Validation of the slaughterhouse porcine heart model for ex-situ heart perfusion studies.

INTRODUCTION: To validate slaughterhouse hearts for ex-situ heart perfusion studies, we compared cold oxygenated machine perfusion in less expensive porcine slaughterhouse hearts (N = 7) to porcine hearts that are harvested following the golden standard in laboratory animals (N = 6). METHODS: All hearts received modified St Thomas 2 crystalloid cardioplegia prior to 4 hours of cold oxygenated machine perfusion. Hearts were perfused with homemade modified Steen heart solution with a perfusion pressure of 20-25 mmHg to achieve a coronary flow between 100-200 mL/min. Reperfusion and testing was performed for 4 hours on a normothermic, oxygenated diluted whole blood loaded heart model. Survival was defined by a cardiac output above 3 L with a mean aortic pressure above 60 mmHg. RESULTS: Both groups showed 100% functional survival, with laboratory hearts displaying superior cardiac function. Both groups showed similar decline in function over time. CONCLUSION: We conclude that the slaughterhouse heart can be used as an alternative to laboratory hearts and provides a cost-effective method for future ex-situ heart perfusion studies.

HIPK1 Inhibition Protects against Pathological Cardiac Hypertrophy by Inhibiting the CREB-C/EBPß Axis.

Inhibition of pathological cardiac hypertrophy is recognized as an important therapeutic strategy for heart failure, although effective targets are still lacking in clinical practice. Homeodomain interacting protein kinase 1 (HIPK1) is a conserved serine/threonine kinase that can respond to different stress signals, however, whether and how HIPK1 regulates myocardial function is not reported. Here, it is observed that HIPK1 is increased during pathological cardiac hypertrophy. Both genetic ablation and gene therapy targeting HIPK1 are protective against pathological hypertrophy and heart failure in vivo. Hypertrophic stress-induced HIPK1 is present in the nucleus of cardiomyocytes, while HIPK1 inhibition prevents phenylephrine-induced cardiomyocyte hypertrophy through inhibiting cAMP-response element binding protein (CREB) phosphorylation at Ser271 and inactivating CCAAT/enhancer-binding protein ß (C/EBPß)-mediated transcription of pathological response genes. Inhibition of HIPK1 and CREB forms a synergistic pathway in preventing pathological cardiac hypertrophy. In conclusion, HIPK1 inhibition may serve as a promising novel therapeutic strategy to attenuate pathological cardiac hypertrophy and heart failure.

Gut microbiome mediates the protective effects of exercise after myocardial infarction.

BACKGROUND: Gut microbiota plays important roles in health maintenance and diseases. Physical exercise has been demonstrated to be able to modulate gut microbiota. However, the potential role of gut microbiome in exercise protection to myocardial infarction (MI) remains unclear. RESULTS: Here, we discovered exercise training ameliorated cardiac dysfunction and changed gut microbial richness and community structure post-MI. Moreover, gut microbiota pre-depletion abolished the protective effects of exercise training in MI mice. Furthermore, mice receiving microbiota transplants from exercised MI mice had better cardiac function compared to mice receiving microbiota transplants from non-exercised MI mice. Mechanistically, we analyzed metabolomics in fecal samples from exercised mice post-MI and identified 3-Hydroxyphenylacetic acid (3-HPA) and 4-Hydroxybenzoic acid (4-HBA), which could be applied individually to protect cardiac dysfunction post-MI and apoptosis through NRF2. CONCLUSIONS: Together, our study provides new insights into the role of gut microbiome in exercise protection to MI, offers opportunities to modulate cardiovascular diseases by exercise, microbiome and gut microbiota-derived 3-HPA and 4-HBA. Video Abstract.

Exercise downregulates HIPK2 and HIPK2 inhibition protects against myocardial infarction.

BACKGROUND: Exercise can protect myocardial infarction (MI) and downregulate cardiac Homeodomain-Interacting Protein Kinase 2 (HIPK2). However, the role of HIPK2 in MI is unclear. METHODS: HIPK2-/- mice and miR-222-/- rats, HIPK2 inhibitor (PKI1H) and adeno-associated virus serotype 9 (AAV9) carrying miR-222 were applied in the study. Animals were subjected to running, swimming, acute MI or post-MI remodeling. HIPK2 inhibition and P53 activator were used in neonatal rat cardiomyocytes (NRCMs) and human embryonic stem cell-derived cardiomyocytes (hESC-CMs) subjected to oxygen glucose deprivation/reperfusion (OGD/R). Serum miR-222 levels were analyzed in healthy people and MI patients that were survival or readmitted to the hospital and/or died. FINDINGS: Cardiac HIPK2 protein levels were reduced by exercise while increased in MI. In vitro, HIPK2 suppression by lentiviral vectors or inhibitor prevented apoptosis induced by OGD/R in NRCMs and hESC-CMs. HIPK2 inhibitor-treated mice and HIPK2-/- mice reduced infarct size after acute MI, and preserved cardiac function in MI remodeling. Mechanistically, protective effect against apoptosis by HIPK2 suppression was reversed by P53 activators. Furthermore, increasing levels of miR-222, targeting HIPK2, protected post-MI cardiac dysfunction, whereas cardiac dysfunction post-MI was aggravated in miR-222-/- rats. Moreover, serum miR-222 levels were significantly reduced in MI patients, as well as in MI patients that were readmitted to the hospital and/or died compared to those not. INTERPRETATION: Exercise-induced HIPK2 suppression attenuates cardiomyocytes apoptosis and protects MI by decreasing P-P53. Inhibition of HIPK2 represents a potential novel therapeutic intervention for MI. FUNDING: This work was supported by the grants from National Key Research and Development Project (2018YFE0113500 to JJ Xiao), National Natural Science Foundation of China (82020108002, 81722008, and 81911540486 to JJ Xiao, 81400647 to MJ Xu, 81800265 to YJ Liang), Innovation Program of Shanghai Municipal Education Commission (2017-01-07-00-09-E00042 to JJ Xiao), the grant from Science and Technology Commission of Shanghai Municipality (18410722200 and 17010500100 to JJ Xiao), the "Dawn" Program of Shanghai Education Commission (19SG34 to JJ Xiao), Shanghai Sailing Program (21YF1413200 to QL Zhou). JS is supported by Horizon2020 ERC-2016-COG EVICARE (725229).

COVID-19-related cardiac complications from clinical evidences to basic mechanisms: opinion paper of the ESC Working Group on Cellular Biology of the Heart.

The pandemic of coronavirus disease (COVID)-19 is a global threat, causing high mortality, especially in the elderly. The main symptoms and the primary cause of death are related to interstitial pneumonia. Viral entry also into myocardial cells mainly via the angiotensin converting enzyme type 2 (ACE2) receptor and excessive production of pro-inflammatory cytokines, however, also make the heart susceptible to injury. In addition to the immediate damage caused by the acute inflammatory response, the heart may also suffer from long-term consequences of COVID-19, potentially causing a post-pandemic increase in cardiac complications. Although the main cause of cardiac damage in COVID-19 remains coagulopathy with micro- (and to a lesser extent macro-) vascular occlusion, open questions remain about other possible modalities of cardiac dysfunction, such as direct infection of myocardial cells, effects of cytokines storm, and mechanisms related to enhanced coagulopathy. In this opinion paper, we focus on these lesser appreciated possibilities and propose experimental approaches that could provide a more comprehensive understanding of the cellular and molecular bases of cardiac injury in COVID-19 patients. We first discuss approaches to characterize cardiac damage caused by possible direct viral infection of cardiac cells, followed by formulating hypotheses on how to reproduce and investigate the hyperinflammatory and pro-thrombotic conditions observed in the heart of COVID-19 patients using experimental in vitro systems. Finally, we elaborate on strategies to discover novel pathology biomarkers using omics platforms.

Erratum: Crucial Role of miR-433 in Regulating Cardiac Fibrosis: Erratum.

[This corrects the article DOI: 10.7150/thno.15007.].

Myocardial infarction affects Cx43 content of extracellular vesicles secreted by cardiomyocytes.

Ischemic heart disease has been associated with an impairment on intercellular communication mediated by both gap junctions and extracellular vesicles. We have previously shown that connexin 43 (Cx43), the main ventricular gap junction protein, assembles into channels at the extracellular vesicle surface, mediating the release of vesicle content into target cells. Here, using a comprehensive strategy that included cell-based approaches, animal models and human patients, we demonstrate that myocardial ischemia impairs the secretion of Cx43 into circulating, intracardiac and cardiomyocyte-derived vesicles. In addition, we show that ubiquitin signals Cx43 release in basal conditions but appears to be dispensable during ischemia, suggesting an interplay between ischemia-induced Cx43 degradation and secretion. Overall, this study constitutes a step forward for the characterization of the signals and molecular players underlying vesicle protein sorting, with strong implications on long-range intercellular communication, paving the way towards the development of innovative diagnostic and therapeutic strategies for cardiovascular disorders.

The anti-microbial peptide LL-37/CRAMP levels are associated with acute heart failure and can attenuate cardiac dysfunction in multiple preclinical models of heart failure.

Rationale: Biomarkers for the diagnosis of heart failure (HF) are clinically essential. Circulating antimicrobial peptides LL-37 has emerged as a novel biomarker in cardiovascular disease, however, its relevance as a biomarker for acute HF are undetermined. Methods: Acute HF patients were enrolled in this study and the serum levels of LL-37/CRAMP (cathelicidin-related antimicrobial peptide) were measured by ELISA. The receiver-operator characteristic (ROC) curve was used to determine if serum LL-37 could be a biomarker for acute HF. Mouse CRAMP (mCRAMP, mouse homolog for human LL-37) was also determined in both heart and serum samples of, transverse aortic constriction (TAC)- and isoproterenol (ISO)-induced HF mice models, and phenylephrine (PE) and angiotensin II (AngII)-induced neonatal mouse cardiomyocytes (NMCMs) hypertrophic models, both intracellular and secreted, by ELISA. The protective effects of mCRAMP were determined in TAC, ISO, and AngII-induced HF in mice while whether HF was exacerbated in AngII-infused animals were checked in mCRAMP knockout mice. The underlying mechanism for protective effects of CARMP in pathological hypertrophy was determined by using a NF-κB agonist together with rCRAMP (rat homolog for human LL-37) in AngII or PE treated neonatal rat cardiomyocytes (NRCMs). Results: Serum levels of LL-37 were significantly decreased in acute HF patients (area under the curve (AUC) of 0.616), and negatively correlated with NT-proBNP. We further confirmed that mCRAMP was decreased in both heart and serum samples of TAC- and ISO-induced HF mice models. Moreover, in PE and AngII-induced NMCMs hypertrophic models, both intracellular and secreted mCRAMP levels were reduced. Functionally, mCRAMP could attenuate TAC, ISO, and AngII-induced HF in mice while CRAMP deficiency exacerbated HF. Mechanistically, the anti-hypertrophy effects of CRAMP were mediated by NF-κB signaling. Conclusions: Collectively, serum LL-37 is associated with acute HF and increasing CRAMP is protective against deleterious NF-κB signaling in the rodent.

Lymphocytic subsets play distinct roles in heart diseases.

Heart diseases are one of the leading causes of death for humans in the world. Increasing evidence has shown that myocardial injury induced innate and adaptive immune responses upon early cellular damage but also during chronic phases post-injury. The immune cells can not only aggravate the injury but also play an essential role in the induction of wound healing responses, which means they play a complex role throughout the acute inflammatory response and reparative response after cardiac injury. This review will summarize the current experimental and clinical evidence of lymphocytes, one of the major types of immune cells, participate in heart diseases and try to explain the possible role of these immune cells following cardiac injury.

Lower retention after retrograde coronary venous infusion compared with intracoronary infusion of mesenchymal stromal cells in the infarcted porcine myocardium.

BACKGROUND: Commonly used strategies for cell delivery to the heart are intramyocardial injection and intracoronary (IC) infusion, both having their advantages and disadvantages. Therefore, alternative strategies, such as retrograde coronary venous infusion (RCVI), are explored. The aim of this confirmatory study was to compare cardiac cell retention between RCVI and IC infusion. As a secondary end point, the procedural safety of RCVI is assessed. METHODS: Four weeks after myocardial infarction, 12 pigs were randomised to receive mesenchymal stromal cells, labelled with Indium-111, via RCVI (n=6) or IC infusion (n=6). Four hours after cell administration, nuclear imaging was performed to determine the number of cells retained in the heart both in vivo and ex vivo. Procedure-related safety measures were reported. RESULTS: Cardiac cell retention is significantly lower after RCVI compared with IC infusion (in vivo: RCVI: median 2.89% vs IC: median 13.74%, p=0.002, ex vivo: RCVI: median 2.55% vs IC: median 39.40%, p=0.002). RCVI led to development of pericardial fluid and haematomas on the frontal wall of the heart in three cases. Coronary venous dissection after RCVI was seen in three pigs, of which one also developed pericardial fluid and a haematoma. IC infusion led to no flow in one pig. CONCLUSION: RCVI is significantly less efficient in delivering cells to the heart compared with IC infusion. RCVI led to more procedure-related safety issues than IC infusion, with multiple cases of venous dissection and development of haematomas and pericardial fluid collections.

miR-31a-5p promotes postnatal cardiomyocyte proliferation by targeting RhoBTB1.

A limited number of microRNAs (miRNAs, miRs) have been reported to control postnatal cardiomyocyte proliferation, but their strong regulatory effects suggest a possible therapeutic approach to stimulate regenerative capacity in the diseased myocardium. This study aimed to investigate the miRNAs responsible for postnatal cardiomyocyte proliferation and their downstream targets. Here, we compared miRNA profiles in cardiomyocytes between postnatal day 0 (P0) and day 10 (P10) using miRNA arrays, and found that 21 miRNAs were upregulated at P10, whereas 11 were downregulated. Among them, miR-31a-5p was identified as being able to promote cardiomyocyte proliferation as determined by proliferating cell nuclear antigen (PCNA) expression, double immunofluorescent labeling for α-actinin and 5-ethynyl-2-deoxyuridine (EdU) or Ki-67, and cell number counting, whereas miR-31a-5p inhibition could reduce their levels. RhoBTB1 was identified as a target gene of miR-31a-5p, mediating the regulatory effect of miR-31a-5p in cardiomyocyte proliferation. Importantly, neonatal rats injected with a miR-31a-5p antagomir at day 0 for three consecutive days exhibited reduced expression of markers of cardiomyocyte proliferation including PCNA expression and double immunofluorescent labeling for α-actinin and EdU, Ki-67 or phospho-histone-H3. In conclusion, miR-31a-5p controls postnatal cardiomyocyte proliferation by targeting RhoBTB1, and increasing miR-31a-5p level might be a novel therapeutic strategy for enhancing cardiac reparative processes.

miR-17-3p Contributes to Exercise-Induced Cardiac Growth and Protects against Myocardial Ischemia-Reperfusion Injury.

Limited microRNAs (miRNAs, miRs) have been reported to be necessary for exercise-induced cardiac growth and essential for protection against pathological cardiac remodeling. Here we determined members of the miR-17-92 cluster and their passenger miRNAs expressions in two distinct murine exercise models and found that miR-17-3p was increased in both. miR-17-3p promoted cardiomyocyte hypertrophy, proliferation, and survival. TIMP-3 was identified as a direct target gene of miR-17-3p whereas PTEN was indirectly inhibited by miR-17-3p. Inhibition of miR-17-3p in vivo attenuated exercise-induced cardiac growth including cardiomyocyte hypertrophy and expression of markers of myocyte proliferation. Importantly, mice injected with miR-17-3p agomir were protected from adverse remodeling after cardiac ischemia/reperfusion injury. Collectively, these data suggest that miR-17-3p contributes to exercise-induced cardiac growth and protects against adverse ventricular remodeling. miR-17-3p may represent a novel therapeutic target to promote functional recovery after cardiac ischemia/reperfusion.

Crucial Role of miR-433 in Regulating Cardiac Fibrosis.

Dysregulation of microRNAs has been implicated in many cardiovascular diseases including fibrosis. Here we report that miR-433 was consistently elevated in three models of heart disease with prominent cardiac fibrosis, and was enriched in fibroblasts compared to cardiomyocytes. Forced expression of miR-433 in neonatal rat cardiac fibroblasts increased proliferation and their differentiation into myofibroblasts as determined by EdU incorporation, α-SMA staining, and expression levels of fibrosis-associated genes. Conversely, inhibition of miR-433 exhibited opposite results. AZIN1 and JNK1 were identified as two target genes of miR-433. Decreased level of AZIN1 activated TGF-ß1 while down-regulation of JNK1 resulted in activation of ERK and p38 kinase leading to Smad3 activation and ultimately cardiac fibrosis. Importantly, systemic neutralization of miR-433 or adeno-associated virus 9 (AAV9)-mediated cardiac transfer of a miR-433 sponge attenuated cardiac fibrosis and ventricular dysfunction following myocardial infarction. Thus, our work suggests that miR-433 is a potential target for amelioration of cardiac fibrosis.

Human cardiomyocyte progenitor cells differentiate into functional mature cardiomyocytes: an in vitro model for studying human cardiac physiology and pathophysiology.

To date, there is no suitable in vitro model to study human adult cardiac cell biology. Although embryonic stem cells are able to differentiate into cardiomyocytes in vitro, the efficiency of this process is very low. Other methods to differentiate progenitor cells into beating cardiomyocytes rely on coculturing with rat neonatal cardiomyocytes, making it difficult to study human cardiomyocyte differentiation and (patho)physiology. Here we have developed a method for efficient isolation and expansion of human cardiomyocyte progenitor cells (CMPCs) from cardiac surgical waste or alternatively from fetal heart tissue. Furthermore, we provide a detailed in vitro protocol for efficient differentiation of CMPCs into cardiomyocytes with great efficiency (80-90% of differentiation). Once CMPCs are rapidly dividing ( approximately 1 month after isolation), differentiation can be achieved in 3-4 weeks.