Catecholamines are key modulators of ventricular repolarization patterns in the ball python (Python regius)

Ectothermic vertebrates experience daily changes in body temperature, and anecdotal observations suggest these changes affect ventricular repolarization such that the T-wave in the ECG changes polarity. Mammals, in contrast, can maintain stable body temperatures, and their ventricular repolarization is strongly modulated by changes in heart rate and by sympathetic nervous system activity. The aim of this study was to assess the role of body temperature, heart rate, and circulating catecholamines on local repolarization gradients in the ectothermic ball python (Python regius). We recorded body-surface electrocardiograms and performed open-chest high-resolution epicardial mapping while increasing body temperature in five pythons, in all of which there was a change in T-wave polarity. However, the vector of repolarization differed between individuals, and only a subset of leads revealed T-wave polarity change. RNA sequencing revealed regional differences related to adrenergic signaling. In one denervated and Ringer’s solution–perfused heart, heating and elevated heart rates did not induce change in T-wave polarity, whereas noradrenaline did. Accordingly, electrocardiograms in eight awake pythons receiving intra-arterial infusion of the β-adrenergic receptor agonists adrenaline and isoproterenol revealed T-wave inversion in most individuals. Conversely, blocking the β-adrenergic receptors using propranolol prevented T-wave change during heating. Our findings indicate that changes in ventricular repolarization in ball pythons are caused by increased tone of the sympathetic nervous system, not by changes in temperature. Therefore, ventricular repolarization in both pythons and mammals is modulated by evolutionary conserved mechanisms involving catecholaminergic stimulation.

CineCT imaging platform for in-vivo and ex-vivo measurement of myocardial biomechanics post myocardial infarction and following intramyocardial delivery of theranostic hydrogel

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Institute of Health (NIH) Purpose Myocardial infarction (MI) induces acute regional changes in myocardial strain and stiffness in the infarct and the remote areas of the left ventricle (LV), which lead to adverse changes in LV geometry and function. We hypothesize that cineCT imaging could evaluate these biomechanical changes along with the effects of intramyocardial delivery of theranostic hydrogels. Introduction We present an experimental platform to assess changes in the deformation of the LV myocardium using contrast cineCT (CCT) imaging of the beating porcine heart (active deformation) before and after acute MI and intramyocardial delivery of an imageable theranostic hydrogel. We then assess the acute effects of hydrogel delivery early post-MI on biomechanics (passive deformation) using an ex vivo perfused heart preparation. Methods Contrast cineCT imaging was performed using 64-slice CT on 5 Yorkshire pigs without MI (n = 3) or with MI (n = 2). MI pigs had serial imaging performed before and 1 hour after acute surgical coronary occlusion to induce anterolateral MI. One MI pig was also imaged 1 hour after intramyocardial injection of a novel imageable theranostic iodinated hydrogel within the MI region. Post euthanasia, excised hearts were flushed with chilled UW cardioplegic solution and mounted on a custom inflation apparatus for cineCT imaging during LV inflation by external pump. LV pressure was cycled between 10 and 60 mmHg at 35 bpm. Dilute iohexol was injected into aortic root and UW perfusate (15 ml, 1 ml/sec). CineCT image series were reconstructed, contrast enhanced, resampled to the LV long axis (Z), and exported as a series of 10 CT volumes covering 0-90% of the cardiac/inflation cycle. Volumes were registered incrementally using nonlinear image registration (BioImageSuite) and the calculated displacement at each time point was exported at a resolution of 1 mm. A custom Matlab program was used to fit the displacement field to local trilinear polynomials and then calculate the displacement gradients and 3D Lagrangian strains. To estimate the accuracy of this approach, cardiac volumes were also numerically deformed using a 10 pixel translation and 5% triaxial stretch. Results We successfully acquired serial in-vivo and ex-vivo 3D CineCT images for assessment of the active and passive LV myocardial deformation and tracked deformation through the full cardiac/inflation cycle (Figure 2). Numerical deformation tests showed average tracking errors of < 0.2 mm (1/4 pixel) in the X,Y,Z directions of the volume. These resulted in Lagrangian strain errors of < 0.47% for the in-plane strains EXX and EYY (radial and circumferential plane) and < 0.5% for EZZ (long axis). Conclusions We have developed a novel CineCT imaging platform that allows for high resolution in-vivo and ex-vivo measurement of myocardial biomechanics post-MI and following intramyocardial delivery of imageable theranostic hydrogels, which may improve early active and passive biomechanics.

Preconditioning or Postconditioning with 8-Br-cAMP-AM Protects the Heart against Regional Ischemia and Reperfusion: A Role for Mitochondrial Permeability Transition

The cAMP analogue 8-Br-cAMP-AM (8-Br) confers marked protection against global ischaemia/reperfusion of isolated perfused heart. We tested the hypothesis that 8-Br is also protective under clinically relevant conditions (regional ischaemia) when applied either before ischemia or at the beginning of reperfusion, and this effect is associated with the mitochondrial permeability transition pore (MPTP). 8-Br (10 μM) was administered to Langendorff-perfused rat hearts for 5 min either before or at the end of 30 min regional ischaemia. Ca2+-induced mitochondria swelling (a measure of MPTP opening) and binding of hexokinase II (HKII) to mitochondria were assessed following the drug treatment at preischaemia. Haemodynamic function and ventricular arrhythmias were monitored during ischaemia and 2 h reperfusion. Infarct size was evaluated at the end of reperfusion. 8-Br administered before ischaemia attenuated ventricular arrhythmias, improved haemodynamic function, and reduced infarct size during ischaemia/reperfusion. Application of 8-Br at the end of ischaemia protected the heart during reperfusion. 8-Br promoted binding of HKII to the mitochondria and reduced Ca2+-induced mitochondria swelling. Thus, 8-Br protects the heart when administered before regional ischaemia or at the beginning of reperfusion. This effect is associated with inhibition of MPTP via binding of HKII to mitochondria, which may underlie the protective mechanism.

Glucagon-like peptide-1 directly increases heart rate and shortens atrial refractoriness: an in vivo and ex vivo study in pigs

Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Novo Nordisk Foundation Synergy program Novo Nordisk Foundation Center for Basic Metabolic Research Background Treatment with glucagon-like peptide-1 receptor agonists (GLP-1 RAs) in patients with type 2 diabetes not only reduces hyperglycaemia, but also improves cardiovascular outcomes. However, GLP-1 RA treatment also increases heart rate: an apparent paradox. Purpose Whether the heart rate increase is a direct effect, and whether GLP-1 affects other aspects of cardiac electrophysiology, remain unclear. To answer these questions we investigated the effect of GLP-1 infusion on cardiac electrophysiology in vivo and ex vivo in pigs and pig hearts, respectively, during sinus rhythm and pacing. Methods Anaesthetised pigs (n = 8) received infusions of GLP-1 (10 pmol/kg/min). Electrocardiogram, atrial monophasic action potentials and atrial conduction velocity data were collected and atrial and ventricular effective refractory periods (ERP) were measured. For the ex vivo studies, pig hearts (n = 7) were excised, retrogradely perfused and exposed to consecutive bolus perfusions of 2 and 4 nmol GLP-1, 100 nmol of the GLP-1 receptor antagonist exendin-9-39 and a final 4 nmol bolus of GLP-1. The same electrophysiological parameters were measured. Results In anaesthetised pigs, GLP-1 increased heart rate, cardiac output and diastolic pressure, while systemic vascular resistance was decreased. Infusion of GLP-1 decreased PQ interval in sinus rhythm (P = 0.019, n = 8) and during atrial pacing (P = 0.027, n = 6) with 8 ± 3 % and 12 ± 3 %, respectively. Additionally, GLP-1 decreased atrial ERP at all pacing cycle lengths (P = 0.04, n = 7), while ventricular ERP was unaffected (P = 0.29, n = 7). In the isolated perfused heart, GLP-1 increased heart rate with 13 ± 2 bpm (P = 0.001, n = 7). This increase in heart rate was completely abolished by pre-administration of exendin-9-39. Atrial ERP shortened after GLP-1 perfusion (P = 0.01, n = 7) comparable to the in vivo studies, with an average decrease of 11 ± 2 %. This effect was also abolished by exendin-9-39. Conclusion GLP-1 increases heart rate through activation of the GLP-1 receptor in the isolated perfused heart, suggesting a direct effect of GLP-1 rather than activation through the central nervous system. Additionally, GLP-1 affects atrial electrophysiology, but not ventricular electrophysiology, in vivo and ex vivo independent of the increase in heart rate.

Reciprocalcation of caveolin and HSP-72 on IPC interceded cardio-protection in the orchidectomized rats

Diminished testosterone levels conjoined to cardiovascular risk factor mainly myocardial infarction which broadens the risk of cardiovascular mortality referring to age. Ischemic preconditioning (IPC) is one of the interventions to shield such injury. The present study implicated the possible involvement of caveolin and heat shock protein 72 (HSP-72) during stress in orchidectomy (OCD) challenged rats. OCD was performed in male rats and kept for 6 weeks to observe the reduction in the level of testosterone. Isolated perfused heart of normal and OCD group was subjected to ischemic insult as per IPC cycle. Myocardial infarct size, haemodynamic, enzymatic and oxidative stress parameter were assessed for each heart. Diadzein (DDZ) a caveolin inhibitor was administered before the isolation of heart and it significantly decreases myocardial infarct size, release of lactate dehydrogenase, creatinine kinase and oxidative stress marker. DDZ also potentiated the effect IPC-mediated increase in the heart rate and coronary flow. The effect of caveolin inhibitor was remarkably reduced by quercetin administered before 1 h. of the administration DDZ. The findings of this study revealed that protection of myocardium induced by caveolin inhibitor pretreatment has not been lost in OCD rat heart.

Abstract P046: Variable Effect Of Gstm1 Knockout In Mice

Glutathione S-transferase μ-1 (GSTM1) is an enzyme that has a role in the detoxification of electrophiles, including xenobiotics and products of oxidative stress. In humans, the GSTM1(0) null allele deletion variant is highly prevalent and is associated with both an increase in oxidative stress and an increased risk/severity of a variety of diseases, including cancers, chronic kidney disease, hypertension, and coronary artery disease.Recently, we generated a Gstm1 knockout ( Gstm1 -/- ) mouse line on a 129S6 background. Using these animals, we demonstrated that the loss of GSTM1 resulted in increased oxidative stress and greater kidney injury with either subtotal nephrectomy-induced chronic kidney disease or angiotensin II-induced hypertension, in accordance with clinical data.Following myocardial infarction, reperfusion of the heart results in additional tissue damage that is also mediated by acute oxidative stress. Here, we used an ex vivo Langendorff-perfused heart model of acute cardiac ischemia-reperfusion injury (IRI). Contrary to the expectation that hearts from Gstm1 -/- mice would be more susceptible to IRI, we found that the loss of the antioxidant enzyme GSTM1 was protective in males compared to age-matched wild-type controls. In contrast, the Gstm1 -/- genotype was deleterious in female hearts as expected.To explore the hypothesis that the loss of GSTM1 causes compensatory upregulation of other GSTs and that this effect varies based on both tissue and sex, we examined mRNA expression of α, θ, κ, μ, and π classes of Gst genes via qPCR and corresponding protein expression via HPLC and western blot. We found significant differences between male vs. female and heart vs. kidney in several GSTs both in their expression in wild-type mice and in the change in expression between wild-type and Gstm1 -/- mice. These results suggest that the severity of cardiac IRI may depend on the adaptive response mediated by genes encoding GST enzymes.

Abstract MP124: De-palmitoylation of Cysteine 202 of Cyclophilin-D by Calcium is Important for the Activation of the Permeability Transition Pore

Cyclophilin-D (CypD) is a well-known regulator of the mitochondrial permeability transition pore (PTP), the main effector of cardiac ischemia/reperfusion (I/R) injury characterized by oxidative stress and calcium overload. However, the mechanism by which CypD activates PTP is poorly understood. Cysteine 202 of CypD (C202) is highly conserved across species and can undergo redox-sensitive post-translational modifications, such as S-nitrosylation and oxidation. To study the importance of C202, we developed a knock-in mouse model using CRISPR where CypD-C202 was mutated to a serine (C202S). Hearts from these mice are protected against I/R injury. We found C202 to be abundantly S-palmitoylated under baseline conditions while C202 was de-palmitoylated during ischemia in WT hearts. To further investigate the mechanism of de-palmitoylation during ischemia, we considered the increase of matrix calcium, oxidative stress and uncoupling of ATP synthesis from the electron transport chain. We tested the effects of these conditions on the palmitoylation of CypD in isolated cardiac mitochondria. The palmitoylation of CypD was assessed using a resin-assisted capture (Acyl-RAC). We report that oxidative stress (phenylarsenide) and uncoupling (CCCP) had no effect on CypD palmitoylation (p>0.05, n=3 and n=7 respectively). However, calcium overload led to de-palmitoylation of CypD to the level observed at the end ischemia (1±0.10 vs 0.63±0.09, p=0.012, n=9). To further test the hypothesis that calcium regulates S-palmitoylation of CypD we measured S-palmitoylation of CypD in non-perfused heart lysates from global germline mitochondrial calcium uniporter knock-out mice (MCU-KO), which have reduced mitochondrial calcium and we found an increase in S-palmitoylation of CypD (WT 1±0.04 vs MCU-KO 1.603±0.11, p<0.001, n=6). The data are consistent with the hypothesis that C202 is important for the CypD mediated activation of PTP. Ischemia leads to increased matrix calcium which in turn promotes the de-palmitoylation of CypD on C202. The now free C202 can further be oxidized during reperfusion leading to the activation of PTP. Thus, S-palmitoylation and oxidation of CypD-C202 possibly target CypD to the PTP, making them potent regulators of cardiac I/R injury.

First characterization of glucose flux through the hexosamine biosynthesis pathway (HBP) in ex vivo mouse heart

The hexosamine biosynthesis pathway (HBP) branches from glycolysis and forms UDP-GlcNAc, the moiety for O-linked β-GlcNAc (O-GlcNAc) post-translational modifications. An inability to directly measure HBP flux has hindered our understanding of the factors regulating protein O-GlcNAcylation. Our goals in this study were to (i) validate a LC-MS method that assesses HBP flux as UDP-GlcNAc (13C)-molar percent enrichment (MPE) and concentration and (ii) determine whether glucose availability or workload regulate cardiac HBP flux. For (i), we perfused isolated murine working hearts with [U-13C6]glucosamine (1, 10, 50, or 100 μm), which bypasses the rate-limiting HBP enzyme. We observed a concentration-dependent increase in UDP-GlcNAc levels and MPE, with the latter reaching a plateau of 56.3 ± 2.9%. For (ii), we perfused isolated working hearts with [U-13C6]glucose (5.5 or 25 mm). Glycolytic efflux doubled with 25 mm [U-13C6]glucose; however, the calculated HBP flux was similar among the glucose concentrations at ∼2.5 nmol/g of heart protein/min, representing ∼0.003–0.006% of glycolysis. Reducing cardiac workload in beating and nonbeating Langendorff perfusions had no effect on the calculated HBP flux at ∼2.3 and 2.5 nmol/g of heart protein/min, respectively. To the best of our knowledge, this is the first direct measurement of glucose flux through the HBP in any organ. We anticipate that these methods will enable foundational analyses of the regulation of HBP flux and protein O-GlcNAcylation. Our results suggest that in the healthy ex vivo perfused heart, HBP flux does not respond to acute changes in glucose availability or cardiac workload.