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Advanced echocardiography in adult zebrafish reveals delayed recovery of heart function after myocardial cryoinjury.

Hein SJ, Lehmann LH, Kossack M, Juergensen L, Fuchs D, Katus HA, Hassel D - PLoS ONE (2015)

Bottom Line: We show that functional recovery of cryoinjured hearts occurs in three distinct phases.Importantly, the regeneration process after cryoinjury extends far beyond the proposed 45 days described for ventricular resection with reconstitution of myocardial performance up to 180 days post-injury (dpi).The imaging modalities evaluated here allow sensitive cardiac phenotyping and contribute to further establish adult zebrafish as valuable cardiac disease model beyond the larval developmental stage.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine III, Cardiology, Heidelberg University Hospital, 69120 Heidelberg, Germany and DZHK (German Center for Cardiovascular Research), Partner Site Heidelberg/Mannheim, Heidelberg, Germany.

ABSTRACT
Translucent zebrafish larvae represent an established model to analyze genetics of cardiac development and human cardiac disease. More recently adult zebrafish are utilized to evaluate mechanisms of cardiac regeneration and by benefiting from recent genome editing technologies, including TALEN and CRISPR, adult zebrafish are emerging as a valuable in vivo model to evaluate novel disease genes and specifically validate disease causing mutations and their underlying pathomechanisms. However, methods to sensitively and non-invasively assess cardiac morphology and performance in adult zebrafish are still limited. We here present a standardized examination protocol to broadly assess cardiac performance in adult zebrafish by advancing conventional echocardiography with modern speckle-tracking analyses. This allows accurate detection of changes in cardiac performance and further enables highly sensitive assessment of regional myocardial motion and deformation in high spatio-temporal resolution. Combining conventional echocardiography measurements with radial and longitudinal velocity, displacement, strain, strain rate and myocardial wall delay rates after myocardial cryoinjury permitted to non-invasively determine injury dimensions and to longitudinally follow functional recovery during cardiac regeneration. We show that functional recovery of cryoinjured hearts occurs in three distinct phases. Importantly, the regeneration process after cryoinjury extends far beyond the proposed 45 days described for ventricular resection with reconstitution of myocardial performance up to 180 days post-injury (dpi). The imaging modalities evaluated here allow sensitive cardiac phenotyping and contribute to further establish adult zebrafish as valuable cardiac disease model beyond the larval developmental stage.

No MeSH data available.


Related in: MedlinePlus

Longitudinal echocardiographic evaluation of cardiac function after cryoinjury.(A) Lateral brightfield (top) and fluorescent (bottom) images of hearts derived from sham operated transgenic zebrafish [Tg(myl7:GFP)f1] and after cryoinjury at depicted time points. Dashed lines indicate injured myocardial area (i). (B) PWD recordings from sham (top) and cryoinjured zebrafish at 1dpi (bottom) demonstrating decreased A-wave and increased E-wave amplitudes indicative for diastolic dysfunction. (C) Representative SAX images from sham (upper row) and cryoinjured zebrafish at 1dpi (middle row) and at 30dpi (lower row) with end-diastolic dimensions illustrated in red and end-systolic dimensions in green. (D-I) Quantification of changes in (D) heart rate (HR), (E) fractional shortening (FS), (F) fractional area change (FAC), (G) ejection fraction (EF), (H) E/A ratio and (I) cardiac output (CO) at baseline and at indicated time points during regeneration after myocardial injury. Small number in (D) indicates number of animals analyzed (J) Speckle-tracking analysis of segmental displacement shows akinesia of injured (green, pink and light blue line) as compared to the non-injured segments (yellow, purple and dark blue line). The average curve of all segments is illustrated in black. For color coding of different segments see Fig 2B. Values are expressed as means ± SEM; a, atrium; i, injured area; ot, outflow tract; v, ventricle; AW, anterior wall; PW, posterior wall; *, p<0.05; unpaired student’s t-test and ANOVA with post hoc comparisons by Bonferroni’s multiple comparison test.
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pone.0122665.g004: Longitudinal echocardiographic evaluation of cardiac function after cryoinjury.(A) Lateral brightfield (top) and fluorescent (bottom) images of hearts derived from sham operated transgenic zebrafish [Tg(myl7:GFP)f1] and after cryoinjury at depicted time points. Dashed lines indicate injured myocardial area (i). (B) PWD recordings from sham (top) and cryoinjured zebrafish at 1dpi (bottom) demonstrating decreased A-wave and increased E-wave amplitudes indicative for diastolic dysfunction. (C) Representative SAX images from sham (upper row) and cryoinjured zebrafish at 1dpi (middle row) and at 30dpi (lower row) with end-diastolic dimensions illustrated in red and end-systolic dimensions in green. (D-I) Quantification of changes in (D) heart rate (HR), (E) fractional shortening (FS), (F) fractional area change (FAC), (G) ejection fraction (EF), (H) E/A ratio and (I) cardiac output (CO) at baseline and at indicated time points during regeneration after myocardial injury. Small number in (D) indicates number of animals analyzed (J) Speckle-tracking analysis of segmental displacement shows akinesia of injured (green, pink and light blue line) as compared to the non-injured segments (yellow, purple and dark blue line). The average curve of all segments is illustrated in black. For color coding of different segments see Fig 2B. Values are expressed as means ± SEM; a, atrium; i, injured area; ot, outflow tract; v, ventricle; AW, anterior wall; PW, posterior wall; *, p<0.05; unpaired student’s t-test and ANOVA with post hoc comparisons by Bonferroni’s multiple comparison test.

Mentions: To evaluate our protocol and methodology under disease relevant conditions, we next induced myocardial necrosis in adult zebrafish by application of myocardial cryoinjury as a model of myocardial infarction [28–30]. We used a transgenic zebrafish line expressing green fluorescence protein (GFP) in cardiomyocytes [Tg(myl7:GFP)f1] to visualize cardiomyocyte loss in response to cryoinjury and to follow regeneration (Fig 4A).


Advanced echocardiography in adult zebrafish reveals delayed recovery of heart function after myocardial cryoinjury.

Hein SJ, Lehmann LH, Kossack M, Juergensen L, Fuchs D, Katus HA, Hassel D - PLoS ONE (2015)

Longitudinal echocardiographic evaluation of cardiac function after cryoinjury.(A) Lateral brightfield (top) and fluorescent (bottom) images of hearts derived from sham operated transgenic zebrafish [Tg(myl7:GFP)f1] and after cryoinjury at depicted time points. Dashed lines indicate injured myocardial area (i). (B) PWD recordings from sham (top) and cryoinjured zebrafish at 1dpi (bottom) demonstrating decreased A-wave and increased E-wave amplitudes indicative for diastolic dysfunction. (C) Representative SAX images from sham (upper row) and cryoinjured zebrafish at 1dpi (middle row) and at 30dpi (lower row) with end-diastolic dimensions illustrated in red and end-systolic dimensions in green. (D-I) Quantification of changes in (D) heart rate (HR), (E) fractional shortening (FS), (F) fractional area change (FAC), (G) ejection fraction (EF), (H) E/A ratio and (I) cardiac output (CO) at baseline and at indicated time points during regeneration after myocardial injury. Small number in (D) indicates number of animals analyzed (J) Speckle-tracking analysis of segmental displacement shows akinesia of injured (green, pink and light blue line) as compared to the non-injured segments (yellow, purple and dark blue line). The average curve of all segments is illustrated in black. For color coding of different segments see Fig 2B. Values are expressed as means ± SEM; a, atrium; i, injured area; ot, outflow tract; v, ventricle; AW, anterior wall; PW, posterior wall; *, p<0.05; unpaired student’s t-test and ANOVA with post hoc comparisons by Bonferroni’s multiple comparison test.
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Related In: Results  -  Collection

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pone.0122665.g004: Longitudinal echocardiographic evaluation of cardiac function after cryoinjury.(A) Lateral brightfield (top) and fluorescent (bottom) images of hearts derived from sham operated transgenic zebrafish [Tg(myl7:GFP)f1] and after cryoinjury at depicted time points. Dashed lines indicate injured myocardial area (i). (B) PWD recordings from sham (top) and cryoinjured zebrafish at 1dpi (bottom) demonstrating decreased A-wave and increased E-wave amplitudes indicative for diastolic dysfunction. (C) Representative SAX images from sham (upper row) and cryoinjured zebrafish at 1dpi (middle row) and at 30dpi (lower row) with end-diastolic dimensions illustrated in red and end-systolic dimensions in green. (D-I) Quantification of changes in (D) heart rate (HR), (E) fractional shortening (FS), (F) fractional area change (FAC), (G) ejection fraction (EF), (H) E/A ratio and (I) cardiac output (CO) at baseline and at indicated time points during regeneration after myocardial injury. Small number in (D) indicates number of animals analyzed (J) Speckle-tracking analysis of segmental displacement shows akinesia of injured (green, pink and light blue line) as compared to the non-injured segments (yellow, purple and dark blue line). The average curve of all segments is illustrated in black. For color coding of different segments see Fig 2B. Values are expressed as means ± SEM; a, atrium; i, injured area; ot, outflow tract; v, ventricle; AW, anterior wall; PW, posterior wall; *, p<0.05; unpaired student’s t-test and ANOVA with post hoc comparisons by Bonferroni’s multiple comparison test.
Mentions: To evaluate our protocol and methodology under disease relevant conditions, we next induced myocardial necrosis in adult zebrafish by application of myocardial cryoinjury as a model of myocardial infarction [28–30]. We used a transgenic zebrafish line expressing green fluorescence protein (GFP) in cardiomyocytes [Tg(myl7:GFP)f1] to visualize cardiomyocyte loss in response to cryoinjury and to follow regeneration (Fig 4A).

Bottom Line: We show that functional recovery of cryoinjured hearts occurs in three distinct phases.Importantly, the regeneration process after cryoinjury extends far beyond the proposed 45 days described for ventricular resection with reconstitution of myocardial performance up to 180 days post-injury (dpi).The imaging modalities evaluated here allow sensitive cardiac phenotyping and contribute to further establish adult zebrafish as valuable cardiac disease model beyond the larval developmental stage.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine III, Cardiology, Heidelberg University Hospital, 69120 Heidelberg, Germany and DZHK (German Center for Cardiovascular Research), Partner Site Heidelberg/Mannheim, Heidelberg, Germany.

ABSTRACT
Translucent zebrafish larvae represent an established model to analyze genetics of cardiac development and human cardiac disease. More recently adult zebrafish are utilized to evaluate mechanisms of cardiac regeneration and by benefiting from recent genome editing technologies, including TALEN and CRISPR, adult zebrafish are emerging as a valuable in vivo model to evaluate novel disease genes and specifically validate disease causing mutations and their underlying pathomechanisms. However, methods to sensitively and non-invasively assess cardiac morphology and performance in adult zebrafish are still limited. We here present a standardized examination protocol to broadly assess cardiac performance in adult zebrafish by advancing conventional echocardiography with modern speckle-tracking analyses. This allows accurate detection of changes in cardiac performance and further enables highly sensitive assessment of regional myocardial motion and deformation in high spatio-temporal resolution. Combining conventional echocardiography measurements with radial and longitudinal velocity, displacement, strain, strain rate and myocardial wall delay rates after myocardial cryoinjury permitted to non-invasively determine injury dimensions and to longitudinally follow functional recovery during cardiac regeneration. We show that functional recovery of cryoinjured hearts occurs in three distinct phases. Importantly, the regeneration process after cryoinjury extends far beyond the proposed 45 days described for ventricular resection with reconstitution of myocardial performance up to 180 days post-injury (dpi). The imaging modalities evaluated here allow sensitive cardiac phenotyping and contribute to further establish adult zebrafish as valuable cardiac disease model beyond the larval developmental stage.

No MeSH data available.


Related in: MedlinePlus