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 Multimodal Cardioprotection Strategy During Cardiac Surgery: The ProCCard Study.

OBJECTIVE: The ProCCard study tested whether combining several cardioprotective interventions would reduce the myocardial and other biological and clinical damage in patients undergoing cardiac surgery. DESIGN: Prospective, randomized, controlled trial. SETTING: Multicenter tertiary care hospitals. PARTICIPANTS: 210 patients scheduled to undergo aortic valve surgery. INTERVENTIONS: A control group (standard of care) was compared to a treated group combining five perioperative cardioprotective techniques: anesthesia with sevoflurane, remote ischemic preconditioning, close intraoperative blood glucose control, moderate respiratory acidosis (pH 7.30) just before aortic unclamping (concept of the "pH paradox"), and gentle reperfusion just after aortic unclamping. MEASUREMENTS AND MAIN RESULTS: The primary outcome was the postoperative 72-h area under the curve (AUC) for high-sensitivity cardiac troponin I (hsTnI). Secondary endpoints were biological markers and clinical events occurring during the 30 postoperative days and the prespecified subgroup analyses. The linear relationship between the 72-h AUC for hsTnI and aortic clamping time, significant in both groups (p < 0.0001), was not modified by the treatment (p = 0.57). The rate of adverse events at 30 days was identical. A non-significant reduction of the 72-h AUC for hsTnI (-24%, p = 0.15) was observed when sevoflurane was administered during cardiopulmonary bypass (46% of patients in the treated group). The incidence of postoperative renal failure was not reduced (p = 0.104). CONCLUSION: This multimodal cardioprotection has not demonstrated any biological or clinical benefit during cardiac surgery. The cardio- and reno-protective effects of sevoflurane and remote ischemic preconditioning therefore remain to be demonstrated in this context.

Heat Shock Protein 70 Is Involved in the Efficiency of Preconditioning with Cyclosporine A in Renal Ischemia Reperfusion Injury by Modulating Mitochondrial Functions.

Cyclosporine A (CsA) preconditioning is known to target mitochondrial permeability transition pore and protect renal function after ischemia reperfusion (IR). The upregulation of heat-shock protein 70 (Hsp70) expression after CsA injection is thought to be associated with renal protection. The aim of this study was to test the effect of Hsp70 expression on kidney and mitochondria functions after IR. Mice underwent a right unilateral nephrectomy and 30 min of left renal artery clamping, performed after CsA injection and/or administration of the Hsp70 inhibitor. Histological score, plasma creatinine, mitochondrial calcium retention capacity, and oxidative phosphorylation were assessed after 24 h of reperfusion. In parallel, we used a model of hypoxia reoxygenation on HK2 cells to modulate Hsp70 expression using an SiRNA or a plasmid. We assessed cell death after 18 h of hypoxia and 4 h of reoxygenation. CsA significantly improved renal function, histological score, and mitochondrial functions compared to the ischemic group but the inhibition of Hsp70 repealed the protection afforded by CsA injection. In vitro, Hsp70 inhibition by SiRNA increased cell death. Conversely, Hsp70 overexpression protected cells from the hypoxic condition, as well as the CsA injection. We did not find a synergic association between Hsp70 expression and CsA use. We demonstrated Hsp70 could modulate mitochondrial functions to protect kidneys from IR. This pathway may be targeted by drugs to provide new therapeutics to improve renal function after IR.

Effect of Colchicine on Myocardial Injury in Acute Myocardial Infarction.

BACKGROUND: Inflammation is a key factor of myocardial damage in reperfused ST-segment-elevation myocardial infarction. We hypothesized that colchicine, a potent anti-inflammatory agent, may reduce infarct size (IS) and left ventricular (LV) remodeling at the acute phase of ST-segment-elevation myocardial infarction. METHODS: In this double-blind multicenter trial, we randomly assigned patients admitted for a first episode of ST-segment-elevation myocardial infarction referred for primary percutaneous coronary intervention to receive oral colchicine (2-mg loading dose followed by 0.5 mg twice a day) or matching placebo from admission to day 5. The primary efficacy outcome was IS determined by cardiac magnetic resonance imaging at 5 days. The relative LV end-diastolic volume change at 3 months and IS at 3 months assessed by cardiac magnetic resonance imaging were among the secondary outcomes. RESULTS: We enrolled 192 patients, 101 in the colchicine group and 91 in the control group. At 5 days, the gadolinium enhancement-defined IS did not differ between the colchicine and placebo groups with a mean of 26 interquartile range (IQR) [16-44] versus 28.4 IQR [14-40] g of LV mass, respectively (P=0.87). At 3 months follow-up, there was no significant difference in LV remodeling between the colchicine and placebo groups with a +2.4% (IQR, -8.3% to 11.1%) versus -1.1% (IQR, -8.0% to 9.9%) change in LV end-diastolic volume (P=0.49). Infarct size at 3 months was also not significantly different between the colchicine and placebo groups (17 IQR [10-28] versus 18 IQR [10-27] g of LV mass, respectively; P=0.92). The incidence of gastrointestinal adverse events during the treatment period was greater with colchicine than with placebo (34% versus 11%, respectively; P=0.0002). CONCLUSIONS: In this randomized, placebo-controlled trial, oral administration of high-dose colchicine at the time of reperfusion and for 5 days did not reduce IS assessed by cardiac magnetic resonance imaging. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT03156816.

Impact of Age on Systemic Inflammatory Profile of Patients With ST-Segment-Elevation Myocardial Infarction and Acute Ischemic Stroke.

BACKGROUND: Aging is associated with a chronic low-grade inflammatory state. This condition may affect the acute inflammatory response involved in ST-segment-elevation myocardial infarction (STEMI) or acute ischemic stroke (AIS). We sought to compare the profile of a set of circulating inflammatory markers between young and older patients admitted for STEMI or AIS. METHODS: HIBISCUS-STEMI (Cohort of Patients to Identify Biological and Imaging Markers of Cardiovascular Outcomes in ST Elevation Myocardial Infarction) and HIBISCUS-STROKE (Cohort of Patients to Identify Biological and Imaging Markers of Cardiovascular Outcomes in Stroke) are 2 cohort studies that enrolled patients with STEMI treated with primary percutaneous coronary intervention in the cardiac intensive care unit of Lyon and patients with AIS treated with mechanical thrombectomy in the Lyon Stroke Center, respectively from 2016 to 2019. Patients were classified as older if they were ≥65 years and as young if they were <65 years. In both cohorts, CRP (C-reactive protein), IL (interleukin)-6, IL-8, IL-10, MCP (monocyte chemoattractant protein), sTNF-RI (soluble tumor necrosis factor receptor I), sST2 (soluble form suppression of tumorigenicity 2), and VCAM-1 (vascular cellular adhesion molecule-1) were measured on serum collected at 5 time points using enzyme-linked immunosorbent assay. A multiple logistic regression model was performed to detect an association between area under the curve of circulating inflammatory markers within the first 48 hours and older age. RESULTS: A total of 260 patients with STEMI and 164 patients with AIS were included. Of them, there were 76 (29%) and 105 (64%) older patients with STEMI and AIS, respectively. Following multivariable analysis, a high area under the curve of IL-6 and sTNF-RI, a low lymphocyte count, and a high neutrophil-lymphocyte ratio at 24 hours were associated with older age in patients with STEMI and AIS. CONCLUSIONS: Older patients had higher IL-6 and sTFN-RI levels within the first 48 hours associated with a lower lymphocyte count and a higher neutrophil-lymphocyte ratio at 24 hours in both cohorts.

Molar Sodium Lactate Attenuates the Severity of Postcardiac Arrest Syndrome: A Preclinical Study.

OBJECTIVES: To determine whether continuous IV infusion of molar sodium lactate would limit cardiac arrest-induced neurologic injury and cardiovascular failure. DESIGN: Randomized blinded study (animal model). SETTING: University animal research facility. SUBJECTS: Twenty-four adult male "New Zealand White" rabbits. INTERVENTIONS: Anesthetized rabbits underwent 12.5 minutes of asphyxial cardiac arrest and were randomized to receive either normal saline (control group, n = 12) or molar sodium lactate (molar sodium lactate group, n = 12) at a rate of 5 mL/kg/hr during the whole 120-minute reperfusion period. MEASUREMENTS AND MAIN RESULTS: Pupillary reactivity (primary outcome), levels of S100ß protein, in vitro brain mitochondria functions, cardiovascular function, and fluid balance were assessed. Molar sodium lactate reduced brain injury, with a higher proportion of animals exhibiting pupillary reactivity to light (83% vs 25% in the CTRL group, p = 0.01) and lower S100ß protein levels (189 ± 42 vs 412 ± 63 pg/mL, p < 0.01) at the end of the protocol. Molar sodium lactate significantly prevented cardiac arrest-induced decrease in oxidative phosphorylation and mitochondrial calcium-retention capacity compared with controls. At 120 minutes of reperfusion, survival did not significantly differ between the groups (10/12, 83% in the molar sodium lactate group vs nine of 12, 75% in the control group; p > 0.99), but hemodynamics were significantly improved in the molar sodium lactate group compared with the control group (higher mean arterial pressure [49 ± 2 vs 29 ± 3 mm Hg; p < 0.05], higher cardiac output [108 ± 4 vs 58 ± 9 mL/min; p < 0.05], higher left ventricle surface shortening fraction [38% ± 3% vs 19% ± 3%; p < 0.05], and lower left ventricular end-diastolic pressure [3 ± 1 vs 8 ± 2 mm Hg; p < 0.01]). While fluid intake was similar in both groups, fluid balance was higher in control animals (11 ± 1 mL/kg) than that in molar sodium lactate-treated rabbits (1 ± 3 mL/kg; p < 0.01) due to lower diuresis. CONCLUSIONS: Molar sodium lactate was effective in limiting the severity of the postcardiac arrest syndrome. This preclinical study opens up new perspectives for the treatment of cardiac arrest.

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.

Mild Therapeutic Hypothermia Protects from Acute and Chronic Renal Ischemia-Reperfusion Injury in Mice by Mitigated Mitochondrial Dysfunction and Modulation of Local and Systemic Inflammation.

Renal ischemia-reperfusion (IR) injury can lead to acute kidney injury, increasing the risk of developing chronic kidney disease. We hypothesized that mild therapeutic hypothermia (mTH), 34 °C, applied during ischemia could protect the function and structure of kidneys against IR injuries in mice. In vivo bilateral renal IR led to an increase in plasma urea and acute tubular necrosis at 24 h prevented by mTH. One month after unilateral IR, kidney atrophy and fibrosis were reduced by mTH. Evaluation of mitochondrial function showed that mTH protected against IR-mediated mitochondrial dysfunction at 24 h, by preserving CRC and OX-PHOS. mTH completely abrogated the IR increase of plasmatic IL-6 and IL-10 at 24 h. Acute tissue inflammation was decreased by mTH (IL-6 and IL1-ß) in as little as 2 h. Concomitantly, mTH increased TNF-α expression at 24 h. One month after IR, mTH increased TNF-α mRNA expression, and it decreased TGF-ß mRNA expression. We showed that mTH alleviates renal dysfunction and damage through a preservation of mitochondrial function and a modulated systemic and local inflammatory response at the acute phase (2-24 h). The protective effect of mTH is maintained in the long term (1 month), as it diminished renal atrophy and fibrosis, and mitigated chronic renal inflammation.

A modified calcium retention capacity assay clarifies the roles of extra- and intracellular calcium pools in mitochondrial permeability transition pore opening.

Calcium homeostasis is essential for cell survival and is precisely controlled by several cellular actors such as the sarco/endoplasmic reticulum and mitochondria. Upon stress induction, Ca2+ released from sarco/endoplasmic reticulum stores and from extracellular Ca2+ pools accumulates in the cytosol and in the mitochondria. This induces Ca2+ overload and ultimately the opening of the mitochondrial permeability transition pore (mPTP), promoting cell death. Currently, it is unclear whether intracellular Ca2+ stores are sufficient to promote the mPTP opening. Ca2+ retention capacity (CRC) corresponds to the maximal Ca2+ uptake by the mitochondria before mPTP opening. In this study, using permeabilized cardiomyocytes isolated from adult mice, we modified the standard CRC assay by specifically inducing reticular Ca2+ release to investigate the respective contributions of reticular Ca2+ and extracellular Ca2+ to mPTP opening in normoxic conditions or after anoxia-reoxygenation. Our experiments revealed that Ca2+ released from the sarco/endoplasmic reticulum is not sufficient to trigger mPTP opening and corresponds to ∼50% of the total Ca2+ levels required to open the mPTP. We also studied mPTP opening after anoxia-reoxygenation in the presence or absence of extracellular Ca2+ In both conditions, Ca2+ leakage from internal stores could not trigger mPTP opening by itself but significantly decreased the CRC. Our findings highlight how a modified CRC assay enables the investigation of the role of reticular and extracellular Ca2+ pools in the regulation of the mPTP. We propose that this method may be useful for screening molecules of interest implicated in mPTP regulation.

4TM-TRPM8 channels are new gatekeepers of the ER-mitochondria Ca2+ transfer.

Calcium (Ca2+) release from the endoplasmic reticulum plays an important role in many cell-fate defining cellular processes. Traditionally, this Ca2+ release was associated with the ER Ca2+ release channels, inositol 1,4,5­triphosphate receptor (IP3R) and ryanodine receptor (RyR). Lately, however, other calcium conductances have been found to be intracellularly localized and to participate in cell fate regulation. Nonetheless, molecular identity and functional properties of the ER Ca2+ release mechanisms associated with multiple diseases, e.g. prostate cancer, remain unknown. Here we identify a new family of transient receptor potential melastatine 8 (TRPM8) channel isoforms as functional ER Ca2+ release channels expressed in mitochondria-associated ER membranes (MAMs). These TRPM8 isoforms exhibit an unconventional structure with 4 transmembrane domains (TMs) instead of 6 TMs characteristic of the TRP channel archetype. We show that these 4TM-TRPM8 isoforms form functional channels in the ER and participate in regulation of the steady-state Ca2+ concentration ([Ca2+]) in mitochondria and the ER. Thus, our study identifies 4TM-TRPM8 isoforms as ER Ca2+ release mechanism distinct from classical Ca2+ release channels.

HEXIM1 Diffusion in the Nucleus Is Regulated by Its Interactions with Both 7SK and P-TEFb.

How nuclear proteins diffuse and find their targets remains a key question in the transcription field. Dynamic proteins in the nucleus are classically subdiffusive and undergo anomalous diffusion, yet the underlying physical mechanisms are still debated. In this study, we explore the contribution of interactions to the generation of anomalous diffusion by the means of fluorescence spectroscopy and simulation. Using interaction-deficient mutants, our study indicates that HEXIM1 interactions with both 7SK RNA and positive transcription elongation factor b are critical for HEXIM1 subdiffusion and thus provides evidence of the effects of protein-RNA interaction on molecular diffusion. Numerical simulations allowed us to establish that the proportions of distinct oligomeric HEXIM1 subpopulations define the apparent anomaly parameter of the whole population. Slight changes in the proportions of these oligomers can lead to significant shifts in the diffusive features and recapitulate the modifications observed in cells with the various interaction-deficient mutants. By combining simulations and experiments, our work opens new prospects in which the anomaly α coefficient in diffusion becomes a helpful tool to infer alterations in molecular interactions.

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.

Evolution of the human cold/menthol receptor, TRPM8.

Genes showing versatile functions or subjected to fast expansion and contraction during the adaptation of species to specific ecological conditions, like sensory receptors for odors, pheromones and tastes, are characterized by a great plasticity through evolution. One of the most fascinating sensory receptors in the family of TRP channels, the cold and menthol receptor TRPM8, has received significant attention in the literature. Recent studies have reported the existence of TRPM8 channel isoforms encoded by alternative mRNAs transcribed from alternative promoters and processed by alternative splicing. Since the first draft of the human genome was accomplished in 2000, alternative transcription, alternative splicing and alternative translation have appeared as major sources of gene product diversity and are thought to participate in the generation of complexity in higher organisms. In this study, we investigate whether alternative transcription has been a driving force in the evolution of the human forms of the cold receptor TRPM8. We identified 33 TRPM8 alternative mRNAs (24 new sequences) and their associated protein isoforms in human tissues. Using comparative genomics, we described the evolution of the human TRPM8 sequences in eight ancestors since the origin of Amniota, and estimated in which ancestors the new TRPM8 variants originated. In order to validate the estimated origins of this receptor, we performed experimental validations of predicted exons in mouse tissues. Our results suggest a first diversification event of the cold receptor in the Boreoeutheria ancestor, and a subsequent divergence at the origin of Simiiformes.

FRET Image Correlation Spectroscopy Reveals RNAPII-Independent P-TEFb Recruitment on Chromatin.

Biochemical studies have revealed that the RNA Polymerase II (RNAPII) pause release is triggered by phosphorylation of the transcription machinery by the positive transcription elongation factor b (P-TEFb). However, there are no direct report that P-TEFb and RNA polymerase II interact in single living cells and the biophysical mechanisms mediating this association are still unclear. Förster resonance energy transfer (FRET) detects molecular interactions at the subcellular level. Time domain fluorescence lifetime imaging provides an accurate quantification of FRET efficiency, EFRET, because it is fluorochrome concentration-independent and insensitive to fluorescence bleed-through. However, the way FRET signal is usually analyzed does not provide information about the areas where protein-protein interactions take place. In this work, we developed a method, dubbed FRET image correlation spectroscopy (FICS), which relied on FRET fluorescence lifetime imaging image acquisition and image correlation spectroscopy of EFRET clusters to quantify the spatial distribution of interaction clusters in the nucleus. The combination of high content FRET microscopy with batch image analysis allowed a robust statistical analysis. By applying FICS, we characterized the area and density of interaction clusters between P-TEFb and RNAPII or histone H2A in single living cells. The FICS method applied to cells expressing genetically engineered mutated proteins confirmed that the histidine-rich domain of P-TEFb is required for its interaction with RNAPII. Furthermore, it demonstrated that P-TEFb was also located in close vicinity to histone H2A, independently of its interactions with RNAPII. These results support the hypothesis that P-TEFb dynamics on chromatin regulate its recruitment on RNAPII.

Epidermal TRPM8 channel isoform controls the balance between keratinocyte proliferation and differentiation in a cold-dependent manner.

Deviation of the ambient temperature is one of the most ubiquitous stimuli that continuously affect mammals' skin. Although the role of the warmth receptors in epidermal homeostasis (EH) was elucidated in recent years, the mystery of the keratinocyte mild-cold sensor remains unsolved. Here we report the cloning and characterization of a new functional epidermal isoform of the transient receptor potential M8 (TRPM8) mild-cold receptor, dubbed epidermal TRPM8 (eTRPM8), which is localized in the keratinocyte endoplasmic reticulum membrane and controls mitochondrial Ca(2+) concentration ([Ca(2+)]m). In turn, [Ca(2+)]m modulates ATP and superoxide (O2(·-)) synthesis in a cold-dependent manner. We report that this fine tuning of ATP and O2(·-) levels by cooling controls the balance between keratinocyte proliferation and differentiation. Finally, to ascertain eTRPM8's role in EH in vivo we developed a new functional knockout mouse strain by deleting the pore domain of TRPM8 and demonstrated that eTRPM8 knockout impairs adaptation of the epidermis to low temperatures.

Cold/menthol TRPM8 receptors initiate the cold-shock response and protect germ cells from cold-shock-induced oxidation.

Testes of most male mammals present the particularity of being externalized from the body and are consequently slightly cooler than core body temperature (4-8°C below). Although, hypothermia of the testis is known to increase germ cells apoptosis, little is known about the underlying molecular mechanisms, including cold sensors, transduction pathways, and apoptosis triggers. In this study, using a functional knockout mouse model of the cold and menthol receptors, dubbed transient receptor potential melastatine 8 (TRPM8) channels, we found that TRPM8 initiated the cold-shock response by differentially modulating cold- and heat-shock proteins. Besides, apoptosis of germ cells increased in proportion to the cooling level in control mice but was independent of temperature in knockout mice. We also observed that the rate of germ cell death correlated positively with the reactive oxygen species level and negatively with the expression of the detoxifying enzymes. This result suggests that the TRPM8 sensor is a key determinant of germ cell fate under hypothermic stimulation.-Borowiec, A.-S., Sion, B., Chalmel, F., Rolland, A. D., Lemonnier, L., De Clerck, T., Bokhobza, A., Derouiche, S., Dewailly, E., Slomianny, C., Mauduit, C., Benahmed, M., Roudbaraki, M., Jégou, B., Prevarskaya, N., Bidaux, G. Cold/menthol TRPM8 receptors initiate the cold-shock response and protect germ cells from cold-shock-induced oxidation.

Functional and Modeling Studies of the Transmembrane Region of the TRPM8 Channel.

Members of the transient receptor potential (TRP) ion channel family act as polymodal cellular sensors, which aid in regulating Ca(2+) homeostasis. Within the TRP family, TRPM8 is the cold receptor that forms a nonselective homotetrameric cation channel. In the absence of TRPM8 crystal structure, little is known about the relationship between structure and function. Inferences of TRPM8 structure have come from mutagenesis experiments coupled to electrophysiology, mainly regarding the fourth transmembrane helix (S4), which constitutes a moderate voltage-sensing domain, and about cold sensor and phosphatidylinositol 4,5-bisphosphate binding sites, which are both located in the C-terminus of TRPM8. In this study, we use a combination of molecular modeling and experimental techniques to examine the structure of the TRPM8 transmembrane and pore helix region including the conducting conformation of the selectivity filter. The model is consistent with a large amount of functional data and was further tested by mutagenesis. We present structural insight into the role of residues involved in intra- and intersubunit interactions and their link with the channel activity, sensitivity to icilin, menthol and cold, and impact on channel oligomerization.

Are Orai1 and Orai3 channels more important than calcium influx for cell proliferation?

Transformed and tumoral cells share the characteristic of being able to proliferate even when external calcium concentration is very low. We have investigated whether Human Embryonic Kidney 293 cells, human hepatoma cell Huh-7 and HeLa cells were able to proliferate when kept 72h in complete culture medium without external calcium. Our data showed that cell proliferation rate was similar over a range of external calcium concentration (2µM to 1.8mM). Incubation in the absence of external calcium for 72h had no significant effect on endoplasmic reticulum (ER) Ca(2+) contents but resulted in a significant decrease in cytosolic free calcium concentration in all 3 cell types. Cell proliferation rates were dependent on Orai1 and Orai3 expression levels in HEK293 and HeLa cells. Silencing Orai1 or Orai3 resulted in a 50% reduction in cell proliferation rate. Flow cytometry analysis showed that Orai3 induced a small but significant increase in cell number in G2/M phase. RO-3306, a cdk-1 inhibitor, induced a 90% arrest in G2/M reversible in less than 15min. Our data showed that progression through G2/M phase after release from RO-3306-induced cell cycle arrest was slower in both Orai1 and Orai3 knock-downs. Overexpressing Orai1, Orai3 and the dominant negative non-permeant mutants E106Q-Orai1 and E81Q-Orai3 induced a 50% increase in cell proliferation rate in HEK293 cells. Our data clearly demonstrated that Orai1 and Orai3 proteins are more important than calcium influx to control cell proliferation in some cell lines and that this process is probably independent of ICRAC and Iarc.

Colchicine in acute myocardial infarction: cardiovascular events at 1-year follow up.

OBJECTIVE: In the COVERT-MI randomised placebo-controlled trial, oral administration of high-dose colchicine at the time of reperfusion and for 5 days in acute ST-elevated myocardial infarction did not reduce infarct size but was associated with a significant increase in left ventricular thrombus (LVT) in comparison to placebo. We aimed to assess the 1-year clinical outcomes of the study population. METHODS: This study is a follow-up analysis of the COVERT-MI study on prespecified secondary clinical endpoints at 1 year. The primary endpoint of this study was a composite of major adverse cardiovascular events (MACEs), including all-cause death, acute coronary syndromes, heart failure events, ischaemic strokes, sustained ventricular arrhythmias and acute kidney injury at 1-year follow-up. The quality of life (QOL) and the drug therapy prescription were also assessed. RESULTS: At 1 year, 192 patients (101 patients in the colchicine group, 91 in the placebo group) were followed up. Seventy-six (39.6%) MACEs were reported in the study population. There was no significant difference regarding the number of MACEs between groups: 36 (35.6%) in the colchicine group and 40 (44.1%) in the placebo group (p=0.3). There were no differences in the occurrence of ischaemic strokes between the colchicine group and the control group (3 (3%) vs 2 (2.2%), respectively, p=0.99). There was a trend towards fewer heart failure events in the colchicine group compared with the placebo group (12 (11.9%) vs 18 (19.8%), p=0.20). There was no significant difference in QOL scores at 1 year (75.8±15.7 vs 72.7±16.2 respectively, p=0.18). CONCLUSIONS: There was no significant difference between the colchicine and placebo groups at 1 year regarding MACEs, especially concerning deaths or ischaemic strokes. No excess of ischaemic adverse events was observed despite the initial increase in LVT in the colchicine group. TRIAL REGISTRATION NUMBER: NCT0315681.

Short isoforms of the cold receptor TRPM8 inhibit channel gating by mimicking heat action rather than chemical inhibitors.

Transient receptor potential (TRP) channels couple various environmental factors to changes in membrane potential, calcium influx, and cell signaling. They also integrate multiple stimuli through their typically polymodal activation. Thus, although the TRPM8 channel has been extensively investigated as the major neuronal cold sensor, it is also regulated by various chemicals, as well as by several short channel isoforms. Mechanistic understanding of such complex regulation is facilitated by quantitative single-channel analysis. We have recently proposed a single-channel mechanism of TRPM8 regulation by voltage and temperature. Using this gating mechanism, we now investigate TRPM8 inhibition in cell-attached patches using HEK293 cells expressing TRPM8 alone or coexpressed with its short sM8-6 isoform. This is compared with inhibition by the chemicals N-(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl)piperazine-1-carboxamide (BCTC) and clotrimazole or by elevated temperature. We found that within the seven-state single-channel gating mechanism, inhibition of TRPM8 by short sM8-6 isoforms closely resembles inhibition by increased temperature. In contrast, inhibition by BCTC and that by clotrimazole share a different set of common features.