Abstract
Aims
Interleukin-1β (IL-1β) is an important pathogenic factor in cardiovascular diseases including chronic heart failure (HF). The CANTOS trial highlighted that inflammasomes as primary ...sources of IL-1 β are promising new therapeutic targets in cardiovascular diseases. Therefore, we aimed to assess inflammasome activation in failing hearts to identify activation patterns of inflammasome subtypes as sources of IL-1β.
Methods and results
Out of the four major inflammasome sensors tested, expression of the inflammasome protein absent in melanoma 2 (AIM2) and NLR family CARD domain-containing protein 4 (NLRC4) increased in human HF regardless of the aetiology (ischaemic or dilated cardiomyopathy), while the NLRP1/NALP1 and NLRP3 (NLR family, pyrin domain containing 1 and 3) inflammasome showed no change in HF samples. AIM2 expression was primarily detected in monocytes/macrophages of failing hearts. Translational animal models of HF (pressure or volume overload, and permanent coronary artery ligation in rat, as well as ischaemia/reperfusion-induced HF in pigs) demonstrated activation pattern of AIM2 similar to that of observed in end-stages of human HF. In vitro AIM2 inflammasome activation in human Tohoku Hospital Pediatrics-1 (THP-1) monocytic cells and human AC16 cells was significantly reduced by pharmacological blockade of pannexin-1 channels by the clinically used uricosuric drug probenecid. Probenecid was also able to reduce pressure overload-induced mortality and restore indices of disease severity in a rat chronic HF model in vivo.
Conclusions
This is the first report showing that AIM2 and NLRC4 inflammasome activation contribute to chronic inflammation in HF and that probenecid alleviates chronic HF by reducing inflammasome activation. The present translational study suggests the possibility of repositioning probenecid for HF indications.
Graphical Abstract
Cardioprotective medications are still unmet clinical needs. We have previously identified several cardioprotective microRNAs (termed ProtectomiRs), the mRNA targets of which may reveal new drug ...targets for cardioprotection. Here we aimed to identify key molecular targets of ProtectomiRs and confirm their association with cardioprotection in a translational pig model of acute myocardial infarction (AMI). By using a network theoretical approach, we identified 882 potential target genes of 18 previously identified protectomiRs. The Rictor gene was the most central and it was ranked first in the protectomiR-target mRNA molecular network with the highest node degree of 5. Therefore, Rictor and its targeting microRNAs were further validated in heart samples obtained from a translational pig model of AMI and cardioprotection induced by pre- or postconditioning. Three out of five Rictor-targeting pig homologue of rat ProtectomiRs showed significant upregulation in postconditioned but not in preconditioned pig hearts. Rictor was downregulated at the mRNA and protein level in ischemic postconditioning but not in ischemic preconditioning. This is the first demonstration that Rictor is the central molecular target of ProtectomiRs and that decreased Rictor expression may regulate ischemic postconditioning-, but not preconditioning-induced acute cardioprotection. We conclude that Rictor is a potential novel drug target for acute cardioprotection.
Lipid-lowering drugs have been shown to have cardioprotective effects but may have hidden cardiotoxic properties. Therefore, here we aimed to investigate if chronic treatment with the novel ...lipid-lowering drug bempedoic acid (BA) exerts hidden cardiotoxic and/or cardioprotective effects in a rat model of acute myocardial infarction (AMI). Wistar rats were orally treated with BA or its vehicle for 28 days, anesthetized and randomized to three different groups (vehicle + ischemia/reperfusion (I/R), BA + I/R, and positive control vehicle + ischemic preconditioning (IPC)) and subjected to cardiac 30 min ischemia and 120 min reperfusion. IPC was performed by 3 × 5 min I/R cycles before ischemia. Myocardial function, area at risk, infarct size and arrhythmias were analyzed. Chronic BA pretreatment did not influence cardiac function or infarct size as compared to the vehicle group, while the positive control IPC significantly reduced the infarct size. The incidence of reperfusion-induced arrhythmias was significantly reduced by BA and IPC. This is the first demonstration that BA treatment does not show cardioprotective effect although moderately reduces the incidence of reperfusion-induced arrhythmias. Furthermore, BA does not show hidden cardiotoxic effect in rats with AMI, showing its safety in the ischemic/reperfused heart.
The use of comorbidity models is crucial in cardioprotective drug development. Hypercholesterolemia causes endothelial and myocardial dysfunction, as well as aggravates ischemia/reperfusion ...(I/R)-induced myocardial injury. Endogenous cardioprotective mechanisms against I/R are impaired in hyperlipidemic and hyperglycemic
animal models. Therefore, our aim was to develop a medium throughput comorbidity cell-based test system of myocardial I/R injury, hypercholesterolemia and hyperglycemia that mimics comorbidity conditions.
Cardiac myocytes isolated from neonatal or adult rat hearts were cultured in control or in three different hypercholesterolemic media with increasing cholesterol content (hiChol) or hiChol + hyperglycemic medium, respectively. Each group was then subjected to simulated ischemia/reperfusion (SI/R) or corresponding normoxic condition, respectively. Cholesterol uptake was tested by Filipin staining in neonatal cardiac myocytes. Cell viability, total cell count and oxidative stress, i.e., total reactive oxygen species (ROS) and superoxide level were measured by fluorescent assays.
Neonatal cardiac myocytes took up cholesterol from the different hiChol media at a concentration-dependent manner. In normoxia, viability of hiChol neonatal cardiac myocytes was not significantly changed, however, superoxide levels were increased as compared to vehicle. After SI/R, the viability of hiChol neonatal cardiac myocytes was decreased and total ROS level was increased as compared to vehicle. HiChol combined with hyperglycemia further aggravated cell death and oxidative stress in normoxic as well as in SI/R conditions. Viability of hiChol adult cardiac myocytes was significantly decreased and superoxide level was increased in normoxia and these changes were further aggravated by SI/R. HiChol combined with hyperglycemia further aggravated cell death, however level of oxidative stress increased only in normoxic condition.
HiChol rat cardiac myocytes showed reduction of cell viability and increased oxidative stress, which were further aggravated by SI/R and with additional hyperglycemia. This is the first demonstration that the combination of the current hypercholesterolemic medium and SI/R in cardiac myocytes mimics the cardiac pathology of the comorbid heart with I/R and hypercholesterolemia.
Swiprosin-1 (EFhD2) is a molecule that triggers structural adaptation of isolated adult rat cardiomyocytes to cell culture conditions by initiating a process known as cell spreading. This process ...mimics central aspects of cardiac remodeling, as it occurs subsequent to myocardial infarction. However, expression of swiprosin-1 in cardiac tissue and its regulation in vivo has not yet been addressed. The expression of swiprosin-1 was analyzed in mice, rat, and pig hearts undergoing myocardial infarction or ischemia/reperfusion with or without cardiac protection by ischemic pre- and postconditioning. In mouse hearts, swiprosin-1 protein expression was increased after 4 and 7 days in myocardial infarct areas specifically in cardiomyocytes as verified by immunoblotting and histology. In rat hearts, swiprosin-1 mRNA expression was induced within 7 days after ischemia/reperfusion but this induction was abrogated by conditioning. As in cultured cardiomyocytes, the expression of swiprosin-1 was associated with a coinduction of arrestin-2, suggesting a common mechanism of regulation. Rno-miR-32-3p and rno-miR-34c-3p were associated with the regulation pattern of both molecules. Moreover, induction of swiprosin-1 and ssc-miR-34c was also confirmed in the infarct zone of pigs. In summary, our data show that up-regulation of swiprosin-1 appears in the postischemic heart during cardiac remodeling and repair in different species.
Abstract
Aims
Remote ischaemic preconditioning (RIPC) is a robust cardioprotective intervention in preclinical studies. To establish a working and efficacious RIPC protocol in our laboratories, we ...performed randomized, blinded in vivo studies in three study centres in rats, with various RIPC protocols. To verify that our experimental settings are in good alignment with in vivo rat studies showing cardioprotection by limb RIPC, we performed a systematic review and meta-analysis. In addition, we investigated the importance of different study parameters.
Methods and results
Male Wistar rats were subjected to 20–45 min cardiac ischaemia followed by 120 min reperfusion with or without preceding RIPC by 3 or 4 × 5−5 min occlusion/reperfusion of one or two femoral vessels by clamping, tourniquet, or pressure cuff. RIPC did not reduce infarct size (IS), microvascular obstruction, or arrhythmias at any study centres. Systematic review and meta-analysis focusing on in vivo rat models of myocardial ischaemia/reperfusion injury with limb RIPC showed that RIPC reduces IS by 21.28% on average. In addition, the systematic review showed methodological heterogeneity and insufficient reporting of study parameters in a high proportion of studies.
Conclusion
We report for the first time the lack of cardioprotection by RIPC in rats, assessed in individually randomized, blinded in vivo studies, involving three study centres, using different RIPC protocols. These results are in discrepancy with the meta-analysis of similar in vivo rat studies; however, no specific methodological reason could be identified by the systematic review, probably due to the overall insufficient reporting of several study parameters that did not improve over the past two decades. These results urge for publication of more well-designed and well-reported studies, irrespective of the outcome, which are required for preclinical reproducibility, and the development of clinically translatable cardioprotective interventions.
Graphical Abstract
Graphical Abstract
We tested the cardioprotective efficacy of various RIPC protocols in three study centres in Hungary and the Netherlands. Neither of the applied methodologies resulted in cardioprotection. To verify that our in vivo methodological settings are in accordance with those reported in the literature, a systematic review and meta-analysis was performed. A discrepancy between meta-analysis and our in vivo results was found, despite that major methodological settings were similar to the publications analysed. Systematic review identifies insufficient and highly heterogeneous reporting of the investigated data items that did not improve over time, which could contribute to the discrepancy. RIPC: remote ischaemic preconditioning; I/R: ischaemia/reperfusion; IPC: in situ ischaemic preconditioning; MI: myocardial infarction; IS: infarct size; IQR: inter-quartile range. The figure was created with BioRender.com.
Cardioprotective value of ischemic post- (IPostC), remote (RIC) conditioning in acute myocardial infarction (AMI) is unclear in clinical trials. To evaluate cardioprotection, most translational ...animal studies and clinical trials utilize necrotic tissue referred to the area at risk (AAR) by magnetic resonance imaging (MRI). However, determination of AAR by MRI' may not be accurate, since MRI-indices of microvascular damage, i.e., myocardial edema and microvascular obstruction (MVO), may be affected by cardioprotection independently from myocardial necrosis. Therefore, we assessed the effect of IPostC, RIC conditioning and ischemic preconditioning (IPreC; positive control) on myocardial necrosis, edema and MVO in a clinically relevant, closed-chest pig model of AMI.
Acute myocardial infarction was induced by a 90-min balloon occlusion of the left anterior descending coronary artery (LAD) in domestic juvenile female pigs. IPostC (6 × 30 s ischemia/reperfusion after 90-min occlusion) and RIC (4 × 5 min hind limb ischemia/reperfusion during 90-min LAD occlusion) did not reduce myocardial necrosis as assessed by late gadolinium enhancement 3 days after reperfusion and by ex vivo triphenyltetrazolium chloride staining 3 h after reperfusion, however, the positive control, IPreC (3 × 5 min ischemia/reperfusion before 90-min LAD occlusion) did. IPostC and RIC attenuated myocardial edema as measured by cardiac T2-weighted MRI 3 days after reperfusion, however, AAR measured by Evans blue staining was not different among groups, which confirms that myocardial edema is not a measure of AAR, IPostC and IPreC but not RIC decreased MVO.
We conclude that IPostC and RIC interventions may protect the coronary microvasculature even without reducing myocardial necrosis.
Little is known about the molecular mechanism including microRNAs (miRNA) in hypercholesterolemia-induced cardiac dysfunction. We aimed to explore novel hypercholesterolemia-induced pathway ...alterations in the heart by an unbiased approach based on miRNA omics, target prediction and validation. With miRNA microarray we identified forty-seven upregulated and ten downregulated miRNAs in hypercholesterolemic rat hearts compared to the normocholesterolemic group. Eleven mRNAs with at least 4 interacting upregulated miRNAs were selected by a network theoretical approach, out of which 3 mRNAs (beta-2 adrenergic receptor Adrb2, calcineurin B type 1 Ppp3r1 and calcium/calmodulin-dependent serine protein kinase Cask) were validated with qRT-PCR and Western blot. In hypercholesterolemic hearts, the expression of Adrb2 mRNA was significantly decreased. ADRB2 and PPP3R1 protein were significantly downregulated in hypercholesterolemic hearts. The direct interaction of Adrb2 with upregulated miRNAs was demonstrated by luciferase reporter assay. Gene ontology analysis revealed that the majority of the predicted mRNA changes may contribute to the hypercholesterolemia-induced cardiac dysfunction. In summary, the present unbiased target prediction approach based on global cardiac miRNA expression profiling revealed for the first time in the literature that both the mRNA and protein product of Adrb2 and PPP3R1 protein are decreased in the hypercholesterolemic heart.
Clinical observations are highly inconsistent with the use of the antidiabetic rosiglitazone regarding its associated increased risk of myocardial infarction. This may be due to its hidden ...cardiotoxic properties that have only become evident during post-marketing studies. Therefore, we aimed to investigate the hidden cardiotoxicity of rosiglitazone in ischemia/reperfusion (I/R) injury models. Rats were treated orally with either 0.8 mg/kg/day rosiglitazone or vehicle for 28 days and subjected to I/R with or without cardioprotective ischemic preconditioning (IPC). Rosiglitazone did not affect mortality, arrhythmia score, or infarct size during I/R. However, rosiglitazone abolished the antiarrhythmic effects of IPC. To investigate the direct effect of rosiglitazone on cardiomyocytes, we utilized adult rat cardiomyocytes (ARCMs), AC16, and differentiated AC16 (diffAC16) human cardiac cell lines. These were subjected to simulated I/R in the presence of rosiglitazone. Rosiglitazone improved cell survival of ARCMs at 0.3 μM. At 0.1 and 0.3 μM, rosiglitazone improved cell survival of AC16s but not that of diffAC16s. This is the first demonstration that chronic administration of rosiglitazone does not result in major hidden cardiotoxic effects in myocardial I/R injury models. However, the inhibition of the antiarrhythmic effects of IPC may have some clinical relevance that needs to be further explored.
Little is known about the role of the neuropeptide somatostatin (SST) in myocardial ischemia/reperfusion injury and cardioprotection. Here, we investigated the direct cardiocytoprotective effect of ...SST on ischemia/reperfusion injury in cardiomyocyte cultures, as well as the expression of SST and its receptors in pig and human heart tissues. SST induced a bell-shaped, concentration-dependent cardiocytoprotection in both adult rat primary cardiomyocytes and H9C2 cells subjected to simulated ischemia/reperfusion injury. Furthermore, in a translational porcine closed-chest acute myocardial infarction model, ischemic preconditioning increased plasma SST-like immunoreactivity. Interestingly, SST expression was detectable at the protein, but not at the mRNA level in the pig left ventricles.
SSTR1
and
SSTR2
, but not the other SST receptors, were detectable at the mRNA level by PCR and sequencing in the pig left ventricle. Moreover, remote ischemic conditioning upregulated
SSTR1
mRNA. Similarly, SST expression was also detectable in healthy human interventricular septum samples at the protein level. Furthermore, SST-like immunoreactivity decreased in interventricular septum samples of patients with ischemic cardiomyopathy.
SSTR1, SSTR2,
and
SSTR5
but not
SST
and the other SST receptors were detectable at the mRNA level by sequencing in healthy human left ventricles. In addition, in healthy human left ventricle samples,
SSTR1
and
SSTR2
mRNAs were expressed especially in vascular endothelial and some other cell types as detected by RNA Scope
®
in situ
hybridization. This is the first demonstration that SST exerts a direct cardiocytoprotective effect against simulated ischemia/reperfusion injury. Moreover, SST is expressed in the heart tissue at the peptide level; however, it is likely to be of sensory neural origin since its mRNA is not detectable. SSTR1 and SSTR2 might be involved in the cardioprotective action of SST, but other mechanisms cannot be excluded.
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