Minimally Invasive, Echocardiography‐Guided Mouse Model of Degenerative Mitral Regurgitation Recapitulates Cardiac Remodeling and Arrhythmogenesis

Figures

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  Figure 1. Establishment of a minimally invasive mouse model of mitral regurgitation. A, Schematic illustration of transthoracic, echocardiography‐guided puncture of the anterior mitral leaflet via a 27G needle introduced through the LV apex. B, Representative intraoperative images and echo‐guided needle visualization (red arrow, LV; yellow arrow, MV). C through G, Longitudinal changes in BW, HR, systolic, diastolic and mean blood pressure in sham and MR mice. H, Representative echocardiographic images showing color Doppler‐confirmed regurgitant jet in MR vs sham. I through M, Quantification of LA diameter, VC width, IVSD, LVIDd, and FS. N, Survival curves demonstrating >90% procedural survival. Sham (N=8), MR mice (N=10). Data are expressed using mean±SE. P value determined using Student's t test. AO indicates aorta; BW, body weight; DBP, diastolic blood pressure; FS, fractional shortening; HR, heart rate; IVSD, interventricular septum in diastole; LA, left atrium; LV, left ventricle; LVIDd, left ventricular internal dimension at end‐diastole; MR, mitral regurgitation; MV, mitral valve; SBP, systolic blood pressure; and VC, vena contracta. *P<0.05.

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  Figure 2. Hemodynamic alterations in MR mice assessed by pressure–volume loop analysis. A, Representative PV loop tracings in sham and MR groups. B, Ves, Ved. (C) Pes, and Ped. D, Maximal and minimal rates of LV pressure change (+dP/dt, –dP/dt). E, Ea and (F) diastolic relaxation constant (tau). G, Load‐independent indices: slope of ESPVR and EDPVR. Sham (N=7), MR mice (N=7). Data are expressed using mean±SE. P value determined using Student's t test. Ea, arterial elastance; EDPVR, end‐diastolic pressure–volume relationship; ESPVR, end‐systolic pressure–volume relationship; LV, left ventricular; MR, mitral regurgitation; Ped, end‐diastolic pressure; Pes, end‐systolic pressure; PV, pressure–volume; Ved, end‐diastolic volume; and Ves, end‐systolic volume. **P<0.01.

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  Figure 3. Gross and histological remodeling of MR hearts. A, Gross hearts showing anterior leaflet puncture site (black arrows). B, Increased heart weight–to–body weight and heart weight–to–tibia length ratios in MR compared with sham. C and D, Representative H&E and Masson's trichrome staining of atrial sections, showing atrial enlargement and interstitial fibrosis (blue). E, Quantification of fibrosis percentage, significantly higher in MR mice. Sham (N=6–10), MR mice (N=8–10). F and G, The representative images and quantification of cross‐sectional areas in F‐Actin positive cardiomyocytes in sham and MR mice (WGA: White). Data are expressed using mean ± SE. P value determined using Student's t test. CSA indicates cross‐sectional area; H&E, hematoxylin and eosin; HW/BW, heart weight–to–body weight; MR, mitral regurgitation; and WGA, wheat germ agglutinin. *P<0.05 and ***P<0.001.

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  Figure 4. Transcriptomic profiling of left atrial tissues in MR mice. A, GO enrichment analysis identifying calcium transport, oxidative stress, and cytoskeletal pathways. B, KEGG pathway enrichment showing fluid shear stress, calcium signaling, apoptosis, and lipid metabolism. C, Heatmap clustering of representative apoptosis‐, calcium‐, and shear stress–related genes separating MR from sham. D, Circos plot mapping enriched genes to multiple pathways, illustrating integrated remodeling networks. Sham (N=3), MR mice (N=3). Differential gene expression analysis was performed using DESeq2, and P values shown represent Benjamini–Hochberg–adjusted false discovery rate values. AGE‐RAGE indicates advanced glycation end‐products‐receptor for advanced glycation end‐products; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; and MR, mitral regurgitation.

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  Figure 5. Increased atrial apoptosis in MR mice. A, Representative TUNEL/F‐Actin/DAPI staining demonstrating higher apoptotic cardiomyocyte nuclei (yellow arrowheads) in MR atria. Quantification of TUNEL‐positive (B) cells and (C) cardiomyocytes. (D) Flow cytometry showing increased PI+/troponin+ apoptotic cardiomyocytes in MR atria. E through G, Western blot and quantification of Bax, caspase‐3, and cleaved caspase‐3 demonstrating upregulation in MR tissues. Sham (N=6), MR mice (N=6). Data are expressed using mean±SE. P value determined using Student's t test. MR indicates mitral regurgitation; PI, propidium iodide; and TUNEL, terminal deoxynucleotidyl transferase–mediated dUTP nick‐end labeling. **P<0.01 and ***P<0.001.

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  Figure 6. Atrial fibrillation inducibility and calcium‐handling abnormalities in MR mice. A, Representative ECG tracings showing AF episodes induced by transesophageal burst pacing. B, Increased AF inducibility in MR compared with sham. C, Connexin‐43 immunostaining showing gap junction downregulation in MR atria. D, Quantification of Connexin‐43 expression. E through G, Western blot and quantification of connexin‐43, total RyR2, and phosphorylated RyR2 levels, demonstrating reduced Cx43 and enhanced RyR2 phosphorylation. Sham (N=6), MR mice (N=6). Data are expressed using mean±SE. P value determined using Student's t test. AF indicates atrial fibrillation; MR, mitral regurgitation; and RyR2, ryanodine receptor 2. *P < 0.05 and ***P < 0.001.

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  Figure 7. Proposed schematic of MR‐induced atrial remodeling. Chronic MR causes regurgitant flow stress leading to atrial dilatation, systolic dysfunction, fibrosis, calcium overload, and increased atrial fibrillation susceptibility. LA indicates left atrium; LV, left ventricle; and MR, mitral regurgitation.