**5.2. Mitochondrial dysfunction in right heart failure**

in human HF [76]. As adult cardiac myocytes have limited regenerative capacity, the loss of these cells is often counteracted by replacement with non-myocytes, promoting fibrosis and

The bulk of the research thus far in the field of RV failure and mitochondrial function has focused on PH-associated changes within the pulmonary vasculature (pulmonary artery smooth muscle cells, endothelial cells, and fibroblasts [77]) and metabolic changes that underlie activated inflammatory cells (reviewed in [78, 79]). In addition, a systemic metabolic and mitochondrial hypothesis has been put forward, based on similar changes in mitochondrial function observed in skeletal muscle in models of PH [80]. However, a few RV-specific investigations of changes in metabolism and mitochondrial function exist, and we'll review this

Although the data are less extensive than in the LV, the metabolic switch that occurs during LV hypertrophy and dysfunction also occurs in models of RV dysfunction [81, 82]. Several groups have reported upregulated glycolysis with suppression of FAO, and associated global changes in gene expression favoring glucose oxidation and downregulation of PPARα target genes [62]. Mechanistically, pyruvate dehydrogenase kinase (PDK) has been linked to the metabolic switch. PDK, an inhibitor of pyruvate dehydrogenase, is upregulated in RV hypertrophy. This PDK-mediated metabolic switch is associated with decreased RV myocyte contractility and cardiac output [81]. The shift to aerobic glycolysis has several consequences for the heart. First, greater amounts of lactate are produced, shifting redox status and other homeostatic outcomes, and second, fewer ATP molecules/glucose molecule are produced (32 during glucose oxidation, and 2 during glycolysis). To compensate for increased glycolysis, glucose uptake is accelerated, and can be assessed by positron emission tomography, both in experimental PH and RV dysfunction [83, 84], and in patients with pulmonary arterial

As in left heart failure, researchers have attempted to explain the metabolic switch based on energy production relative to oxygen availability. The pressure overloaded RV is oxygen deprived. In the setting of an oxygen limited hypertrophic RV, energy production which favors

glucose oxidation to generate the same amount of ATP [86]. Some experimental data support this hypothesis, with reports of systolic perfusion gradients limiting coronary artery flow [87], coupled with increased metabolic demands in the hypertrophied heart, which result in a localized RV ischemia. However, the question of oxygen supply in RV hypertrophy is insufficiently answered. While it may be true that angiogenic potential in the failing RV is attenuated, resulting in ischemia [88], methodological difficulties have precluded accurate assessment of RV oxygen supply. Further, other groups have argued that reliance on carbohydrate metabolism

ratio would benefit the working heart, and FAO uses 12% more oxygen than

**5. Molecular mechanisms of right heart failure**

**5.1. Metabolic remodeling in right heart failure**

cardiac dysfunction.

14 Mitochondrial Diseases

evidence below.

hypertension [85].

a high ATP/O2

Similar to the LV, mitochondrial dysfunction is also implicated in RHF, albeit with less literature supporting the mechanistic link, and more conflicting reports depending on the model. MCT and SuHx models of RHF tend to demonstrate more severe mitochondrial dysfunction and depression of mitochondrial biogenesis. Decreased PGC-1α expression and a net loss of mitochondrial protein and oxidative capacity have been reported in SuHx rats, alongside abnormal mitochondrial shape and size by electron microscopy [90]. On the other hand, changes in mitochondrial function in chronic hypoxia models have been particularly discordant and warrant further discussion. Chronic hypoxia decreased ATP synthesis and measurements of mitochondrial number in a rat model of chronic hypoxia in both ventricles, however, the effect was slightly delayed in the right compared to LV [91]. Data from our lab in the neonatal calf model of PH-induced RV dysfunction also demonstrated similar changes to mitochondrial function in both ventricles, with no additional impact of pressure overload on the RV [92]. In these models of PH and RV dysfunction, the hypoxic stimulus is administered systemically using a hypobaric chamber. Thus, the LV experiences similar degrees of hypoxia as the RV, yet does not show signs of contractile or relaxation deficits until much later in disease progression. It's possible that interventricular differences in oxygen supply and demand explain the similar findings in both ventricles, and support the need for better mechanistic understanding of the similarities and differences in oxygen delivery and metabolism in the RV exposed to pathological load.

Mitochondrial dynamics are not well-described in the failing right heart. However, in a study by Marsboom et al., administration of the Drp1 inhibitor Mdivi-1 regressed PH in rodents by arresting proliferation of pulmonary artery smooth muscle cells, resulting in improved exercise capacity and RV function. Therefore, while not described in the RV, modulation of mitochondrial dynamics may be therapeutically viable for right heart failure [93]. Even less has been described on the role of mitophagy in the setting of right heart failure. However, one group has attempted to elucidate the impact of autophagy on remodeling following PAB. p62 and LC3 II/I were both increased in the hypertrophied RV [94]. These data support similar findings in MCT-induced RV dysfunction, with increased autophagy signaling and autophagosome formation by EM [95]. Expression of these markers temporally increased post MCT injection, which the authors suggest indicates a causal role for autophagy during the progression from hypertrophy to failure. Future work is needed to elucidate the changes that occur with mitochondrial dynamics and mitophagy in the failing right heart, and future investigations should test myocyte-specific deletion of Mfn1, Mfn2, and Drp1 in experimental RV failure.

Our understanding of mitochondrial abnormalities in human RV failure is equally as understudied. Although availability of human samples is methodologically limiting, the use of pediatric congenital heart explants has shed some light on changes which underlie human RHF. One study of RV samples obtained from 31 pediatric patients undergoing cardiac surgery for congenital heart disease classified patients based on compensated RV function or failure based on echocardiography, right heart catheterization and MRI. Citrate synthase activity was maintained during hypertrophy, but decreased at failure, while mtDNA content progressively decreased with worsening clinical disease [96]. In addition, adult RV samples of various etiologies demonstrate increased mitochondrial membrane potential which positively correlates with the degree of hypertrophy [97]. A complete understanding of mitochondrial function in human right heart failure is limited by methodological constraints of assessing comprehensive ETC function.

The metabolic switch that occurs in the hypertrophied RV is suggested to be mediated in part through PDK4 [81]. Dichloroacetate (DCA), a small molecule inhibitor of pyruvate dehydrogenase, improves glucose oxidation and has reported improvements in RV stroke volume, cardiac output, and exercise capacity [81, 105]. It is also associated with restoration of RV mitochondrial function and mitochondrial-dependent apoptosis [106]. Though a Phase I clinical trial has been completed in subjects with advanced PH (Clinical Trial Identifier NCT01083524), study results have not been published, and the therapeutic potential of DCA in human RV failure remains unknown. As discussed above, the therapeutic strategy of FAO and improved glucose oxidation lacks consensus [89], and in part is predicated on the balance between ATP production and oxygen availability. While continued efforts to test metabolic mediators in RHF are warranted, a mechanistic understanding of energy production, relative RV ischemia, and the molecular regulators of these processes are necessary for development of targeted

Mitochondria and Metabolism in Right Heart Failure http://dx.doi.org/10.5772/intechopen.70450 17

The causal role of ROS in cardiac dysfunction led to the early belief that antioxidants would attenuate cardiovascular disease. In experimental models, treatments with antioxidants have somewhat inconsistently tended to improve cardiac function [107]. However, clinical trials have largely failed to show benefit of antioxidants in treatment of chronic disease [108]. In light of these disappointing results, many groups have taken fundamentally different approaches to improving redox status. Induction of endogenous antioxidants by a phytochemical supplement preserved RV function and prevented RV fibrosis and capillary loss [109], suggesting activation of cytoprotective transcription factors may more robustly attenuate oxidant stress than traditional vitamin supplements. Other groups use mitochondrially-directed peptides to scavenge free radicals. One such peptide, SS31, accumulates more than 1000-fold in mitochondria [110]. This peptide prevented LV hypertrophy, fibrosis, and diastolic dysfunction [111], and reduced mitochondrial oxidant damage [112]. To date, however, targeted mito-

Interventions which boost mitochondrial function and/or biogenesis have large therapeutic potential in many types of cardiovascular disease, including RV failure. Phytochemical compounds which elicit mitochondrial biogenic properties have received attention lately, such as resveratrol. Resveratrol, the primary polyphenol in red wine, stimulates mitochondrial content, ATP production, and FAO, while inhibiting mitochondrial ROS production in several tissue types and disease contexts [113, 114]. In addition, it is currently in testing as a therapy for chronic obstructive pulmonary disorder (NCT02245932) and non-ischemic heart failure (NCT01914081). Identification of other plant-based or pharmaceutical approaches which stimulate the synthesis of new mitochondria to increase energy production while decreasing

Exercise-induced cardiac hypertrophy has been known to be cardioprotective for decades [115], though the exact mechanisms underlying physiological hypertrophy have remained somewhat

metabolic therapy.

**6.2. Mitochondrial therapeutic interventions**

chondrial peptides have not been tested in models of RV failure.

oxidant damage may have profound impact on the failing right heart.

**6.3. Exercise as right heart therapy**


*Article* **Increasing the Content of Olive Mill Wastewater in Biogas Reactors for a Sustainable Recovery: Methane Productivity and Life Cycle Analyses of the Process**

**Souraya Benalia, Giacomo Falcone, Teodora Stillitano \*, Anna Irene De Luca, Alfio Strano, Giovanni Gulisano, Giuseppe Zimbalatti and Bruno Bernardi**

> Dipartimento di Agraria, Università degli Studi Mediterranea di Reggio Calabria, Località Feo di Vito, 89122 Reggio Calabria, Italy<EMAIL_ADDRESS>(S.B<EMAIL_ADDRESS>(G.F<EMAIL_ADDRESS>(A.I.D.L<EMAIL_ADDRESS>(A.S<EMAIL_ADDRESS>(G.G<EMAIL_ADDRESS>(G.Z<EMAIL_ADDRESS>(B.B.)

**\*** Correspondence<EMAIL_ADDRESS>
**Abstract:** Anaerobic codigestion of olive mill wastewater for renewable energy production constitutes a promising process to overcome management and environmental issues due to their conventional disposal. The present study aims at assessing biogas and biomethane production from olive mill wastewater by performing biochemical methane potential tests. Hence, mixtures containing 0% (blank), 20% and 30% olive mill wastewater, in volume, were experimented on under mesophilic conditions. In addition, life cycle assessment and life cycle costing were performed for sustainability analysis. Particularly, life cycle assessment allowed assessing the potential environmental impact resulting from the tested process, while life cycle costing in conjunction with specific economic indicators allowed performing the economic feasibility analysis. The research highlighted reliable outcomes: higher amounts of biogas (80.22 ± 24.49 NL.kgSV−1) and methane (47.68 ± 17.55 NL.kgSV−1) were obtained when implementing a higher amount of olive mill wastewater (30%) (*v*/*v*) in the batch reactors. According to life cycle assessment, the biogas ecoprofile was better when using 20% (*v*/*v*) olive mill wastewater. Similarly, the economic results demonstrated the profitability of the process, with better performances when using 20% (*v*/*v*) olive mill wastewater. These findings confirm the advantages from using farm and food industry by-products for the production of renewable energy as well as organic fertilizers, which could be used in situ to enhance farm sustainability.

**Keywords:** anaerobic codigestion; biomethane; life cycle assessment (LCA); life cycle costing (LCC); olive mill by-products


**In Silico Study of Rett Syndrome Treatment-Related Genes,** *MECP2***,** *CDKL5***, and** *FOXG1***, by Evolutionary Classification and Disordered Region Assessment**

**Muhamad Fahmi 1, Gen Yasui 1, Kaito Seki 1, Syouichi Katayama 2, Takako Kaneko-Kawano 2, Tetsuya Inazu 2, Yukihiko Kubota 3 and Masahiro Ito 1,3,\***

Received: 30 September 2019; Accepted: 5 November 2019; Published: 8 November 2019

**Abstract:** Rett syndrome (RTT), a neurodevelopmental disorder, is mainly caused by mutations in methyl CpG-binding protein 2 (*MECP2*), which has multiple functions such as binding to methylated DNA or interacting with a transcriptional co-repressor complex. It has been established that alterations in cyclin-dependent kinase-like 5 (*CDKL5*) or forkhead box protein G1 (*FOXG1*) correspond to distinct neurodevelopmental disorders, given that a series of studies have indicated that RTT is also caused by alterations in either one of these genes. We investigated the evolution and molecular features of MeCP2, CDKL5, and FOXG1 and their binding partners using phylogenetic profiling to gain a better understanding of their similarities. We also predicted the structural order–disorder propensity and assessed the evolutionary rates per site of MeCP2, CDKL5, and FOXG1 to investigate the relationships between disordered structure and other related properties with RTT. Here, we provide insight to the structural characteristics, evolution and interaction landscapes of those three proteins. We also uncovered the disordered structure properties and evolution of those proteins which may provide valuable information for the development of therapeutic strategies of RTT.

**Keywords:** Rett syndrome; intrinsically disordered region; phylogenetic profile analysis; post-transcriptional modification; methyl-CpG-binding protein 2; cyclin-dependent kinase-like 5; forkhead box protein G1


8) Antibacterial Test

The antimicrobial activity of the iPP/ZnOc composites was evaluated using *E. coli* DSM 498T (DSMZ, Braunschweig, Germany) as test microorganisms. The evaluation was performed using the ASTM Standard Test Method E 2149-10 [34]. The preparation of the bacterial inoculum required to grow a fresh 18-h shake culture of *E. coli* DSM 498T in a sterile nutrient broth (LB composition for 1 L: 10 g of triptone, 5 g of yeast extract and 10 g of sodium chloride) The colonies were maintained according to good microbiological practice and examined for purity by creating a streak plate. The bacterial inoculum was diluted using a sterile buffer solution (composition for one litre: 0.150 g of potassium chloride, 2.25 g of sodium chloride, 0.05 g of sodium bicarbonate, 0.12 g of CaCl2¨6H2O and pH = 7) until the solution reached an absorbance of 0.3 ˘ 0.01 at 600 nm, as measured spectrophotometrically. This solution, which had a concentration of 1.5 ˆ 108–3.0 ˆ 108 colony forming units/mL (CFUs/mL), was diluted with the buffer solution to obtain a final concentration of 1.5 ˆ 106–3 ˆ 106 CFUs/mL, that was the working bacterial dilution.

The experiments were performed in 50 mL sterilized flasks. One gram of the film was maintained in contact with 10 mL of the working bacterial dilution. After 2 min, 100 mL of the working bacterial dilution was transferred to a test tube, which was followed by serial dilution and plating out on Petri dishes (10 mm ˆ 90 mm) in which the culture media was previously poured. The Petri dishes were incubated at 35 ˝C for 24 h. These dishes represented the *T*0 contact time. The flasks were then placed on a wrist-action shaker for 1 h, 24 h, 48 h, 5 days and 10 days. The bacterial concentration in the solutions at these time points was evaluated by again performing serial dilutions and standard plate counting techniques. Three experiments were performed for each composition. The number of colonies in the Petri dish after incubation was converted into the number of colonies that form a unit per millilitre (CFUs/m) of buffer solution in the flask. The percentage reduction (*R*%) was calculated using the following formula:

$$R\% \text{ (CFU/mL)} = \left[ (B - A) / B \right] \times 100,\tag{1}$$

where *A* = CFUs/mL for the flask containing the sample after the specific contact time and *B* = CFUs/mL at *T*0.

#### **3. Results and Discussion**


*2.3. Test Solutions*

The range of the extract concentrations for the cytotoxicity tests was selected considering the concentration to be incorporated in wine (100 μg/mL). Serial test solutions (0–100 μg/mL) were prepared from stock solution (1000 μg/mL) in dimethyl sulfoxide (DMSO), being the final concentration in DMSO below 0.5%.

Concentrations of the extract used for both oxidative stress assays were calculated based on the cytotoxicity study previously performed. The mean effective concentration (EC50) value obtained (31.18 μg/mL and 20.56 μg/mL in HepG2, and 55.77 μg/mL and 39.02 μg/mL in Caco-2 cells for 24 h and 48 h, respectively) was chosen as the highest exposure concentration, along with the fractions EC50/2 and EC50/4.

To measure protection and reversion abilities, test solutions of the extract to cell viability greater than 75% were selected (EC50/2 and EC50/4).


*2.7. Expression Profiles of MdSAP Genes*

Knowing the patterns of expression in various tissue types can help us understand gene functions. Firstly, we collected different tissues including young roots, stems, fully expanded leaves, flowers, and mature fruit (70 mm, red peel, 150 days after bloom), from apple plants that were five years old after bud grafting. The scion was *Malus domestica* "Golden Delicious", and the rootstock was *M. hupehensis*. Then, we isolated the full-length cDNA, 50 -UTR, and 30 -UTR sequences for 13 *MdSAP* genes and used specific primers for our qRT-PCR assays. These *MdSAP*s were constitutively expressed in the five tissues examined here, albeit at different levels of transcription (Figure 6A). For example, *MdSAP7*, -*8*, -*10*, -*15*, -*19*, and -*29* were most highly expressed in the leaves; *MdSAP12*, -*16*, -*21*, and -*28* were most highly expressed in the fruits; and *MdSAP14*, -*23*, and -*25* were most highly expressed in the roots. To induce a water deficit, irrigation was withheld up to 8 days, while the designated control plants continued to receive normally scheduled irrigation ("Golden Delicious" scions and *M. hupehensis* rootstocks). In response to drought stress, the expression of *MdSAP15*, -*25*, and -*28* was significantly induced from that detected in the non-stressed control plants (Figure 6B), transcripts of *MdSAP7* and -*21* mRNAs were significantly reduced, and expression of the other MdSAP genes remained constant.

**Figure 6.** Tissue-specific expression and drought response of 13 *MdSAP* genes. (**A**) Expression patterns of 13 *MdSAP* genes in apple tissues. (**B**) Expression patterns of 13 *MdSAP* genes in response to drought stress, i.e., normally scheduled irrigation withheld for 0, 4, or 8 days. Three independent replicates were used for calculations. Error bars indicate standard deviation.