Pepducin-mediated cardioprotection via β-arrestin-biased β2-adrenergic receptor-specific signaling

Abstract

Reperfusion as a therapeutic intervention for acute myocardial infarction-induced cardiac injury itself induces further cardiomyocyte death. β-arrestin (βarr)-biased β-adrenergic receptor (βAR) activation promotes survival signaling responses in vitro; thus, we hypothesize that this pathway can mitigate cardiomyocyte death at the time of reperfusion to better preserve function. However, a lack of efficacious βarr-biased orthosteric small molecules has prevented investigation into whether this pathway relays protection against ischemic injury in vivo. We recently demonstrated that the pepducin ICL1-9, a small lipidated peptide fragment designed from the first intracellular loop of β2AR, allosterically engaged pro-survival signaling cascades in a βarr-dependent manner in vitro. Thus, in this study we tested whether ICL1-9 relays cardioprotection against ischemia/reperfusion (I/R)-induced injury in vivo. Methods: Wild-type (WT) C57BL/6, β2AR knockout (KO), βarr1KO and βarr2KO mice received intracardiac injections of either ICL1-9 or a scrambled control pepducin (Scr) at the time of ischemia (30 min) followed by reperfusion for either 24 h, to assess infarct size and cardiomyocyte death, or 4 weeks, to monitor the impact of ICL1-9 on long-term cardiac structure and function. Neonatal rat ventricular myocytes (NRVM) were used to assess the impact of ICL1-9 versus Scr pepducin on cardiomyocyte survival and mitochondrial superoxide formation in response to either serum deprivation or hypoxia/reoxygenation (H/R) in vitro and to investigate the associated mechanism(s). Results: Intramyocardial injection of ICL1-9 at the time of I/R reduced infarct size, cardiomyocyte death and improved cardiac function in a β2AR- and βarr-dependent manner, which led to improved contractile function early and less fibrotic remodeling over time. Mechanistically, ICL1-9 attenuated mitochondrial superoxide production and promoted cardiomyocyte survival in a RhoA/ROCK-dependent manner. RhoA activation could be detected in cardiomyocytes and whole heart up to 24 h post-treatment, demonstrating the stability of ICL1-9 effects on βarr-dependent β2AR signaling. Conclusion: Pepducin-based allosteric modulation of βarr-dependent β2AR signaling represents a novel therapeutic approach to reduce reperfusion-induced cardiac injury and relay long-term cardiac remodeling benefits.

Keywords: Pepducin; cardiac ischemia/reperfusion; cardiomyocyte; β-arrestin; β2-adrenergic receptor.

Figure 1

ICL1-9 attenuates cardiac injury after I/R. (A) WT mice received an intracardiac injection of unlabeled ICL1-9 or TMR-conjugated ICL1-9 (red). Representative heart sections from at least 4 independent mice per timepoint. Sections were stained with troponin I (TNNI, green) to identify cardiomyocytes and DAPI (blue) to label nuclei. n=4 ICL1-9 (Unlabeled), n=5 ICL1-9TMR at each timepoint. (B) Radioligand binding analysis of βAR isoform expression in the LV of WT mice 24 h after intracardiac injection of Scr or ICL1-9 pepducin. n=5 Scr, n=6 ICL1-9, no significant differences observed, two-tailed unpaired t-test for each isoform. (C) Representative Evan's Blue/TTC staining of hearts from mice receiving Scr or ICL1-9 pepducin injection followed by 30 min ischemia/24 h reperfusion (I/R). Quantification of (D) the area at risk (AAR) expressed as a percentage of the total LV area and (E) infarct size expressed as a percentage of the area at risk. n=9 each, * p < 0.05, two-tailed unpaired t-test. (F) Representative TUNEL staining (red) from the AAR of hearts injected with ICL1-9 or Scr control pepducin prior to I/R. Sections were counterstained with troponin I (TNNI, green) to identify cardiomyocytes and DAPI (blue) to label nuclei and are quantified as a percentage of the TUNEL-positive cardiomyocytes in (G). n=9 each, * p < 0.05, two-tailed unpaired t-test. (H) LV fractional shortening (FS) was measured at the short axis from M-mode using VisualSonics Analysis software. n=6 Scr Sham, n=6 ICL1-9 Sham, n=9 Scr I/R, n=10 ICL1-9 I/R, *** p < 0.001, two-way repeated measures ANOVA with Bonferroni post test.

Figure 2

ICL1-9-mediated protection against I/R-induced injury is β2AR- and β-arrestin-dependent. Representative images of hearts from WT controls versus β2ARKO (A) or βarr1KO and βarr2KO (F) mice that received Scr or ICL1-9 pepducin at the time of I/R, stained with Evan's Blue/TTC 24 h following I/R injury. Quantification of (B, G) the area at risk (AAR) expressed as percentage of the total LV area and (C, H) infarct size expressed as a percentage of the AAR for WT controls versus β2ARKO (B-C) or βarr1KO and βarr2KO (G-H) mouse hearts. Representative TUNEL staining (red) within the AAR of WT controls versus β2ARKO (D) or βarr1KO and βarr2KO (I) mouse hearts. Hearts were counterstained for troponin I (TNNI, green) to label cardiomyocytes and DAPI (blue) to identify nuclei. Quantification of TUNEL⁺ cardiomyocytes expressed as a percentage of the total number of cardiomyocytes for WT versus β2ARKO (E) or βarr1KO and βarr2KO (J) mouse hearts. For (A-E): n=7 Scr WT, n=7 ICL1-9 WT, n=10 Scr β2ARKO, n=11 ICL1-9 β2ARKO. For (F-J): n=18 Scr WT, n=10 ICL1-9 WT, n=10 Scr βarr1KO, n=10 ICL1-9 βarr1KO, n=11 Scr βarr2KO, n=10 ICL1-9 βarr2KO. * p < 0.05, ** p < 0.01, one-way ANOVA with Tukey's multiple comparison test.

Figure 3

ICL1-9 reduces fibrotic remodeling following I/R. Cardiac remodeling responses to Scr or ICL1-9 pepducin were assessed via gravimetric and immunohistological analyses 4 weeks post-I/R. (A) Quantification of gravimetric analysis of heart weight normalized to tibia length. n=8 Sham Scr, n=8 Sham ICL1-9, n=12 I/R Scr, n=11 I/R ICL1-9, ns = not significant, ** p < 0.01, one-way ANOVA with Tukey's multiple comparison test. (B) Representative wheat germ agglutinin (WGA) staining of hearts to assess cardiomyocyte cross sectional area. (C) Quantification of cardiomyocyte size from WGA-stained hearts. n=7 Scr Sham, n=8 ICL1-9 Sham, n=12 Scr I/R, n=11 ICL1-9 I/R, ns = not significant, * p < 0.05, one-way ANOVA with Tukey's multiple comparison test. (D) Representative Masson trichrome staining of Scr or ICL1-9 pepducin-injected hearts following I/R injury. (E) Quantification of fibrosis from Masson trichrome-stained hearts, expressed as a percentage of the total LV area. n=8 Sham Scr, n=8 Sham ICL1-9, n=10 I/R Scr, n=10 I/R ICL1-9, ns = not significant, ** p < 0.01, *** p < 0.001, one-way ANOVA with Tukey's multiple comparison test.

Figure 4

ICL1-9 protects against cardiomyocyte death in vitro. ¹²⁵I-cyanopindolol binding analysis of NRVM treated for 1 h (A) or 24 h (B) with Veh, ISO, Scr or ICL1-9 pepducin (10 μM each). n=4 Veh, n=4 ISO, n=5 Scr, n=5 ICL1-9. *p < 0.05, *** p < 0.001 for β1AR density, one-way ANOVA with Tukey's multiple comparison test. (C) Caspase 3/7 activity was measured in NRVM subjected to 24 h with or without serum in the presence of Scr or ICL1-9 pepducin (10 µM each) with values represented as a percentage of no serum Veh control. n=7 Veh, n=7 Veh no serum, n=10 Scr no serum, n=10 ICL1-9 no serum. ns = not significant, ** p < 0.01, *** p < 0.001, one-way ANOVA with Tukey's multiple comparison test. (D) Caspase 3/7 activity was measured in NRVM subjected to 24 h without serum in the presence of Scr or ICL1-9 pepducin (10 µM each) and pretreated with PD98059, LY294002 or AG1478 (10 µM each) with values represented as a percentage of Veh Scr. n=18 Veh Scr, n=20 Veh ICL1-9, n=10 PD98059 Scr and PD98059 ICL1-9, n=8 for all other treatment groups. ns = not significant, ** p < 0.01, *** p < 0.001, one-way ANOVA with Tukey's multiple comparison test.

Figure 5

ICL1-9 activates RhoA in cardiomyocytes and whole heart. RhoA activation was assessed via anti-GTP-bound RhoA pulldown in NRVM pretreated with Veh or 100 nM ICI 118,551 and stimulated with Scr or ICL1-9 pepducin for 5 min (A) or 24 h (B), or in left ventricular lysates of WT or β2ARKO mice 24 h after intracardiac injection (C). Activated RhoA (IP) was normalized to total RhoA/GAPDH (input) and expressed in histograms as fold change from Veh Scr (A-B) or WT Scr (C). For (A): n=7 Veh Scr, n=6 Veh ICL1-9, n=5 ICI Scr and n=6 ICI ICL1-9; for (B): n=9 Veh Scr and Veh ICL1-9, n=4 ICI Scr and ICI ICL1-9; for (C): n=14 WT Scr, n=16 WT ICL1-9, n=13 β2ARKO Scr, n=14 β2ARKO ICL1-9. * p < 0.05, one-way ANOVA with Tukey's multiple comparison test.

Figure 6

RhoA inhibition prevents ICL1-9-mediated cardiomyocyte survival. NRVM underwent serum deprivation for 24 h in the presence or absence of ICL1-9 or Scr pepducin with or without Rhosin (10 µM each). Cells were fixed, stained for TUNEL, DAPI and αSrA (A) and TUNEL-positive cardiomyocytes (arrowheads) were quantified as a percentage of total cardiomyocytes (B). n=27 Veh+Scr, Veh+ICL1-9, n=24 Rhosin+Scr, n=25 Rhosin+ICL1-9 (number of fields of view assessed from 5 plates per treatment and 3 independent primary NRVM isolations). ns = not significant, * p < 0.05, one-way ANOVA with Tukey's multiple comparison test. (C) Caspase 3/7 activity was measured in NRVM subjected to 24 h without serum in the presence of Scr or ICL1-9 pepducin (10 µM each) and pretreated with Rhosin (10 µM) with values represented as a percentage of Veh Scr. n=9 Veh Scr, n=8 for all other groups. ns = not significant, * p < 0.05, one-way ANOVA with Tukey's multiple comparison test.

Figure 7

ICL1-9 decreases H/R-induced caspase 3/7 activity and superoxide formation in cardiomyocytes via RhoA/ROCK activation. NRVM underwent hypoxia for 4 h followed by reoxygenation for 20 h in the presence of Veh, Scr or ICL1-9 pepducin (10 µM each) with or without pretreatment with ICI 118,551 (100 nM), Y-27632 (10µM) or Rhosin (10 µM). Caspase 3/7 activity (A-B) was assessed as in Figure 5 and superoxide formation (C-D) was measured using mitosox red. Values are expressed as a percentage of H/R Veh (A, C) or Veh Scr (B, D). For (A): n=9 for all treatment groups, for (B): n=12 for all treatment groups, for (C): n=8 Veh and H/R Veh, n=14 H/R Scr and H/R ICL1-9, for (C): n=14 for Veh Scr and Veh ICL1-9, n=8 for all other treatment groups. ns = not significant, * p < 0.05, *** p < 0.001, one-way ANOVA with Tukey's multiple test. (E) Representative images of mitoSOX staining (red) in NRVM with or without H/R (left panels) or H/R plus Veh, ICI 118,551 (100 nM), Y-27632 (10µM) or Rhosin (10 µM) and Scr or ICL1-9 pepducin (10 µM each, right panels). Cells were counterstained with DAPI (blue) to detect nuclei. n=5 for Veh No H/R and Veh H/R, n=6 for Veh H/R Scr and Veh H/R ICL1-9, n=4 for all other H/R treatment groups.