Evaluation of general anesthesia protocols for a highly controlled cardiac ischemia-reperfusion model in mice.

BACKGROUND: The aim of our study was to test different anesthetic mixtures in order to identify the most suitable one for a surgical cardiac ischemia-reperfusion model in mice. METHODS: 1) Sixty four mice were submitted to one of the 6 combinations of ketamine or alfaxalone associated to xylazine, medetomidine or midazolam. Depth and quality of anesthesia were evaluated via 5 reflex scores. 2) Impact of analgesic (buprenorphine or butorphanol), anesthesia reversal (with atipamezole) and surgery (cardiac ischemia-reperfusion surgery) have been tested in the selected protocols. 3) infarction size has been measured with TTC (Triphenyl Tetrazolium Chloride) method in mice anesthetized with best protocols. RESULTS: Protocol involving medetomidine induced the longest surgical anesthesia: (median = 120, {interquartile range = 100-125}) min with ketamine and 53 {25-100} min with alfaxalone. Butorphanol substitution with buprenorphine did not alter time-related anesthesia parameters. Atipamezole reversal considerably reduced both recovery and immobilization time (respectively 22 {18-30} min and 98 {88-99} min vs. 55 {40-70} min and 143 {131-149} min, in groups with no reversal, p = 0.001) with no impact on infarction size measurement. CONCLUSION: In this study, the combination alfaxalone/medetomidine/buprenorphine (80/0,3/0,075 mg.kg-1, s.c.) associated with reversal by atipamezole was a reliable anesthetic protocol for murine surgery, particularly for the study of ischemia-reperfusion.

Deleterious Anti-Inflammatory Macrophage Recruitment in Early Post-Infarction Phase: Unraveling the IL-6/MCP-1/STAT3 Axis.

Using a translational approach with an ST-segment myocardial infarction (STEMI) cohort and mouse model of myocardial infarction, we highlighted the role of the secreted IL-6 and MCP-1 cytokines and the STAT3 pathway in heart macrophage recruitment and activation. Cardiac myocytes secrete IL-6 and MCP-1 in response to hypoxic stress, leading to a recruitment and/or polarization of anti-inflammatory macrophages via the STAT3 pathway. In our preclinical model of myocardial infarction, neutralization of IL-6 and MCP-1 or STAT3 pathway reduced infarct size. Together, our data demonstrate that anti-inflammatory macrophages can be deleterious in the acute phase of STEMI.

Oxidation-reduction imaging of myoglobin reveals two-phase oxidation in the reperfused myocardium.

Myocardial infarction (MI) is a serious acute cardiovascular syndrome that causes myocardial injury due to blood flow obstruction to a specific myocardial area. Under ischemic-reperfusion settings, a burst of reactive oxygen species is generated, leading to redox imbalance that could be attributed to several molecules, including myoglobin. Myoglobin is dynamic and exhibits various oxidation-reduction states that have been an early subject of attention in the food industry, specifically for meat consumers. However, rarely if ever have the myoglobin optical properties been used to measure the severity of MI. In the current study, we develop a novel imaging pipeline that integrates tissue clearing, confocal and light sheet fluorescence microscopy, combined with imaging analysis, and processing tools to investigate and characterize the oxidation-reduction states of myoglobin in the ischemic area of the cleared myocardium post-MI. Using spectral imaging, we have characterized the endogenous fluorescence of the myocardium and demonstrated that it is partly composed by fluorescence of myoglobin. Under ischemia-reperfusion experimental settings, we report that the infarcted myocardium spectral signature is similar to that of oxidized myoglobin signal that peaks 3 h post-reperfusion and decreases with cardioprotection. The infarct size assessed by oxidation-reduction imaging at 3 h post-reperfusion was correlated to the one estimated with late gadolinium enhancement MRI at 24 h post-reperfusion. In conclusion, this original work suggests that the redox state of myoglobin can be used as a promising imaging biomarker for characterizing and estimating the size of the MI during early phases of reperfusion.

A simplified and accelerated implementation of SVD for filtering ultrafast power Doppler images.

BACKGROUND AND OBJECTIVE: Ultrafast Power Doppler (UPD) is a growing ultrasound modality for imaging and diagnosing microvasculature disease. A key element of UPD is using singular value decomposition (SVD) as a highly selective filter for tissue and electronic noise. However, two significant drawbacks of SVD are its computational burden and the complexity of its algorithms. These limitations hinder the development of fast and specific SVD algorithms for UPD imaging. This study introduces power SVD (pSVD), a simplified and accelerated algorithm for filtering tissue and noise in UPD images. METHODS: pSVD exploits several mathematical properties of SVD specific to UPD images. In particular, pSVD allows the direct computation of blood-related SVD components from the temporal singular vectors. This feature simplifies the expression of SVD while significantly accelerating its computation. After detailing the theory behind pSVD, we evaluate its performances in several in vitro and in vivo experiments and compare it to SVD and randomized SVD (rSVD). RESULTS: pSVD strongly decreases the running time of SVD (between 5 and 12 times in vivo) without impacting the quality of UPD images. Compared to rSVD, pSVD can be significantly faster (up to 3 times) or slightly slower but gives access to more estimators to isolate tissue subspaces. CONCLUSION: pSVD is highly valuable for implementing UPD imaging in clinical ultrasound and provides a better understanding of SVD for ultrasound imaging in general.

Cardioprotective effects of shock wave therapy: A cardiac magnetic resonance imaging study on acute ischemia-reperfusion injury.

Introduction: Cardioprotection strategies remain a new frontier in treating acute myocardial infarction (AMI), aiming at further protect the myocardium from the ischemia-reperfusion damage. Therefore, we aimed at investigating the mechano-transduction effects induced by shock waves (SW) therapy at time of the ischemia reperfusion as a non-invasive cardioprotective innovative approach to trigger healing molecular mechanisms. Methods: We evaluated the SW therapy effects in an open-chest pig ischemia-reperfusion (IR) model, with quantitative cardiac Magnetic Resonance (MR) imaging performed along the experiments at multiple time points (baseline (B), during ischemia (I), at early reperfusion (ER) (∼15 min), and late reperfusion (LR) (3 h)). AMI was obtained by a left anterior artery temporary occlusion (50 min) in 18 pigs (32 ± 1.9 kg) randomized into SW therapy and control groups. In the SW therapy group, treatment was started at the end of the ischemia period and extended during early reperfusion (600 + 1,200 shots @0.09 J/mm2, f = 5 Hz). The MR protocol included at all time points LV global function assessment, regional strain quantification, native T1 and T2 parametric mapping. Then, after contrast injection (gadolinium), we obtained late gadolinium imaging and extra-cellular volume (ECV) mapping. Before animal sacrifice, Evans blue dye was administrated after re-occlusion for area-at-risk sizing. Results: During ischemia, LVEF decreased in both groups (25 ± 4.8% in controls (p = 0.031), 31.6 ± 3.2% in SW (p = 0.02). After reperfusion, left ventricular ejection fraction (LVEF) remained significantly decreased in controls (39.9 ± 4% at LR vs. 60 ± 5% at baseline (p = 0.02). In the SW group, LVEF increased quickly ER (43.7 ± 11.4% vs. 52.4 ± 8.2%), and further improved at LR (49.4 ± 10.1) (ER vs. LR p = 0.05), close to baseline reference (LR vs. B p = 0.92). Furthermore, there was no significant difference in myocardial relaxation time (i.e. edema) after reperfusion in the intervention group compared to the control group: ΔT1 (MI vs. remote) was increased by 23.2±% for SW vs. +25.2% for the controls, while ΔT2 (MI vs. remote) increased by +24.9% for SW vs. +21.7% for the control group. Discussion: In conclusion, we showed in an ischemia-reperfusion open-chest swine model that SW therapy, when applied near the relief of 50' LAD occlusion, led to a nearly immediate cardioprotective effect translating to a reduction in the acute ischemia-reperfusion lesion size and to a significant LV function improvement. These new and promising results related to the multi-targeted effects of SW therapy in IR injury need to be confirmed by further in-vivo studies in close chest models with longitudinal follow-up.

Adaptive noise reduction for power Doppler imaging using SVD filtering in the channel domain and coherence weighting of pixels.

Objective. Ultrafast power Doppler (UPD) is an ultrasound method that can image blood flow at several thousands of frames per second. In particular, the high number of data provided by UPD enables the use of singular value decomposition (SVD) as a clutter filter for suppressing tissue signal. Notably, is has been demonstrated in various applications that SVD filtering increases significantly the sensitivity of UPD to microvascular flows. However, UPD is subjected to significant depth-dependent electronic noise and an optimal denoising approach is still being sought.Approach. In this study, we propose a new denoising method for UPD imaging: the Coherence Factor Mask (CFM). This filter is first based on filtering the ultrasound time-delayed data using SVD in the channel domain to remove clutter signal. Then, a spatiotemporal coherence mask that exploits coherence information between channels for identifying noisy pixels is computed. The mask is finally applied to beamformed images to decrease electronic noise before forming the power Doppler image. We describe theoretically how to filter channel data using a single SVD. Then, we evaluate the efficiency of the CFM filter for denoisingin vitroandin vivoimages and compare its performances with standard UPD and with three existing denoising approaches.Main results. The CFM filter gives gains in signal-to-noise ratio and contrast-to-noise ratio of up to 22 dB and 20 dB, respectively, compared to standard UPD and globally outperforms existing methods for reducing electronic noise. Furthermore, the CFM filter has the advantage over existing approaches of being adaptive and highly efficient while not requiring a cut-off for discriminating noise and blood signals nor for determining an optimal coherence lag.Significance. The CFM filter has the potential to help establish UPD as a powerful modality for imaging microvascular flows.

Postconditioning by Delayed Administration of Ciclosporin A: Implication for Donation after Circulatory Death (DCD).

Heart transplantation is facing a shortage of grafts. Donation after Circulatory Death (DCD) would constitute a new potential of available organs. In the present work, we aimed to evaluate whether Postconditioning (ischemic or with ciclosporin-A (CsA)) could reduce ischemia-reperfusion injury in a cardiac arrest model when applied at the start of reperfusion or after a delay. An isolated rat heart model was used as a model of DCD. Hearts were submitted to a cardiac arrest of 40 min of global warm ischemia (37 °C) followed by 3 h of 4 °C-cold preservation, then 60 min reperfusion. Hearts were randomly allocated into the following groups: control, ischemic postconditioning (POST, consisting of two episodes each of 30 s ischemia and 30 s reperfusion at the onset of reperfusion), and CsA group (CsA was perfused at 250 nM for 10 min at reperfusion). In respective subgroups, POST and CsA were applied after a delay of 3, 10, and 20 min. Necrosis was lower in CsA and POST versus controls (p < 0.01) whereas heart functions were improved (p < 0.01). However, while the POST lost its efficacy if delayed beyond 3 min of reperfusion, CsA treatment surprisingly showed a reduction of necrosis even if applied after a delay of 3 and 10 min of reperfusion (p < 0.01). This cardioprotection by delayed CsA application correlated with better functional recovery and higher mitochondrial respiratory index. Furthermore, calcium overload necessary to induce mitochondrial permeability transition pore (MPTP) opening was similar in all cardioprotection groups, suggesting a crucial role of MPTP in this delayed protection of DCD hearts.

Quantitative stiffness assessment of cardiac grafts using ultrasound in a porcine model: A tissue biomarker for heart transplantation.

BACKGROUND: Heart transplantation is the definitive treatment for many cardiovascular diseases. However, no ideal approach is established to evaluate heart grafts and it mostly relies on qualitative interpretation of surgeon based on the organ aspect including anatomy, color and manual palpation. In this study we propose to assess quantitatively the Shear Wave Velocity (SWV) using ultrasound as a biomarker of cardiac viability on a porcine model. METHODS: The SWV was assessed quantitatively using a clinical ultrasound elastography device (Aixplorer, Supersonics Imagine, France) linked to a robotic motorized arm (UR3, Universal Robots, Denmark) and the elastic anisotropy was obtained using a custom ultrasound research system. SWV was evaluated as function of time in two porcine heart model during 20h at controlled temperature (4°C). One control group (N = 8) with the heart removed and arrested by cold cardioplegia and immerged in a preservation solution. One ischemic group (N = 6) with the organ harvested after 30 min of in situ warm ischemia, to mimic a donation after cardiac death. Hearts graft were revived at two preservation times, at 4 h (N = 11) and 20 h (N = 10) and the parameters of the cardiac function evaluated. FINDINGS: On control hearts, SWV remained unchanged during the 4h of preservation. SWV increased significantly between 4 and 20h. For the ischemic group, SWV was found higher after 4h (3.04 +/- 0.69 vs 1.69+/-0.19 m/s, p = 0.007) and 20h (4.77+/-1.22 m/s vs 3.40+/-0.75 m/s, p = 0.034) of preservation with significant differences. A good correlation between SWV and cardiac function index was found (r2=0.88) and manual palpation score (r2=0.81). INTERPRETATION: Myocardial stiffness increase was quantified as a function of preservation time and harvesting conditions. The correlation between SWV and cardiac function index suggests that SWV could be used as a marker of graft viability. This technique may be transposed to clinical transplantation for assessing the graft viability during transplantation process. FUNDING: FRM PME20170637799, Agence Biomédecine AOR Greffe 2017, ANR-18-CE18-0015.

A Dynamic Transcriptional Analysis Reveals IL-6 Axis as a Prominent Mediator of Surgical Acute Response in Non-ischemic Mouse Heart.

BACKGROUND: Ischemic heart diseases are a major cause of death worldwide. Different animal models, including cardiac surgery, have been developed over time. Unfortunately, the surgery models have been reported to trigger an important inflammatory response that might be an effect modifier, where involved molecular processes have not been fully elucidated yet. OBJECTIVE: We sought to perform a thorough characterization of the sham effect in the myocardium and identify the interfering inflammatory reaction in order to avoid misinterpretation of the data via systems biology approaches. METHODS AND RESULTS: We combined a comprehensive analytical pipeline of mRNAseq dataset and systems biology analysis to characterize the acute phase response of mouse myocardium at 0 min, 45 min, and 24 h after surgery to better characterize the molecular processes inadvertently induced in sham animals. Our analysis showed that the surgical intervention induced 1209 differentially expressed transcripts (DETs). The clustering of positively co-regulated transcript modules at 45 min fingerprinted the activation of signalization pathways, while positively co-regulated genes at 24 h identified the recruitment of neutrophils and the differentiation of macrophages. In addition, we combined the prediction of transcription factors (TF) regulating DETs with protein-protein interaction networks built from these TFs to predict the molecular network which have induced the DETs. By mean of this retro-analysis of processes upstream gene transcription, we revealed a major role of the Il-6 pathway and further confirmed a significant increase in circulating IL-6 at 45 min after surgery. CONCLUSION: This study suggests that a strong induction of the IL-6 axis occurs in sham animals over the first 24 h and leads to the induction of inflammation and tissues' homeostasis processes.

Comparison Between Multiline Transmission and Diverging Wave Imaging: Assessment of Image Quality and Motion Estimation Accuracy.

High frame rate imaging is particularly important in echocardiography for better assessment of the cardiac function. Several studies showed that diverging wave imaging (DWI) and multiline transmission (MLT) are promising methods for achieving a high temporal resolution. The aim of this study was to compare MLT and compounded motion compensation (MoCo) DWI for the same transmitted power, same frame rates [image quality and speckle tracking echocardiography (STE) assessment], and same packet size [tissue Doppler imaging (TDI) assessment]. Our results on static images showed that MLT outperforms DW in terms of resolution (by 30% on average). However, in terms of contrast, MLT outperforms DW only for the depth of 11 cm (by 40% on average), the result being reversed at a depth of 4 cm (by 27% on average). In vitro results on a spinning phantom at nine different velocities showed that similar STE axial errors (up to 2.3% difference in median errors and up to 2.1% difference in the interquartile ranges) are obtained with both ultrafast methods. On the other hand, the median lateral STE estimates were up to 13% more accurate with DW than with MLT. On the contrary, the accuracy of TDI was only up to ~3% better with MLT, but the achievable DW Doppler frame rate was up to 20 times higher. However, our overall results showed that the choice of one method relative to the other is therefore dependent on the application. More precisely, in terms of image quality, DW is more suitable for imaging structures at low depths, while MLT can provide an improved image quality at the focal point that can be placed at higher depths. In terms of motion estimation, DW is more suitable for color Doppler-related applications, while MLT could be used to estimate velocities along selected lines of the image.

Critical appraisal of STAT3 pattern in adult cardiomyocytes.

The signal transducer and activator of transcription 3, STAT3, transfers cellular signals from the plasma membrane to the nucleus, acting as a signaling molecule and a transcription factor. Reports proposed an additional non-canonical role of STAT3 that could regulate the activity of complexes I and II of the electron transport chain and the opening of the mitochondrial permeability transition pore (PTP) after ischemia-reperfusion in various cell types. The native expression of STAT3 in heart mitochondria, together with a direct versus an indirect transcriptional role in mitochondrial functions, have been recently questioned. The objective of the present study was to investigate the cellular distribution of STAT3 in mouse adult cardiomyocytes under basal and stress conditions, along with assessing its presence and activity in cardiac mitochondria using structural and functional approaches. The analysis of the spatial distribution of STAT3 signal in the cardiomyocytes interestingly showed that it is transversely distributed along the T-tubules and in the nucleus. This distribution was neither affected by hypoxia nor by hypoxia/re­oxygenation conditions. Focusing on the mitochondrial STAT3 localization, our results suggest that serine-phosphorylated STAT3 (PS727-STAT3) and total STAT3 are detected in crude but not in pure mitochondria of mouse adult cardiomyocytes, under basal and ischemia-reperfusion conditions. The inhibition of STAT3, with a pre-validated non-toxic Stattic dose, had no significant effects on mitochondrial respiration, but a weak effect on the calcium retention capacity. Overall, our results exclusively reveal a unique cellular distribution of STAT3 in mouse adult cardiomyocytes, along the T-tubules and in nucleus, under different conditions. They also challenge the expression and activity of STAT3 in mitochondria of these cells under basal conditions and following ischemia-reperfusion. In addition, our results underline technical methods, complemental to cell fractionation, to evaluate STAT3 roles during hypoxia-reoxygenation and at the interface between nucleus and endoplasmic reticulum.

Dose and timing of injections for effective cyclosporine A pretreatment before renal ischemia reperfusion in mice.

BACKGROUND: There is experimental evidence that lethal ischemia-reperfusion injury (IRI) is largely due to mitochondrial permeability transition pore (mPTP) opening, which can be prevented by cyclosporine A (CsA). The aim of our study is to show that a higher dose of CsA (10 mg/kg) injected just before ischemia or a lower dose of CsA (3 mg/kg) injected further in advance of ischemia (1 h) protects the kidneys and improves mitochondrial function. METHODS: All mice underwent a right unilateral nephrectomy followed by 30 min clamping of the left renal artery. Mice in the control group did not receive any pharmacological treatment. Mice in the three groups treated by CsA were injected at different times and with different doses, namely 3 mg/kg 1 h or 10 min before ischemia or 10 mg/kg 10 min before ischemia. After 24 h of reperfusion, the plasma creatinine level were measured, the histological score was assessed and mitochondria were isolated to calculate the calcium retention capacity (CRC) and level of oxidative phosphorylation. RESULTS: Mortality and renal function was significantly higher in the CsA 10 mg/kg-10 min and CsA 3mg/kg-1 h groups than in the CsA 3mg/kg-10 min group. Likewise, the CRC was significantly higher in the former two groups than in the latter, suggesting that the improved renal function was due to a longer delay in the opening of the mPTP. Oxidative phosphorylation levels were also higher 24 h after reperfusion in the protected groups. CONCLUSIONS: Our results suggest that the protection afforded by CsA is likely limited by its availability. The dose and timing of the injections are therefore crucial to ensure that the treatment is effective, but these findings may prove challenging to apply in practice.

Mesenchymal stem cells sense mitochondria released from damaged cells as danger signals to activate their rescue properties.

Mesenchymal stem cells (MSCs) protect tissues against cell death induced by ischemia/reperfusion insults. This therapeutic effect seems to be controlled by physiological cues released by the local microenvironment following injury. Recent lines of evidence indicate that MSC can communicate with their microenvironment through bidirectional exchanges of mitochondria. In particular, in vitro and in vivo studies report that MSCs rescue injured cells through delivery of their own mitochondria. However, the role of mitochondria conveyed from somatic cells to MSC remains unknown. By using a co-culture system consisting of MSC and distressed somatic cells such as cardiomyocytes or endothelial cells, we showed that mitochondria from suffering cells acted as danger-signaling organelles that triggered the anti-apoptotic function of MSC. We demonstrated that foreign somatic-derived mitochondria were engulfed and degraded by MSC, leading to induction of the cytoprotective enzyme heme oxygenase-1 (HO-1) and stimulation of mitochondrial biogenesis. As a result, the capacity of MSC to donate their mitochondria to injured cells to combat oxidative stress injury was enhanced. We found that similar mechanisms - activation of autophagy, HO-1 and mitochondrial biogenesis - occurred after exposure of MSC to exogenous mitochondria isolated from somatic cells, strengthening the idea that somatic mitochondria alert MSC of a danger situation and subsequently promote an adaptive reparative response. In addition, the cascade of events triggered by the transfer of somatic mitochondria into MSC was recapitulated in a model of myocardial infarction in vivo. Specifically, MSC engrafted into infarcted hearts of mice reduced damage, upregulated HO-1 and increased mitochondrial biogenesis, while inhibition of mitophagy or HO-1 failed to protect against cardiac apoptosis. In conclusion, our study reveals a new facet about the role of mitochondria released from dying cells as a key environmental cue that controls the cytoprotective function of MSC and opens novel avenues to improve the effectiveness of MSC-based therapies.

Unacylated ghrelin analog prevents myocardial reperfusion injury independently of permeability transition pore.

Reperfusion injury is responsible for an important part of myocardial infarct establishment due notably to triggering cardiomyocytes death at the first minutes of reperfusion. AZP-531 is an optimized analog of unacylated ghrelin currently in clinical development in several metabolic diseases. We investigated a potential cardioprotective effect of AZP-531 in ischemia/reperfusion (IR) and the molecular underlying mechanism(s) involved in this protection. In vivo postconditioning with AZP-531 in C57BL6 mouse IR model decreased infarct size. Western blot analysis on areas at risk from the different mouse groups showed that AZP-531 activates Akt, ERK1-2 as well as S6 and 4EBP1, mTORC1 effectors. We also showed an inhibition of caspase 3 cleavage and Bax translocation to the mitochondria. AZP-531 also stimulated the expression of antioxidants and was capable of decreasing mitochondrial H2O2 production, contributing to the reduction of ROS accumulation. AZP-531 exhibits cardioprotective effect when administrated for postconditioning in C57BL6 mouse IR model. Treatment with AZP-531 rescued the myocardium from cell death at early reperfusion by stimulating protein synthesis, inhibiting Bax/caspase 3-induced apoptosis as well as ROS accumulation and oxidative stress-induced necrosis. AZP-531 may prove useful in the treatment of IR injury.

Postconditioning with cyclosporine a reduces early renal dysfunction by inhibiting mitochondrial permeability transition.

BACKGROUND: Ischemia-reperfusion (IR) injury leads to mitochondrial permeability transition pore opening, which contributes to cell death. The aim of this study is to determine whether ischemic or pharmacological postconditioning with cyclosporine A (CsA) might protect the kidney from lethal reperfusion injury. METHODS: Male mice underwent a unilateral (right) nephrectomy followed by 30 minutes of contralateral (left) clamping of the renal artery. We studied 4 groups at 20 minutes and 24 hours of reperfusion: a sham group (n = 4), an ischemic group (n = 6), CsA-postconditioned group (postcond-CsA, injection of 3 mg/kg of CsA 5 minutes before the end of ischemia, (n = 6), and an ischemic postconditioning (IPC) group (n = 6), consisting of 3 cycles of 30 seconds of renal ischemia with 30 seconds intervening reperfusion. After 24 hours of reperfusion, we measured plasma creatinine, urea, and histological kidney injury. The kidney mitochondria were isolated to assess the mitochondria calcium retention capacity and oxidative phosphorylation. RESULTS: At 24 hours after reperfusion, serum creatinine decreased in postcond-CsA and IPC compared to ischemic group. The histological score was also significantly improved with postcond-CsA and IPC. At 20 minutes and 24 hours of reperfusion, calcium retention capacity was decreased significantly in the ischemic group. The mitochondrial respiration stay decreased in the ischemic group at 24 hours of reperfusion, whereas the respiration was improved significantly in the postcond-CsA and IPC group. Bax and cleaved caspase 3 decreased in PostCsA and IPC group. CONCLUSIONS: Our results suggest that IPC and CsA, administered immediately before reperfusion, protect the kidney from lethal injury.

Cardiac fibroblasts protect cardiomyocytes against lethal ischemia-reperfusion injury.

Roles of cardiac fibroblasts (CFs) in the regulation of myocardial structure and function have been emphasized in the last decade. Their implications in pathophysiological aspects of chronic heart diseases such as myocardial remodeling and fibrosis are now well established; however their contribution to the acute phase of ischemia-reperfusion injury still remains elusive. We hypothesized that CF may contribute to cardiomyocyte (CM) protection against ischemia-reperfusion injuries. Experiments performed on isolated neonatal rat CF and CM demonstrated that the presence of CF in co-cultures increases CM viability (58 ± 2% versus 30 ± 2% in control) against hypoxia-reoxygenation injury, in a paracrine manner. It was confirmed by a similar effect of hypoxic CF secretome alone on CM viability (51 ± 9% versus 31 ± 4% in untreated cells). These findings were corroborated by in vivo experiments in a mice model of myocardial infarction in which a 25% infarct size reduction was observed in CF secretome treated mice compared to control. Tissue inhibitor of metalloproteinases-1 (TIMPs-1) alone, abundantly detected in CF secretome, was able to decrease CM cell death (35%) and experiments with pharmacological inhibitors of PI3K/Akt and ERK1/2 pathways provided more evidence that this paracrine protection is partly mediated by these signaling pathways. In vivo experiments strengthened that TIMP-1 alone was able to decrease infarct size (37%) and were validated by depletion experiments demonstrating that CF secretome cardioprotection was abolished by TIMP-1 depletion. Our data demonstrated for the first time that CFs participate in cardioprotection during the acute phase of ischemia-reperfusion via a paracrine pathway involving TIMP-1.

Endoplasmic reticulum stress contributes to heart protection induced by cyclophilin D inhibition.

Preventing cyclophilin D (cypD) translocation to the inner mitochondrial membrane can limit lethal reperfusion injury through the inhibition of the opening of the mitochondrial permeability transition pore. Inhibition or loss of function of cypD may also result into an endoplasmic reticulum (ER) stress that has been shown to alter cell survival. We therefore questioned whether ER stress might play a role in the protection induced by CypD deficiency or inhibition. CypD-KO and NIM811 (a CypD inhibitor)-treated mice were subjected to a prolonged ischemia-reperfusion (I/R). Area at risk and infarct size was measured using blue dye and triphenyltetrazolium chloride staining. ER stress markers were measured in the hearts during the reperfusion phase. As expected, cypD-KO mice exhibited a decreased infarct size when compared to wild-type mice (8 ± 1 vs. 20 ± 4% of left ventricular weight; p < 0.01). CypD-deficient mice displayed an increased expression of ER stress proteins such as eukaryotic initiation factor 2α (eIF2α) or glucose regulated protein 78 (Grp78 or Bip). The ER stress inhibitor TUDCA prevented the infarct size reduction afforded by the loss of cypD function (mean infarct size averaged 21 ± 4% of LV weight, p < 0.01 vs. cypD-KO). Similar results were obtained when NIM811, an analog of cyclosporine A, was used to pharmacologically (instead of genetically) inhibit cypD function. This study suggests that the ER stress induced by the inhibition of cypD function plays a key role in protecting the heart against lethal ischemia-reperfusion injury.

Opposite and tissue-specific effects of coenzyme Q2 on mPTP opening and ROS production between heart and liver mitochondria: role of complex I.

Coenzyme Q(2) (CoQ(2)) is known to inhibit mitochondrial permeability transition pore (mPTP) opening in isolated rat liver mitochondria. In this study, we investigated and compared the effects of CoQ(2) on mPTP opening and ROS production in isolated rabbit heart and rat liver mitochondria. Mitochondria were isolated from New Zealand White rabbit hearts and Wistar rat livers. Oxygen consumption, Ca(2+)-induced mPTP opening, ROS production and NADH DUb-reductase activity were measured. Rotenone was used to investigate the effect of CoQ(2) on respiratory complex I activity. CoQ(2) (23 µM) reduced the respiratory control index by 32% and 57% (p<0.01) in heart and liver mitochondria respectively, mainly through an increased oxygen consumption in state 4. CoQ(2) induced a 60% (p<0.05) decrease of calcium retention capacity (CRC) in heart mitochondria and inversely a 46% (p<0.05) increase in liver mitochondria. In basal condition, CoQ(2) induced a 170% (p<0.05) increase of H(2)O(2) production in heart mitochondria and 21% (ns) decrease of H(2)O(2) production in liver mitochondria. Because rotenone, a complex I inhibitor, increases H(2)O(2) production in heart but not in liver mitochondria we investigated the CoQ(2) effect in a dose-response assay of complex I inhibition by rotenone in both mitochondria. CoQ(2) antagonized the effect of rotenone on respiratory complex I activity in liver but not in heart mitochondria. CoQ(2) significantly reduced NADH DUb-reductase activity in liver (-47%) and heart (-37%) mitochondria. In conclusion, our data showed that on the contrary to what was observed in liver mitochondria, CoQ(2) favors mPTP opening and ROS production in heart mitochondria through an opposite effect on respiratory complex I activity.

Determination of the myocardial area at risk with pre- versus post-reperfusion imaging techniques in the pig model.

The purpose of this study was to compare the accuracy of post-reperfusion cardiac magnetic resonance (CMR) and pre-reperfusion multidetector computed tomography (MDCT) imaging to measure the size of the area at risk (AAR), using pathology as a reference technique in a porcine acute myocardial infarction model. Fifteen pigs underwent balloon-induced coronary artery occlusion for 40 min followed by reperfusion. The AAR was assessed with arterial enhanced MDCT performed during occlusion, while two different T2 weighted (T2W) CMR imaging sequences and the contrast-enhanced (ce-) CMR endocardial surface length (ESL) were performed after 90 min of reperfusion. Animals were euthanized and the AAR was assessed by pathology. Additional measurements of the myocardial water content in the AAR, remote and the AAR border zones were performed. AAR by pathology best correlated with measurements made by MDCT (R(2) = 0.88; p < 0.001) with little bias on Bland-Altman plots (bias 2.5%, SD 6.1% LV area). AAR measurements obtained by T2W STIR, T2W ACUTE sequences or the ESL on ce-CMR showed a fair correlation with pathology (R(2) = 0.72, R(2) = 0.65 and R(2) = 0.69, respectively; all p ≤ 0.001), but significantly overestimated the size of the AAR with important bias (17.4 ± 10.8% LV area; 11.7 ± 11.0% LV area; 13.0 ± 10.3% LV area, respectively). The myocardial water content in the AAR border zones was significantly higher than the remote (82.8 vs. 78.8%; p < 0.001). Our data suggest that post-reperfusion imaging methods overestimated the AAR likely due to the presence of edema outside of the boundaries of the AAR. Pre-reperfusion arterial enhanced MDCT showed the greatest accuracy for the assessment of the AAR.

Strain-rate imaging predicts the attenuation of left ventricular remodeling induced by ischemic postconditioning after myocardial infarction in mice.

BACKGROUND: Systolic strain rate (SR) has been shown to correlate with the transmural extent of myocardial infarction (MI). Ischemic postconditioning (PC) decreases MI size. We aimed to determine whether SR may predict the reduction of left ventricular (LV) remodeling induced by PC in a murine model of ischemia-reperfusion. METHODS AND RESULTS: C57BL6 mice underwent 60 minutes of left coronary artery occlusion followed by reperfusion. PC consisted of 3 cycles of 1 minute of reperfusion and 1 minute of ischemia performed immediately after reperfusion. After 24 hours of reperfusion, a first subset of mice was euthanized for determination of infarct size. An additional subset of mice underwent 3 months of reperfusion. Echocardiography and SR were serially assessed at baseline, 3 days, and 1, 2, 3 months after reperfusion. PC decreased the infarct size and increased SR values within infarcted segments as soon as 24 hours after reperfusion as compared with controls (14 ± 1 versus 6 ± 1 s(-1), P<0.001). Systolic SR was correlated with MI size assessed by the area of infarction/area at risk (r=-0.88, P<0.001). At 3 months, PC improved LV ejection fraction (55 ± 4 versus 45 ± 3%; P<0.05) and decreased LV dimensions as compared with controls. Strain rate at day 3 correlated with LV ejection fraction (0.75; P<0.001), end-systolic (r=-0.75; P=0.001), and end-diastolic volumes (r=-0.70; P<0.001) at month 3. CONCLUSIONS: In this murine model of MI, SR accurately assessed the reduction in MI size induced by PC early after reperfusion and the subsequent reduction of LV remodeling.