Permanent Myocardial Infarction Research Articles

Sex Differences in Autoimmune Signatures After Myocardial Infarction in Mice

Myocardial infarction (MI) is caused by ischemic injury to the left ventricle (LV), leading to an acute inflammatory response, scar formation, and loss of LV function. The role of the antibody-mediated response and autoimmune (AI) development during MI is not well understood. AI disease is highly prevalent in females, which increases risk of MI and worsens MI outcomes. Thus, we hypothesized that females would exhibit increased susceptibility to MI-induced development of AI signatures, including B cell activation and autoantibody (AAB) production. Permanent MI was induced by left coronary artery ligation in adult male and female C57BL/6J mice, which were followed for 7, 28, or 56 days, using no MI (D0) as controls. LV function was tracked using echocardiography (VEVO 3100). LV infarct cytokines were measured by qPCR. LV infarct and blood leukocytes were measured by flow cytometry. LV IgG and IgM antibody deposition was measured by immunofluorescence. Plasma AABs (IgG and IgM subclasses) were measured with an autoantigen (AAN) microarray (128 AANs). In both sexes, MI led to progressive LV hypertrophy, increased end-diastolic diameter and volume, anterior/posterior wall thinning, and decreased ejection fraction. Survival was significantly higher in females for each time point (100% vs 50% at D7, 83% vs 57% at D28, 94% vs 39% at D56). MI increased spleen mass/tibia length in D7, 28, and 56 females, but decreased spleen mass in D28 and 56 males. LV inflammatory cytokines (IL-1β, IL-6, IL-18, IFN-γ, TNF, CCL2, IL-10) were increased at D7. In both sexes, IL-6 and IL-18 remained elevated at D28 and 56. Blood and LV myeloid cells were increased at D7 in both sexes, while B and T cells were decreased. At D28, myeloid cells returned to D0 levels; LV activated B cells (CD80+) and T cells (CD3+) were increased. At D56, only B cells remained elevated in the LV. IgG deposition was increased in the female LV at D7 (14.1±1.2% area fraction vs 6.3±1.5% D0) and further at D28 (40.4±3.6%), but only at D28 in males (20.4±4.0% vs 3.1±0.7% D0). Both sexes exhibited increased LV IgM at D7 and 28, however, the IgM response at D7 was higher in females (21.1±2.8% vs 8.3±1.6% in males). In plasma, MI increased AABs against several AANs (p<0.05 vs D0). Compared to D0, D7 females had elevated IgG AABs 1 AAN (dsDNA, a classical AI marker) vs 0 for males, and elevated IgM AABs against 4 different AANs versus 1 for males. D28 females had elevated IgG AABs against 1 AAN vs 4 for males, and elevated IgM AABs against 5 AANs vs 0 in males. D56 females had elevated IgG AABs against 34 AANs vs 9 in males, and elevated IgM AABs against 30 AANs versus 1 for males. For both IgG and IgM AABs in D56 females, several were against AANs highly expressed in the heart, including ACE2, mitochondrion, and myosin. Furthermore, D56 female IgM AABs were increased against IL-6 (9.8±0.8 fold change), which as mentioned earlier, remained elevated in the LV at all post-MI time points. In summary, MI promotes development of AI signatures, including LV B and T cell activation, IgG and IgM antibody deposition, and increased circulating AABs, which is exacerbated in females. NHLBI R01 HL166737 (Mouton), AHA Career Development Award CDA856365 (Mouton), NIH/NHLBI R00 HL146888 (Taylor), U54 HL169191 (Taylor), NIGMS P20 GM104357 (Hall), P30 GM149404 (Hall), U54 GM115428 (Hall), NHLBI R01 HL1630376 (da Silva), NIDDK R01 DK121411 (do Carmo), AHA Postdoctoral Fellowship 835218 (Omoto). 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.

Donepezil attenuates progression of cardiovascular remodeling and improves prognosis in spontaneously hypertensive rats with chronic myocardial infarction.

The acetylcholinesterase inhibitor donepezil restores autonomic balance, reduces inflammation, and improves long-term survival in rats with chronic heart failure (CHF) following myocardial infarction (MI). As arterial hypertension is associated with a significant risk of cardiovascular death, we investigated the effectiveness of donepezil in treating CHF in spontaneously hypertensive rats (SHR). CHF was induced in SHR by inducing permanent MI. After 2 weeks, the surviving SHR were randomly assigned to sham-operated (SO), untreated (UT), or oral donepezil-treated (DT, 5 mg/kg/day) groups, and various vitals and parameters were monitored. After 7 weeks of treatment, heart rate and arterial hypertension reduced significantly in DT rats than in UT rats. Donepezil treatment improved 50-day survival (41% to 80%, P = 0.004); suppressed progression of cardiac hypertrophy, cardiac dysfunction (cardiac index: 133 ± 5 vs. 112 ± 5 ml/min/kg, P < 0.05; left ventricular end-diastolic pressure: 12 ± 3 vs. 22 ± 2 mmHg, P < 0.05; left ventricular +dp/dtmax: 5348 ± 338 vs. 4267 ± 114 mmHg/s, P < 0.05), systemic inflammation, and coronary artery remodeling (wall thickness: 26.3 ± 1.4 vs. 34.7 ± 0.7 μm, P < 0.01; media-to-lumen ratio: 3.70 ± 0.73 vs. 8.59 ± 0.84, P < 0.001); increased capillary density; and decreased plasma catecholamine, B-type natriuretic peptide, arginine vasopressin, and angiotensin II levels. Donepezil treatment attenuated cardiac and coronary artery remodeling, mitigated cardiac dysfunction, and significantly improved the prognosis of SHR with CHF.

Danhong Injection Up-regulates miR-125b in Endothelial Exosomes and Attenuates Apoptosis in Post-Infarction Myocardium.

To investigate the involvement of endothelial cells (ECs)-derived exosomes in the anti-apoptotic effect of Danhong Injection (DHI) and the mechanism of DHI-induced exosomal protection against postinfarction myocardial apoptosis. A mouse permanent myocardial infarction (MI) model was established, followed by a 14-day daily treatment with DHI, DHI plus GW4869 (an exosomal inhibitor), or saline. Phosphate-buffered saline (PBS)-induced ECs-derived exosomes were isolated, analyzed by miRNA microarray and validated by droplet digital polymerase chain reaction (ddPCR). The exosomes induced by DHI (DHI-exo), PBS (PBS-exo), or DHI+GW4869 (GW-exo) were isolated and injected into the peri-infarct zone following MI. The protective effects of DHI and DHI-exo on MI hearts were measured by echocardiography, Masson's trichrome staining, and TUNEL apoptosis assay. The Western blotting and quantitative reverse transcription PCR (qRT-PCR) were used to evaluate the expression levels of miR-125b/p53-mediated pathway components, including miR-125b, p53, Bak, Bax, and caspase-3 activities. DHI significantly improved cardiac function and reduced infarct size in MI mice (P<0.01), which was abolished by the GW4869 intervention. DHI promoted the exosomal secretion in ECs (P<0.01). According to the results of exosomal miRNA microarray assay, 30 differentially expressed miRNAs in the DHI-exo were identified (28 up-regulated miRNAs and 2 down-regulated miRNAs). Among them, DHI significantly elevated miR-125b level in DHI-exo and DHI-treated ECs, a recognized apoptotic inhibitor impeding p53 signaling (P<0.05). Remarkably, treatment with DHI and DHI-exo attenuated apoptosis, elevated miR-125b expression level, inhibited capsase-3 activity, and down-regulated the expression levels of proapoptotic effectors (p53, Bak, and Bax) in post-MI hearts, whereas these effects were blocked by GW4869 (P<0.05 or P<0.01). DHI and DHI-induced exosomes inhibited apoptosis, promoted the miR-125b expression level, and regulated the p53 apoptotic pathway in post-infarction myocardium.

Twelve-week high-fat diet for improving adverse ventricular remodeling post-myocardial infarction by alleviating local inflammation.

Many studies have examined how to achieve better outcomes in myocardial infarction (MI) patients with mild obesity or who are overweight. However, the influence of a high-fat diet (HFD) and the underlying mechanisms by which it can affect ventricular remodeling following MI are poorly understood. This study investigated the impact of a 12-week HFD on the left ventricular (LV) remodeling of permanent MI models and immune cell involvement. Male C57BL/6J mice were fed HFD or normal diet (ND). After 8 weeks of feeding, mice underwent cardiac left anterior descending coronary artery ligation, and the same diet was continued for a further 4 weeks. Cardiac structure and function were detected using echocardiography. Cardiac fibrosis was evaluated using histological staining at 7 and 28 days post-MI. Infiltration of various immune cells was examined using flow cytometry and immunofluorescence at 7 days post-MI. Compared with a ND, the 12-week HFD feeding significantly alleviated ventricular remodeling following MI. HFD mice showed reduced infiltration of neutrophils, a higher proportion of M2/M1 macrophages, decreased conventional and monocyte-derived dendritic cells (moDCs) in the injured myocardium, and elevated levels of regulatory T cells (Tregs). Further investigation of dendritic cells (DCs) phenotypes indicated downregulated expression of major histocompatibility complex class II (MHCII). It also showed costimulatory molecules CD40 and CD86 on conventional and moDCs in mediastinal lymph nodes (mLNs). This study demonstrated the protective effect of a 12-week HFD on ventricular remodeling following MI via the alleviation of local inflammation.

Haematopoietic and cardiac GPR55 synchronize post-myocardial infarction remodelling

While classical cannabinoid receptors are known to crucially impact on myocardial infarction (MI) repair, a function of the cannabinoid-sensitive receptor GPR55 herein is poorly understood. We investigated the role of GPR55 in cardiac physiology and post-MI inflammation and remodelling. Global GPR55−/− and wildtype (WT) mice were basally characterized or assigned to 1, 3 or 28 days permanent MI and subsequently analysed via pro-inflammatory and pro-hypertrophic parameters. GPR55−/− deficiency was basally associated with bradycardia, increased diastolic LV volume and sarcomere length and a subtle inflammatory phenotype. While infarct size and myeloid cell infiltration were unaffected by GPR55 depletion, acute cardiac chemokine production was prolonged post-MI. Concurrently, GPR55−/− hearts exhibited a premature expansion of pro-reparative and phagocytic macrophages paralleled by early up-regulation of extracellular matrix (ECM) factors 3 days post-MI, which could be mimicked by sole haematopoietic GPR55 depletion. Moreover, global GPR55 deficiency mitigated MI-induced foetal gene re-programming and cardiomyocyte hypertrophy, culminating in aggravated LV dilatation and infarct expansion. GPR55 regulates cardiac homeostasis and ischaemia responses by maintaining adequate LV filling and modulating three crucial processes post-MI: wound healing kinetics, cardiomyocyte hypertrophy and maladaptive remodelling.

Donepezil as a novel therapy for suppressing the progression of cardiovascular remodeling in obesity-induced hypertensive rats with reperfused myocardial infarction

Abstract Introduction Acetylcholinesterase inhibition by donepezil has been shown to improve long-term survival in permanent myocardial infarction (MI)-induced chronic heart failure rats. This study examined whether donepezil is applicable to the treatment of obesity-induced hypertension with reperfused MI (RMI). Methods Four-week-old SD rats were fed a high-fat diet (57% kcal as fat) for 15 weeks. We implanted a blood pressure (BP) telemetry into the animals (8-week-old) for monitoring artery pressure. After a 1-week recovery, RMI was created by occluding the left coronary artery (30min) followed by reperfusion. Surviving animals were randomly assigned to untreated (UT, n=16) or donepezil treated (DT, n=16, 3 mg/kg/day) group. After a 10-week treatment, the effects of donepezil were evaluated by hemodynamics, blood biomarkers, immunohistochemistry, and morphology. Results The high-fat diet caused obesity and hypertension (9-week-old: systolic BP = 134±4 mmHg; diastolic BP = 92±2 mmHg) in the normal rats. Compared with UT, DT significantly decreased the heart rate (296±5 vs. 318±8 bpm, P&amp;lt;0.05). DT significantly prevented the progression of cardiac remodeling and dysfunction [cardiac index: 91±4 vs. 73±9 ml/min/kg, P&amp;lt;0.01; left ventricular (LV) end-diastolic pressure: 11±1 vs. 20±2 mmHg, P&amp;lt;0.01; LV dp/dt max: 5347±206 vs. 3637±433 mmHg/sec, P&amp;lt;0.01], through increasing capillary density (+120%/field, P&amp;lt;0.001), reducing cardiac fibrosis (−50%, P&amp;lt;0.01) and myocardial infarcted area (17±2 vs. 24±2%, P&amp;lt;0.05), suppressing cardiac hypertrophy (2.35±0.04 vs. 2.70±0.14 g/kg, P&amp;lt;0.01) and coronary artery remodeling (wall thickness: 30±1 vs. 37±2 mm, P&amp;lt;0.01; media-to-lumen ratio: 2.3±0.2 vs. 6.2±1.6, P&amp;lt;0.001). Additionally, DT not only decreased plasma levels of insulin, norepinephrine, BNP, angiotensin II, but also improved the systemic inflammation. Conclusions Donepezil treatment significantly suppressed the progression of cardiovascular remodeling and dysfunction following RMI in obesity-induced hypertensive rats, suggesting that donepezil may be used as a potential candidate for post-RMI therapy in obesity-induced hypertensive patients. Funding Acknowledgement Type of funding source: None

Kanglexin, a novel anthraquinone compound, protects against myocardial ischemic injury in mice by suppressing NLRP3 and pyroptosis.

Pyroptosis is a form of inflammatory cell death that could be driven by the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation following myocardial infarction (MI). Emerging evidence suggests the therapeutic potential for ameliorating MI-induced myocardial damages by targeting NLRP3 and pyroptosis. In this study, we investigated the myocardial protection effect of a novel anthraquinone compound (4,5-dihydroxy-7-methyl-9,10-anthraquinone-2-ethyl succinate) named Kanglexin (KLX) in vivo and in vitro. Male C57BL/6 mice were pre-treated either with KLX (20, 40 mg· kg−1per day, intragastric gavage) or vehicle for 7 consecutive days prior to ligation of coronary artery to induce permanent MI. KLX administration dose-dependently reduced myocardial infarct size and lactate dehydrogenase release and improved cardiac function as compared to vehicle-treated mice 24 h after MI. We found that MI triggered NLRP3 inflammasome activation leading to conversion of interleukin-1β (IL-1β) and IL-18 into their active mature forms in the heart, which could expand the infarct size and drive cardiac dysfunction. We also showed that MI induced pyroptosis, as evidenced by increased DNA fragmentation, mitochondrial swelling, and cell membrane rupture, as well as increased levels of pyroptosis-related proteins, including gasdermin D, N-terminal GSDMD, and cleaved caspase-1. All these detrimental alterations were prevented by KLX. In hypoxia- or lipopolysaccharide (LPS)-treated neonatal mouse ventricular cardiomyocytes, we showed that KLX (10 μM) decreased the elevated levels of terminal deoxynucleotidyl transferase dUTP nick end labeling- and propidium iodide-positive cells, and pyroptosis-related proteins. We conclude that KLX prevents MI-induced cardiac damages and cardiac dysfunction at least partly through attenuating NLRP3 and subsequent cardiomyocyte pyroptosis, and it is worthy of more rigorous investigations for its potential for alleviating ischemic heart disease.

2420Effects of a selective small-molecule formyl peptide receptor 2 agonist on post myocardial inflammation and left ventricular structure-function relationships

Abstract Background/Introduction Dysregulated inflammation following myocardial infarction (MI) can lead to maladaptive infarct healing, myocardial damage and heart failure. Formyl peptide receptor 2 (FPR2) plays an important role in the ligand-dependent regulation of inflammation resolution. Stimulation of resolution via FPR2 activation is hypothesized to preserve left ventricular (LV) structure-function relationships thereby preventing pathological cardiac remodeling, and heart failure. Purpose We evaluated a selective 4-phenylpyrrolidinone FPR2 agonist in rodent MI models by assessing the impact on LV and infarct scar remodeling and cardiac function. Methods The FPR2 agonist was evaluated in phagocytosis, chemotaxis and cytokine response assays. In vivo, following permanent occlusion of the left anterior descending (LAD) artery, C57BL/6 mice or Sprague-Dawley rats were treated with the FPR2 agonist or vehicle; PO gavage, QD. Treatment began 24 hours after occlusion (0.3 and 3 mg/kg) and continued for three days to assess early inflammation or four weeks to evaluate LV and infarct structure and function. Rats subjected to permanent MI were treated 48 hours after occlusion (0.01, 0.1. 1, 10 mg/kg) for six weeks to assess structure-function relationships. A parallel study in rats evaluated compound treatment (0.01, 0.1. 1, 10 mg/kg) following 60 minutes occlusion and reperfusion of the LAD artery. Results The FPR2 agonist enhanced cellular phagocytosis and chemotaxis, and stimulated IL-10 and MCP-1 gene expression in isolated human whole blood. In mice, FPR2 agonist treatment improved survival post MI, reduced LV chamber area and infarct size (26% and 55% vs. vehicle, respectively, P&lt;0.05) and preserved infarct wall thickness (59% vs. vehicle, P&lt;0.05). Treatment increased macrophage arginase 1 levels three days post-MI in the infarct border zone and CD206 levels in the whole heart, indicating a shift towards a pro-resolution phenotype. In rats, FPR2 agonist treatment preserved infarct wall thickness (maximal at 10 mg/kg, 96% vs. vehicle, P&lt;0.05) and increased LV ejection fraction at all doses (+9% vs. vehicle, P&lt;0.05). Following occlusion and reperfusion of the LAD artery, treatment preserved viable myocardium across the infarct wall at multiple doses (25–41%, P&lt;0.05) resulting in increased ejection fraction (14% and 19% vs vehicle at 0.01 and 1 mg/kg, respectively, P&lt;0.05). Conclusion(s) Improvements in cardiac structure-function versus vehicle treated animals support the concept that agonism of FPR2 improves post-MI wound healing, limiting adverse post-MI LV remodeling, thereby preserving cardiac function. These preclinical results suggest targeting FPR2 may present an innovative approach towards development of effective drug therapies to prevent heart failure post-MI. Acknowledgement/Funding Bristol-Myers Squibb Company

Validation of a preclinical model of heart failure with reduced ejection fraction by a dual angiotensin receptor-neprilysin inhibitor and an angiotensin-converting enzyme inhibitor

Heart failure (HF) patients often suffer from several co-existing diseases and co-morbidities. Among them, hypertension and myocardial infarction (MI) are two of the most frequent causes of HF1. Thus, as expected, experimental induction of MI in Spontaneously Hypertensive Rats (SHR) provokes severe left ventricular dysfunction and is associated with signs of HF2. Therapeutic effects of Angiotensin-converting enzyme inhibitor (ACEi) and Angiotensin receptor-neprilysin inhibitor (ARNi) have been demonstrated in HF patients with reduced ejection fraction (HFrEF). The aim of the present study was to validate the translational value of the HF preclinical model SHR PMI using ARNi (Valsartan/Sacubitril, LCZ696) and ACEi (perindopril) treatments recommended by the ESC guidelines for HFrEF patients. Permanent MI (PMI) was induced by coronary ligation in SHR (8-week-old; N = 30) randomized in three groups: –placebo, LCZ696 (68 mg/kg/d); –Perindopril (1 mg/kg/d); –compared to a sham operated group ( N = 10). Three months post-MI, LV function was assessed by echocardiography and hemodynamic study. HF signs were assessed by LV end-diastolic pressure (LVEDP, mmHg), lung and right ventricle (RV) weights (mg). Two vehicle-treated SHR PMI died during the follow-up, no death was observed in LCZ696 or Perindopril treated groups. Post-MI, both ARNi or ACEi treatments had beneficial effects on the adverse LV remodeling and seemed to prevent the mortality and signs of HF (further studies required to confirm these results). In summary, the SHR PMI model has a good predictive value and can be used as preclinical model for the evaluation of new therapeutic strategy in HFrEF [1] , [2] .

Myocardial glycogen depletion leads to improved left ventricular remodeling in a mouse MI model

High glycogen (GLN) content is generally considered deleterious in prolonged ischemia, however, the exact role of GLN during permanent myocardial infarction (MI) and left ventricular (LV) remodeling post-MI, remains poorly understood. This study aimed to investigate whether myocardial GLN depletion can attenuate adverse LV remodeling post-MI. We used a transgenic knock-in (KI) mouse, in which the allosteric pathway of GLN synthesis is inhibited in muscle, leading to dramatically suppressed myocardial GLN levels. WT and KI male mice (10–12 weeks old) were subjected to MI by left anterior descending (LAD) coronary artery permanent ligation. Heart function was measured by 2D-echo (Vevo 2100, Visualsonics). Mice were sacrificed 56 days post-MI and hearts were collected to measure myocardial GLN content and perform IHC and mRNA studies. Basal GLN content was dramatically depressed in KI hearts. We demonstrated a robustly reduced LV dilatation in KI mice 56 days post-MI, as evidenced by lower end-diastolic volume (WT: 167.69 ± 13.42 vs KI: 138.00 ± 4.62 μl; P < 0.05) and end-systolic volume (WT: 147.62 ± 14.58 vs KI: 123.15 ± 4.05 μl; P < 0.05). Moreover, KI mice exhibited attenuated global LV fibrosis (WT: 61.69 ± 11.10 vs KI: 37.42 ± 6.90%; P < 0.05) and a significant decrease in border zone fibrosis (WT: 58.59 ± 12.11 vs KI: 30.33 ± 4.94%; P < 0.05). Surprisingly, KI MI mice demonstrated a dramatic decrease in myocardial rupture incidence (WT: 40.00 vs KI: 6.25%) and improved survival 56 days post-MI (WT: 42.86% vs KI: 60%). GLN depletion in KI hearts attenuates post-MI adverse LV remodeling and improves survival compared to WT. There is a strong correlation between the reduction of cardiac remodeling and a drastic effect on fibrosis deposition, especially in the border zone. Further studies are required, in order to shed light onto the association between low GLN content, cardioprotection and fibrosis.

Mouse Mast Cell Protease 4 Deletion Protects Heart Function and Survival After Permanent Myocardial Infarction.

Chymase, a mast cell serine protease involved in the generation of multiple cardiovascular factors, such as angiotensin II and endothelin-1 (ET-1), is elevated and participates in tissue degeneration after permanent myocardial infarction (PMI). Anesthetized 4-month old male wild-type (WT) C57BL/6J mice and mouse mast cell protease-4 knockout (mMCP-4 KO) congeners were subjected to ligation of the left anterior descending (LAD) coronary artery. A group of mice was then subjected to Kaplan-Meier 28-day survival analysis. In another group of mice, 18F-fluorodeoxyglucose positron emission tomography (PET) was performed to evaluate heart function and the infarcted zone 3 days post-PMI surgery. Cardiac morphology following PMI was evaluated on formalin-fixed heart slices and glycoproteomic analysis was performed using mass spectrometry. Finally, cardiac and lung tissue content of immunoreactive ET-1 was determined. PMI caused 60% mortality in WT mice, due to left ventricular wall rupture, and 7% in mMCP-4 KO mice. Cardiac PET analysis revealed a significant reduction in left ventricular volume (systolic and diastolic) and preserved the ejection fraction in mMCP-4 KO compared to WT animals. The infarcted area, apoptotic signaling and wall remodeling were significantly decreased in mMCP-4 KO mice compared to their WT congeners, while collagen deposition was increased. Glycoproteomic analysis showed an increase in apolipoprotein A1, an established chymase substrate in mMCP-4 KO mice compared to WT mice post-PMI. ET-1 levels were increased in the lungs of WT, but not mMCP-4 KO mice, 24 h post-PMI. Thus, the genetic deletion of mMCP-4 improved survival and heart function post-PMI.

Defective branched chain amino acid catabolism contributes to cardiac dysfunction and remodeling following myocardial infarction

Cardiac metabolic remodeling is a central event during heart failure (HF) development following myocardial infarction (MI). It is well known that myocardial glucose and fatty acid dysmetabolism contribute to post-MI cardiac dysfunction and remodeling. However, the role of amino acid metabolism in post-MI HF remains elusive. Branched chain amino acids (BCAAs) are an important group of essential amino acids and function as crucial nutrient signaling in mammalian animals. The present study aimed to determine the role of cardiac BCAA metabolism in post-MI HF progression. Utilizing coronary artery ligation-induced murine MI models, we found that myocardial BCAA catabolism was significantly impaired in response to permanent MI, therefore leading to an obvious elevation of myocardial BCAA abundance. In MI-operated mice, oral BCAA administration further increased cardiac BCAA levels, activated the mammalian target of rapamycin (mTOR) signaling, and exacerbated cardiac dysfunction and remodeling. These data demonstrate that BCAAs act as a direct contributor to post-MI cardiac pathologies. Furthermore, these BCAA-mediated deleterious effects were improved by rapamycin cotreatment, revealing an indispensable role of mTOR in BCAA-mediated adverse effects on cardiac function/structure post-MI. Of note, pharmacological inhibition of branched chain ketoacid dehydrogenase kinase (BDK), a negative regulator of myocardial BCAA catabolism, significantly improved cardiac BCAA catabolic disorders, reduced myocardial BCAA levels, and ameliorated post-MI cardiac dysfunction and remodeling. In conclusion, our data provide the evidence that impaired cardiac BCAA catabolism directly contributes to post-MI cardiac dysfunction and remodeling. Moreover, improving cardiac BCAA catabolic defects may be a promising therapeutic strategy against post-MI HF.

Delayed coronary reperfusion is ineffective at impeding the dynamic increase in cardiac efferent sympathetic nerve activity following myocardial ischemia.

Acute myocardial infarction (MI) is associated with an adverse and sustained increase in cardiac sympathetic nerve activity (SNA), triggering potentially fatal ventricular arrhythmias. While myocardial reperfusion undoubtedly improves patient prognosis, it remains unknown whether reperfusion therapy also attenuates the dangerous increase in SNA. This study aimed to investigate the effect of time-dependent coronary reperfusion therapy on cardiac SNA following acute MI. Electrophysiological recordings of cardiac efferent SNA were performed in urethane-anaesthetized rats following ligation of the left anterior descending coronary artery (i.e., MI) for either 15 or 45 min, followed by 'early' or 'delayed' reperfusion, respectively. Another group of rats had permanent ischemia with no reperfusion. Forty-five minutes of ischemia induced a 55 % increase in efferent SNA. Subsequent 'delayed' reperfusion was ineffective at ameliorating further increases in SNA (maximal 153 % increase), so that MI-induced increases in SNA mirrored that observed in rats with permanent MI. Although SNA did not increase during 15 min of ischemia, it did significantly increase, albeit delayed, during the subsequent reperfusion period (max. 75 % increase). Importantly, however, this increase in SNA, which tended to be lower in the 'early'-reperfusion group, was matched with a lower incidence of arrhythmias and mortality rate, compared to the 'delayed'-reperfusion and permanent-MI groups. These results highlight that 'prompt' coronary reperfusion, before SNA becomes activated, may provide a crucial window of opportunity for improving outcome. Further research is essential to identify the mechanisms that underpin, not only sympathetic activation, but also importantly sympathetic deactivation as a potential therapeutic target for MI.

Ablation of C/EBP homologous protein increases the acute phase mortality and doesn't attenuate cardiac remodeling in mice with myocardial infarction

Endoplasmic reticulum stress is a proapoptotic and profibrotic stimulus. Ablation of C/EBP homologous protein (CHOP) is reported to reverse cardiac dysfunction by attenuating cardiac endoplasmic reticulum stress in mice with pressure overload or ischemia/reperfusion, but it is unclear whether loss of CHOP also inhibits cardiac remodeling induced by permanent-infarction. In mice with permanent ligation of left coronary artery, we found that ablation of CHOP increased the acute phase mortality. For the mice survived to 4 weeks, left ventricular anterior (LV) wall thickness was larger in CHOP knockout mice than in the wildtype littermates, while no difference was noted on posterior wall thickness, LV dimensions, LV fractional shortening and ejection fraction. Similarly, invasive assessment of LV hemodynamics, morphological analysis of heart and lung weight indexes, myocardial fibrosis and TUNEL-assessed apoptosis showed no significant differences between CHOP knockout mice and their wildtype ones, while in mice with ischemia for 45 min and reperfusion for 1 week, myocardial fibrosis and apoptosis in the infarct area were significantly attenuated in CHOP knockout mice. These findings indicate that ablation of CHOP doesn't ameliorate cardiac remodeling induced by permanent-myocardial infarction, which implicates that early reperfusion is a prerequisite for ischemic myocardium to benefit from CHOP inhibition.

Protective effects of TREM-1 modulation during experimental myocardial infarction

Purpose: Myocardial healing after infarction is hampered by reperfusion injury and ventricular remodeling, partly mediated by detrimental inflammatory responses. We hypothesized that modulation of the Triggering Receptor Expressed on Myeloid cells-1 (TREM-1), an amplifier of the innate inflammatory response, by the use of a small synthetic peptide, LR12, could improve myocardial injury in a rodent model of myocardial infarction (MI). Methods: For hemodynamic assessment, we used Wistar rats which underwent either permanent occlusion of the left coronary artery or transient ischemia for one hour followed by reperfusion. They were randomly assigned to receive LR12 or vehicle for 5 days. Cardiac function and dimensions were assessed at baseline through positron emission tomography (PET) imaging and 6 weeks after infarction by PET and conductance catheter. For biological investigations, we used male Balb/c mouse with permanent MI and infusion of LR12 or vehicle from 1 hour after MI to day 5. TREM-1, protease activity and others myocardial inflammatory mediators tissue expression were quantified by western-blot, Elisa, and qRT-PCR. Leukocytes populations were studied in heart and blood samples. Results: Following permanent ischemia, LR12 reduced ventricular remodeling, as assessed by a lesser increase of end-diastolic volume measured by PET, and improved the systolic function evaluated by conductance catheter derived load-independent parameters (ESPVR: 0.98±0.43 vs 1.45±0.49 mmHg.μL-1, p=0.04; PRSW: 55±23 vs 80±34 mmHg.μL-1, p=0.03). During transient ischemia, LR12 infusion just before and for 5 days after reperfusion also improved myocardial contractility (PRSW: 64±25 vs 96±26, p=0.02; ESPVR 1.00±0.30 vs 1.61±0.69, p=0.05). TREM-1 was expressed in myocardial tissue, especially in infarcted areas, as early as 6 hours after MI, peaking at 24h. LR12 administration modulated myocardial inflammatory response with a reduction of p38 and ERK1/2 phosphorylation, iNOS and Cox2 expression, while Akt, GSK3β and Socs3 were up-regulated. LR12 modulated the expression of many chemokines (IL-6, -13, -17, TNF-α, ICAM-1), and decreased protease activity in myocardial infarcted areas (MMP9/TIMP1). LR12 profoundly modified leukocytes populations, with an increased number of circulating and myocardial non classical monocytes (Gr1-low) and LT4 at 96h post MI. Conclusion: LR12, by inhibiting TREM-1, improves ventricular remodeling and myocardial contractility in various experimental models of myocardial infarction. These effects are mediated by a profound switch in leukocytes recruitment to the healing myocardium.

A Collagen α2(I) Mutation Impairs Healing after Experimental Myocardial Infarction

Collagen breakdown and de novo synthesis are important processes during early wound healing after myocardial infarction (MI). We tested the hypothesis that collagen I, the main constituent of the extracellular matrix, affects wound healing after MI. The osteogenesis imperfecta mouse (OIM), lacking procollagen-α2(I) expression, represents a model of the type III form of the disease in humans. Homozygous (OIM/OIM), heterozygous (OIM/WT), and wild-type (WT/WT) mice were subjected to a permanent myocardial infarction protocol or sham surgery. Baseline functional and geometrical parameters determined by echocardiography did not differ between genotypes. After MI but not after sham surgery, OIM/OIM animals exhibited significantly increased mortality, due to early ventricular rupture between day 3 and 7. Echocardiography at day 1 demonstrated increased left ventricular dilation in OIM/OIM animals. Less collagen I mRNA within the infarct area was found in OIM/OIM animals. At 2 days after MI, MMP-9 expression in the infarct border zone was higher in OIM/OIM than in WT/WT animals. Increased granulocyte infiltration into the infarct border zone occurred in OIM/OIM animals. Neither granulocyte depletion nor MMP inhibition reduced mortality in OIM/OIM animals. In this murine model, deficiency of collagen I leads to a myocardial wound-healing defect. Both structural alterations within pre-existing collagen matrix and impaired collagen de novo expression contribute to a high rate of early myocardial rupture after MI.

Hypoxic preconditioning enhances bone marrow mesenchymal stem cell migration via Kv2.1 channel and FAK activation.

Transplantation using stem cells including bone marrow mesenchymal stem cells (BMSCs) is emerging as a potential regenerative therapy after ischemic attacks in the heart and brain. The migration capability of transplanted cells is a critical cellular function for tissue repair. Based on our recent observations that hypoxic preconditioning (HP) has multiple benefits in improving stem cell therapy and that the potassium Kv2.1 channel acts as a promoter for focal adhesion kinase (FAK) activation and cell motility, the present investigation tested the hypothesis that HP treatment can increase BMSC migration via the mechanism of increased Kv2.1 expression and FAK activities. BMSCs derived from green fluorescent protein-transgenic mice were treated under either normoxic (N-BMSC) or hypoxic (0.5% O(2)) (HP-BMSC) conditions for 24 h. Western blot analysis showed HP selectively upregulated Kv2.1 expression while leaving other K(+) channels, such as Kv1.5 and Kv1.4, unaffected. Compared with normoxic controls, significantly larger outward delayed rectifier K(+) currents were recorded in HP-BMSCs. HP enhanced BMSC migration/homing activities in vitro and after intravenous transplantation into rats subjected to permanent myocardial infarction (MI). The HP-promoted BMSC migration was inhibited by either blocking K(+) channels or knocking down Kv2.1. Supporting a relationship among HP, Kv2.1, and FAK activation, HP increased phosphorylation of FAK(397) and FAK(576/577), and this effect was antagonized by blocking K(+) channels. These findings provide novel evidence that HP enhances the ability of BMSCs to migrate and home to the injured region; this effect is mediated through a regulatory role of Kv2.1 on FAK phosphorylation/activation.

Single high-dose intramyocardial administration of erythropoietin promotes early intracardiac proliferation, proves safety and restores cardiac performance after myocardial infarction in rats☆

Various studies demonstrate erythropoietin (EPO) as a cardioprotective growth hormone. Recent findings reveal EPO in addition might induce proliferation cascades inside myocardium. We aimed to evaluate whether a single high-dose intramyocardial EPO administration safely elevates early intracardiac cell proliferation after myocardial infarction (MI). Following permanent MI in rats EPO (3000 U/kg) in MI EPO-treatment group (n=99) or saline in MI control group (n=95) was injected along the infarction border. Intramyocardial EPO injection activated the genes of cyclin D1 and cell division cycle 2 kinase (cdc2) at 24 h after MI (n=6, P<0.05) evaluated by real time-PCR. The number of Ki-67+ intracardiac cells analyzed following immunohistochemistry was significantly enhanced by 45% in the peri-infarction zone at 48 h after EPO treatment (n=6, P<0.001). Capillary density was significantly enhanced by 17% as early as seven days (n=6, P<0.001). After six weeks, left ventricular performance assessed by conductance catheters was restored under baseline and dobutamine induced stress conditions (n=11-14, P<0.05). No thrombus formation was observed in the heart and in distant organs. No deleterious systemic adverse effects were apparent. Single high-dose intramyocardial EPO delivery proved safety and promoted early intracardiac cell proliferation, which might in part have contributed to an attenuated myocardial functional decline.

Thioredoxin-1 Gene Delivery Induces Heme Oxygenase-1 Mediated Myocardial Preservation After Chronic Infarction in Hypertensive Rats

Hypertension, the major risk factor for many cardiovascular diseases, is a result of multiple causes along with excessive generation of reactive oxygen species (ROS) resulting in imbalance of redox status. In this study, we investigated the therapeutic potential of Adenoviral-Thioredoxin-1 (Adeno-Trx-1) in spontaneous hypertensive rats (SHRs) at a dosage of 1 x 10(9) pfu. The rats were assigned as normotensive Wistar-Kyoto (WKY), SHR, SHR + Adeno-Lac-Z (SHRLac-Z), and SHR + Adeno-Trx-1 (SHRTrx-1). Echo-guided injection of adeno virus was done 48 h before permanent myocardial infarction (MI) by left anterior descending coronary artery (LAD) occlusion. Decreased infarct size (52 +/- 4.1% vs. 67 +/- 6.1%), number of apoptotic cardiomyocytes (161 +/- 14.8 vs. 240 +/- 22.2), left ventricular inner diameter (7 +/- 0.33 vs. 9 +/- 0.46 mm), increased ejection fraction (52 +/- 6.3 vs. 42 +/- 3.3%), and fractional shortening (28 +/- 1.8 vs. 22 +/- 2.04 %) was observed in the SHRTrx-1 compared to SHR. Western Blot and immunohistochemical analysis demonstrated increased expression of Trx-1, HO-1, and Bcl-2 in the SHRTrx-1 compared to SHR. In addition, increased HO-1 activity was also observed in SHRTrx-1 as compared to SHR and SHRLac-Z groups. Our study demonstrates that the cardioprotective effect of Adeno-Trx-1 gene therapy in SHR is Trx-1/HO-1/Bcl-2 mediated and may represent future target to develop therapy against hypertension associated cardiac failure.

Transplantation of hypoxia-preconditioned mesenchymal stem cells improves infarcted heart function via enhanced survival of implanted cells and angiogenesis

This study explored the novel strategy of hypoxic preconditioning of bone marrow mesenchymal stem cells before transplantation into the infarcted heart to promote their survival and therapeutic potential of mesenchymal stem cell transplantation after myocardial ischemia. Mesenchymal stem cells from green fluorescent protein transgenic mice were cultured under normoxic or hypoxic (0.5% oxygen for 24 hours) conditions. Expression of growth factors and anti-apoptotic genes were examined by immunoblot. Normoxic or hypoxic stem cells were intramyocardially injected into the peri-infarct region of rats 30 minutes after permanent myocardial infarction. Death of mesenchymal stem cells was assessed in vitro and in vivo after transplantation. Angiogenesis, infarct size, and heart function were measured 6 weeks after transplantation. Hypoxic preconditioning increased expression of pro-survival and pro-angiogenic factors including hypoxia-inducible factor 1, angiopoietin-1, vascular endothelial growth factor and its receptor, Flk-1, erythropoietin, Bcl-2, and Bcl-xL. Cell death of hypoxic stem cells and caspase-3 activation in these cells were significantly lower compared with that in normoxic stem cells both in vitro and in vivo. Transplantation of hypoxic versus normoxic mesenchymal stem cells after myocardial infarction resulted in an increase in angiogenesis, as well as enhanced morphologic and functional benefits of stem cell therapy. Hypoxic preconditioning enhances the capacity of mesenchymal stem cells to repair infarcted myocardium, attributable to reduced cell death and apoptosis of implanted cells, increased angiogenesis/vascularization, and paracrine effects.