Huoxue Qianyang Qutan Recipe limits cardiac remodeling by regulating FUNDC1/IP3R2 signaling pathway in obese hypertensive rats

Figures

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  Fig. 1. HQQR improves metabolic and cardiovascular parameters in OBH rats. (A) Representative images showing body size of SHR and OBH rats. (B–D) Body weight, abdominal circumference, and Lee’s index in different groups (n = 6). (E) SBP, DBP, and MAP after treatment (n = 6). (F,G) Body weight and Lee’s index after treatment (n = 6). (H,I) Liver weight and LW/BW after treatment (n = 6). (J,K) HW/BW and HW/TL after treatment (n = 6). (L) Representative echocardiographic images and quantitative analysis of cardiac function parameters, including LVMI, LVIDD, LVPWD, IVSD, EF, and FS (n = 6). Data are presented as mean ± SD. *, **, *** respectively means P < 0.05, P < 0.01, and P < 0.001. WKY Wistar–Kyoto rats, SHR spontaneously hypertensive rats, OBH obese hypertensive rats, HQQR-L low-dose Huoxue Qianyang Qutan Recipe, HQQR-H high-dose Huoxue Qianyang Qutan Recipe, Val valsartan, LW/BW liver weight/body weight, HW/BW heart weight/body weight, HW/TL heart weight/tibia length, LVMI left ventricular mass index, LVIDD left ventricular internal diameter at end-diastole, LVPWD left ventricular posterior wall thickness in diastole, IVSD interventricular septal thickness, EF ejection fraction, FS…

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  Fig. 2. HQQR alleviated left ventricular hypertrophy and improved MAM function in OBH rats. (A) Representative images of HE, WGA, and TEM of myocardial tissue (n = 6; scale bars = 100 μm for HE and WGA; 5 μm for TEM). (B) Quantification of cardiomyocyte cross-sectional area (n = 6). (C,D) Quantitative analysis of MAM structure, including mitochondrial length and ER–mitochondria distance (n = 6). (E) Quantification of mitochondrial Ca²⁺ levels and mitoROS (n = 6). (F) Representative fluorescence images of mitochondrial Ca²⁺ and mitoROS (n = 6; scale bar = 100 μm). (G) Activities of mitochondrial electron transport chain complexes I–IV (n = 6 per group). Data are presented as mean ± SD. *, **, and *** respectively means P < 0.05, P < 0.01, and P < 0.001. WKY, Wistar–Kyoto rats; SHR, spontaneously hypertensive rats; OBH, obese hypertensive rats; HQQR-L, low-dose Huoxue Qianyang Qutan Recipe; HQQR-H, high-dose Huoxue Qianyang Qutan Recipe; Val, valsartan; HE, hematoxylin and eosin; WGA, wheat germ agglutinin; TEM, transmission electron microscopy; MAM, mitochondria-associated endoplasmic reticulum membrane; DAPI, 4′,6-diamidino-2-phenylindole; mitoROS, mitochondrial reactive oxygen species.

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  Fig. 3. HQQR downregulated the expression of FUNDC1 and IP3R2 while promoting the ubiquitination of IP3R2. (A) Representative blot images and quantitative analysis of FUNDC1 and IP3R2 protein expression in rat myocardial tissue (n = 4). Original blots are presented in Original Blot Fig. 1. (B) Representative blot images and quantitative analysis of FUNDC1 and IP3R2 in primary cardiomyocytes following Ang II stimulation (n = 4). Original blots are presented in Original Blot Fig. 2. (C) ATP content in cardiomyocytes (n = 4). (D,E) Effect of HQQR treatment on IP3R2 ubiquitination levels in vivo and in vitro (n = 4). GAPDH was used as the loading control. Original blots are presented in Original Blot Fig. 3.Data are presented as mean ± SD. *, **, and *** respectively means P < 0.05, P < 0.01, and P < 0.001. WKY Wistar–Kyoto rats; SHR spontaneously hypertensive rats; OBH obese hypertensive rats; HQQR-L low-dose Huoxue Qianyang Qutan Recipe; HQQR-H high-dose Huoxue Qianyang Qutan Recipe; Val valsartan; FUNDC1 FUN14 domain-containing protein 1; IP3R2 inositol 1,4,5-trisphosphate receptor type 2; GAPDH glyceraldehyde-3-phosphate dehydrogenase; Ang II angiotensin II; IB immunoblotting; Ub…

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  Fig. 4. Kockdown of FUNDC1 improved the effects of Ang II on cardiomyocytes. (A) Representative immunoblot images with quantitative analysis of hypertrophy-related proteins, including ANP, BNP, and β-MHC (n = 3). Original blots are presented in Original Blot Fig. 4. (B) Mitochondrial Ca²⁺ levels (n = 3). (C) Quantification of mitoROS levels by flow cytometry (n = 3). (D,E) Mitochondrial membrane potential and ATP levels in primary cardiomyocytes (n = 3). (F) Representative immunoblot images and quantitative analysis of FUNDC1 and IP3R2 protein expression (n = 3). Original blots are presented in Original Blot Fig. 5. (G) Interaction between FUNDC1 and IP3R2 in cardiomyocytes detected by co-immunoprecipitation (n = 3). Original blots are presented in Original Blot Fig. 6. Data are presented as mean ± SD. *, **, and *** respectively means P < 0.05, P < 0.01, and P < 0.001. Ang II angiotensin II; ANP atrial natriuretic peptide; BNP brain natriuretic peptide; β-MHC β-myosin heavy chain; GAPDH glyceraldehyde-3-phosphate dehydrogenase; FUNDC1 FUN14 domain-containing protein 1; IP3R2 inositol 1,4,5-trisphosphate receptor type 2; mitoROS mitochondrial reactive oxygen species; NC negative control;…

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  Fig. 5. HQQR downregulates FUNDC1/IP3R2 expression and improves MAM function in primary cardiomyocytes. (A) Representative immunoblot images and quantitative analysis of hypertrophy-related proteins, including ANP, BNP, and β-MHC (n = 3). Original blots are presented in Original Blot Fig. 7. (B) Mitochondrial Ca²⁺ levels after treatment (n = 3). (C) Quantification of mitoROS after treatment by flow cytometry (n = 3). (D) Mitochondrial membrane potential level after treatment (n = 3). (E) ATP levels in primary cardiomyocytes (n = 3). (F) Representative immunoblot images and quantitative analysis of FUNDC1 and IP3R2 protein expression (n = 3). Original blots are presented in Original Blot Fig. 8. Data are presented as mean ± SD. *, **, and *** respectively means P < 0.05, P < 0.01, and P < 0.001. HQQR-H high-dose Huoxue Qianyang Qutan Recipe; FUNDC1 FUN14 domain-containing protein 1; IP3R2 inositol 1,4,5-trisphosphate receptor type 2; GAPDH glyceraldehyde-3-phosphate dehydrogenase; ANP atrial natriuretic peptide; BNP brain natriuretic peptide; β-MHC β-myosin heavy chain; mitoROS mitochondrial reactive oxygen species; ATP adenosine triphosphate.

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  Fig. 6. HQQR ameliorates myocardial injury in OBH rats via regulating FUNDC1/IP3R2‑mediated MAM function. Left panel: Under the pathological state of OBH, SHR fed a HFD develop aggravated cardiac remodeling, characterized by upregulated hypertrophy markers in cardiomyocytes. Increased expression of FUNDC1 and IP3R2 promotes excessive formation of MAMs, facilitating abnormal Ca²⁺ influx from the ER into mitochondria. This leads to mitochondrial Ca²⁺ overload and mitochondrial dysfunction, ultimately exacerbating myocardial damage. Right panel: HQQR downregulates FUNDC1 and IP3R2 expression and promotes ubiquitination-dependent degradation of IP3R2, thereby normalizing MAM structure and function. This reduces ER-to-mitochondria Ca²⁺ transfer, restores mitochondrial function, attenuates oxidative stress, and alleviates cardiac remodeling in OBH rats. HQQR Huoxue Qianyang Qutan Recipe; OBH obese hypertensive; FUNDC1 FUN14 domain-containing protein 1; IP3R2 inositol 1,4,5-trisphosphate receptor type 2; MAM mitochondria-associated endoplasmic reticulum membrane; SHR spontaneously hypertensive rats; HFD high-fat diet; ANP atrial natriuretic peptide; BNP B-type natriuretic peptide; β-MHC…