Cardiac Pathophysiology Research Articles

Modeling cardiac fibroblast heterogeneity in fibrotic heart using human pluripotent stem cell derived epicardial cells

Abstract Introduction Our understanding of human cardiac pathophysiology is limited due to the scarcity of human heart samples. Model organisms do not faithfully recapitulate human physiology and pathophysiology to the extent that is needed. Therefore, human pluripotent stem cell (hPSC)-derived cardiac cells represent promising sources to overcome these limitations to studying heart diseases. Purpose To generate cardiac organoids mimicking the interaction between cardiomyocytes and non-cardiomyocytes in the developing heart and the postnatal pathological conditions. Method It has been well known that epicardium is a main source of non-cardiac cells in the developing heart. We therefore generated cardiac organoids by mixing hPSC-derived cardiomyocytes and hPSC-derived epicardium and test if they could mimic the developing heart and be applicable for modeling heart failure. Results We established a cardiac organoid system generated from human pluripotent stem cells that models the developing heart's early interactions, specifically the epicardial and non-myocyte development, as well as ventricular cardiomyocyte proliferation. Inside the cardiac organoids, epicardium spontaneously differentiated into CD90-positive cardiac fibroblasts expressing fibroblast markers, FN, COL1 and COL3, and CD90-negative smooth muscle cells (SMC) expressing SMC markers, ACTA2 and MYH11. Additionally, we observed the significant increase in cardiomyocyte proliferation in the cardiac organoids compared to in cardiomyocyte only aggregation (p<0.001). We then mature our cardiac organoids using our unique maturation method, the combination of PPARα agonist, dexamethasone, T3 hormone, and palmitate, generating metabolically mature cardiac organoids. These mature organoids recapitulated the phenotypic changes observed in the failing human heart when exposed to pathological stimuli, including fibrosis, metabolic changes, and increase in heart failure markers. We used single-cell transcriptomics to dissect the cellular heterogeneity of our organoids in a model of heart failure with comparisons to primary human data and revealed our organoids recapitulated in vivo human failing heart. This analysis allowed to confirm that our organoids contain a unique subpopulation of cardiac fibroblasts possessing reparative features, suggesting the recapitulation of in vivo heterogeneity in our model. Conclusions We successfully generated cardiac organoids mimicking the interaction between cardiomyocytes and non-cardiac cells. Our system enables the recapitulation of in vivo heterogeneity, providing a platform for the accurate understanding of heart development and disease in a dish. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Canadian Institutes of Health Research (CIHR)

Defective efferocytosis of vascular cells in heart disease.

The efficient phagocytic clearance of dying cells and apoptotic cells is one of the processes that is essential for the maintenance of physiologic tissue function and homeostasis, which is termed “efferocytosis.” Under normal conditions, “find me” and “eat me” signals are released by apoptotic cells to stimulate the engulfment and efferocytosis of apoptotic cells. In contrast, abnormal efferocytosis is related to chronic and non-resolving inflammatory diseases such as atherosclerosis. In the initial steps of atherosclerotic lesion development, monocyte-derived macrophages display efficient efferocytosis that restricts plaque progression; however, this capacity is reduced in more advanced lesions. Macrophage reprogramming as a result of the accumulation of apoptotic cells and augmented inflammation accounts for this diminishment of efferocytosis. Furthermore, defective efferocytosis plays an important role in necrotic core formation, which triggers plaque rupture and acute thrombotic cardiovascular events. Recent publications have focused on the essential role of macrophage efferocytosis in cardiac pathophysiology and have pointed toward new therapeutic strategies to modulate macrophage efferocytosis for cardiac tissue repair. In this review, we discuss the molecular and cellular mechanisms that regulate efferocytosis in vascular cells, including macrophages and other phagocytic cells and detail how efferocytosis-related molecules contribute to the maintenance of vascular hemostasis and how defective efferocytosis leads to the formation and progression of atherosclerotic plaques.

Deciphering the Basis of Molecular Biology of Selected Cardiovascular Diseases: A View on Network Medicine.

Cardiovascular diseases are the leading cause of death across the world. For decades, researchers have been studying the causes of cardiovascular disease, yet many of them remain undiscovered or poorly understood. Network medicine is a recently expanding, integrative field that attempts to elucidate this issue by conceiving of disease as the result of disruptive links between multiple interconnected biological components. Still in its nascent stages, this revolutionary application of network science facilitated a number of important discoveries in complex disease mechanisms. As methodologies become more advanced, network medicine harbors the potential to expound on the molecular and genetic complexities of disease to differentiate how these intricacies govern disease manifestations, prognosis, and therapy. This is of paramount importance for confronting the incredible challenges of current and future cardiovascular disease research. In this review, we summarize the principal molecular and genetic mechanisms of common cardiac pathophysiologies as well as discuss the existing knowledge on therapeutic strategies to prevent, halt, or reverse these pathologies.

Biomimetic cardiac tissue culture model (CTCM) to emulate cardiac physiology and pathophysiology ex vivo

There is need for a reliable in vitro system that can accurately replicate the cardiac physiological environment for drug testing. The limited availability of human heart tissue culture systems has led to inaccurate interpretations of cardiac-related drug effects. Here, we developed a cardiac tissue culture model (CTCM) that can electro-mechanically stimulate heart slices with physiological stretches in systole and diastole during the cardiac cycle. After 12 days in culture, this approach partially improved the viability of heart slices but did not completely maintain their structural integrity. Therefore, following small molecule screening, we found that the incorporation of 100 nM tri-iodothyronine (T3) and 1 μM dexamethasone (Dex) into our culture media preserved the microscopic structure of the slices for 12 days. When combined with T3/Dex treatment, the CTCM system maintained the transcriptional profile, viability, metabolic activity, and structural integrity for 12 days at the same levels as the fresh heart tissue. Furthermore, overstretching the cardiac tissue induced cardiac hypertrophic signaling in culture, which provides a proof of concept for the ability of the CTCM to emulate cardiac stretch-induced hypertrophic conditions. In conclusion, CTCM can emulate cardiac physiology and pathophysiology in culture for an extended time, thereby enabling reliable drug screening.

Challenges and opportunities for the next generation of cardiovascular tissue engineering.

Engineered cardiac tissues derived from human induced pluripotent stem cells offer unique opportunities for patient-specific disease modeling, drug discovery and cardiac repair. Since the first engineered hearts were introduced over two decades ago, human induced pluripotent stem cell-based three-dimensional cardiac organoids and heart-on-a-chip systems have now become mainstays in basic cardiovascular research as valuable platforms for investigating fundamental human pathophysiology and development. However, major obstacles remain to be addressed before the field can truly advance toward commercial and clinical translation. Here we provide a snapshot of the state-of-the-art methods in cardiac tissue engineering, with a focus on in vitro models of the human heart. Looking ahead, we discuss major challenges and opportunities in the field and suggest strategies for enabling broad acceptance of engineered cardiac tissues as models of cardiac pathophysiology and testbeds for the development of therapies.

Short-Communication: Short-Term Treatment with Taurine Prevents the Development of Cardiac Hypertrophy and Early Death in Hereditary Cardiomyopathy of the Hamster and Is Sex-Dependent.

Premature death due to heart failure is a major health problem. Taurine is a non-essential amino acid that has received much attention. However, although many studies have been carried out on the beneficial effects of taurine in cardiac pathophysiology, no studies have investigated the effect of taurine treatment on the development of hereditary cardiomyopathy (HCM) associated with hypertrophy, heart failure, and early death. This study aims to verify whether short-term treatment (20 days) with taurine in tap water prevents the development of hypertrophy and premature death in hereditary cardiomyopathy of the hamster (HCMH) of the line UM-X7.1 and if its effect is sex-dependent. Our results show that treatment for 20 days with taurine (250 mg/kg/day or 25 mg/animal/day) during the development of the hypertrophic phase (220 days old) significantly decreased (p < 0.01) the heart weight to body weight ratio in male HCMHs without affecting the female. During the 20 days (220–240 days old), there were nearly 40% premature deaths in non-treated males HCMHs and 50% in female HCMHs. Treatment for 20 days wholly and significantly prevented early death in both males and females HCMHs. Our results demonstrate that short-term treatment with taurine prevents the development of cardiac hypertrophy associated with HCM in a sex-dependent manner; however, it prevents early death in a sex-independent fashion. Our results suggest that taurine supplementation could be used to treat HCM.

Can cannabis kill? Characteristics of deaths following cannabis use in England (1998-2020).

Cannabis is the most widely used illegal drug but is rarely considered a causal factor in death. This study aimed to understand trends in deaths in England where cannabinoids were detected at post-mortem, and to evaluate the clinical utility of post-mortem cannabinoid concentrations in coronial investigations. Deaths with cannabinoid detections reported to the National Programme on Substance Abuse Deaths (NPSAD) were extracted and analysed. From 1998 to 2011, on average 7% of all cases reported to NPSAD had a cannabinoid detected (n = 110 deaths per year), rising to 18% in 2020 (n = 350). Death following cannabis use alone was rare (4% of cases, n = 136/3455). Traumatic injury was the prevalent underlying cause in these cases (62%, n = 84/136), with cannabis toxicity cited in a single case. Polydrug use was evident in most cases (96%, n = 3319/3455), with acute drug toxicity the prevalent underlying cause (74%, n = 2458/3319). Cardiac complications were the most cited physiological underlying cause of death (4%, n = 144/3455). The median average Δ9-tetrahydrocannabinol post-mortem blood concentrations were several magnitudes lower than previously reported median blood concentrations in living users (cannabis alone: 4.3 µg/L; cannabis in combination with other drugs: 3.5 µg/L). Risk of death due to cannabis toxicity is negligible. However, cannabis can prove fatal in circumstances with risk of traumatic physical injury, or in individuals with cardiac pathophysiologies. These indirect harms need careful consideration and further study to better elucidate the role cannabis plays in drug-related mortality. Furthermore, the relevance of cannabinoid quantifications in determining cause of death in coronial investigations is limited.

Abstract P1081: Palmitoylation-dependent Regulation Of Rab3gap1, Cardiomyocyte Exocytosis, And Atrial Natriuretic Peptide Secretion

Palmitoylation is the reversible post-translational modification of proteins with fatty acids catalyzed by zDHHC S-acyltransferases that functions as a potent regulatory mechanism controlling protein trafficking and signal transduction. To investigate functions of palmitoylation in cardiac homeostasis and pathophysiology, we generated transgenic mice with cardiomyocyte-specific overexpression of the Golgi enzyme, zDHHC9, which was previously reported as the Ras S-acyltransferase and is associated with X-linked intellectual disability. Although we did not observe altered Ras palmitoylation, zDHHC9 transgenic mice develop age-dependent cardiac hypertrophy that progresses to functional decompensation and cardiomyopathy. We found palmitoylation of Rab3 GTPase activating protein 1 (Rab3gap1) was significantly elevated in zDHHC9 overexpressing hearts prior to disease onset concomitant with enhanced GTP-loading of Rab3a, suggesting zDHHC9-mediated palmitoylation impairs Rab3gap1-dependent nucleotide cycling on Rab3a. Consistent with this, zDHHC9 overexpression resulted in enhanced Rab3gap1 retention at the Golgi and expansion of Rab3a-positive vesicles at the cardiomyocyte periphery. Co-immunoprecipitation analysis in HEK293AD cells confirmed that zDHHC9 elevation diminishes interaction between active Rab3a and Rab3gap1. To interrogate roles of zDHHC9 in cardiomyocyte exocytosis, we assessed secretion of atrial natriuretic peptide (ANP) and surprisingly found that while zDHHC9 overexpression increased intracellular retention and reduced release of ANP, knockdown of zDHHC9 promoted phenylephrine-induced ANP secretion. Taken together, these data suggest zDHHC9-mediated palmitoylation of Rab3gap1 represses ANP secretion by disrupting GTP:GDP cycling on Rab3a necessary for release of ANP from secretory granules. These studies reveal unexpected functions for zDHHC9-mediated palmitoylation in modulation of secretory pathway flux and regulated hormone secretion via downstream impacts on Rab3a-mediated vesicle fusion and exocytosis and suggest novel roles for zDHHC9, Rab3gap1, and Rab3a in natriuretic peptide secretion.

Dual inhibition of MAPK and PI3K/AKT pathways enhances maturation of human iPSC-derived cardiomyocytes.

SummaryHuman induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide great opportunities for mechanistic dissection of human cardiac pathophysiology; however, hiPSC-CMs remain immature relative to the adult heart. To identify novel signaling pathways driving the maturation process during heart development, we analyzed published transcriptional and epigenetic datasets from hiPSC-CMs and prenatal and postnatal human hearts. These analyses revealed that several components of the MAPK and PI3K-AKT pathways are downregulated in the postnatal heart. Here, we show that dual inhibition of these pathways for only 5 days significantly enhances the maturation of day 30 hiPSC-CMs in many domains: hypertrophy, multinucleation, metabolism, T-tubule density, calcium handling, and electrophysiology, many equivalent to day 60 hiPSC-CMs. These data indicate that the MAPK/PI3K/AKT pathways are involved in cardiomyocyte maturation and provide proof of concept for the manipulation of key signaling pathways for optimal hiPSC-CM maturation, a critical aspect of faithful in vitro modeling of cardiac pathologies and subsequent drug discovery.

Human phenotype ontology annotation and cluster analysis for pulmonary atresia to unravel clinical outcomes.

BackgroundPulmonary atresia (PA) is a heterogeneous congenital heart defect and ventricular septal defect (VSD) is the most vital factor for the conventional classification of PA patients. The simple dichotomy could not fully describe the cardiac morphologies and pathophysiology in such a complex disease. We utilized the Human Phenotype Ontology (HPO) database to explore the phenotypic patterns of PA and the phenotypic influence on prognosis.MethodsWe recruited 786 patients with diagnoses of PA between 2008 and 2016 at Fuwai Hospital. According to cardiovascular phenotypes of patients, we retrieved 52 HPO terms for further analyses. The patients were classified into three clusters based on unsupervised hierarchical clustering. We used Kaplan–Meier curves to estimate survival, the log-rank test to compare survival between clusters, and univariate and multivariate Cox proportional hazards regression modeling to investigate potential risk factors.ResultsAccording to HPO term distribution, we observed significant differences of morphological abnormalities in 3 clusters. We defined cluster 1 as being associated with Tetralogy of Fallot (TOF), VSD, right ventricular hypertrophy (RVH), and aortopulmonary collateral arteries (ACA). ACA was not included in the cluster classification because it was not an HPO term. Cluster 2 was associated with hypoplastic right heart (HRH), atrial septal defect (ASD) and tricuspid disease as the main morphological abnormalities. Cluster 3 presented higher frequency of single ventricle (SV), dextrocardia, and common atrium (CA). The mortality rate in cluster 1 was significantly lower than the rates in cluster 2 and 3 (p = 0.04). Multivariable analysis revealed that abnormal atrioventricular connection (AAC, p = 0.011) and persistent left superior vena cava (LSVC, p = 0.003) were associated with an increased risk of mortality.ConclusionsOur study reported a large cohort with clinical phenotypic, surgical strategy and long time follow-up. In addition, we provided a precise classification and successfully risk stratification for patients with PA.

Single-cell transcriptome reveals effects of semaglutide on non-cardiomyocytes of obese mice

Non-cardiomyocytes (nonCMs) play an important part in cardiac fibrosis pathophysiology, but the underlying molecular pathways are unknown. Semaglutide has cardioprotective properties, but it is still unclear whether it helps with cardiac fibrosis and what the processes are. The goal of this study is to use single cell transcriptomics approaches to investigate the molecular mechanism of semaglutide's cardioprotective action in obese mice. We found 15 non-CMs, with fibroblasts making up the majority of them. We found eight DEGs that altered significantly following semaglutide treatment by screening for differentially expressed genes (DEGs). DEGs were shown to have biological activities primarily related to extracellular matrix and collagen synthesis and distribution, with Serpinh1 and Pcolce expression being the most dramatically altered. Serpinh1 and Pcolce were mostly found in fibroblasts, which play a key role in the fibrosis of the heart. Furthermore, we discovered that semaglutide lowered cardiac collagen content and alleviated obesity-induced ventricular wall hypertrophy. As a result, our findings show that Serpinh1 and Pcolce, which are expressed by fibroblasts, may play a role in the development of obese cardiac fibrosis. By reducing Serpinh1 and Pcolce expression and delaying cardiac fibrosis, semaglutide may have a cardioprotective effect.

Perioperative management of patients with prosthetic heart valves-A narrative review.

ABSTRACTWorldwide, about 13% of the 200,000 annual recipients of prosthetic heart valves (PHV) present for various surgical procedures. Also, more and more females are opting for pregnancies after having PHV. All patients with PHV present unique challenges for the anesthesiologists, surgeons and obstetricians (in case of deliveries). They have to deal with the perioperative management of anticoagulation and a host of other issues involved. We reviewed the English language medical literature relevant to the different aspects of perioperative management of patients with PHV, particularly the guidelines of reputed societies that appeared in the last 20 years. Regression of cardiac pathophysiology following valve replacement is variable both in extent and timeline. The extent to which reverse remodeling occurs depends on the perioperative status of the heart. We discussed the perioperative assessment of patients with PHV, including focused history and relevant investigations with the inferences drawn. We examined the need for prophylaxis against infective endocarditis and management of anticoagulation in such patients in the perioperative period and the guidelines of reputed societies. We also reviewed the conduct of anesthesia, including general and regional anesthesia (neuraxial and peripheral nerve/plexus blocks) in such patients. Finally, we discussed the management of delivery in this group of high-risk patients. From the discussion of different aspects of perioperative management of patients with PHV, we hope to guide in formulating the comprehensive plan of management of safe anesthesia in such patients.

Myocardial brain-derived neurotrophic factor regulates cardiac bioenergetics through the transcription factor Yin Yang 1.

Brain-derived neurotrophic factor (BDNF) is markedly decreased in heart failure patients. Both BDNF and its receptor, tropomyosin-related kinase receptor (TrkB), are expressed in cardiomyocytes; however, the role of myocardial BDNF signalling in cardiac pathophysiology is poorly understood. Here, we investigated the role of BDNF/TrkB signalling in cardiac stress response to exercise and pathological stress. We found that myocardial BDNF expression was increased in mice with swimming exercise but decreased in a mouse heart failure model and human failing hearts. Cardiac-specific TrkB knockout (cTrkB KO) mice displayed a blunted adaptive cardiac response to exercise, with attenuated upregulation of transcription factor networks controlling mitochondrial biogenesis/metabolism, including peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α). In response to pathological stress (transaortic constriction, TAC), cTrkB KO mice showed an exacerbated heart failure progression. The downregulation of PGC-1α in cTrkB KO mice exposed to exercise or TAC resulted in decreased cardiac energetics. We further unravelled that BDNF induces PGC-1α upregulation and bioenergetics through a novel signalling pathway, the pleiotropic transcription factor Yin Yang 1. Taken together, our findings suggest that myocardial BDNF plays a critical role in regulating cellular energetics in the cardiac stress response.

Cellular and Engineered Organoids for Cardiovascular Models.

An ensemble of in vitro cardiac tissue models has been developed over the past several decades to aid our understanding of complex cardiovascular disorders using a reductionist approach. These approaches often rely on recapitulating single or multiple clinically relevant end points in a dish indicative of the cardiac pathophysiology. The possibility to generate disease-relevant and patient-specific human induced pluripotent stem cells has further leveraged the utility of the cardiac models as screening tools at a large scale. To elucidate biological mechanisms in the cardiac models, it is critical to integrate physiological cues in form of biochemical, biophysical, and electromechanical stimuli to achieve desired tissue-like maturity for a robust phenotyping. Here, we review the latest advances in the directed stem cell differentiation approaches to derive a wide gamut of cardiovascular cell types, to allow customization in cardiac model systems, and to study diseased states in multiple cell types. We also highlight the recent progress in the development of several cardiovascular models, such as cardiac organoids, microtissues, engineered heart tissues, and microphysiological systems. We further expand our discussion on defining the context of use for the selection of currently available cardiac tissue models. Last, we discuss the limitations and challenges with the current state-of-the-art cardiac models and highlight future directions.

Hedgehog Morphogens Act as Growth Factors Critical to Pre- and Postnatal Cardiac Development and Maturation: How Primary Cilia Mediate Their Signal Transduction.

Primary cilia are crucial for normal cardiac organogenesis via the formation of cyto-architectural, anatomical, and physiological boundaries in the developing heart and outflow tract. These tiny, plasma membrane-bound organelles function in a sensory-integrative capacity, interpreting both the intra- and extra-cellular environments and directing changes in gene expression responses to promote, prevent, and modify cellular proliferation and differentiation. One distinct feature of this organelle is its involvement in the propagation of a variety of signaling cascades, most notably, the Hedgehog cascade. Three ligands, Sonic, Indian, and Desert hedgehog, function as growth factors that are most commonly dependent on the presence of intact primary cilia, where the Hedgehog receptors Patched-1 and Smoothened localize directly within or at the base of the ciliary axoneme. Hedgehog signaling functions to mediate many cell behaviors that are critical for normal embryonic tissue/organ development. However, inappropriate activation and/or upregulation of Hedgehog signaling in postnatal and adult tissue is known to initiate oncogenesis, as well as the pathogenesis of other diseases. The focus of this review is to provide an overview describing the role of Hedgehog signaling and its dependence upon the primary cilium in the cell types that are most essential for mammalian heart development. We outline the breadth of developmental defects and the consequential pathologies resulting from inappropriate changes to Hedgehog signaling, as it pertains to congenital heart disease and general cardiac pathophysiology.

OTUB1: A Key Player in the Adaptive Cardiac Hypertrophy

Deubiquitinases (DUBs), a family enzymes that reverse the process of ubiquitination, participate in diverse cellular processes through regulating protein degradation, intracellular trafficking, cell signaling, and protein‐protein interaction. The functional importance of DUBs in cardiac pathophysiology remains poorly defined. Herein, we report a critical role of OTUB1, one of the most highly expressed DUBs in the heart, in adaptive cardiac remodeling. Analysis of RNA sequencing data revealed that OTUB1 is upregulated in the failing human hearts. OTUB1 is also upregulated and activated in cardiomyocytes (CMs) treated with adrenergic agonists. OTUB1‐deficient CMs were resistant to phenylephrine (PE) ‐ and isoproterenol‐induced CM hypertrophy as characterized by attenuated cell enlargement, decreased fetal gene activation, and diminished protein synthesis. In vivo, CM‐specific deletion of OTUB1 in the developing mouse heart caused dilated cardiomyopathy without a prior development of compensatory cardiac hypertrophy, eventually leading to heart failure and premature lethality of mice by 6 months of age. The severe cardiac phenotype is accompanied by lipid accumulation, ER dilatation, prevalent autophagic vesicles containing ribosomes, as well as dysregulated expression of metabolic and ribosome genes. Delivery of wild‐type OTUB1, but not the enzymatic dead mutant, via AAV9 to OTUB1‐deficient hearts attenuated cardiac dysfunction and prolonged the lifespan of mice. On the molecular level, OTUB1 deficiency blunted mTOR signaling in cultured CMs and mouse hearts. Our findings indicate that OTUB1 is indispensable for the development of adaptive cardiac hypertrophy, possibly through modulating mTOR signaling.

Cardiac disease in pregnancy.

In the developed world, cardiovascular disease has become the most frequent cause of death during pregnancy and postpartum, outnumbering by far obstetric causes of death such as bleeding or thromboembolism. Many factors contribute to this phenomenon, including an increasing age of pregnant women, co-morbidities, and an unhealthy lifestyle. The cardiovascular system is not only significantly challenged by physiological alterations in pregnancy but also by obstetric medication. Depending upon the severity of the underlying condition, pregnant women with cardiovascular disease should be managed by a multidisciplinary heart team in which anaesthesiologists play an important role. Profound knowledge of the cardiac pathophysiology is a prerequisite for the successful anaesthesiologic management of pregnant patients with cardiovascular disease. As there is no difference in general and regional anaesthesia regarding maternal outcomes, neuraxial anaesthesia using incremental techniques should be preferred for labour and (caesarean) delivery if not contraindicated by non-cardiac issues.

HMGB1 in macrophage nucleus protects against pressure overload induced cardiac remodeling via regulation of macrophage differentiation and inflammatory response

Pressure overload induced cardiac remodeling is associated with a complex spectrum of pathophysiological mechanisms. As inflammatory cells, macrophages maintain a critical position in mechanical stress-induced myocardial remodeling. HMGB1 is a highly conserved, ubiquitous protein in various types of cells whose biological roles are closely dependent on subcellular sites. However, whether HMGB1 expressed in macrophages performs the protective or pathological responses in cardiac remodeling is unknown. In this study, we generated the myeloid-specific HMGB1 knockout mice and detected the effects of macrophage HMGB1 in response to pathophysiological stress. Our data showed HMGB1 in macrophages played a protective role against the pressure overload induced cardiac pathophysiology. The deletion of HMGB1 in macrophages gains more differentiation of M1-type pro-inflammatory macrophage during the mechanical stress-induced myocardial remodeling, thereby aggravating the inflammatory response in whole heart, resulting in accelerated deterioration of cardiac function. Moreover, in vitro data also validated HMGB1 got involved in the process of macrophage polarization. Macrophages without HMGB1 are more inclined to differentiate into M1 during the stretch process. In summary, the present results indicated that loss of HMGB1 in macrophages can exacerbate heart failure through increased differentiation of pro-inflammatory macrophages and enhanced inflammatory response.

Methods of mouse cardiomyocyte isolation from postnatal heart

Primary cardiomyocytes (CMs) are invaluable materials used to study in vitro cardiac physiology and pathophysiology. However, there is still a lack of comprehensive, systematic and comparative studies on methods for isolation of primary CMs from postnatal hearts. Here, we aimed to compare and optimize protocols for the isolation of CMs from embryonic to adult stages. We found that the trypsin digestion method was suitable for embryonic mouse CM isolation. The Gentle MACS method yielded high-quality CMs from neonatal hearts (postnatal day 1-day 3, P1-P3). The Langendorff-free perfusion method was applicable for isolation of CMs from mice older than P3. P14 and P56 CMs could also be isolated by the Langendorff perfusion system. The transcriptional profiles and cellular function of the isolated CMs were respectively confirmed by RNA sequencing and Angiotensin II treatment, suggesting the feasibility and effectiveness of the isolation methods. Overall, this study supplies a series of detailed and optimized protocols to isolate CMs at different developmental stages, which provides support for the study of the mechanisms, pathology, and pharmacology of cardiovascular diseases.

Simultaneous triple-parametric optical mapping of transmembrane potential, intracellular calcium and NADH for cardiac physiology assessment

Investigation of the complex relationships and dependencies of multiple cellular processes that govern cardiac physiology and pathophysiology requires simultaneous dynamic assessment of multiple parameters. In this study, we introduce triple-parametric optical mapping to simultaneously image metabolism, electrical excitation, and calcium signaling from the same field of view and demonstrate its application in the field of drug testing and cardiovascular research. We applied this metabolism-excitation-contraction coupling (MECC) methodology to test the effects of blebbistatin, 4-aminopyridine and verapamil on cardiac physiology. While blebbistatin and 4-aminopyridine alter multiple aspects of cardiac function suggesting off-target effects, the effects of verapamil were on-target and it altered only one of ten tested parameters. Triple-parametric optical mapping was also applied during ischemia and reperfusion; and we identified that metabolic changes precede the effects of ischemia on cardiac electrophysiology.