bluuebunny/crossref_metadata_2025 huggingface dataset:
Sarcomere disassembly and transfection efficiency in proliferating human iPSC-derived cardiomyocytes
Authors: Yuan Q , Maas R , Brouwer E , Sluijter J , Van Der Velden J , Buikema J A N W
Published: November 2023

Introduction

Proper contraction of cardiomyocytes (CMs) is dictated by the architecture of the sarcomeres and appears together with polyploidy inversely correlated with limited self-renewal and regeneration of the adult heart. This limited regenerative potential correlates with the decrease in mononuclear diploid cells in adult versus neonatal myocardium. Previous animal studies have shown that the sarcomeres are dissembled during proliferation of neonatal CMs. However, little is known about sarcomere assemblage during human CMs nuclear and/or cell division, which would be relevant information for strategies to boost endogenous heart repair.

Purpose

In this study we used the human induced pluripotent stem cell (hiPSC) model to investigate sarcomere assemblage during mitosis, followed by cytokinesis, multinucleation and/or self-duplication in massively expanding hiPSC-CMs. Furthermore, we examined whether CMs undergoing nuclear and/or cellular duplication would be more susceptible for genomic editing than non-proliferative hiPSC-CMs.

Methods

hiPSC-CMs were massively expanded using our previously described CHIR99021 small molecular treatment and cell-cell contact removal. We then performed immunofluorescence staining with cardiac Troponin T (cTnT) and Ki67 to follow sarcomere organization during cell cycle activity. Next, we performed Time-Lapse imaging in a hiPSC-CM culture system to follow the sequence of cellular and nuclear divisions in hiPSC-CMs. RNA sequencing lookup was performed for proliferation, sarcomere and molecular pathway-related genes. Efficiency of non-viral vector-based fluorescence (mCherry) expression and transfection efficiency was done by flow cytometry analysis and Time-Lapse imaging.

Results

We observed that both mononuclear and binuclear hiPSC-CMs give rise to mononuclear daughter cells or binuclear progeny. Within this source of highly proliferative hiPSC-CMs, treated with CHIR99021 small molecule, we found that Wnt and Hippo signaling were more present when compared to metabolic matured and non-proliferative hiPSC-CMs and adult human heart tissue. Moreover, we found that CHIR99021 treatment increased the efficiency of non-viral vector incorporation in high-proliferative hiPSC-CMs, in which fluorescent transgene expression became present after completion of chromosomal segregation (M-phase).

Conclusion

This study demonstrates that there is a complex landscape in proliferation, multinucleation and self-duplication of human CMs. Moreover, enhanced incorporation of alien non-viral vectors is related to Wnt activation and cell cycle activity of hiPSC-CMs. Altogether, our findings provide insights in duplication of human CMs and tools for molecular gene studies.
Single-cell transcriptomics analysis reveals Cilp+Cd200+ fibroblasts promote mesenchymal endothelial transition (MEndoT) in cardiac injury
Authors: Lin Y , Cai W , Dong W , Wen D , Zeng C , Guan H , Shentu W
Published: November 2023

Background

Cardiac fibroblasts are considered to be highly heterogeneous mesenchymal cells after a cardiac injury. Understanding the heterogeneity of cardiac fibroblasts is of fundamental importance for fibroblast-targeted strategies to treat patients with heart disease. Advances in the single-cell RNA-sequencing (scRNA-Seq) technology have provided a possibility to elaborate the heterogeneity of cells in more depth. However, currently, most studies on scRNA-Seq of cardiac fibroblasts are performed using non-myocyte cells isolated at a certain point in time, which include a very complex, interdependent community of interstitial cells. scRNA-Seq based on lineage-tracing of cardiac fibroblasts is essential for a deeper understanding of cellular heterogeneity of specific cell type populations, which can reveal how a pre-existing cardiac fibroblast population responds to cardiac injury. Our previous studies demonstrated that a subset of cardiac fibroblasts can adopt the endothelial cell phenotype by undergoing mesenchymal–endothelial transition (MEndoT) and contributes to cardiac repair by revascularizing the damaged myocardium.

Purpose

To decipher which subgroup of the preexisting cardiac fibroblasts involved in MEndoT respond to cardiac injury.

Methods and Results

For lineage-tracing-based studies, Col1a2 Cre ERT: R26R td-Tomato transgenic mice were generated for reliable cardiac fibroblast tracking. 10X scRNA-Seq was performed to analyze the diversity of prelabeled cardiac fibroblasts after sham or transverse aortic constriction injury. Using the Seurat R package, the td-Tomato labeled cells were further clustered into 11 distinct populations. In addition, the cell trajectory assay shows both Cilp and Cd200 positive (Cilp+/Cd200+) cluster located in the early state and showing the phenotype of mesenchymal stem cells, indicating that this clusters have high diversity with regard to differentiation and directly contribute to angiogenesis. Besides, we isolated Cilp+/Cd200+ cardiac fibroblasts from wild-type mice by flow sorting and performed matrigel tube formation and acetylated low-density lipoprotein (Ac-LDL) uptake to measure the potential of MEndoT. Interestingly, compared with Cilp-/Cd200- cardiac fibroblasts, both matrigel tube formation and Ac-LDL uptake of the Cilp+/Cd200+ group were significantly increased (∼1.3 fold higher in matrigel tube formation, P<0.05;∼3.3 fold higher in Ac-LDL uptake, P<0.05).

Conclusion

Our findings indicated that Cilp+/Cd200+ is the main group of cardiac fibroblasts involved in MEndoT and plays a major role in the organization of the extracellular matrix and cell differentiation. This study provided new therapeutic strategies for cardiac hypertrophy by accurately regulating the cardiac fibroblasts.
HIF-1 regulates common bidirectional gene expression changes during hypoxia and iron deficiency in human induced pluripotent stem cell derived cardiomyocytes
Authors: Papadopoulou C , Reinhold J , Price J L , Hoole S , Smith J G , Miska E
Published: November 2023

Background

Heart failure regardless of cause is characterised by extensive transcriptional reprogramming and a switch to foetal developmental programmes via unknown mechanisms (1-3). An additional factor that adversely impacts heart failure patients is iron deficiency (ID)(4). ID is also associated with worse prognosis in acute coronary syndromes (5). However, the mechanisms by which ID interferes with clinical outcomes in heart disease remain incompletely understood.

Purpose

The purpose of this study was to investigate gene expression changes in human cardiomyocytes during hypoxia and ID, two important stressors in the context of ischaemia and heart failure.

Methods

In this study, we use human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) derived through a protocol that promotes left ventricle cardiomyocyte differentiation (6). Cells were harvested at different stages of differentiation (day 6 and day 32) and mature (day 32) hiPSC-CMs were treated with 30 μM deferoxamine to induce ID either under normoxic or hypoxic conditions with 1% O2. hiPSC contraction was assessed using musclemotion software and metabolism through use of a Seahorse extracellular flux analyser. RNA was isolated using standard isolation kits and RNA-seq (n=5) was performed. Data was analysed for gene expression by alignment with the human hg19 reference genome using the software STAR. A log2 fold change of 1 and P value of <0.0001 were considered significant.

Results

We show that both hypoxic and ID hiPSC-CMs demonstrate impaired contractility and reduced oxygen consumption, features of cardiomyocytes from failing hearts. Importantly, hiPSC-CMs exposed to hypoxia, ID or the combination of the two exhibit bidirectional gene expression changes with significant overlap (Figure 1). Focusing on the commonly affected gene set, it becomes evident that hypoxia & ID work via similar mechanisms involving activation of the HIF-1 pathway, that in turn orchestrates the metabolic switch to glycolysis and glutamine oxidation similar to foetal cells. Overall, hypoxia promotes a shift towards immature gene programmes, with 45% of dysregulated genes in hypoxia mimicking expression patterns of less mature cells. Interestingly changes in long non-coding RNA (lncRNA) profiles in hypoxia and ID mimic gene expression changes in these conditions (figure 2). Although restoring oxygen levels in hypoxic hiPSC-CMs rescues the hypoxic transcriptional signature, hiPSC-CMs previously exposed to hypoxia can use glycolytic pathways more efficiently upon further exposures to hypoxia.

Conclusion

These results shed light into the mechanisms ID impacts heart disease patients and are suggestive that HIF-1 is a key player in orchestrating the gene switch in ID and hypoxia.
Impact of microtubule detyrosination on subcellular distribution of Nav1.5 in mdx cardiomyocytes
Authors: Casini S , Nasilli G , De Waal T , Marchal G A , Rothenberg E , Delmar M , Remme C A
Published: November 2023

Background

Subcellular trafficking and distribution of the cardiac sodium channel Nav1.5 in cardiomyocytes is dependent on the microtubule network as well as Nav1.5 interacting proteins. Duchenne muscular dystrophy (DMD) is associated with loss of dystrophin, a Nav1.5-interacting protein localized at the lateral membrane of cardiomyocytes, leading to sodium current (INa) reduction in this microdomain. Cardiomyocytes of DMD (mdx) mice also display increased microtubule detyrosination, but its impact on INa and Nav1.5 distribution is unclear.

Purpose

To investigate the effect of reducing microtubule detyrosination by the compound parthenolide (PTL) on INa and subcellular Nav1.5 distribution in mdx cardiomyocytes (CMs).

Methods and Results

Isolated ventricular CMs from wild type (WT) and mdx (DMD) mice were incubated with either 10 µM PTL or DMSO for 3-5 hours. INa properties were assessed using the patch-clamp technique, confocal microscopy was used to investigate microtubule detyrosination, and stochastic optical reconstruction microscopy (STORM) was employed to assess Nav1.5 cluster density and size, as well as Nav1.5 cluster distribution (crest vs groove) at the cardiomyocyte lateral membrane. Compared to WT. mdx CMs displayed increased levels of detyrosinated tubulin and decreased whole-cell INa. Incubation with PTL decreased the fraction of detyrosinated tubulin and significantly increased whole-cell INa magnitude in mdx CMs, but had no effect in WT CMs. No effect of PTL on INa gating properties were observed, indicating that it increased INa by enhancing Nav1.5 membrane trafficking. Indeed, STORM analysis showed that PTL increased Nav1.5 cluster density at both the lateral membrane and intercalated disc region in mdx CMs while it had no effect on WT CMs. Furthermore, Nav1.5-α-actinin cluster distance analysis showed that PTL increased Nav1.5 clusters specifically at the crest of the lateral membrane in mdx CMs.

Conclusions

In mdx CMs, reduction of MT detyrosination rescues INa density and increases Nav1.5 cluster density both at the intercalated disc and lateral membrane. Hence, in addition to loss of the Nav1.5-interacting protein dystrophin, MT remodelling contributes to INa reduction in DMD. The PTL-induced increase in Nav1.5 at the crest of the lateral membrane of mdx CMs is likely due to a dystrophin-independent mechanism since dystrophin is normally absent from this microdomain. Overall, our findings identify MT detyrosination as a potential therapeutic target for modulating INa and subcellular Nav1.5 distribution during pathophysiological conditions.
Regulation of BACH1 expression by hemin improves cardiac function and revascularisation in a mouse model of myocardial infarction
Authors: Slater S , Alvino V , Avolio E , Katare R , Madeddu P
Published: November 2023

Therapies to induce mature neovascularization formation and moderate oxidative stress after myocardial infarction (MI) are urgently needed. The BACH1 transcription factor is known as a repressor of genes involved in the antioxidant response (1). We have demonstrated BACH1 can regulate neovascularization through modulation of pro-angiogenic genes, particularly angiopoietin-1 (2).

We hypothesise expression of BACH1 after MI contributes to suppression of genes which promote reparative angiogenesis. We propose treatment with hemin (a therapeutic compound known to degrade BACH1) will prevent BACH1 repression of target genes, leading to increased expression of proangiogenic factors and increased neovascularisation and cardiac repair in a mouse MI model.

MI was induced in 8-week old C57/Bl6 male mice by permanent ligation of the left anterior descending coronary artery, n=10-14. Expression of Bach1 and the antioxidant Hmox1 in heart tissue was determined by Western blot at day 3 and 10 post MI, whilst a second group were randomized to receive vehicle, 12.5, 25 or 50mg/kg hemin intraperitoneally every 3 days until 28 days post-MI. On day 28, following echocardiography, mice were euthanised and ventricular tissue collected for histological analysis. Data was analysed by 2-way ANOVA with post-test multiple comparisons.

Compared to sham mice, Bach1 expression significantly increased at both day 3 and 10 post MI (P<0.0001 and P<0.01 respectively). Bach1 expression at day 10 was significantly lower compared to day 3, suggesting a transient effect (P<0.01). Hmox1 expression was significantly decreased at both timepoints (both P<0.001 vs. sham) suggesting in the initial period after MI the antioxidant and angiogenic responses are suboptimal. Treatment of mice with 25mg/kg hemin for 28 days post MI caused significant increases in capillary and arteriole density in the peri-infarct zone (P<0.0002 and P<0.0008 vs. vehicle respectively). There was no effect on capillary or arteriole density in the remote zone. In vehicle-treated mice there was thinning of the left ventricular end-diastolic anterior wall thickness (LVAW) corresponding to the infarct area. Interestingly, a significant increase in the thickness of the LVAW was observed after 25mg/kg hemin treatment (P<0.0001 vs. vehicle), whilst LV internal diameter significantly decreased (P<0.0001 vs. vehicle). Ejection fraction (P<0.0001), fractional shortening (P<0.0009) and stroke volume (P<0.0001) all significantly increased in mice treated with hemin compared to vehicle.

Treatment of mice with 25mg/kg hemin for 28 days post MI was sufficient to induce significantly increased capillary and arteriole density in the peri-infarct region. We also observed hemin exerted benefits on volumetric and contractility indexes. This data suggests hemin has potential to be used therapeutically post MI to improve revascularisation and cardiac function.
Paracrine cardioprotective signaling by red blood cells through hypoxia-mediated activation of soluble guanylate cyclase and release of cyclic guanosine monophosphate
Authors: Yang J N , Sundqvist M , Zheng X , Jiao T , Collado A , Mahdi A , Tengbom J , Mergia E , Zhou Z , Carlstrom M , Akaike T , Cortese-Krott M , Weitzberg E , Lundberg J O , Pernow J
Published: November 2023

Background

Red blood cells (RBCs) are known to regulate cardiovascular function under hypoxic conditions and to mediate cardioprotection via nitric oxide (NO)-like bioactivity. However, the molecular signalling behind this effect and the identity of any mediator released by the RBCs remain unknown. Previous studies revealed that NO activates soluble guanylate cyclase (sGC) and increases formation of the second messenger cyclic guanosine monophosphate (cGMP) in RBCs. The functional role of this signalling in RBCs during hypoxia/ischemia is still unclear.

Aim

To determine the functional role of the NO-sGC-cGMP signalling pathway in RBCs during hypoxia and myocardial ischemia.

Method

RBCs collected from wild-type and sGC knockout mice were exposed to 1% hypoxia or normoxia for 1 h. The RBCs or the extracellular supernatant were then administered to isolated Langendorff-perfused mouse hearts subjected to 40 min global ischemia and 60 min reperfusion. Afterwards, left ventricular developed pressure and infarct size were determined. Additional groups of mice and patients with mild hypertension were given oral nitrate (10mM in their drinking water) before the collection of RBCs to further increase NO signalling.

Result

Administration of RBCs or the extracellular supernatant collected from wild-type mouse RBCs exposed to hypoxia to isolated hearts subjected to ischemia-reperfusion improved post-ischemic cardiac function and reduced infarct size (Fig. 1A and B). By contrast, supernatant collected from hypoxic RBCs of sGC knockout mice failed to induce cardioprotection (Fig. 1C). The cardioprotection induced by hypoxic RBCs of wild-type mice was blocked by MK571, an inhibitor of cyclic nucleotide transport (Fig. 1D). Hypoxia increased extracellular export of hate cGMP from mouse RBCs (Fig. 1E), and exogenous cGMP resulted in similar cardioprotection induced by the supernatant. The cardioprotective effect of RBCs was blocked by an inhibitor of cardiac cGMP-dependent protein kinase G (PKG) (Fig. 1F) and was associated with increased cardiac PKG-dependent phosphorylation of vasodilator-stimulated phosphoprotein. Oral administration of nitrate to mice for 4 weeks to increase NO bioactivity further enhanced the cardioprotective effect of hypoxic RBCs. RBCs collected from patients randomized to a 5-week nitrate-rich diet induced cardioprotection in the isolated rat heart via an effect dependent on sGC activation in the RBCs (Fig 2).

Conclusion

RBCs generate and export cGMP as a physiological response to hypoxia mediating cardioprotection via a paracrine effect. This effect can be further augmented by a simple dietary intervention with nitrate suggesting preventive and therapeutic opportunities in ischemic heart disease.Figure 1Figure 2
Mitochondrial succinate transport as a novel therapeutic target for cardiac ischemia/reperfusion injury
Authors: Prag H A , Abe J , Pala L , Schanz S , Caldwell S T , Murphy M P , Hartley R C , Krieg T
Published: November 2023

Background

The accumulation of the mitochondrial metabolite succinate during ischemia and its rapid oxidation by succinate dehydrogenase (SDH) upon reperfusion drives ischemia/reperfusion injury (IRI) in myocardial infarction. Succinate oxidation leads to the production of reactive oxygen species (ROS) by reverse electron transport (RET) at mitochondrial complex I, orchestrating a cascade of events leading to cardiomyocyte death. Modulating succinate metabolism, such as SDH inhibition, is a promising therapeutic strategy. During ischemia, the mitochondria produced succinate is transported into the cytosol, enabling the accumulation of a large succinate pool. On reperfusion, the succinate is transported back into mitochondria and oxidised by SDH, thus we hypothesised that targeting succinate mitochondrial transport could be a novel therapeutic target to prevent cardiac IRI.

Purpose

To assess the role of succinate mitochondrial transport as a novel therapeutic target to prevent cardiac IRI.

Methods

Isolated heart mitochondria were used to assess mitochondrial respiration by Oroboros, RET-ROS production by Amplex Red and butylmalonate delivery. Cultured H9C2 myoblasts and isolated primary adult cardiomyocytes from C57BL/6J mice were used to assess butylmalonate uptake and delivery from esters, as well as their impact on succinate accumulation and oxidation in vitro. LAD ligation and reperfusion in C57BL/6J mice was used as an in vivo MI model. Metabolite levels were measured by LC-MS/MS.

Results

Butylmalonate, an inhibitor of the mitochondrial dicarboxylate carrier (DIC), blocks succinate-dependent respiration and RET-ROS production in isolated mitochondria. However, butylmalonate entry into cells was inefficient and does not impact infarct size after IRI in vivo. We developed novel butylmalonate esters to improve butylmalonate delivery to cardiomyocytes in vivo. We found that diacetoxymethyl butylmalonate (DAB) effectively delivered butylmalonate and could prevent succinate accumulation and oxidation in vitro. DAB could also prevent succinate-mediated respiration and RET-ROS at lower concentrations than butylmalonate alone. Finally, DAB reduced acute infarct size in vivo when administered at reperfusion.

Conclusions

Blocking succinate transport back into mitochondria at reperfusion prevents the damage in IRI by preventing the oxidation of succinate by SDH and downstream RET-ROS production. Therefore, targeting the mitochondrial transport of succinate may be a promising novel strategy in preventing cardiac IRI.
The prolyl-hydroxylase inhibitior Roxadustat upregulates HIF-1alpha and is associated with improved cardiac repair after myocardial infarction in mice
Authors: Staggl S , Messner M , Ghadge S K , Maurer T , Wimmer A , Seiringer H , Poelzl G , Bauer A , Zaruba M M
Published: November 2023

Introduction

Activation of the CXCL12/CXCR4-axis is known to aid myocardial repair by attraction of reparative CXCR4+ cells through ischemia triggered hypoxia-inducible factor-1α (HIF-1α) activation of CXCL12 (1). To prolongate upregulation of CXCL12 we aimed to stabilize HIF-1α by administration of Roxadustat, the only prolyl hydroxylase inhibitor (PHI) clinically approved by the EMA with the purpose to augment myocardial remodelling and improve LV function.

Methods

To analyse the effects of HIF-1α mediated CXCL12 upregulation in vivo (BM & heart), optimal doses (50mg/kg i.p., every 72h) of the PHI Roxadusat (FG-4592) were administered to Bl6/J mice. CXCL12 and CXCR4 expression was analysed in-vitro in human umbilical vascular endothelial cells (HUVEC) after administration of FG-4592 (20µM to 1000µM) at different time points (1 to 24 hrs). Western Blots were performed and effects on HIF-1α -signalling were quantified by densitometry. We induced MI in BL6/J mice at the age of 8-10 weeks by LAD-ligation. Two groups (n=10) were treated with Roxadustat or saline for 28 days before assessment of cardiac function by echocardiography and evaluation by histology. Two other groups of n=5 mice were treated with Roxadustat or saline for one week and analysed regarding apoptotic-index by automated cell count in DAPI & Tunel stained sections.

Results

We were able to show a significant upregulation of HIF-1α after exposure of 50-500 μM Roxadustat for 4-24 hours in-vitro (HUVEC) and in-vivo (murine BL6/J hearts). Moreover, administration of 500 µM Roxadustat induced marked activation of CXCR4 and CXCL12, respectively. Infarct size (19.07%±7.86 vs. 24.14%±12,05; n= 10/8; p = 0.297), as well as LV-wall thickness in the infarcted myocardium (788.27µm±240.10 vs. 651.40µm±231.47; n=10/8; p = 0.240) revealed no significant differences 28 days after MI. Automated cell counting in the borderzone revealed a lowered apoptotic-index of Roxadustat treated animals (3.7%±1.1 vs. 6.3%±2.3; p = 0.071). Echocardiographic analyses showed a significantly preserved ejection fraction (EF) (46.1%±6.0 vs. 30.5%±9.3; n=10/9; p<0.001), reduced diastolic (4.18±0.32 vs. 4.77±0.40; p=0.002) and systolic (3.23±0.31 vs. 3.95±0.43; p<0.001) LV diameters in Roxadustat treated mice 28 days after MI compared to saline treated controls.

Conclusion

The clinically approved PHI Roxadustat revealed increased expression of HIF-1α associated with upregulation of CXCR4 and CXCL12. Administration of Roxadustat after MI revealed attenuated infarct remodelling and functional improvement showing its potential in ischemic heart disease.
The clinically active PARP inhibitor olaparib ameliorates doxorubicin-induced cardiotoxicity in both in vitro and in vivo model
Authors: Chen D , Croft A , Haw T J , Kelly C , Leong A , Sverdlov A , Ngo D
Published: November 2023

Introduction

Inhibition of poly (ADP-ribose) polymerase (PARP) has been shown to be cardioprotective in acute and chronic myocardial injury. Additionally, PARP inhibitor, Olaparib, is a clinically effective anti-cancer agent. Activation of PARP pathway has been suggested to be involved in cardiotoxicity arising from Doxorubicin (DOX, one of the most used anticancer drugs). It is thus, entirely possible that PARP inhibition could be a novel pathway to prevent DOX-induced cardiotoxicity (DIC).

Purpose

To determine the cardioprotective effects of Olaparib in preventing DOX-induced cardiotoxicity in in vitro and in vivo models.

Methods

In vitro: Human cardiomyocytes (HCMs) were treated with DOX at 1uM (EC50) +/- various doses (15.6nM, 1uM, 80uM, 100uM and 150uM) of Olaparib at 72hrs. Cell viability was assessed via CellTiter-Glo®. The mRNA expressions of treated HCMs were performed by qPCR. In vivo: Female C57BL/6 mice (6-8 weeks old) were administered: A) DOX at 5mg/kg/week for 6 weeks via IP injections, B) vehicle of 0.9% saline and 5% DMSO in PBS, C) Olaparib at 50mg/kg was administered 3 times/week, D) Olaparib at 50mg/kg was administered 3 times/week, followed by DOX treatments. Cardiac function was assessed by echocardiography at baseline and end of 6 weeks’ treatment.

Results

For the in vitro model, Doxorubicin induced marked reduction in HCMs cell viability. Concomitant Olaparib treatment at higher doses (80uM, 100uM, 150uM) significantly preserved DOX-induced cell viability after 72 hours of treatment. Molecular analysis in HCMs showed DOX-induced upregulation of mRNA gene expressions for apoptosis: Casp3, DNA damage: BBC3, and cardiac remodelling: TGF-β; all of which were reversed by Olaparib. In vivo: we found significant reduction of left ventricular function and increased ventricular wall thickness in DOX treated group after 6 weeks’ treatment compare to baseline [LVEF%: 52.4±1.3 vs.56.5±2.0, P=0.02; FS%:16.7±1.4 vs. 20.1±1.3, P=0.03; Total Wall thickness (mm):2.1±0.1 vs. 1.9±0.1, P<0.05]; whereas the mice pre-treated with Olaparib in combination with DOX showed preservation of cardiac functions between endpoint and baseline.

Conclusion

Olaparib protected HCMs against DIC and improved cardiac function in DIC mice model. Our findings indicate that Olaparib could provide cardioprotection in DOX-induced cardiotoxicity.
The effect of cardiosphere-derived cell extracellular vesicles embedded in cardiac matrix hydrogel on cardiac remodeling in a porcine model of ischemic cardiomyopathy
Authors: Casado Mayo L , Canadilla V , Aguirre Acosta A , Gomez-Cid L , Sanz R , Ocampo A , Crisostomo V , Fernandez-Santos M E , Gonzalez Mansilla A , Bermejo J , Fernandez-Aviles F , Grigorian L
Published: November 2023

Background

Numerous preclinical studies have confirmed that stem cell-derived extracellular vesicles (EVs) can prevent from negative cardiac remodeling after an ischemic injury, reduce the scar and preserve heart function. It is believed that by increasing the engraftment of the therapeutic product at the target site may bust its efficacy. The use of bioengineering materials such as hydrogels as delivery scaffolds for intramyocardial EVs administration may constitute a minimally invasive therapeutic strategy that improves the efficacy of EVs by increasing their permanence in the tissue.

Objective

To evaluate the therapeutic efficacy of a new regenerative product constituted by the combination of EVs secreted by cardiosphere-derived cell (CDC-EVs) embedded in a hydrogel-based scaffold made from decellularized cardiac matrix (HDM).

Methods

Twelve pigs (50% females) with induced ischemic cardiomyopathy were randomly assigned to receive: phosphate-buffer saline (PBS) as control group (n=4), CDC-EVs alone (n=4) and combination of CDC-EVs and HDM (EV-HDM, n=4). Treatment was administered via percutaneous intramyocardial injection in the scar border zone 1-month after myocardial infarction with NOGA system. Animals were followed-up another month and sacrificed. Hearts were excised for histological analysis performed according to the cardiac area: scar (anterior wall), scar border zone and remote zone (inferior wall). Functional evaluation was performed with cardiac magnetic resonance imaging immediately before- and 1-month after treatment.

Results

Cardiomyocytes size was significantly lower in EV-HDM group compared to control PBS both at scar border (4660± 2088 vs. 5254±2294 pixels; p=0.02) and remote areas (3381±1259 vs. 4237±1256 pixels; p<0.001), while no changes were observed in CDC-EV alone group compared to controls. Interstitial fibrosis was significantly lower in EV-HDM group compared to PBS controls both at scar epicardial side (34% vs. 44%; p=0.01) and remote areas (16% vs. 20%; p<0.01), similarly to CDC-EV vs. PBS (34% at the scar; p<0.01 and 17% at the remote zone; p=0.01). While left ventricular ejection fraction (LVEF) tended to decrease in PBS group from 36% to 32%, it showed an opposite trend in CDC-EV (from 35 % to 38%) and EV-HDM (from 31% to 37%).

Conclusion

The combined product of EV-HDM reduce cardiac fibrosis and hypertrophy, favorably affecting the remodeling process and may thus increase LVEF in ischemic heart disease. The results of our study fail to clearly demonstrate the superiority of EV-HDM over CDC-EVs alone, although it may exist.
TCF21 gene and cardiovascular events in a coronary population
Authors: Sa D , Mendonca M I , Santos M , Temtem M , Sousa F , Henriques E , Borges S , Freitas S , Rodrigues M , Guerra G , Drumond A , Sousa A C , Palma Dos Reis R
Published: November 2023

Background

Recent research showed that TCF21 expression in precursor cells that migrate into the disease lesions contributes to the fibrous cap, stabilizing the lesions and preventing heart attacks. Targeted deletion of the transcription factor encoded by the TCF21 was associated with vascular smooth muscle cell disruption impairing the fibrous cap structure, increasing cardiovascular(CV) disease risk. Its association with CV events is unknown.

Purpose

Analyze the TCF21 rs12190287 variant G>C evaluating its association with atherosclerosis severity and the appearance of cardiovascular (CV) events.

Methods

We performed a prospective study with 1,716 coronary artery disease (CAD) patients (mean age 53.3±7.8 years). TCF21 rs12190287 G>C was genotyped by TaqMan genotyping assay (Applied Biosystems) in all patients. The severity of CAD was graded according to the number of obstructed coronary arteries with at least 70% narrowed lumen. Chi-squared tests were used to determine differences in CAD severity by genotype. We performed a first multivariate logistic regression analysis to assess the independent risk variables associated with CAD severity. After that, we presented a second multivariate Cox regression to evaluate independent variables related to CV events. Kaplan Meier estimated the survival curves.

Results

48.0% of the CC genotype carriers were associated with more than two obstructed coronary arteries compared to the GG wild genotype, which was associated with only 9.2% (p=0.002). Multivariate analysis (logistic regression) showed that the CC genotype had a high risk of CAD severity (OR=2.95; p=0.001) than GG. After Cox regression analysis, which takes into account the time to the first event, the CC genotype remained in the equation with an HR of 1.38; 95%CI 1.02-1.88; p=0.040. The survival events free at ten years is 49.1% in the CAD patients with the GG genotype, then drops to 39.6 % with the CC risk genotype.

Conclusion

This work shows that the GG wild genotype protects against CAD severity. In contrast, the CC genotype is associated with an increased risk of CAD severity. The patients with this allelic variant had a worse survival event free. Future research with the implementation of epigenetic and genetic therapeutics targeted to deleterious genes will allow the eradication of coronary heart disease worldwide.
Diastolic dysfunction is associated with cardiac small vessel disease in ovariectomized females but not in males
Authors: Cornuault L , Grouthier V , Rouault P , Bourguignon C , Foussard N , Chapouly C , Gadeau A P , Couffinhal T , Renault M A
Published: November 2023

Introduction

Coronary microvascular disease has been proposed to be responsible for heart failure with preserved ejection fraction (HFpEF) about 10 years ago. However, to date the role and phenotype of the coronary microvasculature has still been poorly considered and investigated in animal models of HFpEF.

Objective

To characterize the phenotype of the coronary microvasculature in male and female mice with diastolic dysfunction.

Method

We assessed cardiac function and characterized the coronary microvasculature in two mouse models of HFpEF: mice fed with a high fat diet (HFD) + L-NAME regimen and Leptin receptor deficient (Leprdb/db) mice. Notably, our study was done in males, females and ovariectomized (OVX) female mice in order to search for possible sexual dimorphisms.

Results

Upon a HFD + L-NAME regimen, both males and OVX females but not non OVX females develop diastolic dysfunction attested by an increased end diastolic pressure. In Leprdb/db mice, both Male and non OVX female mice develop diastolic dysfunction. Notably female mice have reduced estradiol and progesterone level mice mimicking ovariectomy.

We found that both OVX and non OVX females but not males display increased endothelial activation attested by increased ICAM1 expression, endothelium leakage attested by increased Fibrinogen and IgG extravasation and decreased arteriole diameter suggesting vasoconstriction. The same results were found in Leprdb/db mice.

Conclusion: Diastolic dysfunction is not always associated with cardiac small vessel disease since Leprdb/db males and C57BL/6J males fed with a high fat diet (HFD) + L-NAME regimen develop diastolic dysfunction in the absence of endothelial dysfunction. Also endothelial dysfunction may not be sufficient to induce diastolic dysfunction since non OVX female mice fed with a high fat diet (HFD) + L-NAME regimen display endothelial dysfunction while they do not develop diastolic dysfunction.
Novel endothelial-enriched long non-coding RNA AL590004.3 (IRENE) is dysregulated in HFpEF and regulates endothelial enhancement of cardiomyocyte function
Authors: Juni R P , Phelp P , Busscher D , Belluomo R , Kremer V , Van Bergen A , Jalink L , Boon R A
Published: November 2023

Background

Heart failure with preserved ejection fraction (HFpEF) is the most prevalent form of HF to date. However, there is a lack of medication which stages HFpEF as an unmet clinical need. HFpEF is characterized by cardiac microvascular dysfunction, promoted by co-morbidities-associated low-grade pro-inflammatory state. We have shown that cardiac endothelial cells regulate cardiomyocyte relaxation, which is impaired when endothelial cells are inflamed. Long non-coding RNAs (lncRNAs) have been implicated in various cardiovascular diseases. However, their role in HFpEF is unknown. Here, we discovered a novel lncRNA AL590004.3 which is significantly reduced in the heart of patients with HFpEF. We hypothesized that AL590004.3 is important for cardiac endothelial function, and dysregulation of endothelial AL590004.3 impairs cardiomyocyte relaxation.

Methods and results

We demonstrated that AL590004.3 is significantly downregulated (∼7 fold, p<0.001) in HFpEF as assessed by in silico analysis of RNA sequencing of HFpEF patient cardiac biopsies. To assess the tissue distribution, we performed RNA sequencing comparing cells from various tissues and found the highest expression of AL590004.3 in the cardiac microvascular endothelial cells (CMECs), indicating its importance in cardiac endothelial cell biology. The expression of AL590004.3 was reduced when CMECs were exposed to pro-inflammatory cytokine TNFα and IL1β, indicating the detrimental effect of inflammation, as displayed by patients with HFpEF. Further, replicative senescent of CMECs led to reduced AL590004.3 expression (2 fold, p<0.005), showing the deleterious contribution of ageing, similar to what occurs in HFpEF patients. We showed that AL590004.3 is mostly expressed in the chromatin fraction (∼90%) of CMECs, suggesting its function in chromatin modification. To assess the function of AL590004.3, we performed loss-of-function. Using endothelial-cardiomyocyte co-culture set-up, we showed silencing of AL590004.3 in CMECs impaired endothelial function to enhance cardiomyocyte relaxation (∼1.5 fold, p<0.01). To dissect the molecular mechanism, we performed RNA sequencing of CMECs devoid of AL590004.3 and revealed four protein-coding genes (PXDC1, FAM50, ECI2, PRPF4B) that are differentially co-regulated, indicating their potential as protein-binding partners, which warrant further investigation.

Conclusion

We showed that AL590004.3 is a cardiac endothelial cell-specific lncRNA that is significantly downregulated in cardiac biopsy from HFpEF patients. AL590004.3 is important for cardiac endothelial cell function as loss-of-function in CMECs impaired endothelial enhancement of cardiomyocyte relaxation. Herewith, we name this lncRNA Important for the Regulation of ENdothelial function in HFpEF (IRENE).
The dominant-negative effect of Loss of function Nav 1.5 variants is not a critical determinant of phenotype severity
Authors: Kato K , Tano A , Jinzai H , Kubo T , Ohno S , Nakagawa Y , Horie M
Published: November 2023

Introduction

Loss-of-function (LOF) variants of SCN5A cause various arrhythmic diseases. Recent studies have demonstrated that some LOF-SCN5A variants can exert a dominant-negative (DN) effect through channel dimerization, resulting in higher penetrance of disease phenotypes. However, the association between the DN effect and phenotype severity remains unclear. In this study, we identified a proband with compound heterozygous SCN5A variants, p.G833R and p.T1396P, in a family with bradyarrhythmia. We analyzed the electrophysiological function of these variants and their association with channel-dimerization to elucidate their effects on phenotype severity.

Purpose

The purpose of this study is to investigate the effect of sodium channel dimerization on the phenotype severity in a family with bradyarrhythmia

Methods

We performed functional analysis of the mutant Nav1.5s induced in HEK cells utilizing a whole-cell patch-clamp method. The DN effect was also assessed by co-expressing WT and mutants in each heterozygous combinations, with or without difopein, a peptide working as a channel-coupling canceler. Cell surface expression of mutated channels were measured by flowcytometry.

Results

We identified SCN5A p.T1396P and p.G833R variants segregating in a family suffering from severe bradyarrhythmia. The proband carried both variants in a compound heterozygous manner, while her mother carried only p.T1396P. The clinical phenotype was more severe and had early onset in the proband; the compound heterozygous case.

In the patch-clamp study, HEK 293 cells solely expressing T1396P did not show any measurable sodium current, while those with G833R had comparative current with that of WT channel. Cells expressing WT/T1396P showed significant reduction and delay of inactivation decay, which was normalized by co-expression with difopein. The results implied that loss-of-function variant T1396P manifested DN-effect via channel dimerization.

Cells co-expressing T1396P and G833R did not demonstrate a DN-effect, suggesting that Nav1.5-G833R did not form a dimer. Considering more severe phenotype of compound heterozygous member in the family, the DN-cancelation by monomerized Nav1.5-G833R was not associated with a mild phenotype.

Conclusion

Our result supports the current Nav1.5-dimerization concept and consequent DN-effect by LOF-SCN5A variants. However, the phenotype severity was not simply explainable by the presence of a dominant negative effect. Further multiplanar analyses including assays for channel clustering, localization, and gating modification would be necessary.
Trapped re-entry challenges the binary view on cardiac arrhythmias
Authors: De Coster T , Teplenin A S , Feola I , Bart C I , Ramkisoensing A A , Ypey D L , Panfilov A V , Zeppenfeld K , De Vries A A F , Pijnappels D A
Published: November 2023

Background

There is currently a binary view on cardiac arrhythmias: (1) arrhythmias are present and thereby affect the atria or ventricles as a whole, or (2) they are not present and therefore the heart is in sinus rhythm. However, complex fractionated atrial electrograms (CFAEs) under sinus rhythm (SR) are observed in patients with atrial fibrillation (AF), raising the question whether both cardiac states (arrhythmia and SR) could coexist. Although CFAEs in AF patients have been associated with functional and structural heterogeneities (e.g. dense fibrotic regions), the underlying mechanisms of fractionation under SR remain incompletely understood.

Purpose

To test the hypothesis that an arrhythmia can exist locally with the majority of cardiac tissue in sinus rhythm, made possible through dense local fibrotic regions forming a small electrically isolated re-entrant circuit that can "trap" excitation waves, which can only be "released" under dynamic tissue changes at an isthmus connected to the bulk of the tissue.

Methods

By harnessing the unique possibilities of light-gated depolarizing ion channels (CatCh) to precisely control in vitro cardiac excitability in time and space, complemented with advanced computational modelling of whole human atria, we explored the geometry of such an electrically isolated circuit and assessed its clinical relevance.

Results

Optical mapping studies, in monolayers of CatCh-activated neonatal rat atrial cardiomyocytes (n=8), revealed that re-entry can be established and trapped by creating an electrically isolated pathway with a bulk-connecting isthmus causing source-sink mismatch. A tachyarrhythmia was shown to exist locally with SR prevailing in the bulk of the monolayer. Next, conditions were found under which re-entry could escape this pathway, thereby converting a local dormant arrhythmic source into an active driver with global impact. Escape could be established by overcoming the source-sink mismatch through widening of the isthmus or a reduction of the gap junctional coupling. In a digital twin of the human atria, it was revealed that the conditions for "trapped re-entry" and its release can be realized as well. Unipolar pseudo-electrograms derived from these complementary computational 3D studies showed CFAEs at the site of "trapped re-entry" in coexistence with normal electrograms of SR in the bulk of the atria. Upon release of the re-entry, acute arrhythmia onset occurred, affecting the complete atria as evidenced by wave front and electrogram visualization.

Conclusion

Through the concept of "trapped re-entry", we not only present a new mechanism of acute manifestation of focal tachyarrhythmias, but also provide novel insight into the origin of fractionated electrograms. This insight may provide new rationales for treatment of cardiac arrhythmias, especially for ablation by site-directed targeting.Trapped re-entry in vitro and in silico
Light transmittance in human atrial tissue and transthoracic illumination in rats support translatability of optogenetic cardioversion of AF
Authors: Portero V , Nyns E C A , Harlaar N , Deng S , Tianyi T , Bart C I , Van Brakel T J , Palmen M , Hjortnaes J , Ramkisoensing A A , Zhang G Q , Poelma R H , Ordog B , De Vries A A F , Pijnappels D A
Published: November 2023

Background

Optogenetics could offer a solution to the current lack of an ambulatory method for rapid automated cardioversion of AF, but key translational aspects remain to be studied.

Objective

To investigate whether optogenetic cardioversion of AF is effective in the aged heart and whether sufficient light penetrates the human atrial wall.

Methods

Atria of adult (7 weeks old) and aged (2 years old) rats were optogenetically modified to express light-gated ion channels (i.e. ReaChR), followed by AF induction and atrial illumination to determine effectivity of optogenetic cardioversion. The irradiance level was determined by light transmittance measurements on human atrial tissue.

Results

AF could be effectively terminated in remodelled atria of aged rats (97%, n=6). Next, in terms of ReaChR activation, it was shown that light pulses of 25mW/mm2 are expected to fully penetrate the human atrial wall (∼2 mm). Applying such irradiation onto the chest of adult rats resulted in full transthoracic illumination (also ∼2 mm) as evidenced by optogenetic cardioversion of AF (90%, n=4).

Conclusion

Optogenetic cardioversion of AF is effective in the aged rat heart and can also be realized with irradiation levels compatible with human atrial transmural light penetration.
Cardiomyocyte - stromal cells interplay as a novel platform for mechanistic insights in arrhythmogenic cardiomyopathy
Authors: Maione A S , Iengo L , Sala L , Lodola F , Lippi M , Zaza A , Tondo C , Pompilio G , Sommariva E
Published: November 2023

Background

Arrhythmogenic Cardiomyopathy (ACM) is an inherited heart disorder characterized by a high incidence of sudden death at young age. Mutations linked to ACM occur primarily in desmosomal genes (e.g. PKP2) [1]. Different cell types individually contribute to set the ACM phenotype with either functional abnormalities (cardiomyocytes; CM) [2]; or fibro-fatty substitution (cardiac mesenchymal stromal cells; CMSC) [3]; these two cell types interact and show a reciprocal influence [4]. The relative contribution of the different cell type derangements to determine disease evolution and electrical instability is still unclear.

Purpose

Based on this evidence, we hypothesize that the use of a multicellular cardiac system, modelling the functional interaction between CM and CMSC, will allow the understanding of the relative contribution of the different cell types to the ACM phenotypes.

Methods

Primary stromal cells (carrier of the PKP2 mutation c.2013delC) have been collected from a right ventricle biopsy sample; control stromal cells obtained from a biobank. iPSC reprogrammed from the same ACM patient and the isogenic line in which the PKP2 mutation c.2013delC was corrected [5] were used. Cardiac cocultures (cc) were assembled by combining 85% iPSC-CM and 15% primary CMSC, either mutated or control, in different combination (as in Table 1). Movies of contracting clusters have been collected and analysed by the MUSCLEMOTION tool [6]. Fibro-fatty accumulation have been evaluated by immunofluorescence analysis by confocal microscopy.

Results

Immunofluorescence analysis highlighted that ACM cc (cc 4) accumulate more lipids and collagen than healthy control (HC) ones (cc 3). Since intermediate fibro-fatty levels were observed in ‘mixed cc’ (cc 1 and 4), we conclude that ACM iPSC-CM are able to influence fibro-adipo-commitment of CMSC. Contraction studies showed a high propensity for cc monolayers which include ACM iPSC-CM (cc1 and cc2) to display arrhythmic events. An increase in the percentage of arrhythmic events was triggered when the ACM CMSC were replacing HC CMSC, indicating that ACM CMSC can affect iPSC-CM rhythm.

Conclusion

Overall, our data confirm the importance of the cardiomyocytes and stromal cells interplay during ACM pathogenesis. Furthermore, our novel advanced ACM cell model can recapitulate different phenotypes of ACM and offers a unique opportunity for validating pharmacological therapies to modulate ACM-related characteristics (e.g. fibro-adipose replacement, contractile defects and proarrhythmic events).