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Although irreversible cardiomyocyte injury provokes intracellular Ca2+ ([Ca2+]i) overload, the underlying dynamics of this response and its effects on cellular morphology remain unknown. We therefore visualised rapid-scanning confocal fluo4-[Ca2+]i dynamics and morphology of cardiomyocytes in Langendorff-perfused rat hearts following saponin-membrane permeabilisation. Our data demonstrate that 0.4% saponin-treated myocytes immediately exhibited high-frequency Ca2+ waves (131.3 waves/min/cell) with asynchronous, oscillatory contractions having a mean propagation velocity of 117.8 μm/s. These waves slowly decreased in frequency, developed a prolonged decay phase, and disappeared in 10 min resulting in high-static, fluo4-fluorescence intensity. The myocytes showing these waves displayed contraction bands, i.e., band-like actin-fibre aggregates with disruption of sarcomeric α-actinin. The contraction bands were not attenuated by the abolition of Ca2+ waves under pretreatment with ryanodine plus thapsigargin, but were partially attenuated by the calpain inhibitor MDL28170, while mechanical arrest of the myocytes by 2,3-butanedione monoxime completely attenuated contraction-band formation. The depletion of adenosine 5′-triphosphate by the mitochondrial electron uncoupler carbonyl cyanide 4-trifluoromethoxy phenylhydrazone also attenuated Ca2+ waves and contraction bands. Overall, saponin-induced myocyte [Ca2+]i overload provokes agonal Ca2+ waves and contraction bands. Contraction bands are not the direct consequence of the waves but are caused by cross-bridge interactions of the myocytes under calpain-mediated proteolysis. [ABSTRACT FROM AUTHOR]

The [Na.sup.+]-HC[O.sup.-.sub.3] cotransporter (NBC) plays a key role in intracellular pH (p[H.sub.i] regulation in normal ventricular muscle. However, the state of NBC in nonischemic hypertrophied hearts is unresolved. In this study, we examined functional and molecular properties of NBC in adult rat ventricular myocytes. The cells were enzymatically isolated from both normal and hypertrophied hearts. Ventricular hypertrophy was induced by pressure overload created by suprarenal abdominal aortic constriction of 50% for 7 wk. p[H.sub.i] was measured in single cells using the fluorescent pH indicator 2',7'-bis(2-carboxyethyl)5-(6)carboxy-fluorescein. Real-time PCR analysis was used to quantitatively assess expression of NBC-encoding mRNA, including SLC4A4 (encoding electrogenic NBC, NBCel) and SLC4A7 (electroneutral NBC, NBCnl). Our results demonstrate that: 1) mRNA levels of both the electrogenic NBCel (SLC4A4) and electroneutral NBCnl (SLC4A7) forms of NBC were increased by aortic constriction, 2) the onset of NBC upregulation occurred within 3 days after constriction, 3) normal and hypertrophied ventricles displayed regional differences in NBC expression, 4) acid extrusion via NBC ([J.sub.NBC]) was increased significantly in hypertrophied myocytes, 5) although acid extrusion via [Na.sup.+]/[H.sup.+] exchange was also increased in hypertrophied myocytes, the relative enhancement of [J.sub.NBC] was larger, 6) membrane depolarization markedly increased [J.sub.NBC] in hypertrophied myocytes, and 7) losartan, an ANG II A[T.sub.1] receptor antagonist, significantly attenuated the upregulation of both NBCs induced by 3 wk of aortic constriction. Enhanced NBC activity during hypertrophic development provides a mechanism for intracellular [Na.sup.+] overload, which may render the ventricles more vulnerable to [Ca.sup.2+] overload during ischemia-reperfusion. cardiac myocytes; intracellular pH regulation; messenger ribonucleic acid; polymerase chain reaction

Objectives: To develop a classification system for urine cytology with artificial intelligence (AI) using a convolutional neural network algorithm that classifies urine cell images as negative (benign) or positive (atypical or malignant). Patients and Methods: We collected 195 urine cytology slides from consecutive patients with a histologically confirmed diagnosis of urothelial cancer (between January 2016 and December 2017). Two certified cytotechnologists independently evaluated and labelled each slide; 4637 cell images with concordant diagnoses were selected, including 3128 benign cells (negative), 398 atypical cells, and 1111 cells that were malignant or suspicious for malignancy (positive). This pathologically confirmed labelled dataset was used to represent the ground truth for AI training/validation/testing. Customized CutMix (CircleCut) and Refined Data Augmentation were used for image processing. The model architecture included EfficientNet B6 and Arcface. We used 80% of the data for training and validation (4:1 ratio) and 20% for testing. Model performance was evaluated with fivefold cross‐validation. A receiver‐operating characteristic (ROC) analysis was used to evaluate the binary classification model. Bayesian posterior probabilities for the AI performance measure (Y) and cytotechnologist performance measure (X) were compared. Results: The area under the ROC curve was 0.99 (95% confidence interval [CI] 0.98–0.99), the highest accuracy was 95% (95% CI 94–97), sensitivity was 97% (95% CI 95–99), and specificity was 95% (95% CI 93–97). The accuracy of AI surpassed the highest level of cytotechnologists for the binary classification [Pr(Y > X) = 0.95]. AI achieved >90% accuracy for all cell subtypes. In the subgroup analysis based on the clinicopathological characteristics of patients who provided the test cells, the accuracy of AI ranged between 89% and 97%. Conclusion: Our novel AI classification system for urine cytology successfully classified all cell subtypes with an accuracy of higher than 90%, and achieved diagnostic accuracy of malignancy superior to the highest level achieved by cytotechnologists. [ABSTRACT FROM AUTHOR]