Modeling propofol-induced cardiotoxicity in the isolated-perfused newborn mouse heart

Abstract

Infants and children are vulnerable to developing propofol infusion syndrome (PRIS) and young age is a risk factor. Cardiac involvement is often prominent and associated with death. However, the mechanisms of pediatric PRIS are poorly understood because of the paucity of investigation and lack of a gold standard animal model. Unfortunately, in vivo modeling of PRIS in a newborn mouse is not feasible and would be complicated by confounders. Thus, we focused on propofol-induced cardiotoxicity and aimed to develop an ex-vivo model in the isolated-perfused newborn mouse heart. We hypothesized that the model would recapitulate the key cardiac features of PRIS seen in infants and children and would corroborate prior in vitro observations. Isolated perfused newborn mouse hearts were exposed to a toxic dose of propofol or intralipid for 30-min. Surface electrocardiogram, ventricular contractile force, and oxygen extraction were measured over time. Real-time multiphoton laser imaging was utilized to quantify calcein and tetramethylrhodamine ethyl ester fluorescence. Propidium iodide uptake was assessed following drug exposure. A toxic dose of propofol rapidly induced dysrhythmias, depressed ventricular contractile function, impaired the mitochondrial membrane potential, and increased open probability of the permeability transition pore in propofol-exposed hearts without causing cell death. These features mimicked the hallmarks of pediatric PRIS and corroborated prior observations made in isolated newborn cardiomyocyte mitochondria. Thus, acute propofol-induced cardiotoxicity in the isolated-perfused developing mouse heart may serve as a relevant ex-vivo model for pediatric PRIS.

Keywords: Langendorff preparation; cardiotoxicity; heart; isolated-perfused heart; model; newborn; propofol; propofol infusion syndrome.

FIGURE 1

Propofol impairs heart rate, cardiac rhythm, and ventricular contractile force. (a) Representative tracings of surface electrocardiogram (ECG) and ventricular contractile force (tension) over time. Tracings captured prior to exposure (baseline) and at various time points during exposure are depicted. Heart rate (HR) is provided in beats per minute. Black arrows indicate P waves in the propofol‐exposed ECG tracing while the red arrow indicates a dissociated ventricular beat. (b) Heart rate and ventricular contractile force over time. Values are means ± SD. n = 6–8 per group. p values were calculated by two‐way ANOVA with repeated measures. *p < 0.05, ^† p < 0.01, ^‡ p < 0.001 versus time‐matched intralipid values.

FIGURE 2

Percent myocardial oxygen extraction and oxygen consumption over time. Values are means ± SD. N = 6–8 per group. Significance was assessed with two‐way ANOVA with repeated measures.

FIGURE 3

Propofol compromises ΔΨ and opens the mPTP. Real‐time multiphoton laser imaging of isolated‐perfused newborn hearts was used to measure ΔΨ and determine open probability of the mPTP during propofol or intralipid exposure. Following stabilization, hearts were loaded with calcein‐AM and tetramethylrhodamine ethyl ester (TMRE) and then exposed to either propofol or intralipid for 30 min. Calcein was quenched with cobalt chloride during each exposure. Intramitochondrial calcein (green) and actively respiring mitochondria (TMRE; red fluorescence) were identified within ventricular cardiomyocytes. Nuclei can be seen as circular voids. (a) Representative images obtained at 25× magnification after 15 min of exposure. Scale bar is 100 μm. (b) Representative merged images obtained at 100× magnification after 15 and 30 min of exposure. Scale bar is 50 μm. (c) Graphical quantification of myocardial calcein and TMRE fluorescence over time is shown. Intralipid values at the 15‐min mark were arbitrarily set to 1. n = 3 biological replicates per exposure group per time point. Calcein and TMRE fluorescence within 3–4 imaged fields per replicate were quantified. p values were calculated using two‐way ANOVA with repeated measures. *p < 0.05, ^† p < 0.01 versus intralipid values at the 15‐min mark.

FIGURE 4

Lack of cell death in propofol‐ and lipid‐exposed hearts. Propidium iodide (PI) uptake by cardiomyocyte nuclei was assessed and quantified following exposure to propofol or intralipid. Ischemia‐reperfused hearts served as positive controls. Representative images obtained at 10× magnification are shown (above). Scale bar is 100 μm. Bright punctate PI‐positive nuclei are easily seen. Graphical quantification of the number of myocardial PI‐positive nuclei is shown (below). n = 3 biological replicates per exposure group. PI fluorescence within 3–4 imaged fields per replicate was quantified. p values were calculated using one‐way ANOVA. ^‡ p < 0.001 versus propofol‐ and intralipid‐exposed hearts.