Amlexanox and Forskolin Prevents Isoproterenol-Induced Cardiomyopathy by Subduing Cardiomyocyte Hypertrophy and Maladaptive Inflammatory Responses

Abstract

Chronic catecholamine stress (CCS) induces the occurrence of cardiomyopathy—pathological cardiac hypertrophy (PCH), which is characterized by left ventricular systolic dysfunction (LVSD). Recently, mounting evidence has implicated myocardial inflammation in the exacerbation of pathological cardiac remodeling. However, there are currently no well-defined treatment interventions or regimes targeted at both the attenuation of maladaptive myocardial hypertrophy and inflammation during CCS to prevent PCH. G protein-coupled receptor kinase 5 (GRK5) and adenylyl cyclases (ACs)-cAMP mediates both cardiac and inflammatory responses. Also, GRK5 and ACs are implicated in stress-induced LVSD. Herein, we aimed at preventing PCH during CCS via modulating adaptive cardiac and inflammatory responses by inhibiting GRK5 and/or stimulating ACs. Isoproterenol-induced cardiomyopathy (ICM) was modeled using 0.5 mg/100 g/day isoproterenol injections for 40 days. Alterations in cardiac and inflammatory responses were assessed from the myocardia. Similarities in the immunogenicity of cardiac troponin I (cTnI) and lipopolysaccharide under CCS were assessed, and Amlexanox (35 μM/ml) and/or Forskolin (10 μM/ml) were then employed in vitro to modulate adaptive inflammatory responses by inhibiting GRK5 or activating ACs-cAMP, respectively. Subsequently, Amlexanox (2.5 mg/100 g/day) and/or Forskolin (0.5 mg/100 g/day) were then translated into in vivo during CCS to modulate adaptive cardiac and inflammatory responses. The effects of Amlexanox and Forskolin on regulating myocardial systolic functions and inflammatory responses during CCS were ascertained afterward. PCH mice had excessive myocardial hypertrophy, fibrosis, and aggravated LVSD, which were accompanied by massive CD68+ inflammatory cell infiltrations. In vitro, Forskolin-AC/cAMP was effective than Amlexanox-GRK5 at downregulating proinflammatory responses during stress; nonetheless, Amlexanox and Forskolin combination demonstrated the most efficacy in modulating adaptive inflammatory responses. Individually, the translated Amlexanox and Forskolin treatment interventions were ineffective at subduing the pathological remodeling and sustaining cardiac function during CCS. However, their combination was potent at preventing LVSD during CCS by attenuating maladaptive myocardial hypertrophy, fibrosis, and inflammatory responses. The treatment intervention attained its potency mainly via Forskolin-ACs/cAMP-mediated modulation of cardiac and inflammatory responses, coupled with Amlexanox inhibition of GRK5 mediated maladaptive cascades. Taken together, our findings highlight the Amlexanox and Forskolin combination as a potential therapeutic intervention for preventing the occurrence of pathological cardiac hypertrophy during chronic stress.

Isoproterenol-Induced Cardiomyopathy Recovery Intervention: Amlexanox and Forskolin Enhances the Resolution of Catecholamine Stress-Induced Maladaptive Myocardial Remodeling

“…This study aimed to explore the therapeutic potentials of AMLX in attenuating LVSD and facilitating recovery from ICM, as demonstrated in the AMI model ( 11 ). Also, we sought to, for the first time, explore if FSKN could enhance the recovery rates of the ICM model, as it stimulates the synthesis of cAMP, which is an essential modulator of cardiac and inflammatory responses ( 1 , 2 , 12 , 18 ).…”

“…However, AMLX treatment improved HR and systolic functions, which are consistent with Mo et al's findings ( 11 ). Nonetheless, our previous study found that when AMLX treatments were given simultaneously with ISO injections, AMLX's efficacy in attenuating LVSD was dampened ( 2 ). Hence, it is speculated that for AMLX treatment to be effective at attenuating LVSD in ICM, the sources of stressors must be obliterated.…”

“…Mice (FVB males aged 2–3 months) were randomly grouped for in vivo experiments. 0.5 mg/100 g/day of ISO were injected subcutaneously (s.c.) as done previously ( 2 , 13 ), for 21 days to induce ICM in the mice. The 5% v/v dimethyl sulfoxide equivalents were injected (s.c.) to the placebo (Pb) group.…”

“…Also, AMLX and FSKN combine treatments were employed ( Figure 1A ). The dosages of AMLX and FSKN employed were based on their efficacies demonstrated in previous studies ( 2 , 11 ).…”

“…Subsequently, the cultured PM Φ were treated with 10 μM/ml of ISO and/or 1 μg/ml LPS for 12 h, and the following treatment interventions were employed for the next 12 h. (i) 0.5 % v/v DMSO as placebo (ii) 35 μM/ml of AMLX, (iii) 10 μM/ml FSKN, and (iv) AMLX and FSKN combination, at the respective doses. These treatment interventions were explored in the absence of ISO (stress), and the dosages employed were based on their efficacy in previous studies ( 2 , 16 ). Supernatants and PM Φ were used for ELISA and immunofluorescence, respectively ( Figure 1B ).…”