Up‐regulated TLR4 in cardiomyocytes exacerbates heart failure after long‐term myocardial infarction

It remains unclear whether and how cardiomyocytes contribute to the inflammation in chronic heart failure (CHF). We recently reviewed the capacity of cardiomyocytes to initiate inflammation, by means of expressing certain immune receptors such as toll‐like receptors (TLRs) that respond to pathogen‐ and damage‐associated molecular patterns (PAMP and DAMP). Previous studies observed TLR4‐mediated inflammation within days of myocardial infarction (MI). This study examined TLR4 expression and function in cardiomyocytes of failing hearts after 4 weeks of MI in rats. The increases of TLR4 mRNA and proteins, as well as inflammatory cytokine production, were observed in both the infarct and remote myocardium. Enhanced immunostaining for TLR4 was observed in cardiomyocytes but not infiltrating leucocytes. The injection of lentivirus shRNA against TLR4 into the infarcted heart decreased inflammatory cytokine production and improved heart function in vivo. Accordingly, in cardiomyocytes isolated from CHF hearts, increases of TLR4 mRNA and proteins were detected. More robust binding of TLR4 with lipopolysaccharide (LPS), a PAMP ligand for TLR4, and heat shock protein 60 (HSP60), a DAMP ligand for TLR4, was observed in CHF cardiomyocytes under a confocal microscope. The maximum binding capacity (B_max) of TLR4 was increased for LPS and HSP60, whereas the binding affinity (Kd) was not significantly changed. Furthermore, both LPS and HSP60 induced more robust production of inflammatory cytokines in CHF cardiomyocytes, which was reduced by TLR4‐blocking antibodies. We conclude that the expression, ligand‐binding capacity and pro‐inflammatory function of cardiomyocyte TLR4 are up‐regulated after long‐term MI, which promote inflammation and exacerbate heart failure.     

Alteration of collagenous protein profile in congestive heart failure secondary to myocardial infarction

The rat model of myocardial infarction is characterized by progressive cardiac hypertrophy and failure. Rats with infarcts greater than 30% of the left ventricle exhibited early and moderate, stages of heart failure 4 and 8 weeks after the occlusion of the left coronary artery, respectively. As heart failure is usually associated with remodeling of the extracellular matrix, a histological and biochemical study of cardiac collagenous proteins was carried out using failing hearts. Total collagen content in the right ventricle increased at 2, 4, and 8 weeks following occlusion of the left coronary artery whereas such a change in viable left ventricle was seen after 4 and 8 weeks. Total cardiac hydroxyproline concentration was increased in both right and left ventricular samples from the infarcted animals when compared to those of control; this increase was due to elevation of pepsin-insoluble collagen fraction. The myocardial noncollagenous/collagenous protein ratio was decreased in experimental right and left ventricular samples when compared to control samples. These findings suggest that an increase in cross-linking of cardiac collagen as well as disparate synthesis of collagenous and noncollagenous proteins occurs in this model of congestive heart, failure.     

Circulating LIPCAR is a potential biomarker of heart failure in patients post-acute myocardial infarction

In heart failure (HF) patients with reduced ejection fraction, LIPCAR, a long noncoding RNA is elevated and is associated with left ventricular remodeling and poor prognosis. We studied the role of LIPCAR in patients with HF post-acute myocardial infarction (AMI) to find biomarkers for early detection of HF. We conducted a study of 127 patients with AMI, of which 59 were patients with HF post-AMI. LIPCAR levels were higher in HF patients post-AMI than patients without HF, and LIPCAR had a high predictive value for diagnosis of HF, which was estimated by receiver operating characteristic curves (AUC: 0.985). The results indicate that LIPCAR may be a marker of early HF after AMI.     

Identification of potentially critical genes in the development of heart failure after ST-segment elevation myocardial infarction (STEMI)

Heart failure (HF) remains a common complication after acute ST-segment elevation myocardial infarction (STEMI). Here, we aim to identify critical genes related to the developed HF in patients with STEMI using bioinformatics analysis. The microarray data of GSE59867, including peripheral blood samples from nine patients with post-infarct HF and eight patients without post-infarct HF, were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) between HF and non-HF groups were screened by LIMMA package. Functional enrichment analyses of DEGs were conducted, followed by construction of a protein-protein interaction (PPI) network. The dynamic messenger RNA (mRNA) level of the hub genes during the follow-up was analyzed to further elucidate their role in HF development. A total of 58 upregulated and 75 downregulated DEGs were screen out. They were mainly enriched in biological processes about inflammatory response, extracellular matrix organization, response to cAMP, immune response, and positive regulation of cytosolic calcium ion concentration. Pathway analysis revealed that the DEGs were also involved in hematopoietic cell lineage, pathways in cancer, and extracellular matrix-receptor interaction. In the PPI network consisting of 58 nodes and 72 interactions, CXCL8 (degree = 15), THBS1 (degree = 8), FOS (degree = 7), and ITGA2B (degree = 6) were identified as the hub genes. In the comparison of patients with and without post-infarct HF, the mRNA level of these hub genes were all higher within 30 days but reached similar at 6 months after STEMI. In conclusion, CXCL8, THBS1, FOS, and ITGA2B may play important roles in the development of HF after acute STEMI.     

Endostatin attenuates heart failure via inhibiting reactive oxygen species in myocardial infarction rats

The purpose of the present study was to evaluate whether endostatin overexpression could improve cardiac function, hemodynamics, and fibrosis in heart failure (HF) via inhibiting reactive oxygen species (ROS). The HF models were established by inducing ischemia myocardial infarction (MI) through ligation of the left anterior descending (LAD) artery in Sprague–Dawley (SD) rats. Endostatin level in serum was increased in MI rats. The decrease in cardiac function and hemodynamics in MI rats were enhanced by endostatin overexpression. Endostatin overexpression inhibited the increase in collagen I, collagen III, α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), matrix metalloproteinase (MMP)-2 and MMP9 in the hearts of MI rats. MI-induced cardiac hypertrophy was reduced by endostatin overexpression. The increased levels of malondialdehyde (MDA), superoxide anions, the promoted NAD(P)H oxidase (Nox) activity, and the reduced superoxide dismutase (SOD) activity in MI rats were reversed by endostatin overexpression. Nox4 overexpression inhibited the cardiac protective effects of endostatin. These results demonstrated that endostatin improved cardiac dysfunction and hemodynamics, and attenuated cardiac fibrosis and hypertrophy via inhibiting oxidative stress in MI-induced HF rats.     

Differential cytokine expression in myocytes and non-myocytes after myocardial infarction in rats

The proinflammatory cytokines interleukin (IL)-1 and IL-6 are increased after acute myocardial infarction (MI). Moreover, serum IL-6 level is elevated after MI, but has also been associated with heart failure. In the present study, heart function was monitored in a rat model of chronic MI. Cytokine expression in the infarcted and non-infarcted myocardium as well as in hearts of sham-operated controls was measured by the ribonuclease-protection assay. To identify the cells contributing to the increased cytokine expression, we further analyzed myocytes and non-myocytes isolated in the acute phase as well as during congestive heart failure (CHF) after MI. There was a strong induction in cytokine expression in the myocytes of the infarct area 6 h after MI. In the non-infarcted myocardium, cytokine expression increased only slightly in the non-myocytes after 6 h. This was not different from sham-operated controls and may, therefore, be induced by stress and catecholamines. In CHF, however, cytokine expression level in myocytes was normal. It increased slightly but significantly in the non-myocytes 4 and 8 weeks after MI. In conclusion, we suggest that pro-inflammatory cytokines, produced by the ischemic myocytes may be involved in the initiation of wound healing of the necrotic area, whereas the effect of pro-inflammatory cytokines in CHF, if any, seems not to be crucial.     

Changes in fatty acid compositions of myocardial lipids in rats with heart failure following myocardial infarction

Changes in fatty acid composition of myocardial lipids were examined in rats with heart failure following myocardial infarction. Left ventricular systolic pressure (LVSP) was decreased and left ventricular end-diastolic pressure (LVEDP) was elevated 24 h, 1 and 12 weeks after left coronary artery ligation (CAL), suggesting the development of heart failure at these periods in this model. Hearts were isolated 24 h, 1 week and 12 weeks after the operation. Myocardial lipids in the infarcted scar tissue, non-infarcted remaining left ventricle including interseptum and right ventricle were separated into phospholipid (PL), triacylglycerol (TG), diacylglycerol (DAG) and free fatty acid (FFA) fractions. In the scar tissue PL content markedly decreased whereas TG, DAG and FFA contents increased 24 h after CAL. Despite a marked decrease in constituted fatty acids of PL fraction in the scar tissue the percentage of arachidonic acid in PL was elevated 12 weeks after CAL, suggesting that release of arachidonic acid during PL degradation was suppressed. In the non-infarcted viable left ventricle PL content remained unchanged throughout the experiment whereas TG, DAG and FFA contents were elevated 24 h after CAL. Despite no changes in PL and other lipid contents in the non-infarcted tissue the percentage of linoleic acid in PL was reduced and that of docosahexaenoic acid in PL was elevated 12 weeks after CAL. Our findings showed that myocardial lipid composition of the non-infarcted left ventricle was altered only in an early stage of the development of heart failure and fatty acid compositions of PL was exchanged in a late stage of the development of heart failure. The exchange may be related to cardiac dysfunction or myocardial remodelling in the rat with heart failure.     

Luteolin alleviates post‐infarction cardiac dysfunction by up‐regulating autophagy through Mst1 inhibition

Myocardial infarction (MI), which is characterized by chamber dilation and LV dysfunction, is associated with substantially higher mortality. We investigated the effects and underlying mechanisms of Luteolin on post‐infarction cardiac dysfunction. Myocardial infarction was constructed by left anterior descending coronary artery ligation. In vitro, cultured neonatal cardiomyocytes subjected to simulated MI were used to probe mechanism. Luteolin significantly improved cardiac function, decreased cardiac enzyme and inflammatory cytokines release after MI. Enhanced autophagic flux as indicated by more autophagosomes puncta, less accumulation of aggresomes and P62 in the neonatal cardiomyocytes after hypoxia was observed in the Luteolin pre‐treatment group. Western blot analysis also demonstrated that Luteolin up‐regulated autophagy in the cardiomyocytes subjected to simulated MI injury. Furthermore, Luteolin increased mitochondrial membrane potential, adenosine triphosphate content, citrate synthase activity and complexes I/II/III/IV/V activities in the cardiomyocytes subjected to simulated MI injury. Interestingly, mammalian sterile 20‐like kinase 1 (Mst1) knockout abolished the protective effects of Luteolin administration. Luteolin enhances cardiac function, reduces cardiac enzyme and inflammatory markers release after MI. The protective effects of Luteolin are associated with up‐regulation of autophagy and improvement of mitochondrial biogenesis through Mst1 inhibition.     

Elevation of IGF-2 receptor and the possible underlying implications in end-stage heart failure patients before and after heart transplantation

Up-regulation of insulin-like growth factor 2 receptor (IGF-2R) involved in angiotensin II-induced cell apoptosis in cardiomyoblasts, and correlated with cardiomyocyte apoptosis in hypertensive rat hearts. Here, we detected IGF-2R levels and explored the possible underlying implications in end-stage heart failure (HF) patients before and after heart transplantation. Western blot and immunohistochemistry were used to measure cardiac IGF-2R levels. ELISA was used to detect serum IGF-2R and CD8 levels. Labelling of DNA strand breaks and dihydroethidium detection were used to determine cellular apoptosis and reactive oxygen species, respectively. Cardiac IGF-2R levels increased in end-stage HF patients (n = 11) compared with non-failing control subjects. Leu27-IGF-2, an IGF-2 analogue to activate specially the IGF-2R, could induce apoptosis and reactive oxygen species production in neonatal rat ventricular myocytes. The serum IGF-2R levels were significantly higher in HF patients than those in non-failing control subjects. An unexpected observation is that the serum IGF-2R levels further increased after heart transplantation, peaked at the first month, and gradually reduced close to the levels before heart transplantation at the 6th months after heart transplantation. Serum CD8, a marker of acute rejection, had no change after heart transplantation, but IGF-2R and Granzyme B, as a ligand for the IGF-2R and a marker for CD8 T lymphocyte activation, coexisted in the transplanted hearts. Our preliminary studies suggest that elevation of IGF-2R may participate in pathological process of end-stage HF and involved in the acute cellular rejection after heart transplantation.     

Mitophagy‐regulated mitochondrial health strongly protects the heart against cardiac dysfunction after acute myocardial infarction

Autophagy including mitophagy serves as an important regulatory mechanism in the heart to maintain the cellular homeostasis and to protect against heart damages caused by myocardial infarction (MI). The current study aims to dissect roles of general autophagy and specific mitophagy in regulating cardiac function after MI. By using Beclin1^+/−, Fundc1 knockout (KO) and Fundc1 transgenic (TG) mouse models, combined with starvation and MI models, we found that Fundc1 KO caused more severe mitochondrial and cardiac dysfunction damages than Beclin1^+/− after MI. Interestingly, Beclin1^+/− caused notable decrease of total autophagy without detectable change to mitophagy, and Fundc1 KO markedly suppressed mitophagy but did not change the total autophagy activity. In contrast, starvation increased total autophagy without changing mitophagy while Fundc1 TG elevated total autophagy and mitophagy in mouse hearts. As a result, Fundc1 TG provided much stronger protective effects than starvation after MI. Moreover, Beclin1^+/−/Fundc1 TG showed increased total autophagy and mitophagy to a level comparable to Fundc1 TG per se, and completely reversed Beclin1^+/−‐caused aggravation of mitochondrial and cardiac injury after MI. Our results reveal that mitophagy but not general autophagy contributes predominantly to the cardiac protective effect through regulating mitochondrial function.     

Decreased expression of cardiac sarcoplasmic reticulum Ca2+-pump ATPase in congestive heart failure due to myocardial infarction

Myocardial infarction in rats induced by occluding the left coronary artery for 4, 8 and 16 weeks has been shown to result in congestive heart failure (CHF) characterized by hypertrophy of the viable ventricular myocardial tissue. We have previously demonstrated a decreased calcium transport activity in the sarcoplasmic reticulum (SR) of post-myocardial infarction failing rat hearts. In this study we have measured the steady state levels of the cardiac SR Ca²⁺-pump ATPase (SERCA2) mRNA using Northern blot and slot blot analyses. The relative amounts of SERCA2 mRNA were decreased with respect to GAPDH mRNA and 28 S rRNA in experimental failing hearts at 4 and 8 weeks post myocardial infarction by about 20% whereas those at 16 weeks declined by about 35% of control values. The results obtained by Western blot analysis, revealed that the immunodetectable levels of SERCA2 protein in 8 and 16 weeks postinfarcted animals were decreased by about 20% and 30%, respectively. The left ventricular SR Ca²⁺-pump ATPase specific activity was depressed in the SR preparations of failing hearts as early as 4 weeks post myocardial infarction and declined by about 65% at 16 weeks compared to control. These results indicate that the depressed SR Ca²⁺-pump ATPase activity in CHF may partly be due to decreased steady state amounts of SERCA2 mRNA and SERCA2 protein in the failing myocardium.     

Calhex231 ameliorates myocardial fibrosis post myocardial infarction in rats through the autophagy‐NLRP3 inflammasome pathway in macrophages

The calcium‐sensing receptor (CaSR) is involved in the pathophysiology of many cardiovascular diseases, including myocardial infarction (MI) and hypertension. The role of Calhex231, a specific inhibitor of CaSR, in myocardial fibrosis following MI is still unclear. Using Wistar rats, we investigated whether Calhex231 ameliorates myocardial fibrosis through the autophagy‐NLRP3 inflammasome pathway in macrophages post myocardial infarction (MI). The rats were randomly divided into sham, MI and MI + Calhex231 groups. Compared with the sham rats, the MI rats consistently developed severe cardiac function, myocardial fibrosis and infiltration of inflammatory cells including macrophages. Moreover, inflammatory pathway including activation of NLRP3 inflammasome, IL‐1β and autophagy was significantly up‐regulated in myocardial tissue, infiltrated cardiac macrophages and peritoneal macrophages of the MI rats. These impacts were reversed by Calhex231. In vitro, studies revealed that calindol and rapamycin exacerbated MI‐induced autophagy and NLRP3 inflammasome activation in peritoneal macrophages. Calhex231 and 3‐Methyladenine (a specific inhibitor of autophagy) attenuated both autophagy and NLRP3 inflammasome activation; however, the caspase‐1 inhibitor Z‐YVAD‐FMK did not. Our study indicated that Calhex231 improved cardiac function and ameliorated myocardial fibrosis post MI, likely via the inhibition of autophagy‐mediated NLRP3 inflammasome activation; this provides a new therapeutic target for ventricular remodelling‐related cardiovascular diseases.     

Predictive values of sST2 and IL-33 for heart failure in patients with acute myocardial infarction

Timely prediction of the risk of heart failure in acute myocardial infarction patients is critical for better prognosis. This article aims to evaluate the predictive value of serum soluble growth stimulation expressed gene 2 (sST2) and interleukin-33 in patients with acute myocardial infarction complicated by heart failure. A total of 42 healthy controls and 144 acute myocardial infarction patients were recruited in the study. According to Killip cardiac function classification as the basis for concurrent heart failure, they were distributed into non-heart failure group (n = 76) and heart failure group (n = 68). ELISA was utilized to determine the serum sST2 and interleukin-33 levels, and the diagnostic efficiency was evaluated by receiver operating characteristics curve. sST2 and interleukin-33 levels in patients with acute myocardial infarction were significantly increased when compared with normal healthy controls, and were further enhanced in the heart failure group. With the increased Killip cardiac function classification, interleukin-33 and sST2 levels were gradually elevated. Multivariate analysis indicated that interleukin-33 and sST2 could be used as independent predictors for heart failure combined with acute myocardial infarction.     

PKCβII inhibition attenuates myocardial infarction induced heart failure and is associated with a reduction of fibrosis and pro‐inflammatory responses

Protein kinase C βII (PKCβII) levels increase in the myocardium of patients with end‐stage heart failure (HF). Also targeted overexpression of PKCβII in the myocardium of mice leads to dilated cardiomyopathy associated with inflammation, fibrosis and myocardial dysfunction. These reports suggest a deleterious role of PKCβII in HF development. Using a post‐myocardial infarction (MI) model of HF in rats, we determined the benefit of chronic inhibition of PKCβII on the progression of HF over a period of 6 weeks after the onset of symptoms and the cellular basis for these effects. Four weeks after MI, rats with HF signs that were treated for 6 weeks with the PKCβII selective inhibitor (βIIV5‐3 conjugated to TAT_47–57 carrier peptide) (3 mg/kg/day) showed improved fractional shortening (from 21% to 35%) compared to control (TAT_47–57 carrier peptide alone). Formalin‐fixed mid‐ventricle tissue sections stained with picrosirius red, haematoxylin and eosin and toluidine blue dyes exhibited a 150% decrease in collagen deposition, a two‐fold decrease in inflammation and a 30% reduction in mast cell degranulation, respectively, in rat hearts treated with the selective PKCβII inhibitor. Further, a 90% decrease in active TGFβ1 and a significant reduction in SMAD2/3 phosphorylation indicated that the selective inhibition of PKCβII attenuates cardiac remodelling mediated by the TGF‐SMAD signalling pathway. Therefore, sustained selective inhibition of PKCβII in a post‐MI HF rat model improves cardiac function and is associated with inhibition of pathological myocardial remodelling.