Stimulates Cardiomyocyte Proliferation Research Articles

Fibroblast-specific deletion of ERBB4 impairs cardiac regeneration in neonatal mice

Abstract Background Scarless regeneration of the neonatal mammalian heart is a remarkable but poorly understood process. Previous studies have shown that paracrine actions of neuregulin-1 (NRG1) are indispensable to stimulate cardiomyocyte proliferation in the injured neonatal heart, and are mediated by activation of the ERBB4 receptor. It remains unclear, however, whether NRG1 also contributes to other steps of neonatal cardiac regeneration, e.g. to the control of collagen synthesis by fibroblasts, hence contributing to scarless healing. Purpose To study temporal evolution of the infarct size during 21 days after myocardial infarction (MI) in neonatal fibroblast-specific Erbb4 knock-out (KO) and wild type (WT) mice. We hypothesized that scarless healing would be completed within 21 days in WT mice, and incomplete in KO mice. Methods Fibroblast-specific hemizygous Erbb4 KO (FB-Erbb4-KO) mice were generated (Erbb4F/+ Col1a2-Cre+, C57BL/6 background), because fibroblast-specific nullizygous Erbb4 KO were not viable. Validation of the KO was performed both on RNA (qPCR) and protein (western blot) level. MI was induced in one-day old FB-Erbb4-KO and WT littermates of both sexes by permanent ligation of the left anterior descending (LAD) coronary artery under hypothermic anaesthesia. Pups from both groups were euthanized at 4 (WT, n=9; KO, n=11), 7 (WT, n=12; KO, n=4), 10 (WT, n=11; KO, n=7) and 21 (WT, n=16; KO, n=8) days post-MI. Following dissection, hearts were formalin-fixed and paraffin embedded for histopathological evaluation. Transverse sections were cut at 4 distinct locations from the ligation site to the apex and stained with Masson’s Trichrome for infarct scar measurement. Genotyping was performed post-mortem and unblinded after data analysis. Results Both the transgenic mice model and the LAD ligation technique were successfully validated. No significant differences in the initial infarct scar size were observed between WT and FB-Erbb4-KO mice 4 days post-MI (p=0.6556). 7-days post-MI the infarct scar size was 6-fold smaller compared to 4 days post-MI in both FB-Erbb4-KO and WT mice, with a trend towards a larger infarct scar size in the FB-Erbb4-KO group compared to WT (p=0.0989). At day 10 post-MI, infarct scar size in the FB-Erbb4-KO group was significantly larger (p=0.0499) compared to WT. At 21 days post-MI, the infarct size had almost completely disappeared in the WT group, but remained clearly present in the FB-Erbb4-KO group (Fig. 1, p=0.0099). Infarct scar sizes at all timepoints are graphically shown in Fig. 2. Conclusion Fibroblast-specific deletion of the ERBB4 receptor causes incomplete resolution of the infarct scar in murine neonates 21 days after MI. While the initial infarct scar size 4 days after injury is the same, impaired scar resolution becomes progressively more evident towards day 21 post-MI. These findings indicate that scarless healing of the neonatal mammalian heart requires NRG1/ERBB4 signalling in fibroblasts.Fig. 1Fig. 2

Cell cycle visualization tools to study cardiomyocyte proliferation in real-time.

Cardiomyocytes in the adult human heart are quiescent and those lost following heart injury are not replaced by proliferating survivors. Considerable effort has been made to understand the mechanisms underlying cardiomyocyte cell cycle exit and re-entry, with view to discovering therapeutics that could stimulate cardiomyocyte proliferation and heart regeneration. The advent of large compound libraries and robotic liquid handling platforms has enabled the screening of thousands of conditions in a single experiment but success of these screens depends on the appropriateness and quality of the model used. Quantification of (human) cardiomyocyte proliferation in high throughput has remained problematic because conventional antibody-based staining is costly, technically challenging and does not discriminate between cardiomyocyte division and failure in karyokinesis or cytokinesis. Live cell imaging has provided alternatives that facilitate high-throughput screening but these have other limitations. Here, we (i) review the cell cycle features of cardiomyocytes, (ii) discuss various cell cycle fluorescent reporter systems, and (iii) speculate on what could improve their predictive value in the context of cardiomyocyte proliferation. Finally, we consider how these new methods can be used in combination with state-of-the-art three-dimensional human cardiac organoid platforms to identify pro-proliferative signalling pathways that could stimulate regeneration of the human heart.

Thermosensitive Hydrogel with Programmable, Self‐Regulated HIF‐1α Stabilizer Release for Myocardial Infarction Treatment

AbstractHIF‐1α (hypoxia induced factor‐1α), a vital protective signal against hypoxia, has a short lifetime after myocardial infarction (MI). Increasing HIF‐1α stability by inhibiting its hydroxylation with prolyl hydroxylases inhibitors such as DPCA (1,4‐dihydrophenonthrolin‐4‐one‐3‐carboxylic acid) presents positive results. However, the optimal inhibitor administration profile for MI treatment is still unexplored. Here, injectable, thermosensitive hydrogels with programmable DPCA release are designed and synthesized. Hydrogel degradation and slow DPCA release are coupled to form a feedback loop by attaching pendant DPCA to polymer backbone, which serve as additional crosslinking points through π–π and hydrophobic interactions. Pendant carboxyl groups are added to the copolymer to accelerate DPCA release. Burst release in the acute phase for myocardial protection and extended near zero‐order release across the inflammatory and fibrotic phases with different rates are achieved. All DPCA‐releasing hydrogels upregulate HIF‐1α, decrease apoptosis, promote angiogenesis, and stimulate cardiomyocyte proliferation, leading to preserved cardiac function and ventricular geometry. Faster hydrogel degradation induced by faster DPCA release results in a HIF‐1α expression eight times of healthy control and better therapeutic effect in MI treatment. This research demonstrates the value of precise regulation of HIF‐1α expression in treating MI and other relevant diseases and provides an implantable device‐based modulation strategy.

Cardiomyocyte proliferation and regeneration in congenital heart disease.

Despite advances in prenatal screening and a notable decrease in mortality rates, congenital heart disease (CHD) remains the most prevalent congenital disorder in newborns globally. Current therapeutic surgical approaches face challenges due to the significant rise in complications and disabilities. Emerging cardiac regenerative therapies offer promising adjuncts for CHD treatment. One novel avenue involves investigating methods to stimulate cardiomyocyte proliferation. However, the mechanism of altered cardiomyocyte proliferation in CHD is not fully understood, and there are few feasible approaches to stimulate cardiomyocyte cell cycling for optimal healing in CHD patients. In this review, we explore recent progress in understanding genetic and epigenetic mechanisms underlying defective cardiomyocyte proliferation in CHD from development through birth. Targeting cell cycle pathways shows promise for enhancing cardiomyocyte cytokinesis, division, and regeneration to repair heart defects. Advancements in human disease modeling techniques, CRISPR-based genome and epigenome editing, and next-generation sequencing technologies will expedite the exploration of abnormal machinery governing cardiomyocyte differentiation, proliferation, and maturation across diverse genetic backgrounds of CHD. Ongoing studies on screening drugs that regulate cell cycling are poised to translate this nascent technology of enhancing cardiomyocyte proliferation into a new therapeutic paradigm for CHD surgical interventions.

Engineered nanovesicles mediated cardiomyocyte survival and neovascularization for the therapy of myocardial infarction

Myocardial infarction (MI) leads to substantial cellular necrosis as a consequence of reduced blood flow and oxygen deprivation. Stimulating cardiomyocyte proliferation and angiogenesis can promote functional recovery after cardiac events. In this study, we explored a novel therapeutic strategy for MI by synthesizing a biomimetic nanovesicle (NV). This biomimetic NVs are composed of exosomes sourced from umbilical cord mesenchymal stem cells, which have been loaded with placental growth factors (PLGF) and surface-engineered with a cardiac-targeting peptide (CHP) through covalent bonding, termed Exo-P-C NVs. With the help of the myocardial targeting effect of homing peptides, NVs can be enriched in the MI site, thus improve cardiac regeneration, reduce fibrosis, stimulate cardiomyocyte proliferation, and promote angiogenesis, ultimately resulted in improved cardiac functional recovery. It was demonstrated that Exo-P-C NVs have the potential to offer novel therapeutic strategies for the improvement of cardiac function and management of myocardial infarction.

Checkpoint Kinase 1 Stimulates Endogenous Cardiomyocyte Renewal and Cardiac Repair by Binding to Pyruvate Kinase Isoform M2 C-Domain and Activating Cardiac Metabolic Reprogramming in a Porcine Model of Myocardial Ischemia/Reperfusion Injury.

The regenerative capacity of the adult mammalian hearts is limited. Numerous studies have explored mechanisms of adult cardiomyocyte cell-cycle withdrawal. This translational study evaluated the effects and underlying mechanism of rhCHK1 (recombinant human checkpoint kinase 1) on the survival and proliferation of cardiomyocyte and myocardial repair after ischemia/reperfusion injury in swine. Intramyocardial injection of rhCHK1 protein (1 mg/kg) encapsulated in hydrogel stimulated cardiomyocyte proliferation and reduced cardiac inflammation response at 3 days after ischemia/reperfusion injury, improved cardiac function and attenuated ventricular remodeling, and reduced the infarct area at 28 days after ischemia/reperfusion injury. Mechanistically, multiomics sequencing analysis demonstrated enrichment of glycolysis and mTOR (mammalian target of rapamycin) pathways after rhCHK1 treatment. Co-Immunoprecipitation (Co-IP) experiments and protein docking prediction showed that CHK1 (checkpoint kinase 1) directly bound to and activated the Serine 37 (S37)and Tyrosine 105 (Y105) sites of PKM2 (pyruvate kinase isoform M2) to promote metabolic reprogramming. We further constructed plasmids that knocked out different CHK1 and PKM2 amino acid domains and transfected them into Human Embryonic Kidney 293T (HEK293T) cells for CO-IP experiments. Results showed that the 1-265 domain of CHK1 directly binds to the 157-400 amino acids of PKM2. Furthermore, hiPSC-CM (human iPS cell-derived cardiomyocyte) invitro and invivo experiments both demonstrated that CHK1 stimulated cardiomyocytes renewal and cardiac repair by activating PKM2 C-domain-mediated cardiac metabolic reprogramming. This study demonstrates that the 1-265 amino acid domain of CHK1 binds to the 157-400 domain of PKM2 and activates PKM2-mediated metabolic reprogramming to promote cardiomyocyte proliferation and myocardial repair after ischemia/reperfusion injury in adult pigs.

Upregulation of LncRNA UCA1 promotes cardiomyocyte proliferation by inhibiting the miR-128/SUZ12/P27 pathway

Enhancing cardiomyocyte proliferation is essential to reverse or slow down the heart failure progression in many cardiovascular diseases such as myocardial infarction (MI). Long non-coding RNAs (lncRNAs) have been reported to regulate cardiomyocyte proliferation. In particular, lncRNA urothelial carcinoma-associated 1 (lncUCA1) played multiple roles in regulating cell cycle progression and cardiovascular diseases, making lncUCA1 a potential target for promoting cardiomyocyte proliferation. However, the role of lncUCA1 in cardiomyocyte proliferation remains unknown. This study aimed at exploring the function and underlying molecular mechanism of lncUCA1 in cardiomyocyte proliferation. Quantitative RT-PCR showed that lncUCA1 expression decreased in postnatal hearts. Gain-and-loss-of-function experiments showed that lncUCA1 positively regulated cardiomyocyte proliferation in vitro and in vivo. The bioinformatics program identified miR-128 as a potential target of lncUCA1, and loss of miR-128 was reported to promote cardiomyocyte proliferation by inhibiting the SUZ12/P27 pathway. Luciferase reporter assay, qRT-PCR, western blotting, and immunostaining experiments further revealed that lncUCA1 acted as a ceRNA of miR-128 to upregulate its target SUZ12 and downregulate P27, thereby increasing cyclin B1, cyclin E, CDK1 and CDK2 expression to promote cardiomyocyte proliferation. In conclusion, upregulation of lncRNA UCA1 promoted cardiomyocyte proliferation by inhibiting the miR-128/SUZ12/P27 pathway. Our results indicated that lncUCA1 might be a new therapeutic target for stimulating cardiomyocyte proliferation.

RETRACTED: The novel antibody fusion protein rhNRG1-HER3i promotes heart regeneration by enhancing NRG1-ERBB4 signaling pathway

Stimulating cardiomyocyte proliferation in the adult heart has emerged as a promising strategy for cardiac regeneration following myocardial infarction (MI). The NRG1-ERBB4 signaling pathway has been implicated in the regulation of cardiomyocyte proliferation. However, the therapeutic potential of recombinant human NRG1 (rhNRG1) has been limited due to the low expression of ERBB4 in adult cardiomyocytes. Here, we investigated whether a fusion protein of rhNRG1 and an ERBB3 inhibitor (rhNRG1-HER3i) could enhance the affinity of NRG1 for ERBB4 and promote adult cardiomyocyte proliferation. In vitro and in vivo experiments were conducted using postnatal day 1 (P1), P7, and adult cardiomyocytes. Western blot analysis was performed to assess the expression and activity of ERBB4. Cardiomyocyte proliferation was evaluated using Ki67 and pH 3 immunostaining, while fibrosis was assessed using Masson staining. Our results indicate that rhNRG1-HER3i, but not rhNRG1, promoted P7 and adult cardiomyocyte proliferation. Furthermore, rhNRG1-HER3i improved cardiac function and reduced cardiac fibrosis in post-MI hearts. Administration of rhNRG1-HER3i inhibited ERBB3 phosphorylation while increasing ERBB4 phosphorylation in adult mouse hearts. Additionally, rhNRG1-HER3i enhanced angiogenesis following MI compared to rhNRG1. In conclusion, our findings suggest that rhNRG1-HER3i is a viable therapeutic approach for promoting adult cardiomyocyte proliferation and treating MI by enhancing NRG1-ERBB4 signaling pathway.

Chemokine CCL2 promotes cardiac regeneration and repair in myocardial infarction mice via activation of the JNK/STAT3 axis.

Stimulation of adult cardiomyocyte proliferation is a promising strategy for treating myocardial infarction (MI). Earlier studies have shown increased CCL2 levels in plasma and cardiac tissue both in MI patients and mouse models. In present study we investigated the role of CCL2 in cardiac regeneration and the underlying mechanisms. MI was induced in adult mice by permanent ligation of the left anterior descending artery, we showed that the serum and cardiac CCL2 levels were significantly increased in MI mice. Intramyocardial injection of recombinant CCL2 (rCCL2, 1 μg) immediately after the surgery significantly promoted cardiomyocyte proliferation, improved survival rate and cardiac function, and diminished scar sizes in post-MI mice. Alongside these beneficial effects, we observed an increased angiogenesis and decreased cardiomyocyte apoptosis in post-MI mice. Conversely, treatment with a selective CCL2 synthesis inhibitor Bindarit (30 μM) suppressed both CCL2 expression and cardiomyocyte proliferation in P1 neonatal rat ventricle myocytes (NRVMs). We demonstrated in NRVMs that the CCL2 stimulated cardiomyocyte proliferation through STAT3 signaling: treatment with rCCL2 (100 ng/mL) significantly increased the phosphorylation levels of STAT3, whereas a STAT3 phosphorylation inhibitor Stattic (30 μM) suppressed rCCL2-induced cardiomyocyte proliferation. In conclusion, this study suggests that CCL2 promotes cardiac regeneration via activation of STAT3 signaling, underscoring its potential as a therapeutic agent for managing MI and associated heart failure.

Inhibition of fatty acid oxidation enables heart regeneration in adult mice.

Postnatal maturation of cardiomyocytes is characterized by a metabolic switch from glycolysis to fatty acid oxidation, chromatin reconfiguration and exit from the cell cycle, instating a barrier for adult heart regeneration1,2. Here, to explore whether metabolic reprogrammingcan overcome this barrier and enable heart regeneration, we abrogate fatty acid oxidation in cardiomyocytes by inactivation of Cpt1b. We find that disablement of fatty acid oxidation in cardiomyocytes improves resistance to hypoxia and stimulates cardiomyocyte proliferation, allowing heart regeneration after ischaemia-reperfusion injury. Metabolic studies reveal profound changes in energy metabolism and accumulation of α-ketoglutarate in Cpt1b-mutant cardiomyocytes, leading to activation of the α-ketoglutarate-dependent lysine demethylase KDM5 (ref. 3). Activated KDM5 demethylates broad H3K4me3 domains in genes that drive cardiomyocyte maturation, lowering theirtranscription levels and shifting cardiomyocytes into a less mature state, thereby promoting proliferation. We conclude that metabolic maturation shapes the epigenetic landscape of cardiomyocytes, creating a roadblock for further cell divisions. Reversal of this process allows repair of damaged hearts.

Insulin-like growth factor type 2 is a better survival marker than insulin-like growth factor type 1 in patients after acute decompensated heart failure.

A decreased insulin-like growth factor 1 (IGF-1) level has been found in heart failure (HF). There are no reports assessing IGF-2 in HF, although in vitro research has shown that IGF-2 stimulates cardiomyocyte proliferation more than IGF-1. The study aim was to compare the IGF-1 and IGF-2 concentrations depending on HF exacerbation and annual survival. Among 75 patients hospitalized due to newly diagnosed or exacerbated HF, the following evaluations were performed: anthropometric measurements, basic laboratory tests, heart echocardiography, and IGF-1 and IGF-2 concentrations. The annual survival was assessed. The participants were divided into NYHA II and NYHA III/IV groups. They did not differ in age, gender, body mass index (BMI), waist-hip ratio, glycated hemoglobin (HbA1c), high-density lipoprotein cholesterol, or triglycerides, but differed in echocardiographic parameters, BNP, total cholesterol (TC) and low-density lipoprotein (LDL) cholesterol levels. Nine (12%) patients died during the 12-month follow-up. There were no differences in IGF-1 between NYHA groups and depending on the BMI, carbohydrate metabolism disorders and annual survival. A significantly lower IGF-2 concentration was found in NYHA III/IV vs. NYHA II: 583.71 (162.35) vs. 676.08 (172.09), p = 0.02, and in those who died: 501.47 (172.89) vs. 645.31 (166.17) nmol/l, p = 0.04. There was a positive correlation between IGF-2 and TC: r = 0.28, p = 0.015 and LDL: r = 0.29, p = 0.011 in the whole group and among patients with BMI ≥ 25 kg/m2: respectively for TC (r = 0.31, p = 0.014) and LDL (r = 0.28, p = 0.028). No IGF-1 correlation was found. Reduced IGF-2 concentration is a better marker of patients with more advanced HF and a higher 1-year death risk than low IGF-1. Its secretion may depend on the cholesterol concentration.

Kynurenine promotes neonatal heart regeneration by stimulating cardiomyocyte proliferation and cardiac angiogenesis

Indoleamine 2,3 dioxygenase-1 (IDO1) catalyzes tryptophan-kynurenine metabolism in many inflammatory and cancer diseases. Of note, acute inflammation that occurs immediately after heart injury is essential for neonatal cardiomyocyte proliferation and heart regeneration. However, the IDO1-catalyzed tryptophan metabolism during heart regeneration is largely unexplored. Here, we find that apical neonatal mouse heart resection surgery led to rapid and consistent increases in cardiac IDO1 expression and kynurenine accumulation. Cardiac deletion of Ido1 gene or chemical inhibition of IDO1 impairs heart regeneration. Mechanistically, elevated kynurenine triggers cardiomyocyte proliferation by activating the cytoplasmic aryl hydrocarbon receptor-SRC-YAP/ERK pathway. In addition, cardiomyocyte-derived kynurenine transports to endothelial cells and stimulates cardiac angiogenesis by promoting aryl hydrocarbon receptor nuclear translocation and enhancing vascular endothelial growth factor A expression. Notably, Ahr deletion prevents indoleamine 2,3 dioxygenase -kynurenine–associated heart regeneration. In summary, increasing indoleamine 2,3 dioxygenase-derived kynurenine level promotes cardiac regeneration by functioning as an endogenous regulator of cardiomyocyte proliferation and cardiac angiogenesis.

Stimulation of Cardiomyocyte Proliferation Is Dependent on Species and Level of Maturation.

The heart is one of the least regenerative organs. This is in large part due to the inability of adult mammalian cardiomyocytes to proliferate and divide. In recent years, a number of small molecules and molecular targets have been identified to stimulate cardiomyocyte proliferation, including p38 inhibition, YAP-Tead activation, fibroblast growth factor 1 and Neuregulin 1. Despite these exciting initial findings, a therapeutic approach to enhance cardiomyocyte proliferation in vivo is still lacking. We hypothesized that a more comprehensive in vitro validation using live-cell imaging and assessment of the proliferative effects on various cardiomyocyte sources might identify the most potent proliferative stimuli. Here, we used previously published stimuli to determine their proliferative effect on cardiomyocytes from different species and isolated from different developmental timepoints. Although all stimuli enhanced DNA synthesis and Histone H3 phosphorylation in neonatal rat ventricular cardiomyocytes to similar degrees, these effects varied substantially in mouse cardiomyocytes and human iPSC-derived cardiomyocytes. Our results highlight p21 inhibition and Yap-Tead activation as potent proliferative strategies to induce cultured cardiomyocyte cell cycle activity across mouse, rat and human cardiomyocytes.

Quantifying Cardiomyocyte Proliferation and Nucleation to Assess Mammalian Cardiac Regeneration.

Neonatal mice display a remarkable ability to regenerate their heart following an injury during the first week of life. A key facet of successful cardiac regeneration is the proliferation of cardiomyocytes to replace the lost cells. Stimulating cardiomyocyte proliferation in the adult heart is a very promising approach to restore cardiac structure and function following injury. Here, we outline our approach to assess cardiomyocyte proliferation following a myocardial injury via the cell cycle markers phospho-histone H3 and Aurora B. We additionally discuss how we assess successful regeneration using wheat germ agglutinin to measure cardiomyocyte size, nuclear staining to quantify cardiomyocyte nucleation, and Trichrome staining to identify myocardial regeneration and scarring in the myocardium.

High-Throughput Screening Platform in Postnatal Heart Cells and Chemical Probe Toolbox to Assess Cardiomyocyte Proliferation.

Restoring lost heart muscle is an attractive goal for cardiovascular regenerative medicine. One appealing strategy is the therapeutic stimulation of cardiomyocyte proliferation, which inter alia remains challenging due to available assay technologies capturing the complex biology. Here, a high-throughput-formatted phenotypic assay platform was established using rodent whole heart-derived cells to preserve the cellular environment of cardiomyocytes. Several readouts allowed the quantification of cycling cardiomyocytes, including a transgenic H2B-mCherry system for unequivocal, automated detection of cardiomyocyte nuclei. A chemical genetics approach revealed pronounced species differences and furnished pan-kinase inhibitors 5 and 36 as potent and robust inducers of endoreplication and acytokinetic mitosis. Combined profiling of the commonly used p38 MAPK inhibitors SB203580 (1), SB239063 (2) and a novel set of skepinone-L (6) derivatives pointed to off-target effects beyond p38 that might be critical for effective cardiomyocyte cytokinesis. Kinome-focused screening eventually furnished TG003 (38) as a novel candidate for stimulating cardiomyocyte proliferation.

Exogenous extracellular matrix proteins decrease cardiac fibroblast activation in stiffening microenvironment through CAPG

Controlling fibrosis is an essential part of regenerating the post-ischemic heart. In the post-ischemic heart, fibroblasts differentiate to myofibroblasts that produce collagen-rich matrix to physically stabilize the infarct area. Infarct models in adult mice result in permanent scarring unlike newborn animals which fully regenerate. Decellularized extracellular matrix (dECM) hydrogels derived from early-aged hearts have been shown to be a transplantable therapy that preserves heart function and stimulates cardiomyocyte proliferation and vascularization. In this study, we investigate the anti-fibrotic effects of injectable dECM hydrogels in a cardiac explant model in the context of age-associated tissue compliance. Treatments with adult and fetal dECM hydrogels were tested for molecular effects on cardiac fibroblast activation and fibrosis. Altered sensitivity of fibroblasts to the mechanosignaling of the remodeling microenvironment was evaluated by manipulating the native extracellular matrix in explants and also with elastomeric substrates in the presence of dECM hydrogels. The injectable fetal dECM hydrogel treatment decreases fibroblast activation and contractility and lowers the stiffness-mediated increases in fibroblast activation observed in stiffened explants. The anti-fibrotic effect of dECM hydrogel is most observable at highest stiffness. Experiments with primary cells on elastomeric substrates with dECM treatment support this phenomenon. Transcriptome analysis indicated that dECM hydrogels affect cytoskeleton related signaling including Macrophage capping protein (CAPG) and Leupaxin (LPXN). CAPG was down-regulated by the fetal dECM hydrogel. LPXN expression was decreased by stiffening the explants; however, this effect was reversed by dECM hydrogel treatment. Pharmacological disruption of cytoskeleton polymerization lowered fibroblast activation and CAPG levels. Knocking down CAPG expression with siRNA inhibited fibroblast activation and collagen deposition. Collectively, fibroblast activation is dependent on cooperative action of extracellular molecular signals and mechanosignaling by cytoskeletal integrity.

A Neonatal Mouse Model for Pressure Overload: Myocardial Response Corresponds to Severity.

The heart regeneration after apical resection and myocardial infarction in neonatal mice has been studied for years. However, the response of neonatal mouse heart under pressure overload is seldom explored. This study aimed to induce pressure overload in neonatal mice through a transverse aortic constriction (TAC) with different-gauge needles so as to investigate the effect of pressure overload on cardiomyocyte proliferation and hypertrophy in these mice. Myocardial hypertrophy was evaluated by echocardiographic, pathological, and molecular analyses. Cardiomyocyte proliferation was detected by immune-staining of phospho-histone H3, Ki67, and 5-bromo-2-deoxyuridine. Mild pressure overload induced with a 30-gauge needle stimulated cardiomyocyte proliferation, adaptive hypertrophy, and angiogenesis. The heart function was not hampered even 21 days after the surgery. Moderate pressure overload induced with a 32-gauge needle led to pathological myocardial hypertrophy, fibrosis, and heart failure 7 days after the surgery. The gene and protein expression levels of markers of hypertrophy and fibrosis increased in 32-gauge TAC group compared with that in sham and 30-gauge TAC groups. The mice barely survived after severe pressure overload induced with a 34-gauge needle. The findings of this study might provide new insights into cardiomyocyte proliferation and hypertrophy in neonatal mice under pressure overload.

Effect of Maternal Panadol Extra Exposure on Embryonic Heart of Rat’s Offspring and Possible Protective Effect of Vitamin D

Introduction: excessive use of paracetamol with caffeine in Panadol extra during pregnancy increase the risk of spontaneous abortion. Vitamin D stimulates cardiomyocyte proliferation and protects against injury. Aim of Work: To investigate the effect of maternal exposure to large doses of Panadol extra on the development of the embryonic hearts of rat’s offspring and possible protective effects of vitamin D.Material and Methods: Forty-five albino rats; 30 adult females and 15 adult males were used in this study. The pregnant rats were divided into Group I as a control group, Group II (treated group; Panadol extra) received the maximum daily dose (4000mg paracetamol/ 520 mg caffeine) / kg/ day and Group III (protective group; Panadol extra + vitamin D) received previously mentioned dose of Panadol extra plus vitamin D at a dose of 400 IU/kg/day. All the treatments were given by oral gavage from the 1st day of pregnancy until day 19. Pregnant rats from all groups were sacrificed on day 19 of pregnancy and their pups were extracted & weighted, then sacrificed. Blood samples were taken for biochemical assessment. Sections from the hearts were stained (H&E, Eosin & Mallory’s trichrome stain), Immunohistochemical (alpha-smooth muscles) for histopathological and morphometric studies.Results: group (II) revealed changes of blood gases as PH, increase in PCO2, decrease PO2, high lipid profile, and increase Creatinine kinase-MB. There were different histopathological changes as cardiomyocyte damage, necrosis, and inflammation with increased fibrosis by Mallory stain. The morphometric results revealed an enlarged size of cardiac cells, decreased proliferation, and maturation. All these results were greatly improved in the group (III) when concomitant administration of vitamin D in the last protective group. Conclusion: prolonged and excessive maternal use of Panadol extra greatly affect heart development. Vitamin D has a potential effect on cardiac regeneration and development.

DUSP5 expression in left ventricular cardiomyocytes of young hearts regulates thyroid hormone (T3)-induced proliferative ERK1/2 signaling

Cardiomyocytes of newborn mice proliferate after injury or exposure to growth factors. However, these responses are diminished after postnatal day-6 (P6), representing a barrier to building new cardiac muscle in adults. We have previously shown that exogenous thyroid hormone (T3) stimulates cardiomyocyte proliferation in P2 cardiomyocytes, by activating insulin-like growth factor-1 receptor (IGF-1R)-mediated ERK1/2 signaling. But whether exogenous T3 functions as a mitogen in post-P6 murine hearts is not known. Here, we show that exogenous T3 increases the cardiomyocyte endowment of P8 hearts, but the proliferative response is confined to cardiomyocytes of the left ventricular (LV) apex. Exogenous T3 stimulates proliferative ERK1/2 signaling in apical cardiomyocytes, but not in those of the LV base, which is inhibited by expression of the nuclear phospho-ERK1/2-specific dual-specificity phosphatase, DUSP5. Developmentally, between P7 and P14, DUSP5 expression increases in the myocardium from the LV base to its apex; after this period, it is uniformly expressed throughout the LV. In young adult hearts, exogenous T3 increases cardiomyocyte numbers after DUSP5 depletion, which might be useful for eliciting cardiac regeneration.

Microenvironment stiffness requires decellularized cardiac extracellular matrix to promote heart regeneration in the neonatal mouse heart

The transient period of regeneration potential in the postnatal heart suggests molecular changes with maturation influence the cardiac response to damage. We have previously demonstrated that injury and exercise can stimulate cardiomyocyte proliferation in the adult heart suggesting a sensitivity to exogenous signals. Here, we consider whether exogenous fetal ECM and mechanically unloading interstitial matrix can drive regeneration after myocardial infarction (MI) surgery in low-regenerative hearts of day5 mice. Compared to controls, exogenous fetal ECM increases cardiac function and lowers fibrosis at 3 weeks post-injury and this effect can be augmented by softening heart tissue. In vitro experiments support a mechano-sensitivity to exogenous ECM signaling. We tested potential mechanisms and observed that fetal ECM increases nuclear YAP localization which could be enhanced by pharmacological stabilization of the cytoskeleton. Blocking YAP expression lowered fetal ECM effects though not completely. Lastly we observed mechanically unloading heart interstitial matrix increased agrin expression, an extracellular node in the YAP signaling pathway. Collectively, these data support a combined effect of exogenous factors and mechanical activity in altering agrin expression, cytoskeletal remodeling, and YAP signaling in driving cardiomyocyte cell cycle activity and regeneration in postnatal non-regenerative mice. Statement of SignificanceWith the purpose of developing regenerative strategies, we investigate the influence of the local niche on the cardiac injury response. We conclude tissue stiffness, as anticipated in aging or disease, impairs regenerative therapeutics. Most novel, mechanical unloading facilitates enhanced cardiac regeneration only after cells are pushed into a permissive state by fetal biomolecules. Specifically, mechanical unloading appears to increase extracellular agrin expression that amplifies fetal-stimulation of nuclear YAP signaling which correlates with observed increases of cell cycle activity in cardiomyocytes. The results further suggest the cytoskeleton is critical to this interaction between mechanical unloading and independently actived YAP signaling. Using animal models, tissue explants, and cells, this work indicates that local mechanical stimuli can augment proliferating-permissive cardiomyocytes in the natural cardiac niche.