Effect of Vitamin B2 and Vitamin E on Cancer-Related Sarcopenia in a Mouse Cachexia Model

Cancer-related sarcopenia is associated with impaired energy metabolism and increased oxidative stress production in skeletal muscles. With an aim to treat cancer-related sarcopenia using dietary intervention, we investigated the effects of vitamin B2 (VB2) and vitamin E (VE), which are recognized to have antioxidant effects, on CT26 mouse colon cancer cells and skeletal muscles in vitro and in vivo. VB2 suppressed tumor growth by suppressing cell proliferation and inducing more pronounced apoptosis by increasing the production of adenosine triphosphate (ATP) and reactive oxygen species (ROS). VE suppressed tumor growth by suppressing cell proliferation and increasing apoptosis by decreasing the production of ATP and ROS. In C2C12 mouse skeletal myoblast cells, VB2 treatment increased the production of ATP and ROS and VE treatment decreased the production of ATP and ROS; both treatments suppressed skeletal myoblast maturation. In the mouse model, intraperitoneal inoculation (peritoneal model) resulted in marked macrophage infiltration and elevated blood tumor necrosis factor-α and high-mobility group box-1 inflammatory cytokine levels, leading to cachexia. In contrast, subcutaneous inoculation (subcutaneous model) showed poor macrophage infiltration and low inflammatory cytokine levels, without cachexia. VB2 and VE activated macrophages and exacerbated cancer-related sarcopenia in the peritoneal model, whereas VB2 and VE treatment did not exhibit significant changes in sarcopenia in the subcutaneous model. In order to improve cancer-related sarcopenia by dietary intervention, it is important to consider the effect on inflammatory cytokines.

Autologous skeletal myoblast patch implantation prevents the deterioration of myocardial ischemia and right heart dysfunction in a pressure-overloaded right heart porcine model

Right ventricular dysfunction is a predictor for worse outcomes in patients with congenital heart disease. Myocardial ischemia is primarily associated with right ventricular dysfunction in patients with congenital heart disease and may be a therapeutic target for right ventricular dysfunction. Previously, autologous skeletal myoblast patch therapy showed an angiogenic effect for left ventricular dysfunction through cytokine paracrine effects; however, its efficacy in right ventricular dysfunction has not been evaluated. Thus, this study aimed to evaluate the angiogenic effect of autologous skeletal myoblast patch therapy and amelioration of metabolic and functional dysfunction, in a pressure-overloaded right heart porcine model. Pulmonary artery stenosis was induced by a vascular occluder in minipigs; after two months, autologous skeletal myoblast patch implantation on the right ventricular free wall was performed (n = 6). The control minipigs underwent a sham operation (n = 6). The autologous skeletal myoblast patch therapy alleviated right ventricular dilatation and ameliorated right ventricular systolic and diastolic dysfunction. 11C-acetate kinetic analysis using positron emission tomography showed improvement in myocardial oxidative metabolism and myocardial flow reserve after cell patch implantation. On histopathology, a higher capillary density and vascular maturity with reduction of myocardial ischemia were observed after patch implantation. Furthermore, analysis of mRNA expression revealed that the angiogenic markers were upregulated, and ischemic markers were downregulated after patch implantation. Thus, autologous skeletal myoblast patch therapy ameliorated metabolic and functional dysfunction in a pressure-overloaded right heart porcine model, by alleviating myocardial ischemia through angiogenesis.

Abstract 13717: Autologous Skeletal Myoblast Patch Remodeled Swine Pressure-overloaded Right Heart by Amelioration in Myocardial Ischemia

Introduction: Myocardial ischemia (MI) is majorly seen in the right ventricular (RV) dysfunction in patients with congenital heart disease secondary to residual hemodynamic stressors in form of pressure-overloaded, suggesting therapeutic target for RV dysfunction may be how to control MI. Autologous skeletal myoblast patch showed the angiogenetic effect for left ventricular dysfunction by cytokine paracrine effects, which are expected to be sufficiently effective against pressure-overloaded RV dysfunction. Hypothesis: An autologous skeletal myoblast patch alleviates the MI in a pressure-overloaded right heart in swine model, leading to amelioration of metabolic and functional dysfunction. Methods: Five-month-old mini-pigs underwent pulmonary artery banding. Two months after banding, myoblast patch derived from autologous skeletal muscles were placed on the epicardium of RV free wall. Groups were as follows: control (C, n=6), sheet implantation (S, n=6). Two months after sheet implantation, cardiac function exam and C11-Acetate positron emission tomography (PET) were done, and hearts were dissected for histologic and real-time polymerase chain reaction (RT-PCR) analysis. Results: Two months after sheet implantation, RV dysfunction was significantly ameliorated in group S than group C (RVEF; S 44.9+/-2.2 vs C 31.9+/-2.1 % [p=0.0042]). PET revealed the deterioration of myocardial blood flow (Rest/Stress; S 3.22+/-0.39 vs C 2.13+/-0.92 min -1 [p=0.0421]) and myocardial oxidative metabolism (K mono -Rest/Stress: S 3.17+/-0.69 vs C 2.03+/-0.65 min -1 [p=0.0421]) were suppressed in group S than group C. In histologic analysis, group S presented more angiogenesis in CD31 expression (S 18.3+/-1.5 vs C 10.7+/-2.8 units/cells [p=0.0122]). In RT-PCR analysis, mRNA expression of vascular endothelial growth factor (S 1.28+/-0.35 vs C 0.75+/-0.17 folds [p=0.030]), hepatocyte growth factor (S 1.70+/-0.79 vs C 0.74+/-0.10 folds [p=0.030]), and chemokine stromal cell-derived factor-1 (S 1.49+/-0.97 vs C 0.35+/-0.20 folds [p=0.030]) were upregulated in group S than group C. Conclusions: Autologous skeletal myoblast patch ameliorated metabolic and functional dysfunction in a swine model of pressure-overloaded right heart by alleviation of MI.

Autologous skeletal myoblast sheet prevents cardiomyocyte ischemia and right heart dysfunction in pressure-overloaded right heart porcine model

Introduction Severe heart failure (HF) with congenital heart disease (CHD) have demonstrated life threatening disorder despite of remarkable progress in medical therapies. Autologous skeletal myoblast sheet transplantation therapy showed clinical efficacy for left ventricular dysfunction by cytokine paracrine effects, which are expected to be sufficiently effective against right ventricular (RV) dysfunction which is often seen in end-stage of CHD patients with severe HF. Hypothesis An autologous skeletal myoblast sheet transplantation alleviates RV dysfunction in a pressure-overloaded right heart in a porcine model. Methods Five-to-six-month-old Göttingen mini-pigs underwent pulmonary artery banding with vascular occluding system. To create the porcine model of chronic pressure-overloaded right heart, vascular occluding system was gradually inflated, over a month, to make pulmonary stenosis to banding velocity >3.0 m/s measured by echocardiography (UCG), and then fixed for another month. Two months after banding, autologous skeletal myoblast sheet was placed on the epicardium of the RV free wall and followed for 2 months. Groups were as follows: control (C, n=5), sheet implantation (S, n=5). Cardiac function was measured using UCG, cardiac computed tomography (CT), and cardiac catheterization (Cath). Two months after sheet implantation, hearts were dissected for histologic analysis. Results Before sheet implantation, RV dysfunction was equal in groups; however, 2 months after sheet implantation, RV dysfunction and myocardial ischemia was significantly ameliorated in group S than group C. On CT, RV ejection fraction exacerbation were well controlled in Group S compared to Group C (S 44.9±2.2 vs C 31.9±2.1% [p=0.0042]). UCG and Cath revealed well maintained systolic and diastolic function in Group S compared to Group C (Tei index: S 0.42±0.06 vs C 0.70±0.07 [p=0.0240], Fraction Area Change: S 45.8±7.8 vs C 19.5±1.3% [p=0.0240], Isovolumic Relaxation Time; S 44.3±9.2 vs C 97.3±9.5 ms [p=0.0304]). On C11-Acetate Positron Emission Tomography, myocardial ischemia was more prominent in Group C compared to Group S (K mono-Rest/Stress: S 3.17±0.69 vs C 2.03±0.65 min-1 [p=0.0421], Myocardial Blood Flow-Rest/Stress: S 3.22±0.39 vs C 2.13±0.92 min-1 [p=0.0421]). In histologic analysis, Group S presented less progressed hypertrophic change in periodic acid-Schiff stain (S 13.5±0.9 vs C 18.0±3.0 μg [p=0.0240]), anti-fibrotic changes in picrosirius red stain (S 3.0±0.3 vs C 4.2±0.2% [p=0.0421]), more angiogenesis in CD31 expression (S 18.3±1.5 vs C 10.7±2.8 / 104 μm2 [p=0.0240]), and less production of reactive oxygen species in fluorescent immunostaining (S 5.9±1.7 vs C 18.4±1.7% [p=0.0304]). Conclusion Autologous skeletal myoblast sheet transplantation alleviates cardiomyocyte Ischemia and RV dysfunction in a porcine model of pressure-overloaded right heart. Funding Acknowledgement Type of funding source: None

Elevation of phosphate levels impairs skeletal myoblast differentiation

Hyperphosphatemic conditions such as chronic kidney disease are associated with severe muscle wasting and impaired life quality. While regeneration of muscle tissue is known to be reliant on recruitment of myogenic progenitor cells, the effects of elevated phosphate loads on this process have not been investigated in detail so far. This study aims to clarify the direct effects of hyperphosphatemic conditions on skeletal myoblast differentiation in a murine in vitro model. C2C12 murine muscle progenitor cells were supplemented with phosphate concentrations resembling moderate to severe hyperphosphatemia (1.4–2.9 mmol/l). Phosphate-induced effects were quantified by RT-PCR and immunoblotting. Immunohistochemistry was performed to count nuclear positive cells under treatment. Cell viability and metabolic activity were assessed by XTT and BrdU incorporation assays. Inorganic phosphate directly induced ERK-phosphorylation in pre-differentiated C2C12 myoblast cells. While phosphate concentrations resembling the upper normal range significantly reduced Myogenin expression (− 22.5%, p = 0.015), severe hyperphosphatemic conditions further impaired differentiation (Myogenin − 61.0%, p < 0.0001; MyoD − 51.0%; p < 0.0001). Analogue effects were found on the protein level (Myogenin − 42.0%, p = 0.004; MyoD − 25.7%, p = 0.002). ERK inhibition strongly attenuated phosphate-induced effects on Myogenin expression (p = 0.002). Metabolic activity was unaffected by the treatments. Our data point to a phosphate-induced inhibition of myoblast differentiation without effects on cell viability. Serum phosphate levels as low as the upper normal serum range significantly impaired marker gene expression in vitro. Investigation of cellular effects of hyperphosphatemia may help to better define serum cutoffs and modify existing treatment approaches of phosphate binders, especially in patients at risk of sarcopenia.

Anti-Mycobacterial Peroxides: A New Class of Agents for Development Against Tuberculosis

Background: With few exceptions, existing tuberculosis drugs were developed many years ago and resistance profiles have emerged. This has created a need for new drugs with discrete modes of action. There is evidence that tuberculosis (like other bacteria) is susceptible to oxidative pressure and this has yet to be properly utilised as a therapeutic approach in a manner similar to that which has proven highly successful in malaria therapy. Objective: To develop an alternative approach to the incorporation of bacterial siderophores that results in the creation of antitubercular peroxidic leads for subsequent development as novel agents against tuberculosis. Methods: Eight novel peroxides were prepared and the antitubercular activity (H37Rv) was compared to existing artemisinin derivatives in vitro. The potential for toxicity was evaluated against the L6 rat skeletal myoblast and HeLa cervical cancer lines in vitro. Results: The addition of a pyrimidinyl residue to an artemisinin or, preferably, a tetraoxane peroxidic structure results in antitubercular activity in vitro. The same effect is not observed in the absence of the pyrimidine or with other heteroaromatic substituents. Conclusion: The incorporation of a pyrimidinyl residue adjacent to the peroxidic function in an organic peroxide results in anti-tubercular activity in an otherwise inactive peroxidic compound. This will be a useful approach for creating oxidative drugs to target tuberculosis.