Integrative Physiology

Endocardium Contributes to Cardiac FatNovelty and Significance

Hui Zhang, Wenjuan Pu, Qiaozhen Liu, Lingjuan He, Xiuzhen Huang, Xueying Tian, Libo Zhang, Yu Nie, Shengshou Hu, Kathy O. Lui, Bin Zhou
https://doi.org/10.1161/CIRCRESAHA.115.307202
Circulation Research. 2016;118:254-265
Originally published December 9, 2015

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Abstract

Rationale: Unraveling the developmental origin of cardiac fat could offer important implications for the treatment of cardiovascular disease. The recent identification of the mesothelial source of epicardial fat tissues reveals a heterogeneous origin of adipocytes in the adult heart. However, the developmental origin of adipocytes inside the heart, namely intramyocardial adipocytes, remains largely unknown.

Objective: To trace the developmental origin of intramyocardial adipocytes.

Methods and Results: In this study, we identified that the majority of intramyocardial adipocytes were restricted to myocardial regions in close proximity to the endocardium. Using a genetic lineage tracing model of endocardial cells, we found that Nfatc1+ endocardial cells contributed to a substantial number of intramyocardial adipocytes. Despite the capability of the endocardium to generate coronary vascular endothelial cells surrounding the intramyocardial adipocytes, results from our lineage tracing analyses showed that intramyocardial adipocytes were not derived from coronary vessels. Nevertheless, the endocardium of the postnatal heart did not contribute to intramyocardial adipocytes during homeostasis or after myocardial infarction.

Conclusions: Our in vivo fate-mapping studies demonstrated that the developing endocardium, but not the vascular endothelial cells, gives rise to intramyocardial adipocytes in the adult heart.

Introduction

More than 64% of adults in the United States and more than half of the population in other developed countries are overweight or obese.1 Obesity is among the most significant risk factors in the development of common medical conditions including type 2 diabetes mellitus, cardiovascular diseases, stroke, and fatty liver disease. Given that obesity has become an epidemic and obesity-associated cardiovascular complications contribute to one of the principal causes of death, there has been a growing interest in understanding the developmental origins of adipose tissues.2 Indeed, recognition of cellular ontogeny would provide a necessary context to address issues associated with fat tissue metabolism, cardiovascular diseases, as well as cardiac repair and regeneration.

Fat not only stores up in subcutaneous tissues but also accumulates in ectopic sites such as heart, liver, and muscle, causing some organ-specific diseases. In the heart, the most obvious fat can be found in the epicardium, which forms epicardial adipose tissues. Recent studies based on genetic lineage tracing strategy revealed epicardium as the developmental origin of these epicardial fat cells.3–5 Furthermore, the adult epicardial cells could be reactivated and respond to cardiac injury, giving rise to new adipocytes during myocardial infarction (MI).5 In addition to the epicardial location, fat could also accumulate deep inside the myocardium, which was regarded as intramyocardial adipocytes. Excessive intramyocardial fat depots are associated with many cardiovascular diseases such as cardiac lipotoxicity, arrhythmogenic right ventricular cardiomyopathy, left ventricular tachycardia after MI, inflammation, atherosclerosis, and heart failure.6–9 Nevertheless, it remains largely unknown about the developmental origin of the intramyocardial adipocytes. Therefore, understanding the cellular ontogeny of the intramyocardial fat tissue not only is important for developmental biology but also contributes direct implications on novel treatment options for cardiovascular diseases.2,10,11

Endocardium is a layer of specialized endothelial cells that cover the trabecular myocardium in developing heart. Endocardium represents a unique cardiac lineage derived from Flk+ multipotent cardiovascular progenitors.12 During cardiac valve development, endocardial cells undergo endothelial-to-mesenchymal transition (EMT) to form endocardial cushion, which is essential for the formation and remodeling of valves.13 In addition to valve mesenchymal cell contribution, endocardial cells could also give rise to coronary vascular endothelial cells (VECs).14,15 However, it is still controversial over the magnitude by which the endocardial cells contribute to coronary vessels in the developing heart.14–18 Recent study showed that endocardial cells contribute to coronary vessels in ventricular septum and the inner myocardial wall during late embryonic and early neonatal stages.19 These studies, albeit with difference in quantitative and temporal contribution, confirmed the involvement of endocardium in coronary angiogenesis. It has been reported that the vascular endothelium gives rise to both white and brown adipocytes, suggesting a model to study coordination of adipogenesis and angiogenesis during adipose tissue expansion.20 Indeed, the development of adipose tissues is spatially and temporally associated with angiogenesis,21,22 indicating the possible involvement of coronary angiogenesis during intramyocardial adipocyte formation.

In this study, we reported that most intramyocardial adipocytes were restricted to myocardial regions in close proximity to the endocardium. Genetic lineage tracing analyses showed that the endocardial cells contribute to both intramyocardial adipocytes and coronary VECs. Our fate-mapping studies also revealed that endocardial cells, but not VECs, give rise to intramyocardial adipocytes in the adult heart. The identification of endocardial source of intramyocardial fat will enable further studies on the biochemical and physiological cues regulating fat tissue formation inside the heart.

Methods

Detailed Materials and Methods are provided in the Online Data Supplement. All animal procedures were approved by the Institutional Animal Care and Use Committee of the Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Science. Immunostaining and in situ hybridization were performed according to protocol described previously.23 All data were presented as mean±SEM. Statistical comparisons between data sets were done by a 2-sided unpaired Student t test for comparing differences between 2 groups. P<0.05 was considered to be statistically significant.

Results

Intramyocardial Fat Tissues Are More Restricted to the Myocardium Facing Toward the Endocardium

To understand where the intramyocardial adipocytes located, we performed immunostaining for the lipid droplet-associated protein, perilipin on heart sections of mice at different stages. We collected hearts at embryonic day (E) 15.5, postnatal day (P) 0, 1 week (P1w), P2w, P3w, P4w, and P8w. Immunostaining of perilipin showed that both intramyocardial and epicardial fat tissues appeared at approximately P3w to P4w, and the number of perilipin+ cells increased at P8w (Figure 1A; Online Figure IA). We confirmed this phenotype of intramyocardial adipocytes by oil red staining (Online Figure IB). In P26w adult heart, these intramyocardial oil red+ adipocytes were in close proximity to the innermost layer of the endocardium with some entirely attached to the endocardium (Figure 1B). In addition, costaining of perilipin and the cardiomyocyte-specific marker troponin I, cardiac 3 revealed that perilipin+ adipocytes in the epicardial layer were restricted to atrioventricular groove (Figure 1C, arrowheads), whereas the intramyocardial perilipin+ adipocytes lied among cardiomyocytes located in close proximity to the endocardium (Figure 1C and 1D, arrows).

Figure 1.
Figure 1.

Intramyocardial fat is highly enriched in regions close to the endocardium of adult heart. A, Immunohistochemical staining of the lipid droplet-associated protein, perilipin (PLIN, brown) on embryonic and postnatal hearts at different stages. Intramyocardial PLIN+ adipocytes (arrows) start to appear at the postnatal (P) 4 wk (w), P4w. B, Oil red staining on heart sections of P26w-old mice. Oil red+ fat cells are more restricted to regions close to the endocardium (arrows) and epicardium (arrowheads). B1 and B2 are magnified images showing fat cells. C, Immunostaining of PLIN and troponin I, cardiac 3 (TNNI3) in P31w heart. Nuclei are stained with DAPI (4′,6-diamidino-2-phenylindole). Arrowheads indicate epicardial fat in the atrioventricular groove. D, Magnified image shows restricted location of PLIN+ adipocytes (arrows) in the TNNI3+ myocardium close to the endocardium. E, Quantification of the percentage of fat cells and distance away from the endocardium in adult mice hearts. Distance of 0 µm indicates close contact of adipocytes with the endocardium. More than 95% intramyocardial adipocytes are within 100 µm from the endocardium, and <5% intramyocardial adipocytes are >100 µm away from the endocardium. Values are shown as mean±SEM; n=3. LA indicates left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle; and VS, ventricular septum. Scale bars, 1 mm in B and C; 100 µm in A and D.

We also noticed that some intramyocardial adipocytes were not located in close proximity to the innermost layer of endocardium facing the ventricular chamber, but they reside close to the endocardium that lines between 2 blocks of myocardium (Online Figure II). To address whether the endothelial cells lining between the 2 blocks of myocardium were indeed part of the endocardium, we used the coronary VEC-specific marker fatty acid binding protein 4 (FABP4)24,25 to distinguish coronary VECs (FABP4+cadherin 5+) from endocardium (FABP4−cadherin 5+). The endothelial cells lining between the 2 blocks of myocardium were FABP4−cadherin 5+ endocardial cells, and intramyocardial adipocytes were located next to them (Online Figure IIA and IIB). Similarly, we used an additional coronary VEC-specific marker apeline to distinguish endocardial cells (apeline−platelet/endothelial cell adhesion molecule 1 [PECAM]+) and coronary VECs (apeline+PECAM+). We found that a subset of intramyocardial adipocytes were located close to these endocardial cells that lined between the 2 blocks of myocardium (Online Figure IIC and IID). Quantification of the distance between the perilipin+ adipocytes and the endocardium showed that >95% of the intramyocardial adipocytes were within 100 µm from the endocardium (Figure 1E). These data showed that the intramyocardial adipocytes were highly enriched in regions close to the endocardium of the adult heart.

Lineage Tracing Reveals an Endocardial Origin of the Intramyocardial Adipocytes

The above data suggested a locational correlation between intramyocardial adipocytes and the endocardium, implicating that the endocardial cells could contribute to the adipocytes. To address this hypothesis, we performed nuclear factor of activated T cell (Nfatc1) promoter-driven lineage tracing experiments. Nfatc1 was specifically expressed in the endocardium of early developing heart at E9.5; however, its expression was significantly reduced at later embryonic and neonatal stages (Online Figure IIIA). Moreover, previous lineage tracing experiments using Nfatc1-IRES-Cre knockin mouse line showed that the Nfatc1-IRES-Cre labeled endocardial cells in developing embryos.15 Therefore, we generated similar mouse line by knocking Dre recombinase into the ninth exon of Nfatc1 with a linking peptide 2A via homologous recombination (Figure 2A). Dre recombinase is a Cre-like site-specific recombinase that catalyzes recombination at the rox sites rather than the loxp sites.26,27 We crossed Nfatc1-Dre with rox-specific reporter Rosa26-rox-stop-rox-red fluorescence protein (RFP), which is named as R26-RSR-RFP throughout this study.28 In Nfatc1-expressing cells, Dre-Rox recombination led to RFP labeling of Nfatc1+ cells and their descendants (Figure 2B). We found that Nfatc1-Dre specifically labeled endocardial cells of the developing heart by costaining of RFP and PECAM on E9.5 heart sections (Figure 2C and 2D). Consistent with our previous work that the endocardium gives rise to coronary VECs,19 we detected that Nfatc1+ endocardial cells contribute to the majority of coronary VECs in the inner myocardium wall by costaining PECAM or coronary VECs-specific marker FABP429 with RFP (Online Figure IIIB and IIIC).

Figure 2.
Figure 2.

Nuclear factor of activated T cells (Nfatc1)-Dre–labeled cells contribute to adipocytes. A, Schematic diagram showing the knockin strategy of Nfatc1-Dre by homologous recombination. B, Schematic diagram showing Dre-Rox recombination for genetic lineage tracing of Nfatc1+ cells. C, Whole mount image of an embryonic day (E) 9.5 Nfatc1-Dre;R26-RSR-RFP embryo. D, Immunostaining of red fluorescence protein (RFP) and endothelial cell–specific marker platelet/endothelial cell adhesion molecule 1 (PECAM) on sections of E9.5 embryo. Arrowheads indicate RFP+PECAM+ endocardial cells. E, Immunostaining of RFP and perilipin (PLIN) on heart sections of postnatal day (P) 12 wk (w) Nfatc1-Dre;R26-RSR-RFP mice. Arrowheads indicate RFP+PLIN+ fat cells. *Region encircled by endocardium. F and G, Lipid accumulation detection by BODIPY493/503 staining and oil red staining on consecutive sections from E. *Region encircled by endocardium. Scale bars, 1 mm in C, 100 µm in D–F. LV indicates left ventricle.

To prove whether the labeled endocardial cells contribute to intramyocardial adipocytes, we costained RFP with the adipocyte marker perilipin and found that a subset of RFP+ cells also adopted the perilipin+ adipocyte fate in both left and right ventricles (Figure 2E; Online Figure IIID). By performing staining of another fat tissue marker BODIPY493/503, or oil red in the consecutive heart sections, we confirmed that these intramyocardial RFP+perilipin+ cells were fat tissues (Figure 2F and 2G; Online Figure IIIE and IIIF). We also found that a subset of RFP+ cells expressed adipogenic transcription factors peroxisome proliferator activated receptor γ and CCAAT/enhancer binding protein (C/EBP), α (Online Figure IV), which were previously used to detect intramyocardial adipocytes.6 To address the percentage of intramyocardial adipocytes derived from the endocardium, we quantified perilipin+RFP+ cells among all intramyocardial adipocytes and found that endocardial cells contributed to 41.38±5.75 % fat cells using the Dre-Rox fate-mapping model. Together, these data indicate that Nfatc1+ endocardial cells contributed to intramyocardial adipocytes in adult heart.

Inducible Tracing Confirms the Endocardial Origin of the Intramyocardial Adipocytes

One caveat in interpreting the results of genetic lineage tracing is that the expression of a reporter or fate-mapping largely depends on the expression map of the Cre or Dre recombinase. Utilization of the constitutively active recombinase driven by the Nfatc1 promoter may not be able to precisely map the cell fate of endocardium because of possible activation of the recombinase at unexpected times or locations.18,30 Therefore, the constitutively active recombinase, despite being highly efficient, might sometimes lead to overinterpretation of the results of lineage tracing studies. To address this issue, we used an inducible Cre under the control of Nfatc1 promoter (Nfatc1-CreER) and crossed it with Rosa26-loxp-stop-loxp-RFP (Rosa26-RFP) mice for mapping the cell fate of endocardial cells. Our previous study showed that this transgenic line specifically labeled the endocardial cells when tamoxifen was administered at an early embryonic stage.19 We performed tamoxifen injection at E8.5 and found specific labeling of endocardial cells at E9.5 (Online Figure VA). Although the activity of inducible Cre was not as efficient as constitutively active recombinase under the control of Nfatc1 promoter, it labeled the majority of endocardial cells (≈70%) and also their derivatives, including the coronary VECs in the inner myocardial wall.19 Furthermore, we followed up the endocardial cell fate till the adult stage and found that these endocardial cells formed a substantial number of FABP4+ coronary VECs in the inner myocardial wall (Online Figure VB). In addition to coronary VECs, we found that the Nfatc1-derived cells (RFP+) expressed the fat cell marker perilipin (Figure 3B and 3C). Quantification of RFP+perilipin+ adipocytes showed that ≈38% to 40% intramyocardial fat cells in both right and left ventricles were derived from Nfatc1-CreER–labeled endocardial cells (Figure 3D). To further confirm that the labeled RFP+perilipin+ cells are fat tissues, we collected series of heart sections and performed both oil red staining and immunostaining for RFP and perilipin on consecutive sections. These RFP+perilipin+ cells were also oil red+ cells (Figure 3E), verifying that these endocardium-derived cells were indeed adipocytes. Immunostaining for perilipin and the brown fat-specific marker uncoupling protein 1 showed that intramyocardial adipocytes were the perilipin+uncoupling protein 1− white fat but not the perilipin+uncoupling protein 1+ brown fat (Online Figure VI). Although endocardial cells contribute to intramyocardial adipocytes, we ask whether their endothelial cell marker remained after lineage conversion. Therefore, costaining of RFP, perilipin, and endothelial cell–specific marker PECAM or endomucin was performed on Nfatc1-CreER;Rosa26-RFP heart sections; we found that endocardium-derived adipocytes (RFP+perilipin+) no longer expressed endothelial cell markers PECAM or endomucin (Online Figure VII). To independently test the adipogenic potential of the endocardial cells, we isolated endocardial cells from Nfatc1-CreER;Ai47 (a GFP reporter line) and cultured in vitro for induction of adipogenesis. Oil red staining showed that these endocardial cells could differentiate into adipocytes in response to adipogenic stimuli in vitro (Online Figure VIIIA–VIIID). We also isolated endocardial cells for single-cell culture and expansion to score for adipogenesis in vitro. Our results of oil red staining showed that clones from single endocardial cell could differentiate into adipocytes in response to adipogenic stimuli in vitro (Online Figure VIIIE and VIIIF).

Figure 3.
Figure 3.

Endocardial (Endo) cells contribute to adipocytes in subendocardium of adult heart. A, Schematic diagram showing the strategy of endocardial cell labeling and lineage tracing analysis using the nuclear factor of activated T cell (Nfatc1)-CreER;Rosa26-RFP mouse model at postnatal day (P) 12 wk (w) to P28w. B, Immunostaining of red fluorescence protein (RFP) and perilipin (PLIN) on heart sections derived from P21w Nfatc1-CreER;Rosa26-RFP mice. Arrows indicate PLIN+ fat tissue inside heart; arrowhead points to epicardial (Epi) fat in the atrioventricular groove; dotted lines demarcate epicardium. C, Magnified image of merged channels showing RFP+PLIN+ cells (arrows). D, Quantification of the percentage of genetically labeled PLIN+ cells among adipocytes in the right and left ventricle (RV and LV, respectively), n=5. E, Oil red staining and immunostaining of serial heart sections derived from the Nfatc1-CreER;Rosa26-RFP mice. Oil red+ indicates fat cells. Arrows indicate costaining of Nfatc1-derived cells (RFP+) and fat cells (PLIN+). Scale bars, 1 mm in B; 100 µm in C and E.

To further illustrate the endocardial contribution to intramyocardial adipocytes in vivo, we administered low dosage of tamoxifen to Nfatc1-CreER;Rosa26-RFP mice and performed clonal analysis of labeled cells in vivo to focus on the fates of coronary VECs and adipocytes. We found some endocardial cells differentiated into adipocytes (type I clone), coronary VECs (type II clone), or the intermediate of both adipocytes and coronary VECs (type III clone) in the adult heart (Online Figure IX). By examining 23 hearts via immunostaining for RFP, perilipin, and PECAM on serial heart sections, we detected 5 type I clones, 218 type II clones, and 2 type III clones (Online Figure IX). The results from this clonal analysis suggested that most endocardial cells were unipotent and capable of differentiating into either coronary VECs or adipocytes. Only few endocardial cells were bipotent and capable of differentiate into both coronary VECs and adipocytes.

VECs Are Not the Source of Intramyocardial Adipocytes

In the heart, endocardial cells and coronary VECs are 2 distinct types of endothelial cells with different morphology and molecular signatures.19 During both fetal and neonatal heart growth, endocardial cells contribute to a substantial number of coronary VECs in the myocardial wall.15,19 Previous study has documented that VECs in the adipose tissue are capable of giving rise to adipocytes,20 so we asked whether the endocardium-derived coronary VECs might also transdifferentiate into intramyocardial adipocytes. To directly address this possibility, we took advantage of the molecular marker apeline that is specifically expressed by VECs but not by the endocardial cells.14,16,31 Our previous work also showed that apeline-CreER specifically and efficiently labeled coronary VECs but not the endocardium.16 We generated the apeline-CreER;Rosa26-RFP mouse line and performed triple tamoxifen treatment to label almost all coronary VECs in the developing heart (Figure 4A). By costaining RFP with the pan-endothelial cell marker PECAM, we found that apeline-CreER labeled coronary VECs (PECAM+RFP+) but not endocardial cells (PECAM+RFP−) in the neonatal P7 heart (Online Figure XA). We confirmed this result by costaining RFP with an additional VECs marker FABP4, and found that the endocardial cells were RFP−FABP4− whereas almost all coronary VECs were FABP4+RFP+ (Online Figure XB). We then followed the fate of coronary VECs in adult stage when intramyocardial adipocytes have developed. Immunostaining of RFP with endothelial cell marker cadherin 5 on adult heart sections showed that most coronary VECs were cadherin 5+RFP+, whereas endocardial cells were RFP−cadherin 5+ (Figure 4B–4D), indicating that the labeling of coronary VECs in our apeline-CreER;Rosa26-RFP mouse line was efficient and specific. By immunostaining of RFP and perilipin on these sections from adult apeline-CreER;Rosa26-RFP mice, we did not detect any RFP+perilipin+ cells in the right ventricle, ventricular septum, or left ventricle (Figure 4E), indicating that coronary VECs did not contribute to intramyocardial adipocytes in adult heart.

Figure 4.
Figure 4.

Coronary vascular endothelial cells (VECs) do not contribute to intramyocardial adipocytes. A, Schematic diagram showing the labeling strategy of coronary VECs using the apeline (Apln)-CreER;Rosa26-RFP mouse model. B, A cartoon figure showing the labeling of coronary VECs (red), but not of endocardium (green) of the adult heart. C, Immunostaining of red fluorescence protein (RFP), cadherin 5 (CDH5), and DAPI on heart sections from postnatal day (P) 26 wk (w) adult mice. D, Interpretation of immunostaining images from C using pseudocolors. Most RFP(+)CDH5(+) cells are coronary VECs (red pseudocolor) and most RFP(−)CDH5(+) are endocardial cells (green pseudocolor). Almost all coronary VECs are labeled in the Apln-CreER;Rosa26-RFP mice. E, Perilipin (PLIN)+ intramyocardial fat cells are not RFP+, suggesting that they are not derived from the RFP+ coronary VECs. F, A cartoon figure showing endocardial cells differentiate into intramyocardial adipocytes and coronary VECs. Coronary VECs do not contribute to intramyocardial adipocytes. Scale bars, 100 µm. LV indicates left ventricle; RA, right atrium; RV, right ventricle; and VS, ventricular septum.

Other Sources of Intramyocardial Adipocytes

To study the other possible sources of intramyocardial adipocytes, we performed cardiomyocyte-specific lineage tracing. The cardiomyocyte-specific myosin heavy chain 6, cardiac muscle, α-MerCreMer mouse line32 was crossed with the Rosa26-RFP reporter line to examine if labeled cardiomyocyte differentiated into intramyocardial adipocytes in the adult heart (Online Figure XIA). We treated myosin heavy chain 6, cardiac muscle, α-MerCreMer;Rosa26-RFP mice with tamoxifen at the late embryonic stage and found that most cardiomyocytes were labeled in the adult heart (Online Figure XIA and XIB). Our results from immunostaining for RFP and perilipin showed that RFP+ cardiomyocytes did not contribute to perilipin+ intramyocardial adipocytes in the adult heart (Online Figure XIC). Neither did these labeled cardiomyocytes adopt an epicardial adipocyte cell fate in the adult heart (Online Figure XID). Therefore, it is not likely that cardiomyocytes could give rise to the intramyocardial or epicardial fat in the adult heart during homeostasis.

Nevertheless, because previous works have showed that the epicardium and its derivatives, the epicardium-derived cells, contribute to adipocytes during both development and post injury,3–5 we ask whether epicardial-derived cells could also give rise to intramyocardial adipocytes. We crossed the epicardial Wt1-CreER line33 with the Rosa26-RFP reporter line to trace the cell fate of epicardial-derived cells in the adult heart. We treated Wt1-CreER;Rosa26-RFP mice with tamoxifen at an early embryonic stage (E10.5) and found efficient labeling of perivascular cells in the adult heart (Online Figure XIIA and XIIB). Immunostaining for RFP and perilipin showed that a subset of perilipin+ intramyocardial adipocytes were RFP+ (Online Figure XIIC), confirming that in addition to the endocardium, the epicardium also contributed to intramyocardial adipocytes during normal heart development. To examine whether the endocardium, in reverse, contribute to the epicardial fat, we performed immunostaining for RFP and perilipin in the Nfatc1-CreER;Rosa26-RFP mice. We did not detect any perilipin+ epicardial adipocytes that coexpressed RFP (Online Figure XIID), indicating that the endocardium-derived cells did not contribute to the epicardial fat.

Lineage Tracing of Adult Endocardial Cells in Homeostasis and After MI

Our previous work has showed that adult epicardial cells could give rise to new adipocytes after MI.5 Similarly, we asked whether the adult endocardial cells still possess this adipogenic potential in homeostasis and after MI. Because there are no inducible Cre lines for postnatal endocardial labeling, we took advantage of the inducible cadherin 5-CreER line and labeled both the endocardium and the coronary VECs.34 We treated the cadherin 5-CreER;Rosa26-RFP mice with tamoxifen in the neonatal stage and examined the cell fate of RFP+ cells in the adult heart (Figure 5A). We first collected samples at week 3 post tamoxifen treatment to confirm that our labeling strategy for endothelial cells was efficient and specific. Costaining of RFP with PECAM showed that most endothelial cells including the endocardial cells were labeled as RFP+ (Figure 5B). Examination of both ventricles and the interventricular septum showed that the RFP+ cells were PECAM+ endothelial cells but not the perilipin+ intramyocardial adipocytes (Figure 5C and 5D). These data suggested that adult endocardial cells did not differentiate into fat cells in homeostasis.

Figure 5.
Figure 5.

Postnatal endocardial (ENDO) cells do not contribute to adipocytes. A, Schematic showing strategy for tamoxifen induction at postnatal 1 week (P1w) and analysis at P12w to P36w. B, Immunostaining for red fluorescence protein (RFP) and platelet/endothelial cell adhesion molecule 1 (PECAM) on heart sections of P3w Cdh5-CreER;Rosa26-RFP mice. Tamoxifen is injected at 2 wk after birth. C and D, Immunostaining for RFP, perilipin (PLIN), and PECAM on heart sections of adult Cdh5-CreER;Rosa26-RFP mice. C1, C2, and D1 are magnified images of boxed regions in C and D, respectively. RFP+ cells adopt an endothelial cell fate but not the adipocyte cell fate. LV indicates left ventricle; RA, right atrium; RV, right ventricle; and VS, ventricular septum. Scale bars, 100 µm. Each figure is a representative of 4 to 5 individual samples.

To test whether endocardium could be reactivated and differentiated into adipocytes after injury, we induced MI by permanent ligation of left anterior descending coronary artery. We treated the cadherin 5-CreER;Rosa26-RFP mice with tamoxifen 2 weeks before MI, and found that most endothelial cells were labeled as RFP+ (Figure 6A and 6B). Four weeks after MI, large fibrotic tissue formed in the left ventricle. Moreover, we observed a few intramyocardial adipocytes that remained as RFP− in the infarct region (Figure 6C). In both the border region of infarcted left ventricle and the remote right ventricle, we did not find any perilipin+ adipocytes that were RFP+. Unlike the adult epicardial cells, our results suggested that the adult endocardial cells did not give rise to adipocytes in homeostasis or after MI. Nevertheless, whether adult endocardial cells generate new adipocytes in other forms of cardiovascular diseases, such as obesity-associated cardiomyopathy, requires further investigation.

Figure 6.
Figure 6.

Adult endocardial (ENDO) cells do not contribute to intramyocardial adipocytes after myocardial infarction (MI). A, Schematic diagram showing strategy for tamoxifen induction, MI, and analysis of the Cdh5-CreER;Rosa26-RFP lineage tracing mice. B, Immunostaining for red fluorescence protein (RFP) and cadherin 5 (CDH5) on heart sections before MI. C–E, Immunostaining for RFP and perilipin (PLIN) on heart sections of Cdh5-CreER;Rosa26-RFP adult mice. Cdh5-CreER labels both endocardial cells and vascular endothelial cells, and they do not contribute to PLIN+ adipocytes (white arrowheads). Scale bars, 100 µm. LV indicates left ventricle; and RV, right ventricle; Representative images are derived from 3 individual samples.

Discussion

In this study, we presented the endocardial origin of intramyocardial adipocytes in adult heart. Endocardium is a common origin of both coronary VECs and intramyocardial adipocytes. Previous study showed that VECs give rise to both white and brown adipocytes during development.20 However, our VEC-specific lineage tracing studies showed that these intramyocardial adipocytes were not derived from coronary VECs. Although most of these coronary VECs in the inner myocardial wall were derived from the endocardium, which could be a common progenitor for both coronary VECs and intramyocardial adipocytes, the endocardial cells adopt a unique developmental program for adipocyte formation (Figure 4F). It would be important to understand the molecular regulators that mediated the transition of endocardial cells to adipocytes, which requires further study in the future.

Endocardial cell is a unique type of endothelial cells that play crucial roles in many aspects of cardiovascular development. The single layer of endocardial cells lining the developing heart provides a substantial portion of mesenchyme for cardiac cushion formation through endocardial-to-mesenchymal transition.13 In addition to mesenchymal cells, endocardial cells also contribute to VECs during both embryonic development and postnatal heart growth.14,15,19 However, there is still controversy over the magnitude of endocardial contribution in the embryonic heart.14–17 Moreover, endothelial cells (including both endocardium and blood vessels) give rise to fibroblasts in the adult injured heart through an EMT.35 Recent studies suggested that most of these fibroblasts derived from EMT originate, in fact, from the endocardium during embryonic development,36 suggesting that endocardial cells also contribute to fibroblast formation. During cardiac injury such as MI, endocardium is a site for endogenous arteriogenesis with a good source of endothelial cells for neovascularization.37 These studies suggested that adult endocardial cells could be driven to undergo EMT for a fibroblast fate or coronary vascular cell fate. Moreover, our work showed that endocardial cells could also generate adipocytes in the heart. Altogether, these studies suggest that endocardial cells could differentiate into multiple lineages during heart development, homeostasis, and after injury, including coronary VECs, fibroblasts, and adipocytes. Because it has been reported that mesenchymal cells can also be differentiated into adipocytes,38 it is also possible that these endocardial cells directly transdifferentiate into adipocytes or form mesenchymal-like cells in the postnatal heart. The latter has been recently demonstrated in disease models such as endocardial fibroelastosis, in which postnatal endocardial cells undergo excessive EMT.39 Our work provided important evidence that endocardial cells contributed to adipocytes, but it remains elusive at present if this lineage conversion was direct or indirect. As endocardial cells give rise to mesenchymal cells and some type of mesenchymal cells have been reported to differentiate into adipocytes, it is also possible that the endocardial-to-mesenchymal transition is an intermediate stage for endocardium-to-fat transition. The differentiation of adipocytes should involve principal adipogenic factors such as peroxisome proliferator activated receptor γ and C/EBPa, which require further investigation.

Currently, it is not clear what functions are mediated by those intramyocardial adipocytes in normal adult heart. Previous work has suggested that adipocytes could secrete some adipocytokines, such as adiponectin and C1q and tumor necrosis factor–related protein 9, which have been shown to exert salutary effects on the survival of cardiomyocytes and vascular functions. Adiponectin protects against inflammation and injury in autoimmune myocarditis,40 reduces cardiac oxidative stress, and ameliorates cardiomyocyte hypertrophy in diabetic cardiomyopathy.41 C1q and tumor necrosis factor–related protein 9 secreted by fat cells is also cardioprotective, proangiogenic, and antiapoptotic,42 and increased C1q and tumor necrosis factor–related protein 9 could restore cardiac function, attenuate cardiomyocyte apoptosis, and increase neovascularization in hypoxia-induced cardiomyocyte injury.43 Therefore, it is likely that intramyocardial adipocytes in the normal heart exert a protective role for cardiomyocytes and coronary vessels through paracrine mechanism, which merits further investigation in the future. However, excessive adipocytes might also be associated with cardiovascular diseases, such as arrhythmogenic right ventricular cardiomyopathy. In this genetic disease, excessive adipose tissues are found to replace myocardium of the right ventricle, which is then clinically manifested as ventricular arrhythmias and sudden cardiac death.6 Generation of lineage tracing tools and mouse models to address specific cardiomyopathies would enhance our understanding in the fate and function of these intramyocardial fat particularly during the adult disease states of the heart.

Unraveling the developmental origin of intramyocardial adipocytes would have implications to the understanding of pathophysiological processes of cardiovascular disease. Excessive adipocytes in the heart is one of the phenotypic hallmarks for some types of cardiomyopathy. In arrhythmogenic right ventricular cardiomyopathy, adipocytes replace cardiac myocytes in the right ventricle. Adult cardiomyocytes are terminally differentiated, which are less likely to dedifferentiate into adipocytes. Recent elegant genetic fate-mapping studies using Mef2C and Nkx2.5 Cre lineage tracing models show that the adipocytes of the arrhythmogenic right ventricular cardiomyopathy are derived from the second heart field progenitor cells.6 Because Mef2C and Nkx2.5 Cre lines label the endocardium, VECs, and smooth muscle in addition to the myocardium,44,45 it remains possible that other cell types (including endocardial cells or VECs) might give rise to adipocytes. Our study using the endocardium-specific lineage tracing model suggested that some of the intramyocardial adipocytes arise from the endocardium, raising the possibility that excessive deposit of fat in the myocardium could also be of the endocardial origin in the arrhythmogenic right ventricular cardiomyopathy. In the setting of MI, collagen has been regarded as the principal structural substrate of ventricular tachycardia. However, recent studies showed that formation of intramyocardial adipocytes in the infarcted left ventricle could contribute a significant risk factor associated with altered electrophysiological properties in ventricular tachycardia.7 The developmental process of epicardium-to-fat transition could be recapitulated after MI,5 but the endocardium-to-fat transition during normal development was not deployed again in the injured heart. It remains unknown whether endocardial cells could give rise to excessive adipocytes in other types of cardiovascular diseases such as arrhythmogenic right ventricular cardiomyopathy6 or endocardial fibroelastosis,39 which require further studies. Indeed, additional origins, such as the second heart field progenitor cells, also provide an important source of adipocytes in arrhythmogenic right ventricular cardiomyopathy.6 Nevertheless, our work has demonstrated an alternative source of intramyocardial adipocytes, and more research is needed to identify the pathophysiological signals that determine lineage conversion of cardiac adipocytes via endocardium-to-fat transition, paving the way for a better understanding in cardiovascular diseases including the increased incidence of obesity-associated cardiomyopathy.

Acknowledgments

We thank Ralf Adams of Max Plank Institute for providing the Cdh5-CreER mice, and Hongkui Zeng of the Allen Institute for Brain Science for providing the Ai66 and Ai47 mice.

Sources of Funding

This work was supported by the National Science Foundation of China (91339104, 31271552, and 31222038 to B. Zhou; 31301188 and 31571503 to X. Tian; 31501172 to H. Zhang; 81430006 to S. Hu), Ministry of Science and Technology (2012CB945102 and 2013CB945302 to B. Zhou), Shanghai Basic Research Key Project (14JC1407400 to B. Zhou) and Zhangjiang Stem Cell Project (ZJ2014-ZD-002), Shanghai Institutes for Biological Sciences (SIBS) President Fund, Sanofi-SIBS Fellowship (X. Tian, H. Zhang), SIBS Postdoc Fund (2013KIP311 to X. Tian, 2014KIP314 to H. Zhang), Youth Innovation Promotion Association of CAS (2015218 to X. Tian), Astrazeneca, Shanghai Yangfan Project (15YF1414000 to H. Zhang) and Qimingxing Project (15QA1404300 to X. Tian) and Research Grants Council of Hong Kong (24110515 to K.O. Lui).

Disclosures

None.

Footnotes

  • In November 2015, the average time from submission to first decision for all original research papers submitted to Circulation Research was 15.52 days.

  • The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.115.307202/-/DC1.

  • Nonstandard Abbreviations and Acronyms
    EMT
    endothelial-to-mesenchymal transition
    EPDCs
    epicardial-derived cells
    FABP4
    fatty acid binding protein 4
    MI
    myocardial infarction
    Nfatc1
    nuclear factor of activated T cells
    PECAM
    platelet/endothelial cell adhesion molecule 1
    RFP
    red fluorescence protein
    VECs
    vascular endothelial cells

  • Received July 13, 2015.
  • Revision received December 3, 2015.
  • Accepted December 9, 2015.

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Novelty and Significance

What Is Known?

  • Excessive cardiac fat is the phenotypic hallmark for some types of cardiomyopathy.

  • Second heart field progenitor cells is a source of fat cells in arrhythmogenic right ventricular cardiomyopathy.

  • Endocardium contributes to vascular endothelium.

  • Vascular endothelium of the adipose tissue gives rise to fat cells.

What New Information Does This Article Contribute?

  • Most intramyocardial adipocytes of the heart reside in the myocardial region close to endocardium.

  • Developing endocardial cells contribute to these intramyocardial adipocytes.

  • Cell fate change of endocardial cells to adipocytes does not involve an intermediate step via the vascular endothelium.

  • Epicardium also contributes to intramyocardial adipocytes.

Unraveling the developmental origins of cardiac fat provides novel insights into treatment options for obesity-associated cardiovascular diseases. In this study, we found that most intramyocardial fat cells are located in the myocardial region close to endocardium. By using the genetic lineage tracing technology, we uncovered that the developing endocardium contributes to intramyocardial fat cells. The finding of endocardium-to-fat transition has important implications in the cause and pathophysiology of the obesity-associated cardiomyopathy, such as arrhythmogenic right ventricular cardiomyopathy.

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  • Endocardium Contributes to Cardiac FatNovelty and Significance
    Hui Zhang, Wenjuan Pu, Qiaozhen Liu, Lingjuan He, Xiuzhen Huang, Xueying Tian, Libo Zhang, Yu Nie, Shengshou Hu, Kathy O. Lui and Bin Zhou
    Circulation Research. 2016;118:254-265, originally published December 9, 2015
    https://doi.org/10.1161/CIRCRESAHA.115.307202
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