Volume 24, Issue 6, Pages 1639-1652 (August 2018) Deficiency of PKD2L1 (TRPP3) Exacerbates Pathological Cardiac Hypertrophy by Augmenting NCX1-Mediated Mitochondrial Calcium Overload  Zongshi Lu, Yuanting Cui, Xing Wei, Peng Gao, Hexuan Zhang, Xiao Wei, Qiang Li, Fang Sun, Zhencheng Yan, Hongting Zheng, Gangyi Yang, Daoyan Liu, Zhiming Zhu  Cell Reports  Volume 24, Issue 6, Pages 1639-1652 (August 2018) DOI: 10.1016/j.celrep.2018.07.022 Copyright © 2018 The Authors Terms and Conditions

Cell Reports 2018 24, 1639-1652DOI: (10.1016/j.celrep.2018.07.022) Copyright © 2018 The Authors Terms and Conditions

Figure 1 PKD2L1 Is Involved in Cardiac Hypertrophy Induced by HSD or Ang II Infusion (A) Representative western blots of PKD2L1 and hypertrophic markers β-MHC, ANP, and BNP in H9c2 cells treated with NaCl (20 mM) or Ang II (100 nM) for 48 hr. The quantitative results are shown below. (B) Representative western blots of PKD2L1 and hypertrophic markers β-MHC, ANP, and BNP in the hearts of mice fed with HSD for 3 months or infused with Ang II for 4 weeks. The quantitative results are shown below (n = 6). Data are expressed as mean ± SD. ∗p < 0.05 and ∗∗p < 0.01 versus control group. Cell Reports 2018 24, 1639-1652DOI: (10.1016/j.celrep.2018.07.022) Copyright © 2018 The Authors Terms and Conditions

Figure 2 PKD2L1 Gene Knockout Exaggerates High Salt-Induced Cardiac Hypertrophy (A and B) Ratio of heart weight (HW) to body weight (BW) (A) and HW to tibia length (TL) (B) in the indicated groups (n = 6). (C) Histological analysis of heart sections from WT and PKD2L1−/− mice fed with ND or HSD. Heart cross-sections were stained with H&E to analyze hypertrophic growth (top row; scale bars, 1 mm). (D–G) Histological analyses of the H&E staining (D), WGA (wheat germ agglutinin) staining (E), and PSR (picrosirius red) staining indicating the interstitial (F) or perivascular (G) fibrosis of heart sections from PKD2L1−/− and WT mice fed with ND or HSD for 24 weeks. (H and I) Representative western blots (H) of β-MHC, ANP, and BNP in heart tissues from PKD2L1−/− and WT mice fed with ND or HSD for 24 weeks. The quantitative protein expression levels are shown in (I) (n = 6). Data are expressed as mean ± SD. ∗p < 0.05 and ∗∗p < 0.01 versus ND group; #p < 0.05 and ##p < 0.01 versus WT mice. See also Figure S1. Cell Reports 2018 24, 1639-1652DOI: (10.1016/j.celrep.2018.07.022) Copyright © 2018 The Authors Terms and Conditions

Figure 3 PKD2L1 Knockout Exaggerates Cardiac Dysfunction and Impaired Metabolism by HSD (A) Representative images of mouse hearts by M-mode echocardiography. (B) Posterior wall thickness of left ventricle (LVPW), interventricular septum (IVS) thickness, left ventricular (LV) fractional shortening (% FS), and LV ejection fraction (% EF) are determined using echocardiomyography (n = 9 or 10). (C and D) Treadmill endurance capacity and energy expenditure in WT and PKD2L1−/− mice. Endurance capacity test protocol (C) and the quantitative results of endurance capacity (D) are shown (n = 11). (E) Oxygen consumption, respiratory exchange ratio, energy expenditure, and heat production of WT and PKD2L1−/− mice on ND and HSD (n = 15). (F) Physical activity of WT and PKD2L1−/− mice on ND and HSD (n = 15). Data are shown as mean ± SD. ∗p < 0.05 and ∗∗p < 0.01 versus ND; #p < 0.05 versus WT group. See also Figure S2. Cell Reports 2018 24, 1639-1652DOI: (10.1016/j.celrep.2018.07.022) Copyright © 2018 The Authors Terms and Conditions

Figure 4 Cardiac Mitochondrial Respiratory Chain Function and Cardiac Mitochondrial Enzymatic Activities in WT and PKD2L1−/− Mice (A–F) Parameters indicating complex I leak (CI leak) (A), complex I + complex II leak (CI+II leak) (B), complex I oxidative phosphorylation (CI OXPHOS) (C), complex II OXPHOS (CII OXPHOS) (D), complex I + complex II OXPHOS (CI+II OXPHOS) (E), and complex I + complex II electron transport (CI+II ETS) (F) in cardiac mitochondrial respiratory chain from WT and PKD2L1−/− mice fed with ND or HSD for 24 weeks (n = 10). (G and H) Enzymatic activity of complex I (mOD/min) (G) and complex II (mOD/min) (H) of heart tissue from WT and PKD2L1−/− mice fed with ND or HSD for 24 weeks (n = 6). Data are presented as mean ± SD. ∗p < 0.05 and ∗∗p < 0.01 versus ND; #p < 0.05 and ##p < 0.01 versus WT. Cell Reports 2018 24, 1639-1652DOI: (10.1016/j.celrep.2018.07.022) Copyright © 2018 The Authors Terms and Conditions

Figure 5 PKD2L1 Is Localized in Mitochondria and Regulates Mitochondrial Ca2+ Uptake (A) Confocal immunofluorescent staining with PKD2L1 or ATP5A antibody in neonatal rat cardiomyocytes. The nucleus was stained by DAPI. Scale bar, 50 μm. (B) Localization of PKD2L1 on plasma membrane and mitochondrial fractions of mouse heart. Markers used: ORAI1, plasma membrane (PM); GAPDH, cytoplasm; IP3R, endothelial reticulum (ER); VDAC, mitochondria. (C and D) Thapsigargin-induced SR Ca2+ release and Ca2+ uptake via store-operated channels (SOCs) after SR Ca2+ depletion in H9c2 treated with si-con or siPkd2l1 with or without 20 mM NaCl (C). Cells were incubated in a Ca2+-free buffer in the presence of Fura-2 (1 μM). Peak Fx/F0 following thapsigargin (1 mM) stimulation and after reintroducing 2mM extracellular Ca2+. The quantitative results are shown in (D) (n = 6 independent experiments). (E and F) Mitochondrial Ca2+ dynamics changes in digitonin-permeabilized si-con or siPkd2l1-treated H9c2 cells with or without 20 mM NaCl treated with 200 μM ATP (E) or 10 μM histamine (F) in a Ca2+-free extracellular solution. The quantitative results are shown on the right (n = 6 independent experiments). (G) Reduction of medium Ca2+ by isolated heart mitochondria from WT and PKD2L1−/− mice on ND or HSD. Purified heart mitochondria were incubated in a Ca2+-free buffer in the presence of Rhod (1 μM). Ca2+ was added at a final concentration of 50 or 300 μM, and intramitochondrial Ca2+ was monitored by Rhod-2 fluorescence for 120 s. The quantitative results are shown on the right (n = 6 independent experiments). (H and I) Cytosolic and mitochondrial ROS detected by DHE (H) and Mitosox (I), respectively (n = 6 per group). Data are presented as mean ± SD. ∗p < 0.05 and ∗∗p < 0.01 versus si-con group in (D)–(G) and con group in (H) and (I); #p < 0.05 versus si-con group in (H) and (I). Cell Reports 2018 24, 1639-1652DOI: (10.1016/j.celrep.2018.07.022) Copyright © 2018 The Authors Terms and Conditions

Figure 6 Inhibition of NCX1 Reduces the Pro-hypertrophic Effect of PKD2L1 Knockout (A and B) Representative western blots of mitochondrial ion channels, NCX1, NHE1, LETM1, and MCU in hearts of PKD2L1−/− mice and WT controls (A). The quantitative results are shown in (B) (n = 6). (C and D) Representative western blots of NCX1 and hypertrophic markers β-MHC, ANP, and BNP in H9c2 cells treated with NaCl (20 mM), siPkd2l1, and/or NCX1 inhibitor KB-R7943 for 48 hr (C). The quantitative results are shown in (D) (n = 3 independent experiments). Data are presented as mean ± SD. ∗p < 0.05 and ∗∗p < 0.01 versus WT group in (B) and 0 mM group in (D); #p < 0.05 and ##p < 0.01 versus si-con group in (D). (E) The localization of NCX1 on plasma membrane and mitochondrial fractions of mouse heart. The immunoblots for ORAI1, GAPDH, IP3R, and VDAC were the same ones used in Figure 5B. (F) Confocal immunofluorescent staining with PKD2L1 or NCX1 antibody in neonatal rat cardiomyocytes. The nucleus was stained by DAPI. Scale bar, 50 μm. See also Figure S3. Cell Reports 2018 24, 1639-1652DOI: (10.1016/j.celrep.2018.07.022) Copyright © 2018 The Authors Terms and Conditions

Figure 7 Knockdown of PKD2L1 Increases H3K27ac Level on the Promoter of NCX1 by Inactivating AMPK (A) ChIP-qPCR results of the relative binding level of H3K27ac or H3K27me3 on the promoters of genes encoding Ncx1, Nhe1, and Mcu in NaCl- and/or siPkd2l1-treated H9c2 cells (n = 3 independent experiments). (B) Representative western blots of PKD2L1, NCX1 and levels of hypertrophic marker proteins in H9c2 cells treated with NaCl, siPkd2l1, and/or curcumin. Results shown are representative of three blots. GAPDH served as a loading control. (C) ChIP-qPCR results of the relative binding level of H3K27ac or H3K27me3 on the promoters of genes encoding Ncx1 in NaCl-, curcumin-, and/or siPkd2l1-treated H9c2 cells (n = 3 independent experiments). (D) Representative western blots of levels of phosphorylated and total AMPK in heart tissues from indicated mice fed with ND or HSD (n = 6 mice per group). Results shown are representative of six blots. GAPDH served as a loading control. (E) Representative western blots of PKD2L1, NCX1 and levels of phosphorylated and total AMPK, hypertrophic marker proteins in H9c2 cells treated with NaCl, siPkd2l1, and/or AICAR. Results shown are representative of three blots. GAPDH served as a loading control. (F) ChIP-qPCR results of the relative binding level of H3K27ac or H3K27me3 on the promoters of genes encoding Ncx1 in NaCl-, AICAR-, and/or siPkd2l1-treated H9c2 cells (n = 3 independent experiments). Data in (A), (C), and (D) are mean ± SD. ∗p < 0.05 and ∗∗p < 0.01 versus 0 mM NaCl con group; #p < 0.05 and ##p < 0.01 versus si-con group. See also Figure S4. Cell Reports 2018 24, 1639-1652DOI: (10.1016/j.celrep.2018.07.022) Copyright © 2018 The Authors Terms and Conditions