Discussion
In this study, we demonstrated that, although β2AR is crucial for mediating stimuli in cardiac and immune cells for their proper functioning during CCS (Kim MH et al., 2014), its effector Gαs – ACs and receptor kinase – GRKs expressions are altered under extreme stress. AC5 and AC6 expressed in cardiomyocytes exhibited a negative correlation under stress. While AC5 was being depleted in a stress intensity-dependent manner, AC6 was being upregulated in the same manner. This also goes to prove that AC6 mainly handles stress besides calcium channel modulation (Wu YS et al., 2017; Tang T et al., 2008). Like AC5, the immune cells-specific isoform AC7 was also downregulated in PCH mice. The decrease in the expression of AC5 and AC7 in PCH mice decrease their cAMP concentration level. This implied a decrease in cardiac function and the abolishment of cAMP-dependent anti-inflammatory effects on immune cells (Paur H et al., 2012; Raker VK et al., 2016). The downregulation of cAMP also terminated its adaptive regulation of the TFs; NFAT, MEF2, and NF-κB in both immune cells and cardiomyocytes (Kipanyula MJ et al, 2013; Pereira L et al., 2015; Murphy JG et al., 2019; Gerlo S et al., 2011; Raker VK et al., 2016).
GRKs are observed to be fairly expressed in Ctrl and Vhl mice but, they are overexpressed in PCH mice to phosphorylate, desensitize and down-regulate hypersensitize βARs. GRK2 typically phosphorylated βARs to desensitize the receptor (Premont RT et al., 2007). Meanwhile, mounting evidence has shown that upregulated GRK5 facilitates a non-canonical GPCR-independent stimulus signaling that progresses adverse cardiac hypertrophy. As previously reported, besides GRK5 upregulation, we also found the overexpression of ANP, BNP and ERK1/2, and TFs: GATA4, NFAT, MEF2, and NF-κB in the PCH mice. The ability of GRK5 to translocate from the cytosol into the nuclei enables it to directly induce nuclear activities by phosphorylating the inflammatory and myocyte TFs (Hullmann JE et al., 2014; Martini JS et al., 2008; Islam KN et al., 2013; Sorriento D et al., 2018). Therefore, GRK5 induces both excessive myocyte hypertrophy and necrosis, and proinflammatory response.
To set the basis for our latter translational in vivo models, we first investigated and compared the immune responses elicited by DAMPs from necrotic cardiomyocytes and LPS, both under chronic stress. The excessive cardiomyocytes necrosis occurring in the hearts of PCH mice induced increased secretions of proinflammatory cytokines, IL-1β, IL-6, TNFα, and IFNγ, while the anti-inflammatory cytokine IL-10 was dampened (Fig. 2a). Although we are not the first to demonstrate the hyperactive inflammatory responses of immune cells to either cDAMP or LPS during CCS (Laukova M et al., 2018; Zimmer A et al., 2019), we are to first to profile and compare the two to show the similarity in their proinflammatory and anti-inflammatory cytokine expressions (Fig. 2b). It could be argued that LPS still induces a proinflammatory response from the macrophages in the absence of CCS. Nonetheless, our obtained data, as well as other researchers, have suggested that the proinflammatory responses induced by LPS are heightened during stress than at the physiological state (Laukova M et al., 2018; Liu YZ et al., 2017; Maydych, V, 2019).
Interestingly, hyperactivation and modulation of the inflammatory response by necrotic cardiomyocytes and LPS are mediated by GRK5 (Patial S et al., 2011; Packiriswamy N et al., 2015). This implied inhibiting GRK may exert an anti-inflammatory effect. As such, we hypothesized that inhibiting GRK5 in PMɸ while directly stimulating ACs to synthesis cAMP to adaptively regulate inflammatory TFs may attenuate the hyperactive response of PMɸ to LPS during chronic stress.
Herein, we explored the inhibitory effects of Amlexanox on GRK5. Also, we utilized Forskolin to stimulate AC-cAMP synthesis, independent of βARs-Gαs directly. Both were done in attempts to halt the maladaptive inflammatory response of PMɸ to LPS during CCS. The data obtained from this in vitro experiment supported our earlier hypothesis, along with some unexpected outcomes. ALX single treatment of the LPS-challenged stressed PMɸ was unable to effectively attenuated its hyper proinflammatory response. However, the dosage used in the study was within the range that had been reported earlier to halt inflammatory response.18 Comparatively, FSK single treatment performed better than the ALX. Meanwhile, treatment of the LPS-challenged stressed PMɸ with the combination of ALX and FSK successfully attenuated excessive secretion of proinflammatory cytokines; IL-1β, IL-6, and TNFα while, anti-inflammatory IL-10 was secreted in abundance.
By using immunofluorescence to ascertain the locations of GRK5 in PMɸ across all the groups, we explored the possible mode of actions that resulted in the combination therapy being the most potent treatment. We demonstrated in (Fig. 4) that the ALX single therapy inhibited GRK5 expression and prevented it from translocating into the nuclei. Implying that the maladaptive inflammatory responses which still resulted might be due to cancellation of the cAMP-dependent modulation of adaptive inflammatory responses during CCS, just as suggested (Raker VK et al., 2016; Wehbi VL et al., 2016; Bopp T et al., 2009). As shown here (Fig. 4), although GRK5 still translocated into the nuclei of PMɸ-LPS-CCS after treatment with FSK as much as it did without any therapies, inflammatory responses were not aggravated as it did with both ALX single therapy and no therapy groups. Therefore, it is suggested that the combination therapy of ALX and FSK attained its potency mostly via FSK – ACs – cAMP-mediated immunoregulation, coupled with ALX inhibiting GRK5-mediated immune response activation.
These intriguing outcomes led us to hypothesize that rather than the single therapies of either FSK or ALX, the combination therapy of ALX and FSK, if translated in vivo, may attenuate maladaptive inflammatory response occurring during chronic CCS which drives the adverse remodeling of hearts into pathological cardiac hypertrophy.
We translated the treatments of ALX and FSK in vivo, and at the end of all models, echocardiogram results revealed that the combination of ALX and FSK had effectively preserved cardiac function during CCS with ejection fractions above 65% and fraction shortenings above 35% (Fig. 5b, 5c and Supplementary Fig. S4). Again, ALX alone failed to maintain proper function, and although FSK tried to some extent, the echocardiogram of mice treated with only FSK showed all forms of arrhythmias. Protein analysis from apical myocardium was done to elucidate the probable mechanism used by combination therapy to preserve cardiac function during CCS. In summary, we found that even though ALX single therapy successfully inhibited GRK5, cardiac hypertrophy and inflammatory TFs, GATA4, NFAT, MEF2, and NF-κB were still overexpressed during CCS (Fig. 7). This phenomenon also goes to prove Hullmann et al.’s suggestion in 2014, that the inhibition of GRK5 would not halt the activation of NFAT It is also logical then to speculate that the inhibition of GRK5 might not prevent the activation of GATA4, MEF2, and NF-κB in vivo, based on our results and the fact that their upregulations and activations interactions with one another. Even so, FSK single therapy decreased the expression of these TFs by upregulating the expression of cAMP (Fig. 6b).
PCH mice had dilated left and right heart chambers, excessive cardiomyocyte hypertrophy, and marked deposits of collagen. On the contrary, cardiomyocyte hypertrophy was attenuated by the single treatment with ALX during CCS, although GATA4, NFAT, MEF2, and NF-κB were upregulated. Many others have reported ALX’s ability to attenuated myocyte hypertrophy during pressure-over in vitro (Lieu M et al., 2019; Homan KT et al., 2014). However, we are the first to demonstrated that even though single treatment with ALX attenuated cardiac hypertrophy in vivo, neither does it prevent massive collagen deposits nor preserve proper cardiac function during CCS. Also, we demonstrated that FSK single therapy was unable to prevent cardiomyocyte hypertrophy and collagen deposits in the myocardium during CCS, although it did not perform as bad as ALX in the latter. We also set the pace to illustrate that the combination of ALX and FSK, has the therapeutic potential to prevent maladaptive cardiomyocyte hypertrophy and massive interstitial collagen deposits during CCS.
Furthermore, by utilizing CD68 as a biomarker to ascertain the rate of infiltration of macrophages and other mononuclear cells into the myocardium during CCS, we showed that PCH mice had enormous immune cells infiltrating their myocardia in response to myocyte necrosis. Compared to the single therapies of ALX and FSK, their combined therapy was most effective in minimizing the mononuclear cell infiltrations (Fig. 9a). This indicates that the combined therapy prevented of myocyte necrosis and subdued adverse inflammatory responses.
Also, inflammatory cytokine assessment also confirmed that although single treatments with ALX and FSK failed, their combination kept the gap between proinflammatory and anti-inflammatory responses close to a homeostatic immune state. This prevented biased prolonged proinflammatory responses, which could have exacerbated myocyte necrosis and aggravated interstitial collagen deposits. The efficacy of the combination of ALX and FSK in preventing hyperactive inflammatory response and adverse cardiac remodeling during CCS in vivo may be due to the combined efforts of the anti-inflammatory effects they exert individually (Quan MY et al., 2019; Raker VK et al., 2016).