Muscarinic and nicotinic receptors stimulation by vagus nerve stimulation ameliorates trastuzumab-induced cardiotoxicity via reducing programmed cell death in rats.

Despite its efficacy in human epidermal growth factor receptor 2 positive cancer treatment, trastuzumab-induced cardiotoxicity (TIC) has become a growing concern. Due to the lack of cardiomyocyte regeneration and proliferation in adult heart, cell death significantly contributes to cardiovascular diseases. Cardiac autonomic modulation by vagus nerve stimulation (VNS) has shown cardioprotective effects in several heart disease models, while the effects of VNS and its underlying mechanisms against TIC have not been found. Forty adult male Wistar rats were divided into 5 groups: (i) control without VNS (CSham) group, (ii) trastuzumab (4 mg/kg/day, i.p.) without VNS (TSham) group, (iii) trastuzumab + VNS (TVNS) group, (iv) trastuzumab + VNS + mAChR blocker (atropine; 1 mg/kg/day, ip, TVNS + Atro) group, and (v) trastuzumab + VNS + nAChR blocker (mecamylamine; 7.5 mg/kg/day, ip, TVNS + Mec) group. Our results showed that trastuzumab induced cardiac dysfunction by increasing autonomic dysfunction, mitochondrial dysfunction/dynamics imbalance, and cardiomyocyte death including apoptosis, autophagic deficiency, pyroptosis, and ferroptosis, which were notably alleviated by VNS. However, mAChR and nAChR blockers significantly inhibited the beneficial effects of VNS on cardiac autonomic dysfunction, mitochondrial dysfunction, cardiomyocyte apoptosis, pyroptosis, and ferroptosis. Only nAChR could counteract the protective effects of VNS on cardiac mitochondrial dynamics imbalance and autophagy insufficiency. Therefore, VNS prevented TIC by rebalancing autonomic activity, ameliorating mitochondrial dysfunction and cardiomyocyte death through mAChR and nAChR activation. The current study provides a novel perspective elucidating the potential treatment of VNS, thus also offering other pharmacological therapeutic promises in TIC patients.

Acetylcholinesterase inhibition protects against trastuzumab-induced cardiotoxicity through reducing multiple programmed cell death pathways.

BACKGROUND: Trastuzumab (Trz)-induced cardiotoxicity (TIC) is one of the most common adverse effects of targeted anticancer agents. Although oxidative stress, inflammation, mitochondrial dysfunction, apoptosis, and ferroptosis have been identified as potential mechanisms underlying TIC, the roles of pyroptosis and necroptosis under TIC have never been investigated. It has been shown that inhibition of acetylcholinesterase function by using donepezil exerts protective effects in various heart diseases. However, it remains unknown whether donepezil exerts anti-cardiotoxic effects in rats with TIC. We hypothesized that donepezil reduces mitochondrial dysfunction, inflammation, oxidative stress, and cardiomyocyte death, leading to improved left ventricular (LV) function in rats with TIC. METHODS: Male Wistar rats were randomly assigned to be Control or Trz groups (Trz 4 mg/kg/day, 7 days, I.P.). Rats in Trz groups were assigned to be co-treated with either drinking water (Trz group) or donepezil 5 mg/kg/day (Trz + DPZ group) via oral gavage for 7 days. Cardiac function, heart rate variability (HRV), and biochemical parameters were evaluated. RESULTS: Trz-treated rats had impaired LV function, HRV, mitochondrial function, and increased inflammation and oxidative stress, leading to apoptosis, ferroptosis, and pyroptosis. Donepezil co-treatment effectively decreased those adverse effects of TIC, resulting in improved LV function. An in vitro study revealed that the cytoprotective effects of donepezil were abolished by a muscarinic acetylcholine receptor (mAChR) antagonist. CONCLUSIONS: Donepezil exerted cardioprotection against TIC via attenuating mitochondrial dysfunction, oxidative stress, inflammation, and cardiomyocyte death, leading to improved LV function through mAChR activation. This suggests that donepezil could be a novel intervention strategy in TIC.

GSDMD-mediated pyroptosis dominantly promotes left ventricular remodeling and dysfunction in post-myocardial infarction: a comparison across modes of programmed cell death and mitochondrial involvement.

BACKGROUND: Myocardial infarction (MI) has recently accounted for more than one-third of global mortality. Multiple molecular pathological pathways, such as oxidative stress, inflammation, and mitochondrial dysfunction, have been recognized as possible mechanisms in the development of MI. Furthermore, different phases of ischemic injury following the progression of MI were also associated with multiple types of programmed cell death (PCDs), including apoptosis, necroptosis, ferroptosis, and pyroptosis. However, it remains unknown whether which types of PCDs play the most dominant role in post-myocardial infarction (post-MI). METHOD: In this study, we used a preclinical rat model of MI induced by permanent left anterior descending coronary artery (LAD) ligation (n = 6) or a sham operated rat model (n = 6). After a 5-week experiment, cardiac function and morphology, mitochondrial studies, and molecular signaling analysis of PCDs were determined. RESULTS: Herein, we demonstrated that post-MI rats had considerably impaired cardiac geometry, increased oxidative stress, myocardial injuries, and subsequently contractile dysfunction. They also exhibited worsened cardiac mitochondrial function and dynamic imbalance. More importantly, we found that post-MI mediated abundant myocardial cell death through multiple PCDs, including apoptosis, necroptosis, and pyroptosis, but not ferroptosis. CONCLUSION: In this study, we provide the first insights into the mechanism of PCDs by pyroptosis, which is leveraged as the most dominant mode of cell death after MI.

Ferroptosis inhibitor improves cardiac function more effectively than inhibitors of apoptosis and necroptosis through cardiac mitochondrial protection in rats with iron-overloaded cardiomyopathy.

Iron overload cardiomyopathy (IOC) is the leading cause of death in cases of iron overload in patients. Previous studies demonstrated that iron overload led to cardiomyocyte dysfunction and death through multiple pathways including apoptosis, necroptosis and ferroptosis. However, the dominant cell death pathway in the iron-overloaded heart needs clarification. We tested the hypothesis that ferroptosis, an iron-dependent cell death, plays a dominant role in IOC, and ferroptosis inhibitor exerts greater efficacy than inhibitors of apoptosis and necroptosis on improving cardiac function in iron-overloaded rats. Iron dextran was injected intraperitoneally into male Wistar rats for four weeks to induce iron overload. Then, the rats were divided into 5 groups: treated with vehicle, apoptosis inhibitor (z-VAD-FMK), necroptosis inhibitor (Necrostatin-1), ferroptosis inhibitor (Ferrostatin-1) or iron chelator (deferoxamine) for 2 weeks. Cardiac function, mitochondrial function, apoptosis, necroptosis and ferroptosis were determined. The increased expression of apoptosis-, necroptosis- and ferroptosis-related proteins, were associated with impaired cardiac and mitochondrial function in iron-overloaded rats. All cell death inhibitors attenuated cardiac apoptosis, necroptosis and ferroptosis in iron-overloaded rats. Ferrostatin-1 was more effective than the other drugs in diminishing mitochondrial dysfunction and Bax/Bcl-2 ratio. Moreover, both Ferrostatin-1 and deferoxamine reversed iron overload-induced cardiac dysfunction as indicated by restored left ventricular ejection fraction and E/A ratio, whereas z-VAD-FMK and Necrostatin-1 only partially improved this parameter. These results indicated that ferroptosis could be the predominant form of cardiomyocyte death in IOC, and that inhibiting ferroptosis might be a potential novel treatment for IOC.

Acute administration of myeloid differentiation factor 2 inhibitor and N-acetyl cysteine attenuate brain damage in rats with cardiac ischemia/reperfusion injury.

Inflammation and oxidative stress are mechanisms which potentially underlie the brain damage that can occur after cardiac ischemic and reperfusion (I/R) injury. 2i-10 is a new anti-inflammatory agent, acting via direct inhibition of myeloid differentiation factor 2 (MD2). However, the effects of 2i-10 and the antioxidant N-acetylcysteine (NAC) on pathologic brain in cardiac I/R injury are unknown. We hypothesized that 2i-10 and NAC offer similar neuroprotection levels against dendritic spine reduction through attenuation of brain inflammation, loss of tight junction integrity, mitochondrial dysfunction, reactive gliosis, and suppression of AD protein expression in rats with cardiac I/R injury. Male rats were allocated to either sham or acute cardiac I/R group (30 min of cardiac ischemia and 120 min of reperfusion). Rats in cardiac I/R group were given one of following treatments intravenously at the onset of reperfusion: vehicle, 2i-10 (20 or 40 mg/kg), and NAC (75 or 150 mg/kg). The brain was then used to determine biochemical parameters. Cardiac I/R led to cardiac dysfunction with dendritic spine loss, loss of tight junction integrity, brain inflammation, and mitochondrial dysfunction. Treatment with 2i-10 (both doses) effectively reduced cardiac dysfunction, tau hyperphosphorylation, brain inflammation, mitochondrial dysfunction, dendritic spine loss, and improved tight junction integrity. Although both doses of NAC effectively reduced brain mitochondrial dysfunction, treatment using a high dose of NAC reduced cardiac dysfunction, brain inflammation, and dendritic spine loss. In conclusion, treatment with 2i-10 and a high dose of NAC at the onset of reperfusion alleviated brain inflammation and mitochondrial dysfunction, consequently reducing dendritic spine loss in rats with cardiac I/R injury.

Therapeutic potential of a single-dose melatonin in the attenuation of cardiac ischemia/reperfusion injury in prediabetic obese rats.

Although acute melatonin treatment effectively reduces cardiac ischemia/reperfusion (I/R) injury in lean rats by modulating melatonin receptor 2 (MT2), there is no information regarding the temporal effects of melatonin administration during cardiac I/R injury in prediabetic obese rats. Prediabetic obese rats induced by chronic consumption of a high-fat diet (HFD) were used. The rats underwent a cardiac I/R surgical procedure (30-min of ischemia, followed by 120-min of reperfusion) and were randomly assigned to receive either vehicle or melatonin treatment. In the melatonin group, rats were divided into 3 different subgroups: (1) pretreatment, (2) treatment during ischemic period, (3) treatment at the reperfusion onset. In the pretreatment subgroup either a nonspecific MT blocker (Luzindole) or specific MT2 blocker (4-PPDOT) was also given to the rats prior to melatonin treatment. Pretreatment with melatonin (10 mg/kg) effectively reduced cardiac I/R injury by reducing infarct size, arrhythmia, and LV dysfunction. Reduction in impaired mitochondrial function, mitochondrial dynamic balance, oxidative stress, defective autophagy, and apoptosis were observed in rats pretreated with melatonin. Unfortunately, the cardioprotective benefits were not observed when 10-mg/kg of melatonin was acutely administered to the rats after cardiac ischemia. Thus, we increased the dose of melatonin to 20 mg/kg, and it was administered to the rats during ischemia or at the onset of reperfusion. The results showed that 20-mg/kg of melatonin effectively reduced cardiac I/R injury to a similar extent to the 10-mg/kg pretreatment regimen. The MT2 blocker inhibited the protective effects of melatonin. Acute melatonin treatment during cardiac I/R injury exerted protective effects in prediabetic obese rats. However, a higher dose of melatonin is required when given after the onset of cardiac ischemia. These effects of melatonin were mainly mediated through activation of MT2.

Vagus nerve stimulation exerts cardioprotection against doxorubicin-induced cardiotoxicity through inhibition of programmed cell death pathways.

The aberration of programmed cell death including cell death associated with autophagy/mitophagy, apoptosis, necroptosis, pyroptosis, and ferroptosis can be observed in the development and progression of doxorubicin-induced cardiotoxicity (DIC). Vagus nerve stimulation (VNS) has been shown to exert cardioprotection against cardiomyocyte death through the release of the neurotransmitter acetylcholine (ACh) under a variety of pathological conditions. However, the roles of VNS and its underlying mechanisms against DIC have never been investigated. Forty adults male Wistar rats were divided into 5 experimental groups: (i) control without VNS (CSham) group, (ii) doxorubicin (3 mg/kg/day, i.p.) without VNS (DSham) group, (iii) doxorubicin + VNS (DVNS) group, (iv) doxorubicin + VNS + mAChR antagonist (atropine; 1 mg/kg/day, ip, DVNS + Atro) group, and (v) doxorubicin + VNS + nAChR antagonist (mecamylamine; 7.5 mg/kg/day, ip, DVNS + Mec) group. Our results showed that doxorubicin insult led to left ventricular (LV) dysfunction through impaired cardiac autonomic balance, decreased mitochondrial function, imbalanced mitochondrial dynamics, and exacerbated cardiomyocyte death including autophagy/mitophagy, apoptosis, necroptosis, pyroptosis, and ferroptosis. However, VNS treatment improved cardiac mitochondrial and autonomic functions, and suppressed excessive autophagy, apoptosis, necroptosis, pyroptosis, and ferroptosis, leading to improved LV function. Consistent with this, ACh effectively improved cell viability and suppressed cell cytotoxicity in doxorubicin-treated H9c2 cells. In contrast, either inhibitors of muscarinic (mAChR) or nicotinic acetylcholine receptor (nAChR) completely abrogated the favorable effects mediated by VNS and acetylcholine. These findings suggest that VNS exerts cardioprotective effects against doxorubicin-induced cardiomyocyte death via activation of both mAChR and nAChR.

Blood metabolomes as non-invasive biomarkers and targets of metabolic interventions for doxorubicin and trastuzumab-induced cardiotoxicity.

This study aimed to identify the alterations of blood metabolome levels and their association with cardiac dysfunction and cardiac injury following treatment with doxorubicin and trastuzumab. Eight-week-old male Wistar rats were divided into four groups (n = 6 per group) to receive intraperitoneal injection with either: (1) 1 mL of normal saline solution (NSS) at days 0, 4, 8, 15, 22, and 29 (control group for doxorubicin); (2) 3 mg/kg/day of doxorubicin at days 0, 4, 8, 15, 22, and 29 (doxorubicin group); (3) 1 mL of NSS at days 0-6 (control group for trastuzumab); or (4) 4 mg/kg/day of trastuzumab at days 0-6 (trastuzumab group). Four days after the last injected dose, cardiac function was determined. The rats were then euthanized to collect venous blood and the heart for the quantification of 107 serum and 100 cardiac metabolomes using mass spectrometry-based targeted metabolomics. We observed strong relationships between 72 cardiac versus 61 serum metabolomes in doxorubicin and trastuzumab groups. Moreover, significant correlations between cardiac function and the cardiac injury biomarker versus 28 and 58 serum metabolomes were revealed in doxorubicin and trastuzumab-treated rats, respectively. Interestingly, the patterns of both serum and cardiac metabolome alterations differed between doxorubicin and trastuzumab groups. Our findings emphasize the potential role of the constituents of the blood metabolome as non-invasive biomarkers to assess severity and prognosis of heart failure induced by doxorubicin and trastuzumab. These findings may contribute to the development of metabolic-targeted therapy specific for cardioprotection during different phases of cancer treatment.

Therapeutic potentials of cell death inhibitors in rats with cardiac ischaemia/reperfusion injury.

Growing evidence demonstrated that cell death pathways including ferroptosis, apoptosis and necroptosis contribute to cardiac ischaemia/reperfusion (I/R) injury. We hypothesized that ferroptosis, apoptosis and necroptosis contribute differently to myocardial damage during acute cardiac I/R injury. Rats underwent cardiac I/R or sham operation. I/R-operated rats were divided into 4 groups: vehicle, apoptosis (Z-vad), ferroptosis (Fer-1) and necroptosis (Nec-1) inhibition. Rats in each cell death inhibitor group were subdivided into 3 different dose regimens: low, medium and high. Infarct size, left ventricular (LV) function, arrhythmias and molecular mechanism were investigated. Cardiac I/R caused myocardial infarction, LV dysfunction, arrhythmias, mitochondrial dysfunction, mitochondrial dynamic imbalance, inflammation, apoptosis and ferroptosis. Infarct size, LV dysfunction, mitochondrial dysfunction, apoptosis and ferroptosis were all reduced to a similar extent in rats treated with Z-vad (low and medium doses) or Fer-1 (medium and high doses). Fer-1 treatment also reduced mitochondrial dynamic imbalance and inflammation. No evidence of necroptosis was found in association with acute I/R injury, therefore Nec-1 treatment could not be assessed. Apoptosis and ferroptosis, not necroptosis, contributed to myocardial damage in acute I/R injury. Inhibitors of these 2 pathways provided effective cardioprotection in rats with I/R injury though modulation of mitochondrial function and attenuated apoptosis and ferroptosis.

Effectiveness of high cardiorespiratory fitness in cardiometabolic protection in prediabetic rats.

BACKGROUND: Caloric restriction and exercise are lifestyle interventions that effectively attenuate cardiometabolic impairment. However, cardioprotective effects of long-term lifestyle interventions and short-term lifestyle interventions followed by weight maintenance in prediabetes have never been compared. High cardiorespiratory fitness (CRF) has been shown to provide protection against prediabetes and cardiovascular diseases, however, the interactions between CRF, prediabetes, caloric restriction, and exercise on cardiometabolic health has never been investigated. METHODS: Seven-week-old male Wistar rats were fed with either a normal diet (ND; n = 6) or a high-fat diet (HFD; n = 30) to induce prediabetes for 12 weeks. Baseline CRF and cardiometabolic parameters were determined at this timepoint. The ND-fed rats were fed continuously with a ND for 16 more weeks. The HFD-fed rats were divided into 5 groups (n = 6/group) to receive one of the following: (1) a HFD without any intervention for 16 weeks, (2) 40% caloric restriction for 6 weeks followed by an ad libitum ND for 10 weeks, (3) 40% caloric restriction for 16 weeks, (4) a HFD plus an exercise training program for 6 weeks followed by a ND without exercise for 10 weeks, or (5) a HFD plus an exercise training program for 16 weeks. At the end of the interventions, CRF and cardiometabolic parameters were re-assessed. Then, all rats were euthanized and heart tissues were collected. RESULTS: Either short-term caloric restriction or exercise followed by weight maintenance ameliorated cardiometabolic impairment in prediabetes, as indicated by increased insulin sensitivity, improved blood lipid profile, improved mitochondrial function and oxidative phosphorylation, reduced oxidative stress and inflammation, and improved cardiac function. However, these benefits were not as effective as those of either long-term caloric restriction or exercise. Interestingly, high-level baseline CRF was correlated with favorable cardiac and metabolic profiles at follow-up in prediabetic rats, both with and without lifestyle interventions. CONCLUSIONS: Short-term lifestyle modification followed by weight maintenance improves cardiometabolic health in prediabetes. High CRF exerted protection against cardiometabolic impairment in prediabetes, both with and without lifestyle modification. These findings suggest that targeting the enhancement of CRF may contribute to the more effective treatment of prediabetes-induced cardiometabolic impairment.

Promoting mitochondrial fusion in doxorubicin-induced cardiotoxicity: a novel therapeutic target for cardioprotection.

Changes in mitochondrial dynamics have been recognized as being one of the mechanisms related to cardiotoxicity following a high cumulative dose of doxorubicin (DOX). A mitochondrial division inhibitor-1 (Mdivi-1) and fusion promoter (M1) have been shown to be cardioprotective in a variety of cardiovascular settings, however, their anticardiotoxic efficacy against DOX therapy remains unclear. We therefore investigated whether treatment with Mdivi-1 and M1 protects the heart against DOX-induced cardiotoxicity via mitochondria-targeted pathways. Male Wistar rats (n=40) received DOX (3 mg/kg, six doses, n=32) or 3% dimethylsulfoxide (DMSO) in the normal saline solution (NSS) (n=8) as a control. DOX-injected rats were given one of four treatments beginning with the first DOX injection via intraperitoneal injection: 1) 3% DMSO in NSS (n=8), 2) Mdivi-1 (1.2 mg/kg per day, n=8), 3) M1 (2 mg/kg per day, n=8), and 4) Mdivi-1+M1 (n=8) for 30 days. Cardiac function, mitochondrial function, oxidative stress, myocardial injury, and protein expression associated with inflammation, autophagy, mitophagy, apoptosis, and mitochondrial dynamics were determined. DOX caused a significant deterioration in mitochondrial function and dynamic regulation, and an increase in markers of oxidative stress, inflammation, myocardial injury, apoptosis, autophagy, and mitophagy, resulting in impaired cardiac function. Cotreatment of DOX with Mdivi-1, M1, or a combination of the two mitigated these detrimental effects of DOX. These findings imply that either inhibiting fission or promoting fusion of mitochondria protects the heart from DOX-induced myocardial damage. Modulation of mitochondrial dynamics could be a novel therapeutic target in alleviating DOX-induced cytotoxic effects without compromising its anticancer efficacy.

An apoptosis inhibitor suppresses microglial and astrocytic activation after cardiac ischemia/reperfusion injury.

OBJECTIVE: Microglial hyperactivation and apoptosis were observed following myocardial infarction and ischemia reperfusion (I/R) injury. This study aimed to test the hypothesis that the apoptosis inhibitor, Z-VAD, attenuates microglial and astrocytic hyperactivation and brain inflammation in rats with cardiac I/R injury. MATERIALS AND METHODS: Rats were subjected to either sham or cardiac I/R operation (30 min-ischemia followed by 120-min reperfusion), rats in the cardiac I/R group were given either normal saline solution or Z-VAD at 3.3 mg/kg via intravenous injection 15 min prior to cardiac ischemia. Left ventricular ejection fraction (% LVEF) was determined during the cardiac I/R protocol. The brain tissues were removed and used to determine brain apoptosis, brain inflammation, microglial and astrocyte morphology. RESULTS: Cardiac dysfunction was observed in rats with cardiac I/R injury as indicated by decreased %LVEF. In the brain, we found brain apoptosis, brain inflammation, microglia hyperactivation, and reactive astrogliosis occurred following cardiac I/R injury. Pretreatment with Z-VAD effectively increased %LVEF, reduced brain apoptosis, attenuated brain inflammation by decreasing IL-1ß mRNA levels, suppressed microglial and astrocytic hyperactivation and proliferation after cardiac I/R injury. CONCLUSION: Z-VAD exerts neuroprotective effects against cardiac I/R injury not only targeting apoptosis but also microglial and astrocyte activation.

The temporal impact of erythropoietin administration on mitochondrial function and dynamics in cardiac ischemia/reperfusion injury.

Erythropoietin (EPO) has been shown to provide protection against ischemia/reperfusion (I/R) injury in various experimental models. However, clinical trials revealed unsatisfactory results when EPO was given to patients with myocardial infarction following reperfusion. The timing of EPO administration and its relation to mitochondrial function may largely involve in this controversy. We hypothesized that EPO given at different time points exert varying cardioprotective effects in terms of myocardial infarct size, left ventricular (LV) function, arrhythmia, apoptosis, mitochondrial function, and mitochondrial dynamics in rats with cardiac I/R injury. Male Wistar rats were subjected to either sham (n = 6) or cardiac I/R operation (n = 48). Rats undergoing cardiac I/R operation (30-min ischemia, followed by 120-min reperfusion) were allotted into 4 subgroups (n = 12/group): vehicle, EPO pretreatment, EPO given during ischemia, and EPO given at reperfusion. EPO was administered intravenously at 5000 unit/kg. Arrhythmia and LV function were monitored throughout the protocol. Next, the hearts were collected to determine infarct size, mitochondrial function, mitochondrial dynamics, gap junction protein, and apoptosis. Cardiac I/R promoted arrhythmias, LV dysfunction, infarct size expansion, apoptosis, mitochondrial dysfunction and increased mitochondrial fission. EPO given either before or during ischemia, but not at reperfusion, attenuated arrhythmia scores, LV dysfunction, infarct size, and apoptosis. Only EPO given either before or during ischemia alleviated mitochondrial swelling, mitochondrial depolarization, and reduced the levels of p-Drp1ser616/Drp1. Data from in vitro study also confirmed that EPO directly attenuated mitochondrial dysfunction in H9c2 cells subjected to hypoxia/reoxygenation. In conclusion, the EPO administration, either before or during ischemia, exerted cardioprotection against I/R injury by attenuating mitochondrial dynamic imbalance, mitochondrial dysfunction, and apoptosis, leading to reduced infarct size and improved LV function.

Not only metformin, but also D-allulose, alleviates metabolic disturbance and cognitive decline in prediabetic rats.

BACKGROUND: Prediabetes can be characterized as obesity with metabolic disturbance, leading to cognitive decline and brain pathologies. D-allulose administration in obese animals decreased metabolic disturbance. However, the comparative effects of D-allulose and metformin on cognition and brain functions in the diet-induced prediabetic condition are unclear. We assume that both D-allulose and metformin equally restore cognition and brain functions in prediabetic rats to an equal extent. MATERIALS AND METHODS: Fifty-six rats were randomly divided into two groups: a control and diet-induced prediabetic group which had received a normal diet (ND) and a high-fat diet (HFD) for 24 weeks, respectively. After dietary protocol had been followed for 12 weeks, ND rats were given solely drinking water daily for 12 weeks. HFD-prediabetic rats randomly received drinking water with either D-allulose (1.9 g/kg/day of D-allulose) or metformin (300 mg/kg/day of metformin) for 12 weeks. Following this, cognition and brain parameters were determined. RESULTS: Brain oxidative stress, mitochondrial dysfunction, microglial hyper-activation, apoptosis, brain insulin insensitivity, hippocampal synaptic dysfunction, and cognitive decline were observed in prediabetic rats. D-allulose and metformin equally attenuated brain oxidative stress, brain mitochondrial ROS production, hippocampal apoptosis, brain insulin insensitivity, hippocampal synaptic dysfunction, resulting in improved learning process in prediabetic rats. Metformin conferred greater advantage on the amelioration of brain mitochondrial dysfunction and brain microglial hyper-activation than D-allulose, resulting in improvement in both learning and memory processes in prediabetic rats. CONCLUSIONS: Not only metformin, but also D-allulose, has beneficial effects on the enhancement of brain function and cognition in prediabetic condition.

Acetylcholinesterase inhibitor ameliorates doxorubicin-induced cardiotoxicity through reducing RIP1-mediated necroptosis.

Doxorubicin is an effective chemotherapeutic drug, but causes cardiotoxicity which limits its use. Oxidative stress, mitochondrial dysfunction, and inflammation are closely implicated in doxorubicin-induced cardiotoxicity (DIC). Necroptosis, a new form of programmed cell death, was also upregulated by doxorubicin, leading to cardiomyocyte death and cardiac dysfunction. Donepezil, an acetylcholinesterase inhibitor, exerted cardioprotection against various heart diseases. However, its cardioprotective effects in DIC are still unknown. We hypothesized that donepezil reduces reactive oxygen species (ROS) production, mitochondrial dysfunction, mitochondrial dynamics imbalance, necroptosis, and apoptosis in DIC rats. Male Wistar rats were assigned to receive either normal saline solution (n = 8) or doxorubicin (3 mg/kg, 6 doses, n = 16) via intraperitoneal injection. The doxorubicin-treated rats were further subdivided to receive either sterile drinking water (n = 8) or donepezil (5 mg/kg/day, p.o., n = 8) for 30 days. At the end of the experiment, the left ventricular (LV) function was determined. Serum and heart tissue were collected to evaluate histological and biochemical parameters. Doxorubicin-treated rats exhibited higher levels of inflammatory cytokines and ROS production. Doxorubicin also impaired mitochondrial function, mitochondrial dynamics balance, mitophagy, and autophagy, which culminated in apoptosis. Furthermore, doxorubicin increased necroptosis as evidenced by increased phosphorylation of receptor-interacting protein kinase 1, receptor-interacting protein kinase 3, and mixed-lineage kinase domain-like. All of these mechanisms led to LV dysfunction. Interestingly, donepezil alleviated mitochondrial injury, mitophagy, autophagy, and cardiomyocyte death, leading to improved LV function in DIC. In conclusion, donepezil attenuated DIC-induced LV dysfunction by reducing mitochondrial damage, mitophagy, autophagy, apoptosis, and necroptosis.

PCSK9 inhibitor and atorvastatin reduce cardiac impairment in ovariectomized prediabetic rats via improved mitochondrial function and Ca2+ regulation.

Post-menopausal women have a higher risk of developing cardiometabolic dysfunction. Atorvastatin attenuates dyslipidaemia and cardiac dysfunction but it can have undesirable effects including increased risk of diabetes and myalgia. Currently, the proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor efficiently reduces low-density lipoprotein cholesterol (LDL-C) levels more effectively than atorvastatin. We have been suggested that PCSK9 inhibitor attenuated cardiometabolic impairment more effectively than atorvastatin in ovariectomized prediabetic rats. Female Wistar rats (n = 48) were fed a normal diet (ND) or high-fat diet (HFD) for 12 weeks. Then, HFD rats were assigned to a sham-operated (Sham) or ovariectomized (OVX) group. Six weeks after surgery, the OVX group was subdivided into 4 treatment groups: vehicle (HFOV), atorvastatin (HFOA) (40 mg/kg/day; s.c.), PCSK9 inhibitor (HFOP) (4 mg/kg/day; s.c.) and oestrogen (HFOE2 ) (50 µg/kg/day; s.c.) for an additional 3 weeks. Metabolic parameters, cardiac and mitochondrial function, and [Ca2+ ]i transients were evaluated. All HFD rats became obese-insulin resistant. HFS rats had significantly impaired left ventricular (LV) function, cardiac mitochondrial function and [Ca2+ ]i transient dysregulation. Oestrogen deprivation (HFOV) aggravated all of these impairments. Our findings indicated that the atorvastatin, PCSK9 inhibitor and oestrogen shared similar efficacy in the attenuation in cardiometabolic impairment in ovariectomized prediabetic rats.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor exerts greater efficacy than atorvastatin on improvement of brain function and cognition in obese rats.

The accumulation of lipid as a result of long-term consumption of a high-fat diet (HFD) may lead to metabolic and brain dysfunction. Atorvastatin, a recommended first-line lipid-lowering agent, has shown beneficial effects on metabolic and brain functions in several models. Recently, proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor was approved as an effective therapeutic drug for dyslipidemia patients. However, few studies have reported on the effect of this PCSK9 inhibitor on brain function. In addition, the comparative efficacy on the improvement of metabolic and brain functions between PCSK9 inhibitor and atorvastatin in obese models have not been elucidated. We hypothesized that PCSK9 inhibitor improves metabolic and brain functions in an obese model to a greater extent than atorvastatin. Thirty-two female rats were fed with either a normal diet (ND) or HFD for 15 weeks. At week 13, ND rats were given normal saline and HFD rats were given either normal saline, atorvastatin (40 mg/kg/day) or PCSK9 inhibitor (4 mg/kg/day) for 3 weeks. Oxidative stress, blood brain barrier breakdown, microglial hyperactivity, synaptic dysplasticity, apoptosis, amyloid proteins production in the hippocampus and cognitive decline were found in HFD-fed rats. Atorvastatin and PCSK9 inhibitor therapies equally attenuated hippocampal apoptosis and amyloid protein production in HFD-fed rats. Interestingly, PCSK9 inhibitor had the greater efficacy than atorvastatin on the amelioration of hippocampal oxidative stress, blood brain barrier breakdown, microglial hyperactivity, synaptic dysplasticity in the hippocampus and cognitive decline. These findings suggest that PCSK9 inhibitor may be another drug of choice for improving brain function in the obese condition with discontinued statin therapy.

The comparative effects of high dose atorvastatin and proprotein convertase subtilisin/kexin type 9 inhibitor on the mitochondria of oxidative muscle fibers in obese-insulin resistant female rats.

The present study aimed to compare the effects of high dose atorvastatin and a proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor on the mitochondrial function in oxidative muscle fibers in obese female rats. Female Wistar rats were fed with either a normal diet (ND: n = 12) or a high-fat diet (HFD: n = 36) for a total of 15 weeks. At week 13, ND-fed rats received a vehicle, and HFD-fed rats were divided to three groups to receive either a vehicle, 40 mg/kg/day of atorvastatin, or 4 mg/kg/day of PCSK9 inhibitor (SBC-115076) for 3 weeks. Soleus muscles were investigated to assess mitochondrial ROS, membrane potential, swelling, mitochondrial-related protein expression, and level of malondialdehyde (MDA). The results showed that HFD-fed rats with vehicle developed obese-insulin resistance and dyslipidemia. Both atorvastatin and PCSK9 inhibitor reduced obesity and dyslipidemia, as well as improved insulin sensitivity in HFD-fed rats. However, the efficacy of PCSK9 inhibitor to increase weight loss and reduce dyslipidemia in HFD-fed rats was greater than those of atorvastatin. An increase in MDA level, ratio of p-Drp1ser616/total Drp1 protein, CPT1 protein, mitochondrial ROS, and membrane depolarization in the soleus muscle were observed in HFD-fed rats with vehicle. PCSK9 inhibitor enabled the restoration of all these parameters to normal levels. However, atorvastatin facilitated restoration of some parameters, including MDA level, p-Drp1ser616/total Drp1 ratio, and CPT1 protein expression. These findings suggest that PCSK9 inhibitor is superior to atorvastatin in instigating weight loss, cholesterol reduction, and attenuation of mitochondrial oxidative stress in oxidative muscle fibers of obese female rats.

Mitochondrial Fusion Promoter Given During Ischemia Has Greater Neuroprotective Efficacy Than When Given at Onset of Reperfusion in Rats with Cardiac Ischemia/Reperfusion Injury.

BACKGROUND: Cardiac ischemia/reperfusion (I/R) injury has been shown to impose deleterious effects not only on the heart but also on the brain. Our previous study demonstrated that pretreatment with a mitochondrial fusion promoter (M1) provided central neuroprotective effects following cardiac I/R injury. OBJECTIVE: To investigate the effects of M1 given during the ischemic phase and M1 given at the beginning of reperfusion on brain pathologies following cardiac I/R. METHODS: Male Wistar rats were randomly divided into either a sham operation (n = 6) or cardiac I/R injury (n = 18) group. Rats with cardiac I/R injury were then randomly divided into 3 subgroups: 1) Control, 2) M1 treatment during cardiac ischemia (2 mg/kg, intravenous (i.v.)), and 3) M1 treatment at the beginning of reperfusion (2 mg/kg, i.v.). After euthanasia, the brain of each rat was removed for further analysis. RESULTS: Cardiac I/R injury caused brain mitochondrial dynamic imbalance, brain mitochondrial dysfunction, brain apoptosis, microglial dysmorphology, brain inflammation, tau hyperphosphorylation, and synaptic dysplasticity. M1 treatment at both time points effectively improved these parameters. M1 given during the ischemic phase had greater efficacy with regard to preventing brain mitochondrial dysfunction and suppressing brain inflammation, when compared to M1 given at the beginning of reperfusion. CONCLUSIONS: Our findings suggest that treatment with this mitochondrial fusion promoter prevents mitochondrial dynamic imbalance in the brain of rats with cardiac I/R injury, thereby attenuating brain pathologies. Interestingly, giving the mitochondrial fusion promoter during the ischemic phase exerted greater neuroprotection than if given at the beginning of reperfusion.