Selective blockade of Cav1.2 (α1C) versus Cav1.3 (α1D) L-type calcium channels by the black mamba toxin calciseptine.

L-type voltage-gated calcium channels are involved in multiple physiological functions. Currently available antagonists do not discriminate between L-type channel isoforms. Importantly, no selective blocker is available to dissect the role of L-type isoforms Cav1.2 and Cav1.3 that are concomitantly co-expressed in the heart, neuroendocrine and neuronal cells. Here we show that calciseptine, a snake toxin purified from mamba venom, selectively blocks Cav1.2 -mediated L-type calcium currents (ICaL) at concentrations leaving Cav1.3-mediated ICaL unaffected in both native cardiac myocytes and HEK-293T cells expressing recombinant Cav1.2 and Cav1.3 channels. Functionally, calciseptine potently inhibits cardiac contraction without altering the pacemaker activity in sino-atrial node cells, underscoring differential roles of Cav1.2- and Cav1.3 in cardiac contractility and automaticity. In summary, calciseptine is a selective L-type Cav1.2 Ca2+ channel blocker and should be a valuable tool to dissect the role of these L-channel isoforms.

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.

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.

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.

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.

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.