Mitochondrial Responses to Sublethal Doxorubicin in H9c2 Cardiomyocytes: The Role of Phosphorylated CaMKII.

Background: Doxorubicin (Dox) is effective against different types of cancers, but it poses cardiotoxic side effects, frequently resulting in irreversible heart failure. However, the complexities surrounding this cardiotoxicity, especially at sublethal dosages, remain to be fully elucidated. We investigated early cellular disruptions in response to sublethal Dox, with a specific emphasis on the role of phosphorylated calcium/calmodulin-dependent protein kinase II (CaMKII) in initiating mitochondrial dysfunction. Methods: This study utilized the H9c2 cardiomyocyte model to identify a sublethal concentration of Dox and investigate its impact on mitochondrial health using markers such as mitochondrial membrane potential (MMP), mitophagy initiation, and mitochondrial calcium dynamics. We examined the roles of and interactions between CaMKII, dynamin-related protein 1 (Drp1), and the mitochondrial calcium uniporter (MCU) in Dox-induced mitochondrial disruption using specific inhibitors, such as KN-93, Mdivi-1, and Ru360, respectively. Results: Exposure to a sublethal dose of Dox reduced the MMP red-to-green fluorescence ratio in H9c2 cells by 40.6% compared with vehicle, and increased the proportion of cells undergoing mitophagy from negligible levels compared with vehicle to 62.2%. Mitochondrial calcium levels also increased by 8.7-fold compared with the vehicle group. Notably, the activation of CaMKII, particularly its phosphorylated form, was pivotal in driving these mitochondrial changes, as inhibition using KN-93 restored MMP and decreased mitophagy. However, inhibition of Drp1 and MCU functions had a limited impact on the observed mitochondrial disruptions. Conclusion: Sublethal administration of Dox is closely linked to CaMKII activation through phosphorylation, emphasizing its pivotal role in early mitochondrial disruption. These findings present a promising direction for developing therapeutic strategies that may alleviate the cardiotoxic effects of Dox, potentially increasing its clinical efficacy.

[Gut microbiota and cardiovascular disease-insights into the potential and challenges of drug discovery].

Cardiovascular disease is a major cause of death worldwide, with high prevalence and morbidity. Recent advances in technology have reported that abnormalities in the gut microbiota are associated with a variety of diseases, including cardiovascular diseases. The gut microbiota is a complex ecosystem that plays an important role in maintaining host health. It has been reported that the imbalance of gut microbiota causes changes in the production of substances derived from gut bacteria, such as short-chain fatty acids, trimethylamine-N-oxide, and lipopolysaccharide, and contributes to the development of cardiovascular diseases. In the drug discovery, it is a promising approach to prevention and therapy of the cardiovascular disease to focus on the relation between gut and heart, such as gut bacteria. However, there are challenges that must be overcome to convert this approach into effective therapy. In this review, we focus on cardiovascular diseases, particularly atherosclerotic disease, heart failure, and atrial fibrillation, and discuss the relationship between gut bacteria and substances derived from gut bacteria in cardiovascular disease. We also discuss the challenges and potential of drug discovery targeting the gut-heart relationship for the treatment and prevention of cardiovascular disease.

Inhibition of the uric acid efflux transporter ABCG2 enhances stimulating effect of soluble uric acid on IL-1ß production in murine macrophage-like J774.1 cells.

Soluble uric acid (UA) absorbed by cells through UA transporters (UATs) accumulates intracellularly, activates the NLRP3 inflammasome and thereby increases IL-1ß secretion. ABCG2 transporter excludes intracellular UA. However, it remains unknown whether ABCG2 inhibition leads to intracellular accumulation of UA and increases IL-1ß production. In this study, we examined whether genetic and pharmacological inhibition of ABCG2 could increase IL-1ß production in mouse macrophage-like J774.1 cells especially under hyperuricemic conditions. We determined mRNA and protein levels of pro-IL-1ß, mature IL-1ß, caspase-1 and several UATs in culture supernatants and lysates of J774.1 cells with or without soluble UA pretreatment. Knockdown experiments using an shRNA against ABCG2 and pharmacological experiments with an ABCG2 inhibitor were conducted. Extracellularly applied soluble UA increased protein levels of pro-IL-1ß, mature IL-1ß and caspase-1 in the culture supernatant from lipopolysaccharide (LPS)-primed and monosodium urate crystal (MSU)-stimulated J774.1 cells. J774.1 cells expressed UATs of ABCG2, GLUT9 and MRP4, and shRNA knockdown of ABCG2 increased protein levels of pro-IL-1ß and mature IL-1ß in the culture supernatant. Soluble UA increased mRNA and protein levels of ABCG2 in J774.1 cells without either LPS or MSU treatment. An ABCG2 inhibitor, febuxostat, but not a urate reabsorption inhibitor, dotinurad, enhanced IL-1ß production in cells pretreated with soluble UA. In conclusion, genetic and pharmacological inhibition of ABCG2 enhanced IL-1ß production especially under hyperuricemic conditions by increasing intracellularly accumulated soluble UA that activates the NLRP3 inflammasome and pro-IL-1ß transcription in macrophage-like J774.1 cells.

Thrombin induces a temporal biphasic vascular response through the differential phosphorylation of endothelial nitric oxide synthase via protease-activated receptor-1 and protein kinase C.

Endothelial nitric oxide synthase (eNOS) is a critical regulatory enzyme that controls vascular tone via the production of nitric oxide. Although thrombin also modulates vascular tone predominantly via the activation of protease-activated receptors (PARs), the time course and mechanisms involved in how thrombin controls eNOS enzymatic activity are unknown. eNOS enzymatic activity is enhanced by the phosphorylation of eNOS-Ser1177 and reduced by the phosphorylation of eNOS-Thr495. In this study, we hypothesized that thrombin regulates vascular tone through the differential phosphorylation of eNOS. Using rat descending aorta, we show that thrombin modulates vascular tone in an eNOS-dependent manner via activated PAR-1. We also show that thrombin causes a temporal biphasic response. Protein kinase C (PKC) is associated with second phase of thrombin-induced response. Western blot analysis demonstrated thrombin phosphorylated eNOS-Ser1177 and eNOS-Thr495 in human umbilical vein endothelial cells. A PKC inhibitor suppressed the thrombin-induced phosphorylation of eNOS-Thr495, but not that of eNOS-Ser1177. Our results suggest that thrombin induces a temporal biphasic vascular response through the differential phosphorylation of eNOS via activated PAR-1. Thrombin causes transient vasorelaxation by the phosphorylation of eNOS-Ser1177, and subsequent attenuation of vasorelaxation by the phosphorylation of eNOS-Thr495 via PKC, leading to the modulation of vascular tone.

Preventive effects of nitrate-rich beetroot juice supplementation on monocrotaline-induced pulmonary hypertension in rats.

Beetroot (Beta vulgaris L.) has a high level of nitrate; therefore, its dietary intake could increase nitric oxide (NO) level in the body, possibly preventing the development of pulmonary hypertension (PH). In this study, we examined the effects of beetroot juice (BJ) supplementation on PH and the contribution of nitrate to such effects using a rat model of monocrotaline (MCT, 60 mg/kg s.c.)-induced PH. Rats were injected subcutaneously with saline or 60 mg/kg MCT and were sacrificed 28 days after the injection. In some rats injected with MCT, BJ was supplemented from the day of MCT injection to the day of sacrifice. First, MCT-induced right ventricular systolic pressure elevation, pulmonary arterial medial thickening and muscularization, and right ventricular hypertrophy were suppressed by supplementation with low-dose BJ (nitrate: 1.3 mmol/L) but not high-dose BJ (nitrate: 4.3 mmol/L). Of the plasma nitrite, nitrate, and their sum (NOx) levels, only the nitrate levels were found to be increased by the high-dose BJ supplementation. Second, in order to clarify the possible involvement of nitrate in the preventive effects of BJ on PH symptoms, the effects of nitrate-rich BJ (nitrate: 0.9 mmol/L) supplementation were compared with those of the nitrate-depleted BJ. While the former exerted preventive effects on PH symptoms, such effects were not observed in rats supplemented with nitrate-depleted BJ. Neither supplementation with nitrate-rich nor nitrate-depleted BJ affected plasma nitrite, nitrate, and NOx levels. These findings suggest that a suitable amount of BJ ingestion, which does not affect systemic NO levels, can prevent the development of PH in a nitrate-dependent manner. Therefore, BJ could be highly useful as a therapy in patients with PH.

Cardioprotective Effects of a Nonsteroidal Mineralocorticoid Receptor Blocker, Esaxerenone, in Dahl Salt-Sensitive Hypertensive Rats.

We investigated the effects of esaxerenone, a novel, nonsteroidal, and selective mineralocorticoid receptor blocker, on cardiac function in Dahl salt-sensitive (DSS) rats. We provided 6-week-old DSS rats a high-salt diet (HSD, 8% NaCl). Following six weeks of HSD feeding (establishment of cardiac hypertrophy), we divided the animals into the following two groups: HSD or HSD + esaxerenone (0.001%, w/w). In survival study, all HSD-fed animals died by 24 weeks of age, whereas the esaxerenone-treated HSD-fed animals showed significantly improved survival. We used the same protocol with a separate set of animals to evaluate the cardiac function by echocardiography after four weeks of treatment. The results showed that HSD-fed animals developed cardiac dysfunction as evidenced by reduced stroke volume, ejection fraction, and cardiac output. Importantly, esaxerenone treatment decreased the worsening of cardiac dysfunction concomitant with a significantly reduced level of systolic blood pressure. In addition, treatment with esaxerenone in HSD-fed DSS rats caused a reduced level of cardiac remodeling as well as fibrosis. Furthermore, inflammation and oxidative stress were significantly reduced. These data indicate that esaxerenone has the potential to mitigate cardiac dysfunction in salt-induced myocardial injury in rats.

MicroRNA-494-3p inhibits formation of fast oxidative muscle fibres by targeting E1A-binding protein p300 in human-induced pluripotent stem cells.

MYOD-induced microRNA-494-3p expression inhibits fast oxidative myotube formation by downregulating myosin heavy chain 2 (MYH2) in human induced pluripotent stem cells (hiPSCs) during skeletal myogenesis. However, the molecular mechanisms regulating MYH2 expression via miR-494-3p remain unknown. Here, using bioinformatic analyses, we show that miR-494-3p potentially targets the transcript of the E1A-binding protein p300 at its 3'-untranslated region (UTR). Myogenesis in hiPSCs with the Tet/ON-myogenic differentiation 1 (MYOD1) gene (MyoD-hiPSCs) was induced by culturing them in doxycycline-supplemented differentiation medium for 7 days. p300 protein expression decreased after transient induction of miR-494-3p during myogenesis. miR-494-3p mimics decreased the levels of p300 and its downstream targets MYOD and MYH2 and myotube formation efficiency. p300 knockdown decreased myotube formation efficiency, MYH2 expression, and basal oxygen consumption rate. The binding of miR-494-3p to the wild type p300 3'-UTR, but not the mutated site, was confirmed using luciferase assay. Overexpression of p300 rescued the miR-494-3p mimic-induced phenotype in MyoD-hiPSCs. Moreover, miR-494-3p mimic reduced the levels of p300, MYOD, and MYH2 in skeletal muscles in mice. Thus, miR-494-3p might modulate MYH2 expression and fast oxidative myotube formation by directly regulating p300 levels during skeletal myogenesis in MyoD-hiPSCs and murine skeletal muscle tissues.

Involvement of γ-Glutamyl Transpeptidase in Ischemia/Reperfusion-Induced Cardiac Dysfunction in Isolated Rat Hearts.

GGsTop is a highly potent and specific, and irreversible γ-glutamyl transpeptidase (GGT) inhibitor without any influence on glutamine amidotransferases. The aim of the present study was to investigate the involvement of GGT in ischemia/reperfusion-induced cardiac dysfunction by assessing the effects of a treatment with GGsTop. Using a Langendorff apparatus, excised rat hearts underwent 40 min of global ischemia without irrigation and then 30 min of reperfusion. GGT activity was markedly increased in cardiac tissues exposed to ischemia, and was inhibited by the treatment with GGsTop. Exacerbation of cardiac functional parameters caused by ischemia and reperfusion, namely the reduction of left ventricular (LV) developed pressure and the maximum and negative minimum values of the first derivative of LV pressure, and the increment in LV end-diastolic pressure was significantly attenuated by GGsTop treatment. The treatment with GGsTop suppressed excessive norepinephrine release in the coronary perfusate, a marker for myocardial dysfunction, after ischemia/reperfusion. In addition, oxidative stress indicators in myocardium, including superoxide and malondialdehyde, after ischemia/reperfusion were significantly low in the presence of GGsTop. These observations demonstrate that enhanced GGT activity contributes to cardiac damage after myocardial ischemia/reperfusion, possibly via increased oxidative stress and subsequent norepinephrine overflow. GGT inhibitors have potential as a therapeutic strategy to prevent myocardial ischemia/reperfusion injury in vivo.

Effects of Beet Juice Supplementation on Monocrotaline-Induced Pulmonary Hypertension in Rats.

BACKGROUND: Recently, attention has been focused on the cardiovascular protective effects of beet juice (BJ) with high amounts of nitrate. In this study, we examined the effect of BJ supplementation in a rat model of monocrotaline (MCT)-induced pulmonary hypertension (PH). METHODS: MCT (60 mg/kg) was subcutaneously administered to rats, and BJ (prepared by dissolving BJ powder at a concentration of 1 g/l or 10 g/l in drinking water) supplementation was started from the day of, 1 week before, and 2 weeks after MCT injection. Saline-injected rats given drinking water were used as controls. RESULTS: Low-dose BJ supplementation starting from the day of MCT injection exerted protective effects on the MCT-induced elevation of right ventricular systolic pressure, right ventricular hypertrophy, and pulmonary arterial remodeling, without causing a significant increase in plasma nitrite plus nitrate (NOx) levels. On the other hand, such beneficial effects were not observed with high-dose BJ supplementation, although the NOx levels were slightly higher than those in the low-dose group. In addition, low-dose BJ supplementation starting from 1 week before MCT injection did not improve PH symptoms, as described above. Furthermore, low-dose BJ supplementation starting from 2 weeks after MCT injection was ineffective against functional and morphological alterations in pulmonary circulation associated with MCT-induced PH. CONCLUSIONS: Habitual ingestion of a suitable amount of BJ could be a potential option for preventing PH. However, beneficial effects cannot be expected when PH has developed to some degree.

Stimulation of nitric oxide-sensitive soluble guanylate cyclase in monocrotaline-induced pulmonary hypertensive rats.

AIMS: In this study, we examined whether a disruption in the balance between nitric oxide (NO)-sensitive and -insensitive soluble guanylate cyclase (sGC) is observed in pulmonary hypertension (PH) and whether treatment with NO-enhancing drugs can halt disease progression. MAIN METHODS: Rats were injected subcutaneously with saline or 60 mg/kg monocrotaline (MCT). At 14 days after injection, the vascular reactivity of isolated extralobar pulmonary arteries was assessed by organ chamber technique. In a separate experiment, isosorbide mononitrate (0.3 or 1 g/L) or sodium nitrite (30 or 300 mg/L) was administered in drinking water for the last 14 days (from day 15 to day 28), and their therapeutic potential was evaluated. KEY FINDINGS: The NO-sensitive sGC stimulant BAY 41-2272 and the NO-insensitive sGC stimulant BAY 60-2770 both relaxed the pulmonary arteries, which was comparable between saline- and MCT-injected rats. Treatment with isosorbide mononitrate suppressed the MCT-induced right ventricular systolic pressure (RVSP) elevation and pulmonary arterial medial thickening but not right ventricular hypertrophy. However, the beneficial effects on RVSP and pulmonary vascular remodeling were not observed when a high dose was administered. The same results were obtained following the sodium nitrite treatment. Interestingly, NO-enhancing drugs did not increase plasma nitrite plus nitrate levels at a dose that provided the greatest therapeutic advantage. SIGNIFICANCE: These findings suggest that the balance between NO-sensitive and -insensitive sGC is not disrupted in the early stage of MCT-induced PH. Furthermore, supplementation with an adequate amount of NO may be a useful therapy to prevent the progression of PH.

Establishment of Imaging Spectroscopy of Nuclear Gamma-Rays based on Geometrical Optics.

Since the discovery of nuclear gamma-rays, its imaging has been limited to pseudo imaging, such as Compton Camera (CC) and coded mask. Pseudo imaging does not keep physical information (intensity, or brightness in Optics) along a ray, and thus is capable of no more than qualitative imaging of bright objects. To attain quantitative imaging, cameras that realize geometrical optics is essential, which would be, for nuclear MeV gammas, only possible via complete reconstruction of the Compton process. Recently we have revealed that "Electron Tracking Compton Camera" (ETCC) provides a well-defined Point Spread Function (PSF). The information of an incoming gamma is kept along a ray with the PSF and that is equivalent to geometrical optics. Here we present an imaging-spectroscopic measurement with the ETCC. Our results highlight the intrinsic difficulty with CCs in performing accurate imaging, and show that the ETCC surmounts this problem. The imaging capability also helps the ETCC suppress the noise level dramatically by ~3 orders of magnitude without a shielding structure. Furthermore, full reconstruction of Compton process with the ETCC provides spectra free of Compton edges. These results mark the first proper imaging of nuclear gammas based on the genuine geometrical optics.

Characterization of Ruminococcus albus cellodextrin phosphorylase and identification of a key phenylalanine residue for acceptor specificity and affinity to the phosphate group.

Ruminococcus albus has the ability to intracellularly degrade cello-oligosaccharides primarily via phosphorolysis. In this study, the enzymatic characteristics of R. albus cellodextrin phosphorylase (RaCDP), which is a member of glycoside hydrolase family 94, was investigated. RaCDP catalyzes the phosphorolysis of cellotriose through an ordered 'bi bi' mechanism in which cellotriose binds to RaCDP before inorganic phosphate, and then cellobiose and glucose 1-phosphate (Glc1P) are released in that order. Among the cello-oligosaccharides tested, RaCDP had the highest phosphorolytic and synthetic activities towards cellohexaose and cellopentaose, respectively. RaCDP successively transferred glucosyl residues from Glc1P to the growing cello-oligosaccharide chain, and insoluble cello-oligosaccharides comprising a mean of eight residues were produced. Sophorose, laminaribiose, ß-1,4-xylobiose, ß-1,4-mannobiose and cellobiitol served as acceptors for RaCDP. RaCDP had very low affinity for phosphate groups in both the phosphorolysis and synthesis directions. A sequence comparison revealed that RaCDP has Gln at position 646 where His is normally conserved in the phosphate binding sites of related enzymes. A Q646H mutant showed approximately twofold lower apparent K(m) values for inorganic phosphate and Glc1P than the wild-type. RaCDP has Phe at position 633 corresponding to Tyr and Val in the +1 subsites of cellobiose phosphorylase and N,N'-diacetylchitobiose phosphorylase, respectively. A F633Y mutant showed higher preference for cellobiose over ß-1,4-mannobiose as an acceptor substrate in the synthetic reaction than the wild-type. Furthermore, the F633Y mutant showed 75- and 1100-fold lower apparent Km values for inorganic phosphate and Glc1P, respectively, in phosphorolysis and synthesis of cellotriose.

Role of superoxide production in post-ischemic cardiac dysfunction and norepinephrine overflow in rat hearts.

Reactive oxygen species and norepinephrine are known as physiological active substances which cause cell damage and cardiac dysfunction in myocardial ischemia/reperfusion injury. We investigated the role of reactive oxygen species, especially superoxide (O2(-)), in ischemia-induced norepinephrine overflow and cardiac dysfunction using superoxide scavengers tempol and tiron. According to the Langendorff technique, isolated rat hearts were subjected to 40-min global ischemia followed by 30-min reperfusion. Tempol (10 and 100 µM) and tiron (100 and 500 µM) were perfused 15 min before ischemia and during reperfusion. Cardiac levels of oxidative stress markers such as O2(-) and malondialdehyde were notably increased during ischemia and following reperfusion, which were suppressed by the administration of tempol or tiron. These agents significantly improved ischemia/reperfusion-induced cardiac dysfunction such as decreased left ventricular developed pressure and the maximum and minimum value of the first derivative of left ventricular pressure and increased left ventricular end-diastolic pressure. Furthermore, norepinephrine overflow in the coronary effluent after ischemia/reperfusion was significantly suppressed by the administration of each agent. These results suggest that endogenously increased O2(-) is involved in norepinephrine overflow and cardiac dysfunction after myocardial ischemia/reperfusion.