Weeks Of Reperfusion Research Articles

Rapsyn overexpression improves recovery of the neuromuscular junction from tourniquet-induced ischemia-reperfusion injury in mouse hindlimb

The neuromuscular junction (NMJ) is an important factor to influence skeletal muscle contractile function. Tourniquet-induced ischemia-reperfusion (IR) results in the fragmentation of nicotinic acetylcholine receptors (nAChR) clusters in the NMJ and impairs neural controlled skeletal muscle contraction. Rapsyn, a cytoplasmic nAChR-associated protein concentrates and anchors nAChRs in the postsynaptic membrane and participates in the nAChR assembly at NMJ. Here, we observed the effect of overexpression of Rapsyn on morphological and functional recovery of the NMJ from tourniquet-induced IR injuries in mouse hindlimb. Unilateral hindlimb of C57/BL6 mice were subjected to 3 hours of ischemia by placing a rubber band and followed 6 weeks of reperfusion. Ad-m-RAPSN (5.2 x 1010 PFU/ml) was injected into gastrocnemius muscle (50 μl) by Hamilton microliter syringe after 2 weeks of tourniquet-induced IR. After 6 weeks of IR, the sciatic nerve-stimulated gastrocnemius muscle contraction and endplate potential (EPP) were recorded in in-situ muscle samples; the NMJs were labelled with alpha-bungarotoxin (BTX) and nerofilment-200 (NF-200) + synaptophysin (Syn) antibodies, and NMJ images were captured with Zeiss LSM 800. After 6 weeks of IR, NMJs in the gastrocnemius muscle showed significantly fragmented nAChR clusters (9.3 ± 0.7 vs. sham 1.7 ± 0.1 for numbers of fragments) with low rapsyn expression, and the EPP (15.29 ± 0.93 mV vs. sham 26.87 ± 0.89 mV) and nerve-stimulated muscle contraction (117.2 ± 8.5 g vs. sham 157.9 ± 4.9 g) were reduced. Ad-m-RAPSN injection increased rapsyn expression, mitigated fragmentation of nAChR clusters (3.2 ± 0.3), increased the EPP (20.35 ± 0.86) and nerve-stimulated muscle contraction (138.1 ± 3.5 g). The data suggest that tourniquet-related IR induces AChR cluster fragmentation and impairs NMJ function, and rapsyn overexpression improves the morphological and functional recovery of the NMJ from tourniquet-induced IR injuries.. This research is supported by the NIH/NIGMS 1R01GM145736 to Y-LL. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

THU583 Cardioprotection By Nitric Oxide Is Mediated Via Mitochondrial Function In Rodents

Abstract Disclosure: K. Islam: None. R. Islam: None. A. Draper: None. Background: Reduced nitric oxide (NO)bioavailability and decreased expression of mitochondrial transcription factor A (TFAM) occurs in the setting of heart failure (HF). TFAM promotes transcription of mitochondrial DNA, mitochondrial biogenesis, respiratory chain activity, and ATP synthesis. We investigated the effects of oral NaNO2therapy to increase NO in chronic HF following acute myocardial infarction and examined the effects of NaNO2 on the activation of TFAM, mitophagy, and antioxidant signaling pathways. Methods: Male C57/BL6J mice underwent 60 minutes of myocardial ischemia (MI) induced by left coronary artery (LCA) occlusion followed by 4 weeks of reperfusion. NaNO2 or saline vehicle (VEH)was administered at a dose of 165 µg/kg at reperfusion and then daily for 4weeks in the drinking water (100 mg/L). Left ventricular ejection fraction (LVEF) was determined at baseline and at 4 weeks of reperfusion. At 4 weeks of reperfusion myocardial tissue samples were collected and analyzed by RT-qPCR, immunoblot, oxidative stress assay, and protein sulfhydration assay. Results: NaNO2 administration markedly preserved left ventricular ejection fraction (LVEF) and improved left ventricular diastolic and systolic dimensions at 4 weeks as compared to VEH. Mitochondrial antioxidant proteins, SOD2 (MnSOD), peroxiredoxin-3 (Prx-3) and thioredoxin 2 (TXN2) (p < 0.05) were markedly increased in NaNO2treated mice compared to VEH. Expression of mitophagic markers, p62 (p <0.05) and Map1lc3 (p < 0.05) were significantly increased in NaNO2treated HF mice as compared to VEH. Expression of mitochondrial quality control such as PINK1 and PARKIN2 were also markedly increased HF mice after treated with nitrite therapy as compared with VEH. Marked induction of sulfhydration of interferon regulator factor 1 (IRF-1) as well as upregulation of mitochondrial transcription factor A (TFAM) (p < 0.05) and suppression of DNA methyltransferase3a (Dnmt-3a) (p < 0.05) were observed in NaNO2 treated ischemic HF mice as compared with VEH. Conclusions: Our results clearly demonstrate thatNaNO2 therapy improves left ventricular function during ischemic HF through induction of antioxidant signaling, mitophagy, sulfhydration of IRF-1and TFAM resulting in increased mitochondrial biogenesis. Presentation: Thursday, June 15, 2023

Preclinical model to investigate heart failure development following ischemia-reperfusion injury

Heart failure is major public health problem with ischemic cardiomyopathy as the main etiology. Despite therapeutic advances, the high mortality of patients highlights that development of new therapeutic strategies is urgently needed. In this context, the establishment of a pre-clinical model mimicking the clinical pathology would be an asset to characterize the functional mechanisms responsible for the development of heart failure following ischemia-reperfusion injury. In this study, male and female C57Bl/6J mice (8–12 weeks old) underwent left anterior coronary artery ligation for 90 minutes followed by 12 weeks reperfusion (n = 18) or no surgical intervention (CTRL; n = 8). Ejection fraction (EF), left ventricle internal diameter (LVID), E-wave deceleration time (EDT) and the isovolumic relaxation time (IVRT) were assessed by echocardiography during the 12 weeks of post-ischemic reperfusion and cellular Ca2 + (dys)regulation mechanisms in failing isolated cardiomyocytes were investigated at 8 weeks with the imaging Ionoptix system. Our results showed that, despite a significant alteration of EF at day 2 post-infarction (due to ischemic stress by itself), ischemic mice developed a chronical heart dysfunction characterized, on one hand, by a systolic dysfunction with a significant decrease of EF averaging 26% and a LV dilatation around 36% from the day 2 to the week 8 of reperfusion (P < 0.05), and on the other hand, associated with a significant diastolic dysfunction at 12 weeks of reperfusion. Mechanistically, Ca2+ phenotyping showed a significant alteration in excitation/contraction coupling and a contractility defect in failing cardiomyocytes isolated at 8 weeks reperfusion (P < 0.05 vs. CTRL). Our experimental conditions show that 90 minutes ischemia followed by 8 weeks reperfusion seems to be a good algorithm to mimic development of heart failure after an ischemia-reperfusion stress, which is characterized by both systolic and diastolic cardiac dysfunctions.

Cardiac fibroblast heat shock protein 47 aggravates cardiac fibrosis post myocardial ischemia-reperfusion injury by encouraging ubiquitin specific peptidase 10 dependent Smad4 deubiquitination.

Despite complications were significantly reduced due to the popularity of percutaneous coronary intervention (PCI) in clinical trials, reperfusion injury and chronic cardiac remodeling significantly contribute to poor prognosis and rehabilitation in AMI patients. We revealed the effects of HSP47 on myocardial ischemia-reperfusion injury (IRI) and shed light on the underlying molecular mechanism. We generated adult mice with lentivirus-mediated or miRNA (mi1/133TS)-aided cardiac fibroblast-selective HSP47 overexpression. Myocardial IRI was induced by 45-min occlusion of the left anterior descending (LAD) artery followed by 24h reperfusion in mice, while ischemia-mediated cardiac remodeling was induced by four weeks of reperfusion. Also, the role of HSP47 in fibrogenesis was evaluated in cardiac fibroblasts following hypoxia-reoxygenation (HR). Extensive HSP47 was observed in murine infarcted hearts, human ischemic hearts, and cardiac fibroblasts and accelerated oxidative stress and apoptosis after myocardial IRI. Cardiac fibroblast-selective HSP47 overexpression exacerbated cardiac dysfunction caused by chronic myocardial IRI and presented deteriorative fibrosis and cell proliferation. HSP47 upregulation in cardiac fibroblasts promoted TGFβ1-Smad4 pathway activation and Smad4 deubiquitination by recruiting ubiquitin-specific peptidase 10 (USP10) in fibroblasts. However, cardiac fibroblast specific USP10 deficiency abolished HSP47-mediated fibrogenesis in hearts. Moreover, blockage of HSP47 with Col003 disturbed fibrogenesis in fibroblasts following HR. Altogether, cardiac fibroblast HSP47 aggravates fibrosis post-myocardial IRI by enhancing USP10-dependent Smad4 deubiquitination, which provided a potential strategy for myocardial IRI and cardiac remodeling.

Human Muse cells reduce myocardial infarct size and improve cardiac function without causing arrythmias in a swine model of acute myocardial infarction.

We recently reported that multilineage-differentiating stress enduring (Muse) cells intravenously administered after acute myocardial infarction (AMI), selectively engrafted to the infarct area, spontaneously differentiated into cardiomyocytes and vessels, reduced the infarct size, improved the left ventricular (LV) function and remodeling in rabbits. We aimed to clarify the efficiency of Muse cells in a larger animal AMI model of mini-pigs using a semi-clinical grade human Muse cell product. Mini-pigs underwent 30 min of coronary artery occlusion followed by 2 weeks of reperfusion. Semi-clinical grade human Muse cell product (1x107, Muse group, n = 5) or saline (Vehicle group, n = 7) were intravenously administered at 24 h after reperfusion. The infarct size, LV function and remodeling were evaluated by echocardiography. Arrhythmias were evaluated by an implantable loop recorder. The infarct size was significantly smaller in the Muse group (10.5±3.3%) than in the Vehicle group (21.0±2.0%). Both the LV ejection fraction and fractional shortening were significantly greater in the Muse group than in the Vehicle group. The LV end-systolic and end-diastolic dimensions were significantly smaller in the Muse group than in the Vehicle group. Human Muse cells homed into the infarct border area and expressed cardiac troponin I and vascular endothelial CD31. No arrhythmias and no blood test abnormality were observed. Muse cell product might be promising for AMI therapy based on the efficiency and safety in a mini-pig AMI.

Abstract 10526: Surface Located Adiponectin On Adipocytes-derived Exosomes Mediates Adipocytes/cardiomyocytes Communication And Contributes To Cardioprotection

Introduction and Hypothesis: Increasing evidence emphasizes the roles of adipocyte-cardiomyocyte (ADp-CM) communication plays in health and diabetes. We recently reported that diabetes switched ADp-derived Exosomes (ADp-Exo) from carrying cardioprotective molecules to delivering cardiotoxic molecules to CM. However, the molecular mechanisms mediating ADp-Exo cardioprotection and its alteration by diabetes remain unknown. Methods and Results: ADp-Exo was isolated from epididymal white adipocytes of WT mice. Western Blot demonstrated high level of adiponectin (APN), a potent cardioprotective adipokine, present in ADp-Exo. More interestingly, APN was detected on the surface of ADp-Exo by flow cytometry using Exo-flow kit (System Biosciences). To determine whether surface-located APN on ADp-Exo protect heart, WT ADp-Exo were intramyocardially injected. Mice were subjected to 90 minutes coronary occlusion, followed by 4 weeks of reperfusion. Cardiac function was evaluated by echocardiograph weekly. WT ADp-Exo significantly reduced MI/R injury in vivo , as evidenced by improved LVEF, radial and longitudinal myocardial Strain and SR (vs vehicle group, p&lt;0.05). Importantly, pre-incubation of ADp-Exo with an APN neutralization antibody blocked ADp-Exo cardioprotection. To determine the role of CM AdipoR1, ex vivo and in vitro experiments were performed. WT ADp-Exo activated AdipoR1 pathway (increased phosphorylation of ACC, ERK and AMPK) in CM and protected against cell injury and death (MTT and LDH assay). However, surface APN blocked WT ADp-Exo or APN -/- ADp-Exo failed to active this signaling and protection. WT ADp-Exo failed to activate cell salvage kinases in AdipoR1 -/- CM. Finally, overexpression of GRK2, a pathogenic molecule that we reported to cause AdipoR1 S205 phosphorylation, blocked WT ADp-Exo cardioprotection. AAV9-mediated overexpression of a phosphorylation resistant AdipoR1 (AdipoR1 S205A ) restored ADp-Exo protective action. Conclusions: We demonstrate for the first time that ADp-Exo surface located APN mediates ADp-CM communication via CM AdipoR1 pathway, promoting cardioprotection. This protective system is blocked by GRK2-induced AdipoR1 phosphorylation in diabetic heart, promoting MI/R injury.

Mitochondrial Telomerase Reverse Transcriptase Protects From Myocardial Ischemia/Reperfusion Injury by Improving Complex I Composition and Function.

The catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), has protective functions in the cardiovascular system. TERT is not only present in the nucleus but also in mitochondria. However, it is unclear whether nuclear or mitochondrial TERT is responsible for the observed protection, and the appropriate tools are missing to dissect this. We generated new mouse models containing TERT exclusively in the mitochondria (mitoTERT mice) or the nucleus (nucTERT mice) to finally distinguish between the functions of nuclear and mitochondrial TERT. Outcome after ischemia/reperfusion, mitochondrial respiration in the heart, and cellular functions of cardiomyocytes, fibroblasts, and endothelial cells, as well, were determined. All mice were phenotypically normal. Although respiration was reduced in cardiac mitochondria from TERT-deficient and nucTERT mice, it was increased in mitoTERT animals. The latter also had smaller infarcts than wild-type mice, whereas nucTERT animals had larger infarcts. The decrease in ejection fraction after 1, 2, and 4 weeks of reperfusion was attenuated in mitoTERT mice. Scar size was also reduced and vascularization increased. Mitochondrial TERT protected a cardiomyocyte cell line from apoptosis. Myofibroblast differentiation, which depends on complex I activity, was abrogated in TERT-deficient and nucTERT cardiac fibroblasts and completely restored in mitoTERT cells. In endothelial cells, mitochondrial TERT enhanced migratory capacity and activation of endothelial nitric oxide synthase. Mechanistically, mitochondrial TERT improved the ratio between complex I matrix arm and membrane subunits, explaining the enhanced complex I activity. In human right atrial appendages, TERT was localized in mitochondria and there increased by remote ischemic preconditioning. The telomerase activator TA-65 evoked a similar effect in endothelial cells, thereby increasing their migratory capacity, and enhanced myofibroblast differentiation. Mitochondrial, but not nuclear TERT, is critical for mitochondrial respiration and during ischemia/reperfusion injury. Mitochondrial TERT improves complex I subunit composition. TERT is present in human heart mitochondria, and remote ischemic preconditioning increases its level in those organelles. TA-65 has comparable effects ex vivo and improves the migratory capacity of endothelial cells and myofibroblast differentiation. We conclude that mitochondrial TERT is responsible for cardioprotection, and its increase could serve as a therapeutic strategy.

The therapeutic effects of masitinib in complex regional pain syndrome in a mouse model of tourniquet‐induced extremity ischemia‐reperfusion

Tourniquet, as a life‐saving device, is widely used to stop extremity hemorrhage in civilian medicine and on the battlefield. However, tourniquet use and subsequent release can induce a long‐term increase in pain sensitivity called complex regional pain syndrome. Complex regional pain syndrome is a form of chronic pain triggered by inflammation and nerve dysfunction, with a great resistance to different treatments for relieving pain. Increasing evidence showed a pivotal role of neuroinflammation in the formation of hyperalgesia and allodynia. In our study, we investigated the therapeutic effects of masitinib, an anti‐neuroinflammatory drug, on ischemia‐reperfusion (IR) increased pain sensitivity in a mouse model of tourniquet‐induced hindpaw IR. Unilateral hindpaw of C57/BL6 mice were subjected to 3 hours of ischemia by placing a rubber band at ankle joint and followed 4 weeks of reperfusion. Masitinib treatment (28 mg/kg/day, i.p.) began on the day of IR induction and last for 4 weeks. Tourniquet‐induced IR caused nerve damage in the skin and abolished the paw sensation to 2 g of mechanical stimulation before the 3rd day of reperfusion. In the mice with tourniquet‐induced IR, mechanical allodynia appeared at day 7 (0.54 ± 0.10 g, compared to 0.83 g ± 0.10 in sham mice; p&lt;0.05), reached to the maximum reaction at 2 weeks (0.19 g ± 0.04 g), and lasted 4 weeks. In the mice with masitinib plus tourniquet‐induced IR, 33% (4/12) of mice at day 1 and 40% (6/15) of mice at day 3 responded to 2 g of stimulation. Treatment with masitinib significantly alleviated IR‐induced mechanical allodynia from 2 weeks to 4 weeks. Additionally, paw thickness, a marker for hindpaw edema, was observed. Treatment with masitinib also attenuated IR‐induced expansion of the hindpaw. Our data provide the evidence that treatment with masitinib effectively alleviates complex regional pain syndrome in tourniquet‐induced hindpaw IR.

Male, but not female, spontaneously hypertensive rats (SHR) have sustained renal injury following a single ischemic insult progressing to chronic kidney disease (CKD).

Renal ischemia‐reperfusion (IR) injury is a major cause of acute kidney injury (AKI), which is an independent risk factor for the development of CKD and all‐cause mortality. Clinical and pre‐clinical studies further support sex differences in incidence and mortality rates of AKI and a higher tendency to develop AKI to CKD transition than females. Hypertension is a common co‐morbid condition associated with AKI and CKD. Despite the prevalence of hypertension and the importance of blood pressure (BP) control in maintaining renal health, little is known regarding the impact of hypertension on AKI recovery and AKI to CKD progression. The goal of the current study was to test the hypothesis that hypertensive males will have greater IR injury than hypertensive females resulting in more rapid CKD progression. 13 wk old male and female SHR were subjected to sham or 30‐minute warm bilateral ischemia followed by reperfusion (n=5–6). Blood, a 24 hr urine sample and kidneys were collected 1 week post‐IR to assess renal injury. Systolic blood pressure (BP) measured weekly by tail cuff method. Plasma creatinine (Pcr) and urine protein creatinine ratio (UPCR) remained elevated at 1‐week post IR in male SHR compared sham (Pcr PIR=0.03, Psex=0.05; PIR*sex=0.04; ACR PIR=0.005, Psex=0.0005; PIR*sex=0.005); Pcr and UPCR returned to baseline in SHR females. Consistent with this finding, histological examination of SHR kidneys 7 days post‐IR showed increases in vascular congestion (Psex*IR=0.002) and tubular damage (Psex*IR=0.001) compared to sham only in males. However, glomerular filtration rate (GFR) and systolic BP was not altered in both male and female SHR at 1‐week post IR. To determine if this was sustained dysfunction or simply delayed recovery, additional 13 wk old male and female SHR were subjected to sham or 30‐minute warm bilateral ischemia followed by 20 weeks of reperfusion. Plasma and urine were collected weekly (n=3–4). Male SHR showed progressive increases in UPCR (PIR&lt;0.05) and systolic BP up to 20 weeks post‐IR compared to respective shams (PIR=0.001). Whereas in SHR female, UPCR remains at baseline up to 16‐week post IR compared to respective sham. However, at 20 weeks of post IR, both male and female SHR exhibit increase in UPCR compared to respective sham control; although the increase in UPCR was greater in males (PIR=0.0002, Psex=0.01; PIR*sex=0.09). Male SHR also exhibited decreases in glomerular filtration rate (GFR) (PIR=0.007, Psex=0.02; PIR*sex=0.12) and increases in systolic BP (PIR=0.008, Psex=0.0001; PIR*sex=0.27) compared to SHR female at 20 weeks post‐IR. In conclusion, our data demonstrated that impaired renal recovery following IR in SHR males results in exaggerated progression towards CKD compared females.Support or Funding InformationWork was supported by R01 HL127091 and P01 HL134604. Kidney function and systolic BP in male and female SHR at 1 and 20‐week of post‐IR. Male SHR UPCR GFR (mL/min) Systolic BP (mmHg) 1 week post IR Sham 1.14 ± 0.23 2.95±0.33 187±8 IR 3.44±1.37 2.55±0.28 192±6 20 week post IR Sham 1.70±0.26 2.58±0.5 205±10 IR 4.50±0.85 1.10±0.67 225±4 Female SHR 1 week post IR Sham 0.77±0.47 3.24±0.34 164±10 IR 0.79±0.34 3.35±0.68 166±3 20 week post IR Sham 1.10±0.54 2.96±1.09 167±9 IR 2.72±0.46 2.83±0.44 177±9 ACR= Urine Protein Creatinine Ratio; GFR=Glomerular Filtration Rate; BP= Blood Pressure.

Myocardial hypothermia induced after reperfusion does not prevent adverse left ventricular remodeling nor improve cardiac function

AimsThe purpose of the study was to determine whether late therapeutic hypothermia (LTH), administered after reperfusion, could prevent adverse left ventricular (LV) remodeling and improve cardiac function in the rat myocardial ischemia/reperfusion model. Main methodsRats were randomized to normothermia (n = 10) or LTH (initiated at 1 min after coronary artery reperfusion, n = 10) and subjected to 30 min of coronary occlusion followed by 6 weeks of reperfusion. Hypothermia was induced by pumping cold saline over the anterior surface of the LV until the temperature cooled to <32 °C. In the normothermic group, the heart was bathed in saline at 38 °C. Key findingsAfter 6 weeks of recovery, fractional shortening of the LV was comparable in the LTH (20.2 ± 0.6%) and normothermic group (20.0 ± 2.1%; p = 0.918). Postmortem LV volume (0.47 ± 0.04 ml in LTH and 0.44 ± 0.05 ml in normothermic group) and lung wet/dry weight ratio were similar in both groups. There were no significant differences in scar size, scar thickness, infarct expansion index, LV cavity or transmurality (%) between groups. This data contrasts with our previous study showing that hypothermia administered during the ischemic phase significantly reduced the scar size; decreased LV cavity, infarct expansion index and transmurality (%), and improved the scar thickness. SignificanceLTH did not prevent adverse LV remodeling nor improve cardiac function in the rat myocardial ischemia/reperfusion model. To have a long term benefit on remodeling, hypothermia must be administered during the ischemic phase and not just the reperfusion phase.

Characterization of Klotho/FGF23 signaling in cardiorenal syndrome‐induced cardiac hypertrophy

IntroductionIn diseases that generate an inflammatory situation, such as cardiorenal syndrome, different clinical conditions promote cardiac and renal dysfunction. There are type 3 and 4 which are characterized by renal insults that subject the heart to dysfunctions; Among the changes that renal injury can lead, we can mention the modulation in Klotho levels, a gene that is negatively regulated by fibroblast growth factor 23 (FGF23). The objective of this study is to evaluate the participation of this axis in cardiac hypertrophy induced by renal ischemiaMethodsC57BL/6 male mice were subjected to surgical occlusion of left renal pedicle for 60 min followed by reperfusion in acute term (24, 48 and 72h), short term (for 8 and 15 days) and long term (4,6 and 8 weeks). Total heart RNA was extracted for gene expression of Klotho, FGF23 and hypertrophy markers using real‐time PCR.ResultsThe renal injury was confirmed by decreasing of left kidney weight and urea/creatinine levels starting at the 15th day of reperfusion. Cardiac hypertrophy was confirmed by increased a‐actin levels at day 15 and 4 weeks of renal reperfusion; however, heart weight has maintained increased until the 8th week of reperfusion. Concerning the FGF23/Klotho axis, both genes were modulated during the cardiorenal syndrome type 3. FGF23 gene expression was mostly increased in heart tissue after 8 days of reperfusion, having its peak during the 4th week. In renal tissue, FGF23 did not present a pattern, although it shows a peak in the first 24 hours, in the 8th day and 15th day (the same timepoints which the heart tissue is affect which electrophysiological and immunological changes). By the other side, Klotho gene expression was mostly reduced in renal tissue after 48h of reperfusion, evidencing even more the renal injury (once some authors consider Klotho is a renal injury biomarker). In heart tissue, klotho expression is reduced since the first 24h, but this is attenuated around the 4th week of reperfusion.ConclusionWe found that cardiac FGF23/Klotho axis dynamics are affected by renal I/R indicating a possible interaction between these two responses during cardiorenal syndrome.Support or Funding InformationFinancial Support: FAPESP 2015/19107‐5 and 2018/03089‐6.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Remote ischemic perconditioning attenuates adverse cardiac remodeling and preserves left ventricular function in a rat model of reperfused myocardial infarction

AimsRemote ischemic conditioning (RIC) is considered a potential clinical approach to reduce myocardial infarct size and ameliorate adverse post-infarct left ventricular (LV) remodeling, however the mechanisms are unknown. The aim was to clarify the impact of RIC on Neuregulin-1 (NRG-1)/ErbBs expression, inflammation and LV hemodynamic function. Methods and resultsMale Sprague-Dawley rats were subjected to 30 min occlusion of the left coronary artery (LCA) followed by 2 weeks of reperfusion and separated into three groups: (1) sham operated (without LCA occlusion); (2) Myocardial ischemia/reperfusion (MIR) and (3) remote ischemic perconditioning group (MIR + RIPerc). Cardiac structural and functional changes were evaluated by echocardiography and on the isolated working heart system. The level of H3K4me3 at the NRG-1 promoter, and both plasma and LV tissue levels of NRG-1 were assessed. The expression of pro-inflammatory cytokines, ECM components and ErbB receptors were assessed by RT-qPCR. MIR resulted in a significant decrease in LV function and enlargement of LV chamber. This was accompanied with a decrease in the level of H3K4me3 at the NRG-1 promoter. Consequently NRG-1 protein levels were reduced in the infarcted myocardium. Subsequently, an upregulated influx of CD68+ macrophages, high expression of MMP-2 and -9 as well as an increase of IL-1β, TLR-4, TNF-α, TNC expression were observed. In contrast, RIPerc significantly decreased inflammation and improved LV function in association with the enhancement of NRG-1 levels and ErbB3 expression. ConclusionsThese findings may reveal a novel anti-remodeling and anti-inflammatory effect of RIPerc, involving activation of NRG-1/ErbB3 signaling.

IL-23R Signaling Plays No Role in Myocardial Infarction

Ischemic heart diseases are the most frequent diseases in the western world. Apart from Interleukin (IL-)1, inflammatory therapeutic targets in the clinic are still missing. Interestingly, opposing roles of the pro-inflammatory cytokine IL-23 have been described in cardiac ischemia in mice. IL-23 is a composite cytokine consisting of p19 and p40 which binds to IL-23R and IL-12Rβ1 to initiate signal transduction characterized by activation of the Jak/STAT, PI3K and Ras/Raf/MAPK pathways. Here, we generate IL-23R-Y416FΔICD signaling deficient mice and challenged these mice in close- and open-chest left anterior descending coronary arteria ischemia/reperfusion experiments. Our experiments showed only minimal changes in all assayed parameters in IL-23R signaling deficient mice compared to wild-type mice in ischemia and for up to four weeks of reperfusion, including ejection fraction, endsystolic volume, enddiastolic volume, infarct size, gene regulation and α smooth muscle actin (αSMA) and Hyaluronic acid (HA) protein expression. Moreover, injection of IL-23 in wild-type mice after LAD ischemia/reperfusion had also no influence on the outcome of the healing phase. Our data showed that IL-23R deficiency has no effects in myocardial I/R.

Combined Angiotensin Receptor-Neprilysin Inhibitors Improve Cardiac and Vascular Function Via Increased NO Bioavailability in Heart Failure.

BackgroundThere is a paucity of data about the mechanisms by which sacubitril/valsartan (also known as LCZ696) improves outcomes in patients with heart failure. Specifically, the effects of sacubitril/valsartan on vascular function and NO bioavailability have not been investigated. We hypothesized that sacubitril/valsartan therapy increases circulating NO levels and improves vascular function in the setting of heart failure.Methods and ResultsMale spontaneously hypertensive rats underwent myocardial ischemia/reperfusion surgery to induce heart failure and were followed for up to 12 weeks with serial echocardiography. Rats received sacubitril/valsartan (68 mg/kg), valsartan (31 mg/kg), or vehicle starting at 4 weeks after reperfusion. At 8 or 12 weeks of reperfusion, animals were euthanized and tissues were collected for ex vivo analyses of NO bioavailability, aortic vascular reactivity, myocardial and vascular histology, and cardiac molecular assays. Left ventricular structure and function were improved by both valsartan and sacubitril/valsartan compared with vehicle. Sacubitril/valsartan resulted in superior cardiovascular benefits, as evidenced by sustained improvements in left ventricular ejection fraction and end‐diastolic pressure. Ex vivo vascular function, as measured by aortic vasorelaxation responses to acetylcholine and sodium nitroprusside, was significantly improved by valsartan and sacubitril/valsartan, with more sustained improvements afforded by sacubitril/valsartan. Furthermore, myocardial NO bioavailability was significantly enhanced in animals receiving sacubitril/valsartan therapy.ConclusionsSacubitril/valsartan offers superior cardiovascular protection in heart failure and improves vascular function to a greater extent than valsartan alone. Sacubitril/valsartan‐mediated improvements in cardiac and vascular function are likely related to increases in NO bioavailability and explain, in part, the benefits beyond angiotensin receptor blockade.

CGMP-Elevating Compounds and Ischemic Conditioning Provide Cardioprotection Against Ischemia and Reperfusion Injury via Cardiomyocyte-Specific BK Channels

The nitric oxide-sensitive guanylyl cyclase/cGMP-dependent protein kinase type I signaling pathway can afford protection against the ischemia/reperfusion injury that occurs during myocardial infarction. Reportedly, voltage and Ca2+-activated K+ channels of the BK type are stimulated by cGMP/cGMP-dependent protein kinase type I, and recent ex vivo studies implicated that increased BK activity favors the survival of the myocardium at ischemia/reperfusion. It remains unclear, however, whether the molecular events downstream of cGMP involve BK channels present in cardiomyocytes or in other cardiac cell types. Gene-targeted mice with a cardiomyocyte- or smooth muscle cell-specific deletion of the BK (CMBK or SMBK knockouts) were subjected to the open-chest model of myocardial infarction. Infarct sizes of the conditional mutants were compared with litter-matched controls, global BK knockout, and wild-type mice. Cardiac damage was assessed after mechanical conditioning or pharmacological stimulation of the cGMP pathway and by using direct modulators of BK. Long-term outcome was studied with respect to heart functions and cardiac fibrosis in a chronic myocardial infarction model. Global BK knockouts and CMBK knockouts, in contrast with SMBK knockouts, exhibited significantly larger infarct sizes compared with their respective controls. Ablation of CMBK resulted in higher serum levels of cardiac troponin I and elevated amounts of reactive oxygen species, lower phosphorylated extracellular receptor kinase and phosphorylated AKT levels and an increase in myocardial apoptosis. Moreover, CMBK was required to allow beneficial effects of both nitric oxide-sensitive guanylyl cyclase activation and inhibition of the cGMP-degrading phosphodiesterase-5, ischemic preconditioning, and postconditioning regimens. To this end, after 4 weeks of reperfusion, fibrotic tissue increased and myocardial strain echocardiography was significantly compromised in CMBK-deficient mice. Lack of CMBK channels renders the heart more susceptible to ischemia/reperfusion injury, whereas the pathological events elicited by ischemia/reperfusion do not involve BK in vascular smooth muscle cells. BK seems to permit the protective effects triggered by cinaciguat, riociguat, and different phosphodiesterase-5 inhibitors and beneficial actions of ischemic preconditioning and ischemic postconditioning by a mechanism stemming primarily from cardiomyocytes. This study establishes mitochondrial CMBK channels as a promising target for limiting acute cardiac damage and adverse long-term events that occur after myocardial infarction.

Abstract 141: Inhibition of Coagulation Factor Xa Attenuates Myocardial Ischemia Reperfusion Injury in Mice

Background: Ischemic/reperfusion (I/R) injury substantially effects the outcome of myocardial infarction (MI). Current reperfusion therapy does not sufficiently prevent injury caused by microvascular thrombo-inflammation. Coagulation proteases mediate inflammation via protease activated receptors. FXa induced thrombin generation is the key step in the coagulation cascade. We hypothesize that inhibition of FXa by rivaroxaban attenuates I/R injury after MI. Methods: Male WT c57BL/6 mice (age 8-9 weeks, n=8 per group) underwent surgical ligation of the left anterior descending coronary artery 7 days prior to experimentation. Next, the ligature was tightened for 1h to induce ischemia and loosened either at 4h (early), or at 4 weeks (late), to allow reperfusion. The intervention consisted of 2 rivaroxaban (1.6 mg/kg) i.v.-injections or placebo (0.9%NaCl) after 15min of ischemia and 5min of reperfusion. In the early model. the area at risk (AAR) was visualized through Evans blue and differentiated from the area of infarction (AOI) through triphenyl tetrazolium chloride staining. Plasma cardiovascular markers were quantified using Luminex Multiplex. In the late model, LVEF was measured 10min pre-ischemia and 4 weeks’ post-reperfusion utilizing echocardiography. Results: The rivaroxaban treatment group showed signs of diminished myocardial damage as indicated by reduced median AOI/AAR (41%[IQR34-48] vs. control 62%[IQR52-67] p&lt;0.001). This was supported by a better preserved LVEF after 4 weeks of reperfusion (25%[IQR19-31] vs. control (16%[IQR12-21]). Although not significantly different, plasma E-Selectin, PECAM-1, PAI-1, proMMP9, and thrombomodulin showed a trend to increased levels upon treatment with rivaroxaban. Conclusion: FXa inhibition by rivaroxaban significantly reduces myocardial I/R injury in mice and may provide long term preservation of LVEF. Raised cardiovascular markers suggest increased tissue remodeling and phenotypical alteration of endothelial cells after rivaroxaban treatment. These results suggest that coagulation proteases (i.e. FXa) play a relevant role in I/R injury during MI, most likely through activation of protease activated receptors.

Opening of calcium-activated potassium channels improves long-term left-ventricular function after coronary artery occlusion in mice

BackgroundOpening of mitochondrial calcium-activated potassium channels (BKCa) reduces infarct size after myocardial ischemia/reperfusion injury (I/R). It is unknown if targeting BKCa-channels improves cardiac performance in the long-term after I/R. MethodsExperiments were conducted in compliance with institutional and national guidelines in C57BL/6 mice (n=7–8/group). Animals were randomized into two groups. Preconditioning was induced by intraperitoneal application of NS1619 (NS, 1μg/g bw) 10min before ischemia, control animals (Con) received the vehicle. All animals underwent 45min of myocardial ischemia and four weeks of reperfusion. Transthoracal Echocardiography (TTE) was conducted one and four weeks after ischemia (TTEW1/TTEW4) and additionally a cardiac MRI was done in week four. At the end of experiments the infarction scar was determined by AZAN staining. ResultsTTE revealed that NS1619 improved ejection fraction one week (Con: 36±4%, NS: 45±4%; P<0.05) and four weeks after I/R (Con: 33±11%, NS: 46±8%; P<0.05). Preconditioning with NS1619 reduced end-diastolic volume at both time points (TTEW1: Con: 60±12μl, NS: 45±8μl; TTEW4: Con: 82±31μl, NS: 44±8μl; each P<0.05) and increased fractional shortening after four weeks (TTEW4: Con: 12±6%, NS: 24±8%; P<0.05). MRI-analysis after four weeks confirmed the echocardiographic results. NS1619 increased ejection fraction by 45% (MRI: Con: 29±6%, NS: 42±9%; P<0.05 vs. Con) and reduced end-diastolic and -systolic volume (EDV, ESV) compared to control (MRI: EDV: Con: 110±19μl, NS: 88±16μl; ESV: Con: 79±19μl, NS: 53±18μl; each P<0.05). Preconditioning reduced infarction scar after four weeks by 25% (Con: 12±3%, NS: 9±2%; P<0.05). ConclusionsPreconditioning by opening of BKCa-channels with NS1619 improves cardiac performance after four weeks of reperfusion and reduces myocardial infarction scar.

Morphological regeneration and functional recovery of neuromuscular junction after ischemic/reperfusion injury induced by tourniquet in mouse hind limb

BackgroundTourniquet‐induced ischemia and subsequent reperfusion cause serious acute ischemia‐reperfusion (IR) injury in the neuromuscular junction (NMJ) and skeletal muscle. These injuries are associated with permanent neurological deficiency, and long‐term skeletal muscle contractile dysfunction, even complete limb paralysis. Monitoring structural and functional repair of the NMJ and skeletal muscle after tourniquet‐induced IR is beneficial to exploring the mechanisms responsible for tourniquet‐induced IR injury and finding effective therapeutic interventions. Here, we observed long‐term morphological and functional alterations of the NMJ in tourniquet‐induced hindlimb IR.MethodUnilateral hindlimb of C57/BL6 mice was subjected to 3 hours of ischemia by placing an orthodontic rubber band and followed different periods of reperfusion (1 day, 3 days, 1 weeks, 2 wekks, 4 weeks, and 6 weeks). At each time point of reperfusion, the gastrocnemius muscle was isolated for measurement of the NMJ morphology and function. Motor nerve terminals and nicotinic acetylcholine receptors (nAChRs) in the NMJ were measured by immunofluorescence staining. The end plate potential (EPP) was recorded by the intracellular recording technique.ResultsIn the NMJ of sham mice, the structure of the nAChR clusters remained intact pretzel‐like shape and motor nerve terminals innervated the nAChR clusters to form synapses for signal transmission. The number of fragments in one nAChR cluster was 1.83 ± 0.07 and all nAChR clusters were innervated by motor nerve terminals (100%). The amplitude of EPPs was 29.84 ± 1.02 mV. At 1 day and 3 days of reperfusion, the motor nerve terminals disappeared, the AChR clusters kept normal shape, and no EPPs were recorded. From 1 week to 6 weeks of reperfusion, the motor nerve terminals gradually reinnervated (46.88%, 88.05%, 99.2%, and 100% for 1, 2, 4, and 6 weeks of reperfusion, respectively). However, the AChR clusters were gradually fragmented and the number of fragments in one nAChR cluster increased with longer period of reperfusion (2.92 ± 0.14, 5.50 ± 0.30, 8.94 ± 0.67, and 6.88 ± 0.50 for for 1, 2, 4, and 6 weeks of reperfusion, respectively; p&lt;0.05 vs. sham). The amplitude of EPPs also increased gradually with longer period of reperfusion, but the amplitude of EPPs at 6 weeks of reperfusion didn't restore towards the level seen in sham mice (13.92 ± 1.11 mV, p&lt;0.05 vs. sham).ConclusionTourniquet‐induced ischemia and subsequent reperfusion impaired the NMJ. The morphological change of motor nerve terminals was faster than that of the nAChR clusters. The slow recovery of fragmented nAChR clusters could attenuate neuromuscular transmission.

Nitrite Therapy Ameliorates Myocardial Dysfunction via H 2 S and Nuclear Factor‐Erythroid 2‐Related Factor 2 (Nrf2)‐Dependent Signaling in Chronic Heart Failure

BackgroundBioavailability of nitric oxide (NO) and hydrogen sulfide (H2S) is reduced in heart failure (HF). Recent studies suggest cross‐talk between NO and H2S signaling. We previously reported that sodium nitrite (NaNO 2) ameliorates myocardial ischemia‐reperfusion injury and HF. Nuclear factor‐erythroid‐2‐related factor 2 (Nrf2) regulates the antioxidant proteins expression and is upregulated by H2S. We examined the NaNO 2 effects on endogenous H2S bioavailability and Nrf2 activation in mice subjected to ischemia‐induced chronic heart failure (CHF).Methods and ResultsMice underwent 60 minutes of left coronary artery occlusion and 4 weeks of reperfusion. NaNO 2 (165 μg/kgic) or vehicle was administered at reperfusion and then in drinking water (100 mg/L) for 4 weeks. Left ventricular (LV), ejection fraction (EF), LV end diastolic (LVEDD) and systolic dimensions (LVESD) were determined at baseline and at 4 weeks of reperfusion. Myocardial tissue was analyzed for oxidative stress and respective gene/protein‐related assays. We found that NaNO 2 therapy preserved LVEF, LVEDD and LVSD at 4 weeks during ischemia‐induced HF. Myocardial malondialdehyde and protein carbonyl content were significantly reduced in NaNO 2‐treated mice as compared to vehicle, suggesting a reduction in oxidative stress. NaNO 2 therapy markedly increased expression of Cu,Zn‐superoxide dismutase, catalase, and glutathione peroxidase during 4 weeks of reperfusion. Furthermore, NaNO 2 upregulated the activity of Nrf2, as well as H2S‐producing enzymes, and ultimately increased H2S bioavailability in ischemia‐induced CHF in mice as compared with vehicle.ConclusionsOur results demonstrate that NaNO 2 therapy significantly improves LV function via increasing H2S bioavailability, Nrf2 activation, and antioxidant defenses.

Abstract 233: DJ-1 Gene Therapy Opposes the Development of Ischemic Heart Failure

Background: DJ-1 is a cytoprotective protein that is activated in response to various pathological stimuli. The ability of DJ-1 to perform its cellular actions is dependent on a conformational change, as evidenced by previous findings that it is cleaved by oxidative stress and that this cleavage activates its cytoprotective function. Previously, we demonstrated that the loss of functional DJ-1 lead to the enhancement of myocardial infarction and exacerbation of cardiac dysfunction following the onset of acute myocardial ischemia-reperfusion (I/R) injury. Currently, it is not known if the gain of functional DJ-1 has cardioprotective effects. Here, we investigated if the delivery of the cleaved form of DJ-1 using a gene therapy approach protected against the development of ischemic-induced heart failure. Methods and Results: We generated a viral vector to express the active form of DJ-1 (AAV9-DJ1WTΔC). To test the efficiency, specificity, and dose-dependence of AAV9-mediated transduction, we injected increasing doses of AAV9-GFP into the femoral vein of mice. A dose-dependent increase in GFP levels was observed in the heart and our analysis found that the 2x10 11 vg/mL dose achieved maximal delivery to the heart with minimal delivery to other tissue. GFP-stained sections of heart indicated that a substantial proportion of cardiomyocytes expressed the transduced gene. Next, we evaluated the protein expression of the full-length and active form of DJ-1 2 weeks after the administration of AAV9-DJ1WTΔC. No changes in the expression of the full-length form of DJ-1 were observed, but a 2-fold increase in the active form of DJ-1 was observed. Finally, mice administered AAV9-DJ1WTΔC displayed significant less left ventricular dilatation, left ventricular dysfunction, and hypertrophy compared to the AAV9-Empty treated following mice 60 minutes of myocardial ischemia and 4 weeks of reperfusion. Conclusion: These results indicated that DJ-1 is an endogenous cytoprotective protein that protects against the development of ischemic heart failure. Importantly, these results demonstrate the feasibility of using a gene therapy approach to deliver the cleaved form of DJ-1 to the heart as a therapeutic strategy to protect against ischemic heart disease.