Increased protein phosphatase 5 expression in inflammation-induced left ventricular dysfunction in rats

Abstract

Background: Titin phosphorylation contributes to left ventricular (LV) diastolic dysfunction. The independent effects of inflammation on the molecular pathways that regulate titin phosphorylation are unclear.

Methods: We investigated the effects of collagen-induced inflammation and subsequent tumor necrosis factor-α (TNF-α) inhibition on mRNA expression of genes involved in regulating titin phosphorylation in 70 Sprague-Dawley rats. LV diastolic function was assessed with echocardiography. Circulating inflammatory markers were quantified by enzyme-linked immunosorbent assay and relative LV gene expression was assessed by Taqman® polymerase chain reaction. Differences in normally distributed variables between the groups were determined by two-way analysis of variance (ANOVA), followed by Tukey post-hoc tests. For non-normally distributed variables, group differences were determined by Kruskal-Wallis tests.

Results: Collagen inoculation increased LV relative mRNA expression of vascular cell adhesion molecule 1 (VCAM1), pentraxin 3 (PTX3), and inducible nitric oxide synthase (iNOS) compared to controls, indicating local microvascular inflammation. Collagen inoculation decreased soluble guanylate cyclase alpha-2 (sGCα2) and soluble guanylate cyclase beta-2 (sGCβ2) expression, suggesting downregulation of nitric oxide-soluble guanylate cyclase-cyclic guanosine monophosphate (NO-sGC-cGMP) signaling. Inhibiting TNF-α prevented collagen-induced changes in VCAM1, iNOS, sGCα2 and sGCβ2 expression. Collagen inoculation increased protein phosphatase 5 (PP5) expression. Like LV diastolic dysfunction, increased PP5 expression was not prevented by TNF-α inhibition.

Conclusion: Inflammation-induced LV diastolic dysfunction may be mediated by a TNF-α-independent increase in PP5 expression and dephosphorylation of the N2-Bus stretch element of titin, rather than by TNF-α-induced downregulation of NO-sGC-cGMP pathway-dependent titin phosphorylation. The steady rise in number of patients with inflammation-induced diastolic dysfunction, coupled with low success rates of current therapies warrants a better understanding of the systemic signals and molecular pathways responsible for decreased titin phosphorylation in development of LV diastolic dysfunction. The therapeutic potential of inhibiting PP5 upregulation in LV diastolic dysfunction requires investigation.

Keywords: Left ventricular diastolic dysfunction; Microvascular inflammation; Protein phosphatase 5; Titin phosphorylation; Tumor necrosis factor-alpha inhibitor.

Fig. 1

LV expression of markers of microvascular inflammation and immune cell activation. Relative mRNA expression of VCAM1 (a), PTX3 (b) and iNOS (c) in left ventricular tissues of control, CIA and CIA + anti-TNFα. Results are expressed in arbitrary units normalized for the expression of the endogenous reference gene, HPRT1, using the ∆∆Ct method. Data are presented as mean ± SEM for VCAM1 and median and interquartile range for PTX3 and iNOS; **p < 0.01 versus control, ***p < 0.001 versus control, ****p < 0.0001 versus control, ^#p < 0.05 versus CIA

Fig. 2

LV expression of sGC subunit genes. Relative mRNA expression of sGCα2 (a) and sGCβ2 (b) in left ventricular tissues of control, CIA, and CIA + anti-TNF-α. Results are expressed in arbitrary units normalized for the expression of the endogenous reference gene, HPRT1, using the ∆∆Ct method. Data expressed as mean ± SEM; *p < 0.05 versus control; ^#p < 0.05 versus CIA

Fig. 3

LV expression of protein phosphatases. Relative mRNA expression of PP1γ (a) and PP5 (b) in left ventricular tissues of control, CIA and CIA + anti-TNF-α. Results are expressed in arbitrary units normalized for the expression of the endogenous reference gene, HPRT1, using the ∆∆Ct method. Data expressed as mean ± SEM; * p < 0.05 versus control group