Initial Despair and Current Hope of Identifying a Clinically Useful Treatment of Myocardial Reperfusion Injury: Insights Derived from Studies of Platelet P2Y12 Antagonists and Interference with Inflammation and NLRP3 Assembly.

Myocardial necrosis following the successful reperfusion of a coronary artery occluded by thrombus in a patient presenting with ST-elevation myocardial infarction (STEMI) continues to be a serious problem, despite the multiple attempts to attenuate the necrosis with agents that have shown promise in pre-clinical investigations. Possible reasons include confounding clinical risk factors, the delayed application of protective agents, poorly designed pre-clinical investigations, the possible effects of routinely administered agents that might unknowingly already have protected the myocardium or that might have blocked protection, and the biological differences of the myocardium in humans and experimental animals. A better understanding of the pathobiology of myocardial infarction is needed to stem this reperfusion injury. P2Y12 receptor antagonists minimize platelet aggregation and are currently part of the standard treatment to prevent thrombus formation and propagation in STEMI protocols. Serendipitously, these P2Y12 antagonists also dramatically attenuate reperfusion injury in experimental animals and are presumed to provide a similar protection in STEMI patients. However, additional protective agents are needed to further diminish reperfusion injury. It is possible to achieve additive protection if the added intervention protects by a mechanism different from that of P2Y12 antagonists. Inflammation is now recognized to be a critical factor in the complex intracellular response to ischemia and reperfusion that leads to tissue necrosis. Interference with cardiomyocyte inflammasome assembly and activation has shown great promise in attenuating reperfusion injury in pre-clinical animal models. And the blockade of the executioner protease caspase-1, indeed, supplements the protection already seen after the administration of P2Y12 antagonists. Importantly, protective interventions must be applied in the first minutes of reperfusion, if protection is to be achieved. The promise of such a combination of protective strategies provides hope that the successful attenuation of reperfusion injury is attainable.

Gamma secretase activating protein promotes end-organ dysfunction after bacterial pneumonia.

Pneumonia elicits the production of cytotoxic beta amyloid (Aß) that contributes to end-organ dysfunction, yet the mechanism(s) linking infection to activation of the amyloidogenic pathway that produces cytotoxic Aß is unknown. Here, we tested the hypothesis that gamma-secretase activating protein (GSAP), which contributes to the amyloidogenic pathway in the brain, promotes end-organ dysfunction following bacterial pneumonia. First-in-kind Gsap knockout rats were generated. Wild-type and knockout rats possessed similar body weights, organ weights, circulating blood cell counts, arterial blood gases, and cardiac indices at baseline. Intratracheal Pseudomonas aeruginosa infection caused acute lung injury and a hyperdynamic circulatory state. Whereas infection led to arterial hypoxemia in wild-type rats, the alveolar-capillary barrier integrity was preserved in Gsap knockout rats. Infection potentiated myocardial infarction following ischemia-reperfusion injury, and this potentiation was abolished in knockout rats. In the hippocampus, GSAP contributed to both pre- and postsynaptic neurotransmission, increasing the presynaptic action potential recruitment, decreasing neurotransmitter release probability, decreasing the postsynaptic response, and preventing postsynaptic hyperexcitability, resulting in greater early long-term potentiation but reduced late long-term potentiation. Infection abolished early and late long-term potentiation in wild-type rats, whereas the late long-term potentiation was partially preserved in Gsap knockout rats. Furthermore, hippocampi from knockout rats, and both the wild-type and knockout rats following infection, exhibited a GSAP-dependent increase in neurotransmitter release probability and postsynaptic hyperexcitability. These results elucidate an unappreciated role for GSAP in innate immunity and highlight the contribution of GSAP to end-organ dysfunction during infection.NEW & NOTEWORTHY Pneumonia is a common cause of end-organ dysfunction, both during and in the aftermath of infection. In particular, pneumonia is a common cause of lung injury, increased risk of myocardial infarction, and neurocognitive dysfunction, although the mechanisms responsible for such increased risk are unknown. Here, we reveal that gamma-secretase activating protein, which contributes to the amyloidogenic pathway, is important for end-organ dysfunction following infection.

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.

Society for Cardiovascular Magnetic Resonance 2019 Case of the Week series.

The Society for Cardiovascular Magnetic Resonance (SCMR) is an international society focused on the research, education, and clinical application of cardiovascular magnetic resonance (CMR). The SCMR web site ( https://www.scmr.org ) hosts a case series designed to present case reports demonstrating the unique attributes of CMR in the diagnosis or management of cardiovascular disease. Each clinical presentation is followed by a brief discussion of the disease and unique role of CMR in disease diagnosis or management guidance. By nature, some of these are somewhat esoteric, but all are instructive. In this publication, we provide a digital archive of the 2019 Case of the Week series as a means of further enhancing the education of those interested in CMR and as a means of more readily identifying these cases using a PubMed or similar search engine.

Cytoprotective Effect of Vitamin D on Doxorubicin-Induced Cardiac Toxicity in Triple Negative Breast Cancer.

BACKGROUND: Doxorubicin (Dox) is a first-line treatment for triple negative breast cancer (TNBC), but its use may be limited by its cardiotoxicity mediated by the production of reactive oxygen species. We evaluated whether vitamin D may prevent Dox-induced cardiotoxicity in a mouse TNBC model. METHODS: Female Balb/c mice received rodent chow with vitamin D3 (1500 IU/kg; vehicle) or chow supplemented with additional vitamin D3 (total, 11,500 IU/kg). the mice were inoculated with TNBC tumors and treated with intraperitoneal Dox (6 or 10 mg/kg). Cardiac function was evaluated with transthoracic echocardiography. The cardiac tissue was evaluated with immunohistochemistry and immunoblot for levels of 4-hydroxynonenal, NAD(P)H quinone oxidoreductase (NQO1), C-MYC, and dynamin-related protein 1 (DRP1) phosphorylation. RESULTS: At 15 to 18 days, the mean ejection fraction, stroke volume, and fractional shortening were similar between the mice treated with vitamin D + Dox (10 mg/kg) vs. vehicle but significantly greater in mice treated with vitamin D + Dox (10 mg/kg) vs. Dox (10 mg/kg). Dox (10 mg/kg) increased the cardiac tissue levels of 4-hydroxynonenal, NQO1, C-MYC, and DRP1 phosphorylation at serine 616, but these increases were not observed with vitamin D + Dox (10 mg/kg). A decreased tumor volume was observed with Dox (10 mg/kg) and vitamin D + Dox (10 mg/kg). CONCLUSIONS: Vitamin D supplementation decreased Dox-induced cardiotoxicity by decreasing the reactive oxygen species and mitochondrial damage, and did not decrease the anticancer efficacy of Dox against TNBC.

Caspase-1 inhibition by VX-765 administered at reperfusion in P2Y12 receptor antagonist-treated rats provides long-term reduction in myocardial infarct size and preservation of ventricular function.

Patients with acute myocardial infarction receive a P2Y12 receptor antagonist prior to reperfusion, a treatment that has reduced, but not eliminated, mortality, or heart failure. We tested whether the caspase-1 inhibitor VX-765 given at reperfusion (a requirement for clinical use) can provide sustained reduction of infarction and long-term preservation of ventricular function in a pre-clinical model of ischemia/reperfusion that had been treated with a P2Y12 receptor antagonist. To address, the hypothesis open-chest rats were subjected to 60-min left coronary artery branch occlusion/120-min reperfusion. Vehicle or inhibitors were administered intravenously immediately before reperfusion. With vehicle only, 60.3 ± 3.8% of the risk zone suffered infarction. Ticagrelor, a P2Y12 antagonist, and VX-765 decreased infarct size to 42.8 ± 3.3 and 29.2 ± 4.9%, respectively. Combining ticagrelor with VX-765 further decreased infarction to 17.5 ± 2.3%. Similar to recent clinical trials, combining ticagrelor and ischemic postconditioning did not result in additional cardioprotection. VX-765 plus another P2Y12 antagonist, cangrelor, also decreased infarction and preserved ventricular function when reperfusion was increased to 3 days. In addition, VX-765 reduced infarction in blood-free, isolated rat hearts indicating at least a portion of injurious caspase-1 activation originates in cardiac tissue. While the pro-drug VX-765 only protected isolated hearts when started prior to ischemia, its active derivative VRT-043198 provided the same amount of protection when started at reperfusion, indicating that even in blood-free hearts, caspase-1 appears to exert its injury only at reperfusion. Moreover, VX-765 decreased circulating IL-1ß, prevented loss of cardiac glycolytic enzymes, preserved mitochondrial complex I activity, and decreased release of lactate dehydrogenase, a marker of pyroptosis. Our results are the first demonstration of a clinical-grade drug given at reperfusion providing additional, sustained infarct size reduction when added to a P2Y12 receptor antagonist.

The impact of irreproducibility and competing protection from P2Y12 antagonists on the discovery of cardioprotective interventions.

Scientists and clinicians have been concerned by the lack of a clinically suitable strategy for cardioprotection in patients with acute myocardial infarction despite decades of intensive pre-clinical investigations and a surprising number of clinical trials based on those observations which have uniformly been disappointing. However, it would be a mistake to abandon this search. Rather it would be useful to examine these past efforts and determine reasons for the multiple failures. It appears that earlier clinical trials were often based on results from a single experimental laboratory, thus minimizing the importance of establishing reproducibility in multiple laboratories by multiple scientists and in multiple models. Clinical trials should be discouraged unless robust protection is demonstrated in pre-clinical testing. After approximately 2005 a loading dose of a platelet P2Y12 receptor antagonist became increasingly widespread in patients with acute myocardial infarction prior to revascularization and quickly became standard-of-care. These agents are now thought to be a cause of failure of recent clinical trials since these pleiotropic drugs also happen to be potent postconditioning mimetics. Thus, introduction of an additional cardioprotective strategy such as ischemic postconditioning which uses the same signaling pathway as these P2Y12 antagonists would be redundant and doomed to failure. Additive cardioprotection could be achieved only if the second intervention had a different mechanism of cardioprotection. This concept has been demonstrated in experimental animals. So lack of reproducibility of earlier studies and failure to examine interventions in experimental animals also treated with anti-platelet agents could well explain past failures. These realizations should clear the way for development of interventions which can be translated into successful clinical treatments.

Cangrelor-Mediated Cardioprotection Requires Platelets and Sphingosine Phosphorylation.

In animal models platelet P2Y12 receptor antagonists put the heart into a protected state, not as a result of suppressed thrombosis but rather through protective signaling, similar to that for ischemic postconditioning. While both ischemic postconditioning and the P2Y12 blocker cangrelor protect blood-perfused hearts, only the former protects buffer-perfused hearts indicating that the blocker requires a blood-borne constituent or factor to protect. We used an anti-platelet antibody to make thrombocytopenic rats to test if that factor resides within the platelet. Infarct size was measured in open-chest rats subjected to 30-min ischemia/2-h reperfusion. Infarct size was not different in thrombocytopenic rats showing that preventing aggregation alone is not protective. While ischemic preconditioning could reduce infarct size in thrombocytopenic rats, the P2Y12 inhibitor cangrelor could not, indicating that it protects by interacting with some factor in the platelet. Ischemic preconditioning is known to require phosphorylation of sphingosine. In rats treated with dimethylsphingosine to block sphingosine kinase, cangrelor was no longer protective. Thus cangrelor's protective mechanism appears to also involve sphingosine kinase revealing yet another similarity to conditioning's mechanism.

Mitochondrially targeted Endonuclease III has a powerful anti-infarct effect in an in vivo rat model of myocardial ischemia/reperfusion.

Recent reports indicate that elevating DNA glycosylase/AP lyase repair enzyme activity offers marked cytoprotection in cultured cells and a variety of injury models. In this study, we measured the effect of EndoIII, a fusion protein construct that traffics Endonuclease III, a DNA glycosylase/AP lyase, to the mitochondria, on infarct size in a rat model of myocardial ischemia/reperfusion. Open-chest, anesthetized rats were subjected to 30 min of occlusion of a coronary artery followed by 2 h of reperfusion. An intravenous bolus of EndoIII, 8 mg/kg, just prior to reperfusion reduced infarct size from 43.8 ± 1.4% of the risk zone in control animals to 24.0 ± 1.3% with no detectable hemodynamic effect. Neither EndoIII's vehicle nor an enzymatically inactive EndoIII mutant (K120Q) offered any protection. The magnitude of EndoIII's protection was comparable to that seen with the platelet aggregation inhibitor cangrelor (25.0 ± 1.8% infarction of risk zone). Because loading with a P2Y12 receptor blocker to inhibit platelets is currently the standard of care for treatment of acute myocardial infarction, we tested whether EndoIII could further reduce infarct size in rats treated with a maximally protective dose of cangrelor. The combination reduced infarct size to 15.1 ± 0.9% which was significantly smaller than that seen with either cangrelor or EndoIII alone. Protection from cangrelor but not EndoIII was abrogated by pharmacologic blockade of phosphatidylinositol-3 kinase or adenosine receptors indicating differing cellular mechanisms. We hypothesized that EndoIII protected the heart from spreading necrosis by preventing the release of proinflammatory fragments of mitochondrial DNA (mtDNA) into the heart tissue. In support of this hypothesis, an intravenous bolus at reperfusion of deoxyribonuclease I (DNase I) which should degrade any DNA fragments escaping into the extracellular space was as protective as EndoIII. Furthermore, the combination of EndoIII and DNase I produced additive protection. While EndoIII would maintain mitochondrial integrity in many of the ischemic cardiomyocytes, DNase I would further prevent mtDNA released from those cells that EndoIII could not save from propagating further necrosis. Thus, our mtDNA hypothesis would predict additive protection. Finally to demonstrate the toxicity of mtDNA, isolated hearts were subjected to 15 min of global ischemia. Infarct size doubled when the coronary vasculature was filled with mtDNA fragments during the period of global ischemia. To our knowledge, EndoIII and DNase are the first agents that can both be given at reperfusion and add to the protection of a P2Y12 blocker, and thus should be effective in today's patient with acute myocardial infarction.

Automated segmentation of the left-ventricle from MRI with a fully convolutional network to investigate CTRCD in breast cancer patients.

Purpose: The goal of this study was to develop a fully convolutional network (FCN) tool to automatedly segment the left-ventricular (LV) myocardium in displacement encoding with stimulated echoes MRI. The segmentation results are used for LV chamber quantification and strain analyses in breast cancer patients susceptible to cancer therapy-related cardiac dysfunction (CTRCD). Approach: A DeepLabV3+ FCN with a ResNet-101 backbone was custom-designed to conduct chamber quantification on 45 female breast cancer datasets (23 training, 11 validation, and 11 test sets). LV structural parameters and LV ejection fraction (LVEF) were measured, and myocardial strains estimated with the radial point interpolation method. Myocardial classification validation was against quantization-based ground-truth with computations of accuracy, Dice score, average perpendicular distance (APD), Hausdorff-distance, and others. Additional validations were conducted with equivalence tests and Cronbach's alpha (C-α) intraclass correlation coefficients between the FCN and a vendor tool on chamber quantification and myocardial strain computations. Results: Myocardial classification results against ground-truth were Dice=0.89, APD=2.4 mm, and accuracy=97% for the validation set and Dice=0.90, APD=2.5 mm, and accuracy=97% for the test set. The confidence intervals (CI) and two one-sided t-test results of equivalence tests between the FCN and vendor-tool were CI=-1.36% to 2.42%, p-value < 0.001 for LVEF (58±5% versus 57±6%), and CI=-0.71% to 0.63%, p-value < 0.001 for longitudinal strain (-15±2% versus -15±3%). Conclusions: The validation results were found equivalent to the vendor tool-based parameter estimates, which show that accurate LV chamber quantification followed by strain analysis for CTRCD investigation can be achieved with our proposed FCN methodology.

Triple therapy greatly increases myocardial salvage during ischemia/reperfusion in the in situ rat heart.

BACKGROUND: Cangrelor, a P2Y12 receptor blocker, administered just prior to reperfusion reduced but did not eliminate myocardial infarction in rabbits. Combining cangrelor with ischemic postconditioning offered no additional protection suggesting they protected by a similar mechanism. To determine if cangrelor's protection might be additive to other cardioprotective interventions we tested cangrelor in combination with ischemic preconditioning, cariporide, a sodium-hydrogen exchange blocker, and mild hypothermia. METHODS: Open-chest rats underwent 30-min coronary occlusion/2-h reperfusion. RESULTS: Cangrelor, administered as a bolus (60 µg/kg) 10 min before reperfusion and continued as an infusion (6 µg/kg/min) for the duration of the experiment, decreased infarction from 45.3 % of risk zone in control hearts to 25.0 %. Combining cangrelor and ischemic preconditioning offered no additional protection. Mild hypothermia (32-33 °C) instituted by peritoneal lavage with cold saline just prior to coronary occlusion resulted in 25.2 % infarction, and combining cangrelor and hypothermia nearly halved infarction to 14.1 % of risk zone. Cariporide (0.5 mg/kg) just prior to occlusion resulted in 27.2 % infarction and 15.8 % when combined with cangrelor. Combining cangrelor, hypothermia and cariporide further halved infarction to only 6.3 %. We also tested another P2Y12 inhibitor ticagrelor which is chemically similar to cangrelor. Ticagrelor (20 mg/kg) fed 1 h prior to surgery reduced infarct size by an amount similar to that obtained with cangrelor (25.6 % infarction), and this protective effect was abolished by chelerythrine and wortmannin, thus implicating participation of PKC and PI3-kinase, resp., in signaling. CONCLUSIONS: Cardioprotection from a P2Y12 receptor antagonist can be combined with at least 2 other strategies to magnify the protection. Combining multiple interventions that use different cardioprotective mechanisms could provide powerful protection against infarction in patients with acute coronary thrombosis.

Two classes of anti-platelet drugs reduce anatomical infarct size in monkey hearts.

BACKGROUND: Recent studies in rabbits have demonstrated that platelet P2Y12 receptor antagonists are cardioprotective, and that the mechanism is surprisingly not related to blockade of platelet aggregation but rather to triggering of the same signal transduction pathway seen in pre- and postconditioning. We wanted to determine whether this same cardioprotection could be documented in a primate model and whether the protection was limited to P2Y12 receptor antagonists or was a class effect. METHODS: Thirty-one macaque monkeys underwent 90-min LAD occlusion/4-h reperfusion. RESULTS: The platelet P2Y12 receptor blocker cangrelor started just prior to reperfusion significantly decreased infarction by an amount equivalent to that seen with ischemic postconditioning (p < 0.001). For any size of risk zone, infarct size in treated hearts was significantly smaller than that in control hearts. OM2, an investigational murine antibody against the primate collagen receptor glycoprotein (GP) VI, produced similar protection (p < 0.01) suggesting a class effect. Both cangrelor and OM2 were quite effective at blocking platelet aggregation (94 % and 97 %, respectively). CONCLUSIONS: Thus in a primate model in which infarct size could be determined directly platelet anti-aggregatory agents are cardioprotective. The important implication of these investigations is that patients with acute myocardial infarction who are treated with platelet anti-aggregatory agents prior to revascularization may already be in a postconditioned state. This hypothesis may explain why in recent clinical trials postconditioning-mimetic interventions which were so protective in animal models had at best only a modest effect.

Can global longitudinal strain (GLS) with magnetic resonance prognosticate early cancer therapy-related cardiac dysfunction (CTRCD) in breast cancer patients, a prospective study?

PURPOSE: To determine if Artificial Intelligence-based computation of global longitudinal strain (GLS) from left ventricular (LV) MRI is an early prognostic factor of cancer therapy-related cardiac dysfunction (CTRCD) in breast cancer patients. The main hypothesis based on the patients receiving antineoplastic chemotherapy treatment was CTRCD risk analysis with GLS that was independent of LV ejection fraction (LVEF). METHODS: Displacement Encoding with Stimulated Echoes (DENSE) MRI was acquired on 32 breast cancer patients at baseline and 3- and 6-month follow-ups after chemotherapy. Two DeepLabV3+ Fully Convolutional Networks (FCNs) were deployed to automate image segmentation for LV chamber quantification and phase-unwrapping for 3D strains, computed with the Radial Point Interpolation Method. CTRCD risk (cardiotoxicity and adverse cardiac events) was analyzed with Cox Proportional Hazards (PH) models with clinical and contractile prognostic factors. RESULTS: GLS worsened from baseline to the 3- and 6-month follow-ups (-19.1 ± 2.1%, -16.0 ± 3.1%, -16.1 ± 3.0%; P < 0.001). Univariable Cox regression showed the 3-month GLS significantly associated as an agonist (hazard ratio [HR]-per-SD: 2.1; 95% CI: 1.4-3.1; P < 0.001) and LVEF as a protector (HR-per-SD: 0.8; 95% CI: 0.7-0.9; P = 0.001) for CTRCD occurrence. Bivariable regression showed the 3-month GLS (HR-per-SD: 2.0; 95% CI: 1.2-3.4; P = 0.01) as a CTRCD prognostic factor independent of other covariates, including LVEF (HR-per-SD: 1.0; 95% CI: 0.9-1.2; P = 0.9). CONCLUSIONS: The end-point analyses proved the hypothesis that GLS is an early, independent prognosticator of incident CTRCD risk. This novel GLS-guided approach to CTRCD risk analysis could improve antineoplastic treatment with further validation in a larger clinical trial.

Direct left-ventricular global longitudinal strain (GLS) computation with a fully convolutional network.

This study's purpose was to develop a direct MRI-based, deep-learning semantic segmentation approach for computing global longitudinal strain (GLS), a known metric for detecting left-ventricular (LV) cardiotoxicity in breast cancer. Displacement Encoding with Stimulated Echoes cardiac image phases acquired from 30 breast cancer patients and 30 healthy females were unwrapped via a DeepLabV3 + fully convolutional network (FCN). Myocardial strains were directly computed from the unwrapped phases with the Radial Point Interpolation Method. FCN-unwrapped phases of a phantom's rotating gel were validated against quality-guided phase-unwrapping (QGPU) and robust transport of intensity equation (RTIE) phase-unwrapping. FCN performance on unwrapping human LV data was measured with F1 and Dice scores versus QGPU ground-truth. The reliability of FCN-based strains was assessed against RTIE-based strains with Cronbach's alpha (C-α) intraclass correlation coefficient. Mean squared error (MSE) of unwrapping the phantom experiment data at 0 dB signal-to-noise ratio were 1.6, 2.7 and 6.1 with FCN, QGPU and RTIE techniques. Human data classification accuracies were F1 = 0.95 (Dice = 0.96) with FCN and F1 = 0.94 (Dice = 0.95) with RTIE. GLS results from FCN and RTIE were -16 ± 3% vs. -16 ± 3% (C-α = 0.9) for patients and -20 ± 3% vs. -20 ± 3% (C-α = 0.9) for healthy subjects. The low MSE from the phantom validation demonstrates accuracy of phase-unwrapping with the FCN and comparable human subject results versus RTIE demonstrate GLS analysis accuracy. A deep-learning methodology for phase-unwrapping in medical images and GLS computation was developed and validated in a heterogeneous cohort.

Does mild hypothermia protect against reperfusion injury? The debate continues.

Mild hypothermia (32-35°C) salvages ischemic myocardium and reduces infarct size in hearts undergoing ischemia/reperfusion. It is clear that a cardioprotective effect is evident when the heart is cooled during ischemia, and the protection is greater as the duration of normothermic ischemia is increasingly limited. The effect of cooling just before and at reperfusion is more controversial. Multiple experimental studies have revealed no effect of mild hypothermia on myocardial infarction when cooling was initiated in the waning minutes of ischemia. But Götberg et al. have demonstrated a small effect in pigs cooled with cold intravenous saline and a venous thermode, although the effect of cooling during ischemia continued to be more prominent. Clinical studies have been disappointing, and possible explanations are offered. Götberg's new data are encouraging, but it is questioned whether this is the correct time to conduct a new large-scale clinical trial.

Cardioprotection by mild hypothermia during ischemia involves preservation of ERK activity.

Cooling the ischemic heart by just a few degrees protects it from infarction without affecting its mechanical function, but the mechanism of this protection is unknown. We investigated whether signal transduction pathways might be involved in the anti-infarct effect of mild hypothermia (35°C). Isolated rabbit hearts underwent 30 min of coronary artery occlusion/2 h of reperfusion. They were either maintained at 38.5°C or cooled to 35°C just before and only during ischemia. Infarct size was measured. The effects of the protein kinase C inhibitor chelerythrine, the nitric oxide synthase inhibitor N (ω)-nitro-L: -arginine methyl ester (L: -NAME), the phosphatidylinositol 3-kinase antagonist wortmannin, or either of the mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitors PD98059 or U0126 on cooling's protection were examined. Myocardial ATP assays were performed and the level of phosphorylation of extracellular signal-regulated kinase (ERK) and MEK was examined by western blotting. To investigate an effect of cooling on protein phosphatase (PPase), a PPase inhibitor cantharidin was tested in the infarct model and the effect of mild hypothermia on PP2A activity in vitro was measured. Infarct size was 34.4 ± 2.2% of the ischemic zone in normothermic (38.5°C) hearts, but only 15.6 ± 8.7% in hearts cooled to 35°C during ischemia. Mechanical function was unaffected. Neither chelerythrine, L: -NAME, nor wortmannin had any effect, but both PD98059 and U0126 completely eliminated protection. Ischemia rather than reperfusion was the critical time when ERK had to be active to realize protection. Phosphorylation of ERK and MEK fell during normothermic ischemia, but during hypothermic ischemia phosphorylation of ERK remained high while that of MEK was increased. Cooling only slightly delayed the rate at which ATP fell during ischemia, and ERK inhibition did not affect that attenuation suggesting ATP preservation was unrelated to protection. Cantharidin, like cooling, also protected during ischemia but not at reperfusion, and its protection was dependent on ERK phosphorylation. However, mild hypothermia had a negligible effect on PP2A activity in an in vitro assay. Hence, mild hypothermia preserves ERK and MEK activity during ischemia which somehow protects the heart. While a PPase inhibitor mimicked cooling's protection, a direct effect of cooling on PP2A could not be demonstrated.

Evidence for an intracellular localization of the adenosine A2B receptor in rat cardiomyocytes.

Protection achieved by ischemic preconditioning is dependent on A(2B) adenosine receptors (A(2B)AR) in rabbit and mouse hearts and, predictably, an A(2B)AR agonist protects them. But it is controversial whether cardiomyocytes themselves actually express A(2B)AR. The present study tested whether A(2B)AR could be demonstrated on rat cardiomyocytes. Isolated rat hearts experienced 30 min of ischemia and 120 min of reperfusion. The highly selective, cell-permeant A(2B)AR agonist BAY60-6583 (500 nM) infused at reperfusion reduced infarct size from 40.4 ± 2.0% of the risk zone in control hearts to 19.9 ± 2.8% indicating that A(2B)AR are protective in rat heart as well. Furthermore, BAY60-6583 reduced calcium-induced mitochondrial permeability transition in isolated rat cardiomyocytes. A(2B)AR protein could be demonstrated in isolated cardiomyocytes by western blotting. In addition, message for A(2B)AR was found in individual cardiomyocytes using quantitative RT-PCR. Surprisingly, immunofluorescence microscopy did not show A(2B)AR on the cardiomyocyte's sarcolemma but rather at intracellular sites. Co-staining with MitoTracker Red in isolated cardiomyocytes revealed A(2B)AR are localized to mitochondria. Western blot analysis of a mitochondrial fraction from either rat heart biopsies or isolated cardiomyocytes revealed a strong A(2B)AR band. Thus, the present study demonstrates that activation of A(2B)AR is strongly cardioprotective in rat heart and suppresses transition pores in isolated cardiomyocytes, and A(2B)AR are expressed in individual cardiomyocytes. However, surprisingly, A(2B)AR are present in or near mitochondria rather than on the sarcolemma as are other adenosine receptors. Because A(2B)AR signaling is thought to result in inhibition of mitochondrial transition pores, this convenient location may be important.

A deep-learning semantic segmentation approach to fully automated MRI-based left-ventricular deformation analysis in cardiotoxicity.

Left-ventricular (LV) strain measurements with the Displacement Encoding with Stimulated Echoes (DENSE) MRI sequence provide accurate estimates of cardiotoxicity damage related to breast cancer chemotherapy. This study investigated an automated LV chamber quantification tool via segmentation with a supervised deep convolutional neural network (DCNN) before strain analysis with DENSE images. Segmentation for chamber quantification analysis was conducted with a custom DeepLabV3+ DCNN with ResNet-50 backbone on 42 female breast cancer datasets (22 training-sets, eight validation-sets and 12 independent test-sets). Parameters such as LV end-diastolic diameter (LVEDD) and ejection fraction (LVEF) were quantified, and myocardial strains analyzed with the Radial Point Interpolation Method (RPIM). Myocardial classification was validated against ground-truth with sensitivity-specificity analysis, the metrics of Dice, average perpendicular distance (APD) and Hausdorff-distance. Following segmentation, validation was conducted with the Cronbach's Alpha (C-Alpha) intraclass correlation coefficient between LV chamber quantification results with DENSE and Steady State Free Precession (SSFP) acquisitions and a vendor tool-based method to segment the DENSE data, and similarly for myocardial strain analysis in the chambers. The results of myocardial classification from segmentation of the DENSE data were accuracy = 97%, Dice = 0.89 and APD = 2.4 mm in the test-set. The C-Alpha correlations from comparing chamber quantification results between the segmented DENSE and SSFP data and vendor tool-based method were 0.97 for LVEF (56 ± 7% vs 55 ± 7% vs 55 ± 6%, p = 0.6) and 0.77 for LVEDD (4.6 ± 0.4 cm vs 4.5 ± 0.3 cm vs 4.5 ± 0.3 cm, p = 0.8). The validation metrics against ground-truth and equivalent parameters obtained from the SSFP segmentation and vendor tool-based comparisons show that the DCNN approach is applicable for automated LV chamber quantification and subsequent strain analysis in cardiotoxicity.