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Background and Aims Fibrotic tissue formed after myocardial infarction (MI) can be as detrimental as MI itself. However, current in vitro cardiac fibrosis models fail to recapitulate the complexities of post‐MI tissue. Moreover, although MI and subsequent fibrosis is most prominent in the aged population, the field suffers from inadequate aged tissue models. Herein, an aged human post‐MI tissue model, representing the native microenvironment weeks after initial infarction, is engineered using three‐dimensional bioprinting via creation of individual bioinks to specifically mimic three distinct regions: remote, border, and scar. Methods The aged post‐MI tissue model is engineered through combination of gelatin methacryloyl, methacrylated hyaluronic acid, aged type I collagen, and photoinitiator at variable concentrations with different cell types, including aged human induced pluripotent stem cell‐derived cardiomyocytes, endothelial cells, cardiac fibroblasts, and cardiac myofibroblasts, by introducing a methodology which utilizes three printheads of the bioprinter to model aged myocardium. Then, using cell‐specific proteins, the cell types that comprised each region are confirmed using immunofluorescence. Next, the beating characteristics are analyzed. Finally, the engineered aged post‐MI tissue model is used as a benchtop platform to assess the therapeutic effects of stem cell‐derived extracellular vesicles on the scar region. Results As a result, high viability (>74%) was observed in each region of the printed model. Constructs demonstrated functional behavior, exhibiting a beating velocity of 6.7 μm/s and a frequency of 0.3 Hz. Finally, the effectiveness of hiPSC‐EV and MSC‐EV treatment was assessed. While hiPSC‐EV treatment showed no significant changes, MSC‐EV treatment notably increased cardiomyocyte beating velocity, frequency, and confluency, suggesting a regenerative potential. Conclusion In conclusion, we envision that our approach of modeling post‐MI aged myocardium utilizing three printheads of the bioprinter may be utilized for various applications in aged cardiac microenvironment modeling and testing novel therapeutics.

Coronary artery disease (CAD), is a global cardiovascular disease that is characterized by myocardial ischemia and hypoxia caused by coronary artery occlusion. Circular RNAs (CircRNAs) is a particular kind of endogenous non-coding RNA, which can affect the occurrence and development of CAD. Concurrently, several circRNAs display stable persistence in CAD patients, attributable to their exceptional exonuclease resistance, thereby harboring the capacity to evolve into a biomarker for CAD diagnosis and prognosis. This article endeavors to clarify the pivotal role of circRNAs in the intricate pathophysiological processes underlying CAD patients or CAD disease models based on their unique biological characteristics and functionalities, and further discuss their prospects in clinical applications of CAD.

Background: Myocardial ischemia injury is one of the leading causes of death and disability worldwide. Cardiac fibroblasts (CFs) have central roles in modulating cardiac function under pathophysiological conditions. Activating transcription factor 3 (ATF3) plays a self-protective role in counteracting CF dysfunction. However, the precise function of CF-specific ATF3 during myocardial infarction (MI) injury/repair remains incompletely understood. The aim of this study was to determine whether CF-specific ATF3 affected cardiac repair after MI. Methods: Fifteen male C57BL/6 wild-type mice were performed with MI operation to observe the expression of ATF3 at 0, 0.5, 1.0, 3.0, and 7.0 days postoperation. Model for MI was constructed in ATF3TGfl/flCol1a2-Cre+ (CF-specific ATF3 overexpression group, n = 5) and ATF3TGfl/flCol1a2-Cre− male mice (without CF-specific ATF3 overexpression group, n = 5). In addition, five mice of ATF3TGfl/flCol1a2-Cre+ and ATF3TGfl/flCol1a2-Cre− were subjected to sham MI operation. Heart function was detected by ultrasound and left ventricular remodeling was observed by Masson staining (myocardial fibrosis area was detected by blue collagen deposition area) at the 28th day after MI surgery in ATF3TGfl/flCol1a2-Cre+ and ATF3TGfl/flCol1a2-Cre− mice received sham or MI operation. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect cell proliferation/cell cycle-related gene expression in cardiac tissue. BrdU staining was used to detect fibroblast proliferation. Results: After establishment of an MI model, we found that ATF3 proteins were increased in the heart of mice after MI surgery and dominantly expressed in CFs. Genetic overexpression of ATF3 in CFs (ATF3TGfl/flCol1a2-Cre+ group) resulted in an improvement in the heart function as indicated by increased cardiac ejection fraction (41.0% vs. 30.5%, t = 8.610, P = 0.001) and increased fractional shortening (26.8% vs. 18.1%, t = 7.173, P = 0.002), which was accompanied by a decrease in cardiac scar area (23.1% vs. 11.0%, t = 8.610, P = 0.001). qRT-PCR analysis of CFs isolated from ATF3TGfl/flCol1a2-Cre+ and ATF3TGfl/flCol1a2-Cre− ischemic hearts revealed a distinct transcriptional profile in ATF3-overexpressing CFs, displaying pro-proliferation properties. BrdU-positive cells significantly increased in ATF3-overexpressing CFs than control CFs under angiotensin II stimuli (11.5% vs. 6.8%, t = 31.599, P = 0.001) or serum stimuli (31.6% vs. 20.1%, t = 31.599, P = 0.001). The 5(6)-carboxyfluorescein N-hydroxysuccinimidyl ester assay showed that the cell numbers of the P2 and P3 generations were higher in the ATF3-overexpressing CFs at 24 h (P2: 91.6% vs. 71.8%, t = 8.465, P = 0.015) and 48 h (P3: 81.6% vs. 51.1%, t = 9.029, P = 0.012) after serum stimulation. Notably, ATF3 overexpression-induced CF proliferation was clearly increased in the heart after MI injury. Conclusions: We identify that CF-specific ATF3 might contribute to be MI repair through upregulating the expression of cell cycle/proliferation-related genes and enhancing cell proliferation.

To investigate the role of cardiomyocyte-derived BDNF as an endogenous regulator to decrease cardiac fibrosis, its underlying mechanism and therapeutic potential. Single-nuclei RNA sequencing (snRNA-seq), KEGG, Gene Ontology and cell‒cell interaction analyses were performed to identify changes in cardiac cells, cardiac functions and pathways due to the conditional knockout of cardiomyocyte-derived BDNF (cardiomyocyte-BDNF-KO). Protein C-terminal sequencing, qPCR, WB, CCK8 assays, flow cytometry, BDNF-AAV9 treatment and histological staining were performed to investigate the roles of BDNF and the BDNF mimic 7,8-DHF (7,8-DHF) in cardiac fibroblasts cardiac myofibroblasts and cardiac fibrosis, and the cross-inhibition of the TGF-β and BDNF-TrkB-FL pathway. snRNA-seq and bioinformatics analysis revealed that cardiomyocyte-BDNF-KO significantly increased the percentage of CFs, decreased the number of cardiomyocytes, and increased the activity of the TGF-β pathway in CFs. Functional studies confirmed that compared with those in wild-type hearts, the expression levels of key signaling molecules in the TGF-β pathway in cardiomyocyte-BDNF-KO CFs in the mouse heart were significantly higher. CFs and CMFs expressed the BDNF receptor TrkB-FL but not BDNF, and treatment with BDNF and 7,8-DHF decreased the expression of key signaling molecules in the TGF-β pathway in CFs and CMFs. BDNF inhibited CF and CMF proliferation, inhibited CF activation and transformation into CMFs, promoted CMF apoptosis, the accumulation of cells in S phase of the cell cycle and TrkB-FL phosphorylation in CFs and CMFs, increased Smad7 expression in CMFs, and inhibited the activity of the TGF-β/Smad2/3/α-SMA pathway. 7,8-DHF had the same effects as BDNF, as documented above. Furthermore, BDNF-AAV9 therapy for cardiomyocyte-BDNF-KO hearts increased Smad7 expression and decreased the activity of the TGF-β/Smad2/3 pathway and the expression of fibrotic effectors, which ameliorated cardiac fibrosis. Cardiomyocyte-derived BDNF acts as an endogenous mediator to restrict cardiac fibrosis by inhibiting CF and CMF proliferation, CF activation and transformation into CMFs, and increasing arrest in S phase of the cell cycle in CFs and CMFs and the apoptosis of CMFs. The BDNF-TrkB-FL pathway cross-inhibits the activity of the TGF-β/Smad2/3/α-SMA pathway and increases the expression of Smad7. BDNF and 7,8-DHF have therapeutic potential for treating cardiac fibrosis.

Fibrosis is defined as the excessive deposition of extracellular matrix (ECM) proteins in the interstitium. It is an essential pathological response to chronic inflammation. ECM protein deposition is initially protective and is critical for wound healing and tissue regeneration. However, pathological cardiac remodeling in excessive and continuous tissue damage with subsequent ECM deposition results in a distorted organ architecture and significantly impacts cardiac function. In this review, we summarized and discussed the histologic features of cardiac fibrosis with the signaling factors that control it. We evaluated the origin and characteristic markers of cardiac fibroblasts. We also discussed lymphatic vessels, which have become more important in recent years to improve cardiac fibrosis.

IntroductionHeart Failure with preserved Ejection Fraction (HFpEF) affects over 50% of patients with HF, majority of whom are females with conditions such as hypertension, obesity and diabetes. Our in vivo animal models of these conditions show increased fibrosis and oxidized Calcium-calmodulin dependent protein kinase II (CaMKII) activity. At the cellular level, mitochondrial health is known to be implicated in HF, and our in vitro models have signified the impact of female cardiac fibroblasts (CFs) on altered calcium (Ca2+) signalling in cardiac myocytes (CMs), during co-culture. However, knowledge on the characteristics of CFs in HFpEF still remains obscure. Here, we investigated altered Ca2+ signalling, mitochondrial Reactive Oxygen Species (ROS) production, and the therapeutic potential of inhibiting CaMKII activity in CFs, during hyperglycaemia and hypertension.MethodsAdult human CFs (Promocell) sourced from both female and male donors were cultured under hyperglycaemic (22 mM Glucose), hypertensive (200nM Angiotensin II) or HFpEF-like (hyperglycaemic plus hypertensive) conditions, in the absence or presence of a CaMKII inhibitor (5µM KN93), for 48 hours. Following pathological conditioning of CFs, cells were loaded with Cal520AM calcium indicator or MitoSOX red mitochondrial superoxide indicator. Live cell fluorescence imaging was utilised to assess Ca2+ activity and mitochondrial ROS production in CFs.ResultsFemale CFs treated under hyperglycaemic conditions showed a greater Endothelin-1-induced Ca2+ transient amplitude (ΔF/F0) relative to control and hypertensive conditioning [Control: 0.184±0.008; Diabetes: 0.305±0.026; Hypertension: 0.208±0.030; HFpEF: 0.241±0.008, n=4passages]. Mitochondrial superoxide (AU) levels were elevated only in female CFs, in both hyperglycaemic and hypertensive conditions [Control: 18.0±2.0; Diabetes: 36.7±3.4; Hypertension: 29.5±4.5; HFpEF: 22.7±2.0, n=4passages]. These alterations in Ca2+ transient amplitude and superoxide production were impeded by the presence of KN93 [Ca2+ Transient Amplitude- Control: 0.197±0.020; Diabetes: 0.167±0.022; Hypertension: 0.199±0.037; HFpEF: 0.205±0.023, Mitochondrial superoxide- Control: 16.6±2.4; Diabetes: 14.7±2.6; Hypertension: 13.3±1.1; HFpEF: 18.1±2.6, [n=4passages].ConclusionsThese results indicate CaMKII is important in mitochondrial oxidative stress in female CFs, in hyperglycaemic and hypertensive conditions. Further work is needed to investigate the importance of these processes in the development of fibrosis in HFpEF.Abstract BS42 Figure 1Endothelin-1-induced calcium transient amplitude in female CFs. * p<0.05 and ** p<0.01Abstract BS42 Figure 2Mitochondrial superoxide production in female CFs. * p<0.05, ** p<0.01 and *** p<0.001Conflict of InterestNone

Diabetic cardiomyopathy (DCM) is one of the leading causes of heart failure in patients with diabetes mellitus, with limited effective treatments. The cardioprotective effects of sodium‐glucose cotransporter 2(SGLT2) inhibitors have been supported by amounts of clinical trials, which largely fills the gap. However, the underlying mechanism still needs to be further explored, especially in terms of its protection against cardiac fibrosis, a crucial pathophysiological process during the development of DCM. Besides, endothelial‐to‐mesenchymal transition (EndMT) has been reported to play a pivotal role in fibroblast multiplication and cardiac fibrosis. This study aimed to evaluate the effect of SGLT2 inhibitor dapagliflozin (DAPA) on DCM especially for cardiac fibrosis and explore the underlying mechanism. In vivo, the model of type 2 diabetic rats was built with high‐fat feeding and streptozotocin injection. Untreated diabetic rats showed cardiac dysfunction, increased myocardial fibrosis and EndMT, which was attenuated after treatment with DAPA and metformin. In vitro, HUVECs and primary cardiac fibroblasts were treated with DAPA and exposed to high glucose (HG). HG‐induced EndMT in HUVECs and collagen secretion of fibroblasts were markedly inhibited by DAPA. Up‐regulation of TGF‐β/Smad signalling and activity inhibition of AMPKα were also reversed by DAPA treatment. Then, AMPKα siRNA and compound C abrogated the anti‐EndMT effects of DAPA in HUVECs. From above all, our study implied that DAPA can protect against DCM and myocardial fibrosis through suppressing fibroblast activation and EndMT via AMPKα‐mediated inhibition of TGF‐β/Smad signalling.

Cardiac fibrosis is a pathological process associated with the development of heart failure. TGF-β and WNT signaling have been implicated in pathogenesis of cardiac fibrosis, however, little is known about molecular cross-talk between these two pathways. The aim of this study was to examine the effect of exogenous canonical WNT3a and non-canonical WNT5a in TGF-β-activated human cardiac fibroblasts. We found that WNT3a and TGF-β induced a β-catenin-dependent response, whereas WNT5a prompted AP-1 activity. TGF-β triggered profibrotic signatures in cardiac fibroblasts, and co-stimulation with WNT3a or co-activation of the β-catenin pathway with the GSK3β inhibitor CHIR99021 enhanced collagen I and fibronectin production and development of active contractile stress fibers. In the absence of TGF-β, neither WNT3a nor CHIR99021 exerted profibrotic responses. On a molecular level, in TGF-β-activated fibroblasts, WNT3a enhanced phosphorylation of TAK1 and production and secretion of IL-11 but showed no effect on the Smad pathway. Neutralization of IL-11 activity with the blocking anti-IL-11 antibody effectively reduced the profibrotic response of cardiac fibroblasts activated with TGF-β and WNT3a. In contrast to canonical WNT3a, co-activation with non-canonical WNT5a suppressed TGF-β-induced production of collagen I. In conclusion, WNT/β-catenin signaling promotes TGF-β-mediated fibroblast-to-myofibroblast transition by enhancing IL-11 production. Thus, the uncovered mechanism broadens our knowledge on a molecular basis of cardiac fibrogenesis and defines novel therapeutic targets for fibrotic heart diseases.

Cardiovascular diseases remain the leading cause of global mortality. Cellular senescence has recently been implicated in the pathogenesis of various cardiovascular diseases. Our group has previously shown that the matricellular protein CCN5 is a potent anti-fibrotic molecule capable of inhibiting and reversing cardiac fibrosis. In this study, we investigated whether CCN5 can modulate cellular senescence in the heart utilizing three readouts: western blotting for p53 and p21, staining for senescence-associated β-galactosidase, and microscopic analysis of γH2AX-foci. CCN5 effectively inhibited doxorubicin-induced cellular senescence in both H9c2 cardiac myoblasts and fibroblasts. In addition, CCN5 suppressed cellular senescence in H9c2 cardiac myoblasts induced by the senescence-associated secretory phenotype factors secreted from cardiac fibroblast, and vice versa. CCN5 also restored the apoptotic response of senescent cells. Finally, CCN5 attenuated myocardial infarction-induced cellular senescence in mice. Collectively, our findings provide novel insights into the potential role of CCN5 in the development of anti-senescence therapies.

The deleterious effects of diabetes in the heart are increasingly attributed to inflammatory signaling through the NLRP3 (NOD, LRR and PYD domains-containing protein 3) inflammasome. Thrombin antagonists reduce cardiac remodeling and dysfunction in diabetic mice, in part by suppressing fibrin-driven inflammation. The role of cellular thrombin receptor subtypes in this context is not known. We sought to determine the causal involvement of protease-activated receptors (PAR) in inflammatory signaling of the diabetic heart. Mice with diet-induced diabetes showed increased abundance of pro-caspase-1 and pro-interleukin (IL)-1β in the left ventricle (LV), indicating transcriptional NLRP3 inflammasome priming, and augmented cleavage of active caspase-1 and IL-1β, pointing to canonical NLRP3 inflammasome activation. Caspase-11 activation, which mediates non-canonical NLRP3 inflammasome signaling, was not augmented. Formation of the plasma membrane pore-forming protein N-terminal gasdermin D (GDSMD), a prerequisite for IL-1β secretion, was also higher in diabetic vs. control mouse LV. NLRP3, ASC and IL-18 expression did not differ between the groups, nor did expression of PAR1 or PAR2. PAR3 was nearly undetectable. LV abundance of PAR4 by contrast increased with diabetes and correlated positively with active caspase-1. Genetic deletion of PAR4 in mice prevented the diet-induced cleavage of caspase-1, IL-1β and GDSMD. Right atrial appendages from patients with type 2 diabetes also showed higher levels of PAR4, but not of PAR1 or PAR2, than non-diabetic atrial tissue, along with increased abundance of cleaved caspase-1, IL-1β and GSDMD. Human cardiac fibroblasts maintained in high glucose conditions to mimic diabetes also upregulated PAR4 mRNA and protein, and increased PAR4-dependent IL-1β transcription and secretion in response to thrombin, while PAR1 and PAR2 expressions were unaltered. In conclusion, PAR4 drives caspase-1-dependent IL-1β production through the canonical NLRP3 inflammasome pathway in the diabetic heart, providing mechanistic insights into diabetes-associated cardiac thromboinflammation. The emerging PAR4-selective antagonists may provide a feasible approach to prevent cardiac inflammation in patients with diabetes.