PMC6500747_01
Clinical features of patients.. (A) Table showing medical history of healthy controls (with or without CV risk factors), DCM control, and PPCM patients. (B) Echocardiogram of PPCM patient showing stably reduced LV systolic function without any significant changes. (C) Angiogram of PPCM patient showing coronaries without any significant obstruction.
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F1
Review of the chart showed no apparent epigenetic insults till the time of her transplant to negatively influence her coronary vascular function and she was free of conventional cardiovascular risk factors including dyslipidemia, obesity, diabetes, hypertension, and substance abuse (Figure 1A). Despite no pre-existing heart condition, her echocardiogram showed marked LV dilatation with severely reduced LV ejection fraction (<20%) (Figure 1B). Importantly, her angiogram revealed coronaries without significant obstructive disease (Figure 1C).
Vascular reactivity in PPCM heart.. (A) Explanted PPCM heart, dissected coronary arteries and wire-myograph. (B) Endothelium function in PPCM heart. Isometric tension recordings of relaxation to Acetylcholine (10muM), Carbachol (10muM), and NO donor SNP (3muM) upon pre-constriction with U46619. (C) Tension recordings of PPCM LAD segments showing less contraction at basal tone upon application of 10muM linopirdine when compared to DCM and healthy control (left panel); impaired adenosine response in PPCM or in presence of 10muM linopirdine (right panel). Statistical analyses were done on three segments (n=3) from one coronary vessel per patient (N=1) using two-way ANOVA. *p< 0.5, **p< 0.05, ***p< 0.01.
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F2
Using isometric tension recordings of vessel reactivity, we investigated the coronary vasculature ex vivo utilizing explanted hearts from the PPCM patient (Figure 2A), an age/gender-matched DCM patient, and a gender-matched healthy donor patient. Wire-myograph measurements found impaired endothelial cell function and nitric oxide production in PPCM and DCM as compared to the healthy control (Figure 2B). Also, responses to linopirdine failed to elicit vasoconstriction in PPCM LADs at basal tone as compared to both healthy control and DCM LADs (Figure 2C, left panel). In healthy LADs, adenosine induced ~30% dilation, but this effect was absent in PPCM coronaries incubated with linopirdine (Figure 2C, right panel).
Functional characteristics of primary endothelial cells from PPCM heart.. (A) Representative images of primary endothelial cells isolated from healthy and PPCM hearts. (B) Representative images of capillary-like networks formed by primary ECs showing impaired tube formation by PPCM ECs compared to healthy controls. (C) Bar graph showing quantification of the number of tubes formed by the primary ECs. (D) Representative fluorescent images of Ac-LDL uptake by primary ECs showing reduced capacity to incorporate Ac-LDL by PPCM ECs compared to healthy controls. (E) Bar graph showing quantification of Ac-LDL fluorescence intensity in primary ECs. All data represented as mean +- SEM, n=3, *p<0.05. Statistical analyses were done using standard Student t test and Mann-Whitney nonparametric test.
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F3
For this, we isolated primary ECs from the control and PPCM heart's coronary vasculature (Figure 3A), and then subjected them to functional assays to assess their endothelial phenotype. Consistent with our wire myography data, primary ECs isolated from the PPCM patient's LADs showed a decreased capacity to form networks of tubular structures when placed on matrigel compared to primary ECs isolated from healthy controls (Figure 3B-C). Similarly, the PPCM patient's ECs incorporated significantly less Ac-LDL when compared to control patient (Figure 3D-E).