Myocardial growth response to fetal Intralipid infusion in sheep

Abstract

Cardiomyocyte proliferative maturation and metabolic maturation occur in the same perinatal period. Interauterine conditions influence developmental trajectories of both processes, however their interconnectedness is unknown. Circulating fetal lipid levels are typically low, but the heart may be prematurely exposed to elevated lipids. We experimentally increased fetal lipid levels to determine the impact on cardiomyocyte proliferative maturation. Fetal sheep were surgically instrumented with catheters. After recovery, Intralipid 20^® or Lactated Ringer’s Solution were infused according to a clinical g/kg schedule for 8 days until 133±1 days of gestation. A left ventricular biopsy was fixed, and remaining cardiomyocytes enzymatically dissociated. Myocardial composition was measured from Masson’s Trichrome-stained sections. Cardiomyocyte length, width, nucleation and Ki-67+ were studied in dispersed cells. Myocardial composition, cardiomyocyte dimensions, and Ki-67+ were not found to be different between groups. Cardiomyocytes of the Intralipid-treated fetuses were 14% more terminally differentiated than Controls (P=0.025). Early developmental exposure to circulating lipid accelerates fetal cardiomyocyte terminal differentiation, and may contribute to fewer cardiomyocytes for life.

INTRODUCTION

Around the time of birth, the heart ceases to grow by cardiomyocyte proliferation, and its primary energy source changes from carbohydrates to lipids. Intrauterine conditions such as placental insufficiency [1–5], exposure to thyroid hormone [6, 7], and maternal obesity [8, 9] alter the developmental trajectories of both cardiomyocyte proliferation and metabolism. Although the processes of terminal differentiation [10–13] and metabolic maturation [14–17] appear coordinated, it is unclear if they are co-regulated [18]. We have shown that preterm exposure to elevated lipids changes cardiomyocyte metabolism [19], but whether lipids cause cardiomyocyte terminal differentiation is unknown.

Fetal cardiomyocyte proliferation establishes the peak number for life, after which replacement proliferation may slow the decline in number but is insufficient to repair damage due to disease [11, 12, 20]. Terminal differentiation, marked in sheep and other species by the appearance of two nuclei per cell, is the end of proliferative growth for a cardiomyocyte [10–12]. If lipid exposure promotes cardiomyocyte terminal differentiation, it may reduce the peak cardiomyocyte number.

Circulating fetal lipid levels are typically very low. Dysregulation of maternal lipid metabolism, such as in gestational diabetes or in polycystic ovarian syndrome, elevates fetal lipid levels relative to a normal developmental timeline [21–24]. Preterm birth also exposes immature hearts to elevated circulating lipid levels with the onset of parenteral nutrition (PN)[25–29]. In order to understand how early introduction of lipids alters growth and maturation of cardiomyocytes, we infused late gestation fetal sheep with Intralipid 20^® at at clinically-appropriate dose for 8 days, and then studied changes within the myocardium. We hypothesized that Intralipid 20^® exposure would accelerate cardiomyocyte terminal differentiation and hypertrophy.

METHODS

Animals

Oregon Health & Science University’s Institutional Animal Care and Use Committee approved and oversaw all animal experiments. Experimental methodologies for fetal surgeries and experiments, including fetal blood pressure and chemistry are published [30]. Briefly, fetuses were surgically catheterized and received Intralipid 20^® per manufacturer’s recommendations for premature human infants. Control fetuses received an equal volume of Lactated Ringer’s Solution. Triglyceride levels achieved were comparable to premature infants given continuous lipid infusion, and do not indicate lipid intolerance [30, 31]. Fetuses were 125±1 days of gestational age (dGA; term is 147 dGA) on day 0 and euthanized at 133±1 dGA. A full-thickness left ventricular midventricular section was removed for fixation in freshly made 4% formaldehyde and the heart was enzymatically digested into individual cardiomyocytes as previously described [32].

Cell and myocardial measurements

Cardiomyocyte cell cycle activity, terminal differentiation and size were measured from isolated cells as previously described [33].

Following fixation, the left ventricular midwall biopsy was embedded in paraffin, sectioned to 5 μm, and stained with Masson’s Trichrome. Relative quantities of the main constituent parts of the left ventricular myocardium were counted from electronic photomicrograph images using an intersecting grid counting technique as previously described [34].

Statistics

Fisher’s exact test was used to assess dichotomous variables in GraphPad Prism (v.10.1.0). All other statistical analyses were carried out in SPSS (v.29.0.0.0). Myocyte parameters were assessed by mixed measures three-way ANOVA (in vivo treatment, sex, ventricular wall) with the Greenhouse-Geisser correction for sphericity. was used to assess Normality of continuous data (Shapiro-Wilk’s test) andhomogeneity of variances (Levene’s test for equality of variances) were assessed. Outliers were assessed in boxplots as data points 1.5 box-lengths or more from the edge of the box. Multiple comparisons used the Šidák correction. Myocardial composition was assessed by two-way ANOVA (in vivo treatment, sex). Significance was defined at α=0.05.

RESULTS

Prior study in this cohort found that heart weight and relative heart weight were not different between Intralipid-treated fetuses and Controls [30].

Cardiomyocyte growth and maturation

Cell cycle activity was not different between groups (Figure 1A, Table 1). Cardiomyocyte binucleation following treatment was 14% higher in the Intralipid group compared to Controls (P=0.025; Figure 1B). Intralipid treatment did not change cardiomyocyte size compared to Controls.

Figure 1. Cardiomyocyte proliferation, terminal differentiation, and size.

Isolated cardiomyocytes were assessed for A) nuclear ki-67 positivity as an index of cell cycle activity (at least 1000 cells per ventricle per animal), and B) binucleation as an index of terminal differentiation (at least 500 cells per ventricle per animal). Isolated cardiomyocytes (at least 100 cells per ventricle per animal) were measured to determine cell C) mononucleate length, D) mononucleate width, E) binucleate length, and F) binucleate length. Raw data with mean. Control female n=7, male n=4; Intralipid female n=4, male n=7. Data were assessed by 3-way ANOVA. Significant P-values (<0.05) shown for differences by treatment and ventricle; there were no differences by sex.

Table 1.

P-values from statistical analyses of cardiac myocyte measurements.

					Main Effects
	3-Way Interaction	2-Way Interactions			Test only if 2- and 3-way interactions are NS.
	Treatment×Sex× Ventricular wall	Test only if 3-way interaction is NS.						Subgroup characteristics
		Treatment×Sex	Treatment× Ventricular wall	Sex× Ventricular wall	Treatment	Sex	Ventricular wall	Normal distribution	Homogeneity of variances	Outliers
Cell cycle activity	0.109	0.560	0.683	0.370	0.324	0.304	0.093	11/12a	3/3	1c
Binucleation	0.734	0.645	0.853	0.141	0.025 d	0.159	0.027 e	11/12a	3/3	2c
Mononucleate length	0.878	0.620	0.254	0.929	0.711	0.831	<0.001 f	12/12	2/3b	1c
Mononucleate width	0.921	0.160	0.081	0.501	0.094	0.517	<0.001 g	11/12a	3/3	0
Binucleate length	0.635	0.736	0.328	0.648	0.908	0.990	<0.001 h	10/12a	2/3b	2c
Binucleate width	0.823	0.978	0.592	0.775	0.261	0.677	<0.001 i	11/12a	3/3	3c

Number for Control=11 (female=7, male=4), Intralipid=10 (female=4, male=6). Mixed measures three-way ANOVA (in vivo treatment, sex, ventricular wall) with the Greenhouse-Geisser correction for sphericity. Left ventricle (LV), not significant (NS), right ventricle (RV)

^(a)Indicated proportion of subgroups were normally distributed. Although ANOVAs are fairly robust to deviations from normality, interpret results with caution.

^(b)Only indicated proportion of subgroups had homogeneity of variances. Although ANOVAs are fairly robust to heterogeneity of variance, interpret results with caution.

^(c)Number of number of outliers found and determined to be biologically relevant and included in analysis (out of N=63).

P-values for multiple comparisons using Šidák correction following significant main effects:

^(d)Control vs. Intralipid=0.025, Cohen’s d=−0.91 (“large”)

^(e)LV vs. Septum=0.005, Cohen’s d=0.48 (“small”).

^(f)RV vs. LV=0.003, Cohen’s d=1.19 (“large”); RV vs. Septum=0.015, Cohen’s d=0.87 (“large”).

^(g)RV vs. LV < 0.001, Cohen’s d=1.48 (“large”); RV vs. Septum < 0.001, Cohen’s d=1.30 (“large”).

^(h)RV vs. LV=0.002, Cohen’s d=1.08 (“large”); RV vs. Septum=0.027, Cohen’s d=0.74 (“large”); LV vs. Septum=0.035, Cohen’s d=−0.58 (“medium”).

⁽ⁱ⁾RV vs. LV < 0.001, Cohen’s d=1.32 (“large”); RV vs. Septum < 0.001, Cohen’s d=1.41 (“large”).

Myocardial composition

Cardiac myocytes comprised the majority of myocardium (79.6%). Connective tissue was the second-most abundant category (11.7%), while blood vessels and non-myocyte cells were the least (9.9%). There were no differences between groups.

DISCUSSION

We found high circulating lipids increased cardiomyocyte terminal differentiation in late-gestation sheep. There were no changes in cell cycle activity, a process which supports both proliferation and the process of terminal differentiation. Given binucleated cardiomyocytes are approximately twice the size of mononucleated cardiomyocytes, the hearts being similar in size between the groups, and unchanged myocardial composition, we infer there were fewer cardiomyocytes generated in the hearts exposed to high lipid levels. The consequence of early terminal differentiation due to preterm hyperlipidemia may thus be reduced cardiomyocyte number at the conclusion of the fetal period.

Maternal obesity has also been associated with cardiomyocyte hypertrophy in offspring [35, 36], which can follow from early terminal differentiation. Consistent with our study, fetal mice exposed to a maternal obesigenic diet did not have decreased cell cycle activity but had small left ventricles, suggesting fewer myocytes [37].

Potentially important candidate signaling molecules for linking cardiomyocyte metabolism to proliferative maturation include the peroxisome proliferator–activated receptors (PPAR), the Hippo-YAP pathway, and transcription factors Meis1 and FOXO1 [38–42]. The Warburg effect may also play a role [43].

In conclusion, high circulating lipid levels prior to the developmentally appropriate timepoint of term birth alters cardiac development. Ventricular ardiomyocytes undergo terminal differentiation prematurely, and fewer cardiomyocytes are created. Further studies will be required to determine the regulatory mechanisms. These changes may contribute to pathology related to hyperlipidemic pregnancy or preterm birth.

Figure 2. Myocardial composition.

A) Left ventricular myocardium was assessed (480 samples per animal) to determine relative quantities of cardiac myocyte cell bodies and nuclei, connective tissue, and blood vessel walls and lumens or other cells. Raw data with mean. Control female n=7, male n=4; Intralipid female n=3, male n=7. Data were assessed by 2-way ANOVA. There were no differences by treatment or sex. B) Representative image from a Control heart. C) Representative image from an Intralipid heart.

Table 2.

P-values from statistical analyses of myocardial composition.

	2-Way Interaction	Main Effects		Subgroup data characteristics
		Test only if 2-way interaction is NS.		Normal distribution	Heterogeneity of variances	Outliers
	Treatment×Sex	Treatment	Sex
Cardiomyocyte	0.253	0.632	0.830	3/4 a	0.300	1 b
Vasculature	0.486	0.455	0.832	4/4	0.383	0
Connective tissue	0.165	0.623	0.539	3/4 a	0.165	1 b

Number for Control=11 (female=7, male=4), Intralipid=10 (female=4, male=6). Two-way ANOVA. Not significantly different (NS).

^(a)Indicated proportion of subgroups were normally distributed. Although ANOVAs are fairly robust to deviations from normality, interpret results with caution.

^(b)Number of number of outliers found and determined to be biologically relevant and included in analysis (out of N=21).

DATA SHARING

Data available in the manuscript and upon reasonable request of the corresponding author.