Metabolic Acidosis Results in Sexually Dimorphic Response in the Heart Tissue

Figures

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  Figure 1. (A) Schematic of study design. Oral administration of NH4Cl, using stepwise dosing increase, induces murine acidosis. Urine pH was measured at baseline (D-7), D0, D3, D6, D9, D12, and D14. Blood gas was measured at baseline (D-7) and D14 for both control (n = 4) and acidosis (n = 4) groups. (B) Blood gas measurement. B1, pH, HCO3−, BE, Be(b) and AGapK levels in the blood; B2, pCO2, pO2, cTCO2, cSO2, cHgb, Hct level in blood; B3, Glu, lac and BUN level in blood. (C) urine pH, (D) body weight. Two tailed student’s t-test was used to determine differences between acidosis and control. * < 0.05; ** < 0.01, n.s. (non-significant). Actual bicarbonate (cHCO3−; mmol/L), base excess of extracellular fluid (BE(ecf); mmol/L), base excess of blood (BE(b); mmol/L), Anion gap (AGapK, mmol/L), Carbon dioxide partial pressure (pCO2; mmHg), oxygen partial pressure (pO2; mmHg), total carbon dioxide (cTCO2; mmol/L), oxygen saturation (cSO2; %), hematocrit (Hct; %PCV), hemoglobin (cHgb; %), Glucose (Glu; mg/dL), lactate (Lac; mmol/L), Blood urea nitrogen (BUN; mmol/L).

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  Figure 2. (A) Venn diagram of differentially-expressed genes in male and female acidosis heart vs. their respective controls. (B) Unbiased hierarchical clustering using z-scores of all expressed genes show four main clusters. Acidosis results in upregulation of genes in Cluster 3 and 4, while Cluster 1 is upregulated in control mice hearts. (C) Ontology analysis on genes in the four main clusters. (D) Quadrant map of canonical pathways that are up/downregulated in both male and female acidosis model. (E) Canonical pathways that are oppositely regulated in male vs. female acidosis mice model. (F) Quadrant map of transcriptional factors (TransFac) activated/inhibited in male vs. female acidosis mice model. The canonical pathways and transcription factors were calculated using IPA analysis of differentially expressed genes.

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  Figure 3. (A,F) The top enriched gene ontologies were very different between male and female samples, acccording to non-parametric Gene Set Enrichment Analysis (GSEA). (B–E) GSEA showing the leading edge genes in response to alkaloid, cell activation in immune response, hormone transport, and glycolysis in acidosis female mice compared to control female mice. (G–J) GSEA showing the leading edge genes in response to anion transmembrane transport, inorganic anion transport, wp1396 non-odorant GPCRs, and WP65 insulin signaling in acidosis male mice compared to control male mice.

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  Figure 4. (A) Blood electrolytes. Acidosis compared to control; two-tailed student’s t-test was used to determine the significance. * < 0.05; *** < 0.001; n.s. (non-significant). Sodium (Na, mmol/L), potassium (K, mmol/L), calcium (Ca, mmol/L), chlorine (Cl, mmol/L). (B) GSEA analysis of differentially expressed genes in male and female acidosis models, (B1) Targeted GSEA analysis and negative logpVal of relevant/cardiac ontologies. (B2) enrichment scores of the relevant/cardiac ontologies in GSEA analysis. GSEA and bubble plot of differentially expressed genes in DCM ontology (C,D), Cardiac muscle contractility (E,F), and Na/K/ATPase-Src signaling (G,H). Bubble plot of differential genes in regulation of cellular pH (I). GSEA and bubble plot of differentially expressed genes in estrogen response early ontology (J,K). GSEA and bubble plots are color coded with male acidosis vs. male control (blue) and female acidosis vs. female control (pink). Size of bubble or bar plot is determined with either pVal (p-value), logpVal or−−log10 of pVal.

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  Figure 5. Summary of key transcriptional and electrolyte changes in male vs. female mice with acidosis. Differential regulation of transcriptional factors causes activation (Up arrow) or inhibition (down arrow) of key cardiac specific signaling pathways in male vs female mice. The blood chemistry was similar in both mice groups.