Cardioprotective Molecules Research Articles

Identification of candidate long noncoding RNAs associated with left ventricular hypertrophy.

Long noncoding RNAs (lncRNAs) constitute an emerging group of noncoding RNAs, which regulate gene expression. Their role in cardiac disease is poorly known. Here, we investigated the association between lncRNAs and left ventricular hypertrophy. Wild-type and adenosine A2A receptor overexpressing mice (A2A-Tg) were subjected to transverse aortic constriction (TAC) and expression of lncRNAs in the heart was investigated using genome-wide microarrays and an analytical pipeline specifically developed for lncRNAs. Microarray analysis identified two lncRNAs up-regulated and three down-regulated in the hearts of A2A-Tg mice subjected to TAC. Quantitative PCR showed that lncRNAs 2900055J20Rik and Gm14005 were decreased in A2A-Tg mice (3.5- and 1.8-fold, p < 0.01). We found from public microarray dataset that 2900055J20Rik and Gm14005 were increased in TAC mice compared to sham-operated animals (1.8- and 1.4-fold, after 28 days, p < 0.01). Interestingly, in this public dataset, cardioprotective drug JQ1 decreased 2900055J20Rik and Gm14005 expression by 2.2- and 1.6-fold (p < 0.01). First, we have shown that data on lncRNAs can be obtained from gene expression microarrays. Second, expression of lncRNAs 2900055J20Rik and Gm14005 is regulated after TAC and can be modulated by cardioprotective molecules. These observations motivate further investigation of the therapeutic value of lncRNAs in the heart.

Exercise protects against chronic β-adrenergic remodeling of the heart by activation of endothelial nitric oxide synthase.

Extensive data have shown that exercise training can provide cardio-protection against pathological cardiac hypertrophy. However, how long the heart can retain cardio-protective phenotype after the cessation of exercise is currently unknown. In this study, we investigated the time course of the loss of cardio-protection after cessation of exercise and the signaling molecules that are responsible for the possible sustained protection. Mice were made to run on a treadmill six times a week for 4 weeks and then rested for a period of 0, 1, 2 and 4 weeks followed by isoproterenol injection for 8 days. Morphological, echocardiographic and hemodynamic changes were measured, gene reactivation was determined by real-time PCR, and the expression and phosphorylation status of several cardio-protective signaling molecules were analyzed by Western-blot. HW/BW, HW/TL and LW/BW decreased significantly in exercise training (ER) mice. The less necrosis and lower fetal gene reactivation induced by isoproterenol injection were also found in ER mice. The echocardiographic and hemodynamic changes induced by β-adrenergic overload were also attenuated in ER mice. The protective effects can be sustained for at least 2 weeks after the cessation of the training. Western-blot analysis showed that the alterations in the phosphorylation status of endothelial nitric oxide synthase (eNOS) (increase in serine 1177 and decrease in threonine 495) continued for 2 weeks after the cessation of the training whereas increases of the phosphorylation of Akt and mTOR disappeared. Further study showed that L-NG-Nitroarginine methyl ester (L-NAME) treatment abolished the cardio-protective effects of ER. Our findings demonstrate that stimulation of eNOS in mice through exercise training provides acute and sustained cardioprotection against cardiac hypertrophy.

Pentraxin-3 and galectin-1 are key mediators of the cardioprotective paracrine effects exerted by fetal mesenchymal stem cells isolated from human placenta

Background and purpose: We have data showing that the cytoprotective paracrine effects exerted by human mesenchymal stem cells (MSC) are negatively influenced by donor age. We also showed that MSC of fetal origin (F-MSC) isolated from human placenta exert remarkable cardioprotective paracrine effects. Our aim was to unravel putative cardioprotective paracrine molecules using proteomics. Methods: Human F-MSC were isolated from amniotic membranes while adult MSC from bone marrow of elderly donors (oBM-MSC; age >65) undergoing routine orthopaedic surgery. Total proteins from F-MSC and oBM-MSC were analyzed by liquid chromatography-Fourier transform mass spectrometry (LC-FTMS). RNA small interference (siRNA) was used to generate specific F-MSC in which Pentraxin-3 (sPTX3) and Galectin-1 (sGAL1) were knocked down (sPTX3-F-MSC and sGAL1-F-MSC, respectively). Rat cardiomyocytes (CMC) were exposed to hypoxia/reoxygenation (H/R) in the presence of control medium (CTRL-M) or medium conditioned by naive F-MSC (F-CM), sPTX3-F-MSC (sPTX3-F-CM), sGAL1-F-MSC (sGAL1-F-CM) and PTX3 or GAL1 recombinant proteins. CMC viability was quantified by MTS assay, apoptosis by Caspase-3 activation and cytotoxicity by LDH release. Results: LC-FTMS detected 129 proteins significantly up-regulated in F-MSC vs oBM-MSC, including secreted proteins. Among them, PTX3 and GAL1 emerged as putative cytoprotective mediators. Recombinant PTX3 or GAL1 administration significantly improved survival, reduced apoptosis and LDH release of CMC exposed to H/R at 10 ng/mL and 3 ng/mL concentration, respectively. sPTX3-F-MSC and sGAL1-F-MSC were efficiently generated by siRNA technology as confirmed at transcriptional and secretion level. Naive F-CM significantly preserved CMC viability from H/R injury (+44% vs CTRL-M, p<0.001). PTX3 or GAL1 knockdowns significantly blunted F-CM protective effect (sPTX3-F-CM -33% vs F-CM, p<0.01; sGAL1-F-CM -46% vs F-CM, p<0.001; sPTX3-F-CM or sGAL1-F-CM vs CTRL-M, p=ns). Both sPTX3-F-CM and sGAL1-F-CM failed in preventing Caspase-3 activation after H/R (sPTX3-F-CM and sGAL1-F-CM vs CTRL-M, p=ns). Conclusions: By comparing the proteomic profile of adult and fetal MSC, we identified PTX3 and GAL1 as mediators of the powerful cardioprotective effects exerted by F-MSC. Our results will help to further clarify the mechanisms of stem cell action in cardiac repair.

Cardioprotective molecules are enriched in beating cardiomyocytes derived from human embryonic stem cells

BackgroundCardiomyocytes derived from human embryonic stem cells (hESC-CMs) have attracted attention because of their cardiac regenerative potential in vivo. Differentiated CMs can be distinguished into two different phenotypic populations: beating and non-beating CMs. A thorough understanding of the different molecular conditions of beating and non-beating CMs would provide valuable information for other potential applications and cell therapy. MethodsIn this study, we generated a comparative protein profiles using proteomic analysis and western blotting, to compare the specific protein expression patterns of beating and non-beating hESC-CMs. ResultsAbundantly 72 upregulated proteins are involved in different biological processes such as stimulus response, cellular catabolism and cell motility. Among these proteins, such as HSPs and other antioxidant molecules, are known to be proteins that potentially play an important role in cardioprotection through the enhancement of cell survival in hypoxic and ischemic conditions present in the injured heart. ConclusionAs a first step toward understanding the different molecular conditions of beating and non-beating hESC-CMs, we sought to study their differential expression patterns and discuss their relevance to in vivo functioning in cardiac injury repair. Thus, the results of this study could provide further evidence supporting a cardiac regenerative approach using an optimized cell source derived from hESCs.

Abstract P319: Protein Modulation in Mouse Heart Under Acute and Chronic Hypoxia

Exploring cellular mechanisms underlying beneficial adaptive processes and detrimental responses to hypoxia represents the object of the present study. Signaling molecules controlling adaptation to hypoxia (HIF-1α), energy balance (AMPK), mitochondrial biogenesis, (PGC-1α) and the regulation of autophagic/apoptotic processes, combined with proteomic dysregulation were assessed. Responses to normobaric acute (48 hrs FiO 2 = 0.08) and chronic (10 days FiO 2 = 0.08) hypoxia in mouse heart proteome were detected applying 2-D DIGE, mass spectrometry and antigen antibody reactions. Both in acute (AH) and chronic (CH) hypoxia, results indicated a deregulation of proteins related to sarcomere stabilization and muscle contraction. Neither in AH nor in CH HIF-1α stabilization was detected. In AH, the metabolic adaptation to oxygen lack is controlled by AMPK activation and sustained by the massive up-regulation of adenosylhomocysteinase and of acetyl coenzyme A synthetase. AH is also characterized by an up-regulation of Bnip 3 which is known to induce mitophagy and decreasing oxidative damage. PGC-1α, a master regulator of mitochondrial biogenesis, was down- regulated. CH was characterized by up-regulation of enzymes involved in antioxidant defense, in aldehyde bio-product detoxification and in misfolded protein degradation. In addition, a massive down-regulation of enzymes controlling anaerobic metabolism was observed. After 10 days of hypoxia, cardioprotective molecules were substantially decreased whereas pro-apoptotic signals were correlated with the increment of apoptotic program and sustained by the down-regulation of specific target proteins. In conclusion, differential analysis provided new insight into target molecules sensing lack of oxygen in vivo and could open new possibilities for protection from damages induced by long term hypoxia exposure.

Protein modulation in mouse heart under acute and chronic hypoxia

Exploring cellular mechanisms underlying beneficial and detrimental responses to hypoxia represents the object of the present study. Signaling molecules controlling adaptation to hypoxia (HIF-1α), energy balance (AMPK), mitochondrial biogenesis (PGC-1α), autophagic/apoptotic processes regulation and proteomic dysregulation were assessed. Responses to acute hypoxia (AH) and chronic hypoxia (CH) in mouse heart proteome were detected by 2-D DIGE, mass spectrometry and antigen-antibody reactions. Both in AH and CH, the results indicated a deregulation of proteins related to sarcomere stabilization and muscle contraction. Neither in AH nor in CH the HIF-1α stabilization was observed. In AH, the metabolic adaptation to lack of oxygen was controlled by AMPK activation and sustained by an up-regulation of adenosylhomocysteinase and acetyl-CoA synthetase. AH was characterized by the mitophagic protein Bnip 3 increment. PGC-1α, a master regulator of mitochondrial biogenesis, was down-regulated. CH was characterized by the up-regulation of enzymes involved in antioxidant defense, in aldehyde bio-product detoxification and in misfolded protein degradation. In addition, a general down-regulation of enzymes controlling anaerobic metabolism was observed. After 10 days of hypoxia, cardioprotective molecules were substantially decreased whereas pro-apoptotic molecules increased accompained by down-regulation of specific target proteins.

Hypoxia inducible factor 1 (HIF-1) and cardioprotection

Since its discovery in early 1990s, hypoxia inducible factor 1 (HIF-1) has been increasingly recognized for its key role in transcriptional control of more than a hundred genes that regulate a wide-spectrum of cellular functional events, including angiogenesis, vasomotor control, glucose and energy metabolism, erythropoiesis, iron homeostasis, pH regulation, cell proliferation and viability. Evidence accumulated during the past 7 years suggests a critical role for HIF-1alpha in mediating cardioprotection. The purpose of our present article is to provide an updated overview on this important regulator of gene expression in the cellular stress-responsive and adaptive process. We have particularly emphasized the involvement of HIF-1 in the induction of cardioprotective molecules, such as inducible nitric oxide synthase (iNOS), hemeoxygenase 1 (HO-1), and erythropoietin (EPO), which in turn alleviate myocardial damages caused by harmful events such as ischemia-reperfusion injury. Despite these advances, further in-depth studies are needed to elucidate the possible coordination or interaction between HIF-1alpha and other key transcription factors in regulating protein expression that leads to cardioprotection.

Long-term thyroid hormone administration improves myocardial performance after myocardial infarction in rats

Thyroid hormone (TH) is critical for tissue differentiation at early stages of development, induces physiological hypertrophy and regulates the expression of important contractile proteins. Furthermore, TH controls the response to stress by regulating important cardioprotective molecules such as heat shock proteins (HSPs). Thus, the present study investigated whether TH administration immediately after acute myocardial infarction can favorably remodel the postinfarcted myocardium. Acute myocardial infarction was induced in rats by coronary artery ligation (AMI, n=10) while SHAM operated animals served as controls (SHAM, n=8). TH was administered for 13 weeks (AMI-THYR, n=9). Cardiac contractile function and left ventricular (LV) chamber remodeling was assessed by serial echocardiography and in Langendorff heart preparations. AMI significantly reduced LV ejection fraction (EF%); 30.0 (s.e.m, 2.3) vs. 73.8 (1.8) in SHAM, p<0.05. +dp/dt and −dp/dt (in mm Hg/s) were 4051 (343) and 2333 (118) respectively for SHAM, 2102 (290) and 1368 (181) for AMI, p<0.05. With TH treatment, EF% was increased to 49.5 (2.7) in AMI-THYR, p<0.05 while +dp/dt and −dp/dt (in mm Hg/s) were 3708 (231) and 2035 (95) for AMI-THYR, p<0.05 vs. AMI. A marked elevation of the expression of β-MHC was found in the viable myocardium of AMI hearts which was prevented by TH. HSP70 myocardial content was decreased in AMI hearts and significantly increased after TH treatment. An ellipsoidal reshaping of LV chamber was observed with TH; cardiac sphericity index, (ratio of long/short axis, SI), was 1.98 (0.03) for SHAM, 1.52 (0.05) for AMI and 1.72 (0.02) for AMI-THYR, p<0.05. In conclusion, long-term TH administration immediately after AMI results in sustained improvement of cardiac haemodynamics.

Long-term thyroid hormone administration reshapes left ventricular chamber and improves cardiac function after myocardial infarction in rats

Thyroid hormone (TH) is critical for tissue differentiation at early stages of development, induces physiological hypertrophy and regulates the expression of important contractile proteins such as myosin heavy chain (MHC) isoform and calcium cycling proteins. Furthermore, TH seems to control the response to stress by regulating important cardioprotective molecules such as heat shock proteins (HSPs). Thus, the present study investigated whether TH administration immediately after acute myocardial infarction can favourably remodel the post-infarcted myocardium. Acute myocardial infarction was induced in rats by coronary artery ligation (AMI, n=10), while SHAM-operated animals served as controls (SHAM, n = 8). TH was administered for 13 weeks (AMI-THYR, n = 9). Cardiac contractile function and left ventricular (LV) chamber remodelling was assessed by serial echocardiography and in Langendorff heart preparations. AMI significantly reduced LV ejection fraction (EF%); 30.0 (s.e.m, 2.3) Vs. 73.8 (1.8) in SHAM, P < 0.05. In addition, +dp/dt and -dp/dt (in mmHg/s) were 4,051 (343) and 2,333 (118) respectively for SHAM Vs. 2,102 (290) and 1,368 (181) for AMI, P < 0.05. With TH treatment, EF% was increased to 49.5 (2.7) in AMI-THYR, P < 0.05, while +dp/dt and -dp/dt (in mmHg/s) were 3,708 (231) and 2,035 (95) for AMI-THYR, P < 0.05 Vs. AMI. A marked elevation of the expression of beta-MHC and a reduced ratio of SERCA/Phospholamban were found in viable myocardium of AMI hearts, which was prevented by TH. Furthermore, heat shock protein 70 myocardial content was decreased in AMI hearts and was significantly increased after TH treatment. An ellipsoidal reshaping of LV chamber was observed with TH; cardiac sphericity index, (ratio of long/short axis, SI), was 1.98 (0.03) for SHAM, 1.52 (0.05) for AMI and 1.72(0.02) for AMI-THYR, P < 0.05. In conclusion, long-term TH administration immediately after AMI results in sustained improvement of cardiac haemodynamics.

Essential fatty acids and their metabolites could function as endogenous HMG-CoA reductase and ACE enzyme inhibitors, anti-arrhythmic, anti-hypertensive, anti-atherosclerotic, anti-inflammatory, cytoprotective, and cardioprotective molecules.

Lowering plasma low density lipoprotein-cholesterol (LDL-C), blood pressure, homocysteine, and preventing platelet aggregation using a combination of a statin, three blood pressure lowering drugs such as a thiazide, a β blocker, and an angiotensin converting enzyme (ACE) inhibitor each at half standard dose; folic acid; and aspirin-called as polypill- was estimated to reduce cardiovascular events by ~80%. Essential fatty acids (EFAs) and their long-chain metabolites: γ-linolenic acid (GLA), dihomo-GLA (DGLA), arachidonic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) and other products such as prostaglandins E1 (PGE1), prostacyclin (PGI2), PGI3, lipoxins (LXs), resolvins, protectins including neuroprotectin D1 (NPD1) prevent platelet aggregation, lower blood pressure, have anti-arrhythmic action, reduce LDL-C, ameliorate the adverse actions of homocysteine, show anti-inflammatory actions, activate telomerase, and have cytoprotective properties. Thus, EFAs and their metabolites show all the classic actions expected of the "polypill". Unlike the proposed "polypill", EFAs are endogenous molecules present in almost all tissues, have no significant or few side effects, can be taken orally for long periods of time even by pregnant women, lactating mothers, and infants, children, and adults; and have been known to reduce the incidence cardiovascular diseases including stroke. In addition, various EFAs and their long-chain metabolites not only enhance nitric oxide generation but also react with nitric oxide to yield their respective nitroalkene derivatives that produce vascular relaxation, inhibit neutrophil degranulation and superoxide formation, inhibit platelet activation, and possess PPAR-γ ligand activity and release NO, thus prevent platelet aggregation, thrombus formation, atherosclerosis, and cardiovascular diseases. Based on these evidences, I propose that a rational combination of ω-3 and ω-6 fatty acids and the co-factors that are necessary for their appropriate action/metabolism is as beneficial as that of the combined use of a statin, thiazide, a β blocker, and an angiotensin converting enzyme (ACE) inhibitor, folic acid, and aspirin. Furthermore, appropriate combination of ω-3 and ω-6 fatty acids may even show additional benefits in the form of protection from depression, schizophrenia, Alzheimer's disease, and enhances cognitive function; and serve as endogenous anti-inflammatory molecules; and could be administered from childhood for life long.

Novel antioxidants in anthracycline cardiotoxicity

It has been suggested nitroxides and their amine precursors prevent incidence of damage caused by superoxide and hydroxyl radicals formed during the oxidative metabolism of doxorubicin (DOX) and daunorubicin (DAU). Both doxorubicin and daunorubicin have been associated with cardiac toxicity in both adults and children. The authors herein suggest that cardioprotective molecules modified by nitroxides and their secondary amine precursors can prevent or diminish the anthracycline-induced cardiomyopathy by accumulating in cardiomiocytes.

Preclinical assessment of anthracycline cardiotoxicity in laboratory animals: Predictiveness and pitfalls

Doxorubicin is one of the most prescribed anticancer drugs, due to its important activity in hematological malignancies as in solid tumors. However, its important cardiac toxicity still limits its long-term use and prevents from reaching optimal benefits. Numerous ways have been proposed to avoid cardiac toxicity, such as protracted infusions or special formulations, development of less cardiotoxic analogues and of cardioprotectors. There is a need for preclinical models able to screen rapidly these various approaches and to provide rational bases for clinical trials. The first model is the long-term rabbit model. Weanling rabbits given weekly injections of doxorubicin for 4 months developed a cardiomyopathy which was obvious from a clinical (cardiac failure) and from a pathological point of view. This model has been widely used afterwards for the discovery of cardioprotective molecules. Models in other animals such as rats or mice were similarly implemented, also with long-term exposures to the drug, resulting in cardiac failure and severe pathological alterations which could be graded for comparison. Starting from the evidence that the damage caused by anthracyclines on cardiomyocytes was immediate after each injection and that the functional efficiency of the myocardium should be affected by the anthracyclines long before the morphological alterations become detectable, we developed a short-term model studying the cardiac performances of isolated perfused hearts of rats that had been treated within 12 days by repetitive administrations of the molecule(s) to be tested. This model appeared easy to implement and provided the data expected from clinical experience: epirubicin appeared less cardiotoxic than doxorubicin; liposomal formulations appeared less cardiotoxic than free drug formulations; dexrazoxane strongly protected against doxorubicin cardiotoxicity. We were then to show that paclitaxel could potentiate doxorubicin cardiotoxicity, but that docetaxel did not so; or that a high dose of dexrazoxane brought significantly higher protection than a conventional dose. Based upon these various contributions, we can encourage the use of the short-term model of isolated perfused rat heart to screen the preclinical cardiotoxicity of anthracycline molecules, formulations and combinations.