Activation of a GPCR, ORL1 Receptor: A Novel Therapy to Prevent Heart Failure Progression

Abstract

The number of ischemic heart failure (HF) patients is growing dramatically worldwide. However, there are at present no preventive treatments for HF. Our previous study showed that Gata4 overexpression improved cardiac function after myocardial infarction in rat hearts. We also found that Gata4 overexpression significantly increased the expression of a Pnoc gene, an endogenous ligand for the cell membrane receptor ORL1. We hypothesized that the activation of the ORL1 receptor would suppress HF in a rat ischemic heart model. Adult Sprague Dawley rats (8 weeks old, six males and six females) underwent left anterior descending coronary artery ligation. Three weeks later, normal saline or MCOPPB (ORL1 activator, 2.5 mg/kg/day) intraperitoneal injection was started, and continued 5 days a week for 3 months. Echocardiography was performed six times: pre-operative, 3 days after coronary artery ligation, pre-MCOPPB or saline injection, and 1, 2, and 3 months after saline or MCOPPB injection started. Animals were euthanized after 3 months' follow-up and the hearts were harvested for histological analysis. The ORL1 activator, MCOPPB, significantly improved cardiac function after myocardial infarction in rats (ejection fraction, MCOPPB vs. saline at euthanasia, 67 ± 3% vs. 43 ± 2%, p < 0.001). MCOPPB also decreased fibrosis and induced angiogenesis. Thus, the ORL1 activator, MCOPPB, may be a novel treatment for preventing HF progression.

Keywords: MCOPPB; ORL1 receptor activation; prevention of ischemic heart failure progression.

Figure 1

(A) Volcano plot of RNAseq analysis. Cardiac fibroblasts were treated with lentivirus encoding Gata4 or GFP (each 20 MOI) for 14 days (n = 3). PNOC gene was significantly upregulated by Gata4. (B) Chromatography assay for MCOPPB detection in the heart. Rats received MCOPPB 2.5 mg/kg/day or saline intraperitoneally for 4 days (n = 3/group) and were euthanized immediately after the last injection. The heart tissues were examined with mass spectrometry. MCOPPB was detected in the heart tissue of all MCOPPB-receiving rats but no MCOPPB in the saline-receiving rats. Representative chromatography of an MCOPPB-receiving rat is shown. (C) Schematic showing the experimental design of the MCOPPB study. Twelve adult Sprague Dawley rats were enrolled. Three weeks after the coronary artery ligation, they were treated with saline or MCOPPB for 3 months; 6 rats (3 males and 3 females) for saline, and another 6 rats (3 males and 3 females) for MCOPPB. Echocardiography was performed six times, 1: pre-op, 2: post-op, 3: pre-treatment, 4: four weeks after treatment started, 5: eight weeks after treatment started, and 6: twelve weeks after treatment started. (D) Fibrosis analysis. The excised heart was cut transversally and sectioned with two (2 to 3 mm thick) slices obtained, with one immediately cephalad and another one immediately caudad to the transverse centerline of the infarct region, which was readily identifiable by gross inspection. After paraffin embedding of these slices, 14 sections per animal (at a 120 μm interval between each section) were stained with Masson’s trichrome to assess the extent of fibrosis (D). The fibrotic area (blue) and the nonfibrotic region (red) were outlined in the LV wall including the septum, using Adobe Photoshop CS5 software, version 22.1.1. (D) and then quantified with MATLAB and Simulink software, version 7.12.0.635 (MathWorks, Inc., Natick, MA, USA). The percent fibrosis was calculated as: (total of blue pixels from all sections/total of blue plus red pixels from all sections) × 100. (E) Three regions where cardiomyocyte diameter was measured: The slide demonstrating the greatest area of fibrosis, as identified by Masson’s Trichrome staining, was selected for each animal. In each slide, 20 longitudinally oriented cardiomyocytes from each of the 3 regions, anterior, lateral, and posterior, were examined, and the diameters were defined. The mean value of 20 measurements represented 1 sample from each position in each animal. A representative photo shows how to measure with a white bar.

Figure 1

(A) Volcano plot of RNAseq analysis. Cardiac fibroblasts were treated with lentivirus encoding Gata4 or GFP (each 20 MOI) for 14 days (n = 3). PNOC gene was significantly upregulated by Gata4. (B) Chromatography assay for MCOPPB detection in the heart. Rats received MCOPPB 2.5 mg/kg/day or saline intraperitoneally for 4 days (n = 3/group) and were euthanized immediately after the last injection. The heart tissues were examined with mass spectrometry. MCOPPB was detected in the heart tissue of all MCOPPB-receiving rats but no MCOPPB in the saline-receiving rats. Representative chromatography of an MCOPPB-receiving rat is shown. (C) Schematic showing the experimental design of the MCOPPB study. Twelve adult Sprague Dawley rats were enrolled. Three weeks after the coronary artery ligation, they were treated with saline or MCOPPB for 3 months; 6 rats (3 males and 3 females) for saline, and another 6 rats (3 males and 3 females) for MCOPPB. Echocardiography was performed six times, 1: pre-op, 2: post-op, 3: pre-treatment, 4: four weeks after treatment started, 5: eight weeks after treatment started, and 6: twelve weeks after treatment started. (D) Fibrosis analysis. The excised heart was cut transversally and sectioned with two (2 to 3 mm thick) slices obtained, with one immediately cephalad and another one immediately caudad to the transverse centerline of the infarct region, which was readily identifiable by gross inspection. After paraffin embedding of these slices, 14 sections per animal (at a 120 μm interval between each section) were stained with Masson’s trichrome to assess the extent of fibrosis (D). The fibrotic area (blue) and the nonfibrotic region (red) were outlined in the LV wall including the septum, using Adobe Photoshop CS5 software, version 22.1.1. (D) and then quantified with MATLAB and Simulink software, version 7.12.0.635 (MathWorks, Inc., Natick, MA, USA). The percent fibrosis was calculated as: (total of blue pixels from all sections/total of blue plus red pixels from all sections) × 100. (E) Three regions where cardiomyocyte diameter was measured: The slide demonstrating the greatest area of fibrosis, as identified by Masson’s Trichrome staining, was selected for each animal. In each slide, 20 longitudinally oriented cardiomyocytes from each of the 3 regions, anterior, lateral, and posterior, were examined, and the diameters were defined. The mean value of 20 measurements represented 1 sample from each position in each animal. A representative photo shows how to measure with a white bar.

Figure 2

(A) Cardiac function was improved with MCOPPB administration. Echocardiography results show longitudinal plot of changes in average ejection fraction (graph on left) and individual relative percent changes in ejection fraction (graph on right) associated with saline (black line) and MCOPPB (orange line) administration, started at day 21 after myocardial infarction induction. Ejection fraction significantly improved in the MCOPPB group (MCOPPB vs. saline at 2 months follow-up, 58 ± 3 vs. 45 ± 2, p < 0.001, MCOPPB vs. saline at euthanasia, 67 ± 3 vs. 43 ± 2, p < 0.001). Two-tailed ANOVA with Bonferroni post hoc test was used. n = 6/group. (B) Systolic wall thickness increased in the MCOPPB group. LV wall thickness was measured by echocardiography with short-axis M-mode images at the interventricular wall (left) and posterior wall (right). Both the end-systolic interventricular septum (IVS) (left) and end-systolic left ventricular posterior wall (LVPW) (right) were significantly greater in the MCOPPB group compared to the saline group at euthanasia (end-systolic IVS, MCOPPB vs. saline, 2.2 ± 0.5 mm vs. 1.1 ± 0.3 mm, p < 0.01; end-systolic LVPW, MCOPPB vs. saline, 2.7 ± 0.2 mm vs. 2.1 ± 0.3 mm, p < 0.05). A two-tailed ANOVA with Bonferroni post hoc test was applied. n = 6/group. Longitudinal changes in average systolic interventricular septum thickness and average systolic LV posterior wall thickness were further plotted, separated by gender. LV posterior wall thickness was higher in the female MCOPPB group at euthanasia (MCOPPB vs. saline at euthanasia, 2.7 ± 0.3 vs. 1.8 ± 0.2, p < 0.05). A two-tailed ANOVA with Bonferroni post hoc test was used. n = 3/group. Orange line: MCOPPB group; black line: saline group. (C) End-systolic volume was significantly decreased in the MCOPPB-receiving group at euthanasia. End-systolic volume was calculated with VenoLAB software (comes pre-packaged for applications in the echomachine, Vevo 770 Imaging System): (7.0/(2.4 + LVIDs)) × LVIDs³, LVIDs = left ventricular internal diameter end systole). End-systolic volume was significantly decreased in the MCOPPB-receiving group at euthanasia (MCOPPB vs. saline, 140 ± 30 μL vs. 280 ± 44 μL, p < 0.001). A two-tailed ANOVA with Bonferroni post hoc test was used. n = 6/group. Longitudinal changes in average left ventricular systolic volume and average diastolic volume were further plotted, separated by gender. Systolic left ventricular volume was lower in the male MCOPPB group at euthanasia (MCOPPB vs. saline at euthanasia, 157 ± 32 vs. 300 ± 31, p < 0.05). A two-tailed ANOVA with Bonferroni post hoc test was used. n = 3/group. Orange line: MCOPPB group; black line: saline group. (D) Stroke volume was significantly increased in the MCOPPB-receiving group at euthanasia. Stroke volume was calculated as [(end-diastolic volume) − (end-systolic volume)]. Stroke volume was significantly increased in the MCOPPB-receiving group at euthanasia (MCOPPB vs. saline, 281 ± 25 μL vs. 213 ± 37 μL, p < 0.05). Two-tailed ANOVA with Bonferroni post hoc test was used. n = 6/group. Longitudinal changes in average left ventricular systolic volume and average diastolic volume were further plotted. (E) Representative images obtained by M-mode echocardiography. Left: saline received animal at pre-treatment and pre-euthanasia. Right: MCOPPB received animal at pre-treatment and pre-euthanasia.

Figure 2

(A) Cardiac function was improved with MCOPPB administration. Echocardiography results show longitudinal plot of changes in average ejection fraction (graph on left) and individual relative percent changes in ejection fraction (graph on right) associated with saline (black line) and MCOPPB (orange line) administration, started at day 21 after myocardial infarction induction. Ejection fraction significantly improved in the MCOPPB group (MCOPPB vs. saline at 2 months follow-up, 58 ± 3 vs. 45 ± 2, p < 0.001, MCOPPB vs. saline at euthanasia, 67 ± 3 vs. 43 ± 2, p < 0.001). Two-tailed ANOVA with Bonferroni post hoc test was used. n = 6/group. (B) Systolic wall thickness increased in the MCOPPB group. LV wall thickness was measured by echocardiography with short-axis M-mode images at the interventricular wall (left) and posterior wall (right). Both the end-systolic interventricular septum (IVS) (left) and end-systolic left ventricular posterior wall (LVPW) (right) were significantly greater in the MCOPPB group compared to the saline group at euthanasia (end-systolic IVS, MCOPPB vs. saline, 2.2 ± 0.5 mm vs. 1.1 ± 0.3 mm, p < 0.01; end-systolic LVPW, MCOPPB vs. saline, 2.7 ± 0.2 mm vs. 2.1 ± 0.3 mm, p < 0.05). A two-tailed ANOVA with Bonferroni post hoc test was applied. n = 6/group. Longitudinal changes in average systolic interventricular septum thickness and average systolic LV posterior wall thickness were further plotted, separated by gender. LV posterior wall thickness was higher in the female MCOPPB group at euthanasia (MCOPPB vs. saline at euthanasia, 2.7 ± 0.3 vs. 1.8 ± 0.2, p < 0.05). A two-tailed ANOVA with Bonferroni post hoc test was used. n = 3/group. Orange line: MCOPPB group; black line: saline group. (C) End-systolic volume was significantly decreased in the MCOPPB-receiving group at euthanasia. End-systolic volume was calculated with VenoLAB software (comes pre-packaged for applications in the echomachine, Vevo 770 Imaging System): (7.0/(2.4 + LVIDs)) × LVIDs³, LVIDs = left ventricular internal diameter end systole). End-systolic volume was significantly decreased in the MCOPPB-receiving group at euthanasia (MCOPPB vs. saline, 140 ± 30 μL vs. 280 ± 44 μL, p < 0.001). A two-tailed ANOVA with Bonferroni post hoc test was used. n = 6/group. Longitudinal changes in average left ventricular systolic volume and average diastolic volume were further plotted, separated by gender. Systolic left ventricular volume was lower in the male MCOPPB group at euthanasia (MCOPPB vs. saline at euthanasia, 157 ± 32 vs. 300 ± 31, p < 0.05). A two-tailed ANOVA with Bonferroni post hoc test was used. n = 3/group. Orange line: MCOPPB group; black line: saline group. (D) Stroke volume was significantly increased in the MCOPPB-receiving group at euthanasia. Stroke volume was calculated as [(end-diastolic volume) − (end-systolic volume)]. Stroke volume was significantly increased in the MCOPPB-receiving group at euthanasia (MCOPPB vs. saline, 281 ± 25 μL vs. 213 ± 37 μL, p < 0.05). Two-tailed ANOVA with Bonferroni post hoc test was used. n = 6/group. Longitudinal changes in average left ventricular systolic volume and average diastolic volume were further plotted. (E) Representative images obtained by M-mode echocardiography. Left: saline received animal at pre-treatment and pre-euthanasia. Right: MCOPPB received animal at pre-treatment and pre-euthanasia.

Figure 2

(A) Cardiac function was improved with MCOPPB administration. Echocardiography results show longitudinal plot of changes in average ejection fraction (graph on left) and individual relative percent changes in ejection fraction (graph on right) associated with saline (black line) and MCOPPB (orange line) administration, started at day 21 after myocardial infarction induction. Ejection fraction significantly improved in the MCOPPB group (MCOPPB vs. saline at 2 months follow-up, 58 ± 3 vs. 45 ± 2, p < 0.001, MCOPPB vs. saline at euthanasia, 67 ± 3 vs. 43 ± 2, p < 0.001). Two-tailed ANOVA with Bonferroni post hoc test was used. n = 6/group. (B) Systolic wall thickness increased in the MCOPPB group. LV wall thickness was measured by echocardiography with short-axis M-mode images at the interventricular wall (left) and posterior wall (right). Both the end-systolic interventricular septum (IVS) (left) and end-systolic left ventricular posterior wall (LVPW) (right) were significantly greater in the MCOPPB group compared to the saline group at euthanasia (end-systolic IVS, MCOPPB vs. saline, 2.2 ± 0.5 mm vs. 1.1 ± 0.3 mm, p < 0.01; end-systolic LVPW, MCOPPB vs. saline, 2.7 ± 0.2 mm vs. 2.1 ± 0.3 mm, p < 0.05). A two-tailed ANOVA with Bonferroni post hoc test was applied. n = 6/group. Longitudinal changes in average systolic interventricular septum thickness and average systolic LV posterior wall thickness were further plotted, separated by gender. LV posterior wall thickness was higher in the female MCOPPB group at euthanasia (MCOPPB vs. saline at euthanasia, 2.7 ± 0.3 vs. 1.8 ± 0.2, p < 0.05). A two-tailed ANOVA with Bonferroni post hoc test was used. n = 3/group. Orange line: MCOPPB group; black line: saline group. (C) End-systolic volume was significantly decreased in the MCOPPB-receiving group at euthanasia. End-systolic volume was calculated with VenoLAB software (comes pre-packaged for applications in the echomachine, Vevo 770 Imaging System): (7.0/(2.4 + LVIDs)) × LVIDs³, LVIDs = left ventricular internal diameter end systole). End-systolic volume was significantly decreased in the MCOPPB-receiving group at euthanasia (MCOPPB vs. saline, 140 ± 30 μL vs. 280 ± 44 μL, p < 0.001). A two-tailed ANOVA with Bonferroni post hoc test was used. n = 6/group. Longitudinal changes in average left ventricular systolic volume and average diastolic volume were further plotted, separated by gender. Systolic left ventricular volume was lower in the male MCOPPB group at euthanasia (MCOPPB vs. saline at euthanasia, 157 ± 32 vs. 300 ± 31, p < 0.05). A two-tailed ANOVA with Bonferroni post hoc test was used. n = 3/group. Orange line: MCOPPB group; black line: saline group. (D) Stroke volume was significantly increased in the MCOPPB-receiving group at euthanasia. Stroke volume was calculated as [(end-diastolic volume) − (end-systolic volume)]. Stroke volume was significantly increased in the MCOPPB-receiving group at euthanasia (MCOPPB vs. saline, 281 ± 25 μL vs. 213 ± 37 μL, p < 0.05). Two-tailed ANOVA with Bonferroni post hoc test was used. n = 6/group. Longitudinal changes in average left ventricular systolic volume and average diastolic volume were further plotted. (E) Representative images obtained by M-mode echocardiography. Left: saline received animal at pre-treatment and pre-euthanasia. Right: MCOPPB received animal at pre-treatment and pre-euthanasia.

Figure 2

(A) Cardiac function was improved with MCOPPB administration. Echocardiography results show longitudinal plot of changes in average ejection fraction (graph on left) and individual relative percent changes in ejection fraction (graph on right) associated with saline (black line) and MCOPPB (orange line) administration, started at day 21 after myocardial infarction induction. Ejection fraction significantly improved in the MCOPPB group (MCOPPB vs. saline at 2 months follow-up, 58 ± 3 vs. 45 ± 2, p < 0.001, MCOPPB vs. saline at euthanasia, 67 ± 3 vs. 43 ± 2, p < 0.001). Two-tailed ANOVA with Bonferroni post hoc test was used. n = 6/group. (B) Systolic wall thickness increased in the MCOPPB group. LV wall thickness was measured by echocardiography with short-axis M-mode images at the interventricular wall (left) and posterior wall (right). Both the end-systolic interventricular septum (IVS) (left) and end-systolic left ventricular posterior wall (LVPW) (right) were significantly greater in the MCOPPB group compared to the saline group at euthanasia (end-systolic IVS, MCOPPB vs. saline, 2.2 ± 0.5 mm vs. 1.1 ± 0.3 mm, p < 0.01; end-systolic LVPW, MCOPPB vs. saline, 2.7 ± 0.2 mm vs. 2.1 ± 0.3 mm, p < 0.05). A two-tailed ANOVA with Bonferroni post hoc test was applied. n = 6/group. Longitudinal changes in average systolic interventricular septum thickness and average systolic LV posterior wall thickness were further plotted, separated by gender. LV posterior wall thickness was higher in the female MCOPPB group at euthanasia (MCOPPB vs. saline at euthanasia, 2.7 ± 0.3 vs. 1.8 ± 0.2, p < 0.05). A two-tailed ANOVA with Bonferroni post hoc test was used. n = 3/group. Orange line: MCOPPB group; black line: saline group. (C) End-systolic volume was significantly decreased in the MCOPPB-receiving group at euthanasia. End-systolic volume was calculated with VenoLAB software (comes pre-packaged for applications in the echomachine, Vevo 770 Imaging System): (7.0/(2.4 + LVIDs)) × LVIDs³, LVIDs = left ventricular internal diameter end systole). End-systolic volume was significantly decreased in the MCOPPB-receiving group at euthanasia (MCOPPB vs. saline, 140 ± 30 μL vs. 280 ± 44 μL, p < 0.001). A two-tailed ANOVA with Bonferroni post hoc test was used. n = 6/group. Longitudinal changes in average left ventricular systolic volume and average diastolic volume were further plotted, separated by gender. Systolic left ventricular volume was lower in the male MCOPPB group at euthanasia (MCOPPB vs. saline at euthanasia, 157 ± 32 vs. 300 ± 31, p < 0.05). A two-tailed ANOVA with Bonferroni post hoc test was used. n = 3/group. Orange line: MCOPPB group; black line: saline group. (D) Stroke volume was significantly increased in the MCOPPB-receiving group at euthanasia. Stroke volume was calculated as [(end-diastolic volume) − (end-systolic volume)]. Stroke volume was significantly increased in the MCOPPB-receiving group at euthanasia (MCOPPB vs. saline, 281 ± 25 μL vs. 213 ± 37 μL, p < 0.05). Two-tailed ANOVA with Bonferroni post hoc test was used. n = 6/group. Longitudinal changes in average left ventricular systolic volume and average diastolic volume were further plotted. (E) Representative images obtained by M-mode echocardiography. Left: saline received animal at pre-treatment and pre-euthanasia. Right: MCOPPB received animal at pre-treatment and pre-euthanasia.

Figure 3

(A) Body weight was increased in both the saline- and MCOPPB-receiving groups. Body weight was compared between pre-treatment and pre-euthanasia. All four groups, (1) males receiving saline, (2) males receiving MCOPPB, (3) females receiving saline, and (4) females receiving MCOPPB, gained weight. No changes were statistically significant. (B) The percentage of body weight gain was lower in the MCOPPB group than the saline group for males. Body weight change was compared via percentage increase in body weight, [(body weight at euthanasia − body weight at first treatment)/body weight at first treatment] × 100; the male MCOPPB group gained weight less than the male saline group (male saline group, 22 ± 3; male MCOPPB group, 13 ± 0.6; female saline group, 9 ± 7; female MCOPPB group, 13 ± 5) (male saline vs. male MCOPPB, p < 0.05). (C) Adjusted heart weight by body weight was increased in MCOPPB-receiving animals. The whole heart was analyzed. The heart was harvested after euthanasia and weight was measured. Then, the weight was adjusted by body weight (heart weight/body weight). Adjusted heart weight was increased in MCOPPB-receiving animals in all groups and the increase in the female MCOPPB group was statistically significant (saline group, 0.0029 ± 0.0001; MCOPPB group, 0.0034 ± 0.0002, p < 0.05) (C).

Figure 4

(A) MCOPPB decreased the extent of left ventricular wall fibrosis. The paraformaldehyde-fixed heart was cut transversally and sectioned with 2 (2 to 3 mm) slices obtained: one immediately cephalad and the other one immediately caudad to the transverse centerline of the infarct region, which was readily identifiable by gross inspection (Figure 1D). After paraffin embedding of these slices, 14 sections per animal (at a 120 μm interval between each section) were stained with Masson’s Trichrome to assess the extent of fibrosis. Representative images are shown in Figure 1D. The fibrotic area (blue) and the nonfibrotic region (red) were outlined in LV wall including the septum, using Adobe Photoshop CS5 software, (Figure 1D) and then quantified with MATLAB and Simulink software (MathWorks, Inc.). The percentage of fibrosis was calculated as: (total of blue pixels from all sections/total of blue plus red pixels from all sections) × 100. The fibrosis area significantly decreased in the MCOPPB group (% fibrosis area, MCOPPB vs. saline, 14 ± 2 vs. 29 ± 10, p < 0.05). A two-tailed t-test was used. (B) Cardiomyocyte diameter increased in the MCOPPB group. Cardiomyocyte diameter was measured at 400x magnification of cardiomyocytes found in the peri-infarct (anterior, lateral) regions subtended by the ligated left anterior descending coronary artery and the non-infarcted (posterior) LV regions (Figure 1E). The slide demonstrating the greatest area of fibrosis, as identified by Masson’s Trichrome staining, was selected for each animal. In each slide, 20 longitudinally oriented (long-axis) cardiomyocytes from each of the 3 regions, anterior, lateral, and posterior, were examined, and the diameters were defined. The mean value of 20 measurements represented 1 sample from each position in each animal. The left image indicates 3 counting positions (20 cardiomyocytes/position). (MCOPPB vs. saline; anterior, 18 ± 3 μm vs. 13 ± 2 μm, p < 0.01, lateral, 19 ± 3 μm vs. 11 ± 1 μm, p < 0.001, posterior, 19 ± 3 μm vs. 12 ± 2 μm, p < 0.001). The right graphs depict cardiomyocyte diameter quantification. (C) MCOPPB increased angiogenesis in infarcted hearts. For angiogenesis analysis, two sections per animal, in which infarction size was largest in the transverse section, were stained with CD31 (R&D systems, AF3628). First, the stained sections were searched for CD31-positive cells by five random microscopic fields per slide at ×200 magnification and the highest number identified in the peri-infarct region was chosen as the number of CD31-positive cells for each slide. EVOS M5000 microscopy was used for immunohistochemical analysis. The MCOPPB group had significantly higher vessel counts in the border zone (44 ± 12 vs. 16 ± 4, p < 0.01). The graph shows the number of vessels/field. n = 6/group. A two-tailed t-test was used. Photo-images are representative slides from the saline and MCOPPB groups.

Figure 5

(A) ORL1 was identified in cardiac fibroblasts and cardiomyocytes. Primary adult rat brain tissue lysates, adult rat cardiac fibroblasts, neonatal rat cardiomyocytes (p2), and H9C2 cells (ATCC-CRL-1446) were immunostained with anti-nociceptin receptor antibody (alomone labs, AOR-015). Another set of the four cell groups were pre-incubated with ORL1-blocking peptide (alomone labs, BLP-OR015). (B) NPPA and NPPB were downregulated by MCOPPB. Neonatal rat cardiomyocytes were treated with a nociception agonist, MCOPPB (0.5 μM), or MCOPPB (0.5 μM) + anti-ORL1 (ORL1 antagonist, [Nphe1]Nociceptin(1-13)NH2]) (10 μM). All groups received ET-1 (100 nM). qPCR shows that NPPA and NPPB, which are downstream transcription genes of the NFAT signaling pathway and related to pathological hypertrophy, were downregulated by MCOPPB. Significantly, those downregulations by MCOPPB were diminished by adding the ORL1 antagonist. A two-tailed ANOVA with Bonferroni post hoc test was used. Primers for NPPA: forward, 5′-CGTATACAGTGCGGTGTCCAAC-3′; reverse, 5′-CATCTTCTCCTCCAGGTGGTCTAG-3′. Primers for NPPB: forward, 5′-AAGTCCTAGCCAGTCTCCAGAACA-3′. Reverse, 5′-TTGAGAGCTGTCTCTGAGCCATT-3′. (C) ORL1 inhibitor increased nuclear dephosphorylated NFAT. H9C2 cells were treated with MCOPPB (0.5 μM), ET-1 (100 nM), and anti-ORL1 (10 μM). Nuclear fractions of cells were extracted using an NE-PER nuclear reagents kit and analyzed by Western blotting with NFATc4 antibody (Santa Cruz Biotechnology—SC 271597). Lamin b (Santa Cruz Biotechnology—SC 374015) was used as a loading control. NFATc4 expression was significantly increased by adding the ORL1 antagonist. (D) Cardiomyocyte cell size was not changed with MCOPPB administration. Neonatal mouse cardiomyocytes (p3) were treated with MCOPPB (0.5 μM) at day 1 and day 2, and immunostained with troponin T at day 3. Cardiomyocyte imaging and an analysis of the images were conducted using the high-content imaging instrument Cytation 7 (BioTek). Average cardiomyocyte size was no treatment, 33.8 ± 3.4 μm² vs. MCOPPB group, 30.6 ± 1.1 μm², (p = 0.20, 3 plates/group). Representative images are shown (×200).

Figure 5

(A) ORL1 was identified in cardiac fibroblasts and cardiomyocytes. Primary adult rat brain tissue lysates, adult rat cardiac fibroblasts, neonatal rat cardiomyocytes (p2), and H9C2 cells (ATCC-CRL-1446) were immunostained with anti-nociceptin receptor antibody (alomone labs, AOR-015). Another set of the four cell groups were pre-incubated with ORL1-blocking peptide (alomone labs, BLP-OR015). (B) NPPA and NPPB were downregulated by MCOPPB. Neonatal rat cardiomyocytes were treated with a nociception agonist, MCOPPB (0.5 μM), or MCOPPB (0.5 μM) + anti-ORL1 (ORL1 antagonist, [Nphe1]Nociceptin(1-13)NH2]) (10 μM). All groups received ET-1 (100 nM). qPCR shows that NPPA and NPPB, which are downstream transcription genes of the NFAT signaling pathway and related to pathological hypertrophy, were downregulated by MCOPPB. Significantly, those downregulations by MCOPPB were diminished by adding the ORL1 antagonist. A two-tailed ANOVA with Bonferroni post hoc test was used. Primers for NPPA: forward, 5′-CGTATACAGTGCGGTGTCCAAC-3′; reverse, 5′-CATCTTCTCCTCCAGGTGGTCTAG-3′. Primers for NPPB: forward, 5′-AAGTCCTAGCCAGTCTCCAGAACA-3′. Reverse, 5′-TTGAGAGCTGTCTCTGAGCCATT-3′. (C) ORL1 inhibitor increased nuclear dephosphorylated NFAT. H9C2 cells were treated with MCOPPB (0.5 μM), ET-1 (100 nM), and anti-ORL1 (10 μM). Nuclear fractions of cells were extracted using an NE-PER nuclear reagents kit and analyzed by Western blotting with NFATc4 antibody (Santa Cruz Biotechnology—SC 271597). Lamin b (Santa Cruz Biotechnology—SC 374015) was used as a loading control. NFATc4 expression was significantly increased by adding the ORL1 antagonist. (D) Cardiomyocyte cell size was not changed with MCOPPB administration. Neonatal mouse cardiomyocytes (p3) were treated with MCOPPB (0.5 μM) at day 1 and day 2, and immunostained with troponin T at day 3. Cardiomyocyte imaging and an analysis of the images were conducted using the high-content imaging instrument Cytation 7 (BioTek). Average cardiomyocyte size was no treatment, 33.8 ± 3.4 μm² vs. MCOPPB group, 30.6 ± 1.1 μm², (p = 0.20, 3 plates/group). Representative images are shown (×200).