FIGURE SUMMARY
Title

Use of Echocardiography Reveals Reestablishment of Ventricular Pumping Efficiency and Partial Ventricular Wall Motion Recovery upon Ventricular Cryoinjury in the Zebrafish

Authors
González-Rosa, J.M., Guzmán-Martínez, G., Marques, I.J., Sánchez-Iranzo, H., Jiménez-Borreguero, L.J., Mercader, N.
Source
Full text @ PLoS One

Echocardiographic image acquisition and basal fractional volume shortening (FVS) quantification.

(A) Schematic representation of animal positioning for image acquisition and picture of the set up. (i) Animals are positioned ventrally and (ii) are immobilized in the same way as for surgical procedures, in a Petri dish, and are covered with fish water containing anaesthetic solution. This positioning allows for a transducer to be placed directly over the body wall at the level of the heart (iii). The transducer is attached to a holder to allow a stable position during acquisition (iv). (B,C) Details from representative 2D echocardiography images from an uninjured zebrafish heart showing maximal ventricular dilatation (B, diastole) and maximal ventricular contraction (C, systole). The diastolic (red) and systolic (green) ventricular areas are outlined and the length of the apical image long axis is also indicated (L). Red and green lines in B and C highlight ventricular border in diastole and systole, respectively. Yellow lines indicate the bulbus arteriosus (BA). (D) FVS obtained in basal conditions (n = 47, mean ± SD = 39 ± 5). (E) Comparison of FVS in basal conditions at two different days, with an interval of 7 days. Shown are means ± SD. The relative FVS (RFVS) of BASAL2 versus BASAL1 within the same animal are statistically comparable (p = 0.1099, Wilcoxon matched-pairs signed rank test). (F) FVS measured in basal conditions with different dosages of anesthesia and throughout time in the same animal. Initial anesthesia conditions are the same as for all acquisitions (60 µM tricaine/3 mM isoflurane). The final acquisition was taken 20 minutes later and the final anesthesia dose was 60 µM tricaine/15 mM isoflurane. Differences in the average FVS are not statistically significant (p = 0.1094, Wilcoxon matched-pairs signed rank test). A, atrium; ba, bulbus arteriosus; FVS, fractional volume shortening; L, length of the apical image long axis; RFVS, relative fractional volume shortening; v, ventricle.

Correlation between histology of imaged hearts and echocardiographic analysis.

(A,B) Groups of animals in which cryoinjury was confirmed by histological AFOG staining after echocardiography. In 24 out of 28 fish, cryoinjury was diagnosed at 7 dpi by measurement of a drop of the RFVS e 20% compared to the equivalent basal measurement. Only one from 7 sham-operated fish presented a drop in RFVS e 20%. (C) Subsequent histological staining however did not support an alteration in the cardiac morphology or injury in any of the sham operated animals. BA, bulbus arteriosus; IA, injured area; dpi, days postinjury; dpm, days postmanipulation; V, ventricle. Size bars, 200 µm.

Cryoinjury induces local, long-term alterations in myocardial organization.

(A,B) Immunohistochemistry on sagittal sections of control (A,A′) and cryoinjured (B-B′′) hearts at 130 dpi from the Tg(myl7:nucDsRed) line. A′-B′′ are zoomed images of boxed areas in A and B, additionally showing autofluorescence to reveal tissue organization. (A-A′) In control hearts, one or two cells constitute the thickness of the compact myocardium (CM). (B-B′′) At 130 dpi, the injured wall (IW) shows an abnormal increase in the number and distribution of cardiomyocytes compared with the contralateral wall (CLW). (C) Quantification of the nuclear density relative to the compact tissue reveals an increase in cardiomyocyte density in the IW compared to the CLW. Graph represents mean values and SD (*** p = 0.006, two tailed Student′s t-test; 100–150 cells counted per section, 3 sections per heart, n = 3 animals analyzed). (D) qPCR from ventricular RNA samples reveal induction of the natriuretic peptide encoding gene nppa upon cryoinjury. Graph represents mean values and SD, n = 4-5 replicates, Expressions levels were normalized to that of ef1α and rps11 and further normalized to that of the uninjured sample. (* p<0.05; one-way ANOVA followed by Tukey′s honest significant difference test). (E-H) Sections of cryoinjured hearts at the indicated times post-injury hybridized with a riboprobe for nppa mRNA. Yellow arrows mark areas of strong nppa expression. (E) In control hearts, nppa is highly expressed in the atrium (yellow arrow) and at lower levels in the trabecular myocardium (white arrow). (F-G) Shortly after injury, nppa is strongly upregulated in the ventricular myocardium. (H) At 90 dpi, the levels of nppa expression are similar to those detected in control hearts. Observe the increase in thickness of the compact layer of the injured wall (asterisk) revealed by no expression of nppa. AT, atrium; BA, bulbus arteriosus, CLW, contralateral wall; CM, compact myocardium; hpi, hours postinjury; dpi, days postinjury; IA, injured area; IW, injured wall; V, ventricle. Bars, 200 µm (full views), 50µm (magnifications).

Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ PLoS One