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Front Physiol
2019 Jan 01;10:1197. doi: 10.3389/fphys.2019.01197.
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Quantitative Phenotyping of Xenopus Embryonic Heart Pathophysiology Using Hemoglobin Contrast Subtraction Angiography to Screen Human Cardiomyopathies.
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Congenital heart disease (CHD) is a significant cause of mortality in infants and adults. Currently human genomic analysis has identified a number of candidate genes in these patients. These genes span diverse categories of gene function suggesting that despite the similarity in cardiac lesion, the underlying pathophysiology may be different. In fact, patients with similar CHDs can have drastically different outcomes, including a dramatic decrease in myocardial function. To test these human candidate genes for their impact on myocardial function, we need efficient animals models of disease. For this purpose, we paired Xenopus tropicalis with our microangiography technique, hemoglobin contrast subtraction angiography (HCSA). To demonstrate the gene-teratogen-physiology relationship, we modeled human cardiomyopathy in tadpoles. First we depleted the sarcomeric protein myosin heavy chain 6 (myh6) expression using morpholino oligos. Next, we exposed developing embryos to the teratogen ethanol and in both conditions showed varying degrees of cardiac dysfunction. Our results demonstrate that HCSA can distinguish biomechanical phenotypes in the context of gene dysfunction or teratogen. This approach can be used to screen numerous candidate CHD genes or suspected teratogens for their effect on cardiac function.
Figure 1. Physiological quantification of embryoheart function in MYH6 morphants. For this experiment 29 control, 39 morphants-−1 ng morpholino injected and 13 morphants-−2 ng morpholino injected analyzed. Measurements derived from manual segmentation include surface area at the end of diastole and systole and the change in ventricle area. (A) Before-after graph presented with extra columns pointing mean with SEM in red. Green line between means flattens as change in surface area diminishes with increasing MO dose. (B,C) As the MO dose is increased, the change in ventricle area is reduced, and the ventricle sizes are smaller. (D) Measurements derived from HCSA application include Hb blush at the end of diastole and systole and the change in blush area. Before-after graph presented with extra columns pointing mean with SEM in red. Green line between means flattens as change in blush area diminishes with increasing MO dose. (E,F) Following HCSA application, Hb blush is quantified at the end of diastole and systole. Ejection Fraction is derived from these measurments. Morphants demonstrated a dose dependent diminished ejection fraction and (G) stroke area. (H) Myocardial mass index estimates the amount of cardiac mass within the manually segmented heart. Myocardial mass index is affected in 2 ng morphants but remains within normal limits in 1 ng morphants. SEM, standard error of the mean; Hb Blush, Hemoglobin-containing pixels; tEDA, total end-diastolic area; tESA, total end-systolic area; ΔtA, change in total area; EDBA, end diastolic blood area; ESBA, end-systolic blood area; EF, ejection fraction; MMI, myocardiac mass index; HCSA, Hb contrast subtraction angiography. ****p < 0.0001, **p < 0.01, and *p < 0.05.
Figure 2. Physiological quantification of embryoheart function in EtOH treated tadpoles. For this experiment 30 controls and 30 EtOH exposed tadpoles analyzed. Measurements derived from manual segmentation includes surface area at the end of diastole and systole and the change in ventricle area. (A) Before-after graph presented with extra columns pointing mean with SEM in red. Green line between means flattens as change in surface area diminishes in EtOH group. (B,C) Ventricles of the ethanol exposed tadpoles were slightly smaller in size but had diminished change in the ventricle area. Following HCSA application, Hb blush quantified at the end of diastole and systole. Ejection fraction is derived from these measurements. (D) Before-after graph presented with extra columns pointing mean with SEM in red. Green line between means flattens as change in blush area diminishes in EtOH group. (E,F) Ethanol exposed tadpoles had less blood at the end of diastole and blood is not propelled efficiently leading an increase in end systolic Hb blush, diminished ejection fraction, and diminished (G) stroke area. (H) Myocardial Mass Index. In ethanol exposed tadpoles myocardial mass is diminished. SEM, standard error of the mean; Hb Blush, Hemoglobin-containing pixels; tEDA, total end-diastolic area; tESA, total end-systolic area; ΔtA, change in total area; EDBA, end diastolic blood area; ESBA, end-systolic blood area; EF, ejection fraction; MMI, myocardiac mass index; HCSA, Hb contrast subtraction angiography. ****p < 0.0001, ***p < 0.001, and **p < 0.01.
S1. Quantitative Phenotyping of Xenopus Embryonic Heart Pathophysiology Using Hemoglobin Contrast Subtraction Angiography to Screen Human Cardiomy
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