Cardiac Oxidative Signaling and Physiological Hypertrophy in the Na/K-ATPase α1s/sα2s/s Mouse Model of High Affinity for Cardiotonic Steroids

Abstract

The Na/K-ATPase is the specific receptor for cardiotonic steroids (CTS) such as ouabain and digoxin. At pharmacological concentrations used in the treatment of cardiac conditions, CTS inhibit the ion-pumping function of Na/K-ATPase. At much lower concentrations, in the range of those reported for endogenous CTS in the blood, they stimulate hypertrophic growth of cultured cardiac myocytes through initiation of a Na/K-ATPase-mediated and reactive oxygen species (ROS)-dependent signaling. To examine a possible effect of endogenous concentrations of CTS on cardiac structure and function in vivo, we compared mice expressing the naturally resistant Na/K-ATPase α1 and age-matched mice genetically engineered to express a mutated Na/K-ATPase α1 with high affinity for CTS. In this model, total cardiac Na/K-ATPase activity, α1, α2, and β1 protein content remained unchanged, and the cardiac Na/K-ATPase dose-response curve to ouabain shifted to the left as expected. In males aged 3-6 months, increased α1 sensitivity to CTS resulted in a significant increase in cardiac carbonylated protein content, suggesting that ROS production was elevated. A moderate but significant increase of about 15% of the heart-weight-to-tibia-length ratio accompanied by an increase in the myocyte cross-sectional area was detected. Echocardiographic analyses did not reveal any change in cardiac function, and there was no fibrosis or re-expression of the fetal gene program. RNA sequencing analysis indicated that pathways related to energy metabolism were upregulated, while those related to extracellular matrix organization were downregulated. Consistent with a functional role of the latter, an angiotensin-II challenge that triggered fibrosis in the α1^r/rα2^s/s mouse failed to do so in the α1^s/sα2^s/s. Taken together, these results are indicative of a link between circulating CTS, Na/K-ATPase α1, ROS, and physiological cardiac hypertrophy in mice under baseline laboratory conditions.

Keywords: Na/K-ATPase; cardiotonic steroids; hypertrophy; isoform; reactive oxygen species.

Figure 1

Increased Na/K-ATPase sensitivity to ouabain in α1^s/sα2^s/s hearts: (a) Representative Western blots and quantitative analysis for Na/K-ATPase α1, α2, and β1 isoforms in heart homogenates (n = 5–6 hearts/genotype). (b) Maximal ATPase activity in crude membrane fractions was measured using a colorimetric assay for Pi release (n = 6 hearts/genotype). (c) Na/K-ATPase dose–response curve to the cardiotonic steroids (CTS) ouabain. Maximal ATPase activity in crude membrane fractions was measured using a colorimetric assay for Pi release in the presence of the ionophore alamethicin and the indicated concentrations of ouabain. The monophasic curve obtained for the α1^s/sα2^s/s is shown in red along with the biphasic curve obtained in the wild-type (α1^r/rα2^s/s) mouse heart (n = 3 hearts/genotype). ns: non-significant, p > 0.05.

Figure 2

Cardiac hypertrophy in α1^s/sα2^s/s mice: (a) Cross-section of whole left ventricles stained with Masson’s trichrome (top); scale bar = 1 mm. Heart weight/tibia length (HW/TL) quantification (bottom) (n = 14–16 hearts/genotype). (b) Wheat germ agglutinin (WGA) staining of left ventricle cross-sections from α1^r/rα2^s/s (top left) and α1^s/sα2^s/s (bottom left) mice; scale bar = 50 μm. Average cardiomyocyte cross-sectional area (top right) was automatically quantified in over 1000 cells from at least five different random fields (n = 4–7 hearts/genotype). Cardiomyocyte density (bottom right) was determined by the number of cardiomyocytes per mm². * p < 0.05; ** p < 0.01.

Figure 3

Absence of cardiac fibrosis or change in the fetal gene program in α1^s/sα2^s/s mice: (a) Representative histological sections of left ventricles from α1^r/rα2^s/s and α1^s/sα2^s/s (top) mice stained with Masson’s trichrome; scale bar = 200 μm. Fibrosis area quantification (bottom) (n = 5 hearts/genotype). (b) Collagen-1 mRNA levels in the left ventricle determined by RT-qPCR (n = 4–5 hearts/genotype). (c) mRNA levels of α-skeletal actin (α-sk actin), β-heavy myosin chain (β-MHC), and brain natriuretic peptide (BNP) in left ventricles determined by RT-qPCR (n = 6 hearts/genotype). ns: non-significant, p > 0.05.

Figure 4

RNA-seq analysis comparing the transcriptome of α1^r/rα2^s/s and α1^s/sα2^s/s hearts: (a) Volcano plot of gene expression in α1^r/rα2^s/s and α1^s/sα2^s/s hearts plotting -log10 of adjusted p-value on y-axis and log2 fold change on x-axis. Red dots are downregulated (left) and upregulated (right) genes with a log2 fold change greater than 0.5. Blue dots represent those genes with a log2 fold change less than 0.5. Green and black dots represent genes with an adjusted p-value greater than 0.05. (b) The enriched pathways in the category of Cellular Component of the Gene Ontology database. (c) Pathway enrichment using the Reactome database. Pathway enrichment analysis were performed with Gene Set Enrichment Analysis (GSEA) as described in the Materials and Methods section. Blue bars represent upregulated pathways and orange bars represent downregulated pathways.

Figure 4

RNA-seq analysis comparing the transcriptome of α1^r/rα2^s/s and α1^s/sα2^s/s hearts: (a) Volcano plot of gene expression in α1^r/rα2^s/s and α1^s/sα2^s/s hearts plotting -log10 of adjusted p-value on y-axis and log2 fold change on x-axis. Red dots are downregulated (left) and upregulated (right) genes with a log2 fold change greater than 0.5. Blue dots represent those genes with a log2 fold change less than 0.5. Green and black dots represent genes with an adjusted p-value greater than 0.05. (b) The enriched pathways in the category of Cellular Component of the Gene Ontology database. (c) Pathway enrichment using the Reactome database. Pathway enrichment analysis were performed with Gene Set Enrichment Analysis (GSEA) as described in the Materials and Methods section. Blue bars represent upregulated pathways and orange bars represent downregulated pathways.

Figure 5

Increased protein carbonylation in α1^s/sα2^s/s hearts: Representative Western blot analysis of protein carbonylation and Ponceau S staining as a loading control in left ventricle homogenates and associated quantitative data (n = 7–8 hearts/group). *** p < 0.001; DNP: 2,4-dinitrophenyl.

Figure 6

Angiotensin-II does not induce cardiac fibrosis in α1^s/sα2^s/s mice: The left panels show representative histological sections stained with Masson’s trichrome, and the right panels show the quantitative data of fibrosis analyzed using ImageJ (n = 4–8 hearts/group). Scale Bar = 100 µm. *** p < 0.001; ns: non-significant, p > 0.05.