Abstract 14412: Activation of Autophagic Flux Maintains Mitochondrial Homeostasis During Cardiac Ischemia/reperfusion Injury

Background and Hypothesis: Reperfusion injury accounts for ~50% of myocardial infarct size, and clinically efficacious therapies are lacking. Histone deacetylase (HDAC) inhibition enhances cardiomyocyte autophagic activity, mitochondria biogenesis, and blunts ischemia/reperfusion (I/R) injury when given at the time of reperfusion. However, as HDAC inhibition has pleiotropic effects, we will test whether augmentation of autophagic flux using a specific autophagy-inducing peptide, Tat-Beclin (TB), is cardioprotective. Methods: 8-12-week-old, wild-type, C57BL6 mice were randomized into three groups: vehicle control, Tat-Scrambled (TS) peptide, or Tat-Beclin (TB) peptide. Each group was subjected to I/R surgery (45min ischemia, 24h reperfusion). Infarct size, systolic function, and mitochondrial dynamics were assayed. Cultured neonatal rat ventricular myocytes (NRVMs) were used to test for cardiomyocyte specificity. Conditional cardiomyocyte ATG7 knockout (ATG7 KO) mice and ATG7 knockdown by siRNA in NRVMs were used to evaluate the role of autophagy. Results: TB treatment at reperfusion reduced infarct size by 20.1±6.3% (n=23, p<0.02) and improved systolic function. Increased autophagic flux and reduced reactive oxygen species (ROS) were observed in the infarct border zone. The cardioprotective effects of TB were abolished in ATG7 KO mice. TB increased mtDNA content in the border zone significantly. In NRVMs subjected to I/R, TB reduced cell death by 41±6% (n=12, p<0.001), decreased ROS, and increased mtDNA content significantly by ~50%. Moreover, TB promoted expression of PGC1α (a major driver of mitochondrial biogenesis) both in the infarct border zone and NRVMs subjected to I/R by ~40%, and increased levels of mitochondrial dynamics gene transcripts Drp1, Fis1, and MFN1 / 2. Conversely, ATG7 knockdown in NRVMs and cardiac ATG7 KO abolished the beneficial effects of TB on mitochondria DNA content. Conclusions: Autophagic flux is an essential process to mitigate myocardial reperfusion injury acting, at least in part, by inducing PGC1α-mediated mitochondrial biogenesis. Augmentation of autophagic flux may emerge as a viable clinical therapy to reduce reperfusion injury in myocardial infarction.

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Activation of Autophagic Flux Blunts Cardiac Ischemia/Reperfusion Injury

Circulation Research ◽

10.1161/circresaha.120.318601 ◽

2021 ◽

Author(s):

Min Xie ◽

Geoffrey W Cho ◽

Yongli Kong ◽

Dan L Li ◽

Francisco Altamirano ◽

...

Keyword(s):

Cell Death ◽

Infarct Size ◽

Reperfusion Injury ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Neonatal Rat ◽

Border Zone ◽

Relative Reduction ◽

Autophagic Flux ◽

Hdac Inhibition

Rationale: Reperfusion injury accounts for up to half of myocardial infarct size, and meaningful clinical therapies targeting it do not exist. We have reported previously that autophagy is reduced during reperfusion and that HDAC inhibition enhances cardiomyocyte autophagy and blunts ischemia/reperfusion (I/R) injury when administered at the time of reperfusion. However, whether inducing autophagy per se, as opposed to other effects triggered by HDAC inhibition, is sufficent to protect against reperfusion injury is not clear. Objective: We set out to test whether augmentation of autophagy using a specific autophagy-inducing peptide, Tat-Beclin, protects the myocardium through reduction of reactive oxygen species (ROS) during reperfusion injury. Methods and Results: Eight to twelve-week-old, wild-type, C57BL6 mice and drug-inducible cardiomyocyte-specific ATG7 knockout mice (to test the dependency on autophagy) were randomized into two groups: exposed to a control Tat-Scrambled (TS) peptide or a Tat-Beclin (TB) peptide. Each group was subjected to I/R surgery (45min coronary ligation, 24h reperfusion). Infarct size, systolic function, autophagic flux, and ROS were assayed. Cultured neonatal rat ventricular myocytes (NRVMs) were exposed to TB during simulated ischemia/reperfusion injury. ATG7 knockdown by siRNA in NRVMs was used to evaluate the role of autophagy. TB treatment at reperfusion reduced infarct size by 20% (absolute reduction; 50% relative reduction) and improved contractile function. Improvement correlated with increased autophagic flux in the border zone with less oxidative stress. ATG7 KO mice did not manifest TB-promoted cardioprotection during I/R. In NRVMs subjected to I/R, TB reduced cell death by 41% and reduced I/R-induced ROS generation. Conversely, ATG7 knockdown in NRVMs abolished these beneficial effects of TB on cell death and ROS reduction. Conclusions: Induction of autophagy at the time of reperfusion is sufficient to mitigate myocardial reperfusion injury by reducing ROS and cell death. Maintenance of appropriate autophagic flux may emerge as a viable clinical therapy to reduce reperfusion injury in acute myocardial infarction.

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Abstract 342: Cortical Bone Stem Cell Therapy Alters Macrophage Phenotype and Reduces Cardiac Cell Death After Myocardial Infarction

Circulation Research ◽

10.1161/res.127.suppl_1.342 ◽

2020 ◽

Vol 127 (Suppl_1) ◽

Author(s):

Alexander R Hoachlandr-Hobby ◽

Remus M Berretta ◽

Yijun Yang ◽

Eric Feldsott ◽

Hajime Kubo ◽

...

Keyword(s):

Myocardial Infarction ◽

Immune Response ◽

Infarct Size ◽

Reperfusion Injury ◽

Cortical Bone ◽

Myocardial Injury ◽

Border Zone ◽

Paracrine Factors ◽

Infarct Border Zone ◽

Infarct Border

Acute injuries to the heart, like myocardial infarction (MI), contribute to the development and pathology of heart failure (HF). Reperfusion of the ischemic heart greatly increases survival but results in reperfusion injury that can account for up to 50% of the final infarct size. The inflammatory response to MI-induced myocardial injury is thought to be responsible for the propagation of reperfusion injury into the infarct border zone, expanding myocardial damage. We have previously shown in a swine model of MI that intramyocardial injections of cortical bone-derived stem cells (CBSCs) into the infarct border zone has no acute cardioprotective effect but reduces scar size by half and prevents the decline of ejection fraction and LV dilation 3 months after MI. Our new preliminary data show that CBSCs have potent immunoregulatory capabilities. Therefore, we hypothesize that CBSC treatment has an effect on the immune response to MI that improves the wound healing response to myocardial injury and mitigates LV remodeling and infarct size 3 months later. To test this hypothesis, we characterized the effects of CBSC paracrine factors on macrophages in vitro and found that CBSC-treated macrophages express higher levels of CD206, produce more IL-1RA and IL-10, and phagocytose apoptotic myocytes more efficiently. In addition, macrophages were increased in CBSC-treated swine hearts 7 days after MI compared to controls with a corresponding increase in IL-1RA and TIMP-2. Apoptosis was decreased overall and in macrophages specifically in CBSC-treated animals. From these data we conclude CBSCs may exert an acute pro-reparative effect on the immune response after MI, reducing reperfusion injury and adverse remodeling resulting in improved functional outcomes at later time points.

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Abstract 357: Activation of Autophagic Flux Blunts Cardiac Ischemia/Reperfusion Injury

Circulation Research ◽

10.1161/res.121.suppl_1.357 ◽

2017 ◽

Vol 121 (suppl_1) ◽

Author(s):

Geoffrey W Cho ◽

Min Xie ◽

Yongli Kong ◽

Dan L Li ◽

Xiang L Luo ◽

...

Keyword(s):

Oxidative Stress ◽

Infarct Size ◽

Reperfusion Injury ◽

Molecular Mechanisms ◽

Contractile Function ◽

Ischemia Reperfusion ◽

Neonatal Rat ◽

Border Zone ◽

Autophagic Flux ◽

Cardiomyocyte Autophagy

Background: Reperfusion injury accounts for a significant portion of myocardial damage in acute coronary syndromes. Autophagy, a process of cell catabolism, plays a vital role in the heart’s response to stress. We have reported that re-induction of ischemia/reperfusion (I/R)-suppressed cardiomyocyte autophagy with histone deacetylase (HDAC) inhibitors affords significant cardioprotection. However, as HDACs govern many processes and may have off-target effects, we set out to modulate autophagy in a manner independent of HDAC activity. Here, we hypothesized that induction of autophagy with a novel agent, Tat-Beclin, at the time of reperfusion, will reduce I/R injury and rescue cardiac function. Methods: Wild type and ATG7 (protein required for autophagic flux) knockout mice were randomized among 3 treatment groups prior to surgical I/R injury [45 min LAD artery ligation; 24h reperfusion]: vehicle control (VC), Tat-Scrambled (TS), or Tat-Beclin (TB). Each agent was delivered at coronary reperfusion. To define molecular mechanisms, cultured adult and neonatal rat ventricular cardiomyocytes (ARVMs/NRVMs) were subjected to simulated I/R. Results: Induction of cardiomyocyte autophagy at reperfusion reduced infarct size 20.1% (±6.3%, n=23, p<0.02 vs VC). This treatment was associated with improved systolic function (declines in fractional shortening: 19.8±3.7% VC; 18.7±2.1% TS; 8.5±1.7% TB, n=11, p<0.01 vs VC). In NRVMs subjected to I/R injury, cell death was reduced 41% (±6%, n=12, p<0.001 vs VC). Improvements correlated with increased autophagic flux measured by the marker LC3-II, particularly at the infarct border zone. Additional data suggested that autophagy rescues I/R injury through reduction of oxidative stress. ATG7 KO mice or NRVM depleted of ATG7 (RNAi) manifested significantly less cardioprotection. Conclusion: Direct induction of cardiomyocyte autophagy reduces infarct size and declines in contractile function. Autophagy rescues I/R injury in part through reduction of oxidative stress. Critically, this cardioprotection was observed when intervention occurred at the time of reperfusion, the clinically relevant context.

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MK5 haplodeficiency decreases collagen deposition and scar size during post-myocardial infarction wound repair

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00532.2017 ◽

2019 ◽

Vol 316 (6) ◽

pp. H1281-H1296 ◽

Cited By ~ 2

Author(s):

Sherin Ali Nawaito ◽

Pramod Sahadevan ◽

Marie-Élaine Clavet-Lanthier ◽

Philippe Pouliot ◽

Fatiha Sahmi ◽

...

Keyword(s):

Myocardial Infarction ◽

Infarct Size ◽

Collagen Content ◽

Post Myocardial Infarction ◽

Border Zone ◽

Wild Type ◽

Serine Threonine Kinase ◽

Threonine Kinase ◽

Infarct Border Zone ◽

Infarct Border

MK5 is a protein serine/threonine kinase activated by p38, ERK3, and ERK4 MAPKs. MK5 mRNA and immunoreactivity are detected in mouse cardiac fibroblasts, and MK5 haplodeficiency attenuates the increase in collagen 1-α1 mRNA evoked by pressure overload. The present study examined the effect of MK5 haplodeficiency on reparative fibrosis following myocardial infarction (MI). Twelve-week-old MK5+/− and wild-type littermate (MK5+/+) mice underwent ligation of the left anterior descending coronary artery (LADL). Surviving mice were euthanized 8 or 21 days post-MI. Survival rates did not differ significantly between MK5+/+ and MK5+/− mice, with rupture of the LV wall being the primary cause of death. Echocardiographic imaging revealed similar increases in LV end-diastolic diameter, myocardial performance index, and wall motion score index in LADL-MK5+/+ and LADL-MK5+/− mice. Area at risk did not differ between LADL-MK5+/+ and LADL-MK5+/− hearts. In contrast, infarct size, scar area, and scar collagen content were reduced in LADL-MK5+/− hearts. Immunohistochemical analysis of mice experiencing heart rupture revealed increased MMP-9 immunoreactivity in the infarct border zone of LADL-MK5+/− hearts compared with LADL-MK5+/+. Although inflammatory cell infiltration was similar in LADL-MK5+/+ and LADL-MK5+/− hearts, angiogenesis was more pronounced in the infarct border zone of LADL-MK5+/− mice. Characterization of ventricular fibroblasts revealed reduced motility and proliferation in fibroblasts isolated from MK5−/− mice compared with those from both wild-type and haplodeficient mice. siRNA-mediated knockdown of MK5 in fibroblasts from wild-type mice also impaired motility. Hence, reduced MK5 expression alters fibroblast function and scar morphology but not mortality post-MI. NEW & NOTEWORTHY MK5/PRAK is a protein serine/threonine kinase activated by p38 MAPK and/or atypical MAPKs ERK3/4. MK5 haplodeficiency reduced infarct size, scar area, and scar collagen content post-myocardial infarction. Motility and proliferation were reduced in cultured MK5-null cardiac myofibroblasts.

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Electromechanical analysis of infarct border zone in chronic myocardial infarction

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00423.2005 ◽

2005 ◽

Vol 289 (3) ◽

pp. H1099-H1105 ◽

Cited By ~ 52

Author(s):

Hiroshi Ashikaga ◽

Steven R. Mickelsen ◽

Daniel B. Ennis ◽

Ignacio Rodriguez ◽

Peter Kellman ◽

...

Keyword(s):

Myocardial Infarction ◽

Systolic Function ◽

Left Ventricular ◽

Border Zone ◽

Electrical Activation ◽

Mapping Technique ◽

Infarct Zone ◽

Infarct Border Zone ◽

Longitudinal Strains ◽

Infarct Border

To test the hypothesis that alterations in electrical activation sequence contribute to depressed systolic function in the infarct border zone, we examined the anatomic correlation of abnormal electromechanics and infarct geometry in the canine post-myocardial infarction (MI) heart, using a high-resolution MR-based cardiac electromechanical mapping technique. Three to eight weeks after an MI was created in six dogs, a 247-electrode epicardial sock was placed over the ventricular epicardium under thoracotomy. MI location and geometry were evaluated with delayed hyperenhancement MRI. Three-dimensional systolic strains in epicardial and endocardial layers were measured in five short-axis slices with motion-tracking MRI (displacement encoding with stimulated echoes). Epicardial electrical activation was determined from sock recordings immediately before and after the MR scans. The electrodes and MR images were spatially registered to create a total of 160 nodes per heart that contained mechanical, transmural infarct extent, and electrical data. The average depth of the infarct was 55% (SD 11), and the infarct covered 28% (SD 6) of the left ventricular mass. Significantly delayed activation (>mean + 2SD) was observed within the infarct zone. The strain map showed abnormal mechanics, including abnormal stretch and loss of the transmural gradient of radial, circumferential, and longitudinal strains, in the region extending far beyond the infarct zone. We conclude that the border zone is characterized by abnormal mechanics directly coupled with normal electrical depolarization. This indicates that impaired function in the border zone is not contributed by electrical factors but results from mechanical interaction between ischemic and normal myocardium.

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Abstract 848: Growth-differentiation Factor-15 Promotes Anti-inflammatory Effects And Enhances Survival After Experimental Myocardial Infarction

Circulation ◽

10.1161/circ.116.suppl_16.ii_165-a ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Tibor Kempf ◽

Christian Widera ◽

Udo Bavendiek ◽

Christian Willenbockel ◽

Mortimer Korf-Klingebiel ◽

...

Keyword(s):

Myocardial Infarction ◽

Wound Healing ◽

Systolic Function ◽

Border Zone ◽

Rank Test ◽

Coronary Syndrome ◽

Differentiation Factor ◽

Anti Inflammatory ◽

Infarct Border Zone ◽

Infarct Border

Growth-differentiation factor (GDF)-15 is a stress-responsive TGF-ß related cytokine. GDF-15 plasma levels are increased and provide independent prognostic information in patients with acute coronary syndrome. After coronary ligation in mice, GDF-15 protein expression is rapidly induced and remains elevated within the infarcted myocardium for at least 7 days. We therefore postulated that GDF-15 is involved in wound healing post MI. The left descending coronary artery was ligated in male GDF-15 knock-out (KO, n=36), heterozygous (+/-, n=72) and wild-type mice (WT, n=26). Six-week mortality was significantly enhanced in KO (81%) and +/- (67%), as compared to WT mice (35%, P=0.004 by log-rank test). Four days after MI, no differences in infarct size (H.E. staining), cardiac dimensions, wall thickness, and systolic function (echocardiography) were observed (n=8 –12 per group). However, cardiac rupture was more frequent in KO (31%) as compared to WT mice (4%, P<0.01). Immunohistochemistry on day 4 demonstrated significantly elevated numbers of granulocytes (KO, 514±61/mm 2 vs. WT, 181±22/mm 2 ; P=0.003) and macrophages (KO, 176±19/mm 2 vs. WT, 89±11/mm 2 ; P=0.002) within the infarct border zone of KO mice (n=8–12 per group). This was paralleled by an increase in MMP-9 activity (in-gel zymography) within the infarct border zone (KO, 246±33% vs. WT, 100±14%; n=8–10; P=0.004); no differences in TIMP1 expression were noted between both genotypes. In vitro, pre-stimulation with GDF-15 dose-dependently inhibited the migration of human granulocytes towards the chemoattractant fMLP (10 −8 mol/L) in transwell migration chambers (3 μm pore size), control 100%; GDF-15 0.5ng/mL 78±5%, 5ng/mL 47±10%, 50ng/mL 34±10% (P<0.001). Our data identify GDF-15 as an endogenous anti-inflammatory cytokine that inhibits leukocyte infiltration, and promotes wound healing and survival after myocardial infarction.

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Cardiac MRI of myocardial salvage at the peri-infarct border zones after primary coronary intervention

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00011.2009 ◽

2009 ◽

Vol 297 (1) ◽

pp. H340-H346 ◽

Cited By ~ 37

Author(s):

Declan P. O'Regan ◽

Rizwan Ahmed ◽

Clare Neuwirth ◽

Yvonne Tan ◽

Giuliana Durighel ◽

...

Keyword(s):

Infarct Size ◽

Cardiac Mri ◽

Coronary Intervention ◽

Border Zone ◽

Wall Thickening ◽

Myocardial Salvage ◽

Border Zones ◽

The Mean ◽

Infarct Border Zone ◽

Infarct Border

The purpose of this study was to use cardiac MRI to define the morphology of the reversibly injured peri-infarct border zone in patients treated with primary percutaneous coronary intervention (PPCI) for acute ST elevation myocardial infarction. In 15 patients, T2-weighted myocardial edema imaging was used to identify the ischemic bed or area at risk (AAR), and late gadolinium enhancement imaging was used to measure infarct size. Images were coregistered, and the boundaries of edema and necrosis were defined using an edge-detection methodology. We observed that infarction always involved the subendocardium but showed variable transmural extension within the AAR. The mean infarct size was 22 ± 19% (range: 8–48%), and the mean AAR was 34 ± 12% (range: 20–57%). The infarcted myocardium was always smaller than the ischemic AAR and involved between 34% and 99% (mean 72 ± 21%) of the ischemic bed primarily due to variation in transmural infarct extension. Although a lateral border zone of potentially viable myocardium was often present, its extent was limited (range: 0–11 mm, mean: 5 ± 4 mm). As a result of this, infarcts occupied the majority (range: 70–100%, mean: 82 ± 13%) of the width of the AAR. The mean fractional wall thickening in the infarcted, peri-infarcted, and remote myocardium was 3.6 ± 16.0%, 40.5 ± 26.4%, and 88.2 ± 39.3%, respectively. These findings demonstrate that myocardial salvage is largely determined by epicardial limitation of the infarct within the ischemic AAR after PPCI. The lateral boundaries of necrosis approximate to the lateral extent of the ischemic bed and systolic wall motion abnormalities extend well beyond the infarct border zone.

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Abstract 4117: A Comparison of Cardiac MRI Peri-Infarct Border Zone Quantification Strategies for the Prediction of Ventricular Tachycardia Inducibility

Circulation ◽

10.1161/circ.118.suppl_18.s_832-c ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Jason C Rubenstein ◽

Daniel Lee ◽

Edwin Wu ◽

Alan Kadish ◽

Rod S Passman ◽

...

Keyword(s):

Infarct Size ◽

Cardiac Mri ◽

Independent Predictor ◽

Electrophysiological Study ◽

Border Zone ◽

Lv Mass ◽

Risk Stratification Tool ◽

The Mean ◽

Infarct Border Zone ◽

Infarct Border

Peri-infarct border zone (BZ) as quantified by delayed-enhancement cardiac MRI (CMR) has been proposed as a risk stratification tool, and is associated with increased mortality, but has been measured by various methods in the literature. We assessed which BZ analysis best predicts inducible arrhythmia during electrophysiological study (EPS). Methods: CMR was performed in 47 patients with CAD referred for EPS to assess for ventricular tachyarrhythmias (VT). CMR data was analyzed for BZ quantification by three methods. Method I used pixels 2–3 standard deviations over the mean of normal tissue, expressed as % of LV mass. Method II (Yan) used the same pixels but is expressed as % of infarct size. Method III (Schmidt) used a full-width, half-max method. EPS results were classified as negative (non-inducible) or positive (monomorphic VT). Patients with indeterminate EPS outcomes (VF or polymorphic VT, n=9) were not included in this analysis. Results: There were 38 subjects-age 60.2 ± 10.8 yrs, 68% male. During EPS, 20 patients were non-inducible and 18 had induced monomorphic VT (MVT). EF was not significantly different between non-inducible patients and those with MVT (33.4 ± 11.9% vs. 28.1 ± 9.3, p=0.13). Infarct size was significantly different (15.0 ± 11.6% vs. 26.4 ± 12.0%, p = 0.005). BZ by method I was significantly different (1.4 ± 1.3% vs. 3.1 ± 1.5%, p = 0.001), but not by method II (15.2 ± 21.3% vs. 8.0 ± 4.8%, p = 0.17) or method III (4.1 ± 4.5g vs. 3.7 ± 3.7g, p = 0.756). Multivariate analysis demonstrated that BZ by method I was an independent predictor of EPS outcome after controlling for infarct size (OR 1.97 per % change, 95% CI 1.04 to 3.73, p = 0.04). Method I BZ remained an independent predictor when controlling for both infarct size and EF, (OR 2.12 per % change, 95% CI 1.06 to 34.22, p = 0.03). Correlation between techniques was variable (Method I vs. Method II R=0.77, p=0.001; Method I vs. Method III R=0.03, p=0.86; Method II vs. Method III R=0.15, p<0.32). Conclusion: This study demonstrates significant variability between the published methods for measuring BZ. We also show that large BZ by Method I is a stronger predictor of inducible MVT during EPS than EF and infarct size. BZ may be yet another CMR marker of elevated risk of arrhythmia, but requires more rigorous definition.

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