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Differences in Cell-Type–Specific Responses to Angiotensin II Explain Cardiac Remodeling Differences in C57BL/6 Mouse Substrains

Originally publishedhttps://doi.org/10.1161/HYPERTENSIONAHA.114.04067Hypertension. 2014;64:1040–1046

Abstract

Despite indications that hearts from the C57BL/6N and C57BL/6J mouse substrains differ in terms of their contractility and their responses to stress-induced overload, no information is available about the underlying molecular and cellular mechanisms. We tested whether subacute (48 hours) and chronic (14 days) administration of angiotensin II (500 ng/kg per day) had different effects on the left ventricles of male C57BL/6J and C57BL/6N mice. Despite higher blood pressure in C57BL/6J mice, chronic angiotensin II induced fibrosis and increased the left ventricular weight/body weight ratio and cardiac expression of markers of left ventricular hypertrophy to a greater extent in C57BL/6N mice. Subacute angiotensin II affected a greater number of cardiac genes in C57BL/6N than in C57BL/6J mice. Some of the most prominent differences were observed for markers of (1) macrophage activation and M2 polarization, including 2 genes (osteopontin and galectin-3) whose inactivation was reported as sufficient to prevent angiotensin II–induced myocardial fibrosis; and (2) fibroblast activation. These differences were confirmed in macrophage- and fibroblast-enriched populations of cells isolated from the hearts of experimental mice. When testing F2 animals, the amount of connective tissue present after chronic angiotensin II administration did not cosegregate with the inactivation mutation of the nicotinamide nucleotide transhydrogenase gene from C57BL/6J mice, thus discounting its possible contribution to differences in cardiac remodeling. However, expression levels of osteopontin and galectin-3 were cosegregated in hearts from angiotensin II–treated F2 animals and may represent endophenotypes that could facilitate the identification of genetic regulators of the cardiac fibrogenic response to angiotensin II.

Introduction

The highly used C57BL/6 mouse inbred strain is the preferred choice for mouse transgenic and knockout studies1 and was the first strain whose genome was fully sequenced.2 However, several C57BL6 substrains have emerged over the years, each showing genomic differences because of genetic drift and displaying various phenotypic differences.1,3 One example is the difference between the C57BL/6J and C57BL/6N substrains. The C57BL/6 strain was initially developed at The Jackson Laboratory, and mice from that colony are identified as C57BL/6J. In 1951, some mice were separated from the original colony to initiate a new colony at the National Institutes of Health, the latter being identified as C57BL/6N.1 Despite the recognition that genetic drift between mouse strains may compromise the reproducibility of experimental data over time and place,4 there are still many publications where the substrain of origin of C57BL/6 mice is not mentioned. However, a recent study reported that the cardiac output of C57BL/6N male mice is higher than that of their C57BL/6J counterparts.5 Likewise, the effects of transverse aortic constriction on survival and the remodeling of left ventricles (LV) are much greater in C57BL/6N than in C57BL/6J mice.6 Thus, evidence indicates that genetic drift can significantly alter cardiac phenotypes in the established C57BL/6 inbred strain.

Despite indications that hearts from the C57BL/6N and C57BL/6J mouse substrains differ in terms of their contractility, as well as their responses to stress-induced overload (including survival rate, maintenance of cardiac function, and development of hypertrophy), no information is available about the underlying molecular and cellular mechanisms. We compared the effects of either subacute (48 hours) and chronic (14 days) infusions of angiotensin II (Ang II) on LV remodeling in both substrains. As we observed substrain-specific differences in expression for some genes known to be specific for particular cell-types, we further confirmed our observations by measuring gene expression in cytofluorometry-sorted cell-specific populations. Finally, expression of some differentially expressed genes was further tested in hearts from Ang II–treated individuals from a hybrid F2 C57BL/6J/C57BL/6N population, to test whether they cosegregated in that genetic cross.

Material and Methods

Animals

Experiments were conducted following approval by the animal ethic committee of the Institut de Recherches Cliniques de Montréal (IRCM) and in agreement with the guidelines of the Canadian Council for Animal Care. C57BL6/J and C57BL6/N mice were purchased from The Jackson Laboratory (Bar Harbor, MN) and Harlan (Indianapolis, IN), respectively, and housed in the animal care facility of IRCM. Physiological and genetic procedures are as described in Methods in the online-only Data Supplement.

Isolation and Characterization of Enriched Cell Populations

Enzymatic digestion and cell enrichment procedures are as described in Methods in the online-only Data Supplement.

Statistical Analyses

Data were presented as mean±SEM. To test whether substrain origin interacted with the effects of treatments in C57BL6/J and C57BL6/N mice, data were analyzed by 2-way ANOVA, followed by Sidak post hoc analysis for multiple comparisons. Comparisons of the effects of treatments in multiple strains were performed by 1-way ANOVA, followed by Sidak post hoc analysis for multiple comparisons.

Results

Genotyping with 89 polymorphic markers confirmed the purity of each strain (Table S1 in the online-only Data Supplement). Several end points of remodeling of LV were examined in C57BL/6J and C57BL/6N male mice 14 days after implantation of minipumps delivering either Ang II (500ng/kg per day) or vehicle. Chronic Ang II increased the abundance of both histologically stained connective tissue and Col1a1 mRNA to a greater extent in C57BL/6N male mice than in their C57BL/6J counterparts (Figure 1). Likewise, chronic Ang II increased the LV weight/body weight ratio to a greater extent in C57BL/6N than in C57BL/6J mice, and the LV abundance of Nppa and Myh7 mRNA was increased only in C57BL/6N mice (Figure 1). In contrast, mean arterial pressure was in average 15 mm Hg lower in that strain than in C57BL/6J mice, with Ang II increasing blood pressure to the same extent in both strains and at all times during administration (from 16% to 35% for daytime values and from 6% to 28% for night-time values; Figure S1). Diastolic and systolic pressure showed strain- and time-dependent differences of similar magnitude (results not shown).

Figure 1.

Figure 1. Effects of chronic 14-day treatments with angiotensin II (Ang II) on either connective tissue content (as quantified after Masson Trichrome staining), left ventricle (LV)/body weight (BW) ratios, and LV mRNA abundance of Col1a1, Nppa, and Myh7 (by reverse transcriptase-quantitative polymerase chain reaction). For each gene, values for LV mRNA abundance corresponded to 2(−ΔΔCt) values, representing relative expression vs that of the Rps16 normalizing gene. A, Representative images of histological sections. BF, For each graph, the results of the 2-way ANOVA analysis for either treatment, strain, or the strain×treatment interaction are as indicated. The bars represent mean±SEM. For LV/BW values, n=10; for other variables, n=4 to 5. *P<0.05, **P<0.01 ***P<0.001, ****P<0.0001, by post hoc Sidak comparisons.

Changes in cardiac gene expression have been reported to occur as early as 24 hours after the onset of Ang II administration.7,8 Because differences in early rapid responses may be ultimately responsible for downstream differences in the chronic effects of Ang II, we compared the profiles of gene expression in hearts of C57BL/6N and C57BL/6J mice harvested 48 hours after implantation of minipumps. A total of 2323 genes showed significant responses to subacute Ang II in the C57BL/6N substrain, in contrast to only 127 genes in C57BL/6J mice. When testing the 2-way interaction between treatment and strain, we found that substrain interacted significantly with the effect of Ang II for 372 genes. Among the latter, 344 and 19 genes showed responses that were exclusive to either C57BL/6N or C57BL/6J mice, respectively, whereas another 28 genes showed responses that were significantly different because of either differences in the amplitude or in the direction of the effect. Among these 372 genes, we further examined the 200 ones showing the greatest absolute changes in C57BL/6N mice (Table S4). Functional Annotation Clustering analysis of these genes revealed that the categories with the highest enrichment scores corresponded to (1) blood vessel development; (2) extracellular region; (3) lysosome; and (4) immune responses (Table S5). Among the top 40 of these 200 genes (as ranked on the basis of their responses in C57BL/6N), enrichments were observed mostly for the blood vessel and extracellular region functional clusters (Table S6). Moreover, several of the genes on the top of that list are well known to be associated with specific biological processes: Tissue-inhibitor of metalloprotease-1 (Timp1), Tenascin (Tnc), and Lysyl-oxidase (Lox) are important regulators of fibrosis911; osteopontin (Spp1) is a marker of the activation of monocytes into macrophages12; and arginase-1 (Arg1) and galectin-3 (Lgals3) are prototypical markers of macrophage M2 polarization.13,14 In contrast to these differentially expressed fibrosis- and macrophage-associated genes, subacute Ang II induced the expression of Myh7 and Col1a1 (all well-known markers of hypertrophic cardiac remodeling) to the same extent in both strains. For some of the above genes, the strain-specific effects of Ang II (500 ng/kg per day) were confirmed by reverse transcriptase-quantitative polymerase chain reaction (Figure 2). Additional experiments showed that Ang II increased the expression of Nppa, Lgals3, and Spp1 in a dose–response fashion (from 500 to 1500 ng/kg per day) in C57BL/6J, whereas maximal responses were already observed with the lowest dose of Ang II in C57BL/6N mice (Figure S2). The differences in sensitivity to Ang II did not seem to result from differences in basic components of the Ang II signaling pathway, as we detected no difference in the expression of the genes coding for either angiotensin type 1-receptor or Galpha-q/11 (the latter representing the major G-protein–coupled transducing the signals for angiotensin type 1 receptor–mediated gene regulation; Figure S3).15

Figure 2.

Figure 2. Effects of 2-day treatments with angiotensin II (Ang II) on left ventricular (LV) mRNA abundance of 6 genes. For each graph, the results of the 2-way ANOVA analysis for either treatment, strain, or the strain×treatment interaction are as indicated. The bars represent mean±SEM, n = 4. *P<0.05, **P<0.01 ***P<0.001, by post hoc Sidak comparisons.

In regards to the macrophage-specific genes, differences in their expression level in LV tissue may result from either an increase in the number of these cells or from qualitative changes in their gene expression profiles. By enzymatically digesting LVs and counting cells by flow cytofluorometry, we found that subacute Ang II increased the number of double-positive CD11b–F4/80 cells in LVs from C57BL/6J and C57BL/6N to the same extent in both substrains (Figure S4). To delineate the cellular targets of the Ang II–induced gene response further, we digested LVs from C57BL/6J mice to prepare 4 different populations of cells enriched for specific cardiac cell types. The efficiency of the cell fractionations was verified by reverse transcriptase-polymerase chain reaction for specific cell markers (Table S7). Additional reverse transcriptase-polymerase chain reaction allowed us to confirm further that Spp1, Arg1, and Lgals3 were much more abundantly expressed in CD11b(+) cells than in any other cell population, whereas Timp1, Tnc, and Lox were mostly restricted to fibroblasts (Table S8). We further used our sorted cell populations to test whether the expression of genes within sorted macrophages and fibroblasts themselves was affected by Ang II in a strain-specific manner. We found that subacute Ang II increased the expression of a marker of macrophage activation (Spp1) and 2 markers of M2 polarization (Arg1 and Lgals3) to a much greater extent in myocardial macrophages from C57BL/6N than in C57BL/6J (Figure 3). Likewise, Ang II induced a decrease in the expression of Nos2 in these cells although the effect was of similar magnitude in both strains. Strain-specific effects were observed in fibroblasts as well, as Ang II affected the expression of Timp1, Tnc, and Col1a1 to a greater extent in myocardial fibroblasts from C57BL/6N than in C57BL/6J (Figure 3).

Figure 3.

Figure 3. AD, Abundance of mRNA of Spp1, Lgals3, Arg1, and Nos2 in a macrophage-enriched cell population isolated from left ventricles (LVs) of C57BL/6J and C57BL/6N mice receiving either sham or angiotensin II (Ang II) treatment for 48 hours. EH, Abundance of mRNA of Spp1, Timp1, Tnc, and Col1a1 in a fibroblast-enriched cell population isolated from LVs of C57BL/6J and C57BL/6N mice receiving either sham or Ang II treatment for 48 hours. For all graphs, results of the 2-way ANOVA analysis for either treatment or strain×treatment interaction are as indicated. The bars represent mean±SEM, n=5. *P<0.05, **P<0.01 ***P<0.001, by post hoc Sidak comparisons.

One known major difference between the 2 substrains concerns the inactivation mutation of the Nnt gene in the C57BL/6J strain.1 To test the possible contribution of the Nnt mutation to our observed substrain-specific differences, we produced an F2 progeny from a cross between the parental C57BL/6N and C57BL/6J lines, and tested whether strain-specific differences cosegregated with the Nnt genotypes (either J/J if both alleles originated from C57BL/6J or N/N if both alleles originated from C57BL/6N). The effects of chronic Ang II on induction of Col1a1 expression and myocardial fibrosis in F2 animals were not different from that in C57BL/6J animals, regardless of their genotype at the Nnt locus (Figure S5); likewise, the Nnt genotype had no effect on the abundance of either Spp1 or Lgals3 mRNA after subacute Ang II. Nonetheless, there was a tight and significant correlation in the expression of both genes in hearts from Ang II–treated F2 mice (Figure 4), indicating that post-Ang II expression levels of these 2 genes cosegregated in the F2 progeny. The H2 broad sense heritability index (representing the ratio of genetic:phenotypic variance) for post-Ang II levels of gene expression was calculated to be 0.80 and 0.91 for Spp1 and Lgals3, respectively. We further tested to what extent the response of this gene in C57BL/6N mice differed from that seen in other laboratory mouse strains: subacute Ang II increased the expression of Spp1 to the same extent in LVs from C57BL/6J, A/J, and FVB/N mice, whereas C57BL/6N mice differed from the other 3 strains by a response of much greater amplitude (Figure 4).

Figure 4.

Figure 4. A, Abundance of Spp1 mRNA in left ventricles (LVs) from several inbred mouse strains (C57BL/6J, A/J, FVB/N, and C57BL/6N). All data were obtained by reverse transcriptase-quantitative polymerase chain reaction by calculating −2(ΔΔCT) and normalizing values by that obtained for the S16 housekeeping gene; n=5. B, Linear regression analysis of the relative abundance of Spp1 and Lgals3 mRNA (log2 normalized values) in LVs from 100 F2 mice each receiving subacute Ang II treatment; r2=0.87; P<0.0001.

Discussion

We observed that infusions of Ang II at the dose of 500ng/kg per day had both chronic and subacute effects that differed between the C57BL/6J and C57BL/6N mouse substrains. Chronic Ang II increased LV fibrosis and LV expression of Nppa and Myh7 in C57BL/6N but not in C57BL/6J mice. In contrast, mean arterial pressure was consistently higher in C57BL/6J than in C57BL/6N mice (both before and after Ang II administration), in keeping with a recent report where blood pressure was measured in the same 2 substrains using a tail-based technique.16 It is also likely that these substrain-dependent differences are not limited to the model of Ang II infusion because transverse aortic constriction has recently been reported to induce LV remodeling to a greater extent in C57BL/6N substrains than in their C57BL/6J counterparts.6

Although no strain-dependent differences were observed in the effects of subacute Ang II on LV expression of Myh7 and Col1a1, this treatment induced a C57BL/6N-selective increase in the expression of several genes that were expressed preferentially in noncardiomyocytes, the most prominent ones being (1) for macrophages, Lgals3 (which codes for galectin-3, also known as Mac-2); (2) for fibroblasts, Tnc (which codes for tenascin); and (3) for both cell-types, Spp1 (which codes for osteopontin). These differences occurred despite no evidence for differential expression of basic components of the Ang II signaling pathway. The differential effect of Ang II (500 ng/kg per day) on these genes may be sufficient to explain its substrain-specific differences on LV fibrosis, because (1) inactivation of either Spp117,18 or Lgals319 are sufficient to prevent Ang II–induced myocardial fibrosis; and (2) recombinant galectin-3 induces fibroblast proliferation and collagen production in whole hearts when administered in the pericardial sac.20 In fact, the effects of Spp1 might be mediated by Lgals3 because inactivation of just Spp1 leads to dramatic reductions in myocardial expression of Lgals3.21 At the cellular level, activated macrophages produce galectin-3, whereas fibroblasts have galectin-3–binding sites.20 Our own data confirmed that among several cardiac cell populations, Lgals3 is expressed at highest levels in CD11b(+) cells (ie, macrophages), and that Ang II induced Lgals3 to a greater extent in CD11b(+) cells from C57BL/6N hearts than in their counterparts from C57BL/6J. This was accompanied by proportional differences in the activation of fibroblasts because Ang II induced Tnc (a marker of fibroblast activation) to a greater extent in fibroblast-enriched cells from C57BL/6N mice.

Interestingly, previous studies in another model of Ang II–dependent LV remodeling (the outbred renin-overexpressing Ren-2 rats) have shown that (1) the genetic background is an important determinant of the sensitivity of the LVs to the effects of Ang II; (2) some cardiac genes show differential expression in the hearts of rats progressing to heart failure in comparison with compensated rats; and (3) many of the latter genes (including Spp1 and Lgals3) are in fact the same as the ones responding to a greater extent in the LV remodeling-prone C57BL/6N strain (Table S4).22 It has been argued that these background-dependent differences in the progression to heart failure represent differences in the sensitivity of LVs to Ang II. Accordingly, our own dose–response experiments with Ang II showed that the strain-dependent differences observed with the low dose (500 ng/kg per day) are no longer apparent when using higher doses (1500 ng/kg per day). Of note, mice are less sensitive than rats to administered Ang II exogenously.23 Although Ang II is typically administered to rats at doses averaging 100 to 200 ng/kg per day,24 the doses used in mice have generally been much higher (up to 3 μg/kg per day).17 The dose at which we have observed substrain-dependent differences was chosen as the lowest one causing reliable increases in blood pressure, to avoid doses whose pathophysiological relevance was unclear.

On activation by environmental cues, monocytes and naïve macrophages can differentiate into different types of activated macrophages each having specific properties and functions.25 Within the spectrum of possible forms of macrophage activation, 2 extremes have been defined as either M1 polarized (or classically activated) or M2 polarized (or alternatively activated) macrophages, each expressing specific sets of genes.26 Because Lgals3 expression is considered to be a marker of M2 polarization,14 we further tested the effect of Ang II on the expression of either NOS2 or Arg1 (ie, markers of either M1 or M2 macrophages), respectively.27 Ang II decreased the expression of Nos2 in macrophages from the hearts of both strains, indicating that macrophages departed from the M1 phenotype in both strains. However, polarization into M2 macrophages seemed to be more pronounced in C57BL/6N mice because Ang II induced the expression of Arg1 to a significantly greater extent than in cells from C57BL/6J. Findings from previous studies underscore the critical roles played by macrophages and their precursors in Ang II–induced fibrosis in either hearts8,28 or kidneys.29,30 In our hands, although Ang II increased the number of CD11b(+) cells to the same extent in both C57BL/6J and C57BL/6N hearts, the strains differed in terms of the effects of Ang II on M2 polarization. This indicates that qualitative differences in the characteristics of activated macrophages (rather than quantitative differences in the number of macrophages infiltrating the heart) are responsible from strain-specific differences in LV remodeling. This notion is compatible with the generally held view associating M2 polarized macrophages with tissue remodeling and fibrosis.3133

Because C57BL/6N mice have gene expression responses that set them apart from either C57BL/6J mice and 2 other inbred mouse strains, it is possible that the divergent response of C57BL/6N mice is a consequence of one of the private mutations that have developed in that strain and is not present in either the C57BL/6J or the other laboratory mouse strains.16 Of note, 1 major difference between the 2 substrains concerns the inactivation mutation of the Nnt gene, which seemed to have occurred in C57BL/6J mice at The Jackson Laboratory after 1971.1 The protein encoded by Nnt is located in the inner mitochondrial membrane, where it plays important roles in mitochondrial peroxide metabolism,34 the latter being potentially involved in LV ventricular remodeling.35 However, the possible contribution of the Nnt mutation was discounted on the basis of the observation that the differences in the effects of chronic Ang II on collagen accumulation and of subacute Ang II on Spp1 and Lgals3 expression did not cosegregate with the mutation at the Nnt locus. However, expression levels of Spp1 and Lgals3 were cosegregated in hearts of Ang II–treated individuals from a F2 hybrid cross.

Perspectives

C57BL/6J and C57BL/6N mice show marked differences in their responses to the LV remodeling effects of Ang II, probably as the result of natural genetic variants that have accumulated over time between colonies and differentiate both substrains. Of note, naturally occurring allelic variants in inbred strains have a special interest: unlike genetically modified mouse models (which often represent extreme perturbations, such as complete loss of function of a targeted gene), they may mimic more closely the more subtle variations that are thought to be responsible for many human diseases and may be major contributors to phenotypic diversity.36 Despite their high level of genetic relatedness, recent mouse genome resequencing efforts have revealed the existence of several single nucleotide polymorphisms between the C57BL/6J and C57BL/6N substrains (http://www.sanger.ac.uk/resources/mouse/genomes). As recently demonstrated, these single nucleotide polymorphisms are also sufficiently numerous to allow the discovery functional genetic variants in hybrid F2 C57BL/6J/C57BL/6N crosses.37 Our data show that the differential responses of Spp1 and Lgals3 to subacute Ang II might represent endophenotypes that may facilitate the identification of genetic regulators of the cardiac fibrogenic response to infusions of Ang II in such crosses. Of note, fibrogenesis is increasingly becoming recognized as a major cause of morbidity and mortality in many chronic diseases,38,39 but for which no specific therapies are available yet. The discovery of genetic variants regulating this process would, therefore, be of great clinical interest.

Acknowledgments

We thank Manon Laprise, Éric Massicotte, Julie Lord, and Dominique Lauzier for their expert technical help, and Drs Kumar and Takahashi for their help with the genotyping of our strains.

Footnotes

The online-only Data Supplement is available with this article at http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA.114.04067/-/DC1.

Correspondence to Christian F. Deschepper, IRCM, 110 Ave des Pins Ouest, Montréal, Québec, Canada H2W 1R7. E-mail

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Novelty and Significance

What Is New?

  • Although C57BL/6 mice represent the most commonly used strain for generation and analysis of transgenic and knockout mice, substrains show marked differences in left ventricular remodeling responses to angiotensin II.

  • The differential responses of osteopontin and galectin-3 represent endophenotypes that may facilitate the identification of genetic regulators of the cardiac fibrogenic response to infusions of angiotensin II in C57BL/6J/C57BL/6N crosses.

What Is Relevant?

  • Cardiac fibrosis represents an important component of hypertension-related end-organ damage.

  • Fibrogenesis represents a major cause of morbidity and mortality in many chronic diseases, but no specific therapies are currently available to halt or reverse fibrosis.

Summary

We have identified an animal model and a subphenotype that will facilitate the identification of genetic regulators of the cardiac fibrogenic response.

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