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).