Astaxanthin Alleviates the Process of Cardiac Hypertrophy by Targeting the METTL3/Circ_0078450/MiR-338-3p/GATA4 Pathway.

Astaxanthin (ASX) is a natural antioxidant with preventive and therapeutic effects on various human diseases. However, the role of ASX in cardiac hypertrophy and its underlying molecular mechanisms remain unclear.Cardiomyocytes (AC16) were used with angiotensin-II (Ang-II) to mimic the cardiac hypertrophy cell model. The protein levels of hypertrophy genes, GATA4, and methyltransferase-like 3 (METTL3) were determined by western blot analysis. Cell size was assessed using immunofluorescence staining. The expression of circ_0078450, miR-338-3p, and GATA4 were analyzed by quantitative real-time PCR. Also, the interaction between miR-338-3p and circ_0078450 or GATA4 was confirmed by dual-luciferase reporter and RIP assays, and the regulation of METTL3 on circ_0078450 was verified by MeRIP and RIP assays.ASX reduced the hypertrophy gene protein expression and cell size in Ang-II-induced AC16 cells. Circ_0078450 was promoted under Ang-II treatment, and ASX reduced circ_0078450 expression in Ang-II-induced AC16 cells. Circ_0078450 could sponge miR-338-3p to positively regulate GATA4 expression, and GATA4 overexpression overturned the suppressive effect of circ_0078450 knockdown on Ang-II-induced cardiomyocyte hypertrophy. Also, the inhibitory effect of ASX on Ang-II-induced cardiomyocyte hypertrophy could be reversed by circ_0078450 or GATA4 overexpression. In addition, METTL3 mediated the m6A methylation of circ_0078450 to enhance circ_0078450 expression. Moreover, METTL3 knockdown suppressed Ang-II-induced cardiomyocyte hypertrophy by inhibiting circ_0078450 expression.Our data showed that ASX repressed cardiac hypertrophy by regulating the METTL3/circ_0078450/miR-338-3p/GATA4 axis.

The Impact of Melatonin Supplementation and NLRP3 Inflammasome Deletion on Age-Accompanied Cardiac Damage.

To investigate the role of NLRP3 inflammasome in cardiac aging, we evaluate here morphological and ultrastructural age-related changes of cardiac muscles fibers in wild-type and NLRP3-knockout mice, as well as studying the beneficial effect of melatonin therapy. The results clarified the beginning of the cardiac sarcopenia at the age of 12 months, with hypertrophy of cardiac myocytes, increased expression of ß-MHC, appearance of small necrotic fibers, decline of cadiomyocyte number, destruction of mitochondrial cristae, appearance of small-sized residual bodies, and increased apoptotic nuclei ratio. These changes were progressed in the cardiac myocytes of 24 old mice, accompanied by excessive collagen deposition, higher expressions of IL-1α, IL-6, and TNFα, complete mitochondrial vacuolation and damage, myofibrils disorganization, multivesicular bodies formation, and nuclear fragmentation. Interestingly, cardiac myocytes of NLRP3-/- mice showed less detectable age-related changes compared with WT mice. Oral melatonin therapy preserved the normal cardiomyocytes structure, restored cardiomyocytes number, and reduced ß-MHC expression of cardiac hypertrophy. In addition, melatonin recovered mitochondrial architecture, reduced apoptosis and multivesicular bodies' formation, and decreased expressions of ß-MHC, IL-1α, and IL-6. Fewer cardiac sarcopenic changes and highly remarkable protective effects of melatonin treatment detected in aged cardiomyocytes of NLRP3-/- mice compared with aged WT animals, confirming implication of the NLRP3 inflammasome in cardiac aging. Thus, NLRP3 suppression and melatonin therapy may be therapeutic approaches for age-related cardiac sarcopenia.

Nandrolone administration with or without strenuous exercise increases cardiac fatal genes overexpression, calcium/calmodulin-dependent protein kinaseiiδ, and monoamine oxidase activities and enhances blood pressure in adult wistar rats.

Misuse of anabolic androgenic steroids (AAS) increases prevalence of cardiovascular abnormalities in athletes, and the underlying molecular mechanism involved in those abnormalities continues to be investigated. The aim of this study was to investigate the effect of chronic nandrolone exposure on alpha and beta-myosin heavy chain (MHC) isoforms gene expression transition, blood pressure related parameters, calcium/calmodulin-dependent protein kinaseIIδ (CaMKIIδ), and monoamine oxidase (MAO) activities in rats' hearts. It was also planned to evaluate the effect of strenuous exercise on cardiac abnormalities induced by nandrolone. Thirty-two male wistar rats were assigned into four groups, namely control, nandrolone, nandrolone with strenuous exercise, and strenuous exercise groups. Nandrolone consumption significantly increased systolic, diastolic, pulse and dicrotic pressure, mean arterial pressure, as well as the amplitude of first peak (H1). Moreover, exercise combined with nandrolone completely masked this effect. The mRNA expression of ß-MHC and the ratio of ß -MHC/α -MHC showed a significant increase in the nandrolone and nandrolone with strenuous exercise groups compared to those in the control group. The values of heart tissue calcium/calmoldulin-dependent protein kinase IIδ (CaMKIIδ), and monoamine oxidase (MAO) in the nandrolone, nandrolone with strenuous exercise and exercise groups were significantly higher than those values in the control group. These findings indicate that nandrolone-induced heart and hemodynamic abnormalities may in part be associated with MHC isoform changes and Ca2+ homeostasis changes mediated by increased CaMKIIδ and MAO activities and that these effects can be provoked via strenuous exercise.

Dasatinib induces gene expression of CYP1A1, CYP1B1, and cardiac hypertrophy markers (BNP, ß-MHC) in rat cardiomyocyte H9c2 cells.

Dasatinib is a new selective tyrosine kinase inhibitor that targets certain kinases involved in cellular growth and development. This drug belongs to a novel anticancer therapy aiming to increase the survival in patients with imatinib-resistant mutations. However, the dasatinib toxicity was reported as a side effect leading to arrhythmias and/or heart failure. Here, we investigated the possibility of dasatinib-induced toxicity in rat cardiomyocyte H9c2 cells. Our objectives were to investigate the ability of dasatinib to induce expression of cytochrome P450 (CYP1A1, CYP1B1) and cardiac hypertrophy markers (BNP, ß-MHC) genes in H9c2 cells. To test this hypothesis, H9c2 cells were incubated with dasatinib at two concentrations (20 and 40 µM). Thereafter, CYP1A1, CYP1B1, BNP, and ß-MHC were determined at gene expression level. Our findings showed that dasatinib induces the CYP1A1, CYP1B1, BNP, and ß-MHC mRNA. The involvement of AhR/CYP1A1 pathway in dasatinib toxicity was tested by resveratrol (RES), an AhR antagonist. Interestingly, the increase in mRNA of different genes by dasatinib was not affected by RES, which confirms that these effects are not mediated through AhR. In addition, this was accompanied by a significant inhibition of constitutive expression of these genes by RES. The current work provides the first evidence for the ability of dasatinib to induce hypertrophic markers in H9c2 cells through AhR-independent pathway.

JMJD3 inhibition protects against isoproterenol-induced cardiac hypertrophy by suppressing ß-MHC expression.

Jumonji domain-containing protein D3 (JMJD3), a histone 3 lysine 27 (H3K27) demethylase, has been extensively studied for their participation in development, cellular physiology and a variety of diseases. However, its potential roles in cardiovascular system remain unknown. In this study, we found that JMJD3 played a pivotal role in the process of cardiac hypertrophy. JMJD3 expression was elevated by isoproterenol (ISO) stimuli both in vitro and in vivo. Overexpression of wild-type JMJD3, but not the demethylase-defective mutant, promoted cardiomyocyte hypertrophy, as implied by increased cardiomyocyte surface area and the expression of hypertrophy marker genes. In contrary, JMJD3 silencing or its inhibitor GSK-J4 suppressed ISO-induced cardiac hypertrophy. Mechanistically, JMJD3 was recruited to demethylate H3K27me3 at the promoter of ß-MHC to promote its expression and cardiac hypertrophy. Thus, our results reveal that JMJD3 may be a key epigenetic regulator of ß-MHC expression in cardiomyocytes and a potential therapeutic target for cardiac hypertrophy.

Molecular mechanisms of cardiotoxicity of gefitinib in vivo and in vitro rat cardiomyocyte: Role of apoptosis and oxidative stress.

Gefitinib (GEF) is a multi-targeted tyrosine kinase inhibitor with anti-cancer properties, yet few cases of cardiotoxicity has been reported as a significant side effect associated with GEF treatment. The main purpose of this study was to investigate the potential cardiotoxic effect of GEF and the possible mechanisms involved using in vivo and in vitro rat cardiomyocyte model. Treatment of rat cardiomyocyte H9c2 cell line with GEF (0, 1, 5, and 10µM) caused cardiomyocyte death and upregulation of hypertrophic gene markers, such as brain natriuretic peptides (BNP) and Beta-myosin heavy chain (ß-MHC) in a concentration-dependent manner at the mRNA and protein levels associated with an increase in the percentage of hypertrophied cardiac cells. Mechanistically, GEF treatment caused proportional and concentration-dependent increases in the mRNA and protein expression levels of apoptotic markers caspase-3 and p53 which was accompanied with marked increases in the percentage of H9c2 cells underwent apoptosis/necrosis as compared to control. In addition, oxidative stress marker (heme oxygenase-1, HO-1) and the formation of reactive oxygen species were increased in response to GEF treatment. At the in vivo level, treatment of Wistar albino rats for 21days with GEF (20 and 30mg/kg) significantly increased the cardiac enzymes (CK, CKmb, and LDH) levels associated with histopathological changes indicative of cardiotoxicity. Similarly, in vivo GEF treatment increased the mRNA and protein levels of BNP and ß-MHC whereas inhibited the antihypertrophoic gene (α-MHC) associated with increased the percentage of hypertrophied cells. Furthermore, the mRNA and protein expression levels of caspase-3, p53, and HO-1 genes and the percentage of apoptotic cells were significantly increased by GEF treatment, which was more pronounced at the 30mg/kg dose. In conclusion, GEF induces cardiotoxicity and cardiac hypertrophy in vivo and in vitro rat model through cardiac apoptotic cell death and oxidative stress pathways.

Carfilzomib-induced cardiotoxicity mitigated by dexrazoxane through inhibition of hypertrophic gene expression and oxidative stress in rats.

Carfilzomib (CFZ) is an inhibitor of proteasome that is generally used in the treatment of multiple myeloma but due to its cardiotoxicity clinical use may be limited. Dexrazoxane (DZR), an inhibitor of topoisomerase-II, prevents cardiac damage by reducing the formation of reactive oxygen species and hypertrophic gene expression. This study evaluated the protective effect of DZR on CFZ-induced cardiotoxicity. Thirty-two male Albino rats were randomly divided into four groups (n = 8). Group I received DMSO, Group II received CFZ (4 mg/kg, intraperitoneally [i.p.]) twice weekly up to day 16, Group III received DZR (20 mg/kg, i.p.) for 16 days and CFZ twice weekly for 16, Group IV received DZR (40 mg/kg, i.p.) for 16 days and CFZ twice weekly for 16. CFZ-induced cardiotoxicity was assessed by hematological, biochemical, mRNA expression, oxidative stress and histopathological studies. CFZ-induced significant changes have been observed in blood parameters including red blood cells, white blood cells, hemoglobin and hematocrit concentrations which were associated with increase in cardiac enzymes markers like creatine kinase (CK), CK-MB and lactate dehydrogenase. Treatment with DZR reversed the hematological statistics and the biochemical markers of CFZ-induced cardiotoxicity. Furthermore, DZR also attenuated the effects of CFZ-induced toxic effect on redox markers such as malondialdehyde and reduced glutathione. Above findings were further confirmed by beta-myosin heavy chain (ß-MHC) and alpha-MHC (α-MHC) gene expression. Histopathological reports suggested that DZR ameliorates CFZ-induced changes in cardiac cellular architecture in rats. These results confirm that DZR protects heart from CFZ-induced cardiotoxicity.

Mouse models for the study of postnatal cardiac hypertrophy.

The main objective of this study was to create a postnatal model for cardiac hypertrophy (CH), in order to explain the mechanisms that are present in childhood cardiac hypertrophy. Five days after implantation, intraperitoneal (IP) isoproterenol (ISO) was injected for 7 days to pregnant female mice. The fetuses were obtained at 15, 17 and 19 dpc from both groups, also newborns (NB), neonates (7-15 days) and young adults (6 weeks of age). Histopathological exams were done on the hearts. Immunohistochemistry and western blot demonstrated GATA4 and PCNA protein expression, qPCR real time the mRNA of adrenergic receptors (α-AR and ß-AR), alpha and beta myosins (α-MHC, ß-MHC) and GATA4. After the administration of ISO, there was no change in the number of offsprings. We observed significant structural changes in the size of the offspring hearts. Morphometric analysis revealed an increase in the size of the left ventricular wall and interventricular septum (IVS). Histopathological analysis demonstrated loss of cellular compaction and presence of left ventricular small fibrous foci after birth. Adrenergic receptors might be responsible for changing a physiological into a pathological hypertrophy. However GATA4 seemed to be the determining factor in the pathology. A new animal model was established for the study of pathologic CH in early postnatal stages.

Cardiac-specific Traf2 overexpression enhances cardiac hypertrophy through activating AKT/GSK3ß signaling.

Tumor necrosis factor superfamily ligands provoke a dilated cardiac phenotype signal through a common scaffolding protein termed tumor necrosis factor receptor-associated factor 2 (Traf2); however, Traf2 signaling in the adult mammalian cardiac hypertrophy is not fully understood. This study was aimed to identify the effect of Traf2 on cardiac hypertrophy and the underlying mechanisms. A significant up-regulation of Traf2 expression was observed in mice failing hearts. To further investigate the role of Traf2 in cardiac hypertrophy, we used cultured neonatal rat cardiomyocytes with gain and loss of Traf2 function and cardiac-specific Traf2-overexpressing transgenic (TG) mice. In cultured cardiomyocytes, Traf2 positively regulated angiotensin II (Ang II)-mediated hypertrophic growth, as detected by [(3)H]-Leucine incorporation, cardiac myocyte area, and hypertrophic marker protein levels. Cardiac hypertrophy in vivo was produced by constriction of transverse aortic (TAC) in TG mice and their wild-type controls. The extent of cardiac hypertrophy was evaluated by echocardiography as well as by pathological and molecular analyses of heart samples. Traf2 overexpression in the heart remarkably enhanced cardiac hypertrophy, left ventricular dysfunction in mice in response to TAC. Further analysis of the signaling pathway in vitro and in vivo suggested that these adverse effects of Traf2 were associated with the activation of AKT/glycogen synthase kinase 3ß (GSK3ß). The present study demonstrates that Traf2 serves as a novel mediator that enhanced cardiac hypertrophy by activating AKT/GSK3ß signaling.

Contractile abnormalities and altered drug response in engineered heart tissue from Mybpc3-targeted knock-in mice.

Myosin-binding protein C (Mybpc3)-targeted knock-in mice (KI) recapitulate typical aspects of human hypertrophic cardiomyopathy. We evaluated whether these functional alterations can be reproduced in engineered heart tissue (EHT) and yield novel mechanistic information on the function of cMyBP-C. EHTs were generated from cardiac cells of neonatal KI, heterozygous (HET) or wild-type controls (WT) and developed without apparent morphological differences. KI had 70% and HET 20% lower total cMyBP-C levels than WT, accompanied by elevated fetal gene expression. Under standard culture conditions and spontaneous beating, KI EHTs showed more frequent burst beating than WT and occasional tetanic contractions (14/96). Under electrical stimulation (6Hz, 37°C) KI EHTs exhibited shorter contraction and relaxation times and a twofold higher sensitivity to external [Ca(2+)]. Accordingly, the sensitivity to verapamil was 4-fold lower and the response to isoprenaline or the Ca(2+) sensitizer EMD 57033 2- to 4-fold smaller. The loss of EMD effect was verified in 6-week-old KI mice in vivo. HET EHTs were apparently normal under basal conditions, but showed similarly altered contractile responses to [Ca(2+)], verapamil, isoprenaline and EMD. In contrast, drug-induced changes in intracellular Ca(2+) transients (Fura-2) were essentially normal. In conclusion, the present findings in auxotonically contracting EHTs support the idea that cMyBP-C's normal role is to suppress force generation at low intracellular Ca(2+) and stabilize the power-stroke step of the cross bridge cycle. Pharmacological testing in EHT unmasked a disease phenotype in HET. The altered drug response may be clinically relevant.