ORIGINAL RESEARCH published: 03 May 2022 doi: 10.3389/fphar.2022.891801 Supplementation With Spirulina Reduces Infarct Size and Ameliorates Cardiac Function in a Pig Model of STEMI Gemma Vilahur 1,2†, Pablo Sutelman 1†, Soumaya Ben-Aicha 1, Guiomar Mendieta 1,3, Monika Radiké 1, Leonie Schoch 1, Laura Casaní 1,2, María Borrell-Pagés 1, Teresa Padro 1,2 and Lina Badimon 1,2,4* 1 Cardiovascular Program-ICCC, Research Institute Hospital de la Santa Creu i Sant Pau, IIB-Sant Pau, Barcelona, Spain, 2 CiberCV, Institute Carlos III, Madrid, Spain, 3Department of Cardiology, Clinic Hospital, Barcelona, Spain, 4Autonomous Edited by: University of Barcelona, Barcelona, Spain Lei Xi, Virginia Commonwealth University, United States Background and Aims: Myocardial infarction (MI) is the clinical manifestation of Reviewed by: atherosclerotic coronary artery disease. Spirulina is an algae known to ameliorate Coert J. Zuurbier, Academic Medical Center, cardiometabolic disorders and with proven anti-inflammatory and anti-oxidant effects. Netherlands We investigated, in a highly translatable animal model, whether oral supplementation with Petra Kleinbongard, spirulina protects against the deleterious effects triggered by ST-elevation MI (STEMI). Essen University Hospital, Germany *Correspondence: Methods: Pigs were fed a regular diet supplemented with spirulina (1 g/animal/bid) or Lina Badimon placebo-control for 10 days. Thereafter, animals were subjected to 1.5 h percutaneous lbadimon@santpau.cat † balloon-induced coronary occlusion (STEMI) followed by 2.5 h reperfusion and then These authors have contributed equally to this work sacrificed. We assessed infarct size and cardiac function. Blood samples and infarcted and remote myocardial tissue were obtained. Specialty section: This article was submitted to Results: Spirulina supplementation reduced infarct size by 64%, increased myocardial Translational Pharmacology, salvage by 18%, and improved cardiac function by 30% vs. controls (p < 0.05). These a section of the journal Frontiers in Pharmacology benefits were associated with attenuation in DNA-oxidative damage and apoptotic Received: 08 March 2022 markers and increased iNOS in the infarcted myocardium, higher AMPK activation in Accepted: 14 April 2022 the remote myocardium, and lower myocardial MCP-1 expression. Systemically, spirulina Published: 03 May 2022 attenuated Cox-2 expression in STEMI-activated peripheral blood mononuclear cells and Citation: enhanced TNF-α release acutely post-STEMI. Additionally, spirulina decreased weight gain Vilahur G, Sutelman P, Ben-Aicha S, Mendieta G, Radiké M, Schoch L, progression over time (p < 0.05) without changes in lipids, glucose, liver or kidney Casaní L, Borrell-Pagés M, Padro T parameters. and Badimon L (2022) Supplementation With Spirulina Conclusion: A 10-day supplementation with spirulina exerts cardioprotection in a Reduces Infarct Size and Ameliorates Cardiac Function in a Pig Model preclinical setting of STEMI by limiting cardiac damage and improving ventricular of STEMI. contractility through anti-oxidative, anti-inflammatory, and anti-apoptotic mechanisms. Front. Pharmacol. 13:891801. doi: 10.3389/fphar.2022.891801 Keywords: spirulina, STEMI, cardioprotection, antioxidation, anti-inflammatory, anti-apoptotic, weight management Frontiers in Pharmacology | www.frontiersin.org 1 May 2022 | Volume 13 | Article 891801 Vilahur et al. Spirulina Limits Infarct Size Post-STEMI INTRODUCTION associated with oral daily supplementation with spirulina in a highly translatable preclinical animal model of STEMI. Despite significant improvement in the treatment of atherosclerotic cardiovascular disease (ASCVD), cardiovascular mortality remains the leading cause of death in developed MATERIALS AND METHODS countries (Badimon et al., 2017). Rupture of coronary atherosclerotic plaques and subsequent thrombus formation Animal Procedures leads to coronary vessel occlusion and ensuing myocardial Animal management was performed following European infarction (MI). Following an acute ST-segment elevation Directive 2010/63/EU and in accordance with the ARRIVE 2.0 myocardial infarction (STEMI), the size of the resultant guidelines (Percie du Sert et al., 2020) and complies with the ′3Rs infarction is the major determinant of post-infarct left (Animal Ethics Infolink, 2019). Experimental protocols were ventricular dysfunction and the subsequent development of approved by the Institutional Animal Care and Use Committees. heart failure (Heusch and Gersh, 2017), a condition which exerts a considerable global burden on healthcare and Study Design economic resources (Moran et al., 2014). New treatment The experimental design involved crossbred commercial female strategies are needed in order to limit myocardial injury and swine (n = 12; Landrace × Largewhite) fed a regular pig chow diet improve clinical outcomes in patients presenting with acute randomly supplemented with spirulina (1 g/animal/bid; n = 6) or STEMI. placebo-control (n = 6) for 10 days. Animals were fed a Nutraceuticals are a group of naturally produced bioactive normocholesterolemic diet dosed at 3.5% of pig’s body weight compounds that have proven health benefits besides their adjusted on a daily basis. The regular chow included: gross nutritive properties (Badimon et al., 2010; Vilahur et al., 2018; protein 15.1%; gross fat 3.40%; gross fiber 4.00%; gross ash Carrizzo et al., 2020). In this specific cluster, microalgae have 5.50%; calcium 0.81%; phosphorus 0.57%; sodium 0.15%; stood out as photosynthetic microorganisms capable of lysine 0.89%; methionine 0.24%. All pigs were monitored to generating biofunctional molecules with several cytoprotective ensure intake of their chow and exclude any potential bias due activities. Particularly, spirulina, a filamentous cyanobacterium to a decrease in food intake. Spirulina capsules were composed of which accounts for up to 30% of the overall microalgal biomass 100% spirulina platensis (Pranarom, ACL 7846467). Placebo produced worldwide (Costa et al., 2019). Spirulina is primarily tablets were comprised of standard commercially available comprised of proteins and essential aminoacids providing high gelatin capsules. At the end of the treatment period and 2 h nutritional value, but additionally contains phenolic after the last dosage, pigs were subjected to a 1.5 h experimental phytochemicals including C-phycocyanin, vitamins, STEMI induction followed by 2.5 h phase of complete reperfusion polyunsaturated fatty acids, and an elevated concentration of (i.e., ischemia/reperfusion). Thereafter pigs were sacrificed and beta-carotene, delivering substantial antioxidative, anti- cardiac tissue samples collected. The dosage of spirulina inflammatory, and anti-atherosclerotic properties beyond its administered in this study was selected according to the use in beneficial effects in metabolic-associated cardiovascular both pigs (Yordanova et al., 2015) and clinical trials (Huang et al., disorders (Finamore et al., 2017; DiNicolantonio et al., 2020). 2018). A systematic review including 18 randomized controlled trials has reported that spirulina supplementation is safe and displays positive effects in multiple metabolic syndrome components Closed-Chest Animal Model of ST-Segment (Yousefi et al., 2019). As such, spirulina has shown to exert a Elevation Myocardial Infarction noteworthy weight management capacity eliciting a reduction in STEMI was experimentally induced by fluoroscopy-guided both waist circumference and body mass index in several clinical percutaneous coronary intervention as previously described trials (DiNicolantonio et al., 2020; Grosshagauer et al., 2020). (Vilahur et al., 2009; Mendieta et al., 2019a; Mendieta et al., Besides, several studies with spirulina have shown an 2020) in a preclinical animal model with human resemblance. improvement in insulin sensitivity and glucose uptake Briefly, prior to the procedure, pigs were anesthetized with a mediated by C-phycocyanin activity, and, in some studies combination of intramuscular tiletamine + zolazepam (7 mg/kg) spirulina has displayed hypolipidemic properties (Park et al., + medetomidine (0.07 mg/kg). Animals received a continuous 2008; Mazokopakis et al., 2014b; Serban et al., 2016). On the other and stable flow of oxygen (inspired fraction of 0.5%) during all the hand, cell culture approaches have demonstrated the ability of intervention with permanent control of arterial saturation. spirulina to exhibit anti-atherosclerotic effects by preventing Anesthesia was maintained with inhalatory isofluorane (2%) monocyte migration through direct inhibition of P- and E- throughout the entire procedure (both in the course of selectin adhesion molecules (Vo and Kim, 2013; Ren et al., myocardial infarction induction as well as in the reperfusion 2018), and to effectively inhibit doxorubicin-induced cardiac period). Continuous electrocardiographic monitoring with damage (Khan et al., 2005; Khan et al., 2006a). However, hemodynamic parameters was performed. Coronary occlusion whether oral supplementation with spirulina is able to limit of the mid left anterior descending artery posterior to the infarct size and ameliorate cardiac dysfunction in the setting emergence of the first diagonal branch was induced by balloon of myocardial infarction remains to be addressed. With this in inflation, verifying a Thrombolysis In Myocardial Infarction mind, we sought to examine the potential cardioprotective effects (TIMI) 0 flow downwards. A period of 1.5 h was sustained Frontiers in Pharmacology | www.frontiersin.org 2 May 2022 | Volume 13 | Article 891801 Vilahur et al. Spirulina Limits Infarct Size Post-STEMI with complete coronary obstruction and subsequently arbitrary density units (AU) whereas for gene expression reperfusion was achieved by balloon deflation confirming analyses, the threshold cycle (Ct) values were determined and TIMI 3 flow restoration. normalized to the housekeeping 18SrRNA. Cardiac Function Evaluation A Phillips iE33 echocardiography system equipped with a S5-1 Systemic Inflammatory Response: Sector Array transducer was employed for the attainment of Peripheral Blood Mononuclear Cells and transthoracic echocardiography in every animal prior to STEMI Circulating Cytokine Levels induction, post-STEMI, and after 2.5 h of reperfusion as We assessed the impact of spirulina supplementation on previously performed (Vilahur et al., 2014b). Left ventricle peripheral blood mononuclear cells (PBMCs) activation and ejection fraction (LVEF) was assessed as a surrogate for LV cytokine release. For this purpose, 20 ml of blood were global systolic function. All measurements were acquired by collected into EDTA tubes at the three tested time points an independent operator blinded to treatment group. (prior-STEMI, post-STEMI, and 2.5 h post-reperfusion) from all animals. Blood samples were immediately subjected to Ficoll-paque Plus (Amersham Biosciences, Piscataway, NJ, Cardiac Sample Collection and Myocardial United States) density gradient centrifugations to isolate Damage Assessment PBMCs. PBMC were processed for protein analyses of After the 2.5 h reperfusion period animals were injected with cyclooxygenase 2 (Cox-2) which was normalized to Ponceau Evan’s Blue dye to properly define the area-at-risk (AAR) in order staining. We also analyzed tumor necrosis factor-α (TNF-α) to ensure comparable degree of jeopardized myocardium and interleukin-1β (IL-1β) levels in plasma using commercially (i.e., ischemic heart). Subsequently, pigs were sacrificed during available enzyme-linked immunosorbent assay kits (porcine anesthesia with an intravenous administration of 10 ml KCl 2M. TNF-α and IL-1β from Quantikine Porcine Kits from R&D Hearts were carefully excised and segmented in six transverse Systems). According to the manufacturers, the minimum divisions (1 cm width) in order to alternatively collect slices for detection limits for TNF-α and IL-1β were 23.4 and 39.1 pg/ 2,3,5-triphenyltetrazolium (TTC) staining (infarct size ml−1, respectively. assessment) or molecular/immunohistochemical analyses. In this latter regard, myocardial samples from both infarcted and Biochemical Plasma Analysis remote regions were obtained, frozen, pulverized in liquid Blood samples were obtained at baseline, day 10, prior-STEMI, nitrogen and minced in Tripure® or lysis buffer for RNA and acutely post-STEMI (5 min following coronary occlusion) and protein isolation, respectively. The AAR and TTC staining were after 2.5 h of reperfusion (sacrifice) for haematological (System appraised by an independent blinded observer through 9000, Serono-Baker Diagnostics) and biochemical analysis planimetry assessment employing ImageJ® software analysis (glucose and lipid levels, and kidney and liver function (NIH). Infarct size was expressed as % AAR. parameters; Clima MC-15 RAL Biotechnologies). Myocardial Oxidative Damage Weight Control Paraffin-embedded myocardial tissue from the infarcted region Animals were subjected to weight assessment at baseline and after was cut and placed on poly-L-lysine-coated slides and completion of the 10-day experimental period. deparaffinized. Antigen removal was required before staining for 8-hydroxyguanosine (8OH-G, mouse monoclonal antibody; Statistical Analysis Abcam ab48508), a sensitive biomarker of intracellular DNA Continuous variables were expressed as mean ± standard damage induced by oxidative stress. Four images per animal were deviation (SD). After testing for normal distribution captured by Nikon Eclipse 80i microscope and digitized by Retiga (Shapiro-Wilk test), repeated ANOVA measures and 1300i Fast camera, and staining of the myocardial tissue was paired t-test as appropriate were used to analyze variables quantified and expressed as % stained area. within each group, whereas unpaired t-test was used for all single time-point measurements. A cut-off value of p < 0.05 Molecular Footprint of the Infarcted Heart was used to consider statistical significance. Statistical We assessed in both the infarcted and remote myocardium analyses were performed with the GraphPad Prism protein expression levels of: 1) inducible nitric oxide synthase software package. (iNOS); 2) monocyte chemoattractant protein-1 (MCP-1), an inflammatory chemokine; 3) adenosine monophosphate activated protein kinase (AMPK) phosphorylation, a key RESULTS marker of cardiac metabolism and autophagy; and 4) total and truncated caspase-3, a marker for apoptotic cell death execution. Spirulina Supplementation Limits Weight Besides, we also investigated caspase-3 transcript levels by real- Gain time PCR-7000 Sequence Detection System of ABIPRISM Weight gain was significantly lower in animals receiving spirulina (Applied Biosystems) by an assay-on demand. Protein supplementation as compared to controls (19% vs. 29%, expression was normalized to ß-actin and presented in respectively; p = 0.01; Figure 1). Frontiers in Pharmacology | www.frontiersin.org 3 May 2022 | Volume 13 | Article 891801 Vilahur et al. Spirulina Limits Infarct Size Post-STEMI FIGURE 1 | Effects of Spirulina supplementation on weight.pp < 0.05 vs. control; †p < 0.05 vs. baseline. Data expressed as mean ± SD. FIGURE 2 | Effect of Spirulina supplementation on cardiac damage and function. (A) Effect of Spirulina on infarct size. (B) Effect of Spirulina on cardiac function assessed by echocardiography. AAR, area at risk; LV, left ventricle; LVEF, left ventricular ejection fraction; STEMI, ST-elevation myocardial infarction.pp < 0.05 vs. control. Data expressed as mean ± SD. Frontiers in Pharmacology | www.frontiersin.org 4 May 2022 | Volume 13 | Article 891801 Vilahur et al. Spirulina Limits Infarct Size Post-STEMI FIGURE 3 | Antioxidant effects of Spirulina on the infarcted myocardium. Effects of Spirulina on DNA oxidative damage assessed by 8 hydroxydeoxyguanosine (8OH-dG) staining.pp < 0.05 vs. control. Data expressed as mean ± SD. Arrow heads depict 8-OH-dG statining. Spirulina Supplementation Reduces 0.05; seen in dark-brown in Figure 3). 8OH-dG staining was Myocardial Damage and Improves Cardiac barely detected in the remote myocardium of all pigs. Function Acutely Post-ST-Segment Elevation Myocardial Infarction Effects of Spirulina on Myocardial Both placebo-control and spirulina-treated animals displayed a Molecular Footprint similar extent of jeopardized myocardium (AAR/LV) (67.7% ± We assessed protein levels of iNOS, MCP-1, and P-AMPK. As 3.5% vs. 69.4% ± 4.9%, respectively; p = 0.5) indicating a observed in Figures 4A, D, spirulina supplementation led to a comparable severity of balloon-induced myocardial ischemic significantly higher iNOS protein level in the infarcted region (p < damage (Figure 2A). 0.05 vs. control animals) whereas exerted no impact on the A 10-day supplementation regime with spirulina markedly remote myocardial tissue. Regarding MCP-1, spirulina limited infarct size by 64% as compared to placebo-control supplemented-animals showed a significant reduction in both animals (29.8% ± 11.5% vs. 10.5% ± 3.7% AAR, respectively; p = the infarcted and remote myocardium as compared to the control 0.007). Accordingly, myocardial salvage was 19% greater in the arm (p < 0.05; Figures 4B–D). P-AMPK showed higher levels in spirulina group as compared to placebo-control animals (p < 0.05). the infarcted myocardium of control pigs as compared to the The overall reduction in myocardial damage was associated with a remote myocardial region (p < 0.05) whereas levels were higher in 38% improvement in LVEF vs. controls, an effect already detected post- the entire left ventricle of spirulina supplemented pigs (p < 0.05 STEMI that persisted up to 2.5 h post-reperfusion (p < 0.05; Figure 2B). vs. remote cardiac tissue from control pigs; Figures 4C,D). Spirulina Supplementation Attenuates Spirulina Supplementation Reduces Myocardial DNA-Oxidative Damage Apoptosis Execution in the Infarcted Spirulina supplemented-animals displayed a marked 58% Cardiac Region reduction in oxidative DNA damage in the infarcted Caspase-3 mRNA was found to be upregulated in the infarcted myocardium as compared to placebo-control animals (p < tissue of all animals as compared to remote cardiac regions; Frontiers in Pharmacology | www.frontiersin.org 5 May 2022 | Volume 13 | Article 891801 Vilahur et al. Spirulina Limits Infarct Size Post-STEMI FIGURE 4 | Effect of Spirulina on myocardial molecular footprint. Myocardial protein expression of inducible nitric oxide synthase iNOS; (A) monocyte chemoattractan protein-1 MCP-1; (B) and Phosphorylated AMP protein kinase P-AMPK; (C) in the infarcted and remote myocardium. (D) Representative Western Blot images.pp < 0.05 vs. control; †p < 0.05 vs. remote myocardium. Data expressed as mean ± SD. yet, no differences were detected in caspase-3 protein were detected in IL-1β circulating levels in both groups at all expression (Figures 5A,B). In contrast, spirulina tested time points (Figure 6B). supplementation was associated with a significant lower levels of truncated caspase-3 in the infarcted cardiac region as compared to controls (p < 0.05; Figure 5C). No changes Spirulina Effects on the Metabolic Profile, were observed in active caspase-3 in the remote myocardium and Hematological and Biochemical of both animal groups. Parameters Liver, kidney, lipid and glucose levels and hematological parameters were all within physiological ranges throughout the Spirulina Supplementation Exerts Systemic entire study (Table 1). Anti-Inflammatory Effects Control animals showed an induction of Cox-2 protein expression in PBMCs acutely post-STEMI that was not found DISCUSSION in spirulina supplemented animals (Figure 6A). All animals showed higher circulating levels of TNF-α 2.5 h post- Atherosclerotic plaque rupture is the most common cause of MI reperfusion as compared to baseline, although these levels (Vilahur et al., 2014a). MI-size largely determines patient’s were significantly higher in the spirulina supplemented outcome. Hence, efforts have focused on the identification of subgroup as compared to controls (p < 0.05). No changes strategies able to limit myocardial damage. In the following study Frontiers in Pharmacology | www.frontiersin.org 6 May 2022 | Volume 13 | Article 891801 Vilahur et al. Spirulina Limits Infarct Size Post-STEMI FIGURE 5 | Effect of Spirulina on myocardial apoptosis execution. (A) Caspase-3 transcript levels. (B) Protein expression of total and cleaved caspase-3.pp < 0.05 vs. Spirulina; †p < 0.05 vs. remote myocardium. Data expressed as mean ± SD. we demonstrate for the first time that a 10-day oral reduction in mortality (Khan et al., 2005). In our study we supplementation with spirulina exerts cardioprotection in a further expand spirulina-related cardioprotective properties to preclinical setting of STEMI by enhancing myocardial salvage the setting of coronary artery total occlusion and elucidate the and improving ventricular contractility through antioxidative, mechanisms behind its action. As such, we have evidenced that anti-inflammatory, and anti-apoptotic mechanisms. animals receiving spirulina supplementation have significantly Spirulina is a blue-green algae increasingly employed as a higher iNOS protein levels and a diminished myocardial nutritional supplement because of the health outcomes associated oxidative injury in the infarcted heart and a higher AMPK to its regular intake, particularly regarding metabolic syndrome activation levels in the entire myocardial tissue. Activation of components (Khan et al., 2006b; Stone et al., 2016). Herein, we iNOS/NO signaling in the setting of MI has shown to exert both further prove that a 10-day supplementation with spirulina protective and detrimental effects (Yu et al., 2018). On the one significantly limits coronary artery occlusion-related cardiac hand, there is overwhelming evidence supporting the ability of damage (i.e., infarct size). The size of infarction is strongly NO-derived iNOS to mediate the anti-stunning, vasodilator and related to mortality and heart failure development in STEMI anti-infarct effects in preconditioned hearts (i.e., hearts more patients (Heusch et al., 2014). Despite efforts have focused on resistant to sustained ischemia-related damage because of the testing therapies to promote myocardial salvage, currently there is previous exposure to multiple short episodes of ischemia and no therapy routinely used in the clinical setting (Heusch and reperfusion) (Xi et al., 1999; Bolli, 2001). In fact, iNOS was the Gersh, 2020). first gene identified to mediate late ischemic pre-conditioning So far, the in vivo effects of spirulina on the myocardium have (Madonna et al., 2015), and gene therapy with iNOS, either 3 days been only investigated in a mice model of doxorubicin-induced or up to 2 months prior-MI induction (Li et al., 2006; Li et al., cardiotoxicity where oral spirulina supplementation for 7 weeks 2011), has shown to exert cardioprotection. Yet, it should also be elicited a decrease in lipid peroxidation, normalized antioxidant considered that enhanced iNOS/NO may lead to peroxynitrite enzymes, and limited doxorubicin-induced myocardial formation and subsequently associated oxidative damage. ultrastructural changes leading to an overall significant However, in such scenario, the powerful antioxidant properties Frontiers in Pharmacology | www.frontiersin.org 7 May 2022 | Volume 13 | Article 891801 Vilahur et al. Spirulina Limits Infarct Size Post-STEMI FIGURE 6 | Effect of espirulina on the systemic inflammatory response. (A) Cox-2 protein expression in peripheral blood mononuclear cells (PBMC). Circulating levels of (B) TNF-α and (C) IL-1β.pp < 0.05 vs. control; †p < 0.05 vs. prior-MI. Data expressed as mean ± SD. of spirulina might have blunted iNOS-related oxidative stress indicating that spirulina cardioprotective benefits are not only thereby switching iNOS from detrimental to protective and confined to the infarcted region but to the entire left ventricle. We consequently enhancing the ability of the heart to withstand and others have reported that AMPK signaling, besides being STEMI-related injury. Nevertheless, further research is warranted essential to maintain cardiac energy metabolism under ischemic to determine the mechanism by which spirulina acutely induces a stress, induces autophagy protecting against the progression of higher iNOS expression in the ischemic myocardium in the post-STEMI adverse cardiac remodeling (Kanamori et al., 2011; setting of MI. TNF-α, a cytokine known to acutely protect Vilahur et al., 2016; Mendieta et al., 2019b). A recent study has against infarction and contractile dysfunction, has shown to demonstrated the ability of spirulina to induce mRNA expression regulate iNOS (Lecour et al., 2002). Here, we detect a levels of autophagy-associated genes (i.e., AMPK and ULK1) in significant and acute increase in TNF-α plasma levels with an the liver of growing lambs, supporting our findings in the heart enhanced iNOS expression and reduced oxidative damage in the tissue (Liang et al., 2020). We also identify lower levels of the infarcted heart of spirulina supplemented animals. TNF-α has an inflammatory marker MCP-1 in the entire myocardium of ambivalent role in MI. Transient increase in TNF-a has shown to spirulina supplemented pigs. Furthermore, spirulina anti- protect against ischemia–reperfusion injury by interacting with inflammatory effects are also evident systemically by a reduced myocardial TNF receptor type 2 and consequent downstream Cox-2 expression in the acute phase after coronary occlusion in activation of the cardioprotective SAFE pathway (Lecour et al., PBMCs. Of note, no changes were observed in IL-1β plasma 2005) whereas excessive and long-lasting release of TNF-α has values among all animals at the different tested time points. In shown to exert detrimental effects (Schulz and Heusch, 2009). this regard, a low intrinsic expression of myocardial NLRP3 Moreover, we also evidence higher AMPK activation in the inflammasome (involved in processing of pro-IL-1β into its remote myocardium of spirulina supplemented animals active form) has been detected in the acute phase of infarcted Frontiers in Pharmacology | www.frontiersin.org 8 May 2022 | Volume 13 | Article 891801 Vilahur et al. Spirulina Limits Infarct Size Post-STEMI TABLE 1 | Haematological parameters, (A) liver and kidney function markers, (B) plasma glucose levels and lipid profile (C) at baseline, 10 days, prior-STEMI (ST-elevation myocardial infarction), 5 min post-STEMI and at 2.5 h post-reperfusion (sacrifice). Time Control Spirulina p value A. Haematological parameters Hematocrit (vol %) Baseline 27.0 ± 0.9 25.6 ± 0.5 0.20 10 days 25.8 ± 1.2 24.8 ± 1.4 0.60 Prior-STEMI 25.7 ± 0.1 23.8 ± 0.9 0.09 Post-STEMI 26.9 ± 1.6 22.2 ± 0.9 0.03 Sacrifice 25.8 ± 1.2 24.8 ± 1.4 0.60 Hemoglobin (g/dl) Baseline 9.7 ± 0.6 9.3 ± 0.4 0.63 10 days 9.7 ± 0.54 9.1 ± 0.5 0.45 Prior-STEMI 10.8 ± 0.1 8.9 ± 0.6 0.03 Post-STEMI 9.5 ± 0.5 8.3 ± 0,4 0.08 Sacrifice 9.7 ± 0.5 9.1 ± 0.5 0.45 White blood cells (109/L) Baseline 15.0 ± 1.9 14.4 ± 1.3 0.81 10 days 16.5 ± 2.4 11.3 ± 1.5 0.10 Prior-STEMI 29.2 ± 7.2 14.3 ± 1.8 0.27 Post-STEMI 14.8 ± 2.3 11.9 ± 2.1 0.36 Sacrifice 16.5 ± 2.4 11.3 ± 1.5 0.10 Platelet count (109/L) Baseline 245.5 ± 27.5 354.2 ± 28.2 0.02 10 days 232.1 ± 28.6 324.8 ± 31.6 0.06 Prior-STEMI 291.5 ± 79.5 362.7 ± 33.6 0.53 Post-STEMI 236.8 ± 29.8 317.1 ± 26.7 0.07 Sacrifice 232.1 ± 28.6 324.8 ± 31.6 0.06 B. Kidney and liver function markers Blood urea nitrogen (mg/dl) Baseline 11.0 ± 2.1 17.1 ± 4.0 0.22 10 days 15.7 ± 7.8 15.7 ± 4.7 0.99 Prior-STEMI 24.8 ± 2.8 17,2 ± 2,3 0.14 Post-STEMI 15.0 ± 3.1 17.6 ± 3.1 0.56 Sacrifice 15.7 ± 7.8 15.7 ± 4.7 0.99 Creatinine (mg/dl) Baseline 1.0 ± 0.1 1.2 ± 0.1 0.12 10 days 1.2 ± 0.1 1.3 ± 0.1 0.44 Prior-STEMI 1.0 ± 0.2 1.3 ± 0.1 0.53 Post-STEMI 1.2 ± 0.1 1.5 ± 0.2 0.15 Sacrifice 1.2 ± 0.1 1.3 ± 0.1 0.44 Alanine aminotransferase (U/L) Baseline 39.3 ± 11.8 35.9 ± 2.6 0.78 10 days 37.2 ± 0.3 35.2 ± 17.0 0.91 Prior-STEMI 45.9 ± 2.2 50.0 ± 16.9 0.81 Post-STEMI 27.7 ± 4.4 26.6 ± 4.0 0.85 Sacrifice 37.2 ± 0.3 35.2 ± 17.0 0.91 C. Plasma glucose levels and lipid profile Glucose (mg/dl) Baseline 95.6 ± 11.9 118.5 ± 7.2 0.13 10 days 95.6 ± 11.9 112.8 ± 23.2 0.56 Prior-STEMI 76.2 ± 10.1 108.7 ± 8.6 0.09 Post-STEMI 129.8 ± 14.5 140.2 ± 12.1 0.59 Sacrifice 95.6 ± 11.9 112.8 ± 23.1 0.56 Total cholesterol (mg/dl) Baseline 97.5 ± 3.9 82.4 ± 6.9 0.10 10 days 95.6 ± 12.9 76.7 ± 5.7 0.27 Prior-STEMI 78.5 ± 17.5 84.5 ± 6.2 0.78 Post-STEMI 90.6 ± 7.6 83.1 ± 5.4 0.44 Sacrifice 95.6 ± 12.9 76.7 ± 5.7 0.27 Triglycerides (mg/dl) Baseline 25.8 ± 3.9 35.0 ± 7.3 0.37 10 days 28.3 ± 4.3 32.5 ± 2.5 0.49 Prior-STEMI 22.5 ± 0.5 23.6 ± 3.1 0.83 Post-STEMI 26.8 ±2.4 29.6 ± 3.6 0.67 Sacrifice 28.3 ± 4.3 32.5 ± 2.5 0.49 High-density lipoprotein cholesterol (mg/dl) Baseline 35.9 ± 4.2 47.9 ± 13.2 0.43 10 days 57.8 ± 8.0 64.5 ± 4.4 0.51 Prior-STEMI 21.6 ± 4.7 42.9 ± 7.4 0.06 (Continued on following page) Frontiers in Pharmacology | www.frontiersin.org 9 May 2022 | Volume 13 | Article 891801 Vilahur et al. Spirulina Limits Infarct Size Post-STEMI TABLE 1 | (Continued) Haematological parameters, (A) liver and kidney function markers, (B) plasma glucose levels and lipid profile (C) at baseline, 10 days, prior-STEMI (ST-elevation myocardial infarction), 5 min post-STEMI and at 2.5 h post-reperfusion (sacrifice). Time Control Spirulina p value Post-STEMI 31.0 ± 6.8 47.4 ± 7.5 0.13 Sacrifice 57.8 ± 8.0 64.5 ± 4.4 0.51 Low-density lipoprotein colesterol (mg/dl) Baseline 85.2 ± 16.2 56.2 ± 16.5 0.25 10 days 96.2 ± 12.1 51.0 ± 14.22 0.06 Prior-STEMI 52.3 ± 12.8 58.3 ± 8.7 0.73 Post-STEMI 78.8 ± 17.0 64.1 ± 10.2 0.47 Sacrifice 96.2 ± 12.1 51.0 ± 14.2 0.06 Data are expressed as mean ± SD. p value denotes differences between control pigs and spirulina-treated animals. All values are within physiological ranges based on our in-house data for young healhy pigs. FIGURE 7 | Study design and spirulina’s cardioprotective effects in a pig model of STEMI. hearts (Jong et al., 2014), and could potentially support the anti-oxidative molecules has shown to partly inhibit lipase detected low IL-1β levels. Nevertheless, the overall antioxidant activity while modulating appetite and food intake (Fujimoto and anti-inflammatory effects may explain the reduction in et al., 2012; Hassan and El-Gharib, 2015). Besides, phenylalanine, caspase-3 activation observed in the infarcted myocardium of one of the essential aminoacids comprised in spirulina structure, spirulina supplemented pigs which, in turn, has likely contributed has also shown to directly inhibit the brain appetite center to promote myocardial salvage and limit cardiac dysfunction in (Mazokopakis et al., 2014a). Regarding the hypolipidemic the setting of STEMI. response to spirulina supplementation, this has been observed It has been claimed that spirulina improves several well- to differ in clinical trials based on the dosage, duration of established cardiovascular risk factors providing benefits treatment, and the presence of comorbidities (Finamore et al., around weight loss and hyperlipidaemia. A recent systematic 2017; Hernández-Lepe et al., 2019). A small trial in 30 healthy review and meta-analysis of five randomized clinical trials volunteers with mild hyperlipidaemia demonstrated a notable assessing spirulina effect on weight loss found that spirulina reduction in total cholesterol after an 8 week supplementation supplementation significantly decreases total weight, body fat with 4.2 g spirulina, although no changes were reported for HDL- percentage, and waist circumference in obese subjects (Moradi cholesterol or triglyceride levels (Nakaya and Goto, 1988). et al., 2019). Of note, there is no clinical evidence to support a Another clinical trial in Korean patients demonstrated that a potential modulation of spirulina in food intake (Moradi et al., 12 week supplementation with spirulina 8 g/day significantly 2019) and, in line with our observations, spirulina has shown to displayed lipid-lowering activity in non-obese subjects, while exert no effects in food intake in mice (Zhao et al., 2019). We in the obese subgroup no substantial change in lipid observe that animals supplemented with spirulina for 10 days metabolism was found (Park and Lee, 2016). Moreover, displayed a significant decrease in the rate of weight gain spirulina supplementation for long periods has recently shown progression in comparison to control pigs. Several mechanistic to alter the gut microbiota that ultimately affect lipid metabolism pathways have been proposed to explain spirulina’s ability to and weight (Li et al., 2021). Conversely, a study in obese improve weight management. As such, spirulina high content of individuals observed no changes in lipid profile after a 12- Frontiers in Pharmacology | www.frontiersin.org 10 May 2022 | Volume 13 | Article 891801 Vilahur et al. Spirulina Limits Infarct Size Post-STEMI week supplementation of spirulina 1 g/day despite a reduction in ETHICS STATEMENT weight gain. We do not observe any changes in lipid profile or glucose levels after this short-term spirulina administration at The animal study was reviewed and approved by European doses of 1 gr/animal/bid. Further research is warranted to Directive 2010/63/EU and the Institutional Animal Care and determine the optimal spirulina dose and timing required to Use Committees of the Hospital de la Santa Creu i Sant Pau. improve glucose metabolism and lipid profile in each specific setting and whether these benefits are associated with changes in the gut microbiome. Importantly, it deserves to be stated that AUTHOR CONTRIBUTIONS spirulina supplementation for 10 days confirmed its adequate safety profile, and no adverse events were registered, with kidney Conceptualization: LB, GV, PS, and GM. Methodology: PS, SB-A, and liver function remaining unmodified throughout the entire MR, MB-P, and LC. Formal analysis: GV, PS, SB-A, LC, and TP. experimental period. Investigation: GV, PS, GM, and LS. Resources: LB and GV. Data This study was carried out in young and healthy pigs without curation: GV, PS, and LB. Writing—original draft preparation: GV, comorbidities and/or associated medication. We intended to PS, MR, and LS. Writing—review and editing: PS, GM, MB-P, LS, address spirulina’s cardioprotective activity based on its LC, and TP. Visualization: PS and SB-A. Supervision: LB. Project nutraceutical profile and its consumption as a dietary administration: GV and TP. Funding acquisition: GV and LB. All supplement in subjects with no overt cardiovascular diseases authors have read and agreed to the published version of the exposed to a sudden ischemic event. In fact, spirulina has manuscript. been proposed as a supplementary nutritional additive in healthy subjects with the aim to improve their overall health status (Karkos et al., 2011). Future studies should investigate the FUNDING effects of spirulina in normal healthy hearts as well as in clinical scenarios combining metabolic comorbidities and ischemic heart This work was supported by Grant PID2019-107160RB-I00 to LB disease. and PGC 2018-094025-B-I00 (to GV) funded by MCIN/AEI/ In conclusion, dietary spirulina supplementation for only 10.13039/501100011033 and Fondo Europeo de Desarrollo 10 days has proven to elicit a cardioprotective effect in case of Regional (FFEDER) A way of making Europe; ISCIII [FIS PI19/ presentation of an ischemic event, through antioxidative, anti- 01687] to TP Instituto de Salud Carlos III (CIBERCV CB16/11/00411 inflammatory, and anti-apoptotic mechanisms that leads to a to LB, TERCEL RD16/0011/018 to LB, FIS2020-01282 to MB-P and reduced size of infarction an improved cardiac function FIS PI19/01687 to TP); the Generalitat of Catalunya-Secretaria (Figure 7). Because of the absence of adverse effects, spirulina d’Universitats i Recerca del Departament d’Economia i supplementation may represent a simple way to counteract the Coneixement de la Generalitat (2017SGR1480 to LB) and deleterious mechanism triggered during STEMI and may be a 2016PROD00043 (Agencia Gestión Ayudas Universitarias promising novel strategy to enhance myocardial salvage in the Investigación: AGAUR ); the Spanish Society of Cardiology (FEC setting of MI. 2019 to MB-P and FED 2020 to GV) and the Fundación Investigación Cardiovascular-Fundación Jesus Serra for their continuous support. DATA AVAILABILITY STATEMENT ACKNOWLEDGMENTS The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be We are very grateful for the highly contribution and support of Maria directed to the corresponding author. Angeles Canovas and Pablo Catalina with animal handling and care. Carrizzo, A., Izzo, C., Forte, M., Sommella, E., Di Pietro, P., Venturini, E., et al. REFERENCES (2020). A Novel Promising Frontier for Human Health: The Beneficial Effects of Nutraceuticals in Cardiovascular Diseases. Int. J. Mol. Sci. 21. doi:10.3390/ Animal Ethics Infolink (2019). NSW Department of Primary Industries and ijms21228706 Animal Research Review Panel. Available at: https://www.animalethics.org. Costa, J. A. V., Freitas, B. C. B., Rosa, G. M., Moraes, L., Morais, M. G., and au/three-rs. Mitchell, B. G. (2019). Operational and Economic Aspects of Spirulina-Based Badimon, L., Bugiardini, R., Cenko, E., Cubedo, J., Dorobantu, M., Duncker, D. J., Biorefinery. Bioresour. Technol. 292, 121946. doi:10.1016/j.biortech.2019. et al. (2017). Position Paper of the European Society of Cardiology-Working 121946 Group of Coronary Pathophysiology and Microcirculation: Obesity and Heart DiNicolantonio, J. J., Bhat, A. G., and OKeefe, J. (2020). Effects of Spirulina on Disease. Eur. Heart J. 38, 1951–1958. doi:10.1093/eurheartj/ehx181 Weight Loss and Blood Lipids: A Review. Open Heart 7, e001003. doi:10.1136/ Badimon, L., Vilahur, G., and Padro, T. (2010). Nutraceuticals and Atherosclerosis: openhrt-2018-001003 Human Trials. Cardiovasc. Ther. 28, 202–215. doi:10.1111/j.1755-5922.2010. Finamore, A., Palmery, M., Bensehaila, S., and Peluso, I. (2017). Antioxidant, 00189.x Immunomodulating, and Microbial-Modulating Activities of the Sustainable Bolli, R. (2001). Cardioprotective Function of Inducible Nitric Oxide Synthase and and Ecofriendly Spirulina. Oxid Med. Cell Longev. 2017, 3247528. doi:10.1155/ Role of Nitric Oxide in Myocardial Ischemia and Preconditioning: an Overview 2017/3247528 of a Decade of Research. J. Mol. Cell Cardiol 33, 1897–1918. doi:10.1006/jmcc. Fujimoto, M., Tsuneyama, K., Fujimoto, T., Selmi, C., Gershwin, M. E., and 2001.1462 Shimada, Y. (2012). Spirulina Improves Non-Alcoholic Steatohepatitis, Frontiers in Pharmacology | www.frontiersin.org 11 May 2022 | Volume 13 | Article 891801 Vilahur et al. Spirulina Limits Infarct Size Post-STEMI Visceral Fat Macrophage Aggregation, and Serum Leptin in a Mouse Model of Disorder in High-Sucrose and High-Fat Diet-Fed Rats in Association with Metabolic Syndrome. Dig. Liver Dis. 44, 767–774. doi:10.1016/j.dld.2012.02.002 Intestinal Microbiota. Food Res. Int. 147, 110530. doi:10.1016/j.foodres.2021. Grosshagauer, S., Kraemer, K., and Somoza, V. (2020). The True Value of 110530 Spirulina. J. Agric. Food Chem. 68, 4109–4115. doi:10.1021/acs.jafc.9b08251 Liang, Y., Huang, X., Zhang, Z., Deng, K., An, S., Gao, X., et al. (2020). Spirulina Hassan, H. A., and El-Gharib, N. E. (2015). Obesity and Clinical Riskiness Supplementation Improves Lipid Metabolism and Autophagic Activities in the Relationship: Therapeutic Management by Dietary Antioxidant Liver and Muscle of Hu Lambs Fed a High-Energy Diet. Arch. Anim. Nutr. 74, Supplementation-Aa Review. Appl. Biochem. Biotechnol. 176, 647–669. 476–495. doi:10.1080/1745039X.2020.1820806 doi:10.1007/s12010-015-1602-6 Madonna, R., Cadeddu, C., Deidda, M., Giricz, Z., Madeddu, C., Mele, D., et al. (2015). Hernández-Lepe, M. A., Wall-Medrano, A., López-Díaz, J. A., Juárez-Oropeza, M. Cardioprotection by Gene Therapy: A Review Paper on Behalf of the Working A., Hernández-Torres, R. P., and Ramos-Jiménez, A. (2019). Hypolipidemic Group on Drug Cardiotoxicity and Cardioprotection of the Italian Society of Effect of Arthrospira (Spirulina) Maxima Supplementation and a Systematic Cardiology. Int. J. Cardiol. 191, 203–210. doi:10.1016/j.ijcard.2015.04.232 Physical Exercise Program in Overweight and Obese Men: A Double-Blind, Mazokopakis, E. E., Papadomanolaki, M. G., Fousteris, A. A., Kotsiris, D. A., Randomized, and Crossover Controlled Trial. Mar. Drugs 17. doi:10.3390/ Lampadakis, I. M., and Ganotakis, E. S. (2014). The Hepatoprotective and md17050270 Hypolipidemic Effects of Spirulina (Arthrospira Platensis) Supplementation in Heusch, G., and Gersh, B. J. (2020). Is Cardioprotection Salvageable? Circulation a Cretan Population with Non-alcoholic Fatty Liver Disease: a Prospective Pilot 141, 415–417. doi:10.1161/CIRCULATIONAHA.119.044176 Study. Ann. Gastroenterol. 27 (4), 387–394. Heusch, G., and Gersh, B. J. (2017). The Pathophysiology of Acute Myocardial Mazokopakis, E. E., Starakis, I. K., Papadomanolaki, M. G., Mavroeidi, N. G., and Infarction and Strategies of protection beyond Reperfusion: a Continual Ganotakis, E. S. (2014). The Hypolipidaemic Effects of Spirulina (Arthrospira challenge. Eur. Heart J. 38, 774–784. doi:10.1093/eurheartj/ehw224 Platensis) Supplementation in a Cretan Population: A Prospective Study. J. Sci. Heusch, G., Libby, P., Gersh, B., Yellon, D., Böhm, M., Lopaschuk, G., et al. (2014). Food Agric. 94, 432–437. doi:10.1002/jsfa.6261 Cardiovascular Remodelling in Coronary Artery Disease and Heart Failure. Mendieta, G., Ben-Aicha, S., Casani, L., Badimon, L., Sabaté, M., and Vilahur, G. Lancet 383, 1933–1943. doi:10.1016/S0140-6736(14)60107-0 (2019). Intravenous Statin Administration during Ischemia Exerts Huang, H., Liao, D., Pu, R., and Cui, Y. (2018). Quantifying the Effects of Spirulina Cardioprotective Effects. J. Am. Coll. Cardiol. 74, 475–477. doi:10.1016/j. Supplementation on Plasma Lipid and Glucose Concentrations, Body Weight, jacc.2019.05.020 and Blood Pressure. Diabetes Metab. Syndr. Obes. 11, 729–742. doi:10.2147/ Mendieta, G., Ben-Aicha, S., Casani, L., Badimon, L., Sabate, M., and Vilahur, G. DMSO.S185672 (2019). Molecular Pathways Involved in the Cardioprotective Effects of Jong, W. M., Leemans, J. C., Weber, N. C., Juffermans, N. P., Schultz, M. J., Intravenous Statin Administration During Ischemia. Basic Res. Cardiol. 115, Hollmann, M. W., et al. (2014). Nlrp3 Plays No Role in Acute Cardiac 2. doi:10.1007/s00395-019-0760-z Infarction Due to Low Cardiac Expression. Int. J. Cardiol. 177, 41–43. Mendieta, G., Ben-Aicha, S., Gutiérrez, M., Casani, L., Aržanauskaitė, M., Carreras, doi:10.1016/j.ijcard.2014.09.148 F., et al. (2020). Intravenous Statin Administration during Myocardial Kanamori, H., Takemura, G., Goto, K., Maruyama, R., Tsujimoto, A., Ogino, A., Infarction Compared with Oral Post-Infarct Administration. J. Am. Coll. et al. (2011). The Role of Autophagy Emerging in Postinfarction Cardiac Cardiol. 75, 1386–1402. doi:10.1016/j.jacc.2020.01.042 Remodelling. Cardiovasc. Res. 91, 330–339. doi:10.1093/cvr/cvr073 Moradi, S., Ziaei, R., Foshati, S., Mohammadi, H., Nachvak, S. M., and Rouhani, M. Karkos, P. D., Leong, S. C., Karkos, C. D., Sivaji, N., and Assimakopoulos, D. A. H. (2019). Effects of Spirulina Supplementation on Obesity: A Systematic (2011). Spirulina in Clinical Practice: Evidence-Based Human Applications. Review and Meta-Analysis of Randomized Clinical Trials. Complement. Evid. Based Compl. Alternat Med. 2011, 531053. doi:10.1093/ecam/nen058 Ther. Med. 47, 102211. doi:10.1016/j.ctim.2019.102211 Khan, M., Shobha, J. C., Mohan, I. K., Naidu, M. U., Sundaram, C., Singh, S., et al. Moran, A. E., Forouzanfar, M. H., Roth, G. A., Mensah, G. A., Ezzati, M., Murray, (2005). Protective Effect of Spirulina against Doxorubicin-Induced C. J., et al. (2014). Temporal Trends in Ischemic Heart Disease Mortality in 21 Cardiotoxicity. Phytother Res. 19, 1030–1037. doi:10.1002/ptr.1783 World Regions, 1980 to 2010: the Global Burden of Disease 2010 Study. Khan, M., Varadharaj, S., Ganesan, L. P., Shobha, J. C., Naidu, M. U., Parinandi, N. Circulation 129, 1483–1492. doi:10.1161/CIRCULATIONAHA.113.004042 L., et al. (2006). C-phycocyanin Protects Against Ischemia-Reperfusion Injury Nakaya, Y. H. N., and Goto, Y. (1988). Cholesterol Lowering Effect of Spirulina. of Heart through Involvement of P38 MAPK and ERK Signaling. Am. J. Physiol. Nutr. Rep. Int. Heart Circ. Physiol. 290, H2136–H2145. doi:10.1152/ajpheart.01072.2005 Park, H. J., and Lee, H. S. (2016). The Influence of Obesity on the Effects of Khan, M., Varadharaj, S., Shobha, J. C., Naidu, M. U., Parinandi, N. L., Kutala, V. Spirulina Supplementation in the Human Metabolic Response of Korean K., et al. (2006). C-phycocyanin Ameliorates Doxorubicin-Induced Oxidative Elderly. Nutr. Res. Pract. 10, 418–423. doi:10.4162/nrp.2016.10.4.418 Stress and Apoptosis in Adult Rat Cardiomyocytes. J. Cardiovasc. Pharmacol. Park, H. J., Lee, Y. J., Ryu, H. K., Kim, M. H., Chung, H. W., and Kim, W. Y. (2008). 47, 9–20. doi:10.1097/01.fjc.0000191520.48404.27 A Randomized Double-Blind, Placebo-Controlled Study to Establish the Effects Lecour, S., Smith, R. M., Woodward, B., Opie, L. H., Rochette, L., and Sack, M. N. of Spirulina in Elderly Koreans. Ann. Nutr. Metab. 52, 322–328. doi:10.1159/ (2002). Identification of a Novel Role for Sphingolipid Signaling in TNF Alpha 000151486 and Ischemic Preconditioning Mediated Cardioprotection. J. Mol. Cell Cardiol Percie du Sert, N., Ahluwalia, A., Alam, S., Avey, M. T., Baker, M., Browne, W. J., 34, 509–518. doi:10.1006/jmcc.2002.1533 et al. (2020). Reporting Animal Research: Explanation and Elaboration for the Lecour, S., Suleman, N., Deuchar, G. A., Somers, S., Lacerda, L., Huisamen, B., et al. ARRIVE Guidelines 2.0. PLoS Biol. 18, e3000411. doi:10.1371/journal.pbio. (2005). Pharmacological Preconditioning with Tumor Necrosis Factor-Alpha 3000411 Activates Signal Transducer and Activator of Transcription-3 at Reperfusion Ren, Z., Xie, Z., Cao, D., Gong, M., Yang, L., Zhou, Z., et al. (2018). C-phycocyanin without Involving Classic Prosurvival Kinases (Akt and Extracellular Signal- Inhibits Hepatic Gluconeogenesis and Increases Glycogen Synthesis via Regulated Kinase). Circulation 112, 3911–3918. doi:10.1161/ Activating Akt and AMPK in Insulin Resistance Hepatocytes. Food Funct. 9, CIRCULATIONAHA.105.581058 2829–2839. doi:10.1039/c8fo00257f Li, Q., Guo, Y., Ou, Q., Wu, W. J., Chen, N., Zhu, X., et al. (2011). Gene Transfer as Schulz, R., and Heusch, G. (2009). Tumor Necrosis Factor-Alpha and its Receptors a Strategy to Achieve Permanent Cardioprotection II: rAAV-Mediated Gene 1 and 2: Yin and Yang in Myocardial Infarction? Circulation 119, 1355–1357. Therapy with Heme Oxygenase-1 Limits Infarct Size 1 Year Later without doi:10.1161/CIRCULATIONAHA.108.846105 Adverse Functional Consequences. Basic Res. Cardiol. 106, 1367–1377. doi:10. Serban, M. C., Sahebkar, A., Dragan, S., Stoichescu-Hogea, G., Ursoniu, S., Andrica, 1007/s00395-011-0208-6 F., et al. (2016). A Systematic Review and Meta-Analysis of the Impact of Li, Q., Guo, Y., Tan, W., Stein, A. B., Dawn, B., Wu, W. J., et al. (2006). Gene Spirulina Supplementation on Plasma Lipid Concentrations. Clin. Nutr. 35, Therapy with iNOS Provides Long-Term protection against Myocardial 842–851. doi:10.1016/j.clnu.2015.09.007 Infarction without Adverse Functional Consequences. Am. J. Physiol. Heart Stone, G. W., Selker, H. P., Thiele, H., Patel, M. R., Udelson, J. E., Ohman, E. M., Circ. Physiol. 290, H584–H589. doi:10.1152/ajpheart.00855.2005 et al. (2016). Relationship between Infarct Size and Outcomes Following Li, T. T., Huang, Z. R., Jia, R. B., Lv, X. C., Zhao, C., and Liu, B. (2021). Spirulina Primary PCI: Patient-Level Analysis from 10 Randomized Trials. J. Am. Platensis Polysaccharides Attenuate Lipid and Carbohydrate Metabolism Coll. Cardiol. 67, 1674–1683. doi:10.1016/j.jacc.2016.01.069 Frontiers in Pharmacology | www.frontiersin.org 12 May 2022 | Volume 13 | Article 891801 Vilahur et al. Spirulina Limits Infarct Size Post-STEMI Vilahur, G., Casaní, L., Peña, E., Crespo, J., Juan-Babot, O., Ben-Aicha, S., et al. Spirulina Platensis Supplementation in Pigs. Bulgarian J. Agric. Sci. 21, (2018). Silybum marianum Provides Cardioprotection and Limits Adverse 1054–1059. Remodeling post-myocardial Infarction by Mitigating Oxidative Stress and Yousefi, R., Saidpour, A., and Mottaghi, A. (2019). The Effects of Spirulina Reactive Fibrosis. Int. J. Cardiol. 270, 28–35. doi:10.1016/j.ijcard.2018. Supplementation on Metabolic Syndrome Components, its Liver 06.030 Manifestation and Related Inflammatory Markers: A Systematic Review. Vilahur, G., Casaní, L., Peña, E., Duran, X., Juan-Babot, O., and Badimon, L. Complement. Ther. Med. 42, 137–144. doi:10.1016/j.ctim.2018.11.013 (2009). Induction of RISK by HMG-CoA Reductase Inhibition Affords Yu, X., Ge, L., Niu, L., Lian, X., Ma, H., and Pang, L. (2018). The Dual Role of Inducible Cardioprotection after Myocardial Infarction. Atherosclerosis 206, 95–101. Nitric Oxide Synthase in Myocardial Ischemia/Reperfusion Injury: Friend or Foe? doi:10.1016/j.atherosclerosis.2009.02.009 Oxid Med. Cell Longev. 2018, 8364848. doi:10.1155/2018/8364848 Vilahur, G., Casani, L., Peña, E., Juan-Babot, O., Mendieta, G., Crespo, J., et al. Zhao, B., Cui, Y., Fan, X., Qi, P., Liu, C., Zhou, X., et al. (2019). Anti-Obesity Effects (2014). HMG-CoA Reductase Inhibition Prior Reperfusion Improves of Spirulina Platensis Protein Hydrolysate by Modulating Brain-Liver axis in Reparative Fibrosis post-myocardial Infarction in a Preclinical Experimental High-Fat Diet Fed Mice. PLoS One 14, e0218543. doi:10.1371/journal.pone. Model. Int. J. Cardiol. 175, 528–538. doi:10.1016/j.ijcard.2014.06.040 0218543 Vilahur, G., Gutiérrez, M., Casani, L., Varela, L., Capdevila, A., Pons-Lladó, G., et al. (2016). Protective Effects of Ticagrelor on Myocardial Injury after Conflict of Interest: The authors declare that the research was conducted in the Infarction. Circulation 134, 1708–1719. doi:10.1161/CIRCULATIONAHA. absence of any commercial or financial relationships that could be construed as a 116.024014 potential conflict of interest. Vilahur, G., Badimon, J. J., Bugiardini, R., and Badimon, L. (2014). Perspectives: The burden of Cardiovascular Risk Factors and Coronary Heart Disease in Publisher’s Note: All claims expressed in this article are solely those of the authors Europe and Worldwide. Eur. Heart J. Suppl. 16, A7–A11. doi:10.1093/eurheartj/ and do not necessarily represent those of their affiliated organizations, or those of sut003 the publisher, the editors and the reviewers. Any product that may be evaluated in Vo, T. S., and Kim, S. K. (2013). Down-Regulation of Histamine-Induced this article, or claim that may be made by its manufacturer, is not guaranteed or Endothelial Cell Activation as Potential Anti-atherosclerotic Activity of endorsed by the publisher. Peptides from Spirulina Maxima. Eur. J. Pharm. Sci. 50, 198–207. doi:10. 1016/j.ejps.2013.07.001 Copyright © 2022 Vilahur, Sutelman, Ben-Aicha, Mendieta, Radiké, Schoch, Casaní, Xi, L., Jarrett, N. C., Hess, M. L., and Kukreja, R. C. (1999). Essential Role of Borrell-Pagés, Padro and Badimon. This is an open-access article distributed under Inducible Nitric Oxide Synthase in Monophosphoryl Lipid A-Induced Late the terms of the Creative Commons Attribution License (CC BY). The use, Cardioprotection: Evidence from Pharmacological Inhibition and Gene distribution or reproduction in other forums is permitted, provided the original Knockout Mice. Circulation 99, 2157–2163. doi:10.1161/01.cir.99.16.2157 author(s) and the copyright owner(s) are credited and that the original publication Yordanova, G., Popova, T., Givko, N., Nedeva, R., Marchev, Y., Kistanova, E., in this journal is cited, in accordance with accepted academic practice. No use, et al. (2015). Lipid Profile of M. Longissimus Dorsi in Response to Dietary distribution or reproduction is permitted which does not comply with these terms. Frontiers in Pharmacology | www.frontiersin.org 13 May 2022 | Volume 13 | Article 891801