Deep learning segmentation model for quantification of infarct size in pigs with myocardial ischemia/reperfusion.

Infarct size (IS) is the most robust end point for evaluating the success of preclinical studies on cardioprotection. The gold standard for IS quantification in ischemia/reperfusion (I/R) experiments is triphenyl tetrazolium chloride (TTC) staining, typically done manually. This study aimed to determine if automation through deep learning segmentation is a time-saving and valid alternative to standard IS quantification. High-resolution images from TTC-stained, macroscopic heart slices were retrospectively collected from pig experiments (n = 390) with I/R without/with cardioprotection to cover a wide IS range. Existing IS data from pig experiments, quantified using a standard method of manual and subsequent digital labeling of film-scan annotations, were used as reference. To automate the evaluation process with the aim to be more objective and save time, a deep learning pipeline was implemented; the collected images (n = 3869) were pre-processed by cropping and labeled (image annotations). To ensure their usability as training data for a deep learning segmentation model, IS was quantified from image annotations and compared to IS quantified using the existing film-scan annotations. A supervised deep learning segmentation model based on dynamic U-Net architecture was developed and trained. The evaluation of the trained model was performed by fivefold cross-validation (n = 220 experiments) and testing on an independent test set (n = 170 experiments). Performance metrics (Dice similarity coefficient [DSC], pixel accuracy [ACC], average precision [mAP]) were calculated. IS was then quantified from predictions and compared to IS quantified from image annotations (linear regression, Pearson's r; analysis of covariance; Bland-Altman plots). Performance metrics near 1 indicated a strong model performance on cross-validated data (DSC: 0.90, ACC: 0.98, mAP: 0.90) and on the test set data (DSC: 0.89, ACC: 0.98, mAP: 0.93). IS quantified from predictions correlated well with IS quantified from image annotations in all data sets (cross-validation: r = 0.98; test data set: r = 0.95) and analysis of covariance identified no significant differences. The model reduced the IS quantification time per experiment from approximately 90 min to 20 s. The model was further tested on a preliminary test set from experiments in isolated, saline-perfused rat hearts with regional I/R without/with cardioprotection (n = 27). There was also no significant difference in IS between image annotations and predictions, but the performance on the test set data from rat hearts was lower (DSC: 0.66, ACC: 0.91, mAP: 0.65). IS quantification using a deep learning segmentation model is a valid and time-efficient alternative to manual and subsequent digital labeling.

Activation of the nonneuronal cholinergic cardiac system by hypoxic preconditioning protects isolated adult cardiomyocytes from hypoxia/reoxygenation injury.

Activation of the vagus nerve mediates cardioprotection and attenuates myocardial ischemia/reperfusion (I/R) injury. In response to vagal activation, acetylcholine (ACh) is released from the intracardiac nervous system (ICNS) and activates intracellular cardioprotective signaling cascades. Recently, however, a nonneuronal cholinergic cardiac system (NNCCS) in cardiomyocytes has been described as an additional source of ACh. To investigate whether the NNCCS mediates cardioprotection in the absence of vagal and ICNS activation, we used a reductionist approach of isolated adult rat ventricular cardiomyocytes without neuronal cells, using hypoxic preconditioning (HPC) as a protective stimulus. Adult rat ventricular cardiomyocytes were isolated, the absence of neuronal cells was confirmed, and HPC was induced by 10/20 min hypoxia/reoxygenation (H/R) before subjection to 30/5 min H/R to simulate I/R injury. Cardiomyocyte viability was assessed by trypan blue staining at baseline and after HPC+H/R or H/R. Intra- and extracellular ACh was quantified using liquid chromatography-coupled mass spectrometry at baseline, after HPC, after hypoxia, and after reoxygenation, respectively. In a subset of experiments, muscarinic and nicotinic ACh receptor (m- and nAChR) antagonists were added during HPC or during H/R. Cardiomyocyte viability at baseline (69 ± 4%) was reduced by H/R (10 ± 3%). With HPC, cardiomyocyte viability was preserved after H/R (25 ± 6%). Intra- and extracellular ACh increased during hypoxia; HPC further increased both intra- and extracellular ACh (from 0.9 ± 0.7 to 1.5 ± 1.0 nmol/mg; from 0.7 ± 0.6 to 1.1 ± 0.7 nmol/mg, respectively). The addition of mAChR and nAChR antagonists during HPC had no impact on HPC's protection; however, protection was abrogated when antagonists were added during H/R (cardiomyocyte viability after H/R: 23 ± 5%; 13 ± 4%). In conclusion, activation of the NNCCS is involved in cardiomyocyte protection; HPC increases intra- and extracellular ACh during H/R, and m- and nAChRs are causally involved in HPC's cardiomyocyte protection during H/R. The interplay between upstream ICNS activation and NNCCS activation in myocardial cholinergic metabolism and cardioprotection needs to be investigated in future studies.NEW & NOTEWORTHY The intracardiac nervous system is considered to be involved in ischemic conditioning's cardioprotection through the release of acetylcholine (ACh). However, we demonstrate that hypoxic preconditioning (HPC) protects from hypoxia/reoxygenation injury and increases intra- and extracellular ACh during hypoxia in isolated adult ventricular rat cardiomyocytes. HPC's protection involves cardiomyocyte muscarinic and nicotinic ACh receptor activation. Thus, besides the intracardiac nervous system, a nonneuronal cholinergic cardiac system may also be causally involved in cardiomyocyte protection by ischemic conditioning.

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.

Cardioprotection by post-conditioning with exogenous triiodothyronine in isolated perfused rat hearts and isolated adult rat cardiomyocytes.

Ischemic post-conditioning (iPoCo) by coronary re-occlusion/reperfusion during immediate reperfusion after prolonged myocardial ischemia reduces infarct size. Mechanical manipulation of culprit lesions, however, carries the risk of coronary microembolization which may obscure iPoCo's cardioprotection. Pharmacological post-conditioning with exogenous triiodothyronine (T3) could serve as an alternative conditioning strategy. Similar to iPoCo, T3 may activate cardioprotective prosurvival pathways. We aimed to study T3's impact on infarct size and its underlying signal transduction. Hearts were isolated from male Lewis rats (200-380 g), buffer-perfused and subjected to 30 min/120 min global zero-flow ischemia/reperfusion (I/R). In additional hearts, either iPoCo (2 × 30 s/30 s I/R) was performed or T3 (100-500 µg/L) infused at reperfusion. Infarct size was demarcated with triphenyl tetrazolium chloride staining and calculated as percent of ventricular mass. Infarct size was reduced with iPoCo to 16 ± 7% vs. 36 ± 4% with I/R only. The maximum infarct size reduction was observed with 300 µg/L T3 (14 ± 2%). T3 increased the phosphorylation of protein kinase B and mitogen extracellular-regulated-kinase 1/2, both key enzymes of the reperfusion injury salvage kinase (RISK) pathway. Pharmacological RISK blockade (RISK-BL) during reperfusion abrogated T3's cardioprotection (35 ± 10%). Adult ventricular cardiomyocytes were isolated from buffer-perfused rat hearts and exposed to 30 min/5 min hypoxia/reoxygenation (H/R); reoxygenation was initiated without or with T3, respectively, and without or with RISK-BL, respectively. Maximal preservation of viability was observed with 500 µg/L T3 after H/R (27 ± 4% of all cells vs. 5 ± 3% in time-matched controls). Again, RISK-BL abrogated protection (11 ± 3%). Mitochondria were isolated at early reperfusion from buffer-perfused rat hearts without or with iPoCo or 300 µg/L T3, respectively, at reperfusion. T3 improved mitochondrial function (i.e.: increased respiration, adenosine triphosphate production, calcium retention capacity, and decreased reactive oxygen species formation) to a similar extent as iPoCo. T3 at reperfusion reduces infarct size by activation of the RISK pathway. T3's protection is a cardiomyocyte phenomenon and targets mitochondria.