Paroxetine-mediated GRK2 inhibition reverses cardiac dysfunction and remodeling after myocardial infarction

Abstract

Heart failure (HF) is a disease of epidemic proportion and is associated with exceedingly high health care costs. G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) kinase 2 (GRK2), which is up-regulated in the failing human heart, appears to play a critical role in HF progression in part because enhanced GRK2 activity promotes dysfunctional adrenergic signaling and myocyte death. Recently, we found that the selective serotonin reuptake inhibitor (SSRI) paroxetine could inhibit GRK2 with selectivity over other GRKs. Wild-type mice were treated for 4 weeks with paroxetine starting at 2 weeks after myocardial infarction (MI). These mice were compared with mice treated with fluoxetine, which does not inhibit GRK2, to control for the SSRI effects of paroxetine. All mice exhibited similar left ventricular (LV) dysfunction before treatment; however, although the control and fluoxetine groups had continued degradation of function, the paroxetine group had considerably improved LV function and structure, and several hallmarks of HF were either inhibited or reversed. Use of genetically engineered mice indicated that paroxetine was working through GRK2 inhibition. The beneficial effects of paroxetine were markedly greater than those of β-blocker therapy, a current standard of care in human HF. These data demonstrate that paroxetine-mediated inhibition of GRK2 improves cardiac function after MI and represents a potential repurposing of this drug, as well as a starting point for innovative small-molecule GRK2 inhibitor development.

Fig. 1. Paroxetine treatment reverses LV dysfunction after MI

(A) Representative short-axis M-mode echocardiography recordings from C57BL/6 mice treated with vehicle (DMSO and water), fluoxetine (fluox), or paroxetine (parox) at baseline, 2 weeks (pretreatment) and 4 and 6 weeks after MI compared to noninfarcted (sham) mice treated the same way. (B and C) Serial measures of noted experimental groups for (B) LVEF and (C) FS. (D and E) Serial measures of (D) LVIDd and (E) LVIDs in these mice. (F) Serial measures of LVEDd in these mice. *P = 0.004, **P = 0.001, ***P < 0.0001 as determined by one-way analysis of variance (ANOVA) relative to corresponding MI vehicle. n = 9 to 14 per group.

Fig. 2. Paroxetine treatment after MI enhances in vivo LV hemodynamic function and restores βAR inotropic reserve

.<br>Hemodynamics were recorded from wild-type (WT) mice treated with vehicle, fluoxetine (fluox), or paroxetine (parox) at 6 weeks after MI (after 4 weeks of treatment) compared to sham. (A) Representative LV dP/dt hemodynamic recordings. Arrows mark administration of ISO. (B) Quantification of mean systemic pressure. (C to E) Quantification of (C) HR, (D) LV +dP/dt average maximum (*P = 0.0234 and 0.0130, **P = 0.0008, ***P ≤ 0.0001), and (E) LV −dP/dt average minimum at baseline and with increasing doses of ISO (0.1 to 10 ng) (*P = 0.0105 and 0.0134 for sham, and 0.0280 for MI; **P = 0.0058, 0.0040, and 0.0087, respectively). bpm, beats per minute. (F) Quantification of LVEDP at baseline (no ISO) 6 weeks after sham and MI (**P = 0.005). Statistics are relative to corresponding sham or MI vehicle by two-or one-way ANOVA as appropriate. n = 12 to 15 per group.

Fig. 3. Paroxetine reduces LV dilation and fibrosis after MI

(A to C) Measures of (A) HW normalized to TL, (B) HL normalized to TL, and (C) LW to TL in WT C57BL/6 mice treated with vehicle, fluoxetine (fluox), or paroxetine (parox) at 6 weeks after MI (4 weeks of treatment) compared to noninfarcted (sham) mice treated the same way. ***P ≤ 0.0001 relative to sham and ^tttP = 0.0075 relative to MI vehicle by one-way ANOVA. n = 12 to 19 per group. (D) Representative images of Masson’s trichrome–stained murine heart sections from WT mice treated with vehicle, fluox, or parox at 6 weeks after sham or MI surgery (4 weeks after treatment). (E and F) Graphic representation of (E) infarct length and (F) lumen area in vehicle-, fluox-, or parox-treated mice 6 weeks after MI. *P = 0.004 by one-way ANOVA. n = 7, 6, and 8, respectively, for (D) to (G). (G) Representative images of Masson’s trichrome–stained murine heart sections focusing on the border zone and the infarct area in vehicle-, fluox-, and parox-treated mice 6 weeks after MI.

Fig. 4. Paroxetine reduces SNS overdrive and restores the myocardial βAR system after MI

(A and B) Quantification of serum (A) epinephrine and (B) norepinephrine from WT mice, either sham or MI at 6 weeks, treated with vehicle, fluoxetine (fluox), or paroxetine (parox). (C) βAR density in sham versus 6-week post-MI hearts (B_max values shown as femtomoles of receptor per milligram of sarcolemmal protein) with vehicle or parox treatment. (D) Representative Western blot image of GRK2 and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) protein expression 6 weeks after sham or MI (after 4 weeks of treatment with vehicle, fluox, or parox). *P = 0.0167, 0.0047, 0.0427, and 0.0244, respectively (A and B), and *P = 0.0003 (C) versus sham vehicle and *P = 0.0119 versus MI parox by one-way ANOVA. n = 6 to 9 per group.

Fig. 5. Paroxetine’s efficacy in reversing HF is not additive when GRK2 is inhibited by βARKct but is maintained even in the face of forced GRK2 overexpression

(A) Measures of LVEF at 2 (left) and 6 (right) weeks after MI in TgβARKct or NLC mice treated with vehicle or paroxetine (parox) compared to sham animals. ***P < 0.0001 relative to sham; ^tP = 0.0004 or P < 0.0001 relative to NLC MI vehicle by one-way ANOVA of all 2-week or all 6-week data. (B and C) Measures of (B) HW and (C) LW normalized to TL in TgβARKct or NLC mice 6 weeks after MI compared to sham controls. **P = 0.0027; ***P = 0.0001 by one-way ANOVA. n = 4 to 8 per group. (D) Measures of LVEF at 2 (left) and 6 (right) weeks after MI in TgGRK2 or NLC mice treated with vehicle or parox compared to sham animals. **P = 0.0001, 0.0019, 0.0016, 0.0138, and 0.0019, respectively, relative to NLC MI vehicle or parox; ***P < 0.0001 relative to sham by one-way ANOVA of all 2-week or all 6-week data. ns, not significant. (E and F) Measures of (E) HW/TL and (F) LW/TL in TgGRK2 or NLC mice 6 weeks after MI compared to sham controls. *P = 0.0121 (E), 0.0412 (F); **P = 0.0025; ***P < 0.0001 by one-way ANOVA. n = 5 to 11 per group.

Fig. 6. Paroxetine’s beneficial effect in post-MIHF is maintained after termination of treatment

C57BL/6 mice treated with vehicle (DMSO and water) or paroxetine (parox) at baseline, 2, 4, 6, and 8 weeks after procedure (sham and MI). Two weeks after surgery, these mice were treated with vehicle or paroxetine (parox) for 4 weeks (weeks 2 to 6), followed by an additional 2 weeks of no treatment (weeks 6 to 8). (A) Serial measures of noted experimental groups for LVEF. (B) Quantification of LV +dP/dt average maximum at baseline and with increasing doses of ISO (0.1 to 10 ng) in these mice. (Cand D) Measures of (C) HW/TL and (D) LW/TL in these mice. (E and F) Quantification of serum (E) epinephrine and (F) norepinephrine in these mice. *P = 0.0461; **P ≤ 0.0009 for (B) sham and (E); **P = 0.0072 for (B) MI and 0.0024 for (F); and ***P ≤ 0.001 versus corresponding sham or MI by one- or two-way ANOVA as appropriate. n = 9 to 15 per group [(A) to (D)], n = 4 to 5 per group for serum catecholamines.

Fig. 7. Paroxetine is more effective at reversing post-MI HF than β-blocker therapy alone

(A and B) Serial measures of LVEF (A) and FS (B) from WT mice treated with vehicle, metoprolol (met), paroxetine (parox), or metoprolol and paroxetine concurrently after MI compared to vehicle-treated mice. **P = 0.0011 and ***P < 0.001 by one-way ANOVA for post-MI parox and met + parox or met relative to corresponding MI vehicle and met. (C and D) Serial measures of (C) LVIDd and (D) LVIDs in these animals compared to sham mice. *P = 0.0012 parox and 0.0047 m + p (4 weeks), 0.0116 (6 weeks met); **P = 0.0005 m + p and 0.0015 met; ***P < 0.0001 by one-way ANOVA relative to MI vehicle. (E and F) Measures of (E) HW and (F) LW normalized to TL 6 weeks after MI or sham. *P = 0.0001 (HW), 0.0246 (LW); **P < 0.0001 relative to sham and ^tP = 0.0349; ^ttP = 0.0045 parox and 0.0077 m + p relative to MI vehicle by one-way ANOVA. n = 4 (met, m + p sham), 13 to 18 all other groups.

Fig. 8. Paroxetine decreases molecular markers of HF in post-MI mice

Analysis of WT murine hearts treated with vehicle, fluoxetine (fluox), paroxetine (parox), metoprolol (met), or metoprolol and paroxetine (m + p) at 6 weeks after MI compared to sham mice treated with vehicle or parox. (A) Quantification of GRK2 protein expression normalized to GAPDH from Western blots. *P = 0.0073; ***P = 0.0002 and 0.0004 by one-way ANOVA compared to sham values. n = 9 to 17 hearts per group from seven Western blots. (C to D) Quantification of RT-PCR data showing fold change in (B) ANF *P < 0.0001 except parox = 0.0008 and ^tP = 0.0145 parox and 0.0006 m + p; (C) BNP *P = 0.0458, **P < 0.0001 except met 0.0004, ^tP = 0.0223; and (D) βMHC mRNA expression in *P = 0.0052, **P < 0.0001; ^tP = 0.0383. All * relative to sham vehicle and t versus post-MI vehicle by one-way ANOVA. n = 6 to 14 per group.