Keywords

1 Introduction

Pulmonary hypertension is a condition of increased pulmonary vascular pressure. Increased pulmonary vascular resistance directly influences the right ventricle (RV) that pumps the blood to the pulmonary circulation . The heart ventricles are capable of adapting to chronic conditions of increased resistance by elevating the force of muscle contraction by increasing the sarcomere units, which results in thickening of the single cardiomyocytes in the process of concentric cardiac hypertrophy . This compensatory process, however, eventually leads to decompensation if the condition persists. In the case of pulmonary hypertension, this decompensation results in the inability of the RV to sustain a satisfactory muscle contraction, leading to right heart failure. Right heart failure is a major cause of death among patients with various forms of pulmonary hypertension.

Compared with the left ventricle (LV) , understanding of the RV pathophysiology has been delayed [1]. Moreover, the RV and LV can be viewed as fundamentally different entities because these two ventricles are developed from distinct progenitor cells [2]. The RV is expected to possess more plastic properties since it is subjected to a wide range of blood pressure throughout life (a level of systemic blood pressure in utero and much lower pulmonary circulation pressure after birth). Remarkably, patients with Eisenmenger syndrome can live with the RV pumping blood against systemic blood pressure throughout adulthood [3]. Thus, understanding the biology of the RV is scientifically exciting and undoubtedly important for developing right heart-specific therapeutic strategies to manage patients with pulmonary hypertension to increase their survival.

Our bodies are influenced by reactive oxygen species (ROS) and other biological oxidants in a variety of ways. Exogenously generated oxidants as well as those produced in our bodies can harm various biological molecules and biological processes. Further, it appears that biological oxidants can regulate cell signal transduction [4]. In particular, oxidants can promote both the growth and the death of cells. This is also important in regulating the fate of RV cardiomyocytes in patients with pulmonary hypertension. However, neither the exact mechanism of how biological oxidants regulate cell signaling nor the role of oxidant signaling in the RV are well understood. This book chapter compiles information on the mechanisms of redox signaling in the RV , which were mainly obtained through experiments performed in our laboratory.

2 Redox Regulation of Serotonin Signaling in the RV

Serotonin plays an important role in the pathogenesis of pulmonary hypertension by causing vasoconstriction and the growth of pulmonary artery smooth muscle cells [5, 6]. ROS have been reported to serve as second messengers in pulmonary artery smooth muscle cells [7,8,9]. Serotonin has also been reported to produce ROS in the heart [10, 11]. However, these results were obtained in isolated cardiac myocytes obtained from both the RV and LV. Thus, the difference between the RV and LV in terms of serotonin-mediated ROS generation was unknown when Dr. Lingling Liu started to perform her experiments in our laboratory.

Protein carbonylation is an important consequence of ROS-mediated protein oxidation [12]. Dr. Liu’s experiments revealed that perfusing isolated rat hearts with serotonin caused protein carbonylation in the RV, but not in the LV [13]. As shown in Fig. 1, RV and LV homogenates derivatized with 2,4-dinitrophenylhydrazine (DNPH) exhibited multiple carbonylated proteins in SDS-PAGE gels. A 10-min treatment of isolated rat hearts perfused through a modified Langendorff system, in which vena carvas were also tied to perfuse the RV and large pulmonary arteries, resulted in the significantly increased carbonylation of various proteins in the RV (Fig. 1a). By contrast, the difference in the intensity of carbonylated bands between control and serotonin-treated LVs was minimal and not statistically significant (Fig. 1b).

Fig. 1
figure 1

Effects of serotonin (5-HT) on protein carbonylation in the RV and LV. Isolated rat hearts were subjected to retrograde perfusion on a Langendorff apparatus. After equilibration, the solution containing 5-HT was perfused for 10 or 30 min. (a) RV and (b) LV homogenates were derivatized with DNPH to label carbonyl moieties and subjected to SDS-PAGE and immunoblotting with the antibody, which interacts with DNPH-derivatized proteins. Total protein levels were visualized by Coomassie Blue staining. Bar graphs represent means ± SEM (n = 3). (*) denotes values significantly different from each other at P < 0.05 (Reproduced from [13] with permission)

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The effort to identify the mechanism of this RV/LV difference led to our finding that the monoamine oxidase-A level is lower in the RV compared with the LV [13]. This event was confirmed by monitoring monoamine oxidase-A protein expression (Fig. 2a), monoamine oxidase-A enzymatic activity (Fig. 2b) and monoamine oxidase-A mRNA expression (Fig. 2c). Monoamine oxidase-A, uses serotonin as a substrate to produce hydrogen peroxide; thus, ROS produced by monoamine oxidase-A may mediate serotonin signaling. However, from our observations, we proposed that the ability of monoamine oxidase-A to degrade intracellular serotonin, rather than the production of ROS, mediates serotonin-induced protein carbonylation in the RV [13].

Fig. 2
figure 2

Expression and activity of monoamine oxidase A (MAO-A) in the RV and LV . (a) Western blotting of MAO-A protein (61 kDa) and a loading control GAPDH. (b) MAO-A activity was measured fluorometrically with octopamine as a substrate. (c) Total RNA was isolated and MAO-A mRNA and 28S rRNA levels were monitored by RT-PCR. The bar graph represents means ± SEM. (*) denotes values significantly different from each other at P < 0.05 (Reproduced from [13] with permission)

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3 Carbonylation-Degradation Pathway of Signal Transduction in RV Hypertrophy

The GATA4 transcription factor plays a critical role in the development of cardiac hypertrophy [14]. In the LV, GATA4 is activated through post-translational modification mechanisms [15]. We studied the mechanism of GATA4 activation in the RV by using the chronic hypoxia model of pulmonary hypertension and RV hypertrophy in rats [16]. As expected, GATA4 DNA binding activity was increased in response to chronic hypoxia-induced pulmonary hypertension in the RV. Surprisingly, however, this was also associated with the increased gene expression of GATA4. Since the mechanism of Gata4 gene transcription as well as the promoter sequences of the Gata4 gene were unknown, Hiroko Nagase in our laboratory cloned the mouse Gata4 promoter [17, 18]. Detailed analyses of the Gata4 promoter region revealed that DNA binding to the CCAAT box is important for the chronic hypoxia-induced pulmonary hypertension-mediated activation of Gata4 gene transcription. Dr. Ah-Mee Park in our laboratory discovered that the transcription factor that binds to the CCAAT box during this process is CBF/NF-Y [16].

Since the mechanism of CBF/NF-Y activation is unknown, we screened for proteins that can interact with CBF/NF-Y. One protein we found to interact with CBF/NF-Y during the hypoxic pulmonary hypertension-mediated development of RV hypertrophy is annexin A1. The binding of CBF/NF-Y to the CCAAT box was inhibited by the addition of recombinant annexin A1 in the reaction mixture for band-shift assays, suggesting that annexin A1 is a negative regulator of CBF/NF-Y DNA binding .

The finding that annexin A1 is involved in this mechanism of RV hypertrophy was of great interest to us because we previously reported a novel oxidant signaling mechanism, which involves protein carbonylation and the subsequent proteasome-dependent degradation of annexin A1 [19]. In this study, Dr. Chi-Ming Wong in our laboratory demonstrated that protein carbonylation is promoted in response to ligand/receptor-mediated cell growth signaling in pulmonary artery smooth muscle cells [19]. One protein identified to be carbonylated was annexin A1, using a proteomic approach and mass spectrometry. Further, this carbonylated annexin A1 protein is degraded by proteasomes. From these studies, we proposed the “oxidation/degradation pathway of signal transduction,” which involves the carbonylation of annexin A1 and subsequent degradation of this oxidized protein by proteasomes. At that time, when we were studying pulmonary artery smooth muscle cells, we did not know exactly how the degradation of annexin A1 would promote cell signaling. It was exciting that our investigations of the mechanism of cardiac hypertrophy revealed one mechanism, in which annexin A1 degradation can elicit cell signaling and gene transcription by regulating CBF/NF-Y in the RV [16].

We tested if this annexin A1 carbonylation/degradation cascade may regulate RV hypertrophy. Our experiments showed that hypoxic pulmonary hypertension indeed caused the carbonylation of annexin A1 within 2 h (Fig. 3a) and annexin A1 was downregulated within 6 h (Fig. 3b). Further, the treatment of an inhibitor of metal-catalyzed protein carbonylation, deferoxamine, inhibited hypoxic pulmonary hypertension-induced RV hypertrophy [16].

Fig. 3
figure 3

A trigger of RV hypertrophy promotes annexin A1 carbonylation and degradation. Rats were subjected to hypoxia, and RV homogenates were prepared. (a) Carbonylated annexin A1 was monitored by labeling with DNPH, immunoprecipitated with the antibody for DNPH-derivatized proteins, and Western blotting with annexin A1 antibody. (b) Annexin A1 protein expression was monitored by Western blotting. Values in bar graphs represent means ± SEM. Asterisks indicate significant difference from control (Reproduced from [16] with permission)

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In the RVs of rats subjected to hypoxic PH, annexin A1 is carbonylated and its expression is reduced. This reduced annexin A1 expression should liberate CBF/NF-Y, promoting its DNA binding activity and thus Gata4 gene transcription (Fig. 4).

Fig. 4
figure 4

Proposed mechanism for GATA4 activation in the RV in response to pulmonary hypertension . Pulmonary hypertension exerts pressure overload on the RV, resulting in the generation of ROS, which in turn carbonylate annexin A1 that is bound to the CBF/NF-Y transcription factor. Carbonylated annexin A1 is degraded, resulting in liberated CBF/NF-Y that can bind to the CCAAT box within the Gata4 promoter. CBF/NF-Y binding enhances the gene transcription of Gata4 and increases the level of the GATA4 transcription factor , which in turn promotes the gene expression of hypertrophic regulators [16]

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We proposed two possible mechanisms by which the signaling pathway described in this study may preferentially occur in the RV compared with the LV. We found that the expression of CBF/NF-Y is higher in the RV compared with the LV (Fig. 5). This may increase the sensitivity of the RV to activate CBF/NF-Y by being liberated from annexin A1 as this protein is degraded. As discussed in the previous section of this chapter, we also found that protein carbonylation mediated by serotonin is more pronounced in the RV than in the LV, indicating that the sensitivity to carbonylation signaling might be higher in the RV. We attributed this finding to the differential expressions of monoamine oxidase between the RV and LV. Thus, this is another possible mechanism through which the activation of Gata4 gene transcription in response to pressure overload preferentially occurs in the RV.

Fig. 5
figure 5

CBF/NF-Y expression in the LV and RV. CBF-B protein expression was monitored in the LV and RV homogenates of normal rats. Asterisks indicate significant difference. (Reproduced from [16] with permission)

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4 Oxidative Modifications in the RV of the SU5416/Ovalbumin Model of Pulmonary Arterial Hypertension

In rats, the injection of SU5416, an inhibitor of the VEGF receptor, plus some stimuli such as hypoxia and inflammation can trigger pulmonary arterial hypertension that resembles the human disease [20, 21]. In these models, a severe increase in pulmonary arterial pressure seems to cause right heart failure [22]. The RVs of rats treated with the SU5416 injection and ovalbumin immunization in our laboratory in the same way as described by Mizuno et al. [20] were concentrically hypertrophied and severely fibrotic (collagen volume fraction between 0.15 and 0.20). We found that such RVs are subjected to biological oxidation as assessed by monitoring the nitrotyrosine formation by immunohistochemistry (Fig. 6). By using a proteomics approach, Dr. Xinhong Wang in our laboratory identified some of these nitrotyrosinylated proteins to be heat shock protein-90 and sarcoplasmic reticulum Ca2+-ATPase (SERCA2). Further, Dr. Wang found that S-glutathionylation of heat shock protein-90 and NADH-ubiquinone oxidoreductase was promoted in the RVs of rats with pulmonary hypertension.

Fig. 6
figure 6

Increased nitrotyrosine in the RV of rats treated with SU5416 and ovalbumin to promote pulmonary arterial hypertension. Rats were subjected to ovalbumin immunization and SU5416 injection as depicted in Panel a. At the end of the treatment, animals were euthanized and the heart tissues were immersed in formalin and embedded in paraffin. Paraffin-embedded tissues were cut and mounted on glass slides. Tissue sections were subjected to immunohistochemistry using the nitrotyrosine antibody (Santa Cruz Biotechnology). Magnification ×400

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Total protein carbonylation as detected by derivatizing the carbonyl groups with DNPH was not found to be altered in the RVs of rats with pulmonary hypertension compared with the controls in this model. Moreover, it was interesting to note that peptides that contain susceptible amino acids to be carbonylated are preferentially decreased in the RVs of pulmonary hypertensive rats compared with the controls. Dr. Earl Stadtman and coworkers defined that arginine, lysine, proline, and threonine (20% of amino acid types) are susceptible amino acids to be carbonylated and converted into glutamic semialdehyde, α-aminoadipic semialdehyde, glutamic semialdehyde, and 2-amino-3-ketobutyric acid , respectively [23].

As shown in Table 1, metabolomics analysis revealed that 28 peptides were significantly modulated at least twofold. All of them were decreased in the RVs of pulmonary hypertensive rats compared with the controls. Notably, Phe-Lys-Lys peptide expression was 42-fold lower in the RVs of rats with pulmonary hypertension. All other peptides were two to five-fold lower. Among these 28 peptides, 24 (85.7%) contained at least one of the carbonylation-susceptible amino acids defined by Dr. Stadtman [23]. These 28 peptides contain 112 amino acids. Among them, 51 amino acids are carbonylation-susceptible amino acids (45.5%). This realization may lead to a potentially important area of research. From these results, one could hypothesize that the carbonylation of specific peptides and subsequent degradation may either mediate or be associated with the development of right heart failure. The latter event may be useful to be used as biomarkers of RV failure.

Table 1 Peptides that are differentially expressed between the RVs of rats with pulmonary arterial hypertension and controls
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5 Conclusions and Future Perspectives

The survival of patients with pulmonary hypertension remains low, and right heart failure is the major cause of death among these patients. Drugs designed to treat left heart failure are not effective against right heart failure, and no right heart-specific therapeutic agents are available. Moreover, RV pathophysiology is still relatively under-studied and not well understood. In particular, the research field of the signal transduction biology of the RV is in its infancy. Understanding the molecular signaling mechanisms for the growth and death of RV cardiomyocytes is critical to developing useful therapeutic strategies to treat right heart failure in pulmonary hypertension patients.

Redox signaling has been shown to play important roles in a variety of diseases , and our laboratory has shown that the RV also utilizes redox signaling mechanisms for cell regulation. However, redox regulatory mechanisms are complex, and it is not likely that simply administering antioxidants to globally eliminate ROS will solve health problems.

In the future, we need to first confirm whether the results found in experimental animals can be translated to humans. Once animal models are confirmed to provide useful information, these systems will be useful to study the complex mechanisms of redox signaling. It is likely that different types of oxidative modifications need to be investigated in concert.