n.s., not significant by one-way analysis of variance; *, p<0.05 versus sham group; ✝, p<0.05 versus the TAC 2d Veh group. We also examined RV hypertrophic signaling pathways at this early stage of LV disease, including ERK and calcineurin; the former known to contribute to concentric hypertrophy and the latter to pathologic remodeling [16–18]. For calcineurin activity, we assessed RCAN1 (regulator of calcineurin 1) gene expression levels. RCAN1 is extremely responsive to changes in calcineurin activity in vivo [19] and thus its expression levels have been used as reflecting calcineurin activity [20,21]. ERK was significantly phosphorylated in the RV as well as in the LV (Fig 4A and 4B), and calcineurin activity was also increased in the RV similarly to the LV (Fig 4C). Sildenafil treatment significantly inhibited both these signals in the RV and the LV (Fig 4). These results suggest that the RV undergoes early hypertrophy molecular changes similar to the LV in the absence of RV afterload increase at the very early stage of LV pressure-overload, and that sildenafil treatment inhibits such molecular remodeling process in both ventricles. Quantification results of phosphor/total ratio (p/t ratio) normalized to sham controls are shown in the bar graphs on the right. TAC induced robust phosphorylation of ERK1/2 in RV as well as LV myocardium, and sildenafil prevented this activation in the RV. RCAN1 mRNA expression was increased by TAC in both ventricles, and suppressed by sildenafil.
n.s., not significant by one-way analysis of variance; *, p<0.05 versus sham group; ✝, p<0.05 versus the TAC 2d Veh group. We also examined RV hypertrophic signaling pathways at this early stage of LV disease, including ERK and calcineurin; the former known to contribute to concentric hypertrophy and the latter to pathologic remodeling [16–18]. For calcineurin activity, we assessed RCAN1 (regulator of calcineurin 1) gene expression levels. RCAN1 is extremely responsive to changes in calcineurin activity in vivo [19] and thus its expression levels have been used as reflecting calcineurin activity [20,21]. ERK was significantly phosphorylated in the RV as well as in the LV (Fig 4A and 4B), and calcineurin activity was also increased in the RV similarly to the LV (Fig 4C).
Sildenafil treatment significantly inhibited both these signals in the RV and the LV (Fig 4). These results suggest that the RV undergoes early hypertrophy molecular changes similar to the LV in the absence of RV afterload increase at the very early stage of LV pressure-overload, and that sildenafil treatment inhibits such molecular remodeling process in both ventricles. Quantification results of phosphor/total ratio (p/t ratio) normalized to sham controls are shown in the bar graphs on the right. TAC induced robust phosphorylation of ERK1/2 in RV as well as LV myocardium, and sildenafil prevented this activation in the RV. RCAN1 mRNA expression was increased by TAC in both ventricles, and suppressed by sildenafil.
As inflammation marker genes were up-regulated in both ventricles at this early stage, and were prevented by sildenafil, we further performed an immunohistochemical study and assessed macrophage infiltration in the RV and the LV. We found that F4/80 positive cells were significantly increased in both ventricles of 2day-TAC hearts and that sildenafil significantly inhibited the increase in both ventricles (Fig 5). (A-C) Myocardium stained for F4/80+ cells in the RV and the LV of the Sham mouse (A), the TAC-2d-Veh mouse (B), and the TAC-2d-Sil mouse (C). (D) The number of F4/80+ cells per high-power field. Transverse aortic constriction for two days induced F4/80+ macrophage infiltration into myocardium not only in the LV but also in the RV, which was suppressed by sildenafil. As inflammation marker genes were up-regulated in both ventricles at this early stage, and were prevented by sildenafil, we further performed an immunohistochemical study and assessed macrophage infiltration in the RV and the LV.
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We found that F4/80 positive cells were significantly increased in both ventricles of 2day-TAC hearts and that sildenafil significantly inhibited the increase in both ventricles (Fig 5). (A-C) Myocardium stained for F4/80+ cells in the RV and the LV of the Sham mouse (A), the TAC-2d-Veh mouse (B), and the TAC-2d-Sil mouse (C). (D) The number of F4/80+ cells per high-power field. Transverse aortic constriction for two days induced F4/80+ macrophage infiltration into myocardium not only in the LV but also in the RV, which was suppressed by sildenafil. Studies have documented anti-inflammatory properties of sildenafil [12,22–24], which might be potentially linked to Gq-signal de-activation. We next tested if inflammation might underlie the activation of pathological molecular signaling pathways in the RV during LV pressure-overload. Similar to sildenafil treatment, dexamethasone treatment (20mg/kg/day, intraperitoneally) in two day-TAC hearts inhibited the induction of inflammatory maker genes in the RV as well as in the LV (Fig 6A) and also prevented calcineurin activation and BNP up-regulation(Fig 6B). These results support the potential role for inflammation in this process. (A) mRNA expression of IL1b and IL6, normalized to GAPDH. Dexamethasone inhibited overexpression of IL1b in both RV and LV myocardium induced by transverse aortic constriction (TAC) for 2 days. (B) mRNA expression of RCAN1 and BNP normalized to GAPDH. Dexamethasone suppressed up-regulation of RCAN1 and BNP in RV and LV myocardium induced by two-day TAC.
Using a short-term LV pressure-overload (TAC) model which presents early stage compensated LV hypertrophy without compromised RV hemodynamics, we found that pathological molecular signaling pathways were activated in the RV free wall myocardium, which might be mediated by inflammation. Interventricular interaction has been reported, but in the opposite pathological settings [38,39]. reported that LV expression levels of pyruvate dehydrogenase-4 (PDK4) and B-MHC dynamically change during RV hypertrophy development induced by chronic hypoxia [38]. observed up-regulation of endothelin-1 mRNA in the LV with impaired LV function also in a rat PAH model induced by monocrotaline [39]. However, it is likely that these LV abnormalities might be directly caused by hypoxia or monocrotaline [40,41].
The present data is the first demonstration that RV-LV interventricular interaction occurs in the absence of direct hemodynamic impacts on the RV, and that this might be mediated by inflammatory process. We observed that sildenafil inhibited BNP but not B-MHC expression in both ventricles, though both BNP and B-MHC are hallmark fetal genes that are reactivated in pathological hypertrophy and heart failure. This suggests that their activation mechanisms are under different regulations, consistent with the observation by Kong et al.[42]. De-activation of ERK in both ventricles by sildenafil, in particular, might contribute to the former, given that ERK signaling activation has been well-demonstrated to induce BNP expression by acting on the BNP promoter directly or indirectly through increased GATA4 binding activity [43]. B-MHC up-regulation might occur despite deactivation of both ERK and calcineurin given that neither GATA4 (downstream of ERK) nor NFAT3 (downstream of calcineurin) is involved in direct B-MHC gene regulation [44]. TAC 2d Veh, TAC for 2 days with vehicle treatment; TAC 2d DXM, TAC for 2 days with dexamethasone treatment. Recent meta-analyses revealed that PDE5 inhibitors improve pulmonary hemodynamics and clinical outcomes in systolic heart failure patients with pulmonary hypertension [25,26]. The present study demonstrates that RV molecular alterations occur very early during LV pressure-overload before RV systolic pressure increases and that these molecular derangement in the RV are inhibited by sildenafil through mechanisms potentially involving anti-inflammation. It is therefore tempting to speculate that earlier intervention with PDE5 inhibitors might confer additional clinical benefits in this pathology. cGMP-PKG (cGMP-dependent protein kinase) activation by PDE5 inhibition not only induces pulmonary vasodilation but also has direct beneficial impacts on the heart through multiple mechanisms. We and others have demonstrated that cGMP-activated PKG binds to Regulators of G protein Signaling (RGS) 2 and 4 to their activation, deactivating Gq-related signaling in LV myocardium [8]. have demonstrated that PDE5 inhibition with Tadalafil improves mitochondrial energy metabolism and sildenafil for women LV cardiac function [11]. More recently, anti-inflammatory properties of sildenafil were reported. Sildenafil treatment is associated with reduced circulating cytokines in patients with diabetes [12] or erectile dysfunction [22]. Sildenafil reduces cardiac and renal inflammation in a mouse model of type I diabetes [23], and in a mouse model of neuro-inflammation [24]. Our data demonstrate such anti-inflammatory properties of sildenafil as potential key contributor to ameliorating early pathological molecular derangement in the RV during LV pressure-overload. The anti-inflammatory effect of sildenafil has been demonstrated in pathological conditions of diabetes [23,27], kidney diseases [28] and neuronal disorders [29]. In particular, intensive studies have been performed in diabetes.
More recently, anti-inflammatory properties of sildenafil were reported. Sildenafil treatment is associated with reduced circulating cytokines in patients with diabetes [12] or erectile dysfunction [22]. Sildenafil reduces cardiac and renal inflammation in a mouse model of type I diabetes [23], and in a mouse model of neuro-inflammation [24]. Our data demonstrate such anti-inflammatory properties of sildenafil as potential key contributor to ameliorating early pathological molecular derangement in the RV during LV pressure-overload. The anti-inflammatory effect of sildenafil has been demonstrated in pathological conditions of diabetes [23,27], kidney diseases [28] and neuronal disorders [29].
In particular, intensive studies have been performed in diabetes. Given that endothelial cells play a crucial role by releasing inflammatory mediators, it is reasonable to speculate that reduction of vascular inflammation by sildenafil might be a significant contributor to ameliorating early molecular derangement in both ventricles in the current study, besides its direct cardiac Gq-inhibitory effects from cGMP-PKG signaling. Importantly, dexamethasone, a corticosteroid, virtually normalized early molecular derangement in both ventricles, supporting the key role for inflammation that could involve cardiac myocytes, vasculature, and adipose tissue. Long-term effects of dexamethasone, however, could be different from those of sildenafil [33], considering the former inhibits both protective or detrimental aspects of inflammation. The anti-inflammatory effects might be coupled to the Gq regulatory action by cGMP-PKG, given that Gq activation is closely linked to inflammation.
For example, lack of Gq-inhibitory protein RGS3 in mice reveals exacerbated inflammation in a mouse model of asthma [34]. Also, in a mouse model of chronic kidney disease, RGS2 deficiency results in enhanced fibrogenic and inflammatory response [35]. The precise molecular mechanisms linking Gq signal activation to inflammation and their regulation by cGMP-PKG, however, warrants further investigation. Signaling pathways for cardiac hypertrophy and failure have been intensively investigated and clarified in LV myocardium or LV cardiac myocytes [17,18]; however, there is limited data available with regard to whether and how similar molecular pathways are at work to contribute to RV pathophysiology [36]. Thus far, most studies have utilized rodent models of pulmonary artery hypertension (PAH) or pulmonary artery banding in order to answer this question and have demonstrated several aspects of distinct RV remodeling in response to afterload stress [37]; however, little has been determined about the RV remodeling process that occur due to LV diseases. Given that endothelial cells play a crucial role by releasing inflammatory mediators, it is reasonable to speculate that reduction of vascular inflammation by sildenafil might be a significant contributor to ameliorating early molecular derangement in both ventricles in the current study, besides its direct cardiac Gq-inhibitory effects from cGMP-PKG signaling.
B-MHC gene expression is induced by complex effects of transcription factors, including SRF and MEF2 under control of HDACs (histone deacetylases), and PKC-activated TEF1 [44]. In addition, MHC isoform switch is also coordinated by microRNA 208, encoded within an intron of A-MHC gene [44,45]. Our results suggest that sildenafil might not inhibit PKC or signaling pathways coupled to HDAC regulation in both ventricles at this early stage of the LV disease. In conclusion, we provide the evidence that RV pathological molecular abnormalities associated with LV disease are initiated early even when the LV disease is still at the early stages, and demonstrate that the PDE5 inhibitor sildenafil has potent effects of ameliorating such molecular abnormalities in both ventricles potentially through the anti-inflammatory effects. The study provides a novel insight into our understanding of the RV pathophysiology associated with LV diseases. Importantly, dexamethasone, a corticosteroid, virtually normalized early molecular derangement in both ventricles, supporting the key role for inflammation that could involve cardiac myocytes, vasculature, and adipose tissue. Long-term effects of dexamethasone, however, could be different from those of sildenafil [33], considering the former inhibits both protective or detrimental aspects of inflammation. The anti-inflammatory effects might be coupled to the Gq regulatory action by cGMP-PKG, given that Gq activation is closely linked to inflammation. For example, lack of Gq-inhibitory protein RGS3 in mice reveals exacerbated inflammation in a mouse model of asthma [34]. Also, in a mouse model of chronic kidney disease, RGS2 deficiency results in enhanced fibrogenic and inflammatory response [35]. The precise molecular mechanisms linking Gq signal activation to inflammation and their regulation by cGMP-PKG, however, warrants further investigation.
| Parameter | Description | Typical Value |
|---|---|---|
| Bioavailability | Percent absorbed into bloodstream | ~40% |
| Tmax (time to peak) | Time to reach maximum plasma concentration | 30-120 minutes |
| Half-life | Duration of drug activity | 4-5 hours |
| Metabolism | Main route | Liver (CYP3A4 enzyme) |
| Excretion | How the drug leaves the body | Mainly feces, some urine |
Signaling pathways for cardiac hypertrophy and failure have been intensively investigated and clarified in LV myocardium or LV cardiac myocytes [17,18]; however, there is limited data available with regard to whether and how similar molecular pathways are at work to contribute to RV pathophysiology [36]. Thus far, most studies have utilized rodent models of pulmonary artery hypertension (PAH) or pulmonary artery banding in order to answer this question and have demonstrated several aspects of distinct RV remodeling in response to afterload stress [37]; however, little has been determined about the RV remodeling process that occur due to LV diseases.
Studies have documented anti-inflammatory properties of sildenafil [12,22–24], which might be potentially linked to Gq-signal de-activation. We next tested if inflammation might underlie the activation of pathological molecular signaling pathways in the RV during LV pressure-overload. Similar to sildenafil treatment, dexamethasone treatment (20mg/kg/day, intraperitoneally) in two day-TAC hearts inhibited the induction of inflammatory maker genes in the RV as well as in the LV (Fig 6A) and also prevented calcineurin activation and BNP up-regulation(Fig 6B). These results support the potential role for inflammation in this process. (A) mRNA expression of IL1b and IL6, normalized to GAPDH.
Dexamethasone inhibited overexpression of IL1b in both RV and LV myocardium induced by transverse aortic constriction (TAC) for 2 days. (B) mRNA expression of RCAN1 and BNP normalized to GAPDH. Dexamethasone suppressed up-regulation of RCAN1 and BNP in RV and LV myocardium induced by two-day TAC. TAC 2d Veh, TAC for 2 days with vehicle treatment; TAC 2d DXM, TAC for 2 days with dexamethasone treatment. Recent meta-analyses revealed that PDE5 inhibitors improve pulmonary hemodynamics and clinical outcomes in systolic heart failure patients with pulmonary hypertension [25,26].
The present study demonstrates that RV molecular alterations occur very early during LV pressure-overload before RV systolic pressure increases and that these molecular derangement in the RV are inhibited by sildenafil through mechanisms potentially involving anti-inflammation. It is therefore tempting to speculate that earlier intervention with PDE5 inhibitors might confer additional clinical benefits in this pathology. cGMP-PKG (cGMP-dependent protein kinase) activation by PDE5 inhibition not only induces pulmonary vasodilation but also has direct beneficial impacts on the heart through multiple mechanisms. We and others have demonstrated that cGMP-activated PKG binds to Regulators of G protein Signaling (RGS) 2 and 4 to their activation, deactivating Gq-related signaling in LV myocardium [8]. have demonstrated that PDE5 inhibition with Tadalafil improves mitochondrial energy metabolism and sildenafil for women LV cardiac function [11]. Using a short-term LV pressure-overload (TAC) model which presents early stage compensated LV hypertrophy without compromised RV hemodynamics, we found that pathological molecular signaling pathways were activated in the RV free wall myocardium, which might be mediated by inflammation.
Interventricular interaction has been reported, but in the opposite pathological settings [38,39]. reported that LV expression levels of pyruvate dehydrogenase-4 (PDK4) and B-MHC dynamically change during RV hypertrophy development induced by chronic hypoxia [38]. observed up-regulation of endothelin-1 mRNA in the LV with impaired LV function also in a rat PAH model induced by monocrotaline [39]. However, it is likely that these LV abnormalities might be directly caused by hypoxia or monocrotaline [40,41]. The present data is the first demonstration that RV-LV interventricular interaction occurs in the absence of direct hemodynamic impacts on the RV, and that this might be mediated by inflammatory process. We observed that sildenafil inhibited BNP but not B-MHC expression in both ventricles, though both BNP and B-MHC are hallmark fetal genes that are reactivated in pathological hypertrophy and heart failure. This suggests that their activation mechanisms are under different regulations, consistent with the observation by Kong et al.[42]. De-activation of ERK in both ventricles by sildenafil, in particular, might contribute to the former, given that ERK signaling activation has been well-demonstrated to induce BNP expression by acting on the BNP promoter directly or indirectly through increased GATA4 binding activity [43]. B-MHC up-regulation might occur despite deactivation of both ERK and calcineurin given that neither GATA4 (downstream of ERK) nor NFAT3 (downstream of calcineurin) is involved in direct B-MHC gene regulation [44]. B-MHC gene expression is induced by complex effects of transcription factors, including SRF and MEF2 under control of HDACs (histone deacetylases), and PKC-activated TEF1 [44]. In addition, MHC isoform switch is also coordinated by microRNA 208, encoded within an intron of A-MHC gene [44,45].
Our results suggest that sildenafil might not inhibit PKC or signaling pathways coupled to HDAC regulation in both ventricles at this early stage of the LV disease. In conclusion, we provide the evidence that RV pathological molecular abnormalities associated with LV disease are initiated early even when the LV disease is still at the early stages, and demonstrate that the PDE5 inhibitor sildenafil has potent effects of ameliorating such molecular abnormalities in both ventricles potentially through the anti-inflammatory effects. The study provides a novel insight into our understanding of the RV pathophysiology associated with LV diseases.