Isolated Mouse Hearts Research Articles

Methods for detection of cardiac glycogen-autophagy.

Glycogen-autophagy ('glycophagy') is a selective autophagy process involved in delivering glycogen to the lysosome for bulk degradation. Glycophagy protein intermediaries include STBD1 as a glycogen tagging receptor, delivering the glycogen cargo into the forming phagosome by partnering with the Atg8 homolog, GABARAPL1. Glycophagy is emerging as a key process of energy metabolism and development of reliable tools for assessment of glycophagy activity is an important priority. Here we show that antibodies raised against the N-terminus of the GABARAPL1 protein (but not the full-length protein) detected a specific endogenous GABARAPL1 immunoblot band at 18kDa. A stable GFP-GABARAPL1 cardiac cell line was used to quantify GABARAPL1 lysosomal flux via measurement of GFP puncta in response to lysosomal inhibition with bafilomycin. Endogenous glycophagy flux was quantified in primary rat ventricular myocytes by the extent of glycogen accumulation with bafilomycin combined with chloroquine treatment (no effect observed with bafilomycin or chloroquine alone). In wild-type isolated mouse hearts, bafilomycin alone and bafilomycin combined with chloroquine (but not chloroquine alone) elicited a significant increase in glycogen content signifying basal glycophagy flux. Collectively, these methodologies provide a comprehensive toolbox for tracking cardiac glycophagy activity to advance research into the role of glycophagy in health and disease.

Novel NLRP3 inhibitor INF195: Low doses provide effective protection against myocardial ischemia/reperfusion injury

BackgroundSeveral factors contribute to ischemia/reperfusion injury (IRI), including activation of the NLRP3 inflammasome and its byproducts, such as interleukin-1β (IL-1β) and caspase-1. However, NLRP3 may paradoxically exhibit cardioprotective properties. This study aimed to assess the protective effects of the novel NLRP3 inhibitor, INF195, both in vitro and ex vivo. MethodsTo investigate the relationship between NLRP3 and myocardial IRI, we synthetized a series of novel NLRP3 inhibitors, and investigated their putative binding mode via docking studies. Through in vitro studies we identified INF195 as optimal for NLRP3 inhibition. We measured infarct-size in isolated mouse hearts subjected to 30-min global ischemia/one-hour reperfusion in the presence of three different doses of INF195 (5, 10, or 20-μM). We analyzed caspase-1 and IL-1β concentration in cardiac tissue homogenates by ELISA. Statistical significance was determined using one-way ANOVA followed by Tukey's test. Results and conclusionINF195 reduces NLRP3-induced pyroptosis in human macrophages. Heart pre-treatment with 5 and 10-μM INF195 significantly reduces both infarct size and IL-1β levels. Data suggest that intracardiac NLRP3 activation contributes to IRI and that low doses of INF195 exert cardioprotective effects by reducing infarct size. However, at 20-μM, INF195 efficacy declines, leading to a lack of cardioprotection. Research is required to determine if high doses of INF195 have off-target effects or dual roles, potentially eliminating both harmful and cardioprotective functions of NLRP3. Our findings highlight the potential of a new chemical scaffold, amenable to further optimization, to provide NLRP3 inhibition and cardioprotection in the ischemia/reperfusion setting.

Long-Term Protective Effects of Succinate Dehydrogenase Inhibition during Reperfusion with Malonate on Post-Infarction Left Ventricular Scar and Remodeling in Mice.

Succinate dehydrogenase inhibition with malonate during initial reperfusion reduces myocardial infarct size in both isolated mouse hearts subjected to global ischemia and in in situ pig hearts subjected to transient coronary ligature. However, the long-term effects of acute malonate treatment are unknown. Here, we investigated whether the protective effects of succinate dehydrogenase inhibition extend to a reduction in scar size and adverse left ventricular remodeling 28 days after myocardial infarction. Initially, ten wild-type mice were subjected to 45 min of left anterior descending coronary artery (LAD) occlusion, followed by 24 h of reperfusion, and were infused during the first 15 min of reperfusion with saline with or without disodium malonate (10 mg/kg/min, 120 μL/kg/min). Malonate-treated mice depicted a significant reduction in infarct size (15.47 ± 3.40% of area at risk vs. 29.34 ± 4.44% in control animals, p < 0.05), assessed using triphenyltetrazolium chloride. Additional animals were then subjected to a 45 min LAD ligature, followed by 28 days of reperfusion. Treatment with a single dose of malonate during the first 15 min of reperfusion induced a significant reduction in scar area, measured using Picrosirius Red staining (11.94 ± 1.70% of left ventricular area (n = 5) vs. 23.25 ± 2.67% (n = 9), p < 0.05), an effect associated with improved ejection fraction 28 days after infarction, as determined using echocardiography, and an attenuated enhancement in expression of the pro-inflammatory and fibrotic markers NF-κB and Smad2/3 in remote myocardium. In conclusion, a reversible inhibition of succinate dehydrogenase with a single dose of malonate at the onset of reperfusion has long-term protective effects in mice subjected to transient coronary occlusion.

Insulin and glycolysis dependency of cardioprotection by nicotinamide riboside

Decreased nicotinamide adenine dinucleotide (NAD+) levels contribute to various pathologies such as ageing, diabetes, heart failure and ischemia–reperfusion injury (IRI). Nicotinamide riboside (NR) has emerged as a promising therapeutic NAD+ precursor due to efficient NAD+ elevation and was recently shown to be the only agent able to reduce cardiac IRI in models employing clinically relevant anesthesia. However, through which metabolic pathway(s) NR mediates IRI protection remains unknown. Furthermore, the influence of insulin, a known modulator of cardioprotective efficacy, on the protective effects of NR has not been investigated. Here, we used the isolated mouse heart allowing cardiac metabolic control to investigate: (1) whether NR can protect the isolated heart against IRI, (2) the metabolic pathways underlying NR-mediated protection, and (3) whether insulin abrogates NR protection. NR protection against cardiac IRI and effects on metabolic pathways employing metabolomics for determination of changes in metabolic intermediates, and 13C-glucose fluxomics for determination of metabolic pathway activities (glycolysis, pentose phosphate pathway (PPP) and mitochondrial/tricarboxylic acid cycle (TCA cycle) activities), were examined in isolated C57BL/6N mouse hearts perfused with either (a) glucose + fatty acids (FA) (“mild glycolysis group”), (b) lactate + pyruvate + FA (“no glycolysis group”), or (c) glucose + FA + insulin (“high glycolysis group”). NR increased cardiac NAD+ in all three metabolic groups. In glucose + FA perfused hearts, NR reduced IR injury, increased glycolytic intermediate phosphoenolpyruvate (PEP), TCA intermediate succinate and PPP intermediates ribose-5P (R5P) / sedoheptulose-7P (S7P), and was associated with activated glycolysis, without changes in TCA cycle or PPP activities. In the “no glycolysis” hearts, NR protection was lost, whereas NR still increased S7P. In the insulin hearts, glycolysis was largely accelerated, and NR protection abrogated. NR still increased PPP intermediates, with now high 13C-labeling of S7P, but NR was unable to increase metabolic pathway activities, including glycolysis. Protection by NR against IRI is only present in hearts with low glycolysis, and is associated with activation of glycolysis. When activation of glycolysis was prevented, through either examining “no glycolysis” hearts or “high glycolysis” hearts, NR protection was abolished. The data suggest that NR’s acute cardioprotective effects are mediated through glycolysis activation and are lost in the presence of insulin because of already elevated glycolysis.

Malonate protection against acute infarction in isolated mouse hearts is lost in the presence of insulin

Abstract Background &amp; Objectives Malonate, an inhibitor of mitochondrial succinate dehydrogenase (SDH), is a promising novel protectant against acute ischaemia-reperfusion injury (IRI) through reduction of ischaemic succinate accumulation and ROS production during early reperfusion. Malonate is still protective when given only at reperfusion, making it an ideal agent for treating AMI patients. However, numerous studies demonstrated that the metabolic milieu could affect the potency of cardioprotective treatments. Therefore, in this study, we explored whether and how the severity of cardiac I/R injury and cardioprotection by malonate is affected by the metabolic milieu. We hypothesized that protection by malonate is affected by metabolic milie. Methods Isolated Langendorff-perfused hearts (n=8 to 19 per group) of C57Bl/6N mice were perfused with perfusate to which substrates and hormones were added in a step-wise manner: 1) glucose (G), 2) glucose + glutamine (GG), 3) glucose + glutamine + palmitate (GGP), or 4) glucose + glutamine + palmitate + insulin (GGPI), and subjected to 30 min ischaemia (I) and 90 min reperfusion (R). Heart were treated with malonate (5mM) or vehicle (saline) during the first 5 min of reperfusion only. Cardiac function of the left ventricle was monitored by a balloon in the left ventricle. Coronary effluent was collected at 7 time points of reperfusion to detect the activity of lactate dehydrogenase (LDH) as an indicator of cell death. At the end reperfusion, hearts were used for infarct size determination through TTC staining. Data are presented as mean ± SD. Results Going from glucose-only perfusate to a perfusate containing all examined substrates and hormones, the severity of cardiac I/R injury increased: infarct size (%) went from 31.9 ± 10.9% to 37.7 ± 10.6% to 52.3 ± 14.2%, and finally to 68 ± 9.9 %. Similar trends for increased IRI were observed for LDH, End-Diastolic Pressure (EDP) and recovery of the Rate-Pressure Product (RPP). When compared with vehicle control, malonate significantly decreased IS% (control vs malonate: 31.9 ± 10.9% vs. 23.3 ± 4.8%, 37.7 ± 10.6% vs. 20.9 ± 8.5%, 68 ± 9.9% vs. 40.2 ± 9.2 %), and increased the RRP recovery (64.1 ± 16.6 %vs. 83.3 ± 17.9 %, 55.6 ± 11% vs. 72.3 ± 4%, 39.3 ± 15.6% vs. 55.9 ± 8.3%) in the G, GG and GGP group. The EDP was significantly decreased by malonate in the G and GG group (32.8 ± 14.3 mmHg vs. 20.9 ± 9.8 mmHg, 35.1 ± 8.7 mmHg vs. 22.2 ± 8.6 mmHg ). LDH was only significantly decreased by malonate in the GG group (35.2 ± 12.9 vs. 17.1 ± 7.7 U / GWW). Most importantly, adding insulin to the perfusate nullified protection by malonate for all four outcomes. Conclusions The severity of cardiac I/R injury is lowest with glucose-only perfusate, and increased with increased complexity of the metabolic milieu. Although malonate is a strong protectant against cardiac IRI for most metabolic milieus, in the isolated heart, protection is lost in the presence of insulin.

Nicotinamide riboside cardioprotection is mediated through glycolysis activation

Abstract Purpose Low NAD+ levels are observed in pathologies such as ageing, diabetes, heart failure and ischemia-reperfusion (IR) injury. Nicotinamide riboside (NR) has emerged as promising therapeutic NAD+ precursor [1,2]. NR was recently reported to protect against in vivo cardiac IR injury [3], but underlying mechanisms of protection were unexplored. Here we used the isolated mouse heart allowing metabolic control to examine NR’s metabolic mechanisms of cardiac protection. Methods Isolated C57BL/6N hearts were Langendorff-perfused with 5.5 mM glucose and 0.2 mM fatty acids (FA), and subjected to 35 min global ischemia and 90 min reperfusion. NR (50 mg/L) or vehicle were administered for 25 min before ischemia, and during the first 25 min of reperfusion. Cardiac functional recovery and cell death parameters (lactate dehydrogenase (LDH) release, % infarct size) were determined. To examine NR’s effects on cardiac metabolism, hearts were perfused with 5.5 mM 13C-glucose and 0.2 mM FA and analyzed by metabolomics and fluxomics using LC-MS techniques before ischemia. The dependency of NR’s protection on glycolysis was tested in hearts perfused with glucose replaced by pyruvate and lactate ("no glycolysis" hearts). Finally, NR’s protection and effects on cardiac metabolism were also examined in "high glycolysis" hearts, by adding insulin to the perfusate. Results NR reduced infarct size, LDH release and improved cardiac functional recovery for glucose/FA perfused hearts, demonstrating NR’s cardioprotection. In these glucose/FA hearts, metabolomics analysis showed NR increased NAD+, glycolytic intermediate phosphoenolpyruvate (PEP), Krebs cycle intermediate succinate and pentose phosphate pathway (PPP) intermediates ribose-5P (R5P) and sedoheptulose-7P (S7P) (Fig. 1). Fluxomics analysis demonstrated that NR’s protection was associated with activated glycolysis, without changes in tricarboxylic acid (TCA) cycle or PPP activities. No 13C-labeling of S7P was observed (Fig. 1). NR protection was lost in the "no glycolysis" hearts. In the "high glycolysis" hearts, NR was unable to protect the heart and increase glycolysis and reduced anaerobic glycolysis and ATP with increases in AMP, indicative of NR-induced energetic stress (Fig. 2). Remarkably, in these insulin perfused hearts, NR still increased PPP intermediates, with high 13C-labeling of S7P (Fig. 2). Conclusions NR’s protection against IR injury is only present in hearts with low glycolysis, associated with activation of pre-ischemic glycolysis. When activation of glycolysis was prevented, through either examining "no glycolysis" hearts or "high glycolysis" hearts, NR protection was abolished. NR treatment increased pre-ischemic PPP intermediates in either low or high glycolysis hearts, whereby insulin was necessary to incorporate 13C-glucose into S7P. The data suggest that NR’s acute cardioprotective effects are mediated through the activation of glycolysis.NR effects in low-glycolysis heartsNR effects in high-glycolysis hearts

Simultaneous assessment of mechanical and electrical function in Langendorff-perfused ex-vivo mouse hearts.

The Langendorff-perfused ex-vivo isolated heart model has been extensively used to study cardiac function for many years. However, electrical and mechanical function are often studied separately-despite growing proof of a complex electro-mechanical interaction in cardiac physiology and pathology. Therefore, we developed an isolated mouse heart perfusion system that allows simultaneous recording of electrical and mechanical function. Isolated mouse hearts were mounted on a Langendorff setup and electrical function was assessed via a pseudo-ECG and an octapolar catheter inserted in the right atrium and ventricle. Mechanical function was simultaneously assessed via a balloon inserted into the left ventricle coupled with pressure determination. Hearts were then submitted to an ischemia-reperfusion protocol. At baseline, heart rate, PR and QT intervals, intra-atrial and intra-ventricular conduction times, as well as ventricular effective refractory period, could be measured as parameters of cardiac electrical function. Left ventricular developed pressure (DP), left ventricular work (DP-heart rate product) and maximal velocities of contraction and relaxation were used to assess cardiac mechanical function. Cardiac arrhythmias were observed with episodes of bigeminy during which DP was significantly increased compared to that of sinus rhythm episodes. In addition, the extrasystole-triggered contraction was only 50% of that of sinus rhythm, recapitulating the "pulse deficit" phenomenon observed in bigeminy patients. After ischemia, the mechanical function significantly decreased and slowly recovered during reperfusion while most of the electrical parameters remained unchanged. Finally, the same electro-mechanical interaction during episodes of bigeminy at baseline was observed during reperfusion. Our modified Langendorff setup allows simultaneous recording of electrical and mechanical function on a beat-to-beat scale and can be used to study electro-mechanical interaction in isolated mouse hearts.

Disruption of Ephx2 in cardiomyocytes but not endothelial cells improves functional recovery after ischemia-reperfusion in isolated mouse hearts

Cytochromes P450 metabolize arachidonic acid to epoxyeicosatrienoic acids (EETs) which have numerous effects. After cardiac ischemia, EET-induced coronary vasodilation increases delivery of oxygen/nutrients to the myocardium, and EET-induced signaling protects cardiomyocytes against postischemic mitochondrial damage. Soluble epoxide hydrolase 2 (EPHX2) diminishes the benefits of EETs through hydrolysis to less active dihydroxyeicosatrienoic acids. EPHX2 inhibition or genetic disruption improves recovery of cardiac function after ischemia. Immunohistochemical staining revealed EPHX2 expression in cardiomyocytes and some endothelial cells but little expression in cardiac smooth muscle cells or fibroblasts. To determine specific roles of EPHX2 in cardiac cell types, we generated mice with cell-specific disruption of Ephx2 in endothelial cells (Ephx2fx/fx/Tek-cre) or cardiomyocytes (Ephx2fx/fx/Myh6-cre) to compare to global Ephx2-deficient mice (global Ephx2-/-) and WT (Ephx2fx/fx) mice in expression, EET hydrolase activity, and heart function studies. Most cardiac EPHX2 expression and activity is in cardiomyocytes with substantially less activity in endothelial cells. Ephx2fx/fx/Tek-cre hearts have similar EPHX2 expression, hydrolase activity, and postischemic cardiac function as control Ephx2fx/fx hearts. However, Ephx2fx/fx/Myh6-cre hearts were similar to global Ephx2-/- hearts with significantly diminished EPHX2 expression, decreased hydrolase activity, and enhanced postischemic cardiac function compared to Ephx2fx/fx hearts. During reperfusion, Ephx2fx/fx/Myh6-cre hearts displayed increased ERK activation compared to Ephx2fx/fx hearts, which could be reversed by EEZE treatment. EPHX2 did not regulate coronary vasodilation in this model. We conclude that EPHX2 is primarily expressed in cardiomyocytes where it regulates EET hydrolysis and postischemic cardiac function, whereas endothelial EPHX2 does not play a significant role in these processes.

Lethal Caspase-1/4-Dependent Injury Occurs in the First Minutes of Coronary Reperfusion and Requires Calpain Activity.

To study the relationship between caspase-1/4 and reperfusion injury, we measured infarct size (IS) in isolated mouse hearts undergoing 50 min global ischemia/2 h reperfusion. Starting VRT-043198 (VRT) at reperfusion halved IS. The pan-caspase inhibitor emricasan duplicated VRT's protection. IS in caspase-1/4-knockout hearts was similarly reduced, supporting the hypothesis that caspase-1/4 was VRT's only protective target. NLRC4 inflammasomes activate caspase-1. NLRC4 knockout hearts were not protected, eliminating NLRC4 as caspase-1/4's activator. The amount of protection that could be achieved by only suppressing caspase-1/4 activity was limited. In wild-type (WT) hearts, ischemic preconditioning (IPC) was as protective as caspase-1/4 inhibitors. Combining IPC and emricasan in these hearts or preconditioning caspase-1/4-knockout hearts produced an additive IS reduction, indicating that more protection could be achieved by combining treatments. We determined when caspase-1/4 exerted its lethal injury. Starting VRT after 10 min of reperfusion in WT hearts was no longer protective, revealing that caspase-1/4 inflicted its injury within the first 10 min of reperfusion. Ca++ influx at reperfusion might activate caspase-1/4. We tested whether Ca++-dependent soluble adenylyl cyclase (AC10) could be responsible. However, IS in AC10-/- hearts was not different from that in WT control hearts. Ca++-activated calpain has been implicated in reperfusion injury. Calpain could be releasing actin-bound procaspase-1 in cardiomyocytes, which would explain why caspase-1/4-related injury is confined to early reperfusion. The calpain inhibitor calpeptin duplicated emricasan's protection. Unlike IPC, adding calpain to emricasan offered no additional protection, suggesting that caspase-1/4 and calpain may share the same protective target.

Abstract 14479: Prolonged Decrease in Endothelial Cell (EC)-Specific Mitochondrial ROS Results in Mitochondrial Dysfunction in Coronary EC and Cardiac Hypertrophy in Post-AMI Animals

Introduction: Reducing mitochondrial ROS (mito-ROS) has been a novel approach for improving cardiac function. Here, we evaluated the effect of prolonged decrease in EC-specific mito-ROS on post-acute myocardial infarction (AMI) coronary angiogenesis and cardiac function. Hypothesis: We hypothesized that a prolonged decrease in EC-specific mito-ROS contributes to mitochondrial dysfunction in coronary EC resulting in post-AMI maladaptive cardiac remodeling. Methods: We used an EC-specific, tetracycline-controlled, transgenic MnSOD overexpression (MnSOD-OE) model. After 16 weeks of MnSOD-OE, mice underwent ligation of left anterior coronary artery (AMI). 28 days post-AMI, ejection fraction (EF), fractional shortening (FS), and left ventricle (LV) mass were determined by echocardiography (n=8/group). Isolated mouse heart ECs (MHEC) were evaluated by Seahorse for mitochondrial function and by western blot for cell signaling. Data were analyzed by Student’s t test. A p -value ≤0.05 was considered statistically significant. Results: MnSOD-OE mice showed 33% decrease (44.4±2.3 vs. 29.7±3.5) in EF and 35% decrease (21.7±1.2 vs. 14.2±1.7) in FS, compared to control. LV mass was increased by 51% (76.4±1.8 vs. 115.1±12.33 mg) in MnSOD-OE heart. LV end systolic and diastolic areas were increased in MnSOD-OE, by 2-fold (5.8±0.4 vs. 12.6±1.8) and 1.7-fold (9.8±0.5 vs. 16.8±1.6 mm 2 ), respectively. MnSOD-OE MHEC showed a reduction in mitochondrial maximal and spare respiration capacities, and decreased activation of Akt by 25% and ERK by 23%. Conclusions: Prolonged MnSOD-OE impaired mitochondrial respiration and inhibited Akt and ERK signaling in MHEC. These changes were associated with maladaptive remodeling post-AMI, including cardiac hypertrophy, LV dilatation, and systolic dysfunction. Together, these findings suggest that prolonged decrease in mito-ROS has detrimental effects on coronary EC resulting in maladaptive post-AMI cardiac remodeling.

Abstract P1060: Sarcomeric Protein Phosphorylation Protects Mouse Hearts From Ischemia/reperfusion Injury

Introduction: Post-translational modifications of cardiac troponin I (cTnI), an essential sarcomeric protein, have been known to play a vital role in regulating heart function. Site-specific phosphorylation on cTnI Ser199 is upregulated in human ischemic heart failure, negatively affects diastolic function but preserves ejection fraction in mice in vivo , and promotes contractility recovery after ischemia/reperfusion (I/R) in male isolated mouse hearts. However, its cardioprotective mechanism is largely unknown. Hypothesis: We assessed the hypothesis that cTnI Ser199 hyperphosphorylation protects hearts during I/R via down-regulating reactive oxygen species (ROS). Methods: Due to the current methodology limitation, there is no way to phosphorylate one specific site on cTnI in vivo . We, therefore, generated a transgenic mouse model named TgD that carried Serine (S) to Aspartic Acid (D) mutation at cTnI Ser200 (equivalent to Ser199 in human sequence) to mimic the site-specific hyperphosphorylation, which was a widely accepted method for in vivo studies on sarcomeric protein phosphorylation. The cardiac function was examined using Langendorff isolated hearts with pacing at baseline and after 30-minute global ischemia followed by 2-hour reperfusion. Results: At baseline, TgD hearts (both genders, 7-8 mice/gender, age 3-5 months) showed comparable rate pressure product (RPP), left ventricular developed pressure (LVDevP), and d P /d t max but decreased d P /d t min relative to wildtype (WT) littermates. After I/R, TgD presented significantly better RPP, LVDevP, d P /d t max , and d P /d t min with remarkable contractile function recovery rate compared to WT (i.e., 47% ±2% vs. 24%±2% in RPP, P&lt;0.05) in both genders. The level of total ROS was similar in TgD and WT hearts at baseline. Although the ROS increased after I/R, it remained at a significantly lower level in post-I/R TgD hearts than WT controls, associated with differentially altered activities of antioxidant enzymes. Conclusions: In conclusion, human heart-failure-related cTnI Ser199 hyperphosphorylation protects heart function during global I/R in isolated mouse hearts in both genders . The protective mechanism, at least in part, involves the downregulation of ROS.

K2P1 leak cation channels contribute to ventricular ectopic beats and sudden death under hypokalemia.

Hypokalemia causes ectopic heartbeats, but the mechanisms underlying such cardiac arrhythmias are not understood. In reduced serum K+ concentrations that occur under hypokalemia, K2P1 two-pore domain K+ channels change ion selectivity and switch to conduct inward leak cation currents, which cause aberrant depolarization of resting potential and induce spontaneous action potential of human cardiomyocytes. K2P1 is expressed in the human heart but not in mouse hearts. We test the hypothesis that K2P1 leak cation channels contribute to ectopic heartbeats under hypokalemia, by analysis of transgenic mice, which conditionally express induced K2P1 specifically in hearts, mimicking K2P1 channels in the human heart. Conditional expression of induced K2P1 specifically in the heart of hypokalemic mice results in multiple types of ventricular ectopic beats including single and multiple ventricular premature beats as well as ventricular tachycardia and causes sudden death. In isolated mouse hearts that express induced K2P1, sustained ventricular fibrillation occurs rapidly after perfusion with low K+ concentration solutions that mimic hypokalemic conditions. These observed phenotypes occur rarely in control mice or in the hearts that lack K2P1 expression. K2P1-expressing mouse cardiomyocytes of transgenic mice much more frequently fire abnormal single and/or rhythmic spontaneous action potential in hypokalemic conditions, compared to wild type mouse cardiomyocytes without K2P1 expression. These findings confirm that K2P1 leak cation channels induce ventricular ectopic beats and sudden death of transgenic mice with hypokalemia and imply that K2P1 leak cation channels may play a critical role in human ectopic heartbeats under hypokalemia.

AMPK Activation Alleviates Myocardial Ischemia-Reperfusion Injury by Regulating Drp1-Mediated Mitochondrial Dynamics.

Mitochondrial dysfunction is a salient feature of myocardial ischemia/reperfusion injury (MIRI), while the potential mechanism of mitochondrial dynamics disorder remains unclear. This study sought to explore whether activation of Adenosine monophosphate-activated protein kinase (AMPK) could alleviate MIRI by regulating GTPase dynamin-related protein 1 (Drp1)-mediated mitochondrial dynamics. Isolated mouse hearts in a Langendorff perfusion system were subjected to ischemia/reperfusion (I/R) treatment, and H9C2 cells were subjected to hypoxia /reoxygenation (H/R) treatment in vitro. The results showed that AICAR, the AMPK activator, could significantly improve the function of left ventricular, decrease arrhythmia incidence and myocardial infarction area of isolated hearts. Meanwhile, AICAR increased superoxide dismutase (SOD) activity and decreased malondialdehyde (MDA) content in myocardial homogenate. Mechanistically, AICAR inhibited the phosphorylation of Drp1 at Ser 616 while enhanced phosphorylation of Drp1 at Ser 637. In addition, AICAR reduced the expression of inflammatory cytokines including TNF-ɑ, IL-6, and IL-1β, as well as mitochondrial fission genes Mff and Fis1, while improved the expression of mitochondrial fusion genes Mfn1 and Mfn2. Similar results were also observed in H9C2 cells. AICAR improved mitochondrial membrane potential (MMP), reduced reactive oxygen species (ROS) production, and inhibited mitochondrial damage. To further prove if Drp1 regulated mitochondrial dynamics mediated AMPK protection effect, the mitochondrial fission inhibitor Mdivi-1 was utilized. We found that Mdivi-1 significantly improved MMP, inhibited ROS production, reduced the expression of TNF-a, IL-6, IL-1β, Fis1, and Mff, and improved the expression of Mfn1 and Mfn2. However, the protection effect of Mdivi-1 was not reversed by AMPK inhibitor Compound C. In conclusion, this study confirmed that activation of AMPK exerted the protective effects on MIRI, which were largely dependent on the inhibition of Drp1-mediated mitochondrial fission.

PPARβ/δ preconditioning increased the cardioprotective effects of MSC in IR injury

Mesenchymal stromal/stem cells (MSC) have been widely used for their therapeutic properties in acute myocardial infarction (AMI) due to their pleiotropic anti-inflammatory, anti-apoptotic, anti-fibrotic and pro-angiogenic properties. Despite promising results in animal studies and their safety/efficacy in phase I/II trials, inconsistencies have been reported in phase III. We have recently shown in a mouse model of ischemia-reperfusion (IR) that the cardioprotective effect of MSC administered during reperfusion was dependent on the presence of PPARβ/δ receptors. However, the role of the modulation of PPARβ/δ on MSC anti-apoptotic and cardioprotective properties has never been investigated. This study aims investigating the role of PPARβ/δ activation on MSC therapeutic properties in an ex vivo model of AMI and their anti-apoptotic functions on cardiac cells. Isolated mouse hearts were subjected to 30 minutes of global ischemia and 60 minutes of reperfusion (IR). Murine MSC (naïve MSC), MSCago (pretreated with GW0742 1 μM agonist) MSCantago (pretreated with GSK0660 1 μM) were administered in the perfusion system during the reperfusion period. Specific DNA fragmentation was quantified in MSC or MSCago challenged with 350 μM H2O2 during 4 hours and in H9c2 rat cardiomyoblasts and EAhy926 human endothelial cells challenged with H2O2 and co-cultured with naïve or primed MSC. MSCago injection during reperfusion decreased infarct size by a larger extent than MSC, an effect lost with MSCantago. Accordingly, an increased number of cells of MSCago was detected in the left ventricular wall after 1 hour of reperfusion. In vitro, we observed for MSCago an increased resistance against oxidative resistance and a more potent anti-apoptotic effect on both cardiomyocytes and endothelial cells in vitro than naive MSC. MSC preconditioning via PPARβ/δ enhanced their cardioprotective effect against IR injury ex vivo by both increasing their resistance to oxidative stress and their anti-apoptotic effects on myocytes and endothelial cells. These results could be of major interest to improve MSC efficacy for the cardioprotection of the myocardium in AMI patients.

Hydropersulfides (RSSH) Outperform Post-Conditioning and Other Reactive Sulfur Species in Limiting Ischemia-Reperfusion Injury in the Isolated Mouse Heart.

Hydrogen sulfide (H2S) exhibits protective effects in cardiovascular disease such as myocardial ischemia/reperfusion (I/R) injury, cardiac hypertrophy, and atherosclerosis. Despite these findings, its mechanism of action remains elusive. Recent studies suggest that H2S can modulate protein activity through redox-based post-translational modifications of protein cysteine residues forming hydropersulfides (RSSH). Furthermore, emerging evidence indicates that reactive sulfur species, including RSSH and polysulfides, exhibit cardioprotective action. However, it is not clear yet whether there are any pharmacological differences in the use of H2S vs. RSSH and/or polysulfides. This study aims to examine the differing cardioprotective effects of distinct reactive sulfur species (RSS) such as H2S, RSSH, and dialkyl trisulfides (RSSSR) compared with canonical ischemic post-conditioning in the context of a Langendorff ex-vivo myocardial I/R injury model. For the first time, a side-by-side study has revealed that exogenous RSSH donation is a superior approach to maintain post-ischemic function and limit infarct size when compared with other RSS and mechanical post-conditioning. Our results also suggest that RSSH preserves mitochondrial respiration in H9c2 cardiomyocytes exposed to hypoxia-reoxygenation via inhibition of oxidative phosphorylation while preserving cell viability.

A Recombinant Dimethylarginine Dimethylaminohydrolase-1–Based Biotherapeutics to Pharmacologically Lower Asymmetric Dimethyl Arginine, thus Improving Postischemic Cardiac Function and Cardiomyocyte Mitochondrial Activity

High serum levels of asymmetric dimethyl arginine (ADMA) are associated with cardiovascular disease and mortality. Pharmacological agents to specifically lower ADMA and their potential impact on cardiovascular complications are not known. In this study, we aimed to investigate the effect of specific lowering of ADMA on myocardial response to ischemia-reperfusion injury (I/R) and direct effects on cardiomyocyte function. Effects of recombinant dimethylarginine dimethylaminohydrolase (rDDAH)-1 on I/R injury were determined using isolated mouse heart preparation. Respiration capacity and mitochondrial reactive oxygen species (ROS) generation were determined on mouse cardiomyocytes. Our results show that lowering ADMA by rDDAH-1 treatment resulted in improved recovery of cardiac function and reduction in myocardial infarct size in mouse heart response to I/R injury (control 22.24 ±4.60% versus rDDAH-1 15.90 ±4.23%, P < 0.01). In mouse cardiomyocytes, rDDAH-1 treatment improved ADMA-induced dysregulation of respiration capacity and decreased mitochondrial ROS. Furthermore, in human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes with impaired contractility under hypoxia and high ADMA, rDDAH-1 treatment improved recovery and beating frequency (P < 0.05). rDDAH-1 treatment selectively modified I/R-induced myocardial cytokine expression, resulting in reduction in proinflammatory cytokine IL-17A (P < 0.001) and increased expression of anti-inflammatory cytokines IL-10 and IL-13 (P < 0.01). Further in vitro studies showed that IL-17A was the predominant and common cytokine modulated by ADMA-DDAH pathway in heart, cardiomyocytes, and endothelial cells. These studies show that lowering ADMA by pharmacological treatment with rDDAH-1 reduced I/R injury, improved cardiac function, and ameliorated cardiomyocyte bioenergetics and beating activity. These effects may be attributable to ADMA lowering in cardiomyocytes and preservation of cardiomyocyte mitochondrial function. SIGNIFICANCE STATEMENT: The pathological role of asymmetric dimethyl arginine (ADMA) has been demonstrated by its association with cardiovascular disease and mortality. Currently, pharmacological drugs to specifically lower ADMA are not available. The present study provides the first evidence that lowering of ADMA by recombinant recombinant dimethylarginine dimethylaminohydrolase (rDDAH)-1 improved postischemic cardiac function and cardiomyocyte bioenergetics and beating activity. Our studies suggest that lowering of ADMA by pharmacologic treatment offers opportunity to develop new therapies for the treatment of cardiovascular and renal disease.

Lack of authentic atrial fibrillation in commonly used murine atrial fibrillation models.

The mouse is a useful preclinical species for evaluating disease etiology due to the availability of a wide variety of genetically modified strains and the ability to perform disease-modifying manipulations. In order to establish an atrial filtration (AF) model in our laboratory, we profiled several commonly used murine AF models. We initially evaluated a pharmacological model of acute carbachol (CCh) treatment plus atrial burst pacing in C57BL/6 mice. In an effort to observe micro-reentrant circuits indicative of authentic AF, we employed optical mapping imaging in isolated mouse hearts. While CCh reduced atrial refractoriness and increased atrial tachyarrhythmia vulnerability, the left atrial (LA) excitation patterns were rather regular without reentrant circuits or wavelets. Therefore, the atrial tachyarrhythmia resembled high frequency atrial flutter, not typical AF per se. We next examined both a chronic angiotensin II (Ang II) infusion model and the surgical model of transverse aortic constriction (TAC), which have both been reported to induce atrial and ventricular structural changes that serve as a substrates for micro-reentrant AF. Although we observed some extent of atrial remodeling such as fibrosis or enlarged LA diameter, burst pacing-induced atrial tachyarrhythmia vulnerability did not differ from control mice in either model. This again suggested that an AF-like pathophysiology is difficult to demonstrate in the mouse. To continue searching for a valid murine AF model, we studied mice with a cardiac-specific deficiency (KO) in liver kinase B1 (Cardiac-LKB1), which has been reported to exhibit spontaneous AF. Indeed, the electrocardiograms (ECG) of conscious Cardiac-LKB1 KO mice exhibited no P waves and had irregular RR intervals, which are characteristics of AF. Histological evaluation of Cardiac-LKB1 KO mice revealed dilated and fibrotic atria, again consistent with AF. However, atrial electrograms and optical mapping revealed that electrical activity was limited to the sino-atrial node area with no electrical conduction into the atrial myocardium beyond. Thus, Cardiac-LKB1 KO mice have severe atrial myopathy or atrial standstill, but not AF. In summary, the atrial tachyarrhythmias we observed in the four murine models were distinct from typical human AF, which often exhibits micro- or macro-reentrant atrial circuits. Our results suggest that the four murine AF models we examined may not reflect human AF well, and raise a cautionary note for use of those murine models to study AF.

Bioassays of Humoral Cardioprotective Factors Released by Remote Ischemic Conditioning in Patients Undergoing Coronary Artery Bypass Surgery.

Remote ischemic conditioning (RIC) induces the release of circulating cardioprotective factors and attenuates myocardial ischemia/reperfusion injury. Evidence for such humoral cardioprotective factor(s) is derived from transfer with plasma (derivatives) from one individual undergoing RIC to another individual's heart, even across species. With transfer into an isolated perfused heart, only a single plasma (derivative) sample can be studied with infarct size as endpoint, and therefore the comparison of samples before and after RIC or between RIC and placebo is hampered by the inter-individual variation of infarct sizes in isolated perfused hearts. We therefore developed a preparation of cardiomyocytes from a single mouse heart, where aliquots of the same heart can undergo hypoxia/reoxygenation (H/R) with exposure to buffer, RIC, or placebo samples without or with pharmacological blockade. To validate this approach, we used plasma dialysates taken before and after RIC from patients undergoing coronary bypass grafting who had experienced protection by RIC (troponin release ↓ by 28% vs placebo). The cardiomyocyte bioassay had little variation after H/R with buffer (mean ± standard deviation; 7% ± 2% viable cells) and demonstrated preserved viability after RIC (15% ± 5% vs 6% ± 3% before). For comparison, infarct size in isolated mouse hearts after global ischemia and reperfusion was 22% ± 14% of left ventricular mass after versus 42% ± 14% before RIC. Stattic, an inhibitor of signal transducer and activator of transcription (STAT)3 protein, abrogated protection in the cardiomyocytes. We have thus established a cardiomyocyte bioassay to analyze RIC's protection which minimizes inter-individual variation and the use of animals.

Measuring the Mitochondrial Ubiquinone (Q) Pool Redox State in Isolated Respiring Mitochondria.

The ubiquinone (Q) pool represents a node in the mitochondrial electron transport chain (ETC) onto which the electrons of all respiratory dehydrogenases converge. The redox state of the Q pool correlates closely with the electron flux through the ETC and is thus a parameter of great metabolic value for both the mitochondrial and cellular metabolism. Here, we describe the simultaneous measurement of respiratory rates of isolated mouse heart mitochondria and the redox state of their Q pool using a custom-made combination of a Clark-type oxygen electrode and a Q electrode.

Вплив низьких концентрацій монооксиду вуглецю на метаболізм ізольованого серця в умовах ішемії-реперфузії

The purpose of the study was to determine the effect of different concentrations of carbon monoxide on the metabolism of isolated mice hearts. Materials and methods. To elucidate the effect of low concentrations of carbon monoxide on the myocardium, we performed retrograde perfusion of isolated hearts of laboratory mice with Krebs-Henseleit solution, which was saturated with carbon monoxide for 5, 10, and 30 minutes. We then determined how different concentrations of carbon monoxide affected coronary volumetric flow rate, myocardial glucose and calcium uptake, creatinine release, and aspartate aminotransferase release. During perfusion, R-wave amplitude and R-R interval were measured using an electrocardiograph. To determine the effect of ischemia on the heart muscle during perfusion with solutions of different concentrations, we measured the area of the affected myocardium after staining with 2,3,5-triphenyltetrazolium chloride. Results and discussion. After these studies, it was found that different concentrations of carbon monoxide had a dose-dependent effect on the isolated mouse heart. However, the dependence of the effects does not follow the pattern «lowest concentration – lowest effect». At the same time, an increase in concentration did not mean an increase in adverse effects on the myocardium. Even on the contrary, the smallest concentration led to increased signs of ischemic myocardial damage. In particular, the use of the solution, through which carbon monoxide was passed for 5 minutes, caused vasoconstrictor effect during perfusion. At the end of reperfusion, vasoconstrictor effect was observed after using a solution through which carbon monoxide was passed for 10 minutes. Increased glucose uptake was observed in the group with 30-minute carbon monoxide permeation against the background of the minimal myocardial creatinine release. In this group there was also a decrease in Ca2+ loss at the beginning of reperfusion (immediately after ischemia). The above phenomenon explains the least degree of ischemic myocardial damage in the isolated mouse heart. The obtained data should be expanded. Since it is difficult to accurately determine the dose of carbon monoxide, then the use of donor compounds is promising. Such compounds include CORM-2 and CORM-3. Under physiological conditions, they decompose in a controlled manner, releasing a specific amount of carbon monoxide. Conclusion. The obtained results indicate that at different concentrations of carbon monoxide can differently influence different structures of cardiomyocyte: at one concentration it binds to calcium channels, other concentrations influence ion channels of plasma membrane, which can explain all these dependencies