Arrhythmogenic Hearts in PKD2 Mutant Mice Are Characterized by Cardiac Fibrosis, Systolic, and Diastolic Dysfunctions

Figure 1. The hearts from Pkd2-KO mice are characterized with hypertrophy and fibrosis. (A–C) Masson's trichrome staining was used to analyzed hearts from wild-type (WT) and Pkd2-KO (Pkd2) mice. Representative of left ventricular thickness (hypertrophy) is shown (A) and quantified (B,C). (D,E) Left ventricular (LV) fibrosis is analyzed; arrows indicate fibrosis (D). LV fibrosis was quantified (E). Data show a significant increase in LV myocardial hypertrophy and fibrosis in Pkd2-KO compared to WT mice. Fibrosis revealed by trichrome cooperation in the tissue was quantified with binary masking of the blue-color image (D,E). WGA staining was used for cell surface staining and cell size measurement. Representative of LV cardiomyocyte size (hypertrophy) is shown (F) and quantified (G). N = 4 mice for each WT and Pkd2-KO group at age matched of 6 months old. **p < 0.01; ***p < 0.001.

Figure 2. The sinoatrial node (SAN) Pkd2-KO mice are characterized with fibrosis and fatty infiltration. Masson's trichrome staining was performed in wild-type (WT) and Pkd2-KO (Pkd2) mouse hearts. (A) A significant increase of fibrotic tissue is seen in SAN of right atrium in PKD mouse compared to WT. Boxes indicated SAN, and the left panel is further magnified and shown in the right panel. (B) No significant difference of fibrotic tissue is detected in AVN of right atrium in Pkd2-KO mouse. Boxes indicated AVN, and the left panel is further magnified and shown in the right panel. AO, aorta; AVN, atrioventricular node; CFB, central fibrous body; CT, Crista terminalis; ENC, endocardium; EPC, epicardium; FI, fatty infiltration; IVS, interventricular septum; LV, left ventricle; RA, right atrium; RV, right ventricle; SA, sinoatrial node; SAA, sinoatrial artery. Scale Bar = 20 mm. N = 6–10 mice for each WT and Pkd2-KO group at age matched of 6 months old. ***p < 0.001.

Figure 3. Cell number is decreased in Pkd2-KO mouse atrioventricular node. (A) Heart tissues were stained with anti-HCN4 antibody used as a marker for the myocardial conduction system (green staining). Atrioventricular node (AVN) is identified by both location and HCN4-postive staining indicated by the red circles; RV, right ventricle. (B) The AVN is next identified as region of interest. (C) Number of cell nucleus (DAPI; blue staining) is counted. (D) Data reveal a significantly lower number of cells in AVN of Pkd2-KO mouse hearts. N = 5 mice for each WT and Pkd2-KO group at age matched of 6 months old. **p < 0.01.

Figure 4. The hearts from Pkd2-KO mice with no autonomic input have irregular beats. (A) Representatives of in vivo electrocardiogram (ECG) are shown with averaged frequency of 540–600 beats/min in wild-type (WT) and Pkd2-KO (Pkd2) mice. (B) Representatives of ex vivo ECG with no autonomic input are shown with different preloads of 5, 10, and 15 mmHg. The Pkd2-KO heart ECG shows irregular heartbeat with normal QRS shape. All ECG from Pkd2-KO showed irregular R-R intervals (arrows). N = 3–4 mice for each WT and Pkd2-KO age-matched 6 months old.

Figure 5. The cardiac function of Pkd2-KO mice is not responsive on changes in cardiac stresses. (A) Individual graphs of the relationship between left ventricle pressure (LVP) and left ventricle volume (LVV) heart function are shown before and after injection of adrenaline (4 mg/L) and diltiazem (0.08 mg/L) in wild-type (WT) and Pkd2-KO (Pkd2) hearts. (B) End-systolic-pressure-volume relationship (ESPVR, an index of systolic contractility function), stroke volume, ejection fraction, and cardiac output are used to evaluate the heart responses on positive-stress (adrenaline) and negative-stress (diltiazem). N = 3–4 mice for each WT and Pkd2-KO age-matched 6 months old. Statistical significance between control and treatment is indicated with asterisk (*) sign; between genotypes in hash (#). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 compared to control within the same genotype. #p < 0.05; ###p < 0.001; ####p < 0.0001 compared to the corresponding WT.

Figure 6. The cardiac function of Pkd2-KO mice is not responsive on changes in preloads. (A) Individual graphs of pressure-volume loop show effects of different preloads of 5, 10, and 15 mmHg on heart function. (B) End-systolic-pressure-volume relationship (ESPVR, an index of systolic contractility function), stroke volume, ejection fraction, and cardiac output are used to measure heart functions in response to different pre-load levels. N =3–4 mice for each WT and PKD age-matched 6 months old. Statistical significance between control and treatment is indicated with asterisk (*) sign; between genotypes in hash (#). *p < 0.05; ***p < 0.001; ****p < 0.0001; #p < 0.05; ##p < 0.01; ###p < 0.001; ####p < 0.0001.

Figure 7. The hearts from Pkd2-KO mice demonstrate abnormal fibrotic pathways. (A) ELISA measurements of TGF-β1 were done in circulating plasma (left panel) and heart tissues (right panel). (B,C) Western blot analyses of mouse tissues show significantly higher expressions of TGF-β1 and TGF-β1 receptors in Pkd2-KO (Pkd2) compared to WT mice. (D,E) Western blot analyses of mouse tissues show significantly higher expressions of tSMAD3, pSmad3, and β-catenin in WT compared to Pkd2-KO mice. N = 4–6 mice for each WT and PKD age-matched 6 months old. *p < 0.05; **p < 0.01.

Figure 8. The hearts from Pkd2-KO mice are characterized with upregulated fibrotic pathways. (A,B) Immunohistochemistry staining demonstrated a significant increase of TGF-β1 and TGF-β1 receptor (dark brown color) in Pkd2-KO (Pkd2) hearts compared to the wild-type (WT). A total of 8 independent slides were used for each mouse. **p < 0.01; ****p < 0.0001.

Figure 9. The hearts from Pkd2-KO mice are characterized with a significant increase in nuclear localization of pSMAD3 and β-catenin. (A,B) Immunofluorescence staining of pSmad3 (A) or β-catenin (B) revealed a significant increase in nuclear localization and nucleus to cytoplasm ratio of pSmad3 or β-catenin in Pkd2-KO (Pkd2) compared to wild-type (WT) hearts. N = 4 mice for each group. A total of 8 independent slides were used for each mouse. *p < 0.05; **p < 0.01.

Figure 10. The hearts from Pkd2-KO mice are significantly infiltrated by macrophages. (A,B) Immunofluorescence staining revealed a significant higher number of macrophages M1 and M2. Image was quantified by binary masking of the red or green fluorescence image. N = 4 mice for each WT and Pkd2-KO age-matched 6 months old. *p < 0.05.

Figure 11. Hypothetical diagram summarizes the pathogeneses of PKD hearts. We hypothesize that hearts from Pkd2-KO mice are characterized by the overexpression of TGF-β1 receptor. Local TGF-β1 secretion by infiltrating macrophages further exacerbates the activation of fibrotic pathways resulting in hypertrophy and interstitial fibrosis. Subsequent activation of TGF-β1 receptor results in the activation of pSMAD3 and b-catenin in the myocytes. The fibrosis in the sinoatrial node and surrounding tissue can results in arrhythmogenic heart. All of which will cause systolic and diastolic cardiac dysfunction.