Targeting mitochondrial shape: at the heart of cardioprotection.

There remains an unmet need to identify novel therapeutic strategies capable of protecting the myocardium against the detrimental effects of acute ischemia-reperfusion injury (IRI), to reduce myocardial infarct (MI) size and prevent the onset of heart failure (HF) following acute myocardial infarction (AMI). In this regard, perturbations in mitochondrial morphology with an imbalance in mitochondrial fusion and fission can disrupt mitochondrial metabolism, calcium homeostasis, and reactive oxygen species production, factors which are all known to be critical determinants of cardiomyocyte death following acute myocardial IRI. As such, therapeutic approaches directed at preserving the morphology and functionality of mitochondria may provide an important strategy for cardioprotection. In this article, we provide an overview of the alterations in mitochondrial morphology which occur in response to acute myocardial IRI, and highlight the emerging therapeutic strategies for targeting mitochondrial shape to preserve mitochondrial function which have the future therapeutic potential to improve health outcomes in patients presenting with AMI.

Mechanisms responsible for beneficial and adverse effects of rosiglitazone in a rat model of acute cardiac ischaemia-reperfusion.

Despite debate regarding its cardioprotective and pro-arrhythmic effects, the precise mechanisms of action of rosiglitazone on the heart are still unclear. We determined the mechanistic effects of rosiglitazone on cardiac function, arrhythmias and infarct size during cardiac ischaemia-reperfusion. Twenty-six rats were used. In each rat, either rosiglitazone or saline solution was administered intravenously prior to a 30 min left anterior descending coronary artery ligation and a 120 min reperfusion. Cardiac function, infarct size, myocardial levels of connexin43, Bax/Bcl-2, cytochrome c, caspase-3, caspase-8, Akt, tumour necrosis factor-α and interleukin-4 and cardiac mitochondrial function were determined. Isolated cardiomyocytes were used for studying intracellular calcium. Rosiglitazone did not alter cardiac function during the ischaemia-reperfusion periods, but increased the arrhythmia score and mortality rate, decreased the time to onset of ventricular fibrillation and prolonged the Ca2+ decay rate, in comparison to the saline-injected group (P<0.05). However, the infarct size in the rosiglitazone-injected group was reduced (P<0.05). Rosiglitazone decreased the levels of connexin43 phosphorylation, active caspase-8 and tumour necrosis factor-α, but increased the level of procaspase-3. However, levels of Bax/Bcl-2, cytochrome c, Akt and interleukin-4 and the cardiac mitochondrial function were not different between the two groups. Rosiglitazone simultaneously exerted both beneficial and adverse cardiac effects in the heart exposed to ischaemia-reperfusion. Although it decreased the infarct size via the extrinsic anti-apoptotic pathway and anti-inflammatory effects, rosiglitazone facilitated a fatal arrhythmia by decreasing connexin43 phosphorylation and prolonging the Ca2+ decay rate in ischaemia-reperfusion. The higher mortality rate in the rosiglitazone-injected group suggests that its undesirable effect was more pronounced than its benefit on infarct size reduction.

Heart rate reduction after genetic ablation of L-type Cav1.3 channels induces cardioprotection against ischemia-reperfusion injury.

Background: Acute myocardial infarction (AMI) is the major cause of cardiovascular mortality worldwide. Most ischemic episodes are triggered by an increase in heart rate, which induces an imbalance between myocardial oxygen delivery and consumption. Developing drugs that selectively reduce heart rate by inhibiting ion channels involved in heart rate control could provide more clinical benefits. The Cav1.3-mediated L-type Ca2+ current (ICav1.3) play important roles in the generation of heart rate. Therefore, they can constitute relevant targets for selective control of heart rate and cardioprotection during AMI. Objective: We aimed to investigate the relationship between heart rate and infarct size using mouse strains knockout for Cav1.3 (Cav1.3-/-) L-type calcium channel and of the cardiac G protein gated potassium channel (Girk4-/-) in association with the funny (f)-channel inhibitor ivabradine. Methods: Wild-type (WT), Cav1.3+/-, Cav1.3-/- and Girk4-/- mice were used as models of respectively normal heart rate, moderate heart rate reduction, bradycardia, and mild tachycardia, respectively. Mice underwent a surgical protocol of myocardial IR (40 min ischemia and 60 min reperfusion). Heart rate was recorded by one-lead surface ECG recording, and infarct size measured by triphenyl tetrazolium chloride staining. In addition, Cav1.3-/- and WT hearts perfused on a Langendorff system were subjected to the same ischemia-reperfusion protocol ex vivo, without or with atrial pacing, and the coronary flow was recorded. Results: Cav1.3-/- mice presented reduced infarct size (-29%), while Girk4-/- displayed increased infarct size (+30%) compared to WT mice. Consistently, heart rate reduction in Cav1.3+/- or by the f-channel blocker ivabradine was associated with significant decrease in infarct size (-27% and -32%, respectively) in comparison to WT mice. Conclusion: Our results show that decreasing heart rate allows to protect the myocardium against IR injury in vivo and reveal a close relationship between basal heart rate and IR injury. In addition, this study suggests that targeting Cav1.3 channels could constitute a relevant target for reducing infarct size, since maximal heart rate dependent cardioprotective effect is already observed in Cav1.3+/- mice.

T-type calcium channel blockade improves survival and cardiovascular function in thalassemic mice.

OBJECTIVES: Iron-overload cardiomyopathy is a major cause of morbidity and mortality in patients with thalassemia. However, the precise mechanisms of iron entry and sequestration in the heart are still unclear. Our previous study showed that Fe(2+) uptake in thalassemic cardiomyocytes are mainly mediated by T-type calcium channels (TTCC). Nevertheless, the role of TTCC as well as other transporters such as divalent metal transporter1 (DMT1) and L-type calcium channels (LTCC) as possible portals for iron entry into the heart in in vivo thalassemic mice under an iron-overload condition has not been investigated. METHODS: An iron-overload condition was induced in genetically altered ß-thalassemic mice and adult wild-type mice by feeding them with an iron diet (0.2% ferrocene w/w) for 3 months. Then, blockers for LTCC (verapamil and nifedipine), TTCC (efonidipine), and DMT1 (ebselen) as well as iron chelator desferoxamine (DFO) were given for 1 month with continuous iron feeding. RESULTS: Treatment with LTCC, TTCC, DMT1 blockers, and DFO reduced cardiac iron deposit, cardiac malondialdehyde (MDA), plasma non-transferrin-bound iron, and improved heart rate variability and left ventricular (LV) function in thalassemic mice with iron overload. Only TTCC and DMT1 blockers and DFO reduced liver iron accumulation, liver MDA, plasma MDA, and decreased mortality rate in iron-overloaded thalassemic mice. CONCLUSIONS: DMT1, LTCC, and TTCC played important roles for iron entry in the thalassemic heart under an iron-overloaded condition. Unlike LTCC blocker, TTCC blocker provided all benefits including attenuating iron deposit in both the heart and liver, reduced oxidative stress, and decreased mortality in iron-overloaded mice.

Effects of Kaempferia parviflora Wall. Ex. Baker and sildenafil citrate on cGMP level, cardiac function, and intracellular Ca2+ regulation in rat hearts.

Although Kaempferia parviflora extract (KPE) and its flavonoids have positive effects on the nitric oxide (NO) signaling pathway, its mechanisms on the heart are still unclear. Because our previous studies demonstrated that KPE decreased defibrillation efficacy in swine similar to that of sildenafil citrate, the phosphodiesterase-5 inhibitor, it is possible that KPE may affect the cardiac NO signaling pathway. In the present study, the effects of KPE and sildenafil citrate on cyclic guanosine monophosphate (cGMP) level, modulation of cardiac function, and Ca transients in ventricular myocytes were investigated. In a rat model, cardiac cGMP level, cardiac function, and Ca transients were measured before and after treatment with KPE and sildenafil citrate. KPE significantly increased the cGMP level and decreased cardiac function and Ca transient. These effects were similar to those found in the sildenafil citrate-treated group. Furthermore, the nonspecific NOS inhibitor could abolish the effects of KPE and sildenafil citrate on Ca transient. KPE has positive effect on NO signaling in the heart, resulting in an increased cGMP level, similar to that of sildenafil citrate. This effect was found to influence the physiology of normal heart via the attenuation of cardiac function and the reduction of Ca transient in ventricular myocytes.

Role of cardiac mitofusins in cardiac conduction following simulated ischemia-reperfusion.

Mitochondrial dysfunction induced by acute cardiac ischemia-reperfusion (IR), may increase susceptibility to arrhythmias by perturbing energetics, oxidative stress production and calcium homeostasis. Although changes in mitochondrial morphology are known to impact on mitochondrial function, their role in cardiac arrhythmogenesis is not known. To assess action potential duration (APD) in cardiomyocytes from the Mitofusins-1/2 (Mfn1/Mfn2)-double-knockout (Mfn-DKO) compared to wild-type (WT) mice, optical-electrophysiology was conducted. To measure conduction velocity (CV) in atrial and ventricular tissue from the Mfn-DKO and WT mice, at both baseline and following simulated acute IR, multi-electrode array (MEA) was employed. Intracellular localization of connexin-43 (Cx43) at baseline was evaluated by immunohistochemistry, while Cx-43 phosphorylation was assessed by Western-blotting. Mfn-DKO cardiomyocytes demonstrated an increased APD. At baseline, CV was significantly lower in the left ventricle of the Mfn-DKO mice. CV decreased with simulated-ischemia and returned to baseline levels during simulated-reperfusion in WT but not in atria of Mfn-DKO mice. Mfn-DKO hearts displayed increased Cx43 lateralization, although phosphorylation of Cx43 at Ser-368 did not differ. In summary, Mfn-DKO mice have increased APD and reduced CV at baseline and impaired alterations in CV following cardiac IR. These findings were associated with increased Cx43 lateralization, suggesting that the mitofusins may impact on post-MI cardiac-arrhythmogenesis.

Hydralazine protects the heart against acute ischaemia/reperfusion injury by inhibiting Drp1-mediated mitochondrial fission.

AIMS: Genetic and pharmacological inhibition of mitochondrial fission induced by acute myocardial ischaemia/reperfusion injury (IRI) has been shown to reduce myocardial infarct size. The clinically used anti-hypertensive and heart failure medication, hydralazine, is known to have anti-oxidant and anti-apoptotic effects. Here, we investigated whether hydralazine confers acute cardioprotection by inhibiting Drp1-mediated mitochondrial fission. METHODS AND RESULTS: Pre-treatment with hydralazine was shown to inhibit both mitochondrial fission and mitochondrial membrane depolarisation induced by oxidative stress in HeLa cells. In mouse embryonic fibroblasts (MEFs), pre-treatment with hydralazine attenuated mitochondrial fission and cell death induced by oxidative stress, but this effect was absent in MEFs deficient in the mitochondrial fission protein, Drp1. Molecular docking and surface plasmon resonance studies demonstrated binding of hydralazine to the GTPase domain of the mitochondrial fission protein, Drp1 (KD 8.6±1.0 µM), and inhibition of Drp1 GTPase activity in a dose-dependent manner. In isolated adult murine cardiomyocytes subjected to simulated IRI, hydralazine inhibited mitochondrial fission, preserved mitochondrial fusion events, and reduced cardiomyocyte death (hydralazine 24.7±2.5% vs. control 34.1±1.5%, P=0.0012). In ex vivo perfused murine hearts subjected to acute IRI, pre-treatment with hydralazine reduced myocardial infarct size (as % left ventricle: hydralazine 29.6±6.5% vs. vehicle control 54.1±4.9%, P=0.0083), and in the murine heart subjected to in vivo IRI, the administration of hydralazine at reperfusion, decreased myocardial infarct size (as % area-at-risk: hydralazine 28.9±3.0% vs. vehicle control 58.2±3.8%, P<0.001). CONCLUSION: We show that, in addition to its antioxidant and anti-apoptotic effects, hydralazine, confers acute cardioprotection by inhibiting IRI-induced mitochondrial fission, raising the possibility of repurposing hydralazine as a novel cardioprotective therapy for improving post-infarction outcomes.

Fasting increases susceptibility to acute myocardial ischaemia/reperfusion injury through a sirtuin-3 mediated increase in fatty acid oxidation.

Fasting increases susceptibility to acute myocardial ischaemia/reperfusion injury (IRI) but the mechanisms are unknown. Here, we investigate the role of the mitochondrial NAD+-dependent deacetylase, Sirtuin-3 (SIRT3), which has been shown to influence fatty acid oxidation and cardiac outcomes, as a potential mediator of this effect. Fasting was shown to shift metabolism from glucose towards fatty acid oxidation. This change in metabolic fuel substrate utilisation increased myocardial infarct size in wild-type (WT), but not SIRT3 heterozygous knock-out (KO) mice. Further analysis revealed SIRT3 KO mice were better adapted to starvation through an improved cardiac efficiency, thus protecting them from acute myocardial IRI. Mitochondria from SIRT3 KO mice were hyperacetylated compared to WT mice which may regulate key metabolic processes controlling glucose and fatty acid utilisation in the heart. Fasting and the associated metabolic switch to fatty acid respiration worsens outcomes in WT hearts, whilst hearts from SIRT3 KO mice are better adapted to oxidising fatty acids, thereby protecting them from acute myocardial IRI.

Dried mulberry fruit ameliorates cardiovascular and liver histopathological changes in high-fat diet-induced hyperlipidemic mice.

BACKGROUND AND AIM: Metabolic disease encompasses most contemporary non-communicable diseases, especially cardiovascular and fatty liver disease. Mulberry fruits of Morus alba L. are a favoured food and a traditional medicine. While they are anti-atherosclerotic and reduce hyperlipidemic risk factors, studies need wider scope that include ameliorating cardiovascular and liver pathologies if they are to become clinically effective treatments. Therefore, the present study sought to show that freshly dried mulberry fruits (dMF) might counteract the metabolic/cardiovascular pathologies in mice made hyperlipidemic by high-fat diet (HF). EXPERIMENTAL PROCEDURE: C57BL/6J mice were fed for 3 months with either: i) control diet, ii) HF, iii) HF+100 mg/kg dMF, or iv) HF+300 mg/kg dMF. Body weight gain, food intake, visceral fat accumulation, fasting blood glucose, plasma lipids, and aortic, heart, and liver histopathologies were evaluated. Adipocyte lipid accumulation, autophagy, and bile acid binding were also investigated. RESULTS AND CONCLUSION: HF increased food intake, body weight, visceral fat, plasma total cholesterol (TC) and low-density lipoprotein (LDL), TC/HDL ratio, blood glucose, aortic collagen, arterial and cardiac wall thickness, and liver lipid. Both dMF doses prevented hyperphagia, body weight gain, and visceral fat accumulation, lowered blood glucose, plasma TG and unfavourable TC/HDL and elevated plasma HDL beyond baseline. Arterial and cardiac wall hypertrophy, aortic collagen fibre accumulation and liver lipid deposition ameliorated in dMF-fed mice. Clinical trials on dMF are worthwhile but outcomes should be holistic commensurate with the constellation of disease risks. Here, dMF should supplement the switch to nutrient-rich from current energy-dense diets that are progressively crippling national health systems.

The Role of Redox Dysregulation in the Inflammatory Response to Acute Myocardial Ischaemia-reperfusion Injury - Adding Fuel to the Fire.

BACKGROUND: The inflammatory response to acute myocardial ischaemia/ reperfusion injury (IRI) plays a critical role in determining myocardial infarct (MI) size, and subsequent post-MI left ventricular (LV) remodelling, making it a potential therapeutic target for improving clinical outcomes in patients presenting with an acute myocardial infarction (AMI). Recent experimental studies using advanced imaging and molecular techniques, have yielded new insights into the mechanisms through which reactive oxygen species (ROS) contribute to the inflammatory response induced by acute myocardial IRI - "adding fuel to the fire". The infiltration of inflammatory cells into the MI zone, leads to elevated myocardial concentrations of ROS, cytokine release, and activation of apoptotic and necrotic death pathways. Anti-oxidant and anti-inflammatory therapies have failed to protect the heart against acute myocardial IRI. This may be, in part, due to a lack of understanding of the time course, nature and mechanisms of the inflammation and redox dysregulation, which occur in the setting of acute myocardial IRI. CONCLUSION: In this article, we examine the inflammatory response and redox dysregulation induced by acute myocardial IRI, and highlight potential therapeutic options for targeting redox dysregulation, in order to attenuate the detrimental effects of the inflammatory response following an AMI, so as to reduce MI size and prevent heart failure.

Non-coding RNAs as therapeutic targets for preventing myocardial ischemia-reperfusion injury.

INTRODUCTION: New treatments are required to improve clinical outcomes in patients with acute myocardial infarction (AMI), for reduction of myocardial infarct (MI) size and preventing heart failure. Following AMI, acute ischemia/reperfusion injury (IRI) ensues, resulting in cardiomyocyte death and impaired cardiac function. Emerging studies have implicated a fundamental role for non-coding RNAs (microRNAs [miRNA], and more recently long non-coding RNAs [lncRNA]) in the setting of acute myocardial IRI. Areas covered: In this article, we discuss the roles of miRNAs and lncRNAs as potential biomarkers and therapeutic targets for the detection and treatment of AMI, review their roles as mediators and effectors of cardioprotection, particularly in the settings of interventions such as ischemic pre- and post-conditioning (IPC & IPost) as well as remote ischemic conditioning (RIC), and highlight future strategies for targeting ncRNAs to reduce MI size and prevent heart failure following AMI. Expert opinion: Investigating the roles of miRNAs and lncRNAs in the setting of AMI has provided new insights into the pathophysiology underlying acute myocardial IRI, and has identified novel biomarkers and therapeutic targets for detecting and treating AMI. Pharmacological and genetic manipulation of these ncRNAs has the therapeutic potential to improve clinical outcomes in AMI patients.

Effects of Vagal Nerve Stimulation on Ganglionated Plexi Nerve Activity and Ventricular Rate in Ambulatory Dogs With Persistent Atrial Fibrillation.

OBJECTIVES: This study was designed to test the hypothesis that low-level vagal nerve stimulation (VNS) reduces the ventricular rate (VR) during atrial fibrillation (AF) through the activation of the inferior vena cava (IVC)-inferior atrial ganglionated plexus nerve activity (IAGPNA). BACKGROUND: Increased IVC-IAGPNA can suppress atrioventricular node conduction and slow VR in canine models of AF. METHODS: Persistent AF was induced in 6 dogs and the IVC-IAGPNA, right vagal nerve activity, left vagal nerve activity, and an electrocardiogram were recorded. After persistent AF was documented, VNS was programed to 14 s "on" and 1.1 min "off." After 1 week, the VNS was reprogramed to 3 min off and stimulation continued for another week. Neural remodeling of the stellate ganglion (SG) was assessed with tyrosine hydroxylase staining and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling staining. RESULTS: Average IVC-IAGPNA was increased during both VNS 1.1 min off (8.20 ± 2.25 µV [95% confidence interval (CI): 6.33 to 9.53 µV]; p = 0.002) and 3 min off (7.96 ± 2.03 µV [95% CI: 6.30 to 9.27 µV]; p = 0.001) versus baseline (7.14 ± 2.20 µV [95% CI: 5.35 to 8.52 µV]). VR was reduced during both VNS 1.1 min off (123.29 ± 6.29 beats/min [95% CI: 116.69 to 129.89 beats/min]; p = 0.001) and 3 min off (120.01 ± 4.93 beats/min [95% CI: 114.84 to 125.18 beats/min]; p = 0.001) compared to baseline (142.04 ± 7.93 bpm [95% CI: 133.72 to 150.37]). Abnormal regions were observed in the left SG, but not in the right SG. Terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling-positive neurons were found in 22.2 ± 17.2% [95% CI: 0.9% to 43.5%] of left SG cells and 12.8 ± 8.4% [95% CI: 2.4% to 23.2%] of right SG cells. CONCLUSIONS: Chronic low-level VNS increases IVC-IAGPNA and damages bilateral stellate ganglia. Both mechanisms could contribute to the underlying mechanism of VR control during AF.

Assessing the effects of mitofusin 2 deficiency in the adult heart using 3D electron tomography.

The effects of mitofusin 2 (MFN2) deficiency, on mitochondrial morphology and the mitochondria-junctional sarcoplasmic reticulum (jSR) complex in the adult heart, have been previously investigated using 2D electron microscopy, an approach which is unable to provide a 3D spatial assessment of these imaging parameters. Here, we use 3D electron tomography to show that MFN2-deficient mitochondria are larger in volume, more elongated, and less rounded; have fewer mitochondria-jSR contacts, and an increase in the distance between mitochondria and jSR, when compared to WT mitochondria. In comparison to 2D electron microscopy, 3D electron tomography can provide further insights into mitochondrial morphology and the mitochondria-jSR complex in the adult heart.

Effects of renal sympathetic denervation on the stellate ganglion and brain stem in dogs.

BACKGROUND: Renal sympathetic denervation (RD) is a promising method of neuromodulation for the management of cardiac arrhythmia. OBJECTIVE: We tested the hypothesis that RD is antiarrhythmic in ambulatory dogs because it reduces the stellate ganglion nerve activity (SGNA) by remodeling the stellate ganglion (SG) and brain stem. METHODS: We implanted a radiotransmitter to record SGNA and electrocardiogram in 9 ambulatory dogs for 2 weeks, followed by a second surgery for RD and 2 months SGNA recording. Cell death was probed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. RESULTS: Integrated SGNA at baseline and 1 and 2 months after RD were 14.0 ± 4.0, 9.3 ± 2.8, and 9.6 ± 2.0 µV, respectively (P = .042). The SG from RD but not normal control dogs (n = 5) showed confluent damage. An average of 41% ± 10% and 40% ± 16% of ganglion cells in the left and right SG, respectively, were TUNEL positive in RD dogs compared with 0% in controls dogs (P = .005 for both). The left and right SG from RD dogs had more tyrosine hydroxylase-negative ganglion cells than did the left SG of control dogs (P = .028 and P = .047, respectively). Extensive TUNEL-positive neurons and glial cells were also noted in the medulla, associated with strongly positive glial fibrillary acidic protein staining. The distribution was heterogeneous, with more cell death in the medial than lateral aspects of the medulla. CONCLUSION: Bilateral RD caused significant central and peripheral sympathetic nerve remodeling and reduced SGNA in ambulatory dogs. These findings may in part explain the antiarrhythmic effects of RD.

Ganglionated plexi and ligament of Marshall ablation reduces atrial vulnerability and causes stellate ganglion remodeling in ambulatory dogs.

BACKGROUND: Simultaneous activation of the stellate ganglion (SG), the ligament of Marshall (LOM), and the ganglionated plexi often precedes the onset of paroxysmal atrial tachyarrhythmia (PAT). OBJECTIVE: The purpose of this study was to test the hypothesis that ablation of the LOM and the superior left ganglionated plexi (SLGP) reduces atrial vulnerability and results in remodeling of the SG. METHODS: Nerve activity was correlated to PAT and ventricular rate (VR) at baseline, after ablation of the LOM and SLGP, and after atrial fibrillation. Neuronal cell death was assessed with tyrosine hydroxylase and terminal deoxynucleotidyl transferase dUTP nick end label (TUNEL) staining. RESULTS: There were 4 ± 2 PAT episodes per day in controls. None were observed in the ablation group, even though SG nerve activity and VR increased from 2.2 µV (95% confidence interval [CI] 1.2-3.3 µV) and 80 bpm (95% CI 68-92 bpm) at baseline, to 3.0 µV (95% CI 2.6-3.4 µV, P = .046) and 90 bpm (95% CI 75-108 bpm, P = .026) after ablation, and to 3.1 µV (95% CI 1.7-4.5 µV, P = .116) and 95 bpm (95% CI 79-110 bpm, P = .075) after atrial fibrillation. There was an increase in tyrosine hydroxylase-negative cells in the ablation group and 19.7% (95% CI 8.6%-30.8%) TUNEL-positive staining in both the left and right SG. None were observed in the control group. CONCLUSION: LOM and SLGP ablation caused left SG remodeling and cell death. There was reduced correlation of the VR response and PAT to SG nerve activity. These findings support the importance of SLGP and LOM in atrial arrhythmogenesis.

Intermittent left cervical vagal nerve stimulation damages the stellate ganglia and reduces the ventricular rate during sustained atrial fibrillation in ambulatory dogs.

BACKGROUND: The effects of intermittent open-loop vagal nerve stimulation (VNS) on the ventricular rate (VR) during atrial fibrillation (AF) remain unclear. OBJECTIVE: The purpose of this study was to test the hypothesis that VNS damages the stellate ganglion (SG) and improves VR control during persistent AF. METHODS: We performed left cervical VNS in ambulatory dogs while recording the left SG nerve activity (SGNA) and vagal nerve activity. Tyrosine hydroxylase (TH) staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were used to assess neuronal cell death in the SG. RESULTS: We induced persistent AF by atrial pacing in 6 dogs, followed by intermittent VNS with short ON-time (14 seconds) and long OFF-time (66 seconds). The integrated SGNA and VR during AF were 4.84 mV·s (95% confidence interval [CI] 3.08-6.60 mV·s) and 142 beats/min (95% CI 116-168 beats/min), respectively. During AF, VNS reduced the integrated SGNA and VR, respectively, to 3.74 mV·s (95% CI 2.27-5.20 mV·s; P = .021) and 115 beats/min (95% CI 96-134 beats/min; P = .016) during 66-second OFF-time and to 4.07 mV·s (95% CI 2.42-5.72 mV·s; P = .037) and 114 beats/min (95% CI 83-146 beats/min; P = .039) during 3-minute OFF-time. VNS increased the frequencies of prolonged (>3 seconds) pauses during AF. TH staining showed large confluent areas of damage in the left SG, characterized by pyknotic nuclei, reduced TH staining, increased percentage of TH-negative ganglion cells, and positive TUNEL staining. Occasional TUNEL-positive ganglion cells were also observed in the right SG. CONCLUSION: VNS damaged the SG, leading to reduced SGNA and better rate control during persistent AF.

Dipeptidyl peptidase-4 inhibitor reduces infarct size and preserves cardiac function via mitochondrial protection in ischaemia-reperfusion rat heart.

AIM: We hypothesized that dipeptidyl peptidase (DPP)-4 inhibitor (vildagliptin) reduces fatal arrhythmias, cardiac dysfunction and infarct size caused by ischaemia-reperfusion (I/R) injury via its attenuation of cardiac mitochondrial dysfunction. METHODS: In total, 26 rats were randomized to receive either 1 mL normal saline solution or 2.0 mg/kg vildagliptin intravenously (n = 13/group) 30 min prior to a 30-min left anterior descending coronary artery occlusion, followed by a 120-min reperfusion. Arrhythmia scores, cardiac functions, infarct size and mitochondrial function were evaluated. RESULTS: Vildagliptin reduced the infarct size by 44% and mitigated cardiac dysfunction by preserving cardiac function without altering the incidence of cardiac arrhythmias. Vildagliptin increased expression of Bcl-2 and pro-caspase3 in the ischaemic area, whereas Bax and phosphorylated-connexin43/total-connexin43 were not altered. Vildagliptin attenuated cardiac mitochondrial dysfunction by reducing the reactive oxygen species level and mitochondrial swelling. CONCLUSIONS: DPP-4 inhibitor provides cardioprotection by reducing the infarct size and ameliorating cardiac dysfunction in I/R hearts by attenuating cardiac mitochondrial dysfunction and cardiomyocyte apoptosis.

Combined vildagliptin and metformin exert better cardioprotection than monotherapy against ischemia-reperfusion injury in obese-insulin resistant rats.

BACKGROUND: Obese-insulin resistance caused by long-term high-fat diet (HFD) consumption is associated with left ventricular (LV) dysfunction and increased risk of myocardial infarction. Metformin and vildagliptin have been shown to exert cardioprotective effects. However, the effect of these drugs on the hearts under obese-insulin resistance with ischemia-reperfusion (I/R) injury is unclear. We hypothesized that combined vildagliptin and metformin provide better protective effects against I/R injury than monotherapy in obese-insulin resistant rats. METHODOLOGY: Male Wistar rats were fed either HFD or normal diet. Rats in each diet group were divided into 4 subgroups to receive vildagliptin, metformin, combined vildagliptin and metformin, or saline for 21 days. Ischemia due to left anterior descending artery ligation was allowed for 30-min, followed by 120-min reperfusion. Metabolic parameters, heart rate variability (HRV), LV function, infarct size, mitochondrial function, calcium transient, Bax and Bcl-2, and Connexin 43 (Cx43) were determined. Rats developed insulin resistance after 12 weeks of HFD consumption. Vildagliptin, metformin, and combined drugs improved metabolic parameters, HRV, and LV function. During I/R, all treatments improved LV function, reduced infarct size and Bax, increased Bcl-2, and improved mitochondrial function in HFD rats. However, only combined drugs delayed the time to the first VT/VF onset, reduced arrhythmia score and mortality rate, and increased p-Cx43 in HFD rats. CONCLUSION: Although both vildagliptin and metformin improved insulin resistance and attenuate myocardial injury caused by I/R, combined drugs provided better outcomes than single therapy by reducing arrhythmia score and mortality rate.

Vagus nerve stimulation initiated late during ischemia, but not reperfusion, exerts cardioprotection via amelioration of cardiac mitochondrial dysfunction.

BACKGROUND: We previously reported that vagus nerve stimulation (VNS) applied immediately at the onset of cardiac ischemia provides cardioprotection against cardiac ischemic-reperfusion (I/R) injury. OBJECTIVE: This study aimed to determine whether VNS applied during ischemia or at the onset of reperfusion exerts differential cardioprotection against cardiac I/R injury. METHODS: Twenty-eight swine (25-30 kg) were randomized into 4 groups: Control (sham-operated, no VNS), VNS-ischemia (VNS applied during ischemia), VNS-reperfusion (VNS applied during reperfusion), and VNS-ischemia+atropine (VNS applied during ischemia with 1 mg/kg atropine administration). Ischemia was induced by left anterior descending (LAD) coronary artery occlusion for 60 minutes, followed by 120 minutes of reperfusion. VNS was applied either 30 minutes after LAD coronary artery occlusion or at the onset of reperfusion and continued until the end of reperfusion. Cardiac function, infarct size, myocardial levels of connexin 43, cytochrome c, tumor necrosis factor α, and interleukin 4, and cardiac mitochondrial function were determined. RESULTS: VNS applied 30 minutes after LAD coronary artery occlusion, but not at reperfusion, markedly reduced ventricular fibrillation incidence and infarct size (~59%), improved cardiac function; attenuated cardiac mitochondrial reactive oxygen species production, depolarization, swelling, and cytochrome c release; and increased the amount of phosphorylated connexin 43 and interleukin 4 as compared with the Control group. These beneficial effects of VNS were abolished by atropine. CONCLUSION: VNS could provide significant cardioprotective effects even when initiated later during ischemia, but was not effective after reperfusion. These findings indicate the importance of timing of VNS initiation and warrant the potential clinical application of VNS in protecting myocardium at risk of I/R injury.