Left ventricular hypertrophy and metabolic resetting in the Notch3-deficient adult mouse heart

Del Gaudio, Francesca; Liu, Dongli; Andaloussi Mäe, Maarja; Braune, Eike-Benjamin; Hansson, Emil M.; Wang, Qing-Dong; Betsholtz, Christer; Lendahl, Urban

doi:10.1038/s41598-023-42010-7

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Published: 12 September 2023

Left ventricular hypertrophy and metabolic resetting in the Notch3-deficient adult mouse heart

Francesca Del Gaudio ORCID: orcid.org/0000-0001-8342-1377¹^na1,
Dongli Liu¹^na1^nAff5,
Maarja Andaloussi Mäe²,
Eike-Benjamin Braune¹,
Emil M. Hansson¹,
Qing-Dong Wang³,
Christer Betsholtz^2,4 &
…
Urban Lendahl ORCID: orcid.org/0000-0001-9543-8141¹

Scientific Reports volume 13, Article number: 15022 (2023) Cite this article

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Abstract

The heart depends on a functional vasculature for oxygenation and transport of nutrients, and it is of interest to learn how primary impairment of the vasculature can indirectly affect cardiac function and heart morphology. Notch3-deficiency causes vascular smooth muscle cell (VSMC) loss in the vasculature but the consequences for the heart remain largely elusive. Here, we demonstrate that Notch3^-/- mice have enlarged hearts with left ventricular hypertrophy and mild fibrosis. Cardiomyocytes were hypertrophic but not hyperproliferative, and the expression of several cardiomyocyte markers, including Tnt2, Myh6, Myh7 and Actn2, was altered. Furthermore, expression of genes regulating the metabolic status of the heart was affected: both Pdk4 and Cd36 were downregulated, indicating a metabolic switch from fatty acid oxidation to glucose consumption. Notch3^-/- mice furthermore showed lower liver lipid content. Notch3 was expressed in heart VSMC and pericytes but not in cardiomyocytes, suggesting that a perturbation of Notch signalling in VSMC and pericytes indirectly impairs the cardiomyocytes. In keeping with this, Pdgfb^ret/ret mice, characterized by reduced numbers of VSMC and pericytes, showed left ventricular and cardiomyocyte hypertrophy. In conclusion, we demonstrate that reduced Notch3 or PDGFB signalling in vascular mural cells leads to cardiomyocyte dysfunction.

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Introduction

Notch signalling is an evolutionarily conserved signalling mechanism, which regulates development and homeostasis of most organs in the body, including the cardiovascular system. Activation of Notch signalling ensues when a transmembrane Notch receptor on a signal-receiving cell interacts with transmembrane Notch ligands presented on a juxtaposed signal-sending cell¹. Ligand-receptor interaction results in proteolytic processing of the Notch receptor by cleavage of the receptor just outside the plasma membrane, followed by a final proteolytic cleavage in the plasma membrane, the latter executed by the γ-secretase complex. The final cleavage steps results in the liberation of the C-terminal portion of the Notch receptor, the Notch intracellular domain (Notch ICD), which relocates from the plasma membrane to the cell nucleus. In the nucleus, Notch ICD interacts with the DNA-binding protein CSL (a.k.a. RBPj) and MAML1, forming a ternary transcriptional complex that regulates expression of Notch downstream genes, including the genes encoding Hes and Hey transcription factors².

Mutations in the Notch pathway that cause heart disease have been identified³. Notably, NOTCH1 mutations are observed in bicuspid aortic valve disease⁴ and hypoplastic left heart syndrome⁵, while mutations in the MIB1 gene, encoding an E3 ubiquitin ligase important for Notch ligand presentation, are linked to left ventricle cardiomyopathy⁶. Furthermore, most patients with Alagille syndrome (OMIM No. 118450), a genetic disorder caused by mutations in the Notch ligand Jagged1, exhibit the congenital heart disease Tetralogy of Fallot⁷. Notch signalling is also important for formation of septa and valves during the genesis and development of the heart⁸, and impaired Mib1 function causes left ventricular non-compaction myocardiopathy and bicuspid aortic valve in the mouse⁹.

In addition to its role in heart development, Notch signalling plays a pivotal role for development and homeostasis of the vasculature¹⁰ and the Notch3 gene is particularly important in this regard. Notch3 is predominantly expressed in mural cells (VSMC and pericytes)^11,12,13 and Notch3-deficient mice exhibit a progressive loss of VSMC after birth^14,15. NOTCH3 missense mutations cause the most common monogenic brain small vessel disease CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy; OMIM No. 125310)¹⁶. The vast majority of NOTCH3 CADASIL mutations are cysteine-altering^17,18, but cysteine-sparing NOTCH3 mutations have also been linked to brain vascular disease^19,20, further underscoring the importance of NOTCH3 function for the brain vasculature.

While the progressive loss of VSMC in the Notch3^-/- mice is well established^14,15, less is known about consequences in other organs, including the heart. Here, we report that Notch3^-/- mice exhibit left ventricular hypertrophy, cardiomyocyte hypertrophy and mild fibrosis. Gene expression changes indicating a metabolic switch from fatty acid oxidation to glucose consumption were observed, and the lipid content in the liver was reduced. As Notch3 was expressed in VSMC and pericytes but not in cardiomyocytes, the data suggest that the impairment of the cardiomyocytes is indirect and that specific cellular perturbations in the vasculature are sufficient to produce a cardiomyocyte phenotype.

Results

Left ventricular hypertrophy in Notch3 ^-/- mice

To learn whether the heart was affected by loss of Notch3 function, we measured the weight of the hearts in Notch3^-/- mice. Notch3^-/- hearts were enlarged (Fig. 1A), with an increased heart versus body weight ratio (Fig. 1B) and a trend towards increased heart weight versus tibia length ratio (Fig. 1C). These data corroborate a previous report of cardiac hypertrophy in a different Notch3 knockout mouse model²¹.

Analysis of the morphology of Notch3^-/- and control hearts demonstrated that the left ventricle and papillary muscles from the Notch3^-/- mice were thickened, whereas the size of the septum was not significantly altered (Fig. 1D). Expression of the gene Nppb, which encodes the brain natriuretic peptide (BNP) and is upregulated when the ventricles are subjected to increased filling pressures and left ventricle wall stretch²², was increased in the Notch3^-/- hearts (Fig. 1E), corroborating the notion of left ventricular hypertrophy. Compared to wildtype control mice, mild fibrosis and perivascular and interstitial collagen deposition were observed in the Notch3^-/- hearts (Fig. 1F). In contrast, the weight of Notch3^-/- lungs did not differ from control mice (Fig. 1G) nor exhibited any signs of fibrosis or vascular changes (data not shown). Collectively, these observations suggest that Notch3^-/- mice exhibit left ventricular hypertrophy, combined with mild fibrosis.

Left ventricular hypertrophy in Notch3 ^-/- mice is associated with cardiomyocyte hypertrophy but not cardiomyocyte hyperproliferation

The absolute majority of left ventricular hypertrophy is caused by cardiomyocyte hypertrophy, but there are also reports indicating that proliferation may be induced in postnatal cardiomyocytes in response to injury or perturbation of major signalling pathways^23,24. To distinguish between these two possibilities, Wheat Germ Agglutinin staining, to outline the cardiomyocyte cell membranes, showed an increase in cardiomyocyte cross section area from 200 to 347 μm² in the Notch3^-/- mice (Fig. 2A,B). Proliferation was assessed by Ki-67 immunostaining, and there was no increase in Ki-67 labelling in the Notch3^-/- mice (Fig. 2C,D).

An alternative cause of ventricular hypertrophy is hypertension²⁵, but Notch3^-/- mice have a normal blood pressure unless challenged with angiotensin-II²¹. In line with a normal blood pressure, the expression of the NppA gene, which encodes atrial natriuretic peptide (ANP) and shows increased expression in response to hypertension²⁶, was not upregulated (Supplemental Figure 1A). In sum, we conclude that the left ventricular hypertrophy is associated with an increase in cardiomyocyte size but not with increased cardiomyocyte proliferation.

Cardiomyocyte morphology and marker expression are altered in the Notch3 ^-/- mice

Left ventricular hypertrophy may result from defects in the contractile capacity of the cardiomyocytes, for example because of mutations in sarcomeric genes^27,28,29. The myocardium is built from cardiomyocytes, which harbour myofibrils. The myofibrils contain myosin and actin as well as auxiliary proteins such as tropomyosin and troponins and are built from aligned sarcomeres, a contractile apparatus which anchor to the α-actinin-containing Z-lines in the cardiomyocytes (Fig. 3A). Between the Z-lines at opposing ends of the sarcomere is the M-band, and titin filaments span between the M-band and the Z-lines. The A-band correspond to the myosin part, while the I-band corresponds to the actin filaments between the Z-lines and the A-band^27,28,29 (Fig. 3A).

To learn whether the sarcomeric structure was altered in Notch3^-/- mice, we analysed sections from Notch3^-/- and control hearts by transmission electron microscopy (TEM). This revealed that the organization of mitochondrial tubules in linear arrays along the sarcomere was disturbed in Notch3^-/- hearts, which showed a looser adhesion between individual tubules (Fig. 3B). Furthermore, the I-bands were less pronounced (Fig. 3B) and the distance between the Z-lines was increased in the Notch3^-/- sarcomeres (Fig. 3C). In contrast, the distance between the A-bands was not statistically different in the Notch3^-/- sarcomeres (data not shown).

In line with an aberrant sarcomere structure, expression of genes encoding sarcomeric cardiomyocyte proteins was altered. Notably, expression of TnT2, which codes for cardiac muscle isoform of troponin T (TNNT2) regulating cardiac muscle contraction in response to Ca2 ⁺and mutated in cardiomyopathies³⁰, was increased (Fig. 3D). Myh7, which encodes myosin heavy chain (MHC)-β and is one of the genes mutated in familial hypertrophic myocardiopathy³¹ was similarly expressed at higher levels, while the related Myh6 gene, coding for myosin heavy chain (MHC)-α and in mice mostly expressed in the ventricles^32,33, showed reduced expression (Fig. 3D). Expression of Actn2 (α-actinin-2), which encodes the major component of the Z-disc and associated with hypertrophic myopathy³⁴, was reduced in the Notch3-deficient mice (Fig. 3D). In conclusion, Notch3^-/- cardiomyocytes exhibit an aberrant morphology, accompanied by altered expression of genes involved in sarcomeric function.

Dysregulated fatty acid and glycolytic metabolism in the Notch3 ^-/- heart

The adult heart relies largely on uptake of fatty acids to meet its energy demands, while the fetal heart preferentially uses glycolytic metabolism^35,36; for review see³⁷. Upon heart injury or disease, the adult heart however switches towards a more glycolytic metabolism³⁸. Pdk4, which decreases glucose oxidation and increases fatty acid oxidation³⁹, and Cd36, encoding the long chain free fatty acid transporter CD36⁴⁰, were both downregulated in the Notch3^-/- hearts (Fig. 4A). Expression of peroxisome proliferation-activated receptor-gamma (PPARγ), which enhances glucose metabolism and causes insulin desensitization⁴¹, was slightly, although not significantly upregulated, while there was a tendency towards downregulation for expression of acyl-CoA dehydrogenase long and medium chain (Acadl, Acadm), which regulate beta oxidation of fatty acids⁴² (Supplemental Figure 1B). At the immunohistochemistry level, CD36 immunofluorescence was downregulated while the transporter for uptake of glucose (GLUT1) was upregulated in the adult Notch3^-/- hearts (Supplemental Figure 1C,D). In two-weeks old mice, there was no significant difference in CD36 and GLUT1 immunofluorescence (Supplemental Figure 1E), while CD36 immunofluorescence was decreased in the Notch3^-/- hearts at two months of age, accompanied by an increase in GLUT1 expression (Supplemental Figure 1F). At two months of age, there was also an increase in the cardiomyocyte area (Supplemental Figure 1G). We analysed whether expression of some of the Notch downstream genes important for the heart and vasculature was altered. Expression of the Hey2 gene, which is important for heart development and is linked to Brugada syndrome, a rare form of arrhythmia^43,44, was downregulated in the Notch3^-/- hearts, which as expected also was the case for Notch3 (Supplemental Figure 1H). In contrast, Hey1 expression was not affected (Supplemental Figure 1H).

To extend the gene expression analysis, we performed bulk transcriptomic analysis of Notch3^-/- and control hearts. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of differently expressed genes (DEG) in Notch3^-/- compared to control hearts (Supplemental Table 1) demonstrated that pathways related to for example metabolism and fatty acid degradation were dysregulated in Notch3^-/- hearts (Fig. 4B and Supplemental Table 2). In addition, we found that the DEG are enriched in diseases such as vascular diseases, lipid metabolism disorders and diseases related to liver dysfunction (Supplemental Figure 1 and Supplemental Table 3).

This shift in gene expression towards a more glycolytic profile could be caused by a direct more local effect on the cardiomyocytes mediated by the VSMC in the heart or be a result of a more general altered composition of nutrients in the blood. To address the latter hypothesis, we analysed whether the amount of lipid in the liver was altered, as an indicator of the lipid status in the vasculature. Staining with the lipid tracer BODIPY 493/503, which identifies neutral and non-polar lipids, revealed a reduction in lipid droplets in the Notch3^-/- liver (Fig. 4C), which was confirmed by Oil Red O staining (Fig. 4D). In sum, the data demonstrate a gene expression switch towards glycolytic metabolism in the Notch3^-/- hearts, accompanied by reduced lipid deposition in the Notch3^-/- livers.

Notch3 is expressed in heart vascular smooth muscle cells but not in cardiomyocytes

It is well established that Notch3 is expressed in VSMC and pericytes in the vasculature^15,45,46, but whether it also is expressed in cardiomyocytes has not been established, and to address this is important to understand whether the observed cardiomyocyte phenotype is a consequence of a cell-autonomous (loss of Notch3 function in the cardiomyocytes) or non-autonomous (loss of Notch3 function in mural cells) effect. To assess the expression of Notch3 in the heart, we took advantage of a targeted insertion of the lacZ gene into the Notch3 locus in the Notch3^-/- mice⁴⁷, and Notch3 expression can thus be monitored by beta-galactosidase immunohistochemistry as a proxy for Notch3 expression¹⁵. In line with this notion, beta-galactosidase immunofluorescence was only observed in the Notch3^-/- mice, which harbour the targeted Notch3 alleles, and not in wildtype mice (Supplemental Figure 3). There was no expression of beta-galactosidase in cardiomyocytes (visualized by α-actinin as a marker for cardiomyocytes) (Fig. 5A), indicating that no or only very low levels of the Notch3 receptor is produced in cardiomyocytes.

In contrast to the lack of Notch3 expression in cardiomyocytes, Notch3 was expressed in the VSMC of the heart vasculature: co-staining for beta-galactosidase and the VSMC marker alpha-smooth muscle actin (αSMA; gene name Acta2) demonstrated extensive co-expression of Notch3 and αSMA at the rim of blood vessels (Fig. 5B). Furthermore, beta-galactosidase staining was adjacent to, but not overlapping with cells expressing CD31, a marker for endothelial cells (Fig. 5C), in keeping with expression in VSMC. To corroborate these findings, we next analysed the distribution of Notch3 mRNA expression in different cell types in the Tabula Muris gene expression atlas⁴⁸. Notch3 expression was predominantly observed in smooth muscle cells and valve cells, while expression was negligible in cardiomyocytes (Fig. 5D). As a control, expression of Ttn (Titin), a cardiomyocyte marker, was largely confined to cardiomyocytes (Fig. 5D).

As Notch3-deficiency can lead to structural changes in the vasculature¹⁵, we next assessed whether blood vessel morphology was altered in the Notch3^-/- hearts. Several vessels were more dilated in the Notch3^-/- hearts (Fig. 6A), while there was no difference in the density of the vasculature, as judged by CD31 expression per area (Fig. 6B). The number of arterioles per area was however reduced in the Notch3^-/- hearts (Fig. 6C). In conclusion, Notch3 is expressed in VSMC but not cardiomyocytes in the heart, and loss of Notch3 is associated with morphological changes in the cardiac vasculature.

Pdgfb ^ret/ret mice exhibit left ventricular hypertrophy

Platelet-derived growth factor receptor beta (Pdgfrb) is important for pericyte function⁴⁹. Pdgfrb is also a Notch downstream target gene^12,50 and there were trends towards downregulation of both Pdgfrb mRNA and protein expression in Notch3^-/- hearts (Fig. 7A,B). As Pdgfrb, like Notch3, was expressed in SMC and valve cells in the heart but not in cardiomyocytes (Fig. 7C), we decided to explore whether perturbed signalling via the PDGFRB receptor led to a heart phenotype. To this end, we used the PDGFB retention (Pdgfb^ret/ret) mouse model, which produces a truncated PDGFB ligand⁵¹ and has been extensively used as a tool to explore perturbed PDGF signalling in the vasculature^49,52. Like the Notch3^-/- mice, the Pdgfb^ret/ret mice exhibited enlarged hearts (Fig. 7D), accompanied by left ventricular hypertrophy (Fig. 7E,F). The Pdgfb^ret/ret mice however differed from the Notch3^-/- mice in terms of lipid accumulation in the liver; the BODIPY 493/503 staining area was not altered in the Pdgfb^ret/ret mice (Fig. 7G). In conclusion, the Pdgfb^ret/ret mouse exhibits left ventricular hypertrophy similar to what is observed in the Notch3^-/- mice, but the two mouse models differ with regard to lipid accumulation in the liver.

Discussion

In this report, we show that Notch3^-/- mice, in addition to the well-established progressive loss of VSMC in the vasculature¹⁵, present a heart phenotype: left ventricular hypertrophy, coupled with cardiomyocyte hypertrophy and mild fibrosis. Notch3 was expressed in VSMC and pericytes of the heart but not in cardiomyocytes, leading us to propose that the effects on the heart are indirect and a consequence of the loss of Notch3 function in the VSMC in the vasculature of the heart or elsewhere in the body. The left ventricular hypertrophy in the Notch3^-/- mice was more profound compared to ablation of CSL in VSMC postnatally²¹, suggesting that the onset of Notch3^-/- hypertrophy may occur at an earlier developmental stage. In keeping with this notion, we observed an increase in cardiomyocyte area and dysregulated CD36 and GLUT1 expression already at two months of age in the Notch3^-/- mice.

To gain insights into the Notch3^-/- heart phenotype, we analysed the expression of genes important for cardiac metabolism, and expression of PDK4, which is involved in fatty acid uptake, and GLUT1, responsible for glycolysis, was down- and up-regulated, respectively. This may indicate that the Notch3^-/- heart switches from oxidative phosphorylation towards a more glycolytic metabolism. The adult heart uses mostly fatty acids as energy source, with fatty acids from the blood transported through endothelial cells into the heart⁵³. However, when diseased or injured, the heart reverts to a glycolytic metabolism, which is otherwise observed at embryonic stages^35,36; for review see³⁷. The observed switch towards a glycolytic metabolism in the Notch3^-/- hearts may therefore be a consequence of the left ventricular hypertrophy. Alternatively, Notch3 deletion and the subsequent loss of VSMC may in some way lead to a more systemic lowering of lipid levels in the vasculature, forcing a switch to higher glucose uptake and a glycolytic metabolism. The latter notion is supported by the lower lipid content in the livers of Notch3^-/- mice. It is also interesting to note that when Notch signalling was abrogated in the endothelium through deletion of the CSL gene in endothelial cells, this resulted in left ventricular hypertrophy and metabolic remodelling of the heart with disturbed fatty acid transport across the vessel wall and higher glucose uptake⁵⁴. An intriguing possibility is therefore that some of the secondary effects from loss of Notch3 function in the VSMC occur in the nearby endothelial cells, leading to reduced uptake of fatty acids, but to address this idea will require further experimentation.

Conceptually, how dysregulated Notch signalling in mural cells affects cardiomyocyte function could either be explained by specific effects on vasculature in the heart or by a more systemic effect on the arterial tree. In support of a first “cardio autonomous” model, we noted that the number of arterioles per area was reduced in the Notch3^-/- hearts, in keeping with two previous reports using a different Notch3 knockout model^21,55, while the overall density of the vasculature was not changed. Interestingly, ablation of CSL in endothelial cells in contrast resulted in an increased density of the heart vasculature⁵⁴. Whether the decrease in number of arterioles in the Notch3^-/- hearts contributes to the left ventricular hypertrophy remains to be explored. Alternatively, a “non-cardio autonomous” model would posit that more global changes to the vasculature would lead to systemic effects, presumably through hemodynamic mechanisms, thereby negatively impacting cardiomyocytes of the heart. The best-known example of this would be left ventricular hypertrophy resulting from chronic hypertension²⁵, which is unlikely the cause of left ventricular hypertrophy in Notch3^-/- mice, as they have a normal blood pressure unless subjected to angiotensin-II²¹. Notch3^-/- mice have however been shown to exhibit deficiencies in the vascular tone of cerebral, renal, and tail resistance arteries^56,57, and it is possible that a more general defect in VSMC contractility in resistance arteries would require compensatory hemodynamic mechanisms of the heart to overcome the chronic pressure overload in the Notch3^-/- mice. A compensatory increase in heart rate and/or stroke volume could contribute to the cardiac remodeling seen in the Notch3^-/- mice and would be in line with the observed increase in the interval between Z-lines in the sarcomeres and elevated Nppb expression in the Notch3^-/- mice. Further studies are however required to understand whether local and/or systemic effects on the vasculature contribute to molecular and morphological changes in Notch3-deficient hearts. Irrespective of the nature of the link between perturbed vascular Notch signalling and a heart phenotype, an indirect effect from the vasculature on the cardiomyocytes may explain why heart problems were observed as a side effect of clinical trials using blocking antibodies to Dll4, a Notch ligand expressed predominantly in endothelial cells^58,59.

The notion, based on analysis of the Notch3^-/- mice, that impairment of the heart can be an indirect result of genetic perturbations primarily the vasculature was corroborated by observations from the Pdgfb^ret/ret mice. The Pdgfb^ret/ret mice have reduced PDGF signalling because the truncated PDGF-B ligand lacking the retention motif is more diffusible, leading to lower local PDGF-B concentration around PDGF-B producing endothelial cells⁵¹ and, consequently, less stimulation of neighbouring PDGFRβ–positive VSMC and pericytes. It is noteworthy that the heart phenotype in the Pdgfb^ret/ret mice was partly similar to the Notch3^-/- phenotype, i.e., both models exhibit left ventricular and cardiomyocyte hypertrophy, although the Pdgfb^ret/ret mice did not show a reduced lipid content in the liver. The similarity in heart phenotypes underscores that primary effects in mural cells can indirectly affect cardiomyocytes, and it is also possible that some of the effects observed in the Notch3^-/- mice may in fact be attributed to reduced Pdgfrb expression, as a small decrease in Pdgfrb mRNA and protein expression was noted in the Notch3^-/- mice. In conclusion, we provide evidence from analysis of mouse models with disturbed Notch and PDGF signalling that a primary perturbation in VSMC and pericytes is sufficient to cause a heart phenotype. To gain further insights into the interplay between the VSMC and pericytes and cardiomyocyte function will be important to explore new therapeutic strategies for heart disease caused by primary vascular impairment.

Methods

Mice

The Notch3^-/- mice (C57BL/6J background) were maintained at the Department of Comparative Medicine (KMA), Karolinska Institutet (Stockholm, Sweden). The Pdgfb^ret/ret mice were maintained at the Rudbeck Laboratory, Uppsala University, Sweden. The mice were provided with water and food ad libitum, were maintained in a 12 h light/dark cycle, and housed in enriched cages. All experimental animal procedures were performed in accordance with local regulations and rules and according to ARRIVE guidelines. All animal experiments were approved by the Stockholm’s North (the Notch3^-/- mice) and Uppsala (the Pdgfb^ret/ret mice) Ethical Committees for Animal Research (Ethical permit No 5253–2019 and 5.8.18–03,029/2020 respectively). Mice used in this study were 6–14 months old as indicated in the figure legends, except for Supplementary Fig. 1E-G, where 2 weeks and 2 months old mice were used.

Histology

For the hematoxylin and eosin (HE) staining, mice were anesthetized and transcardially perfused with Hanks buffered salt solution (HBSS) followed by a solution of 4% formaldehyde in phosphate buffered saline (PBS). Tissues were post fixed in 4% formaldehyde for 4 h at 4 °C and transferred to 70% ethanol solution. After sectioning (4 μm sections), HE staining was performed as follows: The slides were baked for 40–60 min at 60 °C followed by deparaffinization in Xylene (Solveco) for 5 min (2 times) and rehydrated in absolute ethanol for 3–5 min (2 times), 95% ethanol for 2 min, 70% ethanol for 2 min. The slides were then rinsed with warm distilled running water for 2 min. The nuclear staining was performed using Mayer Hematoxylin (Histolab) for 10 min at room temperature (RT). The slides were then rinsed with warm distilled running water for 10–15 min followed by the cytoplasm staining (Eosin, Histolab) for 3 min at RT. After wash with warm distilled running water for 10–15 min, rehydration was performed (70% ethanol: 30 s, 95% ethanol: 2 min, Absolute ethanol: 3-5min for two times followed by two steps in Xylene for 5 min each. The slides were mounted with Pertex (Histolab).

For the Oil Red O staining, mice were euthanized, the organs removed and immediately fresh frozen and embedded in OCT (Scigene). 10 μm cryosections were left at RT for 15 min. Slides then were fixed with 4% PFA (Solveco) for 1 min. The slides were then rinsed with running tap water for 2 min followed by ORO staining (Histolab) for 3 min at RT. After 1 wash with running tap water for 2 min, slides were mounted with aqueous mounting medium (Mount it, Vector).

For the Masson’s staining, embedded paraffin blocks were sectioned to 4µm sections and Modified Masson's Trichrome Stain Kit (Sigma Aldrich) was applied according to the manufacturer’s instructions. Images of HE and Oil Red O staining and Masson’s staining were taken with the Olympus IX73 Microscope.

Immunohistochemistry

Adult mice of both sexes (8–10 months) were euthanised and the heart, lung and liver were removed. Organs were postfixed with 4% buffered formaldehyde (Histolab Products AB) for 4 h at RT, washed in PBS and immersed in 30% sucrose/PBS solution until sinking. Organs were mounted for cryosectioning in OCT (Scigen). Cryosections (14µm) were stored at− 80 °C until further use. Cryosections were thawed at RT for 15 min then washed quickly in PBS, dried and a hydrophobic barrier was created using ImmedgeTM Hydrophobic Barrier Pen (Vector Laboratory, United States). Sections were then permeabilised in 0,3% Triton X for 10 min, washed in PBS and blocked for one hour at RT with Serum-Free Protein Block (Dako). Next, the sections were incubated with primary antibody solution at 4°C overnight, followed by 3 washes in PBS and finally secondary antibody solution for one hour at RT. For nuclear staining, sections were incubated with DAPI (Invitrogen) before mounting in ProLong Gold Antifade (Life Technologies). Antibodies were diluted in 0.5% bovine serum albumin, 0.1% Triton X-100 in PBS. All primary and secondary antibodies are described in Supplemental Table 4. Specimens were analysed using a Leica TCS SP8 confocal microscope (Leica Microsystems). All confocal images are represented as maximum intensity projections unless stated otherwise. Image analysis was carried out using Image J (NIH) software.

Wheat germ agglutinin staining

Wheat germ agglutinin staining (WGA) was carried out as follows: Heart tissue was harvested from the mouse and post fixed with 4% buffered formaldehyde (Histolab Products AB) for 4 h at 4°C. The tissue was then immersed in a 30% sucrose solution at 4 °C until sinking. Subsequently, the tissue was embedded in OCT (Scigen) and stored at − 80 °C in Tissue-Tek cryomold (Sakura) until use. Frozen blocks were then cut into 10 μm sections and stored at -80 °C. Frozen sections were allowed to equilibrate at RT for 15 min and then washed in PBS for 5 min. The 1.0 mg/mL WGA conjugate (W11261, Thermo Fisher) stock solution was diluted into 20ug/ml with PBS and heart sections were incubated with WGA for 20 min, at RT in the dark. After 3 washes in PBS, the heart sections were incubated with DAPI (Invitrogen) for 5 min, at RT in darkness and washed in PBS for 5 min at RT. The sections were mounted with Prolong Gold antifade reagent (Invitrogen) and kept at 4 °C in darkness until visualisation with confocal microscopy (see above).

Transmission electron microscopy

The heart tissue was fixed by perfusion using 3% glutaraldehyde and 1% formaldehyde in 0.1M phosphate buffer, pH 7.4. After perfusion, the heart tissue was harvested and allowed to fix at RT prior to storage at + 4 °C. Following fixation, the tissue was washed in 0.1M phosphate buffer and postfixed in 2% osmium tetroxide in 0.1M phosphate buffer at RT for 2 h before stepwise dehydrated in ethanol followed by acetone and finally resin-embedded in LX-112 (Ladd). Ultrathin sections (approximately 80–100 nm) were cut using an EM UC7 ultramicrotome (Leica) and contrasted with uranyl acetate followed by lead citrate. The sections were examined in a Hitachi HT7700 transmission electron microscope (Hitachi High-Technologies) operated at 80 kV and 2xk2x digital images were acquired using a Veleta CCD camera (Olympus Soft Imaging Solutions).

qPCR analysis

Mice were euthanized, the organs removed and immediately fresh frozen. Frozen tissue from the left ventricle was cut on ice and homogenized in 0.5 ml Trizol (Thermo Fisher Scientific) per 25 mg tissue. Metallic beads are added to the mixture and TissueLyzer (Qiagen) was used with the following setting: 50Hz for 2 min twice with a fast spin in between. 0.1 ml of chloroform per 0.5 ml of Trizol was added, samples were shaken vigorously for 15 s and kept for 10 min at RT and then centrifuged for 5 min at 12,000 × g. The transparent aqueous phase was then transferred to a fresh tube. The RNA was precipitated with 250 μl of isopropanol per 0.5 ml of Trizol used for the initial homogenization. Samples were kept on ice for 1 h and centrifuged for 10 min at 4 °C at 12,000 × g. The pellet was washed once with 75% ethanol by flipping the tube without disturbing the pellet and subsequently centrifuged for 10 min at 12,000 × g (4 °C). The ethanol was removed, and the pellet resuspended in 50 μl of RNAse and DNAse free water (Ambion). RNA was converted to cDNA using the iScript Kit (Biorad) and the qPCR mixture was prepared using the SsoAdvanced Universal SYBR Green Supermix (Biorad). The qPCR primer list is provided in the Supplemental Table 5. qPCR was performed using the Applied Biosystems 7500 Real-Time PCR System (ABI 7500; Applied Biosystems). Relative gene expression was determined using the ΔΔCT method (ΔΔCT = ΔCt sample-Δct control) and fold change expression changes were calculated by normalizing first to GAPDH mRNA expression levels, as a housekeeping gene reference. A minimum of three biological replicates (three different animals per genotype) were used. All the graphs and the statistical analysis were produced using Prism Graphpad (ver.8) and differences in mRNA expression levels between samples were analysed using unpaired t-test.

RNA-seq

The RNA was quantified with Qbit (Invitrogen) and loaded in a 384 well plate for subsequent cDNA conversion using a previously described protocol⁶⁰. Briefly, the RNA undergoes a first-strand conversion in a reaction mixture with SuperScript II reverse transcriptase (Invitrogen), RNase inhibitor (Takara), 5X SuperScript II First-Strand buffer (Invitrogen), DTT (Invitrogen), 5M Betaine (Sigma) MgCl2 (Sigma), and custom locked nucleic acid oligonucleotides. Reverse transcription was performed for 10 cycles followed by inactivation at 70°C for 15 min. The total volume of cDNA was added into PCR master mix containing KAPA HiFi HotStart ReadyMix (KAPA Biosystems) and ISPCR primers. The PCR reaction was performed for 22 cycles. Next, PCR products were purified using SeraMag beads (Sigma Aldrich) and 17% PEG, with the final elution in EB buffer (Qiagen). The library quality and size distribution were checked using a High-Sensitivity DNA chip (Agilent Bioanalyzer). For the library preparation, cDNA samples were mixed with 10mM TAPS-NaOH, 5mM MgCl₂ pH 8,5, 10% PEG 8K, Tn5 enzyme and water and incubated at 55°C for 6 min. After this step the samples were incubated with 0,2% SDS for 5 min at RT. Then the samples were pooled after enrichment PCR as described by the manufacturer (Illumina) through the use of a dual-index strategy, referred to as index 1 (i7) and index 2 (i5). The sequencing was then performed using a HiSeq 3000 unit at the Single Cell Core Facility (SICOF) at Karolinska Institute (Sweden).

Bioinformatic analysis

Four and three biological samples of Notch3^+/+ and Notch3^-/- mice, respectively, have been considered for differential gene expression analysis (DEG) and three technical replicates per biological sample were collapsed. DEG analysis was performed using the DESeq2 package version 1.41.4⁶¹. Differentially expressed genes between Notch3^-/- and control (Notch3^+/+) mice with statistical significance were then identified with a minimum fold change 1.5 and a false discovery rate (FDR) lower than 0.05. Shrunken log fold changes were generated using the adaptive shrinkage estimator ashr⁶². For the full list of differentially expressed genes (DEG), see Supplemental Table 1. KEGG pathway enrichment and Jensens disease analysis of the differentially expressed genes were carried out using ShinyGO v0.77 (http://bioinformatics.sdstate.edu/go/) with a FDR cutoff of 0.05 and a minimum pathway size of 10⁶⁴. Data tables with pathway enrichment values, gene information and GO terms were downloaded directly from the website.

Image analysis

For all histology, immunofluorescence and area images (Figs. 1D, 1F, 2A, 4B, 4C, 6B, 6C, 7F, 7G) a minimum of two/ three animals per genotype were used and three/four pictures were randomly taken and analysed. Briefly, the colour channel was split and converted into 8-bit greyscale in Image J (NIH) and subjected to automatic threshold using the Li method. Regions of interest were drawn and % threshold value of the indicated marker was used (Figs. 4B, 4C, 6B, 6C, 7G). For the cardiomyocyte area measurement, the cells per field were counted and the cross area was measured with ImageJ software (Figs. 2A, 7F). For Fig. 1F, random measurements of the left ventricle wall were made. All the graphs and the statistical analysis were performed using GraphPad Prism (ver.8) and unpaired t-test.

Analysis of Tabula Muris

The data in Figs. 5D and 7G were obtained from https://tabula-muris-senis.ds.czbiohub.org/ by selecting the FACS method for heart tissue and searching for the gene of interest.

Data availability

The transcriptomic data have been uploaded at Gene Expression Omnibus (GEO) with Accession Number GSE237040.

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Acknowledgements

The financial support from the Swedish Research Council (2019:00285), the Swedish Cancer Society (2020-2022: 19-0012) and the Swedish Brain Foundation (F2020-0246) (UL) is gratefully acknowledged. FDG acknowledges funding from Stiftelsen för Ålderssjukdomar (2022-01310). The assistance from the SICOF core facility for the transcriptomic experiments is acknowledged.

Funding

Open access funding provided by Karolinska Institute.

Author information

Dongli Liu
Present address: Department of Pediatrics at the First Affiliated Hospital, Guangxi Medical University in Nanning, Guangxi, People’s Republic of China
These authors contributed equally: Francesca Del Gaudio and Dongli Liu.

Authors and Affiliations

Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden
Francesca Del Gaudio, Dongli Liu, Eike-Benjamin Braune, Emil M. Hansson & Urban Lendahl
Department of Immunology, Genetics, and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden
Maarja Andaloussi Mäe & Christer Betsholtz
Bioscience Cardiovascular, Research and Early Development, Cardiovascular, Renal and Metabolism (CVRM), BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden
Qing-Dong Wang
Department of Medicine, Karolinska Institutet, Huddinge, Sweden
Christer Betsholtz

Authors

Francesca Del Gaudio
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Dongli Liu
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Maarja Andaloussi Mäe
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Contributions

U.L., F.D.G., D.L. and C.B. conceived the study and U.L., F.D.G. and D.L. designed the experiments. F.D.G. and D.L. performed the experiments. Data were collected and analysed by F.D.G., D.L., M.A.M., E.B.B., E.M.H., Q.D.W., C.B. and U.L. The manuscript was written by U.L. and F.D.G. with input and reviewed by all authors. All authors have read and approved the final manuscript.

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Correspondence to Francesca Del Gaudio or Urban Lendahl.

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Competing interests

All authors declare that there are no competing interests with respect to the topic of this article. For the purpose of full disclosure, UL holds a research grant from Merck Healthcare KGaA, Darmstadt, Germany, but no personal remuneration. UL also holds a Distinguished Visiting Professorship at Hong Kong University, is a Visiting Scientist at the Children’s Hospital, Harvard Medical School and serves as the chair of the Council of Åke Wibergs Stiftelse.

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Del Gaudio, F., Liu, D., Andaloussi Mäe, M. et al. Left ventricular hypertrophy and metabolic resetting in the Notch3-deficient adult mouse heart. Sci Rep 13, 15022 (2023). https://doi.org/10.1038/s41598-023-42010-7

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Received: 18 July 2023
Accepted: 04 September 2023
Published: 12 September 2023
DOI: https://doi.org/10.1038/s41598-023-42010-7

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