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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

Changes in cardiac performance in response to beta-adrenergic stimulation and blockage.(A) Representative pulsed-wave Doppler (PWD) signals (upper row) acquired from ACX view illustrating reduced and elevated heart rate in Atenolol (middle, AT) and Isoproterenol (right, Iso) treated zebrafish, respectively, compared to controls (left). Representative SAX images (lower row) with demarcated end-diastolic (red) and end-systolic (green) ventricular dimension after Atenolol and Iso treatment. (B-E) Quantification of changes in heart rate (HR) (B), fractional area change (FAC) (C), fractional shortening (FS) (D), and ejection fraction (EF) (E) after AT or Iso treatment compared to controls (C). Small numbers in columns indicate number of animals measured. (F) For speckle-tracking analysis, the ventricle was divided in six segments as indicated with segment 1 and 2 representing the anterior wall, 3 and 4 the apex, and 5 and 6 the posterior wall. Time-to-peak analysis reveals that AT leads to significantly prolonged time-to-peak indices in myocardial velocity of all segments. Small numbers in columns indicate number of animals measured. Values are expressed as mean ± SEM. Ap, Apex; AT, Atenolol; AW, anterior wall; Iso, Isoproterenol; PW, posterior wall; *, p<0.05; unpaired student’s t-test.
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pone.0122665.g003: Changes in cardiac performance in response to beta-adrenergic stimulation and blockage.(A) Representative pulsed-wave Doppler (PWD) signals (upper row) acquired from ACX view illustrating reduced and elevated heart rate in Atenolol (middle, AT) and Isoproterenol (right, Iso) treated zebrafish, respectively, compared to controls (left). Representative SAX images (lower row) with demarcated end-diastolic (red) and end-systolic (green) ventricular dimension after Atenolol and Iso treatment. (B-E) Quantification of changes in heart rate (HR) (B), fractional area change (FAC) (C), fractional shortening (FS) (D), and ejection fraction (EF) (E) after AT or Iso treatment compared to controls (C). Small numbers in columns indicate number of animals measured. (F) For speckle-tracking analysis, the ventricle was divided in six segments as indicated with segment 1 and 2 representing the anterior wall, 3 and 4 the apex, and 5 and 6 the posterior wall. Time-to-peak analysis reveals that AT leads to significantly prolonged time-to-peak indices in myocardial velocity of all segments. Small numbers in columns indicate number of animals measured. Values are expressed as mean ± SEM. Ap, Apex; AT, Atenolol; AW, anterior wall; Iso, Isoproterenol; PW, posterior wall; *, p<0.05; unpaired student’s t-test.

Mentions: The adult zebrafish heart is approximately 1000 fold smaller than a human heart and still 10 fold smaller than mice hearts. The highly trabecularized ventricle measures approximately 1mm in length and is 0.5-1mm wide, with a compact myocardial layer of 50–100μm thickness [43, 44]. To overcome this significant size limitation we deployed a high frequency ultrasound system specifically developed for small animals, with a previously demonstrated axial and lateral resolution of approximately 40μm and 70μm, respectively, hence providing the appropriate resolution for imaging [45, 46]. Importantly, this system was previously deployed for conventional echocardiography measurements in adult zebrafish [21, 24]. We initially defined three examination planes to globally measure cardiac function in adult zebrafish (Fig 1A and 1B). These three planes were specifically adapted to zebrafish anatomy and defined according to standardized echocardiographic evaluation in clinical practice. In a 60° cranial angulated transversal section, circumferential ventricular contractions can be recorded in a short axis view (SAX) using B-Mode imaging (Fig 1A1, 1B1 and 1C1, S1 Fig and Fig 1, see also S1 Movie). From these images systolic and diastolic circumferential ventricular area can be measured and the fractional area change (FAC), as a parameter for circumferential contraction, can be calculated as later shown in Fig 3A (lower row). To achieve a long axis view of the heart (LAX) we used a strictly medial craniocaudal sagittal section (Fig 1A2 and 1B2). In this view, the ventricular as well as the cardiac outflow tract (bulbus arteriosus) performance can be visualized in a sagittal long axis view (LAX) by B-Mode imaging (Fig 1C2, S2 Fig and Fig 2, see also S2 Movie). We used this imaging plane to subsequently assess the systolic and diastolic longitudinal ventricular area to calculate the ejection fraction (EF), fractional shortening (FS) and stroke volume (SV). To further characterize cardiac blood flow velocities, we applied pulsed-wave Doppler (PWD) measurements in a 45° abdominal-cranial angulated transversal plane (ACX) ensuring a parallel alignment of ultrasound beam and cardiac blood flow (Fig 1A3 and see also S3 Movie). Additionally, this plane enables visualization of the ventricular apex and the atrioventricular- and bulboventricular valves. Importantly, PWD signals have been depicted from the area of maximum inflow and outflow velocities, defined by applying C-Mode imaging. The maximum cardiac inflow velocities were usually found at the leaflets region of the atrioventricular valve, whereas the maximum outflow velocity can usually be detected, depending on the animal’s size, 35–50μm more cranial, close to the bulboventricular valve and the upper thoracic aperture. From this, a PWD signal was recorded for at least three seconds to reliably calculate the heart rate and ventricular velocity time integral (V VTI), as a parameter describing cardiac output performance (Fig 1D, see also S3 Movie). From the maximum inflow PWD signal, the heart rate, atrioventricular velocity time integral (A VTI), maximum velocity of A- and E-wave (Amax and Emax) were measured and the E/A ratio, as a parameter of diastolic function was calculated (Fig 1D). The standard parameters assessed from adult zebrafish applying this protocol are presented in Table 1.


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)

Changes in cardiac performance in response to beta-adrenergic stimulation and blockage.(A) Representative pulsed-wave Doppler (PWD) signals (upper row) acquired from ACX view illustrating reduced and elevated heart rate in Atenolol (middle, AT) and Isoproterenol (right, Iso) treated zebrafish, respectively, compared to controls (left). Representative SAX images (lower row) with demarcated end-diastolic (red) and end-systolic (green) ventricular dimension after Atenolol and Iso treatment. (B-E) Quantification of changes in heart rate (HR) (B), fractional area change (FAC) (C), fractional shortening (FS) (D), and ejection fraction (EF) (E) after AT or Iso treatment compared to controls (C). Small numbers in columns indicate number of animals measured. (F) For speckle-tracking analysis, the ventricle was divided in six segments as indicated with segment 1 and 2 representing the anterior wall, 3 and 4 the apex, and 5 and 6 the posterior wall. Time-to-peak analysis reveals that AT leads to significantly prolonged time-to-peak indices in myocardial velocity of all segments. Small numbers in columns indicate number of animals measured. Values are expressed as mean ± SEM. Ap, Apex; AT, Atenolol; AW, anterior wall; Iso, Isoproterenol; PW, posterior wall; *, p<0.05; unpaired student’s t-test.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4390243&req=5

pone.0122665.g003: Changes in cardiac performance in response to beta-adrenergic stimulation and blockage.(A) Representative pulsed-wave Doppler (PWD) signals (upper row) acquired from ACX view illustrating reduced and elevated heart rate in Atenolol (middle, AT) and Isoproterenol (right, Iso) treated zebrafish, respectively, compared to controls (left). Representative SAX images (lower row) with demarcated end-diastolic (red) and end-systolic (green) ventricular dimension after Atenolol and Iso treatment. (B-E) Quantification of changes in heart rate (HR) (B), fractional area change (FAC) (C), fractional shortening (FS) (D), and ejection fraction (EF) (E) after AT or Iso treatment compared to controls (C). Small numbers in columns indicate number of animals measured. (F) For speckle-tracking analysis, the ventricle was divided in six segments as indicated with segment 1 and 2 representing the anterior wall, 3 and 4 the apex, and 5 and 6 the posterior wall. Time-to-peak analysis reveals that AT leads to significantly prolonged time-to-peak indices in myocardial velocity of all segments. Small numbers in columns indicate number of animals measured. Values are expressed as mean ± SEM. Ap, Apex; AT, Atenolol; AW, anterior wall; Iso, Isoproterenol; PW, posterior wall; *, p<0.05; unpaired student’s t-test.
Mentions: The adult zebrafish heart is approximately 1000 fold smaller than a human heart and still 10 fold smaller than mice hearts. The highly trabecularized ventricle measures approximately 1mm in length and is 0.5-1mm wide, with a compact myocardial layer of 50–100μm thickness [43, 44]. To overcome this significant size limitation we deployed a high frequency ultrasound system specifically developed for small animals, with a previously demonstrated axial and lateral resolution of approximately 40μm and 70μm, respectively, hence providing the appropriate resolution for imaging [45, 46]. Importantly, this system was previously deployed for conventional echocardiography measurements in adult zebrafish [21, 24]. We initially defined three examination planes to globally measure cardiac function in adult zebrafish (Fig 1A and 1B). These three planes were specifically adapted to zebrafish anatomy and defined according to standardized echocardiographic evaluation in clinical practice. In a 60° cranial angulated transversal section, circumferential ventricular contractions can be recorded in a short axis view (SAX) using B-Mode imaging (Fig 1A1, 1B1 and 1C1, S1 Fig and Fig 1, see also S1 Movie). From these images systolic and diastolic circumferential ventricular area can be measured and the fractional area change (FAC), as a parameter for circumferential contraction, can be calculated as later shown in Fig 3A (lower row). To achieve a long axis view of the heart (LAX) we used a strictly medial craniocaudal sagittal section (Fig 1A2 and 1B2). In this view, the ventricular as well as the cardiac outflow tract (bulbus arteriosus) performance can be visualized in a sagittal long axis view (LAX) by B-Mode imaging (Fig 1C2, S2 Fig and Fig 2, see also S2 Movie). We used this imaging plane to subsequently assess the systolic and diastolic longitudinal ventricular area to calculate the ejection fraction (EF), fractional shortening (FS) and stroke volume (SV). To further characterize cardiac blood flow velocities, we applied pulsed-wave Doppler (PWD) measurements in a 45° abdominal-cranial angulated transversal plane (ACX) ensuring a parallel alignment of ultrasound beam and cardiac blood flow (Fig 1A3 and see also S3 Movie). Additionally, this plane enables visualization of the ventricular apex and the atrioventricular- and bulboventricular valves. Importantly, PWD signals have been depicted from the area of maximum inflow and outflow velocities, defined by applying C-Mode imaging. The maximum cardiac inflow velocities were usually found at the leaflets region of the atrioventricular valve, whereas the maximum outflow velocity can usually be detected, depending on the animal’s size, 35–50μm more cranial, close to the bulboventricular valve and the upper thoracic aperture. From this, a PWD signal was recorded for at least three seconds to reliably calculate the heart rate and ventricular velocity time integral (V VTI), as a parameter describing cardiac output performance (Fig 1D, see also S3 Movie). From the maximum inflow PWD signal, the heart rate, atrioventricular velocity time integral (A VTI), maximum velocity of A- and E-wave (Amax and Emax) were measured and the E/A ratio, as a parameter of diastolic function was calculated (Fig 1D). The standard parameters assessed from adult zebrafish applying this protocol are presented in Table 1.

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