Seipin Knockout Mice Develop Heart Failure With Preserved Ejection Fraction

Figures

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  Figure 1. The Development of Cardiomegaly in the Lean Diabetic SKO Mice Mice were subjected to phenotypic characterizations according to the protocol (A) and nuclear magnetic resonance (NMR) (B) to measure body weight and lean mass. (C) Blood glucose and serum insulin of mice (18 to 24 weeks old) under basal feeding; (D) the ratio between tissue weight and body weight of mice (∼24 weeks old); and (E) the ratio of left ventricular (LV), left atrial (LA), right ventricular (RV), and right atrial (RA) weight to body weight of mice (41 to 46 weeks old) were analyzed. *p < 0.05, compared with control (Ctrl) mice (n = 6). PV = pressure-volume; SKO = seipin knockout.

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  Figure 2. The Concentric LV Hypertrophy in the Absence of Hypertension in the SKO Mice (A) At 8, 15, 23, and 36 weeks of age, mice were subjected to echocardiography to measure the thickness of the left ventricle anterior wall (LVAW) and left ventricle posterior wall (LVPW), left ventricle internal diameter (LVID) in diastole (d), LV mass, ejection fraction (EF), and fractional shortening (FS) (n = 8 to 10). (B) The heart tissues of adult mice (36 weeks old) were subjected to examine the ratio of heart weight to tibia length (scale bar = 10 mm, n = 12). Representative images of hematoxylin and eosin–stained heart sections (top, longitudinal section, scale bar = 10 mm; bottom, horizontal section, scale bar = 1 mm) were shown. The cross-sectional area of whole heart tissues (n = 8) and cardiomyocytes (scale bar = 100 μm, n = 10) was quantified in each group of mice. (C) The total (t) and phosphorylated (p) signal transducer and activator of transcription 3 (STAT3) and Janus kinase 2 (JAK2) were examined by Western blotting (n = 6), and (D) the gene expression of hypertrophic markers, including atrial natriuretic peptide (Nppa) and alpha-skeletal actin (Acta1), were detected using mouse heart…

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  Figure 3. LV Diastolic Dysfunction, Exercise Intolerance, and LA Enlargement in the SKO Mice (A) The Doppler echocardiography was used to examine LV isovolumic contraction time (IVCT) and isovolumic relaxation time (IVRT), ejection time (ET), and myocardial performance index (MPI) (n = 8 to 10). The LV pressure-volume loops were analyzed to show representative examples of pressure-volume loops of mice (23 weeks old) (B) and measure LV end-diastolic pressure (EDP) and diastolic stiffness coefficient β derived from the end-diastolic pressure-volume relationship (EDPVR) of mice (n = 6) (C). (D) Mouse running distance and time via treadmill running test (n = 6); (E) the histology of LA tissues and ratio of LA weight to tibia length of mice (36 weeks old) were examined (scale bar = 10 mm, n = 10 to 11). *p < 0.05, compared to Ctrl mice. Abbreviations as in Figure 1.

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  Figure 4. Altered Cardiac Titin Phosphorylation in the SKO Mice (A) The quantitative mass spectrometry was used to measure ratio changes of titin phosphosites of mouse LV tissues (23 to 36 weeks old, n = 6). The bold line arrows (red and black) indicate localization of changed phosphosites in the canonical sequence of mouse titin (UniProtKB entry A2ASS6). (B) Multiple sequence alignments showed serine (S) 12884 of murine cardiac titin was a conserved residue. (C) Protein expression of protein kinase C alpha (PRKCA) was examined in mouse heart tissues (23 to 36 weeks old, n = 6). Fold change was calculated for comparison. *p < 0.05, compared with Ctrl mice FnIII = fibronectin type III domain; Ig = immunoglobulin-like; PEVK = proline, glutamate, valine, and lysine; T = threonine. Abbreviations as in Figure 1.

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  Figure 5. LV Reactive Interstitial Fibrosis and Neutrophil Infiltration in the SKO Mouse Myocardium (A) Representative photomicrographs of picrosirius red staining of mouse heart tissues (scale bar = 100 μm). The pixel area of fibrosis was quantified in each group (n = 6). Gene expression of collagen 1 (Col1a1), interleukin 6 (Il6), and intercellular adhesion molecule 1 (Icam1), and (B) protein levels of alpha-smooth muscle actin (ACTA2), transforming growth factor beta family (TGFB), and tumor necrosis factor (TNF) were examined in mouse hearts (∼24 weeks old, n = 6). Fold change was calculated for comparison. (C) Infiltrated neutrophils with positive immunohistochemical staining of antineutrophil–specific elastase were counted in mouse heart sections (scale bar = 20 μm, n = 6). (D) Representative images of in situ neutrophil extracellular traps in mouse hearts were indicated by arrows to show colocalization of deoxyribonucleic acid (4′,6-diamidino-2-phenylindole [DAPI], blue), myeloperoxidase ([MPO], red), and citrullinated histone 3 ([Cit-H3], green) (scale bar = 20 μm). (E) The protein level of Cit-H3 was examined in mouse heart tissues (24 to 30 weeks old, n = 6). Fold change was…

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  Figure 6. The SKO Mouse Is a Lean Diabetic Murine Model of HFpEF The lean diabetic SKO mice recapitulate major clinical manifestations of heart failure with preserved ejection fraction (HFpEF), resembling those observed in Asian populations, including LV concentric hypertrophy with preserved EF, increased left ventricular stiffness, and LA dilatation associated with exercise intolerance. Both cellular (PRKCA-mediated hyperphosphorylation of titin at the PEVK spring elements) and extracellular (reactive interstitial fibrosis) changes lead to left ventricular stiffness, and the latter is associated with massive neutrophil infiltration and neutrophil extracellular traps (NET) in mouse heart tissues. The NET, cellular titin changes, and extracellular interstitial fibrosis could be suggested as potential therapeutic targets in Asian HFpEF patients. Abbreviations as in Figures 1, 2, and 4.