Cardiac Fibroblasts Mediate a Sexually Dimorphic Fibrotic Response to β-Adrenergic Stimulation

Abstract

Background Biological sex is an important modifier of cardiovascular disease and women generally have better outcomes compared with men. However, the contribution of cardiac fibroblasts (CFs) to this sexual dimorphism is relatively unexplored. Methods and Results Isoproterenol (ISO) was administered to rats as a model for chronic β-adrenergic receptor (β-AR)-mediated cardiovascular disease. ISO-treated males had higher mortality than females and also developed fibrosis whereas females did not. Gonadectomy did not abrogate this sex difference. To determine the cellular contribution to this phenotype, CFs were studied. CFs from both sexes had increased proliferation in vivo in response to ISO, but CFs from female hearts proliferated more than male cells. In addition, male CFs were significantly more activated to myofibroblasts by ISO. To investigate potential regulatory mechanisms for the sexually dimorphic fibrotic response, β-AR mRNA and PKA (protein kinase A) activity were measured. In response to ISO treatment, male CFs increased expression of β1- and β2-ARs, whereas expression of both receptors decreased in female CFs. Moreover, ISO-treated male CFs had higher PKA activity relative to vehicle controls, whereas ISO did not activate PKA in female CFs. Conclusions Chronic in vivo β-AR stimulation causes fibrosis in male but not female rat hearts. Male CFs are more activated than female CFs, consistent with elevated fibrosis in male rat hearts and may be caused by higher β-AR expression and PKA activation in male CFs. Taken together, our data suggest that CFs play a substantial role in mediating sex differences observed after cardiac injury.

Keywords: cardiac fibroblasts; cardiac fibrosis; isoproterenol; sex differences.

Figure 1. Male rats increased cardiac hypertrophy and fibrosis in response to ISO treatment compared with female rats.

(A) Illustration of experimental design testing ISO treatment on male and female rats. (B) Cardiac hypertrophy measured by left ventricle weight to tibia length (LV/TL). Male vehicle n=4, Male ISO n=5, Female vehicle n=6, Female ISO n=8. (C) Representative images of picrosirius red staining of hearts from male and female rats treated with vehicle or ISO for 7 days. Scale bar = 4 mm. (D) Quantification of picrosirius red staining at 7 days. Male vehicle n=3, Male ISO n=8, Female vehicle n=8, Female ISO n=6. (E) Hydroxyproline biochemical assay on male and female rat hearts treated with vehicle or ISO for 7 days. Male vehicle n=11, Male ISO n=8, Female vehicle n=6, Female ISO n=9. (F) Shear elastic modulus of left ventricle from male and female rat hearts treated with vehicle or ISO for 7 days. Male vehicle n=6, Male ISO n=8, Female vehicle n=8, Female ISO n=7. Two‐way ANOVA with Bonferroni post hoc applied. *P<0.05, **P<0.01, ***P<0.001. All data reported as ±SEM. ISO indicates isoproterenol; and KPa, kilopascal.

Figure 2. ISO‐induced fibrosis is independent of sex hormones.

(A) Illustration of experimental design testing ISO treatment on CAS and OVX rats. (B) Cardiac hypertrophy measured by left ventricle weight to tibia length (LV/TL). CAS male vehicle n=8. CAS male ISO n=9. OVX female vehicle n=7. OVX female ISO n=7. (C) Hydroxyproline biochemical assay on CAS male and OVX female rat hearts treated with vehicle or ISO for 7 days. CAS male vehicle n=6. CAS male ISO n=6. OVX female vehicle n=6. OVX female ISO n=6. Two‐way ANOVA with Bonferroni post hoc applied. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. All data reported as ±SEM. CAS indicates castrated; ISO, isoproterenol; and OVX, ovariectomized.

Figure 3. Female CFs are more proliferative than male CFs in response to ISO treatment.

(A) Illustration of experimental design testing ISO treatment on CF proliferation in male and female rats during the early (3 day) or late (7 day) response. (B) Proliferation of CFs after 3 days of ISO treatment measured by EdU staining. Representative images and quantification. White arrows indicate cells shown in insets. DAPI = blue, EdU positive cells = Green, vimentin = pink. Large image scale bar = 100 µm, inset scale bar = 10 µm. Male vehicle n=7, Male ISO n=9, Female vehicle n=8, Female ISO n=7. Two‐way ANOVA with Bonferroni post hoc applied. (C) Proliferation of CFs after 7 days of ISO treatment measured by EdU staining. Representative images and quantification. White arrows indicate cells shown in insets. DAPI = blue, EdU positive cells = Green, vimentin = pink. Large image scale bar = 100 µm, inset scale bar = 10 µm. Male vehicle n=6, Male ISO n=6, Female vehicle n=8, Female ISO n=7. Two‐way ANOVA with Bonferroni post hoc applied. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. All data reported as ±SEM. CF indicates cardiac fibroblasts; DAPI, 4′,6‐diamidino‐2‐phenylindole; EdU, 5‐ethynyl‐2’‐deoxyuridine; ISO, isoproterenol; and ROI, region of interest.

Figure 4. Male CFs are larger and express more αSMA than females at baseline and with ISO treatment.

(A) Illustration of experimental design testing ISO treatment on CF activation in male and female rats during the early (3 day) or late (7 day) response. (B) Myofibroblast activation and cell size of CFs after 3 days of ISO treatment measured by αSMA immunostaining. Representative images and quantification. DAPI = blue, αSMA = Green. Scale bar = 100 µm. Male vehicle n=6, Male ISO n=6, Female vehicle n=6, Female ISO n=10. Two‐ANOVA with Bonferroni post hoc applied. (C) Myofibroblast activation and cell size of CFs after 7 days of ISO treatment measured by αSMA immunostaining. Representative images and quantification. DAPI = blue, αSMA = Green. Scale bar = 100 µm. Male vehicle n=6, Male ISO n=8, Female vehicle n=6, Female ISO n=8. Two‐way ANOVA with Bonferroni post hoc applied. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. All data reported as ±SEM. αSMA indicates α smooth muscle actin; CF, cardiac fibroblasts; DAPI, 4′,6‐diamidino‐2‐phenylindole; and ISO, isoproterenol.

Figure 5. ISO causes myofibroblast activation in both male and female CFs but more dramatic response in males.

(A) mRNA expression levels of myofibroblast and fibroblast genes in male and female CFs from vehicle‐treated rats. αSMA, Col1a1, and Postn, and Tcf21 gene expression (normalized to TBP) was measured by RT‐qPCR. Male n=6, Female n=6. Unpaired student’s t‐test applied. (B) mRNA expression levels of myofibroblast and fibroblast genes in male and female CFs from 3‐day ISO‐treated rats. αSMA, Col1a1, and Postn, and Tcf21 gene expression (normalized to TBP) was measured by RT‐qPCR. Values were normalized to vehicle‐treated samples. ISO Male n=6, ISO Female n=10. Unpaired Student’s t test applied. (C) mRNA expression levels of myofibroblast and fibroblast genes in male and female CFs from 7‐day ISO‐treated rats. αSMA, Col1a1, and Postn, and Tcf21 gene expression (normalized to TBP) was measured by RT‐qPCR. Values were normalized to vehicle‐treated samples. ISO Male n=8, ISO Female n=7 (expect Col1a1 n=5). Unpaired Student’s t test applied. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. All data reported as ±SEM. αSMA indicates α smooth muscle actin; CF, cardiac fibroblasts; Col1a1, collagen 1; ISO, isoproterenol; Postn, periostin; RT‐qPCR, reverse transcriptase–polymerase chain reaction; TBP, TATA binding protein; and Tcf21 transcription factor 21.

Figure 6. Male CFs express more adrenergic receptor β1 and β2 at baseline and with ISO treatment than females.

(A) mRNA expression levels of adrenergic receptor β1 (ADRB1) and adrenergic receptor β2 (ADRB2) in male and female CFs from vehicle‐treated rats. ADRB1 and ADRB2 gene expression (normalized to GAPDH) was measured by RT‐qPCR. Male n=12, Female n=10. Unpaired Student’s t test applied. (B & C) mRNA expression levels of ADRB1 and ADRB2 in male and female CFs from vehicle and ISO‐treated rats at early (B) or late (C) time points. ADRB1 and ADRB2 gene expression (normalized to GAPDH) was measured by RT‐qPCR. ADRB1 Early: Male vehicle n=6, Male ISO n=6, Female vehicle n=6, Female ISO n=9. Two statistical outliers were detected but were not removed as this did not affect statistical significance. ADRB2 Early: Male vehicle n=6, Male ISO n=6, Female vehicle n=6, Female ISO n=9. ADRB1 Late: Male vehicle n=6, Male ISO n=7, Female vehicle n=4, Female ISO n=7. A statistical outlier was removed in the ADRB1 late data set because it was far outside of expected values and we suspect due to error (Male ISO: 3.326). If the outlier is not removed, the results indicate that male ISO and male vehicle are significantly different. ADRB2 Late: Male vehicle n=6, Male ISO n=8, Female vehicle n=4, Female ISO n=7. Two‐way ANOVA with Bonferroni post hoc applied. (D) PKA activity in CFs from vehicle or ISO‐treated male and female rats after 3 days of treatment. Male vehicle n=6, Male ISO n=6, Female vehicle n=4, Female ISO n=10. Two‐way ANOVA with Bonferroni post hoc applied. **P<0.01, ****P<0.0001. All data reported as ± SEM. CF indicates cardiac fibroblasts; ISO, isoproterenol; PKA, protein kinase A; and RT‐qPCR, reverse transcriptase–polymerase chain reaction.

Figure 7. Summary of study results and implications.

Here, ISO was used as a mimic for chronic stimulation of β‐adrenergic receptors (β‐ARs) in male and female rats. Results indicate a cellular sexually dimorphic response of cardiac fibroblasts to ISO. Male cardiac fibroblasts express more β‐ARs than females, leading to increased activation of PKA, and positively correlates with increased myofibroblast activation. We posit that increased myofibroblast activation of male cardiac fibroblasts from ISO treatment leads to increased collagen deposition and fibrosis in male hearts, while females are protected. Cardiac fibrosis negatively correlates with survival in patients with cardiovascular disease. ISO indicates isoproterenol; and PKA, protein kinase A.