Red yeast rice-derived MKA ameliorates cardiac hypertrophy in hypertensive rats by inhibiting ERK1/2/c-Fos pathway

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Cardiac hypertrophy is a key pathological process in hypertensive heart failure, 29 yet current antihypertensive therapies do not directly target it. Red yeast rice (RYR), rich in 30 monacolin K β-hydroxy acid (MKA), is known for lipid -lowering effects, but its potential to 31 ameliorate cardiac hypertrophy is unreported. 32 Purpose: To investigate the effects of RYR -derived MKA on cardiac hypertrophy in 33 spontaneously hypertensive rats (SHR) and elucidate its molecular mechanisms. 34

Spontaneously hypertensive rats (SHR) were treated with 0.6% red yeast rice for 8 35 weeks to assess its effects on blood pressure, cardiac function (echocardiography), cardiac 36 hypertrophy and fibrosis (histopathology), and multi -organ toxicity (histopatholo gy). A 37 multigenerational study was conducted to evaluate protective effects in offspring. Network 38 pharmacology and transcriptomic analysis were integrated to predict molecular targets, which 39 were subsequently validated by molecular docking and experiments. 40

Eight-week RYR treatment significantly reduced blood pressure, inhibited cardiac 41 hypertrophy and fibrosis, and improved cardiac function without gender differences. No 42 pulmonary, hepatic, or renal toxicity was observed. Offspring from treated par ents exhibited 43 further reduced hypertrophy upon continued treatment. Mechanistically, MKA bound 44 ERK1/2 with high affinity, inhibiting its phosphorylation and downstream c -Fos expression, 45 thereby downregulating hypertrophy markers. 46

Red yeast ric e improves hypertensive cardiac hypertrophy via MKA -mediated 47 inhibition of the ERK1/2/c -Fos pathway. Its multi -organ safety and transgenerational effects 48 offer a novel dual-therapy strategy for hypertension and cardiac hypertrophy. 49 50

Red yeast rice; MKA; Cardiac hypertrophy; Hypertension; c-Fos; ERK1/2 51 52 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 13, 2026. ; https://doi.org/10.64898/2026.03.10.710945doi: bioRxiv preprint 3 3 Graphic abstract 53 54 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 13, 2026. ; https://doi.org/10.64898/2026.03.10.710945doi: bioRxiv preprint 4 4 Abbreviation 55 ACE Angiotensin converting enzyme, ACEIs Agents acting on the renin -angiotensin system, 56 Ang II Angiotensin II, -MHC Beta-myosin heavy chain, BCL2 B-cell lymphoma-2, c-Fos 57 AP-1 transcription factor subunit , caspase 3 Cysteinyl aspartate specific proteinase 3 , CCBs 58 Calcium channel blockers , Col1 Collagen type 1 , DMEM Dulbecco's modified Eagle's 59 medium, EGFR Epidermal growth factor receptor , ERK1/2 Extracellular signa l-regulated 60 kinase1/2, FBS Fetal bovine serum, FN1 Fibronectin 1, GAPDH Glyceraldehyde 3-phosphate 61 dehydrogenase, H&E Hematoxylin & eosin , HRP Horseradish peroxidase , LDL-C Low-62 density lipoprotein cholesterol, LVEDV Left ventricular end -diastolic volume, LVESV Left 63 ventricular end -systolic volume , LVH Left ventricular hypertrophy , MAP Mean arterial 64 pressures, MAPK Mitogen-activated protein kinases, MKA Monacolin K β-hydroxy acid , 65 MMP9 Matrix metalloprotease 9 , MTOR Mammalian target of rapamycin , non-HDL Non-66 high-density lipoprotein , p-ERK Phosphorylated extracellular signal -regulated kinase , PDB 67 Protein Data Bank , PI3K Phosphatidylinositol‑4,5‑bisphosphate 3‑kinase , PVDF 68 Polyvinylidene fluoride , SHR Spontaneously hypertensive rats , t-ERK Total extracellul ar 69 signal-regulated kinase 70 71 72 73 74 75 76 77 78 79 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 13, 2026. ; https://doi.org/10.64898/2026.03.10.710945doi: bioRxiv preprint 5 5

80 Hypertension is a prevalent chronic condition primarily characterized by elevated systemic 81 arterial blood pressure (Te Riet et al., 2015) . Its global burden is estimated at 874 million 82 patients, with approximately 9.4 million cardiovascular disease deaths annually attributable 83 to the condition (Forouzanfar et al., 2017) . One clinical consequence of hypertension is lef t 84 ventricular hypertrophy and myocardial remodeling, which can precipitate heart failure 85 (González et al., 2018; Yildiz et al., 2020) . Current guidelines recommend antihypertensive 86 medication for treating cardiac hypertrophy in hypertensive patients (Rabi et al., 2020) . 87 Currently available antihypertensive agents include agents acting on the renin -angiotensin 88 system (ACEIs), β -blockers, calcium channel blockers (CCBs), and diuretics. A randomized 89 controlled trial by Leache et al. (2021) found no benefit in cardiovascular event rates or 90 mortality among hypertensive participants with left ventricular hypertrophy when these 91 antihypertensive drugs were added. Co ncurrently, in clinical practice, while these commonly 92 prescribed oral antihypertensive agents alleviate symptoms, they frequently induce adverse 93 reactions: ACEIs may cause irritating dry cough and angioedema (AlQudah et al., 2020) ; β-94 blockers may exacerbate atrioventricular block and offer no benefit to asthma patients 95 (Argulian et al., 2019) ; CCBs may induce headaches and ankle oedema in certain patients; 96 high-dose diuretics affect water, electrolyte, and metabolic balance (Blowey, 2016; Burnier et 97 al., 2019) . Given these limitations and the absence of interventions specifically targeting 98 myocardial hypertrophy, the necessity for exploring novel therapeutic approaches is 99 highlighted. 100 In recent years, an increasing body of research has demonstr ated that traditional Chinese 101 medicine can protect vascular endothelial function while lowering blood pressure, thereby 102 offering unique advantages in preventing and treating complications of hypertension and 103 improving patients' quality of life. Red yeast r ice has been widely employed in traditional 104 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 13, 2026. ; https://doi.org/10.64898/2026.03.10.710945doi: bioRxiv preprint 6 6 Chinese medicine, health supplements, and food products in China. For instance, a 105 randomized controlled trial in myocardial infarction patients demonstrated that Xuezhikang 106 capsules containing red yeast rice significantly reduced the risk of major coronary events by 107 45% (from 10.4% in the placebo group to 5.7%), decreased cardiovascular mortality by 30%, 108 and reduced all -cause mortality by 33%. while improving total cholesterol, low -density 109 lipoprotein cholesterol (LDL-C), and triglyceride levels with good tolerability (Lu et al., 110 2008). Long -term administration of Lipid -Kang reduces coronary heart disease events and 111 improves lipid profiles in Chinese myocardial infarction patients, demonstrating safety and 112 efficacy (Lu et al., 2008) . Chromatographic analysis further i dentifies monacolin K β -113 hydroxy acid (MKA) as the primary active component of red yeast rice (Higa et al., 2021; 114 Huang et al., 2006) . Daily intake of low -dose red yeast rice containing 2mg MKA 115 significantly reduced LDL-C, total cholesterol, apolipoprotein B, and blood pressure levels in 116 patients with mild dyslipidemia, without muscle or hepatic/renal toxicity, indicating its 117 potential for cardiovascular risk management (Minamizuka et al., 2021) . Another study also 118 found that A 60-day treatment with low-dose MKA dietary supplements significantly reduced 119 LDL-C and total cholesterol by 15.6% and 15.3%, respectively, in patients with moderate -to-120 severe dyslipidemia, with non -HDL cholesterol decreasing by 35.4%, without inducing 121 serious adverse events (Benjian et al., 2022). However, no reports exist on whether red yeast 122 rice directly targets cardiomyocytes to improve hypertensive cardiac hypertrophy. 123 This study utilizes animal experiments to clarify red yeast rice's efficacy in reducing 124 hypertension and improving hypertensive cardiac hypertrophy, conducting multidimensional 125 evaluations of its toxic side effects (male and female individuals, parental and off spring 126 generations, treatment duration, multiple organs). Network pharmacology combined with 127 transcriptomics analysis identifies MKA-derived targets and molecular mechanisms from red 128 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 13, 2026. ; https://doi.org/10.64898/2026.03.10.710945doi: bioRxiv preprint 7 7 yeast rice, with animal and cellular experiments validating MKA's molecul ar mechanisms for 129 improving hypertensive myocardial hypertrophy. 130 2. Materials and Methods 131 2.1 Animal Models 132 Wista rats (control) and spontaneously hypertensive rats (SHR) were purchased from 133 ©Charles River. The animal research protocol was reviewed and approved by the XZHMU 134 Animal Care and Use Committee (Ethical Approval No. 202208S058), with all procedures 135 conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals. 136 2.2 Experimental Design 137 Red yeast rice , predominated by the M KA in form of micropowder, purchased from 138 Tongjuntan®, Hangzhou, Zhejiang, China. The total MKA content is around 3.5 mg in per 139 gram red yeast rice (Higa et al., 2021; Huang et al., 2006) . Five animal experiments were 140 designed for in vivo experiments (Figure. S1, n=5 for each group). 141 2.3 Cardiac echocardiography 142 SHR were administered 3% pentobarbital sodium (30 mg/kg) via intraperitoneal injection. 143 Apply coupling agent to the depilated thoracic area. Assessment of cardiac function through 144 transthoracic echocardiography using VINNO 6LAB ultrasound equipment (VINNO 145 Technology Co., Ltd, C hina). Use M -type long axis view to evaluate left ventricular end 146 diastolic volume (LVEDV) ，left ventricular end systolic volume (LVESV) ，left ventricular 147 ejection fraction (EF) and left ventricular fractional shortening (FS). This process adopts a 148 double-blind experiment. 149 2.4 Hemodynamic analysis 150 Blood pressure was measured through Left common carotid artery catheterization , and the 151 amplitude and frequency of the waveform were observed to confirm normality. 152 2.5 Harvest of tissue samples 153 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 13, 2026. ; https://doi.org/10.64898/2026.03.10.710945doi: bioRxiv preprint 8 8 After hemodynamic analysis, rats were then sacrificed. T he hearts, lungs, livers and kidneys 154 are harvested to do the next experiments. 155 2.6 Hematoxylin & eosin and Masson staining 156 Hematoxylin and eosin (H&E) and Masson staining were performed as our previous studies 157 (Cui et al., 2025; Tan et al., 2020) . Each slice was observed and photographed using 158 microscope lenses at 1.25X and 20X magnification, respectively (Olympus B51167 Japan). 159 2.7 Cardiac Tissue RNA Extraction and Transcriptomics 160 Total RNA was extracted from cardiac tissue of 8-week-old male SHR fed a standard diet for 161 4 weeks (SHR group), 8 -week-old adult SHR fed red yeast rice diet for 4 weeks (SHRD 162 group), and 3-week-old SHR fed red yeast rice diet for 9 weeks (SHR-D group) using TRIzol 163 reagent (ThermoFisher Scientific, U SA) (n=4 per group). Library preparation and RNA 164 sequencing were performed by GDIOD (Guangzhou, China). 165 2.8 Network pharmacology analysis and molecular docking analysis 166 Network pharmacology analysis and molecular docking analysis were performed as our 167 previous study (Gilley et al., 2009; Nguyen et al., 2020; Tan et al., 2023). 168 2.10 Edu Analysis 169 H9c2 cells were procured from Shanghai Fuheng Biotechnology Co., Ltd. (Catalogue No. 170 FH1004, China) and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) in 171 a 37°C, 5% CO₂ incubator. H9c2 cells were seeded into 12 -well plates and cultured for 24 172 hours. They were then treated with Angiotensin II (Ang II, 200 nM) and low, medium, or 173 high concentrations of MKA (5 M, 50 M, 500 M) for 24 hours. Subsequently, Edu 174 analysis was performed using EdU Apollo 567 In Vitro Kit (Absin (Shanghai) Biotechnology 175 Co., Ltd., abs50050-200T) according to the manufacturer's instructions. Images were 176 observed and captured under a Leica fluorescence microscope. EdU -positive cells were 177 quantified from images using Image J software. 178 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 13, 2026. ; https://doi.org/10.64898/2026.03.10.710945doi: bioRxiv preprint 9 9 2.11 TUNEL Analysis 179 Cells were fixed with 4% paraf ormaldehyde, permeabilized with 0.3% Triton X -100/PBS 180 (VICMED) for 5 minutes, and processed using the One -step TUNEL In Situ Apoptosis Kit 181 (Red, Elab Fluor® 594) (Elabscience Co., Ltd., Wuhan, E -CK-A322) according to the 182 manufacturer's instructions. Images were observed and captured under a Leica fluorescence 183 microscope. Obtained images were analyzed by ImageJ. 184 2.12 Western blotting analysis 185 Total protein was extracted from H9c2 and rat heart tissues using RIPA lysis buffer 186 containing PMSF and phosphatase inhibitors. Proteins (20 –50 g/well) were electrophoresed 187 on a 10% sodium dodecyl sulphate (SDS) polyacrylamide gel and transferred to 188 polyvinylidene fluoride (PVDF) membrane as described in our prev ious studies (Tan et al., 189 2020; Tan et al., 2023) . Protein bands were visualized with ECL reagent (Vazyme Biotech, 190 Nanjing, China) and analyzed by ImageJ Software. 191 2.13 Statistical Analysis 192 Image results obtained from experiments were analyzed and processed using ImageJ software. 193 Statistical analysis was performed using GraphPad Prism software (version 10.0; GraphPad, 194 USA). Data are presented as mean ± standard error (Mean ± SEM). Comparisons between 195 two groups were performed using t -tests (and non -parametric tests), whil e comparisons 196 among multiple groups were conducted using one -way analysis of variance (ANOVA) 197 followed by Tukey's multiple comparison test. Statistical significance was set at p < 0.05. 198 More details are available in the Online Supplement. 199 3 Results 200 3.1 Manifestations of hypertension and cardiac hypertrophy in SHR 201 Following standard housing for 8, 12, and 16 weeks, systolic pressures and mean arterial 202 pressures (MAP) were markedly elevated in SHR ( Figure 1A,B ). SHR exhibited 203 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 13, 2026. ; https://doi.org/10.64898/2026.03.10.710945doi: bioRxiv preprint 10 10 significantly higher heart-to-body weight ratios and lung -to-body weight ratios compared to 204 Control ( Figure 1C,D ). HE and Masson staining revealed markedly larger myocardial cell 205 areas and fibrosis areas in SHR ( Figure 1E -G). Echocardiography results demonstrated a 206 time-dependent reduction in both the ejection fraction and shortening velocity of the SHR 207 heart ( Figure 1H -J). These findings indicate that 8 -weeks SHR have exhibited significant 208 hypertension, with myocardial hypertrophy and fibrosis progressively worsening over time. 209 3.2 Red yeast rice significantly lowers blood pressure and alleviates cardiac remodeling 210 in SHR 211 Following an 8 -week period of feeding 8 -week-old SHR chow diets (control) or chow diets 212 containing 0.1%, 0.3%, and 0.6% red yeast rice, systolic pressures and MAP decreased 213 markedly in 0.6% red yeast rice group compared with control group ( Figure 2A,B ), the 214 heart-to-body weight ratio and lung -to-body weight ratio were also significantly reduced 215 (Figure 2C, D ). HE and Masson staining revealed that 0.6% red yeast rice inhibited 216 cardiomyocytes hypertrophy and reduced fibrotic area ( Figure 2E -G). Echocardiography 217 revealed that the 0.6% red yeast rice treatment group exhibited significantly increased EF% 218 and FS% ( Figure 2H-J). Collectively, the cardioprotective effects of red yeast rice on SHR 219 exhibited a concentration -dependent pattern, with the 0.6% concentration demonstrating 220 optimal efficacy. 221 Additionally, the therapeutic effects of 0.6% red yeast rice after 4 -week versus 8-week 222 treatment were compared. Results indicated no change in systolic pressure or MAP after 4 223 weeks, whereas they were markedly reduced after 8 weeks ( Figure S2A,B ). However, the 224 cardiac weight -to-body weight ratio had already decreased significantly af ter 4 weeks of 225 treatment ( Figure S2C,D ). HE and Masson staining revealed that cardiomyocytes 226 hypertrophy and fibrosis were inhibited after 8 weeks of treatment ( Figure 2E -G). This 227 suggests that red yeast rice alleviates cardiac remodeling prior to blood pr essure reduction. 228 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 13, 2026. ; https://doi.org/10.64898/2026.03.10.710945doi: bioRxiv preprint 11 11 Consequently, we hypothesize that red yeast rice may directly target cardiac cells to improve 229 myocardial remodeling. 230 3.3 Effects of Red Yeast Rice on blood pressure and cardiac remodeling in male and 231 female SHR 232 It is well established th at cardiovascular diseases exhibit sex differences. To investigate 233 whether red yeast rice exerts distinct effects in male and female SHR, we selected age -234 matched male and female rats as subjects and fed them a 0.6% red yeast rice diet for 8 weeks. 235

demonstrated significant reductions in systolic pressure and MAP in both female and 236 male red yeast rice -treated groups ( Figure. 3A,B ). Heart-to-body weight ratio and lung-to-237 body weight ratio decreased ( Figure 3C,D ), whilst cardiac hypertrophy and fibrosis were 238 suppressed ( Figure 3E-G). No gender -specific differences in red yeast rice treatment 239 efficacy were observed. 240 Echocardiographic findings revealed significantly increased EF% and FS% in both female 241 and male red yeast rice -treated groups ( Figure 3H-J), alongside markedly reduced LVEDV 242 and LVSDV ( Figure 3K,L). The therapeutic effects on cardiac function showed no gender -243 specific differences. 244 To evaluate potential sex -specific toxicological differences in vital organs, histological 245 staining analysis was performed on lung, liver, and kidney tissues. Results demonstrated that 246 red yeast rice significantly ameliorated fibrosis and inflammation in lung, liver, and kidney 247 tissues of both male and female SHR, with no observed sex differences (Figure S3). No other 248 toxicological alterations were detected. 249 3.4 Effects of Red Yeast Rice on blood pressure and cardiac remodeling in progeny SHR 250 Extensive research indicates that the health status, diet, and medication of progenitors 251 influence offspring health. However, whether administering red yeast rice to progenitor SHR 252 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 13, 2026. ; https://doi.org/10.64898/2026.03.10.710945doi: bioRxiv preprint 12 12 may cause adverse effects in their progeny, or whether it confers benefits to offspring blood 253 pressure and cardiac function, warrants investigation. 254 Progenitors were divided into red yeast rice -treated and standard diet groups. Their offspring 255 were categorized as: neither progenitors nor offspring treated (SHR); progenitors untreated, 256 offspring treated (SHR+D); progenitors treated, offspring untreated (SHRD+N); and both 257 progenitors and offspring treated (S HRD+D). At eight weeks of age, offspring underwent 258 hemodynamic testing and tissue section staining. The results revealed significantly reduced 259 systolic pressure and MAP across all treatment groups compared to SHR, with SHRD+D and 260 SHRD+N demonstrating great er reductions than SHR+D ( Figure 4A -B). The SHRD+D 261 group exhibited a marked decrease in the heart -to-body weight ratio ( Figure 4C-D). HE and 262 Masson staining revealed the most pronounced reduction in myocardial cell area and fibrotic 263 area within the SHRD+D group (Figure 4E-G). 264 Echocardiography demonstrated significant increased EF% and FS% alongside reduction in 265 LVEDV in the SHRD+D group compared to other groups ( Figure 4H -J). These findings 266 suggest parental drug administration confers beneficial effects on offspring blood pressure 267 and cardiac function, with sustained treatment yielding superior outcomes. 268 Furthermore, to assess potential adverse effects on other vital organs in offspring, histological 269 staining analyses were conducted on lung, liver, and kidne y tissues. Results demonstrated 270 that red yeast rice significantly ameliorated fibrosis and inflammation in the lungs, liver, and 271 kidneys of offspring SHR, with the SHRD+D group exhibiting the most pronounced effects 272 (Figure S4). No other toxicological alterations were observed. 273 3.5 Transcriptomics combined with network pharmacology to identify the molecular 274 mechanism 275 To further investigate the molecular mechanism by which red yeast rice ameliorates 276 myocardial hypertrophy, we employed transcriptomics combin ed with network 277 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 13, 2026. ; https://doi.org/10.64898/2026.03.10.710945doi: bioRxiv preprint 13 13 pharmacology to screen the targets and signaling pathways of MKA (3 .5mg/g), the primary 278 active component of red yeast rice. The two -dimensional molecular structure of MKA is 279 depicted in Fig. 5A. Following target prediction for MKA using the Swiss-Target Prediction 280 database, 100 potential targets were identified. Extensive research indicates that MKA can 281 significantly reduce LDL-C. However, there are no reports indicating whether MKA directly 282 targets cardiomyocytes to improve hypertensive cardiac hypertrophy. 283 Using the keyword "hypertension", 12,116 hypertens ion-related targets were obtained from 284 the GeneCard database. A total of 635 target genes with a score ≥8 were selected. The Venn 285 diagram revealed 90 common target genes between MKA and h ypertension (Fig. 5A). Using 286 the keyword "myocardial hypertrophy", 4,189 targets associated with myocardial 287 hypertrophy were obtained. A total of 1,719 target genes with a score ≥8 were selected. The 288 Venn diagram revealed 71 common target genes between MKA and myocardial hypertrophy 289 (Figure 5A). Cytoscape analysis identified the top 10 potential targets ( Figure 5B): EGFR, 290 MAPK3, MMP9, SRC, MAPK1, BCL2, HSP90AA1, HSP90AB1, PIK3CA, and MTOR. KEGG 291 pathway analysis revealed key signaling pathways including EGFR, PI3K -Akt, MAPK, and 292 so on ( Figure 5C). Volcano plots and heatmaps derived from transcriptomic analysis reveal 293 that differentially expressed genes upregulated or downregulated in the S HR group compared 294 to the SHR -D/SHRD group ( Figure 5D,E,G,H ). Furthermore, the number of differentially 295 expressed genes markedly increased when comparing the SHR group to the SHR -D group. It 296 is evident that as the duration of drug administration increased, the number of differentially 297 expressed genes in rats at the same gestational age rose, and the magnitude of transcriptional 298 changes became more pronounced. 299 Subsequently, KEGG analysis of the differentially expressed genes yielded the top 20 300 signaling pathways (Figure 5F, I ). Integrating data from both the SHR and SHRD groups 301 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 13, 2026. ; https://doi.org/10.64898/2026.03.10.710945doi: bioRxiv preprint 14 14 and the SHR and SHR -D groups revealed that the MAPK pathway was consistently 302 implicated in both sets of analyses. 303 Therefore, we hypothesize that MKA inhibits hypertension and cardiac hy pertrophy by 304 targeting MAPK1 (ERK2) and MAPK3 (ERK1). Binding energy is considered robust at ≤ -305 5.0 kcal/mol and exceptionally strong at ≤ -7.0 kcal/mol. Molecular docking results 306 demonstrate that MKA exhibits strong binding affinity with both MAPK1 (ERK2) and 307 MAPK3 (ERK1) proteins (affinity values of -9.41 kcal/mol and -9.14 kcal/mol, Figure 5J). 308 These findings indicate that MKA functions by forming stable complexes with common 309 target genes of MAPK1 (ERK2) and MAPK3 (ERK1). 310 3.6 Red Yeast Rice targets ERK1/2 to downregulate c -Fos and improve cardiac 311 hypertrophy markers in SHR 312 The effects of red yeast rice treatment on SHR were investigated using Western blot analysis. 313 Red yeast rice (0.6%) reduced expression of myocardial fibrosis markers Col1α and FN1, 314 cardiac hypertrophy protein β-MHC, and apoptotic protein Caspase 3 (*p < 0.05, **p < 0.01, 315 ***p < 0.001, ****p < 0.0001, Figure 6A -E). To verify that MKA can act on MAPK1 316 (ERK2) and MA PK3 (ERK1) to inhibit hypertension and cardiac hypertrophy, we checked 317 the level of p -ERK1/2 in the cardiac tissue between SHR group and SHRD group. Western 318 blot results demonstrated that after eight weeks of red yeast rice diet administration, the 319 expression levels of p-ERK1/2 in cardiac tissue were markedly suppressed (Figure 6F,G). 320 Transcriptomic analysis further revealed that expression of the transcription factor c -Fos was 321 significantly downregulated in the treatment group ( Figure 6H ). Western blot res ults 322 confirmed that red yeast rice significantly downregulated the expression of c -Fos ( Figure 323 6G). 324 Previous studies have demonstrated that nuclear translocation of phosphorylated ERK1/2 325 promotes the transcriptional activity c -Fos (Ren et al., 2023) . We therefore hypothesize that 326 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 13, 2026. ; https://doi.org/10.64898/2026.03.10.710945doi: bioRxiv preprint 15 15 red yeast rice, through its primary active component MKA, targets ERK1/2 by inhibiting its 327 phosphorylation, suppresses c-Fos expression, ultimately inhibiting downstream hypertrophic 328 gene expression and thereby mitigating cardiac hypertrophy in SHR. 329 3.7 MKA reverses Ang II-induced cardiomyocyte injury by inhibiting ERK1/2/c-Fos 330 Ang II treatment of H9c2 cardiomyocytes is widely employed to simulate an in vitro model 331 of hypertensive cardiac hyper trophy (Tham et al., 2015) . Following 24 -hour treatment of 332 H9c2 cardiomyocytes with 200 nM A ngiotensin II, EDU cell staining analysis revealed that 333 Ang II significantly suppressed the viability of H9c2 cardiomyocytes ( Fig. 7A). Concurrent 334 administration of 1 M, 5 M, and 10 M MKA for 24 hours revealed that the 5 M group 335 exhibited significantly reversed cell viability, indistinguishable from the control group ( Fig. 336 7A,B). TUNEL staining analysis revealed that Ang II significantly induced apoptosis in H9c2 337 cardiomyocytes, while 5 M MKA markedly suppressed Ang II -induced apoptosis ( Figure 338 7A,C). 339 Western blot analysis validated MKA's effects on hypertrophy and apoptosis -related proteins 340 in AngII-treated H9c2 cardiomyocytes. Ang II significantly upregulated β-MHC and caspase-341 3 expression ( Figure 7D -H), which was reversed following 5 M MKA treatment . 342 Furthermore, Ang II markedly increased p -ERK1/2 and c -Fos expression, which was 343 completely reversed by 5 M MKA treatment ( Figure 7F,H). We therefore hypothesize that 344 MKA downregulates c -fos expression by targeting and inhibiting phospho -ERK1/2, leading 345 to significant suppression of β -MHC and caspase -3 expression and ultimately inhibiting 346 myocardial hypertrophy. 347 348 4. Discussion 349 Left ventricular hypertrophy is the most common clinical manifestation of hypertension, 350 resulting from cardiac cellular remodeling and enlargement. It constitutes a potent risk factor 351 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 13, 2026. ; https://doi.org/10.64898/2026.03.10.710945doi: bioRxiv preprint 16 16 for cardiovascular events and all-cause mortality, particularly arrhythmias and sudden cardiac 352 death (Okin et al., 2013). Compared with individuals without left ventricular hypertrophy, the 353 presence of hypertrophy increases the risk of ventricular arrhythmias by 5.5% and that of 354 sudden cardiac death by 1.2%, ventricular tachycardia/ventricular fibrillation by 2.8-fold (Tin 355 et al., 2002) , and atrial fibrillation by 40 –50% (Sirtori, 2014) . Statins are commonly used 356 adjunctive medications for hypertension management. Beyond cholesterol reduction, statins 357 lower blood pressure through multiple mechanisms, including improved vascular endoth elial 358 function, reduced vascular inflammation, and decreased sympathetic nervous system activity. 359 Additionally, statins may improve insulin resistance, thereby contributing to blood pressure 360 reduction (Alonso et al., 2019) . Our research also found that red yeast rice containing MKA 361 can alleviate hypertensive cardiac hypertrophy. 362 Given the marked differences in hypertension between genders, particularly regarding age of 363 onset and medication use, gender represents another crucial determinant requiring assessment. 364 A large meta-analysis of 46 population -based studies across 22 countries, involving 123,143 365 men and 164,858 women aged 20 –59 years, indicated that hypertensive w omen were 1.33 366 times more likely than men to receive drug therapy, with a higher propensity for diuretic use, 367 whereas men more frequently utilized beta -blockers, ACE inhibitors, and calcium channel 368 blockers (Busjahn et al., 1996) . Among patients receiving monotherapy for hypertension, 369 women were less likely than men to b e prescribed beta -blockers, calcium channel blockers, 370 or ACE inhibitors (Os et al., 1994) . One possible explanation for these findings may be 371 gender differences in side effects. Women are more prone to coughing when treated with 372 ACE inhibitors compared to men (Agrawal and Wenger, 2020) . The results of this study 373 indicate no significant gender differences in the therapeutic efficacy of red yeast rice, thereby 374 providing a research basis for the potential administration of identical medications across 375 different genders. 376 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 13, 2026. ; https://doi.org/10.64898/2026.03.10.710945doi: bioRxiv preprint 17 17 377 Research indicates that parental health profoundly and persistently influences numerous 378 aspects of offspring development in utero and subsequent physiological and metabolic 379 outcomes (Bramham et al., 2014) . Among women with chronic hypertension, fetal growth 380 restriction is observed in 10 -20% of pregnancies (Kattah and Garovic, 2013) , higher rates of 381 preterm birth before 37 weeks gestation, lower birth weight, neonatal unit admissions, and 382 perinatal mortality (McCowan et al., 1996) . Two primary oral antihypertensive agents are 383 available for pregnant women: labe talol and nifedipine (Tomar et al., 2024) . However, 384 neither of these drugs can directly improve cardiac hypertrophy . Our study indicated that 385 maternal consumption of red yeast rice during pregnancy does not impair fetal growth and 386 development, while conferring cardiac benefits to the offspring postnatally. Concurrently, 387 research demonstrates that beyond maternal health influencing the fetus, the paternal dietary 388 patterns and health status prior to conception exert significant effects on offspring 389 metabolism (Aiken et al., 2016) . For instance, paternal overweight at conception doubles 390 offspring obesity risk and impairs metabolic health (Xu et al., 2021) . Acute high -fat diet 391 feeding or genetically induced mitochondrial dysfunction in male mice resulted in impaired 392 glucose homeostasis in male offspring (Aiken et al., 2016) . This suggests that the health of 393 hypertensive fathers also affects progeny. Consequently, this study concurrently administered 394 red yeast rice intervention to hypertensive male rats to observe effects on offspring. Results 395 indicate that pre -pregnancy red yeast rice intervention in both parental rats confers cardiac 396 benefits. Continued postnatal adminis tration to offspring further enhances these effects, 397 providing experimental evidence for the safety of red yeast rice consumption during pre -398 pregnancy and lactation in hypertensive parents and its associated benefits for offspring. 399 Increasing evidence indi cates that the MAPK/ERK1/2 signaling pathway is one of the key 400 pathways contributing to cardiac hypertrophy (Dash et al., 2001) , and its targeted inhibition 401 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 13, 2026. ; https://doi.org/10.64898/2026.03.10.710945doi: bioRxiv preprint 18 18 can ameliorate hypertrophy. Song et al. (2024) found that artemisinin alleviated isoproterenol 402 induced cardiac hypertrophy through inhibiting the ERK1/2 signaling pathways. In clinical 403 trials, the MAPK pathway has seen preliminary therapeutic application. Flesch et al. (2001) 404 employed mechanical unloading of the heart via a left ventricular assist device, 405 demonstrating that mechanical unloading of fa iling hearts improves cardiac function through 406 differential regulation of the MAPK pathway. Babu et al. (2000) utilized deoxyepinephrine-407 induced hypertrophic cardiomyocytes to demonstrate that phosphorylated ERK1/2 binds to c -408 Fos, ultimately leading to excessive cardiomyocyte proliferation. These findings collectively 409 highlight the substantial potential of the ERK1/2/c -Fos pathway in ameliorating myocardial 410 hypertrophy. Meantime, our study clarified that red yeast rice derived MKA attenuates 411 cardiac hypertrophy in SHR by targeting the ERK1/2/c -Fos signaling axis. This mechanism 412 transcends the limitations of traditional red yeast rice research, which has primarily focused 413 on lipid -lowering effects, offering fresh perspectives on its cardiovascular protective 414 functions. 415 This study remains subject to several limitations. Firstly, mechanism validation was confined 416 to rat models and H9c2 cells; the conservation of the ERK1/2/c -Fos pathway requires 417 verification in primary human cardiomyocytes or organoid models. Secondly, the mechanism 418 underpinning the tr ansgenerational protective effect remains unclear, necessitating 419 investigation into whether epigenetic regulation (such as DNA methylation or non -coding 420 RNAs) is involved. Thirdly, long -term clinical trials are required to evaluate red yeast rice's 421 efficacy in reversing human left ventricular hypertrophy and its impact on cardiovascular 422 event endpoints. Fourthly, as a traditional Chinese medicine, our study focused solely on the 423 action and molecular mechanisms of MKA, the primary component of red yeast rice , without 424 investigating the effects of other constituents. Fifthly, the long -term safety profile of this 425 herbal medicine warrants further investigation. Sixthly, future research should examine the 426 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 13, 2026. ; https://doi.org/10.64898/2026.03.10.710945doi: bioRxiv preprint 19 19 combined effects of red yeast rice with other cardiovascula r medications to optimize 427 therapeutic regimens. 428

429 This study confirms that MKA derived from red yeast rice improves hypertensive cardiac 430 hypertrophy by blocking c -Fos-mediated hypertrophic gene expression through targeted 431 binding to ERK1/2 and inh ibiting its phosphorylation ( Figure 8 ). Its demonstrated 432 therapeutic stability in animal models, multi -organ safety profile, and transgenerational 433 protective effects provide substantial support for red yeast rice as a dietary intervention 434 strategy for hypertensive cardiac hypertrophy. 435 436 Competing interests 437 The authors declare that there is no conflict of interest. 438 439 CRediT authorship contribution statement 440 Rubin Tan : Writing – original draft, Writing – review & editing, Software, Methodology, 441 Investigation, F ormal analysis, Data curation, Conceptualization , Supervision, 442 Conceptualization, Project administration, Funding acquisition . Dongqi Yang : Writing – 443 original draft, Software, Formal analysis, Data curation. Kuntao Liu: Writing – original draft, 444 Software, Formal analysis, Data curation. Jia Liu: Formal analysis, Data curation, Writing – 445 original draft. Na Li: Formal analysis, Data curation, Writing – original draft. Jie Cui: Data 446 curation. Xiaoqiu Tan : Supervision, Funding acquisition , Writing – review & ed iting. 447 Qinghua Hu: Supervision, Project administration, Funding acquisition , Writing – review & 448 editing. Chunxiang Zhang: Supervision, Project administration, Funding acquisition , 449 Writing – review & editing. All authors read and approved the final manuscript. 450 451 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 13, 2026. ; https://doi.org/10.64898/2026.03.10.710945doi: bioRxiv preprint 20 20 Acknowledgments 452 This work was supported by grants from the National Natural Science Foundation of China 453 (81700055, U23A20398, 82030007, 82270334 and 82470323), Noncommunicable Chronic 454 Diseases-National Science and Technology Major Project (202 4ZD0537707), the Science 455 and Technology Department of Sichuan Province (2025ZNSFSC0052), and the Natural 456 Science Foundation of Jiangsu Province (BK20160229) , and Southwest Medical University 457 Talent Launch Project (25110200005). The authors would like to ex press their gratitude to 458 Kexue Li and Jingxia Kuai at Xuzhou Medical University for their assistance in our research. 459 460 Data Availability Statement 461 The data that support the findings of this study are available from the corresponding author 462 upon reasonable request. 463 464

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Heart -to-608 body weight ratio ( C) and lung -to-body weight ratio ( D) in 16 -week Control rats versus 8 -, 609 12-, and 16 -week SHR rats. HE and Masson's staining ( E), myocardial cell cross -sectional 610 area ( F), and fibrotic area ( G) in 16 -week Control versus 8 -, 12 -, and 16 -week SHR. 611 Representative cardiac ultrasound ( H), ejection fraction (EF%, I), and fractional shortening 612 (FS%, J) in 16-week Control versus 8-, 12-, and 16-week SHR. Data are presented as mean ± 613 SE, comparisons between two groups were performed using t-tests (and non-parametric tests), 614 while comparisons among multiple grou ps were conducted using one -way analysis of 615 variance (ANOVA) followed by Tukey's multiple comparison test. (*p<0.05, **p<0.01, 616 ***p<0.001, ****p<0.0001. n=3-5). 617 618 Figure 2 Cardiac structure and function in SHR fed red yeast rice diets at different 619 concentrations. Systolic pressure ( A), mean arterial pressure (MAP, B), Heart -to-body 620 weight ratio (C), and lung-to-body weight ratio (D) in SHR fed chow diet (SHR) versus SHR 621 fed 0.1%, 0.3%, and 0.6% red yeast rice diets (SHR+0.1%D, SHR+0.3%D, SHR+0.6%D). 622 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 13, 2026. ; https://doi.org/10.64898/2026.03.10.710945doi: bioRxiv preprint 27 27 HE and Masson's staining (E), myocardial cell cross-sectional area (F), and fibrotic area (G). 623 Representative cardiac ultrasound ( H), ejection fraction (EF%, I), and frac tional shortening 624 (FS%, J) in SHR, SHR+0.1%D, SHR+0.3%D, and SHR+0.6%D. Data are presented as mean 625 ± SE, comparisons between two groups were performed using t -tests (and non -parametric 626 tests), while comparisons among multiple groups were conducted using one -way analysis of 627 variance (A NOVA) followed by Tukey's multiple comparison test. (*p<0.05, **p<0.01, 628 ***p<0.001, n=3-5). 629 630 Figure 3 Cardiac structure and function in SHR fed red yeast rice diets at different 631 gender. Systolic pressure ( A), mean arterial pressure (MAP, B), Heart-to-body weight ratio 632 (C), and lung-to-body weight ratio (D) in female SHR rats fed standard diet (F -SHR), female 633 SHR rats fed red yeast rice diet (F -SHRD), male SHR rats fed standard diet (M -SHR), male 634 SHR rats fed red yeast rice diet (M -SHRD). HE and Masson's sta ining (E), myocardial cell 635 cross-sectional area (F), and fibrotic area (G). Representative cardiac ultrasound (H), ejection 636 fraction (EF%, I), fractional shortening (FS%, J), left ventricular end -diastolic volume 637 (LVEDV, K), and left ventricular end-diastolic volume (LVESV, L) in F-SHR, F-SHRD, M-638 SHR, and M -SHRD. Data are presented as mean ± SE (*p<0.05, **p<0.01, ***p<0.001, 639 n=3-5). 640 641 Figure 4 Cardiac structure and function in SHR fed red yeast rice diets at different 642 offspring. Systolic pressure ( A), mean arterial pressure (MAP, B), Heart -to-body weight 643 ratio (C), and lung-to-body weight ratio (D) in parent SHR fed chow diet produced offspring 644 also fed chow diet (SHR+N); parent SHR fed chow diet produced offspring fed red yeast rice 645 diet (SHR+D); parent SHR fed red yeast rice diet produced offspring also fed red yeast rice 646 diet (SHRD+D); parental SHR fed red yeast rice diet produced offspring SHR fed chow diet 647 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 13, 2026. ; https://doi.org/10.64898/2026.03.10.710945doi: bioRxiv preprint 28 28 (SHRD+N). HE and Masson's staining ( E), myocardial cell cross -sectional area ( F), and 648 fibrotic area (G). Representative cardiac ultrasound (H), ejection fraction (EF%, I), fractional 649 shortening (FS%, J), left ventricular end -diastolic volume (LVEDV, K), and left ventricular 650 end-diastolic volume (LVESV, L) in SHR+N, SHR+D, SHRD+D, and SHRD+N. Data are 651 presented as mean ± SE, comparisons between two groups were performed using t -tests (and 652 non-parametric tests), while comparisons among multiple groups were conducted using one -653 way analysis of variance (ANOVA) followed by Tukey's multiple comparison test (* p<0.05, 654 **p<0.01, ***p<0.001, n=3-5). 655 Figure 5 Network pharmacology combined with transcriptomics identifies the 656 molecular mechanism of MKA action in red yeast rice. A. Venn diagram of MKA targets 657 and genes associated with hypertension and myocardial hypertrophy. B. Top 10 central genes 658 among 71 common target genes for MKA and myocardial hypertrophy. C. KEGG pathway 659 analysis results for MKA -targeted cardiac hypertrophy genes. D. Volcano plot of 660 differentially expressed genes comparing 8 -week-old male SHR fe d a standard diet for 4 661 weeks (SHR group), 8-week-old adult SHR fed red yeast rice diet for 4 weeks (SHRD group). 662 E. Heatmap of differentially expressed genes comparing SHR and SHRD groups. F. KEGG 663 pathway analysis results for differentially expressed gene s comparing SHR and SHRD 664 groups. G. Volcano plot of differentially expressed genes comparing SHR and 3 -week-old 665 SHR fed red yeast rice diet for 9 weeks (SHR -D group). H. Heatmap of differentially 666 expressed genes comparing SHR and SHR -D groups. I. KEGG pathway analysis results for 667 differentially expressed genes comparing SHR and SHR-D groups. 668 669 Figure 6 Western blot results for cardiac tissue proteins in SHR following red yeast rice 670 treatment. A-E. Representative Western blot bands and relative protein expression for FN1, 671 β-MHC, COL1α, Caspase -3 and GADPH; F,G. Representative Western blot bands and 672 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 13, 2026. ; https://doi.org/10.64898/2026.03.10.710945doi: bioRxiv preprint 29 29 relative protein expression for p-ERK and t -ERK, H. Transcriptomic results of transcription 673 factor c-Fos. I,J. Representative Western blot bands and relative protein expression for c-Fos, 674 Data are presented as mean ± SE, comparisons between two groups were performed using t -675 tests (and non-parametric tests) (*p < 0.05, **p < 0.01, ***p < 0.001, n=4). 676 677 Figure 7 Effects of MKA on Angiotensin II -induced cardiomyoc yte injury and 678 hypertrophy. Representative images of EDU and TUNEL staining ( A), EDU positive 679 percent ( B) and apoptosis percent ( C) in H9c2 cardiomyocytes treated with different 680 concentrations of MKA (1, 5, 10 M) and 200 nM Ang II for 24 hours. D-H. Representative 681 Western blots and relative protein expression for β -MHC, p -ERK, t -ERK, c -Fos, and 682 Caspase-3. Data are presented as mean ± SE, comparisons among multiple groups were 683 conducted using one -way analysis of variance (ANOVA) followed by Tukey's multiple 684 comparison test (n = 4 per group; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). LM: 685 1M MKA; MM: 5M MKA; HM: 1M MKA; 686 687 Figure 8 The mechanism of red yeast rice derived MKA on hypertension -induced 688 cardiac hypertrophy. 689 690 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 13, 2026. ; https://doi.org/10.64898/2026.03.10.710945doi: bioRxiv preprint 30 30 Figure 1 691 692 693 694 695 696 697 698 699 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 13, 2026. ; https://doi.org/10.64898/2026.03.10.710945doi: bioRxiv preprint 31 31 Figure 2 700 701 702 703 704 705 706 707 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 13, 2026. ; https://doi.org/10.64898/2026.03.10.710945doi: bioRxiv preprint 32 32 Figure 3 708 709 710 711 712 713 714 715 716 717 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 13, 2026. ; https://doi.org/10.64898/2026.03.10.710945doi: bioRxiv preprint 33 33 Figure 4 718 719 720 721 722 723 724 725 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 13, 2026. ; https://doi.org/10.64898/2026.03.10.710945doi: bioRxiv preprint 34 34 Figure 5 726 727 728 729 730 731 732 733 734 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 13, 2026. ; https://doi.org/10.64898/2026.03.10.710945doi: bioRxiv preprint 35 35 Figure 6 735 736 737 738 739 740 741 742 743 744 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 13, 2026. ; https://doi.org/10.64898/2026.03.10.710945doi: bioRxiv preprint 36 36 Figure 7 745 746 747 748 749 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 13, 2026. ; https://doi.org/10.64898/2026.03.10.710945doi: bioRxiv preprint 37 37 Figure 8 750 751 752 753 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 13, 2026. ; https://doi.org/10.64898/2026.03.10.710945doi: bioRxiv preprint