Canonical transient receptor potential channel 1 aggravates myocardial ischemia-and-reperfusion injury by upregulating reactive oxygen species

Fig. 1. Canonical transient receptor potential channel (TRPC) 1 expression and activity are increased post-myocardial ischemia/reperfusion (I/R). (A) Quantitative polymerase chain reaction to assess the expression of Trpcs, Cacna1c, and Na+/Ca2+ exchanger 1 (Ncx1) messenger RNA (mRNA) in area at risk (AAR) dissected from mice underwent myocardial I/R or sham operation. Expression levels were normalized to 18S RNA and expressed as percentage relative to 18S RNA (n = 6 per group). (B) Representative Western blot images and relative quantification of TRPC1 (left) and TRPC6 (right) in the area at risk of mice undergoing I/R or sham operation (n = 4 per group). (C) HL-1 cells were transfected with indicated small interfering RNAs (siRNA) for 48 h and underwent hypoxia/reoxygenation (H/R, 6 h/9 h). TRPC channel-mediated Ca2+ entry was assayed in the presence of TRPC agonist oleyl-acetyl-glycerol (OAG, 10 μM) and cyclopiazonic acid (CPA, 5 μM). Where indicated, SKF96365 (SKF, 5 μM) or the L-type Ca2+ channel inhibitor nifedipine (Nif, 10 μM) were used. (D) HL-1 cells were infected with the Trpc1 plasmid and assayed for TRPC-mediated Ca2+ entry. ∗∗P < 0.01. BLQ: below the limit of…

Fig. 2. Knockout of canonical transient receptor potential channel 1 (Trpc1) attenuates myocardial ischemia/reperfusion (I/R) injury. (A) Representative echocardiographs recorded from mice post-I/R (30 min/24 h). End-diastolic volume (EDV) and end-systolic volume (ESV) were determined by the bi-plane Simpson method requires the length of the ventricle (red) obtained from a parasternal long-axis (PSLAX) view (endocardial tracing is blue), and the left ventricular areas from three orthogonal short-axis (SAX) views (midventricular, apical, and basal; blue disks). (B) Quantitative analysis of cardiac ejection fraction (EF) in wild type (WT), Trpc1−/−, and Trpc6−/− mice underwent sham operation or I/R (30 min/24 h) calculated as (EDV − ESV)/EDV × 100% (n = 5 per group). (C) 2,3,5-triphenyltetrazolium chloride-Evans blue staining from WT, Trpc1−/−, and Trpc6−/− mice underwent sham operation or I/R (30 min/24 h). For I/R mice, white indicates infarct area, red indicates area at risk (AAR), and blue indicates the tissue not subjected to I/R injury. (D) Average data showing the percentages of the infarct area to AAR (n = 5 pairs of mice). (E) Representative hematoxylin and eosin (H&E) staining…

Fig. 3. Silencing of canonical transient receptor potential channel 1 (Trpc1) in cardiomyocytes mitigates myocardial ischemia/reperfusion (I/R) injury. (A) Time course of adeno-associated virus 9 (AAV9)-mediated silencing of Trpc1 in cardiomyocytes. (B) TRPC1 expression in area at risk (AAR) of myocardial I/R mice 3 weeks post AAV9 delivery. (C) Cardiac function was measured by echocardiography post-I/R (30 min/24 h). Cardiac ejection fraction (EF) was calculated as (end-diastolic volume − end-systolic volume)/end-diastolic volume × 100%. Volume was determined by the bi-plane Simpson method (n = 5 per group). (D) 2,3,5-triphenyltetrazolium chloride-Evans blue staining of from mice underwent sham operation or I/R (30 min/24 h). (E) Average data showing the percentages of the infarct area to AAR (n = 4 pairs of mice). (F) Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL, red) and α-actin (green) staining was used to observe the apoptosis myocytes in the AAR after I/R (30 min/24 h). 4′,6-diamidino-2-phenylindole (DAPI) was used to stain the nuclei (blue). (G) Quantification of apoptosis cells in Fig. 3F (n = 4 per group). ∗∗P < 0.01. shCon: shControl; cTnT:…

Fig. 4. Canonical transient receptor potential channel 1 (Trpc1) deficiency attenuates simulated ischemia/reperfusion (I/R) induced injury in primary cultures of cardiomyocytes. (A) Adult mice ventricular myocytes (AMVMs) were isolated from wild-type (WT), Trpc1−/−, and Trpc6−/− mice post-myocardial I/R. TRPC channel-mediated Ca2+ entry was assayed in the presence of oleyl-acetyl-glycerol (OAG, 10 μM) and cyclopiazonic acid (CPA, 5 μM). SKF96365 (SKF, 5 μM) or the L-type Ca2+ channel inhibitor nifedipine (Nif, 10 μM) were used. (B) AMVMs isolated from WT, Trpc1−/−, and Trpc6−/− mice were exposed hypoxia/reoxygenation (H/R, 5 h/1 h) condition. Live/dead assays were used to assess cell viability. (C) Quantitative data of AMVMs death. Total numbers of live cytosol (green) and dead nuclei (red) were counted in low-power fields (n = 4 per group). (D) Relative secreted levels of lactate dehydrogenase (LDH) in the different groups of AMVMs (n = 6 per group). (E) Flow cytometry images of Annexin V-propidium iodide (PI) staining in neonatal mice ventricular myocytes underwent H/R injury. (F) Quantitative analysis of flow cytometry results (n = 3 per group). ∗∗P < 0.01. FITC: fluorescein…

Fig. 5. Canonical transient receptor potential channel 1 (TRPC1) attributes to reactive oxygen species (ROS) generation in area at risk (AAR) after myocardial ischemia/reperfusion (I/R). (A) Scatter plot and (B) heatmap of gene profiles, red points and green points indicate upregulated and downregulated genes respectively (n = 3 per group). (C) The top 10 enriched clusters in the comparison between wild-type (WT)-I/R and Trpc1−/−-I/R mice. (D) Representative images and quantitative analysis of dihydroethidium (DHE, red) fluorescence intensity for heart sections of sham or I/R (30 min/24 h) mice loaded with DHE (10 μmol/L). 4′,6-diamidino-2-phenylindole (DAPI) was used to stain the nuclei (blue) (n = 3 per group). (E) Aconitase activity was determined in the AAR (n = 6 per group). (F) Adult mice ventricular myocytes isolated from WT, and Trpc1−/− mice were exposed hypoxia/reoxygenation (H/R, 5 h/1 h) condition. Mitochondrial reactive oxygen species formation was measured by MitoTracker Red (MTR) (n = 3 per group). (G) Representative images and quantitative analysis of the MTR fluorescence intensity for the HL-1 cells infected with control (Con) or Trpc1 small interfering RNAs (siRNA) then…

Fig. 6. Canonical transient receptor potential channel 1 (TRPC1) increases reactive oxygen species (ROS) generation via regulating oxoglutaratedehydrogenase-like (Ogdhl) expression. (A) Heatmap of the ROS related gene profiles in the area at risk (AAR) of wild-type (WT) and Trpc1−/− mice that underwent myocardial I/R (30 min/24 h). (B–D) The AAR region was separated 24 h after I/R in WT or Trpc1−/− mice. The (B) messenger RNA (mRNA), and (C) protein levels of OGDHL were determined by quantitative polymerase chain reaction (q-PCR) or Western blot, respectively. (D) Oxoglutarate dehydrogenase complex (OGDHc) and pyruvate dehydrogenase phosphatase (PDH) activities were detected by enzyme-linked immunosorbent assay (ELISA) kit (n = 3 per group). (E) ROS generation was determined in the HL-1 cells transfected with a green fluorescent protein targeting control (Con) or Ogdhl,lectingalactosidebindingsoluble 3 (Lgals3), tripartite-motif protein 30 (Trim30d) small interfering RNAs (siRNAs), then underwent hypoxia/reoxygenation (H/R, 6 h/9 h) (n = 3 per group). (F) The mRNA and (G) protein levels of OGDHL and (H) OGDHc activity in WT or Trpc1−/− adult mice ventricular myocytes upon H/R (5 h/1 h)…

Fig. 7. Canonical transient receptor potential channel 1 (TRPC1) regulates oxoglutaratedehydrogenase-like (Ogdhl) via Ca2+/calcineurin (CaN)/nuclear factor kappa-B (NF-κB) signaling pathway. (A, B) Representative Western blot images and relative quantification of p-NF-κB p65/NFκB p65 (p65) (A) and p-IκB-α/IκB-α (B) in wild-type (WT) and Trpc1−/− adult mouse ventricular myocytes upon hypoxia/reoxygenation (H/R) (5 h/1 h) condition (n = 4 per group). (C) The messenger RNA (mRNA) of Ogdhl in the HL-1 cells transfected with control (Con) or NF-κB small interfering RNA (siRNA) upon H/R (6 h/9 h) (n = 3 per group). (D) Representative Western blot images and relative quantification of OGDHL in the HL-1 cells transfected with control or NF-κB siRNA, and then underwent H/R (6 h/9 h) (n = 4 per group). (E) The mRNA of Ogdhl after transfected with the NF-κB expression or control plasmid (n = 3 per group). (F) HEK-293 cells were transfected with NF-κB expression plasmid and Ogdhl promotor luciferase reporter constructs. Luciferase activity was detected 36 h after transfection using a betascope analyzer (n = 3 per group). (G) The association of NF-κB with the Ogdhl promoter was analyzed by a…