Pharmacological Inhibition of GRK2 Improves Cardiac Metabolism and Function in Experimental Heart Failure

Figures

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  Figure 1. The acute effects of GRK2 inh on isolated AMVM and in healthy mice. (A) FS (%) in AMVM challenged with the vehicle, iso (1 μM), GRK2 inh (1 μM), or both (n = 3 independent experiments). Iso produces a significant increase in FS% in both AMVMs from GRK2 fl/fl and MHCcre GRK2 fl/fl (* P < 0.05 vs. vehicle) but more pronounced in MHCcre GRK2 fl/fl (** P < 0.05 vs. GRK2 fl/fl). GRK2 inh induces a significant increase of FS% in AMVMs from GRK2 fl/fl (! P < 0.05 vs. vehicle) and enhances iso effect on cell shortening (!! P < 0.05 vs. iso alone). GRK2 inh does not exert any effects on cell shortening in MHCcre GRK2 fl/fl. AMVM, adult mice ventricular myocyte; FS, fractional shortening; Iso, isoproterenol; GRK2 inh, G protein‐coupled receptor kinase 2 inhibitor. (B–D) Mice subjected to a single ip injection of C7 (2 or 4 mg/kg/100 μL) with or without iso (n = 5 independent experiments). Cardiac functions evaluated by Ultrasound. GRK2 inhibition (2 and 4 mg/kg/100 μL) significantly increases heart rate, systolic function, and reduces LVDd at 10′ and 30′ with the return to baseline at 90′ post injection (*,** P < 0.05 vs. vehicle). Iso increases cardiac function (! P < 0.05 vs. vehicle).…

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  Figure 2. Reduced cardiac dilation and improved function in post‐MI HF mice treated with GRK2 inhibitor. (A, B) Left ventricle diastolic diameter (LVDd) and EF measured by serial echocardiography (n = 12 per group). Induced MI led to a progressive increase of LVDd and reduced EF, becoming significant at 6 weeks post surgery (* vs. sham, P < 0.05). GRK2 inhibitor treatment arrests cardiac dilation and promotes amelioration of systolic cardiac function in HF (! vs. post‐MI HF, P < 0.05). (c–f) Functional response to acute iso infusion in vivo, illustrating heart rate, and ±dp/dt max response to graded concentrations of the catecholamine (n = 6, per group). HR is higher in post‐MI HF mice respect to sham at baseline (* vs. sham, P < 0.05). No significant differences are evident between sham and HF groups treated with GRK2 inh at 2 mg/kg/day. +dp/dt values are reduced upon iso iv infusion with depressed diastolic function and increased left ventricle diastolic pressure in post‐MI HF (* vs. sham, P < 0.05). GRK2 inhibitor treatment improved cardiac contractility in mice with HF along with improved diastolic function and reduced left ventricle pressure (! vs. post‐MI HF). EF, ejection fraction;…

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  Figure 3. Reverse cardiac remodelling induced by the GRK2 inhibitor. (A–C) Cardiac Hypertrophy, left ventricle mass (LVm) corrected by tibia length and RWT were evaluated by HW/BW ratio and echocardiography (n = 12 per group). HW/BW and LVm increase in post‐MI HF treated with vehicle (* vs. sham, P < 0.05) with a significant reduction in the group treated with the GRK2 inhibitor 2 mg/kg/day (! vs. post‐MI HF, P < 0.05). RWT decreases in HF group (* vs. sham, P < 0.05) and normalized by treatment (! vs. post‐MI HF, P < 0.05). (D, E) Fluorescence‐tagged wheat germ agglutinin (n = 4 per group) shows increased cardiac myocyte cross‐sectional area in post‐MI HF mice (* vs. sham, P < 0.05; magnification 9200, scale bar = 15 μm) with significant reduction with GRK2 inhibitor treatment (! vs. post‐MI HF, P < 0.05). Quantification was performed by three blinded investigators (D. S., A. F., and C. D. G.). (F, G) Masson's trichrome staining (n = 4 per group; magnification 9300, scale bar = 15 μm) also revealed an increase in interstitial fibrosis, quantified as described in Methods, in post‐MI HF group (* vs. sham, P < 0.05; n = 3 per group) with a significant reduction induced by GRK2 inhibitor…

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  Figure 4. Mitochondrial alteration and cardiac steatosis in mice with HF. (A–D) Representative transmission electron micrographs showing abnormalities in mitochondrial ultrastructure evident in post‐MI HF samples. Images show the disrupted mitochondrial structure with numerous breaks and loss of cristae structure and matrix in failing myocardium (pointed by arrows). The mitochondrial structure appears recovered by the GRK2 inhibitor in post‐MI HF. Mitochondrial morphometric analyses (scale bar = 1 μm) obtained quantification of mitochondrial abnormalities with evaluation of (B) mitochondrial area, (C) percentage of abnormal mitochondria, (D) cristae density (* vs. sham, P < 0.05; ! vs. post‐MI HF, P < 0.05; ** vs. sham, P < 0.05). Quantification was performed by three blinded investigators (D. S., A. F., and C. D. G.). (E, F) Representative images (magnification 9200, scale bar = 15 μm) and quantification of Oil Red O staining showing cardiac lipid overload in post‐MI HF samples (* vs. sham, P < 0.05) with the significant reduction after treatment with C7 inhibitor (! vs. post‐MI HF, P < 0.05). Quantification was performed by three blinded investigators (D. S., A. F., and C. D. G.). (G)…

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  Figure 5. Cardiac metabolic and mitochondrial alterations in HF. (A–D) Regulators of mitogenesis and electron transport related genes measured by RT‐PCR. Defective expression of PGC1α, NRF1, MTFA, ETC complex can be observed in post‐MI HF group (* vs. sham, P < 0.05). GRK2 inhibitor (2 mg/kg/day) significantly improves genes expression levels in failing hearts (! vs. post‐MI HF, P < 0.05). Data are expressed as relative gene expression (vs. sham). (E) ATP production measured by luminescence assay. ATP content significantly reduced in post‐MI HF (* vs. sham, P < 0.05). GRK2 inhibitor treatment improves ATP levels during HF (! vs. post‐MI HF, P < 0.05). (E, F) Mitochondrial respiration. Mitochondrial state 3 respiration rates evaluated using succinate or pyruvate as substrates are significantly decreased in post‐MI HF versus sham (*, P < 0.05). GRK2 inhibitor peptide significantly recovers state 3 respiration rates in post‐MI HF animals (! vs. post‐MI HF, P < 0.05). ETC, electron transport chain; MTFA, mitochondrial transcription factor A; NRF1, nuclear respiratory factor; PGC1α, peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha; RT‐PCR, real‐time‐polymerase chain…

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  Figure 6. Effects of GRK2 inhibition on βAR signalling. (A) βAR density measured by radioligand binding assay on isolated plasma membrane from heart samples. Samples from post‐MI HF display a decreased βAR density (* vs. sham, P < 0.05) but restored by GRK2 inhibitor treatment at 2 mg/kg/day (! vs. post‐MI HF). (B) cAMP content and production after iso stimulation (0.1 μM) evaluated on heart samples. Post‐MI HF has a decreased cAMP production at both baseline, and iso stimulated conditions (* vs. sham, P < 0.05) but recovered in the presence of GRK2 inhibitor treatment (! vs. post‐MI, P < 0.05). (C, D) Kinase activity evaluated by Rhodopsin phosphorylation. Representative autoradiography image shows significantly increased rhodopsin phosphorylation in heart sample from post‐MI HF group (* vs. sham, P < 0.05) with significant reduction when C7 is present (! vs. post‐MI HF, P < 0.05).

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  Figure 7. Improved systemic haemodynamic in mice with HF. (A, B) 24 h urine volume and Na/K ratio. Urine volume collected in 24 h is significantly reduced in animals with HF, with reduced Na/K ratio in the same sample (* vs. sham, P < 0.05). GRK2 inhibitor treatment (2 mg/kg/day) increases both volume and Na/K ratio in urine collected after 24 h (! vs. post‐MI HF). (C) Interstitial pulmonary oedema is measured by lung to body weight ratio. HF brought to a significant increase in lung weight (* vs. sham, P < 0.05;) but reduced by the GRK2 inhibitor at 2 mg/kg/day (! vs. post‐MI HF, P < 0.05). (D) SP measured in conscious animals by the tail cuff. SP is reduced in post‐MI HF group in both diastolic and systolic values (* vs. sham, P < 0.05) but recovered with treatment (! vs. post‐MI HF, P < 0.05). (E) SERCA2a, BNP, and ANF mRNA level measured by RT‐PCR on heart samples. HF is associated with decreased SERCA2a and increased BNP, ANF mRNA level (* vs. sham, P < 0.05). The expression level of these is normalized by treatment with GRK2 inhibitor (! vs. post‐MI HF, P < 0.05). Significant modifications of SERCA2a and BNP level are also observed in sham animals undergone treatment (** vs. sham, P…