β-Arrestin mediates the Frank-Starling mechanism of cardiac contractility

Abstract

The Frank-Starling law of the heart is a physiological phenomenon that describes an intrinsic property of heart muscle in which increased cardiac filling leads to enhanced cardiac contractility. Identified more than a century ago, the Frank-Starling relationship is currently known to involve length-dependent enhancement of cardiac myofilament Ca²⁺ sensitivity. However, the upstream molecular events that link cellular stretch to the length-dependent myofilament Ca²⁺ sensitivity are poorly understood. Because the angiotensin II type 1 receptor (AT1R) and the multifunctional transducer protein β-arrestin have been shown to mediate mechanosensitive cellular signaling, we tested the hypothesis that these two proteins are involved in the Frank-Starling mechanism of the heart. Using invasive hemodynamics, we found that mice lacking β-arrestin 1, β-arrestin 2, or AT1R were unable to generate a Frank-Starling force in response to changes in cardiac volume. Although wild-type mice pretreated with the conventional AT1R blocker losartan were unable to enhance cardiac contractility with volume loading, treatment with a β-arrestin-biased AT1R ligand to selectively activate β-arrestin signaling preserved the Frank-Starling relationship. Importantly, in skinned muscle fiber preparations, we found markedly impaired length-dependent myofilament Ca²⁺ sensitivity in β-arrestin 1, β-arrestin 2, and AT1R knockout mice. Our data reveal β-arrestin 1, β-arrestin 2, and AT1R as key regulatory molecules in the Frank-Starling mechanism, which potentially can be targeted therapeutically with β-arrestin-biased AT1R ligands.

Keywords: angiotensin II type I receptor; cardiac function; hemodynamics; mechanotransduction; β-arrestin.

Fig. 1.

In vivo testing of the Frank–Starling relationship. (A) Schematic of the volume infusion protocol. Anesthetized mice underwent vagotomy, followed by retroaortic cannulation of a pressure conductance catheter into the left ventricle. (B) Representative serial pressure-volume (PV) loops measured by conductance catheter placed in a WT mouse left ventricle. Continuous PV loop data were recorded. The basal PV loop recording is shown as a solid black line. At 1 min after each colloid bolus, the PV loop is analyzed, denoted here by a dashed line. With each successive bolus, incremental increases in both LV volume and LVEDP are seen, confirming successful ventricular loading. SV in both basal loops (SV) and loops recorded after completion of volume loading (SV′) is denoted by gray arrows. (C) Example of the relationship between volume infusion and LVEDP in WT mice. (D) Example of the relationship between SV and LVEDP in WT mice.

Fig. S1.

Individual responses of LVEDP to 30-μL bolus infusions of 5% albumin (12.5 g human albumin/250 mL aqueous diluent) (Grifols Pharmaceuticals) in WT (A), β-arrestin 1 KO (B), β-arrestin 2 KO (C), and AT1R KO (D) mice. Mice that did not develop an increase in LVEDP of >5 mm Hg were excluded from the contractility analyses.

Fig. S2.

Individual responses of LVEDV to 30-μL bolus infusions of 5% albumin (12.5 g human albumin/250 mL aqueous diluent) (Grifols Pharmaceuticals) in WT (A), β-arrestin 1 KO (B), β-arrestin 2 KO (C), and AT1R KO (D) mice. Mice that did not develop an increase in LVEDV of >10 μL were excluded from the contractility analyses.

Fig. S3.

Individual SV response vs. LVEDP in mice included in the contractility analyses: (A) WT mice; (B) β-arrestin 1 KO mice; (C) β-arrestin 2 KO mice; (D) AT1R KO mice.

Fig. 2.

Effect of β-arrestin 1 and -2 on the cardiac response to volume loading. (A and B) Representative serial PV loops from a β-arrestin 1 KO mouse (A) and a β-arrestin 2 KO (B) mouse during volume infusion protocol. SV in both basal loops (SV) and in loops recorded after completion of volume loading (SV′) is denoted by the gray arrows. (C) Effect of volume infusion on absolute change in SV from baseline in β-arrestin 1 KO and β-arrestin 2 KO mice compared with WT mice. P values for the interaction between LVEDP and change in SV for data comparison were obtained by two-way repeated-measures ANOVA. *P < 0.005 for comparison between genotypes at a given LVEDP value using Bonferroni’s multiple comparison test. (D) Effect of balloon stretch on developed force in an ex vivo hanging heart preparation. Shown is the average percentage increase in LV dP/dt_max from baseline shown for WT, β-arrestin 1 KO, and β-arrestin 2 KO mice. P values for the interaction between heart beat number and genotype for data comparison were obtained by two-way repeated-measures ANOVA. *P < 0.05 for comparison between genotypes at a given heart rate using Bonferroni’s multiple comparison test. Error bars reflect SEM.

Fig. S4.

Hemodilution effect of infusion. Average hematocrit (A) and average hemoglobin (B) in WT, β-arrestin 1 KO, and β-arrestin 2 KO mice that were not infused (NI) or had completed the volume infusion protocol (Inf). *P < 0.05 vs. noninfused of the same genotype using one-way ANOVA with Bonferroni’s multiple comparison test. Error bars reflect SEM.

Fig. 3.

Effect of β-arrestin 1 and -2 on the force–Ca²⁺ relationship. (A) Force–Ca²⁺ relationship in myofilaments harvested from left ventricles of WT mice at 1.9 μm and 2.3 μm. The change in calcium concentration producing one-half maximal tension (ΔpCa₅₀) at 1.9 μm vs. 2.3 μm was significantly different. *P < 0.05 for WT mice (n = 10 mice) at 1.9 μm vs. 2.3 μm by one-way ANOVA with Bonferroni’s multiple comparison test. (B) Force–Ca²⁺ relationship in β-arrestin 1 KO mice (n = 10 mice) at 1.9 μm and 2.3 μm. (C) Force–Ca²⁺ relationship in β-arrestin 2 KO mice (n = 11 mice) at 1.9 μm and 2.3 μm. (D) Average change in calcium concentration producing one-half maximal tension (ΔpCa₅₀) at 1.9 μm vs. 2.3 μm. *P < 0.05 vs. WT average ΔpCa₅₀ by one-way ANOVA with Bonferroni’s multiple comparison test. Error bars reflect SEM.

Fig. S6.

Myofilament phosphoprotein survey of ex vivo stretch left ventricles. (A) Representative phosphoprotein gel image of ex vivo stretch left ventricles. β1KO, β-arrestin 1 KO; β2KO, β-arrestin 2 KO; C, control; Str, ex vivo stretch. (B) Average phosphorylated myosin-binding protein (MyBPC). (C) Average phosphorylated troponin T (TnT). (D) Average phosphorylated tropomyosin (Tm). (E) Average phosphorylated troponin I (TnI). (F) Average phosphorylated myosin light chain 2 (MLC-2). n = 4 separate left ventricles per genotype and condition (control and stretch). Statistical comparisons are made using one-way ANOVA with Bonferroni’s multiple comparison test. No significant differences were noted between the WT and KO mice. Error bars reflect SEM.

Fig. S7.

Myofilament 2D-DIGE ex vivo stretch left ventricles. (A) Representative 2D gels of ex vivo stretch left ventricles. From top to bottom: merged, WT stretch (green), β-arrestin 1 KO stretch (blue), and β-arrestin 2 KO stretch (red). (B–H) Average ratios of phosphorylated to total tropomyosin (P-TM/T-TM) (B), P1 to myosin light chain 2 (P1-MLC-2/MLC-2) (C), P2 to myosin light chain 2 (P2-MLC-2/MLC-2) (D), total P to myosin light chain 2 (P-MLC-2/MLC-2) (E), P1 to troponin T3 (P1-TnT3/TnT3) (F), P1 to troponin T4 (P1-TnT4/TnT4) (G), and P to troponin T (P-TnT/TnT) (H). n = 5 separate left ventricles per genotype and condition (control and stretch). Statistical comparisons were made using one-way ANOVA with Bonferroni’s multiple comparison test. No significant differences were noted between the WT and KO mice. Error bars reflect SEM. βarr1KO, β-arrestin 1 KO; βarr2KO, β-arrestin 2 KO; C, control; Str, ex vivo stretch.

Fig. S8.

Titin isoform switching in ex vivo stretch left ventricles. (A) Representative gel image of ex vivo stretch left ventricles showing Titin N2BA and N2B isoforms. βarr1KO, β-arrestin 1 KO; βarr2KO, β-arrestin 2 KO; Con, control; Str, ex vivo stretch. (B) Average N2BA isoform (N2BA/total). n = 4 separate left ventricles per genotype and condition (control and stretch). Statistical comparisons were made using one-way ANOVA with Bonferroni’s multiple comparison test. No significant differences were noted between the WT and KO mice. Error bars reflect SEM.

Fig. S9.

Kinase phosphorylation with volume infusion. (A) Average phosphorylated ERK (T202/Y204)/total ERK. (B) Average phosphorylated p-38 alpha (T180/Y182)/total p-38 alpha. (C) Average phosphorylated AKT (S473)/total AKT. (D) Average GSK3β (S9)/total GSK3β in WT (noninfused, n = 3; infused, n = 9), β-arrestin 1 KO (βarr1KO) (noninfused, n = 3; infused, n = 12), and β-arrestin 2 KO (βarr1KO) (noninfused, n = 3; infused, n = 12) mice undergoing the in vivo infusion protocol. The fold change in kinase phosphorylation with volume infusion (Inf) was normalized to noninfusion controls (NI) of the same genotype. (Bottom) Representative gel image showing kinase phosphorylation. Statistical comparisons between infused vs. noninfused conditions in the same genotype were made using Wilcoxon’s rank-sum test, where *P < 0.05. Differences between genotypes were assessed using one-way ANOVA with Bonferroni’s multiple comparison test. No significant differences were noted between the WT and KO mice. Error bars reflect SEM.

Fig. 4.

Response of AT1R KO mice to volume loading and the force–Ca²⁺ relationship. (A) Representative serial PV loops of an AT1R KO mouse during the volume infusion protocol. Stroke volume in the both basal loops (SV) and in loops recorded after completion of volume loading (SV′) are denoted by gray arrows. (B) Effect of volume infusion on absolute change in SV in AT1R KO and WT mice reveals marked depression of Frank–Starling force generation in AT1R KO mice. P values for the interaction between LVEDP and genotype for data comparison were provided by two-way repeated-measures ANOVA. *P < 0.05 for comparison between genotypes at a given LVEDP using Bonferroni’s multiple comparison test. (C) Force–Ca²⁺ relationship in myofilaments harvested from global AT1R KO mice (n = 10) at 1.9 μm and 2.3 μm. The ΔpCa₅₀ value was not significantly different at 1.9 μm vs. 2.3 μm by one-way ANOVA with Bonferroni’s multiple comparison test.

Fig. 5.

Effect of ARB on cardiac response to volume loading. (A) Effect of volume infusion on absolute change in SV in WT mice infused for 5 min with saline, TRV120023 at 100 μg⋅kg^–1⋅min^–1, or losartan at 5 mg⋅kg^–1⋅min^–1. The losartan-infused mice had markedly depressed contractility with volume infusion compared with the saline- or TRV120023-treated mice. P values for the interaction of LVEDP and drug were provided for data comparison by two-way repeated-measures ANOVA. *P < 0.05 for saline vs. losartan; ^†P < 0.05 for TRV120023 vs. losartan at a given LVEDP using Bonferroni’s multiple comparison test. (B) Average change in LV systolic pressure before and immediately after infusion of saline, TRV120023, and losartan revealing similar systolic pressure responses after administration of TRV120023 or losartan. (C) Average change in arterial elastance after infusion of saline, TRV120023, and losartan. (D) Average change in SV before and immediately after infusion of saline, TRV120023, and losartan, showing similar prevolume loading SV in all groups. *P < 0.05 vs. saline using one-way ANOVA with Bonferroni’s multiple comparison test. Error bars reflect SEM.

Fig. S5.

Losartan dose finding. The dose of losartan used in drug infusion experiments was based on the ability to block angiotensin II infusion-induced increases in LV systolic pressure, in a manner comparable to TRV120023 at a dose of 100 μg⋅kg^–1⋅min^–1. All mice were given a continuous infusion of angiotensin 100 ng⋅kg^–1⋅min^–1 for 8 min, during which time they were coadministered saline, losartan 2.5 mg⋅kg^–1⋅min^–1, or losartan 5 mg⋅kg^–1⋅min^–1. Error bars reflect SEM.