This study investigated the effects of chronic systemic inflammation, in the absence of metabolic confounders, on expression of genes involved in LV microvascular endothelial activation, NO-sGC-cGMP signaling and titin dephosphorylation, implicated in LV diastolic dysfunction. We show that collagen inoculation increased LV expression of VCAM1, suggesting endothelial activation. Furthermore, we show increased LV expression of PTX3 and iNOS indicating increased local tissue inflammation and leukocyte invasion. Supporting the role of decreased NO-dependent signaling in development of LV diastolic dysfunction, collagen inoculation decreased the expression sGCα2 and sGCβ2, which code for isoforms of the sGC enzyme that initiates NO-stimulated cGMP signaling. The increased expression of VCAM1, PTX3 and iNOS, together with decreased expression of sGC were prevented by treatment with a TNF-α inhibitor. Taken together, these results show that increased circulating TNF-α stimulates vascular inflammation, increases LV tissue inflammation and disrupts NO-sGC-cGMP signaling, which may modulate cardiomyocyte elasticity, likely by phosphorylating the titin N2-Bus. However, the collagen-induced LV diastolic dysfunction is not reversed by blocking circulating TNF-α , suggesting that TNF-α and hence, decreased NO-sGC-cGMP signaling is likely not the primary mechanism of inflammation-induced LV diastolic dysfunction. Collagen inoculation had no effect on expression of PP1γ, but it significantly increased expression of PP5, a major catalyst of titin dephosphorylation . Like the inflammation-induced LV diastolic dysfunction, the inflammation-induced up-regulation of PP5 expression was not reversed by inhibiting circulating TNF-α. These results show that increased expression of PP5 may increase cardiomyocyte stiffness by decreasing the net phosphorylation level of the N2-Bus stretch element of titin, which may partially mediate chronic systemic inflammation-induced LV diastolic dysfunction.
A recent paradigm suggested that in response to increased circulating inflammatory cytokines, microvascular inflammation drives altered myocardial signaling that ultimately results in hypophosphorylation of the titin N2-Bus and consequently decreased myocyte compliance . The paradigm was supported by an obese diabetic rat model of metabolic risk-associated systemic inflammation, which showed evidence of microvascular inflammation and disruption of NO signaling . Our model of collagen-induced systemic inflammation, which is independent of metabolic confounders, showed upregulated LV mRNA expression of VCAM1. VCAM1 is a glycoprotein that is expressed predominantly on the surfaces of endothelial cells and is activated by pro-inflammatory cytokines [10, 28]. Microvascular inflammation resulting from increased expression of cell adhesion molecules, including VCAM1, promotes leukocyte transmigration [29, 30] and enhances ROS production, which are characteristic of an oxidative stress environment [31,32,33]. Supporting this, we previously showed that collagen inoculation increased CD68 expression in LV tissues, showing infiltration of leukocytes . In the present study, PTX3 and iNOS expression was increased in CIA rats, further indicating leukocyte infiltration in the heart tissues and hence local inflammation. PTX3 is an acute-phase protein that is structurally similar to CRP , and is produced by inflammatory cells, such as monocytes and neutrophils, but it is also produced locally by vascular endothelial cells at the sites of inflammation . Leukocytes that express iNOS are known for their production of ROS, which disrupt NO bioavailability by facilitating dissociation of endothelial NOS dimers [36, 37] and by reacting with NO to produce peroxynitrite . Thus, microvascular inflammation causes the transmigration of immune cells that increase local production of ROS, which interfere with NO–sGC signaling by reducing NO bioavailability .
NO diffuses into cardiomyocytes, where it activates sGC to produce cGMP. cGMP, in turn, activates PKG, one of the kinases that decrease sarcomere tension by phosphorylating the titin N2-Bus region. Whereas decreased local NO concentrations would result in decreased NO-sGC-cGMP signaling, it has been reported that ROS also decrease the responsiveness of the NO-sGC-cGMP system to exogenous NO donors, suggesting that decreased activity or expression of sGC may further decrease cGMP signaling . ROS have been shown to disrupt cGMP production by oxidizing the heme required for NO to bind to sGC and by decreasing protein and mRNA expression of sGC subunits [7, 11, 40]. We have shown that systemic inflammation decreased mRNA expression of both the sGCα2 and sGCβ2 genes. This suggests that the decreased sGCα2 and sGCβ2 expression induced by collagen inoculation is likely mediated by increased ROS production and that the decreased expression will result in decreased NO-sGC-cGMP signaling .
In the current study, blocking circulating TNF-α prevented the inflammation-induced upregulation of VCAM1, PTX3 and iNOS and therefore arguably inhibited pathological ROS production. Furthermore, blocking TNF-α also prevented the inflammation-induced downregulation of mRNA expression of sGC genes. Taken together, our results suggest that increased circulating TNF-α increases LV microvascular endothelial activation, which increases ROS production by endothelial cells and via immune cell transmigration. The resultant increased ROS decrease sGC expression and likely also decrease its activity [7, 40]. This will result in decreased cGMP signaling and hence decreased PKG phosphorylation of the titin N2-Bus region. However, despite the impact of TNF-α blockade on microvascular inflammation and NO-dependent cGMP signaling, we have shown that LV diastolic dysfunction induced by collagen inoculation was not reversed by blocking circulating TNF-α . This indicates that TNF-α and hence, decreased cGMP-stimulated phosphorylation of titin, may not be the primary mechanisms of LV diastolic dysfunction induced by systemic inflammation. In this regard, it is important to consider that kinases other than NO-dependent PKG phosphorylate the N2-Bus region of titin  and that the net phosphorylation state of titin is also regulated by the phosphatases that dephosphorylate the residues phosphorylated by the kinases, increasing titin stiffness . Like protein kinases, protein phosphatases are also regulated by systemic and intracellular signaling pathways .
Phosphorylation and dephosphorylation of titin are part of normal cardiomyocyte contraction. Increased expression of protein phosphatases causes decreased net phosphorylation of cardiac proteins . It has been suggested that increased expression of protein phosphatases PP1 and PP2A may contribute to the hypophosphorylation of titin, characteristic of LV diastolic dysfunction . In the present study, we showed that the relative mRNA expression of PP1γ, which codes for a major eukaryotic protein serine/threonine phosphatase that regulates a variety of cellular functions [15, 45, 46], was not altered in response to systemic inflammation or TNF-α inhibitor treatment. This is in contrast to a previous study that reported increased PP1 protein expression in rats that developed LV diastolic dysfunction in a model of obesity-associated inflammation . Considering the well-known impact of metabolic signals on the regulation of PP1 expression [15, 47], the increased PP1 expression in the prior study may be explained by the metabolic dysfunction likely to result from obesity . Therefore, the unchanged expression of PP1γ in our model of systemic inflammation independent of metabolic dysfunction suggests that PP1 expression may not be directly regulated by chronic systemic inflammation.
The N2-Bus region of titin was recently found to also be dephosphorylated by PP5 . PP5 is ubiquitously expressed in eukaryotic cells and its activity is enhanced by ROS [16, 44]. PP5 expression was shown to be increased in tissue from end-stage failing human hearts and from dogs with LV diastolic dysfunction . However, the stimulus for the increased PP5 expression in LV diastolic dysfunction and heart failure was not explored. In the current study we showed that systemic inflammation, independent of metabolic dysfunction, increased relative mRNA expression of PP5 in collagen-inoculated rats with LV diastolic dysfunction. Our results indicate that systemic inflammation-induced LV diastolic dysfunction may, at least in part, be mediated by increased PP5 expression and dephosphorylation of titin. However, other phosphoprotein phosphatases such as PP2C  and PP2A , that were not measured, may also be involved in titin phosphorylation. Furthermore, molecular pathways independent of titin compliance including myocardial fibrosis and calcium handling may contribute to LV diastolic dysfunction in this study [24, 42].
Importantly, in contrast to the NO-sGC-cGMP pathway, the inflammation-induced upregulation of PP5 expression was not reversed by blocking circulating TNF-α. This suggests that the local PP5 expression may be regulated by a circulating inflammatory signal other than TNF-α. Although PP5 expression is known to be regulated by several intracellular signals [16, 17], the extracellular signals that stimulate these pathways to PP5 expression and activity require further investigation. Since both the upregulation of PP5 expression and the accompanying LV diastolic dysfunction are not mediated by TNF-α, our results suggest that LV diastolic dysfunction may, at least in part, result from increased expression of PP5, which increases cardiomyocyte stiffness by decreasing the net phosphorylation level of the N2-Bus stretch element of titin, whereas disruption of the NO-sGC-cGMP pathway is likely less important.
This study has some limitations. The inclusion of both male and female rats may have reduced statistical power. The use of mRNA expression may not reflect protein expression and function, hence direct measures of NO, ROS and titin phosphorylation may have strengthened the interpretations of this study, and should be included in future studies. Although etanercept has been successfully used in previous animal models , it is a human fusion protein, which, itself, may elicit an immunogenic response in rats.