Angélica Ruiz-Ramírez

Background: For a long time, it was thought that estrogen degradation metabolites (EDMs) had no role in the female body, but this view has changed. Despite the progress, there are still unknowns about EDMs and the heart. We found that EDMs affect isolated cardiac mitochondria, which may impact heart function. Therefore, our objective was to explore the effect of two EDMs, Met A (2-hydroxyestrone) and Met D (17β-estradiol-3-methyl-ether), on two experimental models: rat H9c2 cardiomyoblast cells exposed for increasing times to different concentrations of each EDM, and, also, the effects of each metabolite on heart function in intact and oophorectomized (Oopho) rats. Methods: The cell viability, mitochondrial potential, and oxidative stress were assayed in H9c2 cells. Echocardiographic analyses, histological analyses, and an assessment of cardiac damage and oxidative stress in intact and Oopho rats were tested. Results: The strongest negative effects were seen with Met D, where antioxidant capacity decreased and oxidative stress increased significantly. Upon further analysis of the effects of each metabolite on heart function, echocardiographic analysis revealed that cardiac function had not changed in either group, although clear signs of cardiac hypertrophy and fibrosis were observed. Conclusions: Our results suggest EDMs have specific effects on female rats. This study emphasizes the need to thoroughly investigate EDMs’ side effects and impact on the heart.

Background: Systemic arterial hypertension (SAH) induced by Nω-nitro-L-arginine methyl ester (L-NAME) is a well-established model characterized by nitric oxide (NO^●) synthase inhibition and vascular dysfunction. The transient receptor potential vanilloid 1 (TRPV1) regulates Ca²⁺ flux and may contribute to mitochondrial homeostasis. We hypothesized that TRPV1 activation modulates mitochondria function and attenuates cardiac damage during SAH. Methods: Hypertension was induced in Wistar rats by administration of L-NAME (200 mg/L) for 40 days. During the last four days, hypertensive animals received capsaicin (5 mg/kg/day), capsazepine (6 mg/kg/day), or their combination. Cardiac function was evaluated in isolated hearts using the Langendorff perfusion system. Myocardial tissue viability was assessed by triphenyltetrazolium chloride (TTC) staining, and mitochondrial function was evaluated by measuring respiratory control and apoptosis-related proteins. Results: Capsaicin treatment was associated with significant cardioprotective effects in hypertensive rats. Although the findings are consistent with a role of TRPV1 activation in mediating these effects, the partial protection observed with capsazepine suggests that TRPV1-independent mechanisms may also contribute. Conclusions: TRPV1 activation contributes to cardioprotection in SAH, likely through preservation of mitochondrial function and redox balance. However, additional mechanisms beyond TRPV1 modulation may also participate in the observed protective effects. Further studies—including direct assessment of mitochondrial Ca²⁺ flux and the use of more selective or genetic approaches—are currently underway to clarify the underlying mechanisms.

Many factors related to obesity can impact how mitochondria produce ATP, such as the uncoupling of oxidative phosphorylation (OXPHOS) caused by proton leaks from built-up free fatty acids (FFA), the increased levels of uncoupling proteins (UCPs), and changes in the levels of ATPase inhibitory protein factors 1 (IF1). Therefore, the present study aimed to assess the rate of ATP synthesis in mitochondria isolated from skeletal and cardiac muscle from animal models of sucrose diet-induced obesity at different time periods. Short periods of sucrose intake (6 and 12 weeks) are sufficient to induce fat accumulation, hypertriglyceridemia, and high plasma FFA. However, a significant decline in the ATP synthesis rate starts to be obvious in mitochondria from skeletal muscle after 24 weeks of sucrose consumption. This impairment of ATP synthesis is associated with increased FFA in skeletal muscle homogenate. ATP synthesis rates in both skeletal and cardiac muscle were found to be sensitive to oleic acid and GDP, a physiological inhibitor of UCPs that has been shown to increase with aging. In addition, a sucrose diet increases the IF1 content in both skeletal and heart muscle, probably to avoid the hydrolytic activity of ATP synthase. In mitochondria from heart muscle, a decrease in the ATP synthesis rate was only observed according to the age in both groups of rats, and it was not affected by sucrose feeding. Our results suggest that the decline of the ATP synthesis rate in mitochondria from skeletal muscle can be due to the accumulation of FFA in skeletal muscle tissue as uncouplers, and the IF1 overexpression induced by the sucrose diet is a response mechanism to avoid the ATP hydrolysis and to save the energy charge reduced by FFA-uncoupling OXPHOS.

Several factors related to the obesity can affect mitochondrial ATP biosynthesis including the uncoupling of oxidative phosphorylation (OXPHOS) by proton leak induced by accumulated free fatty acids (FFA), the over-expression of uncoupling proteins (UCPs) and altered expression of ATPase inhibitory protein factors 1 (IF1). Therefore, the present work was aimed to evaluate the activity of ATP synthase in isolated mitochondria from skeletal and heart muscle of animal models of obesity induced by sucrose diet at different periods. Short periods of sucrose intake (6 and 12 weeks) are sufficient to induce fat accumulation, hypertriglyceridemia and high plasma FFA. However, a significant decline in the activity of the ATP synthase starts to be obvious in mitochondria from skeletal muscle after 24 weeks of sucrose consumption. This impairment of ATP synthase is associated with increased FFA in skeletal muscle homogenate. Both skeletal and heart muscle ATP synthase activity were found sensitive to oleic acid and to GDP, a physiological inhibitor of UCPs, found enhanced by aging. In addition, sucrose diet increases the IF1 content in both skeletal and heart muscle probably to avoid the hydrolysis activity of ATP synthase. In mitochondria from heart muscle a decrease in the ATP synthase activity was only observed according to the age in both groups of rats and it was not affected by sucrose feeding. Our results suggest that the decline of the ATP synthase activity in mitochondria from skeletal muscle can be due to the accumulation of FFA in skeletal muscle tissue as uncouplers, and the IF1 over-expression induced by sucrose diet is an adaptative mechanism to inhibit the ATP synthase hydrolysis activity.

Myocardial oxidative stress increases under conditions of hyperglycemia and ischemia/reperfusion (I/R) injury, leading to cellular damage. Inhibition of oxidative stress is involved in the cardioprotective effects of hydrogen sulfide (H₂S) during I/R and diabetes, and H₂S has the potential to protect the heart. However, the mechanism by which H₂S regulates the level of cardiac reactive oxygen species (ROS) during I/R and hyperglycemic conditions remains unclear. Therefore, the objective of this study was to evaluate the cytoprotective effect of H₂S in primary cardiomyocyte cultures subjected to hyperglycemia, hypoxia–reoxygenation (HR), or both conditions, by assessing the PPAR-α/Keap1/Nrf2/p47phox/NOX4/p-eNOS/CAT/SOD and the PPAR-γ/PGC-1α/AMPK/GLUT4 signaling pathways. Treatment with NaHS (100 μM) as an H₂S donor in cardiomyocytes subjected to hyperglycemia, HR, or a combination of both increased cell viability, total antioxidant capacity, and tetrahydrobiopterin (BH₄) concentrations, while reducing ROS production, malondialdehyde concentrations, 8-hydroxy-2′-deoxyguanosine, and dihydrobiopterin (BH₂) concentrations. Additionally, the H₂S donor treatment increased the expression and activity of PPAR-α, reversed the reduction in the expression of PPAR-γ, PGC-1α, AMPK, GLUT4, Nrf2, p-eNOS, SOD, and CAT, and decreased the expression of Keap1, p47phox and NOX4. Therefore, the treatment with the H₂S donor protects cardiomyocytes from damage caused by hyperglycemia, HR, or both conditions by reducing oxidative stress markers and improving antioxidant mechanisms, thereby increasing cell viability and “cardiomyocyte ultrastructure”.

The TRPV1 receptor is expressed on various cell types, including cardiomyocytes. Activation of TRPV1 leads to regulation of Ca²⁺ flux across both cell and mitochondrial membranes, which could control the ion flux under experimental systemic arterial hypertension (SAH), particularly in mitochondria, and thus prevent heart damage. In this study, SAH was induced in male Wistar rats using L-NAME, providing a system in which the regulatory action of nitric oxide (NO) is inhibited. The animals were divided into five groups: 1) Control (C), 2) Hypertensive (H), 3) H+Capsaicin (H+CS), 4) H+Capsazepine (H+CZ), and 5) H+CS+CZ. L-NAME was added to drinking water at a concentration of 200 mg/L for 40 days. Four days before the end of the L-NAME treatment, a daily subcutaneous dose of CS (5 mg/kg in the H+CS group), CZ (6 mg/kg in the H+CZ group), or a combination of CS+CZ was administered. We analyzed mitochondrial function and the damage caused by hypertension in the heart. The activation of TRPV1 under SAH conditions improved mitochondrial respiratory control, regulated oxidative stress, and modulated pro-apoptotic proteins. The improvement in mitochondrial metabolism led to the prevention of damage and better heart function.

Under conditions of hyperglycemia and ischemia/reperfusion (I/R) injury, myocardial oxidative stress increases, leading to cellular damage. Inhibition of oxidative stress has been reported to be involved in the cardioprotective effects of hydrogen sulfide (H2S) during I/R and diabetes. Recent studies have shown that H2S has the potential to protect the heart. However, the mechanism by which H2S regulates the level of cardiac reactive oxygen species (ROS) during I/R and hyperglycemic conditions remains unclear. Therefore, the objective of this study was to evaluate the cytoprotective effect of H2S in primary cardiomyocyte cultures subjected to hyperglycemia, hypoxia/reoxygenation (HR), or both conditions, by assessing the PPAR-α/Keap1/Nrf2/p47phox/NOX4/p-eNOS/CAT/SOD signaling pathway and the PPAR-γ/PGC1α/AMPK/GLUT4 signaling pathway. Treatment with NaHS (100 μM) as an H2S donor in cardiomyocytes subjected to hyperglycemia, HR, or a combination of both experimental conditions increased cell viability, total antioxidant capacity, and tetrahydrobiopterin (BH4) concentrations, while reducing ROS production, malondialdehyde concentrations, 8-hydroxy-2´-deoxyguanosine, and dihydrobiopterin (BH2) concentrations. Additionally, H2S donor treatment increased the expression and activity of PPAR-α, reversed the reduction in the expression of PPAR-γ, PGC1α, AMPK, GLUT4, Nrf2, p-eNOS, SOD and CAT, and decreased the expression of Keap1, p47phox y NOX4. Treatment with the H2S donor protects cardiomyocytes from damage caused by hyperglycemia, HR, or both conditions by reducing oxidative stress markers and improving antioxidant mechanisms, thereby increasing cell viability and cardiomyocyte ultrastructure.