Heart rate reduction after genetic ablation of L-type Cav1.3 channels induces cardioprotection against ischemia-reperfusion injury.

Background: Acute myocardial infarction (AMI) is the major cause of cardiovascular mortality worldwide. Most ischemic episodes are triggered by an increase in heart rate, which induces an imbalance between myocardial oxygen delivery and consumption. Developing drugs that selectively reduce heart rate by inhibiting ion channels involved in heart rate control could provide more clinical benefits. The Cav1.3-mediated L-type Ca2+ current (ICav1.3) play important roles in the generation of heart rate. Therefore, they can constitute relevant targets for selective control of heart rate and cardioprotection during AMI. Objective: We aimed to investigate the relationship between heart rate and infarct size using mouse strains knockout for Cav1.3 (Cav1.3-/-) L-type calcium channel and of the cardiac G protein gated potassium channel (Girk4-/-) in association with the funny (f)-channel inhibitor ivabradine. Methods: Wild-type (WT), Cav1.3+/-, Cav1.3-/- and Girk4-/- mice were used as models of respectively normal heart rate, moderate heart rate reduction, bradycardia, and mild tachycardia, respectively. Mice underwent a surgical protocol of myocardial IR (40 min ischemia and 60 min reperfusion). Heart rate was recorded by one-lead surface ECG recording, and infarct size measured by triphenyl tetrazolium chloride staining. In addition, Cav1.3-/- and WT hearts perfused on a Langendorff system were subjected to the same ischemia-reperfusion protocol ex vivo, without or with atrial pacing, and the coronary flow was recorded. Results: Cav1.3-/- mice presented reduced infarct size (-29%), while Girk4-/- displayed increased infarct size (+30%) compared to WT mice. Consistently, heart rate reduction in Cav1.3+/- or by the f-channel blocker ivabradine was associated with significant decrease in infarct size (-27% and -32%, respectively) in comparison to WT mice. Conclusion: Our results show that decreasing heart rate allows to protect the myocardium against IR injury in vivo and reveal a close relationship between basal heart rate and IR injury. In addition, this study suggests that targeting Cav1.3 channels could constitute a relevant target for reducing infarct size, since maximal heart rate dependent cardioprotective effect is already observed in Cav1.3+/- mice.

Delayed postconditioning in the mouse heart in vivo.

BACKGROUND: Reperfusion during acute myocardial infarction remains the best treatment for reducing infarct size. Postconditioning, applied at the onset of reperfusion, reduces myocardial infarction both in animals and humans. The objective of this study was to identify the time delay to apply postconditioning at reperfusion, allowing preservation of cardioprotection in the mouse myocardium. This is a major issue in the management of acute myocardial infarction patients. METHODS AND RESULTS: Mice were subjected to 40 minutes of ischemia and 60 minutes of reperfusion (IR(60')). Postconditioning protocols corresponding to repetitive ischemia (3 cycles of 1 minute of ischemia and 1 minute of reperfusion) were applied during early reperfusion at various time durations (Δt) after reopening of the coronary artery (Δt=10 seconds, 1, 5, 10, 15, 20, 30, and 45 minutes; PostC(Δt)). Infarct size/area at risk was reduced by 71% in PostC(Δ1) compared with IR(60') mice (P=5×10(-6)). There was a linear correlation (r(2)=0.91) between infarct size and Δt, indicating that the cardioprotective effect of delayed postconditioning was progressively attenuated when Δt time increased. The protective effect of PostC(Δ1) and PostC(Δ15) was still effective when the duration of reperfusion was prolonged to 24 hours (IR(24 hours); PostC(Δ1) and PostC(Δ15) versus IR(24 hours), P=0.001). Similar results were obtained for internucleosomal DNA fragmentation and lactate dehydrogenase release. CONCLUSIONS: This study in our in vivo mouse model of myocardial IR shows for the first time that delaying the intervention of postconditioning to 30 minutes does not abrogate the cardioprotective effect of postconditioning. This finding provides evidence that the time window of protection afforded by postconditioning may be larger than initially reported.

RIP140 Represses Intestinal Paneth Cell Differentiation and Interplays with SOX9 Signaling in Colorectal Cancer.

RIP140 is a major transcriptional coregulator of gut homeostasis and tumorigenesis through the regulation of Wnt/APC signaling. Here, we investigated the effect of RIP140 on Paneth cell differentiation and its interplay with the transcription factor SOX9. Using loss of function mouse models, human colon cancer cells, and tumor microarray data sets we evaluated the role of RIP140 in SOX9 expression and activity using RT-qPCR, immunohistochemistry, luciferase reporter assays, and GST-pull down. We first evidence that RIP140 strongly represses the Paneth cell lineage in the intestinal epithelium cells by inhibiting Sox9 expression. We then demonstrate that RIP140 interacts with SOX9 and inhibits its transcriptional activity. Our results reveal that the Wnt signaling pathway exerts an opposite regulation on SOX9 and RIP140. Finally, the levels of expression of RIP140 and SOX9 exhibit a reverse response and prognosis value in human colorectal cancer biopsies. This work highlights an intimate transcriptional cross-talk between RIP140 and SOX9 in intestinal physiopathology.

A novel therapeutic peptide targeting myocardial reperfusion injury.

AIMS: Regulated cell death is a main contributor of myocardial ischaemia-reperfusion (IR) injury during acute myocardial infarction. In this context, targeting apoptosis could be a potent therapeutical strategy. In a previous study, we showed that DAXX (death-associated protein) was essential for transducing the FAS-dependent apoptotic signal during IR injury. The present study aims at evaluating the cardioprotective effects of a synthetic peptide inhibiting FAS:DAXX interaction. METHODS AND RESULTS: An interfering peptide was engineered and then coupled to the Tat cell penetrating peptide (Tat-DAXXp). Its internalization and anti-apoptotic properties were demonstrated in primary cardiomyocytes. Importantly, an intravenous bolus injection of Tat-DAXXp (1 mg/kg) 5 min before reperfusion in a murine myocardial IR model decreased infarct size by 48% after 24 h of reperfusion. In addition, Tat-DAXXp was still efficient after a 30-min delayed administration, and was completely degraded and eliminated within 24 h thereby reducing risks of potential side effects. Importantly, Tat-DAXXp reduced mouse early post-infarction mortality by 67%. Mechanistically, cardioprotection was supported by both anti-apoptotic and pro-survival effects, and an improvement of myocardial functional recovery as evidenced in ex vivo experiments. CONCLUSIONS: Our study demonstrates that a single dose of Tat-DAXXp injected intravenously at the onset of reperfusion leads to a strong cardioprotection in vivo by inhibiting IR injury validating Tat-DAXXp as a promising candidate for therapeutic application.

Anti-apoptotic peptide for long term cardioprotection in a mouse model of myocardial ischemia-reperfusion injury.

Reperfusion therapy during myocardial infarction (MI) leads to side effects called ischemia-reperfusion (IR) injury for which no treatment exists. While most studies have targeted the intrinsic apoptotic pathway to prevent IR injury with no successful clinical translation, we evidenced recently the potent cardioprotective effect of the anti-apoptotic Tat-DAXXp (TD) peptide targeting the FAS-dependent extrinsic pathway. The aim of the present study was to evaluate TD long term cardioprotective effects against IR injury in a MI mouse model. TD peptide (1 mg/kg) was administered in mice subjected to MI (TD; n = 21), 5 min prior to reperfusion, and were clinically followed-up during 6 months after surgery. Plasma cTnI concentration evaluated 24 h post-MI was 70%-decreased in TD (n = 16) versus Ctrl (n = 20) mice (p***). Strain echocardiography highlighted a 24%-increase (p****) in the ejection fraction mean value in TD-treated (n = 12) versus Ctrl mice (n = 17) during the 6 month-period. Improved cardiac performance was associated to a 54%-decrease (p**) in left ventricular fibrosis at 6 months in TD (n = 16) versus Ctrl (n = 20). In conclusion, targeting the extrinsic pathway with TD peptide at the onset of reperfusion provided long-term cardioprotection in a mouse model of myocardial IR injury by improving post-MI cardiac performance and preventing cardiac remodeling.

Cardiac mGluR1 metabotropic receptors in cardioprotection.

AIMS: In a previous study using a genome-wide microarray strategy, we identified metabotropic glutamate receptor 1 (mGluR1) as a putative cardioprotective candidate in ischaemic postconditioning (PostC). In the present study, we investigated the role of cardiac mGluR1 receptors during cardioprotection against myocardial ischaemia-reperfusion injury in the mouse myocardium. METHODS AND RESULTS: mGluR1 activation by glutamate administered 5 min before reperfusion in C57Bl/6 mice subjected to a myocardial ischaemia protocol strongly decreased both infarct size and DNA fragmentation measured at 24 h reperfusion. This cardioprotective effect was mimicked by the mGluR1 agonist, DHPG (10 µM), and abolished when glutamate was coinjected with the mGluR1 antagonist YM298198 (100 nM). Wortmannin (100 nM), an inhibitor of PI3-kinase, was able to prevent glutamate-induced cardioprotection. A glutamate bolus at the onset of reperfusion failed to protect the heart of mGluR1 knockout mice subjected to a myocardial ischaemia-reperfusion protocol, although PostC still protected the mGluR1 KO mice. Glutamate-treatment improved post-infarction functional recovery as evidenced by an echocardiographic study performed 15 days after treatment and by a histological evaluation of fibrosis 21 days post-treatment. Interestingly, restoration of functional mGluR1s by a PostC stimulus was evidenced at the transcriptional level. Since mGluR1s were localized at the surface membrane of cardiomyocytes, they might contribute to the cardioprotective effect of ischaemic PostC as other Gq-coupled receptors. CONCLUSION: This study provides the first demonstration that mGluR1 activation at the onset of reperfusion induces cardioprotection and might represent a putative strategy to prevent ischaemia-reperfusion injury.

Acute and long-term cardioprotective effects of the Traditional Chinese Medicine MLC901 against myocardial ischemia-reperfusion injury in mice.

MLC901, a traditional Chinese medicine containing a cocktail of active molecules, both reduces cerebral infarction and improves recovery in patients with ischemic stroke. The aim of this study was to evaluate the acute and long-term benefits of MLC901 in ischemic and reperfused mouse hearts. Ex vivo, under physiological conditions, MLC901 did not show any modification in heart rate and contraction amplitude. However, upon an ischemic insult, MLC901 administration during reperfusion, improved coronary flow in perfused hearts. In vivo, MLC901 (4 µg/kg) intravenous injection 5 minutes before reperfusion provided a decrease in both infarct size (49.8%) and apoptosis (49.9%) after 1 hour of reperfusion. Akt and ERK1/2 survival pathways were significantly activated in the myocardium of those mice. In the 4-month clinical follow-up upon an additional continuous per os administration, MLC901 treatment decreased cardiac injury as revealed by a 45%-decrease in cTnI plasmatic concentrations and an improved cardiac performance assessed by echocardiography. A histological analysis revealed a 64%-decreased residual scar fibrosis and a 44%-increased vascular density in the infarct region. This paper demonstrates that MLC901 treatment was able to provide acute and long-term cardioprotective effects in a murine model of myocardial ischemia-reperfusion injury in vivo.

PEGylation rate influences peptide-based nanoparticles mediated siRNA delivery in vitro and in vivo.

Small interfering RNAs (siRNAs) present a strong therapeutic potential because of their ability to inhibit the expression of any desired protein. Recently, we developed the retro-inverso amphipathic RICK peptide as novel non-covalent siRNA carrier. This peptide is able to form nanoparticles (NPs) by self-assembling with the siRNA resulting in the fully siRNA protection based on its protease resistant peptide sequence. With regard to an in vivo application, we investigated here the influence of the polyethylene glycol (PEG) grafting to RICK NPs on their in vitro and in vivo siRNA delivery properties. A detailed structural study shows that PEGylation did not alter the NP formation (only decrease in zeta potential) regardless of the used PEGylation rates. Compared to the native RICK:siRNA NPs, low PEGylation rates (≤20%) of the NPs did not influence their cellular internalization capacity as well as their knock-down specificity (over-expressed or endogenous system) in vitro. Because the behavior of PEGylated NPs could differ in their in vivo application, we analyzed the repartition of fluorescent labeled NPs injected at the one-cell stage in zebrafish embryos as well as their pharmacokinetic (PK) profile after administration to mice. After an intra-cardiac injection of the PEGylated NPs, we could clearly determine that 20% PEG-RICK NPs reduce significantly liver and kidney accumulation. NPs with 20% PEGylation constitutes a modular, easy-to-handle drug delivery system which could be adapted to other types of functional moieties to develop safe and biocompatible delivery systems for the clinical application of RNAi-based cancer therapeutics.

Down-regulation of the transcription factor ZAC1 upon pre- and postconditioning protects against I/R injury in the mouse myocardium.

AIMS: Myocardial infarction leads to heart failure and death. Ischaemic preconditioning (PreC) and postconditioning (PostC) reduce infarct size in animal models and human. Zac1 was identified as the only gene related to apoptosis and jointly down-regulated upon PreC and PostC. The aim of our study was to investigate the role of Zac1 down-regulation during ischaemia-reperfusion (I/R) in vivo. METHODS AND RESULTS: C57BL/6 mice were submitted to myocardial I/R injury, PreC, or PostC protocols. QPCR and immunochemistry showed that Zac1 expression was down-regulated both at the transcriptional and the protein levels upon PreC and PostC. Zac1(-/-) Knockout mice (n = 7) developed smaller infarcts (54%) than Zac1(+/+) littermates (n = 8) and decreased apoptosis (61.7%) in the ischaemic part of the left ventricle during I/R (Zac1(-/-), n = 6 vs. Zac1(+/+), n = 7; P = 0.0012). Mutants showed under control conditions a decrease of 53.9% in mRNA of Daxx, a pro-apoptotic protein playing a key role in I/R injuries (4.81 ± 0.77, n = 4 Zac1(-/-) mice vs. 10.44 ± 3.5, n = 7 Zac1(+/+) mice; P = 0.0121). CONCLUSION: Our study shows for the first time that Zac1 is down-regulated both at the transcriptional and protein levels upon PreC and PostC in wild-type mice. Moreover, inactivation of Zac1 in vivo is associated with a decreased amount of Daxx transcripts and, upon I/R injury, decreased infarct size and apoptosis. Altogether, our results show that Zac1 down-regulation plays a key role during cardioprotection against I/R injury and support the concept that cardioprotection regulates a network of interacting pro-apoptotic genes including Zac1 and Daxx.