PMC7717942_01
Study protocol and cardiac phenotyping. (A) Study protocol. (B) Parasternal long-axis view during end-diastole and end-systole before (upper panel) and 2 weeks after ISO treatment (lower panel). Scale bar represents 2 mm. (C) Comparative volumetry indicating increased left ventricular volumes accompanied by reduced stroke volume. (D) Ejection fraction analysis. (E) Normalized heart weights indicating cardiac enlargement. Remaining ISO-treated animals (n = 12) served as reference. EDV, end-diastolic volume; ESV, end-systolic volume.
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F1
Briefly, male 129/Sv mice (6-8 weeks old) received subcutaneous injections of 25 mg/kg ISO or saline as placebo control for four consecutive days according to a standard protocol (Figure 1A). At baseline, the case animal had a left ventricular ejection fraction of 48% without any evidence of regional wall motion abnormalities (Figure 1B and Supplementary Video 1). However, echocardiography 2 weeks after final injection revealed wall thinning and a pronounced aneurysm of the apex of the left ventricle (LV) resulting in massively increased LV volumes and markedly reduced global systolic function (Figures 1B-D). Cardiac enlargement was also evident from indexed heart weight obtained during necropsy (Figure 1E).
Wall motion analyses by conventional and speckle-tracking echocardiography. (A) Reconstructed M-modes at basal (upper panel) and apical level (lower panel) indicating apical akinesia. (B) Three-dimensional reconstruction of radial strain during three cardiac cycles. Three-dimensional Cartesian coordinate system mapping radial strain, time/cardiac cycles, and myocardial segments derived from the cardiac long axis (from anterior base over apex to posterior base). Radial strain in midmyocardial and apical segments (both anterior and posterior) was markedly lower as compared to basal segments.
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Apical segments were akinetic, whereas the base appeared to contract normally (Figure 2A and Supplementary Video 2). During speckle-tracking echocardiography, the same segmental differences were observed in reduced apical but preserved basal radial strain of the LV (Figure 2B).
Histological analysis. (A) Cardiac cross-sections at basal, midmyocardial, and apical level with exemplary region of interest showing transmural scarring (Picrosirius red staining for detection of collagen fibers). (B) Collagen quantification in subendocardium (Endo) and subepicardium (Epi). (C) Detailed histology of the apical scar presented in (A) indicating replacement fibrosis in response to cardiomyocyte loss (hematoxylin-eosin stain). Remaining ISO-treated animals (n = 12) served as reference.
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Histological analyses showed subendocardial fibrosis within basal and midmyocardial sections of the LV (Figure 3A). Fibrotic lesions in the apex exceeded the subendocardial layer resulting in transmural scarring and aneurysmatic wall thinning (Figures 3A,B). In contrast, the remaining ISO-treated animals showed circumscribed subendocardial collagen accumulation, as expected in this model (Figure 3B). Histopathology of the apical scar revealed replacement fibrosis in response to a profound loss of cardiomyocytes (Figure 3C).
DT-MRI analysis. (A) Long-axis mean diffusivity for a representative heart of the ISO group (reference) and the infarcted heart (case). (B) Quantitative analyses of mean diffusivity in the left ventricle. (C) Bull's-eye plot for mean diffusivity of the infarcted heart. (D) Whole-heart volume rendering and respective surface cut for tractography visualization in (E) tractography of the main eigenvector using varying minimal fiber bundle lengths (0.1, 1, 5, 10, 15 mm) as termination criteria. (F) Helix angle distribution in a basal, midcavity, and apical slab of the infarcted animal with zoomed tractography. (G) Ratio of positive to negative voxels in basal, midcavity, and apical segments. Remaining ISO-treated animals (n = 11) served as reference.
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On average, the case showed highly reduced mean diffusivity in the LV when compared to the remaining animals (Figures 4A,B). Mean diffusivity was found to be higher in apical (=infarcted) than in basal (=remote) segments (Figure 4C). Three-dimensional tractography showed that orientation coherence of myocardial fibers was maintained on a submillimeter scale, while increasing minimal tract lengths revealed increasingly sparse tract reconstruction (Figures 4D,E). Compared to the remaining ISO-treated animals, there was a reduced number of voxels with positive helix angle in the midventricular and apical segments resulting in a lower positive-to-negative helix angle ratio, whereas the proportion of fiber tracts with positive helix angle was higher in basal segments (Figures 4F,G).