GLP-1 activates K_ATP channels in coronary pericytes as the effector of brain-gut-heart signalling mediating cardioprotection

Domain: neuroscience
Published: 2026-02-14
Editorial outcome: minor_revision

Abstract
Failure to reperfuse the coronary microvasculature (“no-reflow”) affects up to 50% of patients after unblocking a coronary artery that was causing ischaemia and acute myocardial infarction. This “no-reflow” is associated with reduced left ventricular ejection fraction, increased infarct size and death. We show that the incretin hormone GLP-1 (glucagon-like peptide 1) can be used to protect the heart after ischaemia by activating ATP-sensitive K+ channels on pericytes that constrict coronary capillaries. Coronary capillary dilation can be activated pharmacologically or by vagally-mediated GLP-1 release from the gut evoked by skeletal muscle ischaemia, and is abolished by block or genetic deletion of pericyte KATP channels. These results define a brain-gut-heart pathway mediating cardioprotection and suggest pharmacological therapies to reduce ischaemia-induced coronary no-reflow and improve post-infarct recovery.

Introduction
Partial or complete occlusion of a coronary artery, often following atherosclerotic plaque rupture, leads to cardiac ischaemia with ST-segment elevation on the ECG. Primary percutaneous coronary intervention (PPCI) is the preferred treatment and aims to restore blood flow in the affected artery and minimise damage to the heart muscle. However, this does not guarantee reperfusion of the downstream capillaries supplying the myocardium1,2 and a lack of capillary reperfusion—‘no-reflow’—affects up to 50% of patients3,4.

Infarct size (the amount of damaged heart tissue) is a predictor of adverse events and left ventricular remodelling after a heart attack. Cardioprotection studies frequently focus on reducing infarct size, but recent research suggests that the presence of no-reflow is an independent predictor of adverse outcomes and may be more significant than infarct size itself4. However, while many studies have examined infarct size, only a few have specifically addressed the microvascular occlusion causing no-reflow as a therapeutic target5,6. This is despite a meta-analysis7 demonstrating a robust relationship between microvessel occlusion and mortality or hospitalisation for subsequent heart failure within 1 year, with a 1% increase in vessel occlusion predicting 14 and 11% increases in death and hospitalisation, respectively. Understanding and targeting therapeutically the mechanism(s) leading to vessel occlusion may therefore significantly improve outcome after cardiac ischaemia.

We have previously shown that pericyte-mediated capillary constriction contributes significantly to no-reflow after cardiac ischaemia8. For the brain, where the same mechanism operates, this results from a rise of intracellular calcium concentration, [Ca2+], triggering pericyte contraction during ischaemia, with subsequent pericyte dysfunction or death maintaining the constriction when the arterial blood supply is restored9. Similar events occur following renal ischaemia10. Despite the critical role of pericyte-mediated capillary constriction not yet being broadly known in the cardiac community11, this finding has been independently reproduced for the heart12. We have shown that pericyte-mediated no-reflow can be significantly reduced by infusing adenosine at the time of reperfusion8, and it has also been suggested that remote ischaemic preconditioning (RPc, in which a limb is made transiently ischaemic before the cardiac ischaemia) has a cardioprotective action that is mediated by relaxation of pericytes12.

These results suggest that, if the mechanism by which RPc relaxes coronary pericytes could be defined, it might offer a pharmacological therapeutic approach superior to the use of adenosine, which lowers blood pressure and has other side effects. We have previously discovered that the cardioprotective action of RPc is mediated by a soluble factor released by activity in the motor fibres of the posterior gastric branch of the vagus nerve13,14, which was suggested15 to be glucagon-like peptide 1 (GLP-1).

We now demonstrate, by examining cardiac ischaemia in vivo and imaging pericyte-mediated coronary capillary constriction in the pressurised vasculature of excised ventricle, that the protective effect of RPc on coronary blood flow involves release of GLP-1, which relaxes coronary pericytes by activating KATP channels in these cells. The magnitude of the resulting blood flow increase and cardiac protection can be modulated by agents that alter trafficking of KATP channels to the surface membrane, including AMP kinase, NO and acetylcholine.

Results
GLP-1 receptors are on pericytes on coronary capillaries
The fluorescent GLP-1R-binding compound16 LUXendin555 bound to pericytes (and also some endothelial cells) in the mouse coronary vasculature, suggesting the presence of GLP-1 receptors on pericytes (Fig. 1a). Similarly, an antibody against GLP-1R also labelled mouse and cultured human coronary pericytes (Fig. 1b, c).

No-reflow after coronary ischaemia reflects capillary constriction
Occluding the rat left anterior descending coronary artery (LAD) for 45 min and then allowing 15 min reperfusion induced a long-lasting 40% decrease of coronary blood volume in the part of the left ventricle supplied by the LAD, as compared with sham-operated rats (p=2.2×10−7, Fig. 2a, c). This correlated with an increase in the percentage of capillaries blocked in the core of the area at risk (1–2 mm deep in the left ventricular wall: Fig. 2b, f) from 11.6 ± 1.5% (n=3 hearts) to 73.9 ± 9.0% (n=6, p=0.0001, one-way ANOVA with Tukey’s test). We have previously shown that this capillary block and reduction of perfused blood volume are produced by pericyte-mediated constriction8. As previously, the diameter of capillaries at pericyte somata was greatly reduced (by ~45%) after ischaemia and reperfusion (p=0.017 by one-way ANOVA in 48 capillaries in 3 sham-operated hearts and 48 capillaries in 3 hearts exposed to ischaemia-reperfusion, Fig. 2g).

RPc reduces no-reflow via GLP-1R-mediated pericyte relaxation
Remote ischaemic preconditioning prevented the ischaemia-evoked pericyte-mediated capillary constriction (p=0.009, Fig. 2g), and (presumably as a result) reduced capillary blockage from 73.9 ± 9.0% to 30.7 ± 5.0% of capillaries blocked (p=0.0016, in 5 hearts) compared to coronary ischaemia/reperfusion alone (Fig. 2d–f). RPc also restored the post-ischaemic perfused blood volume (p=1.7×10−7 compared with ischaemia/reperfusion alone) to a level similar to that seen for sham-operated mice (Fig. 2c).

Applying the GLP-1R blocker Exendin (9–39) (Ex9) intravenously, before the ischaemia, prevented RPc-mediated reduction of capillary block (resulting in 69.9 ± 6.1% capillary block, n=7 hearts, p=0.003 compared with RPc+ischaemia/reperfusion, Fig. 2f), and led to an ischaemia-evoked fall of perfusion volume similar to that seen with ischaemia/reperfusion alone (p=1.7×10−5 compared to RPc+Ex9 group, Fig. 2c). If applied with ischaemia/reperfusion in the absence of RPc, Ex9 had no effect (Fig. 2c, f), consistent with it acting solely by preventing the effects of RPc.

GLP-1R-evoked pericyte relaxation is via activation of KATP channels
To investigate the mechanism by which GLP-1 receptor activation protects against the ischaemia/reperfusion-induced pericyte contraction8,12 that blocks capillaries and reduces coronary blood flow, we used an ex vivo preparation. This comprised mouse free right ventricular wall, which we perfused through the vasculature by cannulating the right coronary artery (see ‘Methods’ and Fig. 3a) in addition to superfusing it with extracellular fluid. Capillary diameters were measured at pericytes somata (Fig. 3b). Oxygen and glucose deprivation (OGD) for 25 min evoked pericyte contraction and a constriction of capillaries, with a decrease of capillary diameter by 15.9 ± 1.7% (p=0.0002, at n=21 pericytes in tissue from 6 hearts, compared to the initial diameter, paired t-test, Fig. 3c). In continuous OGD conditions, the capillary diameter continued to gradually decline to 23.0 ± 2.6% less than the initial value (Fig. 3c).

Activating GLP-1R with Exendin-4 (Ex4, 100 nM) in the continued presence of OGD, starting after 25 min of OGD, relaxed pericytes within 25 min and dilated capillaries back to a diameter that was similar to the baseline value before ischaemia/reperfusion (Fig. 3c, constricted by 5.4 ± 1.0% at n=25 pericytes in 7 hearts, compared to 23.0 ± 2.6% in continuous OGD without Ex4 at n=21 pericytes in 6 hearts; p=6.1×10−6, hearts are the statistical unit, one-way ANOVA with Šídák’s test on final-minute values). However, in the presence of the ATP-sensitive K+ channel blocker glibenclamide (20 µM), the GLP-1R-mediated pericyte relaxation was abolished, and capillaries remained constricted by 19.0 ± 1.4% of the initial diameter (at n=23 pericytes in 6 hearts, p=1.2×10−4 compared to Ex4 only, Fig. 3c). This is consistent with RPc-evoked cardiac protection being blocked by glibenclamide in vivo17 and with GLP-1 activating KATP channels in smooth muscle cells18 and pancreatic beta cells19. Similarly, in the presence of the GLP-1R antagonist Exendin (9–39) (Ex9, 100 nM), Ex4 failed to relax cardiac pericytes (capillary diameters remained reduced by 16.2 ± 2.6% of the initial diameter at n=22 pericytes in 5 hearts, p=5.1×10−4 compared to Ex4 only, one-way ANOVA with Šídák’s test on final-minute values, Fig. 3d).

ATP-sensitive K+ channels in vascular mural cells (smooth muscle cells around arterioles and pericytes around capillaries) have previously been shown to play a major role in regulating vascular tone20. Ventricular cardiac pericytes express21 both Kir6.1 (a key component of ATP-sensitive K+ channels20,22) and SUR2 (an essential subunit of coronary pericyte KATP channels23). To confirm the involvement of ATP-sensitive K+ channels in the regulation of capillary diameter by pericytes, pericytes were preconstricted for 20 min with endothelin-1 (ET1, 20 nM, applied via the capillary lumen). ET1 decreased the capillary diameter at pericyte somata by 29.6 ± 3.3% (at 7 pericytes in 2 hearts after 20 min, Fig. 3e). Applying the KATP channel opener pinacidil22 (100 µM, in the capillary lumen) after terminating ET1 application resulted in a relaxation of pericytes and an increase of capillary diameter back to the baseline level within 20 min (Fig. 3e). In contrast, in tissue from mice with Kir6.1 knocked out in pericytes (see ‘Methods’), capillaries remained constricted in the presence of pinacidil (by 31.3 ± 2.9% of the initial diameter, at n=12 pericytes in 4 hearts, compared to 7.5 ± 2.8% in WT, p=0.007, t-test on final-minute values, Fig. 3e). Similarly, the reversal of OGD-induced capillary constriction by Ex4 (Fig. 3f) was inhibited when Kir6.1 was knocked out in pericytes, so that capillaries remained constricted after 50 min of OGD at 21.4 ± 1.1% of the initial diameter, at n=9 pericytes in 3 hearts, p=1.7×10−5 compared to WT, t-test on final-minute values (Fig. 3f). Consistent with these results in isolated ventricles suggesting a role for ATP-sensitive K+ channels in mediating the effects of GLP-1 during RPc, in mice with Kir6.1 knocked out in pericytes, RPc was not protective against ischaemia/reperfusion injury (Fig. 3g).

Modulation of the GLP-1-KATP pathway by AMP kinase, NO and ACh
The ability of GLP-1 released by RPc to relax cardiac pericytes and increase coronary blood flow presumably depends on the activation of KATP channels in the surface membrane of the pericytes, which will hyperpolarise the pericytes and decrease voltage-g voltage-gated Ca2+ entry. AMP-dependent protein kinase (AMPK) contributes to cell survival during cardiac ischaemia24 and is known to promote activation of KATP channels25, at least in part by increasing trafficking of these channels to the surface membrane26. We found that applying compound C (CC, 5 µM), an AMPK blocker27, inhibited the GLP-1R-mediated reversal of OGD-induced pericyte-mediated constriction of coronary capillaries (p=6.4×10−4 at 15 pericytes in 4 hearts, hearts are the statistical unit, one-way ANOVA with Šídák’s test on final-minute values, Fig. 4a). This is consistent with compound C inhibiting the cardiac protection provided by remote ischaemic conditioning, resulting in a larger infarct size28.

The nitric oxide synthase (NOS) blocker L-NAME (100 µM) did not affect the constriction evoked by OGD alone, but blocked the capillary dilation evoked by activating GLP-1 receptors in OGD (p=0.0037 at 19 pericytes in 3 hearts, one-way ANOVA with Šídák’s test on final-minute values, Fig. 4b). This is consistent with NOS block preventing the beneficial effect of RPc on no-reflow in vivo29. NO might be expected to dilate blood vessels directly by relaxing capillary pericytes or preventing them constricting9, but this would be expected to occur even in the absence of GLP-1 receptor activation, predicting a constriction when L-NAME is applied, which is not seen in Fig. 4b. Instead, it is possible that the effect of NOS block reflects a decrease of trafficking of KATP channels to the membrane because NO has been reported to activate AMPK30,31. Atropine (1 µM) also did not affect the constriction evoked by OGD alone, but blocked the capillary dilation evoked by activating GLP-1 receptors in OGD (p=0.0033 at 25 pericytes in 5 hearts, one-way ANOVA with Šídák’s test on final-minute values, Fig. 4c), consistent with atropine blocking RPc-evoked cardiac protection after ischaemia15. Again, the lack of effect on the OGD-evoked constriction but suppression of the Ex4-evoked dilation might reflect acetylcholine promoting trafficking of KATP channels to the membrane, because the type 3 muscarinic receptors that are cardioprotective in RPc15 are reported to increase activation of AMPK32,33.

To assess whether AMPK does indeed regulate KATP channel trafficking, we used an antibody to Kir6.1, which forms an essential subunit of coronary pericyte KATP channels23 (Fig. 4d, e). In mouse hearts subjected to 40 min LAD ligation followed by 10 min reperfusion, the peak fluorescence intensity of Kir6.1 labelling shifted inside the cell in animals administered with the AMPK inhibitor compound C (CC, 10 mg/kg i.p.), consistent with AMPK mediating insertion of these channels into the surface membrane (Fig. 4f, g). This occurred both in ischaemia/reperfusion (1.11 ± 0.03 µm in the IR+CC group compared to 0.75 ± 0.03 µm in the IR control; p=0.001, one-way ANOVA with Šídák’s test), and in the animals pre-treated with Exendin-4 (Ex4, 5 µg/kg i.p.; 1.13 ± 0.06 µm in the IR+CC+Ex4 group vs 0.67 ± 0.07 µm in the IR+Ex4 group; p=0.0004).

Methods
All experiments were performed in accordance with the European Commission Directive 2010/63/EU (European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes) and the UK Home Office Scientific Procedures Act (1986), with project approval from the University College London Animal Welfare and Ethical Review Body (Bloomsbury Campus).

Animals were group-housed and kept on a 12 h light/dark cycle at 24 °C ambient temperature and 55-65% relative humidity (mice) or at 22 °C ambient temperature and 45–65% relative humidity (rats). Normal rodent chow (Teklad 2018 or 7912, Envigo) and tap water were available ad libitum.

The experiments involving human cells comply with the guidelines of the Declaration of Helsinki. Discarded right ventricular tissue from congenital heart defect surgery (pulmonary valve repair) was obtained with patients’ parents or legal guardians informed consent (ethical approval 15/LO/1064 from the North Somerset and South Bristol Research Ethics Committee).

Animal preparation
Adult male Sprague-Dawley rats weighing 220–250 g or transgenic mice (see below) were anaesthetised with pentobarbital sodium (induction 60 mg/kg i.p. for rats, 90 mg/kg i.p. for mice; maintenance 10–15 mg/kg/h i.v.). The right carotid artery and jugular vein were cannulated for blood pressure (BP) measurement and drug administration, respectively. Stable BP and heart rate were maintained, and level of anaesthesia was monitored by paw pinch response. The trachea was cannulated, and animals were mechanically ventilated with room air using a positive pressure ventilator (Harvard rodent ventilator, tidal volume of 1 ml/100 g of body weight, ventilator frequency ∼60 strokes min−1 for rats, ∼150 strokes min−1 for mice). Arterial BP and ECG were recorded using a Power 1401 interface and Spike2 software (Cambridge Electronic Design), and body temperature was maintained at 37.0 ± 0.5 °C. The heart was exposed via a left thoracotomy, and a 4-0 (rats) or 8-0 (mice) Prolene suture was passed around the left anterior descending (LAD) coronary artery to induce a temporary occlusion. Rats were subjected to 45 min of LAD ligation followed by 15 min of reperfusion to study no-reflow phenomena. To determine the effects of Exendin-4 (a GLP-1R agonist) and Compound C (an AMPK inhibitor, 6-[4-(2-Piperidin-1-yl-ethoxy)-phenyl]-3-pyridin-4-yl-pyyr-azolo[1,5-a] pyrimidine) on the localisation of KATP channels in ischaemia, mice were subjected to 40 min of LAD ligation followed by 10 min of reperfusion, with Compound C (Dorsomorphin 2HCl, APExBIO; 10 mg/kg, i.p., administered 1 h before LAD ligation) and/or Exendin-4 (Tocris; 5 µg/kg i.p., given 30 min before LAD ligation). To determine the extent of myocardial ischaemia/reperfusion damage, mice were subjected to 30 min of LAD ligation followed by 90 min reperfusion. Successful LAD occlusion was confirmed by paling of the myocardial tissue distal to the suture, elevation of the ST-segment in the ECG, and an immediate 15–30 mmHg fall in the BP. To establish remote ischaemic preconditioning, blood supply to the lower limbs was interrupted for 15 min by placing clamps on both femoral arteries at the proximal level ∼1 cm below the inguinal ligament 25 min before the onset of coronary ischaemia. The sham-RPc procedure involved dissection of both femoral arteries without occlusion. GLP-1R antagonist Exendin (9–39) (Tocris, Ex(9–39), 50 µg/kg, i.v.) or vehicle control was administered 40 min before the onset of coronary ischaemia. Control (sham-operated) animals underwent the same procedures, except that after the suture was passed around the LAD coronary artery, it was not drawn tight to occlude the vessel. Ischaemia and sham animals were alternately interleaved.

Infarct size measurement
In mice, at the end of reperfusion period, the LAD was re-ligated, and 5% Evans Blue dye solution (100 µl) was infused via jugular vein to determine area at risk. The animal was then given an anaesthetic overdose (pentobarbital sodium, 200 mg/kg, i.v.), the heart was excised, briefly frozen and sectioned into 5–6 transverse slices from apex to base. The area at risk was demarcated by the absence of Evans Blue staining. Heart slices were then incubated with 1% 2,3,5-triphenyltetrazolium chloride (TTC) in Tris buffer (pH 7.4) for 15 min at 37 °C and fixed in 4% formalin for 24 h. Viable myocardium is stained red by TTC, whereas necrotic myocardium appears white. The area at risk and the necrotic area were determined by computerised planimetry, normalised to the weight of each slice, with the degree of necrosis (i.e. infarct size) expressed as the percentage of area at risk, as described13,14.

Animal perfusion and tissue processing for imaging
After the ischaemia/reperfusion procedure, rats were given an overdose of pentobarbital sodium (200 mg/kg, i.v.) and transcardially perfused with 200 ml saline followed by 200 ml 4% paraformaldehyde (PFA) for fixation and then by a solution of 5% gelatine (Sigma-Aldrich, G2625) containing 0.25% FITC-albumin conjugate (Sigma-Aldrich, A9771) for visualisation of the perfused coronary microvasculature. The initial perfusion with calcium-free saline ensures that the heart is stopped in diastole, which is also evident from the large volume of left ventricle visible at cross-section. The hearts were then fixed overnight in 4% PFA, and 150 µm transverse sections were obtained for immunofluorescence staining. The labelling was done using anti-NG2 antibodies (Merck Millipore, AB5320, 1:200) for pericytes and isolectin B4-Alexa Fluor 647 (Molecular Probes, I32450, 1:200) for the capillary basement membrane.

Mice used in Kir6.1 membrane localisation study were given an anaesthetic overdose (pentobarbital sodium, 200 mg/kg, i.p.) at the end of 10 min reperfusion and transcardially perfused with 20 ml saline, followed by 5 ml 4% PFA for fixation, the hearts were fixed overnight in 4% PFA and sliced into 150 µm transverse sections. The labelling was done using anti-PDGFRß antibodies (R&D Systems, AF1042, 1:50) for pericytes, isolectin GS-IB4-biotinylated (Life Technologies, 121414, 1:200) for capillary basement membrane, and anti-Kir6.1 (Alomone Labs, APC-105, 1:100) to localise the KATP channels, following heat-induced epitope retrieval (using 10 mM sodium citrate buffer at pH 6.0 for 20 min at 80 °C).

To visualise GLP-1Rs in mouse pericytes, wild-type mice were injected with Luxendin 555 probe subcutaneously65 (100 pmol/g body weight) and 2 h after Luxendin 555 administration, given an overdose of pentobarbital sodium (200 mg/kg, i.v.) and perfused-fixed (as described above). Tissue was processed as described above; the pericytes and blood vessels were labelled using anti-NG2 antibodies (Abcam, ab275024, 1:200) and isolectin B4-Alexa Fluor 488 (Molecular Probes, I21411, 1:200) respectively (Fig. 1a). In a separate set of experiments, heart tissue from NG2-dsRed mice was processed as described above and stained with anti-GLP-1R antibody (Abcam, ab218532, 1:200) following heat-induced epitope retrieval as above.

To visualise GLP-1Rs in human pericytes, pericytes isolated from cardiac surgery waste tissue (cell isolation methods and characterisation as described previously66) were cultured to semi-confluency, fixed with 4% PFA for 20 min and labelled with the anti-human PDGFRβ antibody (Santa Cruz, sc-374573, 1:50) and Fluorescent Exendin-4 (Fluorescein-Trp25-Exendin-4, FLEX; Eurogentec, AS-63899, 100 nM).

Imaging vessels after in vivo ischaemia experiments
Images of the left ventricular capillary bed in rats were acquired using laser scanning confocal microscopy (Zeiss LSM700) and analysed using FIJI software (ImageJ 1.53c, NIH). To quantify blood volume across the left ventricular myocardium, low-power z-stack images were taken of an entire transverse section of each heart (using a 1× air objective), maximum intensity projected. 12 regions of interest (ROI) were drawn clockwise around the left ventricle (when looked at from above) from the mid-point of the septum8 (as in Fig. 2a, d), and the mean intensity of FITC-albumin signal was recorded for each ROI and normalised to the highest intensity measured in any ROI. These data were averaged over hearts and renormalised so that the mean value in positions 1–3 of Fig. 2c was 1. This signal is assumed to be proportional to the volume of blood perfusing the myocardium. To compare perfusion in the ischaemic zone, we used the plotted values from ROIs 6 to 10.

For quantification of the percentage of capillaries that were perfused, three randomly selected regions of the outer third of the myocardium of the anterior left ventricular wall were imaged (with the capillaries running parallel to the surface of the tissue slice), as this region of myocardium consistently included the ischaemic area (which showed visible pallor and oedema of the myocardium). A line perpendicular to the capillaries was drawn in the central area of the image (±50 µm), the numbers of the blocked and perfused capillaries were calculated, and the percentage of the blocked capillaries was averaged per heart. The person quantifying the images was blinded to the condition that the heart was exposed to. Blockages of flow in the ischaemic area at risk were identified by abrupt terminations in FITC-albumin signal (Fig. 2b, e).

Transgenic mice
Some experiments used NG2-dsRed mice67, kindly provided by Akiko Nishiyama (University of Connecticut) via Dirk Dietrich (University of Cologne), in which pericytes fluoresce red, facilitating their identification. Pericyte-specific Kir6.1 knockout mice were generated by crossing tamoxifen-inducible NG2-CreERT2 knock-in mice68 (kindly donated by Frank Kirchhoff, University of Saarland) and floxed (flx) Kir6.1 mice (kindly donated by Andrew Tinker, QMUL) to allow deletion of Kir6.1 in NG2-expressing pericytes after oral gavage of tamoxifen. Tamoxifen dissolved in corn oil was given at 100 mg/kg body weight by oral gavage once per day for 3 consecutive days to adult >P21 mice. Experiments were performed from 2 weeks after tamoxifen administration.

Genotypes were identified by polymerase chain reaction on genomic DNA from ear snips of NG2-cre+/−/Kir6.1flx/flx, and littermate control animals NG2-cre+/−/Kir6.1−/− using the following primers: for NG2-Cre, WT sense AGAGATCCTGTCCACAGAGTTCT and antisense GCTGGAGCTGACAGCGGGTG and generic Cre, sense CACCCTGT-TACGTATAGCCG and antisense GAGTCATCCTTAGCGCCGTA (NG2 WT band 557 bp, Cre band 330 bp); for Kir6.1, sense GAGTCTTAACTCAGTTCTGGAGGACCAACA and antisense AGCGAAGAAAACTGCTTCC TGTTCATTAAG (Kir6.1 WT band 474 bp, flx band 600 bp).

Live ex vivo tissue imaging
Adult mice (P100-P120) of both sexes were used in the study. The heart of a mouse freshly euthanised with isoflurane overdose was surgically removed and placed into ice-cold Tyrode’s solution containing the following components (mM): 142.5 NaCl, 2.5 KCl, 0.5 MgCl2, 0.33 NaH2PO4, 5 glucose, 2 Na lactate, 0.1 Na pyruvate, 10 Hepes and 1.8 CaCl2 (pH set to 7.4 with NaOH). After being cleaned of connective tissues, the heart was transferred into a Sylgard-coated prechilled (4 °C) chamber containing Tyrode’s solution and pinned down onto the Sylgard layer of the chamber. The right ventricular wall was dissected, and the right coronary artery exposed and cannulated using a fire-polished bent glass pipette prefilled with Tyrode’s solution. The right ventricular wall was used for imaging as it was thinner than the left ventricular wall, which made it easier to flatten under the objective and ensured full immersion of the tissue in the bath superfusion solution. The pipette used to cannulate the artery was then connected to a fluid-filled line, and intralumenal pressure was created by elevating a cylinder connected to the line with Tyrode’s solution to a height equivalent to 80 mmHg (109 cm H2O) (Fig. 3a). The vascular lumen perfusate was Tyrode’s solution (composition as above). The bath superfusate contained a physiological saline solution (PSS; containing (mM) 114.5 NaCl, 2.5 KCl, 1.2 MgSO4, 1.2 NaH2PO4, 24 NaHCO3, 10 glucose, 1 Na lactate and 1.8 CaCl2) was maintained at 32–34 °C with a heater and temperature controller and bubbled with 5% CO2, 20% O2 and 75% N2 to maintain a pH of 7.4. The bath solution was perfused at ∼5 ml/min. Experimental measurements were started after ∼30–60 min stabilisation. To visualise the vessels, Alexa Fluor 488-conjugated WGA (Thermo Fisher Scientific, W11261; 20 µg/mL, 30 min) was included in the luminal buffer during the stabilisation period. For imaging of the tissue from animals not expressing dsRed in pericytes, an additional step of 20 min incubation with isolectin B4-Alexa Fluor 594 (Molecular Probes, I21413, 10 µg/ml in superfusate solution) was employed. To block cardiac myocyte contraction, a modulator of cardiac myosin, Mavacamten69 (MYK-461, 10 µM, MedChemExpress), was added to the luminal solution.

Two-photon excitation used a Newport-Spectra Physics Mai Tai Ti:Sapphire Laser pulsing at 80 MHz, and a Zeiss LSM780 (Oberkochen, Germany) microscope with a 20× water immersion objective (NA 1.0). Fluorescence was excited using 920 nm wavelength for DsRed, and 820 nm for Alexa Fluor 488. Mean laser power under the objective was <35 mW. Z-stack images were acquired every 2 min. Baseline for each condition was 5 min. The ex vivo model of tissue ischaemia was induced by oxygen/glucose deprivation (OGD)—the tissue was exposed to perfusate solution saturated with 95% N2/5% CO2 gas mixture containing 10 mM sucrose instead of glucose. The period of OGD lasted for 25 min, after which GLP-1R agonist was added to the perfusate solution, and OGD continued for another 25 min. Exendin-4 (Ex4; 100 nM, Tocris) was used as a GLP-1R agonist. N(gamma)-nitro-L-arginine methyl ester (L-NAME; 100 µM, Sigma) was used to inhibit nitric oxide synthesis. Atropine sulphate (1 µM, Sigma) was used to block muscarinic receptors. Compound C was used to inhibit AMPK (5 µM, APExBIO). KATP channels were activated with pinacidil (100 µM, AOBIOUS) and blocked with glibenclamide (20 µM, Santa Cruz). Endothelin-1 (20 nM) was applied either through the vascular lumen or in the superfusate, and the constriction evoked did not differ significantly (22.3 ± 7.3% constriction when ET1 was applied in the lumen solution compared to 24 ± 5.4% constriction when ET1 was applied in the bath solution). For mechanistic experiments, ET1 and pinacidil were applied via the capillary lumen, while all other drugs were applied in the superfusion solution.

Image analysis after live ex vivo tissue imaging and immunofluorescence studies
Pericytes were identified by their morphology (spatially isolated cells located outside capillaries) as confirmed by IB4 labelling, or by antibody labelling for their characteristic markers NG2 or PDGFRβ, or by expression of dsRed under the NG2 promoter.

Capillary diameters and Kir6.1 immunofluorescence intensity profiles were analysed using FIJI software (ImageJ 1.53c, NIH) by a person blinded to the experimental condition. Vessel diameter was defined using a line drawn across the vessel as the width of the intraluminal dye fluorescence (WGA-Alexa Fluor 488) at pericytes somata.

To assess the Kir6.1 immunofluorescence intensity profiles, PDGFRβ immunostaining was used as a reference point to localise the cell surface. Between 3 and 5 zoomed-in images per heart were taken to achieve a total number of 5-10 pericytes per heart, and all imaged pericytes were analysed. The selection of the membrane area was based on clearly visible PDGFRβ signal, on the outer side of the capillary. 10 regions of interest (ROI) per pericyte were drawn as lines in the PDGFRβ channel, starting from the cell surface and directed towards the centre of the cell. The ROIs were then transferred to the Kir6.1 channel and the fluorescence intensity profile of each line was measured and averaged over all the lines per pericyte. The values of the resulting distance from the cell membrane to the peak Kir6.1 immunofluorescence intensity were averaged for each heart and then used for the analysis (Fig. 4).

Statistics and reproducibility
Statistical analyses were performed using GraphPad Prism 9 software. Data normality was assessed with Shapiro-Wilk tests. Comparisons of normally distributed data were made using two-tailed Student’s t-tests. Equality of variance was assessed with an F test, and Welch’s t-test was used when variances were unequal. P values were obtained using one-way ANOVA or t-tests and adjusted for multiple comparisons with the Šídák or Tukey method, as appropriate. Corrected p values < 0.05 were considered statistically significant. Numbers of animals are denoted as N, and numbers of pericytes or capillaries as n. In all statistical tests, animals/hearts (N) were the statistical unit. Data are presented as mean ± SEM.

Where representative examples are shown from immunostaining images, the staining was repeated independently at least twice with similar results. Specifically, for Fig. 1a, the LUXendin probe was administered and subsequently visualised in two mice. For Fig. 1b, hearts from two mice were stained with anti-GLP-1R antibodies. For Fig. 1c, cultured human cardiac pericytes from two different patients were labelled with Fluorescein-Trp25-Exendin-4 (FLEX) to visualise GLP-1Rs. For Fig. 2b, e, three areas from 3 to 5 hearts per group were imaged on different experimental days. For Fig. 4d, three hearts per group (WT and KO) were stained with anti-Kir6.1 antibody. For Fig. 4e, 3–5 hearts per group were imaged and analysed.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This an interesting and well-written manuscript providing evidence in support of the idea that remote preconditioning (RPc) protects the heart after ischemia via GLP-1 activation of KATP channels on pericytes, leading to capillary dilation and protection against the no-reflow phenomenon. The data appear to be of high quality and, at face value, provide compelling support. I have a few comments, some relatively minor.\n1. Many channels are indirectly regulated by ATP. To be consistent with the literature, it would be better to use the generally accepted term: ATP-sensitive potassium channels, rather than ATP-gated.\n2. Figs. 2b and d, do not look like blowups of a and c. Are the images inverted somehow? How was it decided where to draw the lines in Fig 2 b and d? Moving them up or down could give very different results in 2f.\n3. Antibodies against KATP channel subunits have been notoriously unspecific. The authors could carry out control experiments on Kir6.1 knockout tissue to confirm the identified channels in Fig. 4.\n4. How strong is the evidence for the gut/CNS part of the proposed model in Fig. 5? GLP1 is synthesized in L-cells, but also in alpha cells. Could it be made elsewhere, and are the RPc effects lost with spinal lesion, for example?", "reviewer_2": "In the present study Mastitskaya and colleagues studied whether coronary microvascular pericytes participate in the previously reported protective vasodilating effects of “endogenous” glucagon-like peptide (GLP-1) following remote ischemic preconditioning. In analogy to these previous studies from Basalay MV, et al. (“Glucagon-like peptide-1 (GLP-1) mediates cardioprotection by remote ischemic conditioning. Cardiovasc Res. 2016;112:669-676. doi: 10.1093/cvr/cvw216”) here the authors used a rat model of myocardial infarction (coronary artery occlusion followed by reperfusion). In such anesthetized rats, remote ischemic preconditioning (RPc) was induced by 15 min occlusion of femoral arteries applied 25 min prior to the myocardial ischemia. The present studies reproduce the effects observed by Basalay et al. showing that RPc attenuates the ischemia-induced reduction of coronary flow, and that this cardioprotective vasodilating effect is attenuated following blockade of GLP-1 receptors (GLP-1R) by Exendin(9-39) (Ex9, administered through systemic i.v. injection before LAD ligation). The novel aspect of the 1st part of the present studies by Mastitskaya et al. is that GLP-1R – mediated coronary vasodilatation involves fine myocardial capillaries covered by NG2+ pericytes (confocal microscopy studies on cardiac sections, ex vivo). These results are in line with recent studies in brain microcirculation (Chen Y, et al. Exendin-4 improves cerebral ischemia by relaxing microvessels, rapidly increasing cerebral blood flow after reperfusion. Basic Res Cardiol. 2025;120:423-441. doi: 10.1007/s00395-025-01096-y).\nIn the 2nd part of the manuscript Mastitskaya and colleagues performed pharmacological studies in isolated mouse right ventricular free wall preparations to dissect the intracellular mechanisms of the relaxing effects of GLP-1. Capillary diameters “at pericyte somata” were measured under ischemic conditions (mimicked by oxygen and glucose deprivation) or after infusion of endothelin-1, a potent microvascular constrictor (2-photon microscopy studies). The vasodilating effect of the GLP-1R agonist Exendin-4 was studied in the presence of specific activators/inhibitors of ATP-sensitive potassium channels (KATP) or AMPK inhibitors. The design of these experiments was based on previously published studies showing that GLP-1 stimulates KATP-channels in vascular smooth muscle cells, as one mechanism mediating its vasodilating effect (Green BD, et al. GLP-1 and related peptides cause concentration-dependent relaxation of rat aorta through a pathway involving KATP and cAMP. Arch Biochem Biophys. 2008;478:136-42). In line with these previous observations, the present studies show that GLP-1R – mediated coronary vasodilatation involves the hyperpolarizing activation of KATP channels and the membrane insertion of these channels triggered by AMPK. Here, the involvement of pericytes is demonstrated by using right ventricles from mice with Kir6.1 deletion in pericytes (Kir6.1 is a subunit of the KATP channel).\nOverall, this study is potentially interesting, although several main informations were already published before by other groups (the GLP-1/GLP-1R pathway participates in protective heart and brain ischemic preconditioning; in vascular cells (and also in cardiomyocytes) this pathway involves the activation of KATP channels).\nFor this reviewer, the most interesting part of the manuscript would be the clear demonstration of a brain-gut-cardiac pericytes axis (as stated in the manuscript title), but this axis is not clear. My major comments are:\n- The manuscript (and previous studies) involve GLP-1 as endocrine circulating hormone. How are the plasma GLP-1 levels in rats/mice after ischemic preconditioning? Are they higher as for instance after a carbohydrate rich meal?!\n- Are intestinal enteroendocrine K cells the only cells involved as source of GLP-1? (GLP-1 is also produced in other locations)\n- To what extend is the myocardial “capillary dilatation at pericyte sites” dependent on the vasodilatation and enhanced flow through the feeding proximal arteries? (the manuscript repeatedly mentions “pericyte relaxation” but only shows the diameter of capillaries).\n- A more direct and clear “dissection of the role of pericytes in cardiac “no-reflow” and ischemic preconditioning could be achieved by generating mice with pericyte-restricted GLP-1R deletion (floxed mice are available; see Kajitani Y, et al. High frequency of germline recombination in Nestin-Cre transgenic mice crossed with Glucagon-like peptide 1 receptor floxed mice. PLoS One. 2023;18:e0296006. doi: 10.1371/journal.pone.0296006). Intravenous, systemic delivery of Ex9 of course blocked GLP-R1 on other types of cells (in and outside the heart), which might be involved in the regulation of coronary perfusion.\n- In figure 1C, the GLP-1R seems to be mostly in the cytosol of pericytes!\n- Finally, it is not clear to this reviewer how/why the GLP-1/GLP-1R pathway activates KATP channels in VSMCs, pericytes (present studies) and cardiomyocytes, while blocking these channels in pancreatic beta cells (Leech CA, et al. Signal transduction of PACAP and GLP-1 in pancreatic beta cells. Ann N Y Acad Sci. 1996;805:81-92). The reference 19 cited in the manuscript in fact describes that the GLP-1/GLP-1R pathway normally blocks KATP channels in beta cells (as is physiologically meaningful), which was only reverted into an “activation” under very artificial conditions.", "reviewer_3": "The authors provide an interesting set of experiments utilising immunofluorescence labelling, antagonists and KO mice to explore the mainly sub cellular signalling pathways that are involved in pericyte constriction mediating no reflow in response to ischaemia (or OGD). They propose that the brain-gut-heart axis via vagally mediated GLP-1 release is important in signalling RIPc protective vasodilatory signals to the heart pericyte with sub cellular signalling that are dependent on KATP channel membrane expression / opening via a AMP kinase dependent signalling - that result in relaxation.\nNo reflow is an important clinical phenomenon and further understanding the pathophysiology is needed to prevent it occurring and to discover new treatments\nGeneral comments:\n1. The experiments are elegantly demonstrated and illustrated with compelling results. However, the focus is mainly on sub cellular signalling which has been previously demonstrated - see work by Kloner RA and others. However, the data on pericytes are novel and thoroughly explored with a logical series of experiments.\n2. The title of the paper and Figure 5a propose the brain-gut-heart signalling, although this was not specifically assessed in whole animal experiments and therefore possibly the title and conclusions drawn in the discussion over-reach, based on the actual data provided. Further whole animal experiments with possible nerve truncation or in vivo blood sampling for GLP-1 would strengthen the manuscript and support the brain-gut-heart signalling hypothesis.\n3. GLP-1 is inherently unstable with a 2min half life due to degradation by DPP-4. Have the authors looked at potentiating the effect with DPP-4 inhibition, rather than only blocking it? How did they know that local GLP-1 released from the gut reaches the heart?\n4. The Kir6.1 trafficking immunofluorescence intensity profiles measured using imageJ (Figure 4) needs further explanation/ verification with control studies. The images of pericytes/ selection of membrane were presumably not random and are possibly open to bias.\n5. The methodology has been adequately described and the reported. However, the assessment of pericyte \"opening\" is only done visually (microscopically) (%open/ measured diameters), rather than employing other methods e.g. haemodynamic assessment of resistance that could add weight to the authors findings. Furthermore, visual assessment again may be open to reporting error/ selection bias.\n6. The translation of these studies to Man has been challenging - with RIPc not showing improvement in microcirculatory function and mixed results in cardio protection although GLP is a vasodilator. This dichotomy between animal studies and studies in Man deserves further attention in the discussion.\nMinor\n1. Clinically important no-reflow is relatively rare (2% rather than 50% as stated) after coronary stenting - please revise introduction\n2. There are typos in Figure 4: OGG rather than OGD"}, "author_response": "MS # NCOMMS-25-56046B\nResponses to referees’ comments\nWe are grateful for the constructive comments of the Reviewers on our original\nsubmission to the Nature Communications and have taken full account of the criticisms\nraised. We are delighted to have an opportunity to re-submit our work. We now provide\na full response to the reviewers’ comments as well as a thoroughly revised manuscript.\nBelow we state the criticisms (\"critique\") and then provide our responses.\nREVIEWER COMMENTS\nReviewer #1\nThis an interesting and well-written manuscript providing evidence in support of the\nidea that remote preconditioning (RPc) protects the heart after ischemia via GLP-1\nactivation of KATP channels on pericytes, leading to capillary dilation and protection\nagainst the no-reflow phenomenon. The data appear to be of high quality and, at face\nvalue, provide compelling support. I have a few comments, some relatively minor.\nResponse: We would like to thank the reviewer for taking the time to review our\nmanuscript and for their overall positive assessment of our work.\nCritique 1:\nMany channels are indirectly regulated by ATP. To be consistent with the literature, it\nwould be better to use the generally accepted term: ATP-sensitive potassium channels,\nrather than ATP-gated.\nResponse: We thank the reviewer for pointing this out. We have now revised the text\nand use the term ATP-sensitive potassium channels throughout.\nCritique 2:\nFigs. 2b and d, do not look like blowups of a and c. Are the images inverted somehow?\nResponse: The areas outlined in red on panels a and c are just a schematic\nrepresentation of the area used for high-power analysis of capillary blockages. The\ndimensions of the imaged area in 2b and 2d is only 309µm x 309µm while the area\noutlined in red on 2a and 2e is approximately 1.5mm x 1.5mm. We now revised the\nfigure legend to describe this more precisely, as follows (page 2):\n(a) Low-power view of heart cross-section after ischaemia/reperfusion (b) a\nzoomed-in image taken from part of the area outlined red in (a), with a line\ndrawn across capillaries to calculate the ratio of blocked to total capillaries.\nWe also corrected the scale bar in figure 2c which should have been 50µm, not 20µm.\nHow was it decided where to draw the lines in Fig 2 b and d? Moving them up or down\ncould give very different results in 2f.\nResponse: Three randomly selected zoomed-in images per heart were taken from the\nischaemic area (part of which is outlined in red), with the capillaries running parallel to\nthe surface of the tissue slice. A line perpendicular to the capillaries was drawn in the\ncentral area of each randomly placed image (±50 µm), the numbers of the blocked and\nperfused capillaries were calculated, and the percentage of the blocked capillaries was\naveraged per heart. The person performing the analysis was blinded to the condition.\nline up or down by 50 µm would change the measured fraction of vessels blocked from\n65% to 64% or 74% (for IR, Fig. 2b) or from 28% to 36-38% (for IR+RPc, Fig. 2e):\nCritique 3: Antibodies against KATP channel subunits have been notoriously unspecific.\nThe authors could carry out control experiments on Kir6.1 knockout tissue to confirm\nthe identified channels in Fig. 4.\nResponse: We performed all the necessary negative control staining and now provide\nthe images of knockout tissue (Fig. 4d).\nCritique 4: How strong is the evidence for the gut/CNS part of the proposed model in\nFig. 5? GLP1 is synthesized in L-cells, but also in alpha cells. Could it be made elsewhere,\nand are the RPc effects lost with spinal lesion, for example?\nResponse: GLP-1 is indeed also produced in the alpha-cells, in type II taste receptor\ncells (the GLP-1R is also there - thus, GLP-1 is supposed to modulate taste), in the\nolfactory bulb and in the PPG neurons in the brainstem, but most likely none of these\nsources provide quantitively enough GLP-1 to increase (peripheral) circulating levels to\nthe point that GLP-1 can have systemic effects. The centrally produced GLP-1 has only\nbeen shown to have effects within the CNS, mostly associated with appetite\ncontrol (particularly satiation after large meals) as well as fluid homeostasis. The\nevidence for the vagally mediated release of GLP-1 from the gut stems from our\nprevious publication (Mastitskaya et al., PlosONE 2016, doi:\n10.1371/journal.pone.0150108) where we performed a series of selective transections\nof different branches of the vagus, and demonstrated that the posterior gastric branch\nof the subdiaphragmatic vagus is responsible for the release of the molecular factor\nmediating RPc. This is all stated in the Introduction to the paper on page 1 (right\ncolumn, lines 49-53) and Discussion (lines 242-244 and 269-272) of the manuscript.\nIn the present study Mastitskaya and colleagues studied whether coronary\nmicrovascular pericytes participate in the previously reported protective vasodilating\neffects of “endogenous” glucagon-like peptide (GLP-1) following remote ischemic\npreconditioning. In analogy to these previous studies from Basalay MV, et al.\n(“Glucagon-like peptide-1 (GLP-1) mediates cardioprotection by remote ischemic\nconditioning”. Cardiovasc Res. 2016;112:669-676. doi: 10.1093/cvr/cvw216) here the\nauthors used a rat model of myocardial infarction (coronary artery occlusion followed\nby reperfusion). In such anesthetized rats, remote ischemic preconditioning (RPc) was\ninduced by 15 min occlusion of femoral arteries applied 25 min prior to the myocardial\nischemia. The present studies reproduce the effects observed by Basalay et al. showing\nthat RPc attenuates the ischemia-induced reduction of coronary flow, and that this\ncardioprotective vasodilating effect is attenuated following blockade of GLP-1 receptors\n(GLP-1R) by Exendin(9-39) (Ex9, administered through systemic i.v. injection before\nLAD ligation). The novel aspect of the 1st part of the present studies by Mastitskaya et\nal. is that GLP-1R – mediated coronary vasodilatation involves fine myocardial\ncapillaries covered by NG2+ pericytes (confocal microscopy studies on cardiac sections,\nex vivo). These results are in line with recent studies in brain microcirculation (Chen Y,\net al. Exendin-4 improves cerebral ischemia by relaxing microvessels, rapidly increasing\ncerebral blood flow after reperfusion. Basic Res Cardiol. 2025;120:423-441. doi:\n10.1007/s00395-025-01096-y).\nResponse: In fact, the lead author of this paper was also second author on the Basalay\net al. paper mentioned above, and this study is indeed a mechanistic analysis of how\nthe effects of the Basalay et al. paper come about. We identify the cell type involved\n(coronary pericytes, as noted in the reviewer’s comments above), and also the ion\nchannels involved as described below. Although the Chen et al. paper reports something\nsimilar for brain mural cells being able to restore blood flow after ischaemia, it does not\nspecifically identify the underlying mechanism (attributing it to a guanylate cyclase\nindependent NO effect and to PgE2). We thus believe that our paper makes an important\nstep forward, certainly for the heart, and in fact probably for pericytes in other vascular\nbeds, thus opening up the possibility of developing a pharmacological approach to mimic\nthe effects of remote ischaemic preconditioning.\nIn the 2nd part of the manuscript Mastitskaya and colleagues performed\npharmacological studies in isolated mouse right ventricular free wall preparations to\ndissect the intracellular mechanisms of the relaxing effects of GLP-1. Capillary diameters\n“at pericyte somata” were measured under ischemic conditions (mimicked by oxygen\nand glucose deprivation) or after infusion of endothelin-1, a potent microvascular\nconstrictor (2-photon microscopy studies). The vasodilating effect of the\nGLP-1R agonist Exendin-4 was studied in the presence of specific activators/inhibitors of\nATP-sensitive potassium channels (KATP) or AMPK inhibitors. The design of these experiments was\nbased on previously published studies showing that GLP-1 stimulates KATP-channels in\nvascular smooth muscle cells, as one mechanism mediating its vasodilating effect\n(Green BD, et al. GLP-1 and related peptides cause concentration-dependent relaxation\nof rat aorta through a pathway involving KATP and cAMP. Arch Biochem Biophys.\n2008;478:136-42). In line with these previous observations, the present studies show\nthat GLP-1R – mediated coronary vasodilatation involves the hyperpolarizing activation\nof KATP channels and the membrane insertion of these channels triggered by AMPK.\nHere, the involvement of pericytes is demonstrated by using right ventricles from mice\nwith Kir6.1 deletion in pericytes (Kir6.1 is a subunit of the KATP channel).\nResponse: As the referee notes, we show that restoration of coronary blood flow by\nremote ischaemic preconditioning is mediated by GLP-1 activating ATP-sensitive K\n3\nchannels, and we show how this depends crucially on signalling pathways that insert\nthese channels into the cell membrane. However, this goes far beyond the Green et al.\npaper mentioned by the referee (which we do cite in the paper), through establishing:\n(i) the role of GLP-1 in mediating the preconditioning evoked coronary blood flow\nincrease;\n(ii) the role of GLP-1 in coronary capillaries (as opposed to arteries or arterioles) which\nare the part of the coronary circulation that cause no-reflow after cardiac ischaemia,\nthus opening up the possibility for developing a pharmacological approach to mimic the\neffects of remote ischaemic preconditioning;\n(iii) that the GLP1R antagonist Exendin(9-39) has an effect opposite to the agonist\nExendin-4 (unlike in the Green et al. paper on the aorta);\n(iv) the modulatory role of other signalling pathways that regulate K channel\nATP\ntrafficking.\nThus, the impression given by the referee’s comments, that our work is not novel, is\nhighly misleading.\nOverall, this study is potentially interesting, although several main informations were\nalready published before by other groups (the GLP-1/GLP-1R pathway participates in\nprotective heart and brain ischemic preconditioning; in vascular cells (and also in\ncardiomyocytes) this pathway involves the activation of KATP channels).\nResponse: We would like to thank the reviewer for their overall positive assessment of\nour work. However, we believe that the major advances described in this manuscript,\nviz. discovering the mechanism of remote ischaemic preconditioning, identifying the\nreceptors and ion channels involved, and defining how this mechanism is regulated by\nother messenger systems, make it far more novel and interesting than the referee is\nstating.\nFor this reviewer, the most interesting part of the manuscript would be the clear\ndemonstration of a brain-gut-cardiac pericytes axis (as stated in the manuscript title),\nbut this axis is not clear.\nResponse: As noted above for Referee 1, the evidence for the vagally mediated release\nof GLP-1 from the gut is provided in our previous publication (Mastitskaya et\nal, PlosONE 2016, doi: 10.1371/journal.pone.0150108), where we performed a series\nof selective transections of different branches of the vagus, and demonstrated that the\nposterior gastric branch of the subdiaphragmatic vagus is responsible for the release of\nthe molecular factor mediating RPc.\nMy major comments are:\nCritique 1: The manuscript (and previous studies) involve GLP-1 as endocrine\ncirculating hormone. How are the plasma GLP-1 levels in rats/mice after ischemic\npreconditioning? Are they higher as for instance after a carbohydrate rich meal?!\nResponse: We did not measure plasma GLP-1 levels in this study, however, we\npublished data on [GLP-1] previously in Basalay et al. (2016, doi:\n10.1093/cvr/cvw216). There we observed a moderate GLP-1 level increase by 1.7 pg/ml\n(or 0.52 pM) in arterial blood 30 min after the RPc stimulus. For comparison, the\nabsolute increase in total GLP-1 in plasma from the retrobulbar plexus (containing both\narterial and venous blood) was only ~0.5 pM in mice 5 min after oral glucose – a value\nsimilar to our findings on RPc effects on plasma levels - and declined below the baseline\nby 20 min (see Fig 2A-B of Smits et al, 2024, reproduced below).\nrecent study even indicates that accurate measurements of active GLP-1 in the blood\nare only possible with in vivo blockade of the degrading enzyme DPPIV (Smits et al,\n2024 doi: 10.2337/db23-0848). Thus, adding DPPIV inhibitors in vivo prior to giving\noral glucose resulted in glucose evoking a temporary 6pM GLP-1 increase (see Fig 4E of\nSmits et al, 2024 doi: 10.2337/db23-0848), reproduced below: intact [unmetabolised]\nGLP-1 levels measured after giving glucose at t=0 are shown in control conditions\nwithout peptidase inhibition (black dashed line, and in the presence of the DPPIV\ninhibitor valine pyrrolidide or of a combination of valine pyrrolidide with the neprilysin\ninhibitor sacubitril (given orally 30 min prior to the glucose) which are shown as the\npurple dashed line and the solid purple line respectively.\nFig 4E of Smits et al, 2024 (relabelled for easier understanding)\nDPPIV inhibitors\nNo DPPIV inhibitors\nGlucose\nCritique 2: Are intestinal enteroendocrine K cells the only cells involved as source of\nGLP-1? (GLP-1 is also produced in other locations)\nResponse: We believe there is a typo here, and the Reviewer means L-cells. As per\nour response to the similar comment from Reviewer #1, GLP-1 is also produced in the\nalpha-cells, in type II taste receptor cells, in the olfactory bulb and in the PPG neurons\nin the brainstem, but most likely none of these sources provide quantitively enough\nGLP-1 to increase (peripheral) circulating levels to the point that GLP-1 can have\nsystemic effects.\nmostly associated with appetite control and particularly satiation after large meals as\nwell as fluid homeostasis. The evidence for the vagally mediated release of GLP-1 from\nthe gut being the major contributor to cardioprotective effects of RPc is given in our\nprevious publication (Mastitskaya et al, PlosONE 2016, doi:\n10.1371/journal.pone.0150108) where we performed a series of selective transections\nof different branches of the vagus, and demonstrated that the posterior gastric branch\nof the subdiaphragmatic vagus is responsible for the release of the molecular factor\nmediating RPc.\nCritique 3: To what extend is the myocardial “capillary dilatation at pericyte sites”\ndependent on the vasodilatation and enhanced flow through the feeding proximal\narteries? (the manuscript repeatedly mentions “pericyte relaxation” but only shows the\ndiameter of capillaries).\nResponse: Pericytes are the only contractile cells on capillaries (Peppiatt et al., 2006,\nNature doi.org/10.1038/nature05193), and if the capillary diameter decreases or\nincreases “actively” it is due to pericyte contraction or relaxation. We have previously\ndemonstrated for the brain that pericyte relaxation in response to neural activity occurs\nprior to changes of the diameter of feeding arteriole (Hall et al., 2014, Nature,\ndoi.org/10.1038/nature13165), and that the resulting dilation occurs mostly near the\ncell soma where most of the pericyte’s circumferential processes are located (Nortley\net al., 2019, Science doi.org/10.1126/science.aav9518). For the brain we estimated\n(based on detailed measurements of the topology of the vascular network by David\nKleinfeld’s group, doi.org/10.1038/nn.3426) that capillary pericytes are responsible for\n~80% of the increase in blood flow that occurs in response to neuronal activity (Hall et\nal., 2014, Nature, doi.org/10.1038/nature13165). A similar topological and quantitative\nanalysis has yet to be performed for the coronary vasculature. The point, however, is\nthat pericytes can actively relax to increase blood flow, and capillaries are not just being\npushed open as a result of upstream arteriole dilation (Hall et al., 2014, Nature,\ndoi.org/10.1038/nature13165, see Figure 3).\nTo explain why we measure capillary diameters at the somata of pericytes, we have\nnow added to the manuscript the figure below (as Fig. 3b) plotting the capillary diameter\nchanges in the cardiac microvasculature as a function of distance from pericyte somata,\nin response to the vasoconstrictor endothelin-1. As in the brain, the diameter change is\nfocused near the pericyte somata, due to most circumferential processes (which have\nthe optimal orientation to change the diameter) being located there (see Supplementary\nFigs 2 and 3 of Nortley et al., 2019, Science doi.org/10.1126/science.aav9518).\n5 µm\nreflow” and ischemic preconditioning could be achieved by generating mice with\npericyte-restricted GLP-1R deletion (floxed mice are available; see Kajitani Y, et al. High\nfrequency of germline recombination in Nestin-Cre transgenic mice crossed with\nGlucagon-like peptide 1 receptor floxed mice. PLoS One. 2023;18:e0296006. doi:\n10.1371/journal.pone.0150108). Intravenous, systemic delivery of Ex9 of course\nblocked GLP-R1 on other types of cells (in and outside the heart), which might be\ninvolved in the regulation of coronary perfusion.\nResponse: Thank you, this is an excellent idea, and we are considering doing it in the\nfuture. However, to do the experiment we would need both the floxed mouse\nmentioned, and a truly pericyte-specific Cre driver line. The problems of obtaining a\ntruly pericyte-specific Cre driver have been described by Guo et al., (2024,\nhttps://pmc.ncbi.nlm.nih.gov/articles/PMC11502228/). The commonly used NG2-Cre\nline can delete floxed proteins from pericytes but also does so in some smooth muscle\ncells and in oligodendrocyte precursor cells. Although there is a mouse driver line that\nis truly specific for pericytes in the brain (Guo et al., cited above) no such line exists for\ncoronary capillary pericytes. Furthermore, even if we used the NG2-Cre mouse, because\nwe currently do not have the floxed GLP-1R mouse, the 6 months needed for mouse\nimport, quarantine and breeding with the Cre mouse make it unfeasible to complete\nthese experiments for this paper in a reasonable timeframe.\nWe would, however, like to point out that we can be absolutely sure that the Exendin-\n4 is affecting the contractile properties of coronary pericytes, because we are directly\nimaging the changes of capillary diameter produced by the pericytes. Furthermore,\nwe\nhave now added to the manuscript a series of additional live tissue imaging\nexperiments, using a similar approach to that in Figure 3c, employing conditions of\noxygen-glucose deprivation to constrict capillaries, and application of Ex4 to activate\ncapillary GLP-1Rs; but instead of blocking KATP channels with glibenclamide, we now\nblocked GLP-1Rs with Exendin(9-39). The results are now included as Figure 3d, and\nshow that the dilating effect of the GLP-1R mimetic Exendin-4 is blocked (as expected)\nby the GLP-1R antagonist Exendin(9-39):\nResponse: For Figure 1C, we used Fluorescein-Trp25-Exendin-4 (FLEX) to label the\nGLP-1 receptors. This is a fluorescently tagged Exendin-4, i.e. a GLP-1R agonist, and\nso internalisation of the FLEX-GLP-1R complex (and hence a cytosolic appearance of the\nlabelling) is not surprising (and has been reported previously by Roed SN et al, 2014\ndoi: 10.1016/j.mce.2013.11.010).\nCritique 6: Finally, it is not clear to this reviewer how/why the GLP-1/GLP-1R pathway\nactivates KATP channels in VSMCs, pericytes (present studies) and cardiomyocytes,\nwhile blocking these channels in pancreatic beta cells (Leech CA, et al. Signal\ntransduction of PACAP and GLP-1 in pancreatic beta cells. Ann N Y Acad Sci.\n1996;805:81-92). The reference 19 cited in the manuscript in fact describes that the\nGLP-1/GLP-1R pathway normally blocks KATP channels in beta cells (as is\nphysiologically meaningful), which was only reverted into an “activation” under very\nartificial conditions.\nResponse: We are aware of this apparent discrepancy, but it can easily be explained\nby the different mechanisms involved. Although this is not the focus of our paper, which\nis on how remote ischaemic preconditioning is mediated by GLP-1 activating K\nATP\nchannels in pericytes, GLP-1 potentiates the closing of K channels in beta-cells for\nATP\nthe following reasons. Canonically, a rise of glucose concentration tends to close K\nATP\nchannels because it raises intracellular [ATP] (McTaggart et al, J Physiology 2010. doi:\n10.1113/jphysiol.2010.191767). This effect is potentiated by GLP-1R activation as a\nresult of downstream activation of PKA and hence EPAC (Exchange Protein directly\nActivated by Cyclic AMP) (Marzook et al, Front Endocrinol 2021 doi:\n10.3389/fendo.2021.678055). EPAC increases the sensitivity of K(ATP) to ATP and\nhydrolyses PIP2 which tends to keep K channels open (Kang et al, J Physiol. 2006\nATP\ndoi: 10.1113/jphysiol.2006.107391; Baukrowitz et al. Science 1998 doi:\n10.1126/science.282.5391.1141). Without the initial glucose-dependent step, GLP-1\ncannot exert its potentiating effect on K channel closure in beta-cells. Therefore,\nATP\nwithout the presence of glucose, GLP-1 has only negligible effects on insulin release,\nwhich is why GLP-1-based therapies are so useful for the treatment of Type 2 diabetes.\nThe K channel in beta-cells is made of Sur1 and Kir6.2 subunits. PKA phosphorylates\nATP\nthe SUR1 subunit which leads to a decrease in the channel's opening probability,\nenhances sensitivity to ATP and inhibits the counteracting (opening) effect of ADP.\nIn contrast, in pericytes, K channels are made of Sur2B and Kir6.1 subunits. The\nATP\ndownstream effect of PKA activation on K channels for these cells differs from in beta\nATP\ncells. PKA phosphorylation of Sur2B activates (opens) K channels and is, for example,\nATP\nresponsible for beta2-adrenergic effects on vascular relaxation (Shi Y et al, 2007 doi:\n10.1152/ajpregu.00337.2007). PKA can also phosphorylate multiple sites of Sur2B\nleading to vasorelaxation (Quinn KV et al, 2004, doi:\n10.1161/01.RES.0000128513.34817.c4).\nThe authors provide an interesting set of experiments utilising immunofluorescence\nlabelling, antagonists and KO mice to explore the mainly sub cellular signalling pathways\nthat are involved in pericyte constriction mediating no reflow in response to ischaemia\n(or OGD). They propose that the brain-gut-heart axis via vagally mediated GLP-1\nrelease is important in signalling RIPc protective vasodilatory signals to the heart\npericyte with sub cellular signalling that are dependent on KATP channel membrane\nexpression / opening via a AMP kinase dependent signalling - that result in relaxation.\nNo reflow is an important clinical phenomenon and further understanding the\npathophysiology is needed to prevent it occurring and to discover new treatments.\nWe thank the referee for these positive comments.\nGeneral comments:\nCritique 1: The experiments are elegantly demonstrated and illustrated with\ncompelling results. However, the focus is mainly on sub cellular signalling which has\nbeen previously demonstrated - see work by Kloner RA and others. However, the data\non pericytes are novel and thoroughly explored with a logical series of experiments.\nResponse: We thank the referee for their positive comment on the pericyte aspect of\nour work. As far as the supposedly previous demonstration of the subcellular signalling\nis concerned, unfortunately, the referee cites no papers, and in PubMed we could find\nonly one editorial (and no original papers) on GLP-1 by Kloner R.A., and a single paper\ndiscounting mitochondrial K channels as modulating myocardial infarct size. As far as\nATP\nwe are aware, our discoveries on the mechanism of how RPc affects the coronary\nmicrovasculature after ischaemia have not been reported previously.\nCritique 2: The title of the paper and Figure 5a propose the brain-gut-heart signalling,\nalthough this was not specifically assessed in whole animal experiments and therefore\npossibly the title and conclusions drawn in the discussion over-reach, based on the\nactual data provided. Further whole animal experiments with possible nerve truncation\nor in vivo blood sampling for GLP-1 would strengthen the manuscript and support the\nbrain-gut-heart signalling hypothesis.\nResponse: Thank you for this comment and the suggestion of the experiments for\ndemonstration of the brain-gut-heart signaling hypothesis. These experiments were\nperformed in our previous studies on cardioprotective effects of GLP-1. Firstly, in a study\non selectively transecting different branches of the vagus (Mastitskaya et al, PlosONE\n2016, https://doi.org/10.1371/journal.pone.0150108), we demonstrated that the\nsubdiaphragmatic posterior branch of the vagus nerve is responsible for RPc-induced\ncardioprotection (the sectioning of this branch prevented cardioprotection, while\nselective electrical stimulation of it resulted in reduced infarct size; with all appropriate\ncontrols in place). Secondly, we sampled the blood in vivo in the rats undergoing\ntemporary limb ischaemia (the equivalent of RPc) and showed that after 30 min of\nischaemia onset, the plasma levels of GLP-1 were elevated (Basalay et al, 2016\nCardiovascular Res doi: 10.1093/cvr/cvw216). In the current paper, we demonstrate in\nexperiments in vivo that GLP-1R antagonists prevent the protective effects of RPc on\nno-reflow in rats (which supports the findings in the Basalay et al, 2016 study), and\nthat protective effects of RPc are absent in mice with selective Kir6.1 KO in pericytes.\ndescribed in the Introduction to the paper on page 1 (right column, lines 49-53) of the\nmanuscript.\nCritique 3: GLP-1 is inherently unstable with a 2min half life due to degradation by\nDPP-4. Have the authors looked at potentiating the effect with DPP-4 inhibition, rather\nthan only blocking it? How did they know that local GLP-1 released from the gut reaches\nthe heart?\nResponse: We deduce that the cardioprotection conferred by RPc is mediated by GLP-\n1 reaching the heart because it is prevented by the GLP-1R antagonist Exendin(9-39)\n(Fig. 2c, f of the manuscript).\nWe have not tested the effect of DPP4 inhibition because it has some effects that are\nnot mediated by the GLP-1 system (including via GIP, neuropeptide Y and SDF-1α).\nIndeed, it is difficult to distinguish the cardioprotective effects of DPP4 inhibitors from\ntheir effects mediated by increased levels of GLP-1. It is well established that DPP4\ninhibitors have prosurvival effects on myocardium via activation of a SDF-1α/CXCR4-\nmediated STAT3 prosurvival pathway, dramatically reducing the infarct size (Kubota et\nal, 2016 doi: 10.1016/j.yjmcc.2015.12.026), while GLP-1R signaling activates a\nPI3K/Akt prosurvival pathway in myocardium.\nCritique 4: The Kir6.1 trafficking immunofluorescence intensity profiles measured\nusing imageJ (Figure 4) needs further explanation/ verification with control studies. The\nimages of pericytes/ selection of membrane were presumably not random and are\npossibly open to bias.\nResponse: As stated in the Methods, the person analysing the images was blinded to\nthe experimental condition, with the aim of removing possible operator bias. Between\n3-5 zoomed-in images per heart were taken to achieve a total number of 5-10 pericytes\nper heart, and all imaged pericytes were analysed. The selection of the membrane area\nwithin each cell was based on having a clearly visible PDGFR signal, on the outer side\nof the capillary, as has now been described in the Methods (lines 647-651).\nCritique 5: The methodology has been adequately described and the reported.\nHowever, the assessment of pericyte \"opening\" is only done visually (microscopically)\n(%open/ measured diameters), rather than employing other methods e.g.\nhaemodynamic assessment of resistance that could add weight to the authors findings.\nFurthermore, visual assessment again may be open to reporting error/ selection bias.\nResponse: There is a strong correlation between the diameter measured at the\npericytes in Figures 2, 3 and 4, and the amount of tissue perfusion in Figure 2, strongly\nsuggesting that it is the pericyte-mediated capillary constriction (or its absence) that\nrestricts (or allows) coronary blood flow. It would be ideal if a computer model of the\ncoronary vasculature could be used to convert our measurements of diameter changes\nat pericytes into a change of coronary blood flow, but such a model does not yet exist\n(unlike for the brain where capillaries are reported to confer 4 times the resistance\nconferred by arterioles to the flow of blood: Gould et al., 2017, JCBFM,\nhttps://doi.org/10.1177/0271678x16671146). Measurement of capillary diameters was\nperformed by an observer blinded to the condition being applied and diameters at all\npericyte locations in the images were measured.\nnot showing improvement in microcirculatory function and mixed results in cardio\nprotection although GLP is a vasodilator. This dichotomy between animal studies and\nstudies in Man deserves further attention in the discussion.\nResponse: Thank you for this comment. We now added a commentary on this issue to\nthe Discussion (lines 352-379). Indeed, the translation of RPc cardioprotective studies\nhas been challenging. Partly, the enthusiasm in the field was significantly compromised\nby the poor design of some major clinical trials on RPc, e.g. where RPc was performed\nin patients undergoing PCI procedures and the anaesthesia included propofol which is\nknown to suppress vagal tone. Given that the vagal pathway is crucial in mediating\ncardioprotection, it is not surprising that the studies performed under propofol\nanaesthesia resulted in neutral outcomes.\nMore importantly, our aim, in defining the mechanism by which RPc is cardioprotective,\nis not to advocate RPc for clinical use, but to identify a signalling mechanism that can\nbe targeted to confer cardioprotection without the need for RPc.\nMinor:\n1. Clinically important no-reflow is relatively rare (2% rather than 50% as stated) after\ncoronary stenting - please revise introduction.\nResponse: The Reviewer cites no reference but is apparently referring to the statistics\non no-reflow visible on angiography. Indeed, the no-reflow of major coronary blood\nvessels is relatively rare. However, we are providing the statistics for microvascular\nno-\nreflow, as discussed in numerous reviews (doi: 10.1055/s-0041-1725979; doi:\n10.1016/j.yjmcc.2011.06.009).\n2. There are typos in Figure 4: OGG rather than OGD.\nResponse: Thank you. We have corrected this.\n11"}, {"round": 2, "reviewer_comments": {"reviewer_1": "No further comments.", "reviewer_2": "The authors answered all my questions. This clarified which aspects of the manuscript are novel, and which parts mostly extend published data. Overall this is an interesting story.\nAs previously commented by reviewers 2 and 3, the title of the manuscript is not appropriate. The present studies do not further characterize an \"endocrine axis\", they provide novel mechanistic insights regarding the target cells and signalling pathways.", "reviewer_3": "The authors have addressed all my comments."}, "author_response": ""}]

Decision letter