u003c/sub\u003e=F/F\u003csub\u003e0\u003c/sub\u003e was used to determine Ca\u003csup\u003e2+\u003c/sup\u003e transient amplitude as the area under the curve on the ImageJ software. Ca\u003csup\u003e2+\u003c/sup\u003e excitation was then obtained by subtracting 1 from the acquired peak values (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eCell migration assays\u003c/h3\u003e\n\u003cp\u003eCell migration assay was then performed on HcFbs seeded into 6-wells plate previously assigned reference scratches on the bottom. The cells were left to grow to grow in hFibroblasts media until 100% confluent. At confluency, media was aspirated from each well, and cells serum-starved for 24 h. Following this, the plate was retrieved and an initial scratch was made inside each well with a 200 uL pipette tip, as much as close to the reference line. SFM was aspirated to wash of the wounds, and 3 mL fresh SFM added to each well before the plate was photographed using Digital Bio JULI\u0026trade; Smart fluorescent cell analyser (Nanoentek Inc., Seoul, Korea) at 10\u0026times; magnification. The cells in the presence of 10% FCS-supplemented media were then treated with dapa accordingly, and incubated for 24 h. The plate was thereafter retrieved and the cells photographed again for comparison. Assay was analysed by measuring the difference in scratch width and converting this into a percentage migration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGene expression analysis\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eSample preparation\u003c/strong\u003e \u003cp\u003e2 mL HcFbs cell suspension was seeded into 3x 6-wells plate and left to grow until 100% confluent. At confluency, media was aspirated from each well, and cells serum-starved for 24 h. Then, the cells in the presence of 10% FCS-supplemented media were treated with dapa in different media conditions, and incubated for 24 h. After this, the media was aspirated, and the cells lysed by adding 175 \u0026micro;L Lysis Buffer RA1 and 1.75 \u0026micro;L β-mercaptoethanol (β-ME) in each well. Scrappers where then used to scrub the bottom of each well to collect cells, and were transferred into sterile 1.5 mL Eppendorf tubes and vortexed vigorously. Total RNA purification from the cultured cells was then carried out using NucleoSpin\u0026reg; RNA Mini Kit for RNA purification (Macherey-Nagel\u0026trade; GmbH \u0026amp; Co. KG, Duren, Germany) according to manufacturer\u0026rsquo;s instructions. Total RNA was then quantified using the Epoch BioTek\u0026reg; (Fisher Scientific, Loughborough, UK) before being stored in the \u0026minus;\u0026thinsp;80 \u003csup\u003eo\u003c/sup\u003eC freezer till the next day.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eReverse \u0026ndash; Transcription\u003c/em\u003e: briefly, the total RNA was subjected to reverse transcription using the commercially available High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Lithuania) according to manufacturer\u0026rsquo;s instructions. First, 10 \u0026micro;L of a master mix containing 2.0\u0026micro;L of 10 x RT buffer, 0.8 \u0026micro;L of 25 x dNTP, 0.8 \u0026micro;L of 10 x random primers, 1.0 \u0026micro;L of Multiscribe and 4.2 \u0026micro;L of Nuclease-free H\u003csub\u003e2\u003c/sub\u003eO was added into PCR tubes with 10 \u0026micro;L RNA, and then placed in the Applied biosystems 2720 Thermal Cycler\u0026reg; (Life Technologies Holdings Pte Ltd., Singapore) with the following conditions: 25 \u003csup\u003eo\u003c/sup\u003eC for 10 mins to maximise primer RNA template binding, 37 \u003csup\u003eo\u003c/sup\u003eC for 120 mins for reverse transcription, and 85 \u003csup\u003eo\u003c/sup\u003eC for 5 mins to deactivate reverse transcription. cDNA was then stored in the \u0026minus;\u0026thinsp;20 \u003csup\u003eo\u003c/sup\u003eC fridge for later use.\u003c/p\u003e \u003cp\u003e \u003cem\u003eQuantitative real-time PCR (qPCR)\u003c/em\u003e: qPCR was performed to measure the expression level of the mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) using EvaGreen\u0026reg; qPCR Supermix 5x master mix and fluorescently tagged primers. The primers for the genes of interest used are shown on Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. qPCR reactions to determine the expression of mRNA of the genes of interest were set up in a 96-well plates in triplicate, and the results were normalized against those of GAPDH, and run using the SYBR method in the ViiA7TM real-time PCR System (Life Technologies, Paisley, UK) in the following conditions: 50 \u003csup\u003eo\u003c/sup\u003eC for 2 min, 95 \u003csup\u003eo\u003c/sup\u003eC for 10 min and 40 cycles of 95 \u003csup\u003eo\u003c/sup\u003eC for 15 secs, 60 \u003csup\u003eo\u003c/sup\u003eC for 1 min. The data (Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) were analysed using the 2\u003csup\u003e\u003cb\u003eΔΔ\u003c/b\u003eCT\u003c/sup\u003e method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eData were analysed using GraphPad Prism 9 (GraphPad Software, San Diego, CA), and presented as mean values\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E.M of n observations (n, representing the number of samples). Comparisons were made by one \u0026ndash; or two-way analysis of variance (ANOVA) followed by either Tukey\u0026rsquo;s or Benferroni\u0026rsquo;s multiple comparison tests as appropriate. In this study, a value of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicated statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eEffect of dapagliflozin on cardiac fibroblast phenotype\u003c/h2\u003e\n \u003cp\u003eHealthy CFs were only established using bright field imaging. In assessing the CFs phenotype in the absence and presence of dapa on different media conditions the cells were subjected to, cells were treated with either the same concentration of drug (1 \u0026micro;M) or without drug for 24 h and then imaged (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e; LG\u0026thinsp;=\u0026thinsp;low glucose; HG\u0026thinsp;=\u0026thinsp;high glucose; P\u0026thinsp;=\u0026thinsp;palmitate; HG\u0026thinsp;+\u0026thinsp;P\u0026thinsp;=\u0026thinsp;high glucose with palmitate). In RcFbs (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA), there was no observed changes in cell phenotype in LG and HG media conditions, and no obvious cell loss. There was no visible shape in cells in P media conditions, but associated cell loss with the formation of vacuole-like structures. While there seem not to be any phenotypic change, there was an obvious cell loss in HG\u0026thinsp;+\u0026thinsp;P media conditions. However, in media conditions treated with dapa, the shape of cells remained intact with no changes, and there was insignificant cell loss in LG and HG media conditions. While P media conditions exhibited obvious cell death with the formation of vacuole-like structures, HG\u0026thinsp;+\u0026thinsp;P media conditions showed decreased cell death and less vacuole formed.\u003c/p\u003e\n \u003cp\u003eIn HcFbs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB), there was no significant alterations in cell phenotype across all media conditions, with or without dapa treatment, CFs appeared flat, spindle shaped with multiple processes.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eEffect of dapagliflozin on RcFbs proliferation\u003c/h2\u003e\n \u003cp\u003eNext, proliferation of RcFbs in different media conditions, in the absence (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA) and presence of 1\u0026micro;M dapa (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB) was investigated. Proliferation assay revealed HG and HG\u0026thinsp;+\u0026thinsp;P in the absence of dapa significantly influenced cell proliferation, with LG showing lowest proliferation of cells. Thus, there was a significant increase in cell proliferation in HG and HG\u0026thinsp;+\u0026thinsp;P media conditions when compared to LG media condition (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA; p*= 0.03, p*** = 0.0001). Dapa however showed no influence on cell proliferation across all media conditions investigated. But, when compared using 2-way ANOVA, there was a significant drop in cell proliferation of cells in HG media condition treated with dapa compared to those without dapa treatment (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC; p* = 0.01). There also seem to be a trend of increasing proliferation in LG without dapa compared to dapa treated LG condition, but this was not significant (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eEffect of dapagliflozin on AngII-induced Ca\u003csup\u003e2+\u003c/sup\u003e mobilization in RcFbs\u003c/h2\u003e\n \u003cp\u003eTo elucidate the effect of dapa on cellular calcium, the influence of dapa on Ca\u003csup\u003e2+\u003c/sup\u003e mobilization in RcFbs in the different media conditions were examined. AngII (0.3\u0026micro;M) was used to induce intracellular Ca\u003csup\u003e2+\u003c/sup\u003e release. Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e shows Ca\u003csup\u003e2+\u003c/sup\u003e mobilization from random cells (n\u0026thinsp;=\u0026thinsp;6) from across different media conditions. In the group not receiving dapa treatment, there was an increase in Ca\u003csup\u003e2+\u003c/sup\u003e release in LG cultured cells compared to those cultured in other media conditions (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA; p***= 0.0007, p**** ˂0.0001). In media conditions treated with dapa (1\u0026micro;M), a significant increase in Ca\u003csup\u003e2+\u003c/sup\u003e mobilization was also observed in HG media compared to all other media conditions (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB; p****˂0.0001). When a group comparison test was performed, result indicated that there was a significant increase in Ca\u003csup\u003e2+\u003c/sup\u003e mobilization observed only in cells cultured in HG media condition subjected to dapa compared to their untreated counterparts (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC; p**= 0.004).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eEffect of dapagliflozin on HcFbs migration\u003c/h2\u003e\n \u003cp\u003eThe effect of dapa on HcFbs migration cultured on different media conditions was examined using a wound healing assay after 24 h. In media conditions without dapa treatment, LG showed significant migration compared to other groups (figure 4A, 4C; p** = 0.0068, p*** = 0.0009). Though there seem to be an increase in migration in HG media cultured cells, this was not statistically significant when compared with other media conditions treated with dapa (figure 4B, 4D). The result of the multiple comparison test shows that cells cultured in HG media treated with dapa displayed significant migration rate, compared to their untreated counterpart (figure 4E; p** = 0.0106) after 24 h.\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eEffect of dapagliflozin on gene expressions of human collagen-1 (col1a2), human calcium/calmodulin-dependent protein kinase II delta (CAMK2D) in HcFbs.\u003c/strong\u003e\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eqPCR primers design of primers\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGenes\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSequences\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMelting temperature T\u003csub\u003em\u003c/sub\u003e/\u003csup\u003eo\u003c/sup\u003eC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMolecular weight /MW\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCol1a2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF: 5\u0026rsquo;-GAG CGG TAA CAA GGG TGA GC -3\u0026rsquo;\u003c/p\u003e\n \u003cp\u003eR: 5\u0026rsquo;-CTT CCC TAG GGC CTC TC -3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e58.3\u003c/p\u003e\n \u003cp\u003e57.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6,256.1\u003c/p\u003e\n \u003cp\u003e5,979.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCAMK2D\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF: 5\u0026rsquo;- AGT CAG AAG AGA CTC GTG TGT \u0026minus;\u0026thinsp;3\u0026rsquo;\u003c/p\u003e\n \u003cp\u003eR: 5\u0026rsquo;- TGA TGG GTA CTG TTG GTG ACC \u0026minus;\u0026thinsp;3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e55.2\u003c/p\u003e\n \u003cp\u003e56.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6,510.3\u003c/p\u003e\n \u003cp\u003e6,508.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGAPDH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF: 5\u0026rsquo;- ACA ACT TTG GTA TCG TGG AAG G -3\u0026rsquo;\u003c/p\u003e\n \u003cp\u003eR: 5\u0026rsquo;- GCC ATC ACG CCA CAG TTT C -3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e55.1\u003c/p\u003e\n \u003cp\u003e57.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6,814.5\u003c/p\u003e\n \u003cp\u003e5,708.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eThe effect of dapa on gene expressions of Col1a2 and CAMK2D in HcFbs in the different media conditions were studied. For Col1a2 analysis, when compared with the control group in media conditions not receiving dapa treatment, Col1a2 expression was significantly reduced in HG, but increased in HG\u0026thinsp;+\u0026thinsp;P (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA; p*** = 0.006, p**** \u0026lt;0.0001) respectively. However, there was a significant decrease in its expression in HG\u0026thinsp;+\u0026thinsp;P and LG media treated with dapa when compared to controls (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB; p* = 0.03, p** = 0.0084). When a 2-way ANOVA was performed, cells cultured in HG media which received dapa exhibited a significant increase in Col1a2 gene expression compared to their untreated counterpart (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC; p** = 0.006). Similarly, in dapa-free media conditions, CAMK2D showed a significant reduction in HG, but increased expression in HG\u0026thinsp;+\u0026thinsp;P cultured cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD; p** = 0.0083, p***\u0026lt;0.0001) respectively. Dapa however showed no significant effect on CAMK2D expression across all media conditions.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003cdiv class=\"colspec\" align=\"char\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eCT values of genes of interest obtained from ViiA7TM real-time PCR System.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGroups\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCol\u003c/p\u003e\n \u003cp\u003e1a2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCol\u003c/p\u003e\n \u003cp\u003e1a2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCol\u003c/p\u003e\n \u003cp\u003e1a2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGAPDH\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGAPDH\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGAPDH\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCAMK\u003c/p\u003e\n \u003cp\u003e2D\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCAMK\u003c/p\u003e\n \u003cp\u003e2D\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCAMK\u003c/p\u003e\n \u003cp\u003e2D\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGAPDH\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGAPDH\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGAPDH\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLG\u003c/p\u003e\n \u003cp\u003e+dapa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e33.868\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e33.184\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e33.837\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e29.572\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.161\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e30.162\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e30.698\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e33.107\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31.066\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.574\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e33.132\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e36.835\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLG (C)\u003c/p\u003e\n \u003cp\u003e-dapa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31.746\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31.920\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31.528\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e29.523\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.312\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31.289\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e28.733\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e30.399\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e28.743\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.069\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e30.418\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31.287\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHG\u0026thinsp;+\u0026thinsp;dapa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32.502\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e34.845\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e34.330\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e33.121\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNot\u003c/p\u003e\n \u003cp\u003edetermined\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e33.145\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e37.137\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32.830\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e35.266\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNot\u003c/p\u003e\n \u003cp\u003edetermined\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32.349\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31.920\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHG\u003c/p\u003e\n \u003cp\u003e-dapa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e33.763\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e33.532\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32.830\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31.115\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30.414\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e30.986\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e30.404\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e30.638\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e29.650\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30.390\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e30.724\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e30.073\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHG\u0026thinsp;+\u0026thinsp;P\u0026thinsp;+\u0026thinsp;dapa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e33.268\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e33.884\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32.962\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e30.566\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.182\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31.285\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e29.990\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e30.187\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e29.658\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.704\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e29.955\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e29.972\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHG\u0026thinsp;+\u0026thinsp;P\u003c/p\u003e\n \u003cp\u003e-dapa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e30.851\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e30.851\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e30.851\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31.659\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.659\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31.659\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e30.478\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e30.478\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e30.478\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.659\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31.659\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31.659\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eCardiac fibrosis is the hallmark of diabetic cardiomyopathy which may be evident even in the absence of any obvious cardiomyocyte damage, indicating a role for cardiac fibroblast in the pathological process, although, mechanism underlying this is unknown (Ling-Yu Zhang et al., 2021). In the present study, the effects of diabetic media conditions in the presence of dapagliflozin on rat and human CFs have been evaluated to aid a better understanding of the profiles of glucose and dapa in CFs physiology and function. Here, the effects of glucose and dapa on CFs cell phenotype, proliferation, wound healing, Ca\u003csup\u003e2+\u003c/sup\u003e mobilization, and gene expressions of col1a2 and CAMK2D were assessed and documented. Palmitate has been used as part of the media conditions in the present study owing to its ability to inhibit and/or retard oxidation reactions by preventing damaging effects of oxidation in animal cells and tissues (NCBI, 2022). The current study demonstrates that the relatively low concentrations of dapa (1\u0026micro;M) and glucose used in the treatment of CFs in the presence of serum was sufficient in evaluating their potential to affect cell phenotype and function as other studies have used similar concentrations in achieving promising results (Tian et al., 2021; Arow et al., 2020; McMullen et al., 2020). Although, a more realistic result would have been obtained if a concentration-response curve of the drug was produced to have a broader idea of the activity of the drug on these cells. Dapa is an SGLT2 inhibitor indicated for the management of type 2 diabetes mellitus (Obermeier et al., 2010) and functions by inhibiting the SGLT2 receptor, thereby preventing the reabsorption of glucose in the kidney, thus reducing circulating blood glucose, and increasing excreted glucose (Albarr\u0026aacute;n and Ampudia-Blasco, 2013). In the group not receiving treated with dapa, RcFbs did not display any alterations in cell phenotype across the media conditions investigated, however, formation of vacuole-like structures and cell viability were evident in the palmitate media conditions, with obvious cell loss in HG\u0026thinsp;+\u0026thinsp;P media condition. Following treatment of cells with dapa, cell phenotype remained unchanged, even though P media conditions exhibited formation of vacuole-like structures. In HcFbs, there were no obvious cell death and no alterations in phenotype as CFs maintained flat, spindle shaped with multiple processes. This therefore suggest that neither dapa nor glucose, nor the combination of both at their respective concentrations induced any phenotypic alterations on these cells.\u003c/p\u003e \u003cp\u003eEven though CFs form the bulk of matrix producing cells in the heart and are responsible for cardiac remodeling and fibrosis (Porter and Turner, 2009), previous studies have concentrated on the effect of hyperglycemia on cardiac myocytes, with few studying CFs. Diabetes, being an independent risk factor for CVD, with diabetic patients predisposed to CV complications including fibrosis, it is pertinent to evaluate the influence of high glucose on fibroblast activation to enhance identification of specific targets that could be beneficial in delaying disease progression. I therefore sought to establish the role of high glucose on proliferation of CFs. Here it was established that in the absence of dapa, HG significantly increased CF proliferation. This observation is in line with other studies where isolated CFs from diabetic myocardium showed more proliferation than those isolated from healthy control myocardium (Shamhart et al., 2009), with high glucose found to be stimulating CFs proliferation in vitro (Asbun and Villarreal, 2006; Neumann et al., 2002). Although, Zhang et al., (2007) had previously reported that CFs proliferation was decreased in high glucose containing medium relative to low glucose medium, this may be attributed to the fact the cells studied were cultured in cover slip coated with collagen. However, in the presence of dapa, HG-induced CFs proliferation was significantly reduced. This suggests that dapa has a role in decreasing CFs proliferation. The effects of glucose on HcFbs migration was also investigated with/without dapa, and it was found that CFs cultured in dapa-free HG media migrated slowly compared to those cultured in LG conditions, which is in agreement with an earlier study which established impediment of wound healing in condition of increased blood sugar levels in diabetic patients (Kido et al., 2017; Lamers et al., 2011; Braiman-Wiksman et al., 2007) as well as a similar observation found in human gingivial fibroblast (Buranasin et al., 2018). However, this observation contradicts Shamhart et al., (2014) which reported that adult CFs stimulated with high glucose migrated faster than fibroblasts in low glucose. However, in the presence of dapa, CFs migration was increased in HG condition, implying that dapa enhances CF wound healing.\u003c/p\u003e \u003cp\u003eDiabetes and hyperglycaemia induce the mobilization of intracellular Na\u003csup\u003e+\u003c/sup\u003e and Ca\u003csup\u003e2+\u003c/sup\u003e (Baartscheer et al., 2017). Thus, rise in Na\u003csup\u003e+\u003c/sup\u003e and Ca\u003csup\u003e2+\u003c/sup\u003e promotes events leading to dysregulation of mitochondrial homeostasis, such as increased ROS production, cardiac hypertrophy, and remodelling (Uthman et al., 2018) and eventual cell death (Odagiri et al., 2009). Ang II plays a significant role in maintaining cardiac performance via the RAAS II system (Urata et al., 1990), and directly binds to G-protein-coupled AngII receptor type 1 (AT\u003csub\u003e1\u003c/sub\u003e), therefore stimulating Ca\u003csup\u003e2+\u003c/sup\u003e/calmodulin-dependent phosphorylation of L-type calcium channels, resulting in an increase in influx of Ca\u003csup\u003e2+\u003c/sup\u003e (Correa et al., 2015; Goette and Lendeckel, 2008). In this current study, I have sought to evaluate Ca\u003csup\u003e2+\u003c/sup\u003e mobilization in the ER of RcFbs with/without dapa in diabetic media conditions. Interestingly, at 1\u0026micro;M concentration, dapa sufficiently enhanced AngII-mediated intracellular Ca\u003csup\u003e2+\u003c/sup\u003e mobilization in HG cultured cells compared to other groups, especially exerting a decreasing effect in intracellular Ca\u003csup\u003e2+\u003c/sup\u003e mobilization in LG cultured cells. To the best of our knowledge, no study has looked into this using CFs, hence, the data suggests that dapa promotes Ca\u003csup\u003e2+\u003c/sup\u003e mobilization in diabetic CFs. However, in a recent study, cardiomyocytes displayed a reduction in amplitude of Ca\u003csup\u003e2+\u003c/sup\u003e transients (Arow et al., 2020). Since no higher concentrations of the drug were used, I are unsure of any exacerbating effect it may have on Ca\u003csup\u003e2+\u003c/sup\u003e-induced CF responses. Although relevant studies have documented the effect of excess Ca\u003csup\u003e2+\u003c/sup\u003e release on CFs apoptosis and necrosis (Li et al., 2010; Pinton et al., 2008).\u003c/p\u003e \u003cp\u003eConsidering the role CFs play in the disposition of ECM and cardiomyocyte function, effects of high glucose on CFs could cause a direct impact on cardiac contraction which could result to impaired diastolic function. Since cardiac fibrosis is a serious occurrence in diabetic cardiomyopathy and pathological remodelling of the heart, and as collagen and CAMKII are well established important molecules in cardiac physiology (Villarreal et al., 2009; Bers, 2008; Beckendorf et al., 2018), and implicated in CF dysfunction (Horn and Trafford 2016; Martin et al., 2014), the effect of dapa on gene expressions of col1a2 and CAMK2D were measured in HcFbs. It was found that HG decreased col1a2 expression but influenced col1a2 expression in HG\u0026thinsp;+\u0026thinsp;P condition not treated with dapa, which is similar to a previous study that saw an increase in col1 expression in hyperglycemic conditions (Shamhart et al., 2014; Asbun et al., 2005; Tang et al., 2007). However, in the presence of dapa, there was decreased expression of col1a2 in HG\u0026thinsp;+\u0026thinsp;P. Interestingly, dapa influenced the expression col1a2 in HG, suggesting that dapa plays a role in CFs collagen expression, especially in diabetic states. The discrepancy in expression between HG and HGP could be due to the presence of palmitate in the HG\u0026thinsp;+\u0026thinsp;P media. Furthermore, in the absence of dapa, there was a significant increase in CAMK2D expression in HG\u0026thinsp;+\u0026thinsp;P (but decrease in HG) conditions, however, no significant improvement was observed when treated with dapa, suggesting that dapa did not influence the CAMK2D expression. To the best of our knowledge, this is the first study that would explore the influence of dapa on CAMK2D gene expression in CFs in diabetic media conditions.\u003c/p\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eSTUDY LIMITATIONS\u003c/h2\u003e \u003cp\u003eIn performing the experiments, some limitations were encountered. First, contamination of the media (which resulted in the death of most cells) and repression of cell growth (for the ones that eventually survived when media was supplemented with growth factor and insulin) was an issue encountered. For this reason, there were not sufficient cells with which to perform all the experiments, especially with the same cell line. Thus, by filter-sterilizing the media using a syringe and 0.2um filter and optimizing the growth factor and insulin supplemented in the media, this could be overcome. Again, CFs from both rat humans were subjected to different experimental conditions, and this hampered on statistical inferences. Secondly, there was no enough time, and samples to check the efficiency of the primers. Even though qPCR worked, it is necessary to first check the efficiency of primers and optimize where necessary, since it\u0026rsquo;s the first time these primers would be used in the lab. Contamination and degradation of RNA may have impacted the results of the gene analysis, as during the extraction process, samples were not kept on ice. Lastly, as HcFbs were used at high passage number (P9), this could have affected the outcome of the result as high passages affect cell properties.\u003c/p\u003e \u003c/div\u003e"},{"header":"CONCLUSSION","content":"\u003cp\u003eDespite the limitations encountered, our study has demonstrated that the concentration of glucose and dapa used in the in vitro experiment was able to potentially affect CFs function, but not phenotype as neither glucose nor the drug brought about any phenotypic alterations of the CFs studied. Without dapa treatment, CFs proliferated more, but migrated slowly in diabetic media conditions, and these effects were reversed following treatment with dapa. Thus, dapa has a potential in wound healing. Also, dapa has been shown to sufficiently enhance Ca\u003csup\u003e2+\u003c/sup\u003e mobilization in the ER of HG cultured cells, as lower mobilization could be detrimental to the cells, however, its role in CFs is not fully understood. While diabetic media condition increased col1a2 gene expression, this expression was impaired in the presence of dapa. The study further concludes that dapa showed no effect on CAMK2D expression in high glucose media. The implication of these findings suggests that dapa could be a potentially therapeutic agent in treating HF as it has shown to improve wound healing (via the scratch assay) and proliferation of cells.\u003c/p\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eFUTURE EXPERIMENT\u003c/h2\u003e \u003cp\u003eSince CFs were not verified in vivo, further investigation into the effect of diabetic media conditions on CFs should be performed using in vivo study, as this will establish strong evidence of the impact of dapa on diabetic CFs. There is need to specifically use CF from HFpEF from patients or models, at earlier passages to investigate the potency of dapa. Also, by using the MTT Assay, it will be possible to check for cell viability and compare how it relates with cell phenotype. α smooth muscle actin (SMA) in myofibroblasts should be assessed, possibly via immunofluorescence staining to ascertain its expression in the possible change from fibroblasts to myofibroblasts.\u003c/p\u003e \u003c/div\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eALBARR\u0026Aacute;N, O. G., \u0026amp; AMPUDIA-BLASCO, F. J., 2013. Dapagliflozin, the first SGLT-2 inhibitor in the treatment of type 2 diabetes. \u003cem\u003eMedicina clinica\u003c/em\u003e, \u003cstrong\u003e141\u003c/strong\u003e\u003cem\u003e(2)\u003c/em\u003e, pp.36\u0026ndash;43. 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Myocardial Stiffness in Patients with Heart Failure and a Preserved Ejection Fraction: Contributions of Collagen and Titin. \u003cem\u003eCirculation (New York, N.Y.); Circulation.\u003c/em\u003e \u003cstrong\u003e131\u003c/strong\u003e(14), pp.1247\u0026ndash;1259. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1161/CIRCULATIONAHA.114.013215\u003c/span\u003e\u003c/span\u003e.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Glasgow Caledonian University","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Heart failure, diabetic, dapagliflozin, cardiac fibroblasts, left ventricle","lastPublishedDoi":"10.21203/rs.3.rs-5461630/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5461630/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHeart failure (HF) is a disease caused by defects in myocardial structure and function which results to impaired ventricular filling of the ejection of blood, and mostly due to reduced myocardial function of the left ventricle, impairment of the myocardium, endocardium, great vessels, pericardium, heart valves, either in combination or alone, presenting symptoms which include fatigue, oedema, shortness of breath, abdominal distention and right hypochondrial pain. HF, which is classed into HF with reduced ejection fraction (HFrEF) and HF with preserved ejection fraction (HFpEF) has been linked to elevated glucose levels seen in diabetic patients, which is also linked to increased fibrosis which impacts on myocardial function. Unfortunately, no effective drug is yet developed for the treatment HFpEF.\u003c/p\u003e \u003cp\u003eIn the current study, I investigated the effect of dapagliflozin (dapa) on cardiac fibroblasts (CFs) physiology in diabetic media conditions using rat cardiac fibroblasts (RcFbs) and human cardiac fibroblasts (HcFbs). Experiments were performed to study the effects of elevated glucose with/without dapa on gene expressions of human collagen-1 (col1a2), calcium/calmodulin-dependent protein kinase II delta (CAMK2D) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using quantitative polymerase chain reaction (qPCR), Ca\u003csup\u003e2+\u003c/sup\u003e release and migration and proliferation assays of CFs were assessed using confocal microscopy and countess counter. Our study demonstrates that dapa has a potential in wound healing, and has been shown to increase Ca\u003csup\u003e2+\u003c/sup\u003e mobilization in diabetic cultured cells. Although dapa impaired col1a2 expression in diabetic condition, it showed no significant change on CAMK2D expression in diabetic conditions. The implication of these findings suggests that dapa could be a potentially therapeutic agent in treating HF as it has been shown to improve wound healing (via the scratch assay), increased proliferation of cells and increased col1a2 expression.\u003c/p\u003e","manuscriptTitle":"Effect of dapagliflozin on rat and human cardiac fibroblasts physiology cultured in diabetic media conditions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-18 11:47:22","doi":"10.21203/rs.3.rs-5461630/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d484a80e-90f1-4f55-8e9f-a0f40e559bb9","owner":[],"postedDate":"November 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":40327085,"name":"Cardiac \u0026 Cardiovascular Systems"},{"id":40327086,"name":"Cardiothoracic Surgery"},{"id":40327087,"name":"Clinical Pharmacology"}],"tags":[],"updatedAt":"2024-11-18T11:47:22+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-18 11:47:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5461630","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5461630","identity":"rs-5461630","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}