Reperfusion therapy is currently the gold standard treatment for acute ischemic stroke. However, reperfusion injuries such as oedema and haemorrhagic transformation largely limit the use of this potent treatment to a narrow time window. Recently, transient receptor potential melastatin 4 (TRPM4) channel has emerged as a potential target for vascular protection in stroke management. Non-specificity and side effects are major concerns for current TRPM4 blockers. The present study was undertaken to develop a novel TRPM4 blocker for stroke management. We report the generation of a TRPM4-specific antibody M4P which binds to a region close to the channel pore. M4P could inhibit TRPM4 current and downregulate TRPM4 surface expression, therefore prevent hypoxia-induced cell swelling. In the rat model of 3-h stroke reperfusion, application of M4P at 2 h after occlusion ameliorated reperfusion injury by improving blood–brain barrier integrity, and enhanced functional recovery. Our results demonstrate that TRPM4 blockade could attenuate reperfusion injury in stroke recanalization. When applied together with reperfusion treatments, TRPM4 blocking antibody has the potential to extend the therapeutic time window for acute ischemic stroke.Electronic supplementary materialThe online version of this article (10.1007/s00424-019-02326-8) contains supplementary material, which is available to authorized users.

The transient receptor potential melastatin 4 (TRPM4) channel has been suggested to play a key role in the treatment of ischemic stroke. However, in vivo evaluation of TRPM4 channel, in particular by direct channel suppression, is lacking. In this study, we used multimodal imaging to assess edema formation and quantify the amount of metabolically functional brain salvaged after a rat model of stroke reperfusion. TRPM4 upregulation in endothelium emerges as early as 2 h post-stroke induction. Expression of TRPM4 channel was suppressed directly in vivo by treatment with siRNA; scrambled siRNA was used as a control. T2-weighted MRI suggests that TRPM4 inhibition successfully reduces edema by 30% and concomitantly salvages functionally active brain, measured by F-FDG-PET. These in vivo imaging results correlate well with post-mortem 2,3,5-triphenyltetrazolium chloride (TTC) staining which exhibits a 34.9% reduction in infarct volume after siRNA treatment. Furthermore, in a permanent stroke model, large areas of brain tissue displayed both edema and significant reductions in metabolic activity which was not shown in transient models with or without TRPM4 inhibition, indicating that tissue salvaged by TRPM4 inhibition during stroke reperfusion may survive. Evans Blue extravasation and hemoglobin quantification in the ipsilateral hemisphere were greatly reduced, suggesting that TRPM4 inhibition can improve BBB integrity after ischemic stroke reperfusion. Our results support the use of TRPM4 blocker for early stroke reperfusion.

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In stroke and other neurological diseases, Transient Receptor Potential Melastatin 4 (TRPM4) has been reported to cause oncotic cell death which is due to an excessive influx of sodium ions. Following stroke, hypoxia condition activates TRPM4 channel, and the sodium influx via TRPM4 is further enhanced by an increased TRPM4 expression. However, the effect of TRPM4 inhibition on oncotic cell death, particularly during the acute stage, remains largely unknown. Recently, we have developed a polyclonal antibody M4P that specifically inhibits TRPM4 channel. M4P blocks the channel via binding to a region close to the channel pore from extracellular space. Using M4P, we evaluated the acute effect of blocking TRPM4 in neurons, astrocytes, and vascular endothelial cells. In a rat stroke model, M4P co-localized with neuronal marker NeuN and endothelial marker vWF, whereas few GFAP positive astrocytes were stained by M4P in the ipsilateral hemisphere. When ATP was acutely depleted in cultured cortical neurons and microvascular endothelial cells, cell swelling was induced. Application of M4P significantly blocked TRPM4 current and attenuated oncosis. TUNEL assay, PI staining and western blot on cleaved Caspase-3 revealed that M4P could ameliorate apoptosis after 24 h hypoxia exposure. In contrast, acute ATP depletion in cultured astrocytes failed to demonstrate an increase of cell volume, and application of M4P or control IgG had no effect on cell volume change. When TRPM4 was overexpressed in astrocytes, acute ATP depletion successfully induced oncosis which could be suppressed by M4P treatment. Our results demonstrate that comparing to astrocytes, neurons, and vascular endothelial cells are more vulnerable to hypoxic injury. During the acute stage of stroke, blocking TRPM4 channel could protect neurons and vascular endothelial cells from oncotic cell death.

TRPM4 is a calcium-activated non-selective monovalent cation channel implicated in diseases such as stroke. Lack of potent and selective inhibitors remains a major challenge for studying TRPM4. Using a polypeptide from rat TRPM4, we have generated a polyclonal antibody M4P which could alleviate reperfusion injury in a rat model of stroke. Here, we aim to develop a monoclonal antibody that could block human TRPM4 channel. Two mouse monoclonal antibodies M4M and M4M1 were developed to target an extracellular epitope of human TRPM4. Immunohistochemistry and western blot were used to characterize the binding of these antibodies to human TRPM4. Potency of inhibition was compared using electrophysiological methods. We further evaluated the therapeutic potential on a rat model of middle cerebral artery occlusion. Both M4M and M4M1 could bind to human TRPM4 channel on the surface of live cells. Prolonged incubation with TRPM4 blocking antibody internalized surface TRPM4. Comparing to M4M1, M4M is more effective in blocking human TRPM4 channel. In human brain microvascular endothelial cells, M4M successfully inhibited TRPM4 current and ameliorated hypoxia-induced cell swelling. Using wild type rats, neither antibody demonstrated therapeutic potential on stroke. Human TRPM4 channel can be blocked by a monoclonal antibody M4M targeting a key antigenic sequence. For future clinical translation, the antibody needs to be humanized and a transgenic animal carrying human TRPM4 sequence is required for in vivo characterizing its therapeutic potential.