A novel Dual-Branch Asymmetric Encoder-Decoder Segmentation Network for accurate colonic crypt segmentation.

Colorectal cancer (CRC) is a leading cause of cancer-related deaths, with colonic crypts (CC) being crucial in its development. Accurate segmentation of CC is essential for decisions CRC and developing diagnostic strategies. However, colonic crypts' blurred boundaries and morphological diversity bring substantial challenges for automatic segmentation. To mitigate this problem, we proposed the Dual-Branch Asymmetric Encoder-Decoder Segmentation Network (DAUNet), a novel and efficient model tailored for confocal laser endomicroscopy (CLE) CC images. In DAUNet, we crafted a dual-branch feature extraction module (DFEM), employing Focus operations and dense depth-wise separable convolution (DDSC) to extract multiscale features, boosting semantic understanding and coping with the morphological diversity of CC. We also introduced the feature fusion guided module (FFGM) to adaptively combine features from both branches using cross-group spatial and channel attention to improve the model representation in focusing on specific lesion features. These modules are seamlessly integrated into the encoder for effective multiscale information extraction and fusion, and DDSC is further introduced in the decoder to provide rich representations for precise segmentation. Moreover, the local multi-layer perceptron (LMLP) module is designed to decouple and recalibrate features through a local linear transformation that filters out the noise and refines features to provide edge-enriched representation. Experimental evaluations on two datasets demonstrate that the proposed method achieves Intersection over Union (IoU) scores of 81.54% and 84.83%, respectively, which are on par with state-of-the-art methods, exhibiting its effectiveness for CC segmentation. The proposed method holds great potential in assisting physicians with precise lesion localization and region analysis, thereby improving the diagnostic accuracy of CRC.

Clustering-Guided Twin Contrastive Learning for Endomicroscopy Image Classification.

Learning better representations is essential in medical image analysis for computer-aided diagnosis. However, learning discriminative semantic features is a major challenge due to the lack of large-scale well-annotated datasets. Thus, how can we learn a well-structured categorizable embedding space in limited-scale and unlabeled datasets? In this paper, we proposed a novel clustering-guided twin-contrastive learning framework (CTCL) that learns the discriminative representations of probe-based confocal laser endomicroscopy (pCLE) images for gastrointestinal (GI) tumor classification. Compared with traditional contrastive learning, in which only two randomly augmented views of the same instance are considered, the proposed CTCL aligns more semantically related and class-consistent samples by clustering, which improved intra-class tightness and inter-class variability to produce more informative representations. Furthermore, based on the inherent properties of CLE (geometric invariance and intrinsic noise), we proposed to regard CLE images with any angle rotation and CLE images with different noises as the same instance, respectively, for increased variability and diversity of samples. By optimizing CTCL in an end-to-end expectation-maximization framework, comprehensive experimental results demonstrated that CTCL-based visual representations achieved competitive performance on each downstream task as well as more robustness and transferability compared with existing state-of-the-art SSL and supervised methods. Notably, CTCL achieved 75.60%/78.45% and 64.12%/77.37% top-1 accuracy on the linear evaluation protocol and few-shot classification downstream tasks, respectively, which outperformed the previous best results by 1.27%/1.63% and 0.5%/3%, respectively. The proposed method holds great potential to assist pathologists in achieving an automated, fast, and high-precision diagnosis of GI tumors and accurately determining different stages of tumor development based on CLE images.

Perilipin 5 deficiency aggravates cardiac hypertrophy by stimulating lactate production in leptin-deficient mice.

BACKGROUND: Perilipin 5 (Plin5) is well known to maintain the stability of intracellular lipid droplets (LDs) and regulate fatty acid metabolism in oxidative tissues. It is highly expressed in the heart, but its roles have yet to be fully elucidated. METHODS: Plin5-deficient mice and Plin5/leptin-double-knockout mice were produced, and their histological structures and myocardial functions were observed. Critical proteins related to fatty acid and glucose metabolism were measured in heart tissues, neonatal mouse cardiomyocytes and Plin5-overexpressing H9C2 cells. 2-NBDG was employed to detect glucose uptake. The mitochondria and lipid contents were observed by MitoTracker and BODIPY 493/503 staining in neonatal mouse cardiomyocytes. RESULTS: Plin5 deficiency impaired glucose utilization and caused insulin resistance in mouse cardiomyocytes, particularly in the presence of fatty acids (FAs). Additionally, Plin5 deficiency increased the NADH content and elevated the expression of lactate dehydrogenase (LDHA) in cardiomyocytes, which resulted in increased lactate production. Moreover, when fatty acid oxidation was blocked by etomoxir or LDHA was inhibited by GSK2837808A in Plin5-deficient cardiomyocytes, glucose utilization was improved. Leptin-deficient mice exhibited myocardial hypertrophy, insulin resistance and altered substrate utilization, and Plin5 deficiency exacerbated myocardial hypertrophy in leptin-deficient mice. CONCLUSION: Our results demonstrated that Plin5 plays a critical role in coordinating fatty acid and glucose oxidation in cardiomyocytes, providing a potential target for the treatment of metabolic disorders in the heart.

Patient-specific iPSC-derived cardiomyocytes reveal aberrant activation of Wnt/ß-catenin signaling in SCN5A-related Brugada syndrome.

BACKGROUND: Mutations in the cardiac sodium channel gene SCN5A cause Brugada syndrome (BrS), an arrhythmic disorder that is a leading cause of sudden death and lacks effective treatment. An association between SCN5A and Wnt/ß-catenin signaling has been recently established. However, the role of Wnt/ß-catenin signaling in BrS and underlying mechanisms remains unknown. METHODS: Three healthy control subjects and one BrS patient carrying a novel frameshift mutation (T1788fs) in the SCN5A gene were recruited in this study. Control and BrS patient-specific induced pluripotent stem cells (iPSCs) were generated from skin fibroblasts using nonintegrated Sendai virus. All iPSCs were differentiated into cardiomyocytes using monolayer-based differentiation protocol. Action potentials and sodium currents were recorded from control and BrS iPSC-derived cardiomyocytes (iPSC-CMs) by single-cell patch clamp. RESULTS: BrS iPSC-CMs exhibited increased burden of arrhythmias and abnormal action potential profile featured by slower depolarization, decreased action potential amplitude, and increased beating interval variation. Moreover, BrS iPSC-CMs showed cardiac sodium channel (Nav1.5) loss-of-function as compared to control iPSC-CMs. Interestingly, the electrophysiological abnormalities and Nav1.5 loss-of-function observed in BrS iPSC-CMs were accompanied by aberrant activation of Wnt/ß-catenin signaling. Notably, inhibition of Wnt/ß-catenin significantly rescued Nav1.5 defects and arrhythmic phenotype in BrS iPSC-CMs. Mechanistically, SCN5A-encoded Nav1.5 interacts with ß-catenin, and reduced expression of Nav1.5 leads to re-localization of ß-catenin in BrS iPSC-CMs, which aberrantly activates Wnt/ß-catenin signaling to suppress SCN5A transcription. CONCLUSIONS: Our findings suggest that aberrant activation of Wnt/ß-catenin signaling contributes to the pathogenesis of SCN5A-related BrS and point to Wnt/ß-catenin as a potential therapeutic target.

Context-aware dynamic filtering network for confocal laser endomicroscopy image denoising.

Objective.As an emerging diagnosis technology for gastrointestinal diseases, confocal laser endomicroscopy (CLE) is limited by the physical structure of the fiber bundle, leading to the inevitable production of various forms of noise during the imaging process. However, existing denoising methods based on hand-crafted features inefficiently deal with realistic noise in CLE images. To alleviate this challenge, we proposed context-aware kernel estimation and multi-scale dynamic fusion modules to remove realistic noise in CLE images, including multiplicative and additive white noise.Approach.Specifically, a realistic noise statistics model with random noise specific to CLE data is constructed and further used to develop a self-supervised denoised model without the participation of clean images. Secondly, context-aware kernel estimation, which improves the representation of features by similar learnable region weights, addresses the problem of the non-uniform distribution of noises in CLE images and proposes a lightweight denoised model (CLENet). Thirdly, we have developed a multi-scale dynamic fusion module that decouples and recalibrates features, providing a precise and contextually enriched representation of features. Finally, we integrated two developed modules into a U-shaped backbone to build an efficient denoising network named U-CLENet.Main Results.Both proposed methods achieve comparable or better performance with low computational complexity on two gastrointestinal disease CLE image datasets using the same training benchmark.Significance.The proposed approaches improve the visual quality of unclear CLE images for various stages of tumor development, helping to reduce the rate of misdiagnosis in clinical decision-making and achieve computer graphics-assisted diagnosis.

SEVERE BURN-INDUCED MITOCHONDRIAL RECRUITMENT OF CALPAIN CAUSES ABERRANT MITOCHONDRIAL DYNAMICS AND HEART DYSFUNCTION.

ABSTRACT: Mitochondrial damage is an important cause of heart dysfunction after severe burn injury. However, the pathophysiological process remains unclear. This study aims to examine the mitochondrial dynamics in the heart and the role of µ-calpain, a cysteine protease, in this scenario. Rats were subjected to severe burn injury treatment, and the calpain inhibitor MDL28170 was administered intravenously 1 h before or after burn injury. Rats in the burn group displayed weakened heart performance and decreased mean arterial pressure, which was accompanied by a diminishment of mitochondrial function. The animals also exhibited higher levels of calpain in mitochondria, as reflected by immunofluorescence staining and activity tests. In contrast, treatment with MDL28170 before any severe burn diminished these responses to a severe burn. Burn injury decreased the abundance of mitochondria and resulted in a lower percentage of small mitochondria and a higher percentage of large mitochondria. Furthermore, burn injury caused an increase in the fission protein DRP1 in the mitochondria and a decrease in the inner membrane fusion protein OPA1. Similarly, these alterations were also blocked by MDL28170. Of note, inhibition of calpain yielded the emergence of more elongated mitochondria along with membrane invagination in the middle of the longitude, which is an indicator of the fission process. Finally, MDL28170, administered 1 h after burn injury, preserved mitochondrial function and heart performance, and increased the survival rate. Overall, these results provided the first evidence that mitochondrial recruitment of calpain confers heart dysfunction after severe burn injury, which involves aberrant mitochondrial dynamics.

Overexpression of KCNJ2 enhances maturation of human-induced pluripotent stem cell-derived cardiomyocytes.

BACKGROUND: Although human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are a promising cell resource for cardiovascular research, these cells exhibit an immature phenotype that hampers their potential applications. The inwardly rectifying potassium channel Kir2.1, encoded by the KCNJ2 gene, has been thought as an important target for promoting electrical maturation of iPSC-CMs. However, a comprehensive characterization of morphological and functional changes in iPSC-CMs overexpressing KCNJ2 (KCNJ2 OE) is still lacking. METHODS: iPSC-CMs were generated using a 2D in vitro monolayer differentiation protocol. Human KCNJ2 construct with green fluorescent protein (GFP) tag was created and overexpressed in iPSC-CMs via lentiviral transduction. The mixture of iPSC-CMs and mesenchymal cells was cocultured with decellularized natural heart matrix for generation of 3D human engineered heart tissues (EHTs). RESULTS: We showed that mRNA expression level of KCNJ2 in iPSC-CMs was dramatically lower than that in human left ventricular tissues. KCNJ2 OE iPSC-CMs yielded significantly increased protein expression of Kir2.1 and current density of Kir2.1-encoded IK1. The larger IK1 linked to a quiescent phenotype that required pacing to elicit action potentials in KCNJ2 OE iPSC-CMs, which can be reversed by IK1 blocker BaCl2. KCNJ2 OE also led to significantly hyperpolarized maximal diastolic potential (MDP), shortened action potential duration (APD) and increased maximal upstroke velocity. The enhanced electrophysiological maturation in KCNJ2 OE iPSC-CMs was accompanied by improvements in Ca2+ signaling, mitochondrial energy metabolism and transcriptomic profile. Notably, KCNJ2 OE iPSC-CMs exhibited enlarged cell size and more elongated and stretched shape, indicating a morphological phenotype toward structural maturation. Drug testing using hERG blocker E-4031 revealed that a more stable MDP in KCNJ2 OE iPSC-CMs allowed for obtaining significant drug response of APD prolongation in a concentration-dependent manner. Moreover, KCNJ2 OE iPSC-CMs formed more mature human EHTs with better tissue structure and cell junction. CONCLUSIONS: Overexpression of KCNJ2 can robustly enhance maturation of iPSC-CMs in electrophysiology, Ca2+ signaling, metabolism, transcriptomic profile, cardiomyocyte structure and tissue engineering, thus providing more accurate cellular model for elucidating cellular and molecular mechanisms of cardiovascular diseases, screening drug-induced cardiotoxicity, and developing personalized and precision cardiovascular medicine.

Boosting few-shot confocal endomicroscopy image recognition with feature-level MixSiam.

As an emerging early diagnostic technology for gastrointestinal diseases, confocal laser endomicroscopy lacks large-scale perfect annotated data, leading to a major challenge in learning discriminative semantic features. So, how should we learn representations without labels or a few labels? In this paper, we proposed a feature-level MixSiam method based on the traditional Siamese network that learns the discriminative features of probe-based confocal laser endomicroscopy (pCLE) images for gastrointestinal (GI) tumor classification. The proposed method is divided into two stages: self-supervised learning (SSL) and few-shot learning (FS). First, in the self-supervised learning stage, the novel feature-level-based feature mixing approach introduced more task-relevant information via regularization, facilitating the traditional Siamese structure can adapt to the large intra-class variance of the pCLE dataset. Then, in the few-shot learning stage, we adopted the pre-trained model obtained through self-supervised learning as the base learner in the few-shot learning pipeline, enabling the feature extractor to learn richer and more transferable visual representations for rapid generalization to other pCLE classification tasks when labeled data are limited. On two disjoint pCLE gastrointestinal image datasets, the proposed method is evaluated. With the linear evaluation protocol, feature-level MixSiam outperforms the baseline by 6% (Top-1) and the supervised model by 2% (Top1), which demonstrates the effectiveness of the proposed feature-level-based feature mixing method. Furthermore, the proposed method outperforms the previous baseline method for the few-shot classification task, which can help improve the classification of pCLE images lacking large-scale annotated data for different stages of tumor development.

Establishment of an induced pluripotent stem cell line (ZJULLi004-A) from a hypertrophic cardiomyopathy patient carrying MYBPC3/c.3764C>A mutation.

Hypertrophic cardiomyopathy (HCM) is an inherited cardiovascular disease characterized by left ventricular hypertrophy and a high risk of sudden death. In this study, a skin biopsy was obtained from a HCM patient harboring a heterozygous missense mutation (c.3764C>A; p.A1225D) in the myosin binding protein C3 (MYBPC3) gene. The isolated fibroblasts were reprogrammed using non-integrated Sendai viral method to establish the patient-specific induced pluripotent stem cell (iPSC) line. The established iPSC line displayed normal morphology and karyotype, expressed pluripotency markers, and can differentiate into three germ layers in vivo.

Generation of an induced pluripotent stem cell line (ZJULLi003-A) from a hypertrophic cardiomyopathy patient carrying MYH7/c.4384G > A mutation.

Hypertrophic cardiomyopathy (HCM) is an autosomal dominant inherited cardiovascular disease characterized by left ventricular hypertrophy and cardiomyocyte disarray. In this study, a skin biopsy was obtained from a HCM patient, who carried a missense mutation (c.4384G > A; p.E1462K) in the myosin heavy chain 7 (MYH7) gene. The skin fibroblasts were subsequently reprogrammed with a non-integrated Sendai viral method to generate a patient-specific induced pluripotent stem cell (iPSC) line. The generated iPSC line showed typical morphology and normal karyotype, expressed pluripotency markers, and was capable to differentiate into three germ layers.

Generation of an induced pluripotent stem cell line from a long QT syndrome patient carrying KCNH2/1956C > A mutation.

Long QT syndrome (LQT) is an inherited primary arrhythmic disorder characterized by prolonged QT interval on the surface electrocardiogram and life-threatening arrhythmia. In this study, a skin biopsy was obtained from an LQT type 2 (LQT2) patient, who carried a nonsense mutation (c.1956C > A; p.Y652X) in the potassium voltage-gated channel subfamily H member 2 (KCNH2) gene. The skin fibroblasts were reprogrammed by non-integrated Sendai viral method to generate a patient-specific induced pluripotent stem cell (iPSC) line. The generated iPSC line showed typical embryonic stem cell-like morphology, exhibited normal karyotype, expressed pluripotency markers, and was capable to differentiate into three germ layers.

Impairment of µ-calpain activation by rhTNFR:Fc reduces severe burn-induced membrane disruption in the heart.

Stress cardiomyopathy is a major clinical complication after severe burn. Multiple upstream initiators have been identified; however, the downstream targets are not fully understood. This study assessed the role of the plasma membrane in this process and its relationship with the protease µ-calpain and tumor necrosis factor-alpha (TNF-α). Here, third-degree burn injury of approximately 40% of the total body surface area was established in rats. Plasma levels of LDH and cTnI and cardiac cell apoptosis increased at 0.5 h post burn, reached a peak at 6 h, and gradually declined at 24 h. This effect correlated well with not only the disruption of cytoskeletal proteins, including dystrophin and ankyrin-B, but also with the activation of µ-calpain, as indicated by the cleaved fragments of α-spectrin and membrane recruitment of the catalytic subunit CAPN1. More importantly, these alterations were diminished by blocking calpain activity with MDL28170. Burn injury markedly increased the cellular uptake of Evans blue, indicating membrane integrity disruption, and this effect was also reversed by MDL28170. Compared with those in the control group, cardiac cells in the burn plasma-treated group were more prone to damage, as indicated by a marked decrease in cell viability and increases in LDH release and apoptosis. Of note, these alterations were mitigated by CAPN1 siRNA. Moreover, after neutralizing TNF-α with rhTNFR:Fc, calpain activity was blocked, and heart function was improved. In conclusion, we identified µ-calpain as a trigger for severe burn-induced membrane disruption in the heart and provided evidence for the application of rhTNFR:Fc to inhibit calpain for cardioprotection.

CaMKII/calpain interaction mediates ischemia/reperfusion injury in isolated rat hearts.

Previous studies indicated that Ca2+/calmodulin-dependent kinase II (CaMKII), a kinase involved in the modulation of ryanodine receptor activity, activates Ca2+-regulated protease µ-calpain to promote myocardial ischemia/reperfusion injury. This study was performed to explore the underlying mechanisms in CaMKII-induced calpain activation to better understand heart injury. To examine the Ca2+ paradox and ischemia/reperfusion injury, isolated rat hearts were subjected to a Ca2+-free solution for 3 min, or left coronary artery occlusion for 40 min, prior to restoration of normal perfusion. Blockade of trans-sarcoplasmic reticulum Ca2+ flux using ryanodine and thapsigargin failed to prevent Ca2+ paradox-induced heart injury. In contrast, the Ca2+ paradox increased CaMKII auto-phosphorylation at Thr287, while the CaMKII inhibitor KN-62 and the Na+/Ca2+ exchanger inhibitor KB-R7943 alleviated heart injury and calpain activity. Intriguingly, the binding of µ-calpain large subunit calpain-1 (CAPN1) to phospho-CaMKII was blunted by both inhibitors. Thus, a Ca2+ leak via the ryanodine receptor is not an essential element in CaMKII-elicited calpain activation. In hearts receiving vector injection, ischemia/reperfusion caused elevated calpain activity and α-fodrin degradation, along with membrane integrity damage, similar to the effects noted in control hearts. Importantly, all these alterations were diminished with delivery of adeno-associated virus expressing mutant CaMKIIδC T287A. Ischemia/reperfusion increased CaMKII auto-phosphorylation and binding of CAPN1 to phospho-CaMKII, and facilitated the translocation of phospho-CaMKII and CAPN1 to the plasma membrane, all of which were reversed by injecting CaMKII mutant. Furthermore, the relocation capacity and the interaction of CaMKII with CAPN1 appeared to be dependent upon CaMKII autophosphorylation, as its mutant delivery increased the level of CaMKII, but did not increase membrane content of CaMKII and CAPN1, or their interactions. Together, CaMKII/calpain interaction represents a new avenue for mediating myocardial ischemia/reperfusion injury, and CaMKII likely serves as both a kinase and a carrier, thereby promoting calpain membrane translocation and activation.

Human induced pluripotent stem cell-derived cardiomyocytes reveal abnormal TGFß signaling in type 2 diabetes mellitus.

Diabetes mellitus is a serious metabolic condition associated with a multitude of cardiovascular complications. Moreover, the prevalence of diabetes in heart failure populations is higher than that in control populations. However, the role of cardiomyocyte alterations in type 2 diabetes mellitus (T2DM) has not been well characterized and the underlying mechanisms remain elusive. In this study, two patients who were diagnosed as T2DM were recruited and patient-specific induced pluripotent stem cells (iPSCs) were generated from urine epithelial cells using nonintegrated Sendai virus. The iPSC lines derived from five healthy subjects were used as controls. All iPSCs were differentiated into cardiomyocytes (iPSC-CMs) using the monolayer-based differentiation protocol. T2DM iPSC-CMs exhibited various disease phenotypes, including cellular hypertrophy and lipid accumulation. Moreover, T2DM iPSC-CMs exhibited higher susceptibility to high-glucose/high-lipid challenge than control iPSC-CMs, manifesting an increase in apoptosis. RNA-Sequencing analysis revealed a differential transcriptome profile and abnormal activation of TGFß signaling pathway in T2DM iPSC-CMs. We went on to show that inhibition of TGFß significantly rescued the hypertrophic phenotype in T2DM iPSC-CMs. In conclusion, we demonstrate that the iPSC-CM model is able to recapitulate cellular phenotype of T2DM. Our results indicate that iPSC-CMs can therefore serve as a suitable model for investigating molecular mechanisms underlying diabetic cardiomyopathies and for screening therapeutic drugs.

CaMKII mediates myocardial ischemia/reperfusion injury­induced contracture in isolated rat heart.

Contracture or diastolic dysfunction is a primary cause of injury following ischemia/reperfusion (IR). The present study examined whether Ca2+/calmodulin­dependent kinase II (CaMKII) is involved in contracture. Isolated rat hearts were subjected to either global IR or Ca2+ paradox (CaP), which is characterized by contracture. Left ventricular end­diastolic pressure, electron microscopy and troponin I  TnI) in coronary effluent were examined to indicate the extent of contracture. Compared with control hearts, both the IR and CaP groups exhibited an increase in necrosis and apoptosis, and a marked depression in contractile function. Western blot analysis showed that IR stimulated the phosphorylation (Thr287) and oxidation (Met281/282) of CaMKII, and the phosphorylation of phospholamban (PLN), a substrate of CaMKII. By contrast, only the phosphorylation of CaMKII was increased in the CaP group. Treatment with either 3 µM KN­62, an inhibitor of CaMKII, or 5 µM KB­R7943, an inhibitor of the Na+/Ca2+ exchanger, mitigated the damage and the post­translational modification of both CaMKII and PLN. Similar to the effect of the negative inotropic agent 2,3­butanedione­monoxime, the increased cell survival after treatment with KN­62 was associated with improved diastolic function. Examination using electron microscopy and a biochemical test showed the development of contraction bands, disruption of the sarcolemmal membrane and an increase in the release of TnI in both IR and CaP hearts; these results indicated the occurrence of contracture. Furthermore, these changes were inhibited by either KN­62 or KB­R7943. Taken together, these data provided evidence that CaMKII mediates reperfusion­elicited contracture, and that the activation of CaMKII via phosphorylation is involved in this process.

Calpain inhibition ameliorates scald burn-induced acute lung injury in rats.

BACKGROUND: The molecular pattern of severe burn-induced acute lung injury, characterized by cell structure damage and leukocyte infiltration, remains unknown. This study aimed to determine whether calpain, a protease involved in both processes, mediates severe burn-induced acute lung injury. METHODS: Rats received full-thickness scald burns covering 30% of the total body surface area, followed by instant fluid resuscitation. MDL28170 (Tocris Bioscience), an inhibitor of calpain, was given intravenously 1 h before or after the scald burn. The histological score, wet/dry weight ratio, and caspase-3 activity were examined to evaluate the degree of lung damage. Calpain activity and its source were detected by an assay kit and immunofluorescence staining. The proteolysis of membrane skeleton proteins α-fodrin and ankyrin-B, which are substrates of calpain, was measured by Western blot. RESULTS: Time-course studies showed that tissue damage reached a peak between 1 and 6 h post-scald burn and gradually diminished at 24 h. More importantly, calpain activity reached peak levels at 1 h and was maintained until 24 h, paralleled by lung damage to some extent. Western blot showed that the levels of the proteolyzed forms of α-fodrin and ankyrin-B correlated well with the degree of damage. MDL28170 at a dose of 3 mg/kg b. w. given 1 h before burn injury not only antagonized the increase in calpain activity but also ameliorated scald burn-induced lung injury, including the degradation of α-fodrin and ankyrin-B. Immunofluorescence images revealed calpain 1 and CD45 double-positive cells in the lung tissue of rats exposed to scald burn injury, suggesting that leukocytes were a dominant source of calpain. Furthermore, this change was blocked by MDL28170. Finally, MDL28170 given at 1 h post-scald burn injury significantly ameliorated the wet/dry weight ratio compared with burn injury alone. CONCLUSIONS: Calpain, a product of infiltrating leukocytes, is a mediator of scald burn-induced acute lung injury that involves enhancement of inflammation and proteolysis of membrane skeleton proteins. Its late effects warrant further study.

CaMKII inhibition mitigates ischemia/reperfusion-elicited calpain activation and the damage to membrane skeleton proteins in isolated rat hearts.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been implicated in myocardial ischemia/reperfusion (IR) injury. The aim of this study was to determine the effect of CaMKII on the damage to membrane skeleton proteins, which is an important cause of IR injury. Isolated rat hearts were subjected to 45-min global ischemia/2-h reperfusion. Both KN-62 and KN-93 were used to inhibit CaMKII. Compared with controls, the hearts in the IR group exhibited remarkable myocardial injury area, LDH release, cell apoptosis and contractile dysfunction, along with an increase in the phosphorylation of CaMKII and its substrate phospholamban. Treatment with either KN-62 or KN-93 mitigated both the heart injury and the phosphorylation of CaMKII and phospholamban. The analysis of cell skeleton proteins revealed that IR injury resulted in an increase in the 150-kDa fragments resulting from the degradation of α-fodrin and dystrophin translocating from the sarcolemmal membrane to the cytosol and a decrease in the 220-kDa isoform of ankyrin-B. As expected, Evans blue dye staining showed an increase in membrane permeability or membrane rupture in the IR group. All of these alterations were alleviated by treatment with either KN-62 or KN-93. In addition, both KN-62 and KN-93 blocked the activity and membrane recruitment of calpain, a key protease responsible for destroying cell skeleton proteins during IR injury. In conclusion, our data provide evidence that damage to membrane skeleton proteins via calpain is a destructive downstream event of CaMKII activation in the setting of myocardial IR injury.

Glutamate protects against Ca(2+) paradox-induced injury and inhibits calpain activity in isolated rat hearts.

This study determined the effects of glutamate on the Ca(2+) paradoxical heart, which is a model for Ca(2+) overload-induced injury during myocardial ischaemia and reperfusion, and evaluated its effect on a known mediator of injury, calpain. An isolated rat heart was retrogradely perfused in a Langendorff apparatus. Ca(2+) paradox was elicited via perfusion with a Ca(2+) -free Krebs-Henseleit (KH) solution for 3 minutes followed by Ca(2+) -containing normal KH solution for 30 minutes. The Ca(2+) paradoxical heart exhibited almost no viable tissue on triphenyltetrazolium chloride staining and markedly increased LDH release, caspase-3 activity, cytosolic cytochrome c content, and apoptotic index. These hearts also displayed significantly increased LVEDP and a disappearance of LVDP. Glutamate (5 and 20 mmol/L) significantly alleviated Ca(2+) paradox-induced injury. In contrast, 20 mmol/L mannitol had no effect on Ca(2+) paradox. Ca(2+) paradox significantly increased the extent of the translocation of µ-calpain to the sarcolemmal membrane and the proteolysis of α-fodrin, which suggests calpain activation. Glutamate also blocked these effects. A non-selective inhibitor of glutamate transporters, dl-TBOA (10 µmol/L), had no effect on control hearts, but it reversed glutamate-induced cardioprotection and reduction in calpain activity. Glutamate treatment significantly increased intracellular glutamate content in the Ca(2+) paradoxical heart, which was also blocked by dl-TBOA. We conclude that glutamate protects the heart against Ca(2+) overload-induced injury via glutamate transporters, and the inhibition of calpain activity is involved in this process.