Introduction
The elevated level of circulating catecholamines during chronic stress is a hallmark for the initiation and progression of adverse cardiac remodeling (Paur H et al., 2012; Adzika GK et al., 2019). Pathological cardiac hypertrophy (PCH) is the irreversible resultant cardiomyopathy if there are no timely preventive measures to subdue the excessively firing of neurohormonal stimuli during chronic stress. Left ventricular (LV) diastolic dysfunction is a clinical characteristic of patients having PCH. This heart malfunction is a result of the abnormal increase in heart size with excessively thickened ventricular walls which, are stiffer due to the massive deposition of collagen. The typical myocardium architecture becomes distorted. The LV becomes dilated, and the heart loses its ability to rapidly replenish blood for the next ejection (Xie M et al., 2013; Hartupee J et al., 2016). The heart harbors resident macrophages which crosstalk between cardiomyocytes and other cellular factions such as fibroblasts to ensure myocardial homeostasis. However, this crosstalk implicates the immune system in playing a pivotal role in the pathological remodeling of the heart (Hartupee J et al., 2016; Kong P et al., 2014). Cardiac resident macrophages are stimulated by cardiac danger-associated molecular patterns (cDAMPs), cardiac troponin, and myosin that being released by necrotic cardiomyocytes. These resident macrophages elicit proinflammatory responses in an attempt to curb further myocyte necrosis (Epelman S et al., 2014; Heidt T et al., 2014). This immune response is aggravated under chronic catecholamine stress (CCS) conditions and a biased hyperactive inflammatory result instead. The secretion of Interleukin (IL) 1β, IL-2, IL-6, tumor necrosis factor-alpha (TNFα), and interferon-gamma (IFN-γ) are excessively while IL-10 secretion becomes downregulated. The aforementioned exacerbates the myocyte necrosis, prolongs myofibroblast activation and increases interstitial fibrosis markedly (Kong P et al., 2014; Hulsmans M et al., 2016). Interestingly, the innate immune response elicited when challenged by cardiac troponin and myosin during CCS is very similar with lipopolysaccharides (LPS)-challenged macrophages under the same stress conditions (Laukova M et al., 2018).
β1-adrenergic receptor (β1AR) and β2-adrenergic receptor (β2AR) expressed in the heart mediate the neuroendocrine stimulates the regulate cardiac function. β1AR modulates chronotropic and inotropic via the typical stimulatory G protein (Gαs) – adenylyl cyclases (ACs) – cyclic adenosine monophosphate (cAMP) pathway. The pleiotropic nature of β2AR enables it to traffick stimuli via Gαs or Gαi in cardiomyocytes, and immune cells. Also, a mounting of evidence indicates that β2AR mediates maladaptive stimuli signaling during cardiovascular diseases (CVDs) (Paur H et al., 2012; Adzika GK et al., 2019; Laukova M et al., 2018). These suggests exploring the post-β2AR proteins and kinases may have therapeutic potentials of attenuating the occurrence of PCH. Furthermore, a negative correlation exists between ACs and GRKs in healthy and failing cardiac and immune systems (Adzika GK et al., 2019). Therefore, this study focuses on ACs and G protein-coupled receptor kinases (GRKs) to exploit their possible therapeutic potentials.
ACs in generally synthesize cAMP. AC isoform AC7 is expressed explicitly in immune cells. It induces anti-inflammatory response via increase cAMP synthesis.2 While expression of the isoforms AC5 and AC6 are cardiac specific (Li Y et al.,2012). cAMP is essential for maintaining homeostasis in the immune system and proper cardiac function of the heart. Besides the activation of protein kinase A (PKA) in cardiomyocytes and immune cells, cAMP adaptively modulates the activities nuclear factor of activated T-cells (NFATs) and NF-kB (Kipanyula MJ et al, 2013; Pereira L et al., 2015; Murphy JG et al., 2019; Gerlo S et al., 2011). However, during CCS, the ACs are uncoupled from Gαs, terminating the synthesis of cAMP and abolishing its modulation of proper cardiac and immune functions.
GRKs modulates the expression of β2AR. GRK2 isoform mainly desensitizes and internalizes β2AR during catecholamine stress. Conversely, GRK5 can translocate from the plasma membrane into the nucleus. As such, its upregulation facilitates its entry into the nucleus to maladaptively phosphorylate transcription factors (TFs) that elicits adverse immune responses and pathological cardiac remodeling (Fig. 1) (Patial S et al., 2011; Gravning J et al., 2013; Hullmann JE et al., 2014). GRK5 blockade impedes the non-canonically activation of immune and cardiac transcription factors, NFATs, myocyte enhancer factor 2(MEF2), GATA4, Csx/Nkx2–5, and NF-kB. Thus, GRK5 inhibition attenuates cardiac hypertrophy and, to some extent, maladaptive immune response during stress (Hullmann JE et al., 2014; Quan MY et al., 2019).
Nonetheless, there is no evidence that neither cardiac function was improved nor attenuation of hyperactive immune responses when GRK5 was knockout or inhibited with amlexanox (ALX) in an attempt to prevent PCH during CCS. Also, besides the known roles AC5, AC6 and AC7, there have been no attempts to directly stimulate their activities by using Forskolin (FSK) to adaptively modulate the immune response and cardiac function to prevent PCH.
Herein, we show that the combination therapy of ALX and FSK can maintain proper cardiac function while sustaining immune homeostasis. Our data from preliminary in vitro experiments revealed that the combination treatment of peritoneal macrophages (PMɸ) with ALX and FSK after being challenged with Isoproterenol (ISO) and LPS, attenuated induction hyperactive proinflammatory responses. However, neither single treatments with ALX nor FSK were able to attain this as much. These were translated in vivo to curb inflammatory responses from exacerbating cardiomyocyte necrosis and adversely remodeling the heart into PCH during CCS.