Neonatal Mouse Ventricular Myocytes Research Articles

Transcriptional regulation of the postnatal cardiac conduction system heterogeneity

The cardiac conduction system (CCS) is a network of specialized cardiomyocytes that coordinates electrical impulse generation and propagation for synchronized heart contractions. Although the components of the CCS, including the sinoatrial node, atrioventricular node, His bundle, bundle branches, and Purkinje fibers, were anatomically discovered more than 100 years ago, their molecular constituents and regulatory mechanisms remain incompletely understood. Here, we demonstrate the transcriptomic landscape of the postnatal mouse CCS at a single-cell resolution with spatial information. Integration of single-cell and spatial transcriptomics uncover region-specific markers and zonation patterns of expression. Network inference shows heterogeneous gene regulatory networks across the CCS. Notably, region-specific gene regulation is recapitulated in vitro using neonatal mouse atrial and ventricular myocytes overexpressing CCS-specific transcription factors, Tbx3 and/or Irx3. This finding is supported by ATAC-seq of different CCS regions, Tbx3 ChIP-seq, and Irx motifs. Overall, this study provides comprehensive molecular profiles of the postnatal CCS and elucidates gene regulatory mechanisms contributing to its heterogeneity.

Sex-dependent phosphorylation of Argonaute 2 reduces the mitochondrial translocation of miR-181c and induces cardioprotection in females

Obesity-induced cardiac dysfunction is growing at an alarming rate, showing a dramatic increase in global prevalence. Mitochondrial translocation of miR-181c in cardiomyocytes results in excessive reactive oxygen species (ROS) production during obesity. ROS causes Sp1, a transcription factor for MICU1, to be degraded via post-translational modification. The subsequent decrease in MICU1 expression causes mitochondrial Ca2+ accumulation, ultimately leading to a propensity for heart failure. Herein, we hypothesized that phosphorylation of Argonaute 2 (AGO2) at Ser 387 (in human) or Ser 388 (in mouse) inhibits the translocation of miR-181c into the mitochondria by increasing the cytoplasmic stability of the RNA-induced silencing complex (RISC). Initially, estrogen offers cardioprotection in pre-menopausal females against the consequences of mitochondrial miR-181c upregulation by driving the phosphorylation of AGO2. Neonatal mouse ventricular myocytes (NMVM) treated with insulin showed an increase in pAGO2 levels and a decrease in mitochondrial miR-181c expression by increasing the binding affinity of AGO2-GW182 in the RISC. Thus, insulin treatment prevented excessive ROS production and mitochondrial Ca2+ accumulation. In human cardiomyocytes, we overexpressed miR-181c to mimic pathological conditions, such as obesity/diabetes. Treatment with estradiol (E2) for 48 h significantly lowered miR-181c entry into the mitochondria through increased pAGO2 levels. E2 treatment also normalized Sp1 degradation and MICU1 transcription that normally occurs in response to miR-181c overexpression. We then investigated these findings using an in vivo model, with age-matched male, female and ovariectomized (OVX) female mice. Consistent with the E2 treatment, we show that female hearts express higher levels of pAGO2 and thus, exhibit higher association of AGO2-GW182 in cytoplasmic RISC. This results in lower expression of mitochondrial miR-181c in female hearts compared to male or OVX groups. Further, female hearts had fewer consequences of mitochondrial miR-181c expression, such as lower Sp1 degradation and significantly decreased MICU1 transcriptional regulation. Taken together, this study highlights a potential therapeutic target for conditions such as obesity and diabetes, where miR-181c is upregulated. New and noteworthyIn this study, we show that the phosphorylation of Argonaute 2 (AGO2) stabilizes the RNA-induced silencing complex in the cytoplasm, preventing miR-181c entry into the mitochondria. Furthermore, we demonstrate that treatment with estradiol can inhibit the translocation of miR-181c into the mitochondria by phosphorylating AGO2. This ultimately eliminates the downstream consequences of miR-181c overexpression by mitigating excessive reactive oxygen species production and calcium entry into the mitochondria.

Monitoring mitochondrial calcium in cardiomyocytes during coverslip hypoxia using a fluorescent lifetime indicator

An increase in mitochondrial calcium via the mitochondrial calcium uniporter (MCU) has been implicated in initiating cell death in the heart during ischemia-reperfusion (I/R) injury. Measurement of calcium during I/R has been challenging due to the pH sensitivity of indicators coupled with the fall in pH during I/R. The development of a pH-insensitive indicator, mitochondrial localized Turquoise Calcium Fluorescence Lifetime Sensor (mito-TqFLITS), allows for quantifying mitochondrial calcium during I/R via fluorescent lifetime imaging. Mitochondrial calcium was monitored using mito-TqFLITS in neonatal mouse ventricular myocytes (NMVM) isolated from germline MCU-KO mice and MCUfl/fl treated with CRE-recombinase to acutely knockout MCU. To simulate ischemia, a coverslip was placed on a monolayer of NMVMs to prevent access to oxygen and nutrients. Reperfusion was induced by removing the coverslip. Mitochondrial calcium increases threefold during coverslip hypoxia in MCU-WT. There is a significant increase in mitochondrial calcium during coverslip hypoxia in germline MCU-KO, but it is significantly lower than in MCU-WT. We also found that compared to WT, acute MCU-KO resulted in no difference in mitochondrial calcium during coverslip hypoxia and reoxygenation. To determine the role of mitochondrial calcium uptake via MCU in initiating cell death, we used propidium iodide to measure cell death. We found a significant increase in cell death in both the germline MCU-KO and acute MCU-KO, but this was similar to their respective WTs. These data demonstrate the utility of mito-TqFLITS to monitor mitochondrial calcium during simulated I/R and further show that germline loss of MCU attenuates the rise in mitochondrial calcium during ischemia but does not reduce cell death.

Differential sensitivity to longitudinal and transverse stretch mediates transcriptional responses in mouse neonatal ventricular myocytes.

To identify how cardiomyocyte mechanosensitive signaling pathways are regulated by anisotropic stretch, micropatterned mouse neonatal cardiomyocytes were stretched primarily longitudinally or transversely to the myofiber axis. Four hours of static, longitudinal stretch induced differential expression of 557 genes, compared with 30 induced by transverse stretch, measured using RNA-seq. A logic-based ordinary differential equation model of the cardiac myocyte mechanosignaling network, extended to include the transcriptional regulation and expression of 784 genes, correctly predicted measured expression changes due to anisotropic stretch with 69% accuracy. The model also predicted published transcriptional responses to mechanical load in vitro or in vivo with 63-91% accuracy. The observed differences between transverse and longitudinal stretch responses were not explained by differential activation of specific pathways but rather by an approximately twofold greater sensitivity to longitudinal stretch than transverse stretch. In vitro experiments confirmed model predictions that stretch-induced gene expression is more sensitive to angiotensin II and endothelin-1, via RhoA and MAP kinases, than to the three membrane ion channels upstream of calcium signaling in the network. Quantitative cardiomyocyte gene expression differs substantially with the axis of maximum principal stretch relative to the myofilament axis, but this difference is due primarily to differences in stretch sensitivity rather than to selective activation of mechanosignaling pathways.NEW & NOTEWORTHY Anisotropic stretch applied to micropatterned neonatal mouse ventricular myocytes induced markedly greater acute transcriptional responses when the major axis of stretch was parallel to the myofilament axis than when it was transverse. Analysis with a novel quantitative network model of mechanoregulated cardiomyocyte gene expression suggests that this difference is explained by higher cell sensitivity to longitudinal loading than transverse loading than by the activation of differential signaling pathways.

Abstract EC.10: Transcriptional Heterogeneity Of The Postnatal Cardiac Conduction System

The cardiac conduction system (CCS) is a network of specialized cardiomyocytes responsible for the coordinated spreading of electrical impulses throughout the heart for synchronized contractions. The CCS is largely made up of the sinoatrial node (SAN), atrioventricular node (AVN) and the fast-conducting ventricular conduction system (VCS), which is further divided into the His bundle, Bundle branches and Purkinje fibers. Although these CCS structures were anatomically discovered >100 years ago, their molecular constituents are not fully known. In this study, CCS cells, making up ~0.05% of myocytes (~500 cells/heart), were isolated from early postnatal hearts (postnatal day 1 to 4) of fluorescent reporter mice (Cntn2-Cre;Rosa26TdTomato), purified using fluorescence-activated cell sorting, and subjected to single-cell RNA-sequencing. Unbiased cluster analysis on ~7000 CCS cells demonstrated distinct transcriptomic profiles in the SAN, AVN, proximal VCS ( His bundle, bundle branches) and distal VCS ( Purkinje fibers). Conforming to previous studies, global conduction markers, such as Cntn2 and Id2, were present throughout the entire CCS, while a VCS-specific transcription factor, Irx3 , was expressed only in the VCS. A comparison between proximal and distal VCS components revealed enrichments of Igfbp7 , Lyz2 and Sfrp5 in the proximal VCS, and Scn10a , Psd3 and Wisp1 in the distal VCS. Sub-clustering analysis followed by validation with immunostaining further identified unique molecular profiles of the SAN ( Shox2 , Vsnl1 ), AVN ( Rspo3 ), His bundle ( S100a6 , Pcp4 ) and bundle branches ( Nppa , Mest ). Data integrating publicly available ATAC-seq in SAN, AV junction, and atrial and ventricular cardiomyocytes as well as Tbx3-ChIP-seq, suggested that these regional markers are possibly regulated by two CCS-specific transcription factors, Irx3 and Tbx3 . More importantly, overexpressing Irx3 and/or Tbx3 in cultured neonatal mouse atrial and ventricular myocytes recapitulated these region-specific expression patterns in a dish. Together, our study provides a comprehensive view of the molecular profiles within the postnatal CCS at a high-resolution and show Irx3 and Tbx3 as genetic regulators establishing heterogeneities of the CCS.

Determinants of iFGF13-mediated regulation of myocardial voltage-gated sodium (NaV) channels in mouse.

Posttranslational regulation of cardiac NaV1.5 channels is critical in modulating channel expression and function, yet their regulation by phosphorylation of accessory proteins has gone largely unexplored. Using phosphoproteomic analysis of NaV channel complexes from adult mouse left ventricles, we identified nine phosphorylation sites on intracellular fibroblast growth factor 13 (iFGF13). To explore the potential roles of these phosphosites in regulating cardiac NaV currents, we abolished expression of iFGF13 in neonatal and adult mouse ventricular myocytes and rescued it with wild-type (WT), phosphosilent, or phosphomimetic iFGF13-VY. While the increased rate of closed-state inactivation of NaV channels induced by Fgf13 knockout in adult cardiomyocytes was completely restored by adenoviral-mediated expression of WT iFGF13-VY, only partial rescue was observed in neonatal cardiomyocytes after knockdown. The knockdown of iFGF13 in neonatal ventricular myocytes also shifted the voltage dependence of channel activation toward hyperpolarized potentials, a shift that was not reversed by WT iFGF13-VY expression. Additionally, we found that iFGF13-VY is the predominant isoform in adult ventricular myocytes, whereas both iFGF13-VY and iFGF13-S are expressed comparably in neonatal ventricular myocytes. Similar to WT iFGF13-VY, each of the iFGF13-VY phosphomutants studied restored NaV channel inactivation properties in both models. Lastly, Fgf13 knockout also increased the late Na+ current in adult cardiomyocytes, and this effect was restored with expression of WT and phosphosilent iFGF13-VY. Together, our results demonstrate that iFGF13 is highly phosphorylated and displays differential isoform expression in neonatal and adult ventricular myocytes. While we found no roles for iFGF13 phosphorylation, our results demonstrate differential effects of iFGF13 on neonatal and adult mouse ventricular NaV channels.

Abstract P3092: Transcriptomic Landscape Of The Postnatal Atrioventricular Conduction System

The atrioventricular conduction system (AVCS) is a network of specialized cardiomyocytes responsible for coordinated spreading of electrical impulses throughout the heart to allow synchronized contractions. Specifically, it is composed of the AV node (AVN), which delays the electrical signal, and the ventricular conduction system (VCS) (i.e. His bundle, bundle branches and Purkinje fibers), which rapidly propagates the signal throughout the ventricles. Although these AVCS structures were discovered >100 years ago, its molecular constituents are not fully known. In this study, AVCS cells, making up ~0.05% of myocytes (~500 cells/heart), were isolated from early postnatal hearts (Postnatal day 1 to 4) of fluorescent reporter mice (Cntn2- Cre;Rosa26TdTomato), purified using fluorescence-activated cell sorting, and were subjected to single-cell RNA-sequencing. Unbiased cluster analysis on ~7000 AVCS cells demonstrated distinct transcriptomic profiles in the AVN, proximal-VCS (i.e. His bundle and bundle branches) and distal-VCS (i.e. Purkinje fibers). Conforming to previous studies, global conduction markers, such as Etv1 and Kcne1, were present throughout the entire AVCS, whereas a VCS-specific TF, Irx3 , was expressed only in the VCS. Additionally, Tbx3, Pcp4, Nppa and Lyz2 were enriched in the proximal, not distal, VCS. Sub-clustering analysis of AVN and proximal-VCS further identified unique molecular profiles, such as Shox2 (in compact AVN) and Rspo3 (likely in lower nodal region connecting AVN and proximal-VCS). Moreover, we uncovered a potential regulatory relationship between Irx3 and Tbx3 in the AVCS, mediating their downstream targets, Gja5 and Gja1 . In Irx3 and Tbx3 -positive proximal-VCS, Gja5 and Gja1 expressions were lower compared to only Irx3 -positive distal-VCS, whereas in only Tbx3 -positive AVN, both Gja5 and Gja1 expressions were undetected. Importantly, this regional difference was recapitulated in cultured neonatal mouse atrial and ventricular myocytes overexpressing Irx3 and/or Tbx3 . Overall, our study provides a comprehensive view of the cellular and molecular heterogeneity within the postnatal AVCS at a high-resolution and reveals novel gene regulatory mechanisms that may govern the development of the AVCS.

A ROCK1 Inhibitior Fasudil Alleviates Cardiomyocyte Apoptosis in Diabetic Cardiomyopathy by Inhibiting Mitochondrial Fission in a Type 2 Diabetes Mouse Model.

Diabetes mellitus (DM) often involves cardiovascular complications; however, treatment regimens are limited. ROCK1 (rho-associated coiled-coil containing protein kinase 1) serves as a pathological factor in several diabetic complications. Herein, we aimed to explore the effect of Fasudil (a ROCK1 inhibitor) on the progress of cardiac dysfunction in type 2 DM (T2DM), and to explore the possible mechanisms. Type II diabetic mice models were established by inducing insulin resistance through a high-fat diet combined with low-dose streptozotocin (STZ) injection. NMCMs (neonatal mouse ventricular cardiac myocytes) in the control group were treated with 5.5 mM glucose, while those in the High Glucose (HG) group were treated with 33 mM glucose and 10 nmol/L insulin. In vivo, we found that type 2 diabetes enhanced the expression and activation of ROCK1 (p < 0.05). The ROCK1 inhibitor, Fasudil, prevented cardiac dysfunction, fibrosis, oxidative stress and myocardial ultrastructural disorders (p < 0.05) in the diabetic mice. In vitro, ROCK1 was upregulated in HG-induced cardiomyocytes, and ROCK1 inhibition using Fasudil reversed the increased apoptosis, consistent with in vivo results. Mechanistically, ROCK1 inhibition abrogated apoptosis, relieved mitochondrial fission, and efficiently attenuated the escalated production of reactive oxygen species in vitro and in vivo. The content of Ser616-phosphorylated dynamin-related protein 1 (Drp1) increased while ROCK1 led to apoptosis in HG-treated cardiomyocytes, which could be partly neutralized by ROCK1 inhibition with Fasudil, consistent with the in vivo results. Fasudil attenuated the cardiac dysfunction in diabetes by decreasing excessive mitochondrial fission via inhibiting Drp1 phosphorylation at serine 616.

Secretion of miRNA-326-3p by senescent adipose exacerbates myocardial metabolism in diabetic mice

BackgroundAdipose tissue homeostasis is at the heart of many metabolic syndromes such as diabetes. Previously it has been demonstrated that adipose tissues from diabetic patients are senescent but whether this contributes to diabetic cardiomyopathy (DCM) remains to be elucidated.MethodsThe streptozotocin (STZ) type 1 diabetic mice were established as animal model, and adult mouse ventricular myocytes (AMVMs) isolated by langendorff perfusion as well as neonatal mouse ventricular myocytes (NMVMs) were used as cell models. Senescent associated β galactosidase (SA-β-gal) staining and RT-qPCR were used to identify the presence of adipose senescence in diabetic adipose tissue. Senescent adipose were removed either by surgery or by senolytic treatment. Large extracellular vesicles (LEVs) derived from adipose tissue and circulation were separated by ultracentrifugation. Cardiac systolic and diastolic function was evaluated through cardiac ultrasound. Cardiomyocytes contraction function was evaluated by the Ionoptix HTS system and live cell imaging, mitochondrial morphology and functions were evaluated by transmission electron microscope, live cell fluorescent probe and seahorse analysis. RNA-seq for AMVMs and miRNA-seq for LEVs were performed, and bioinformatic analysis combined with RT-qPCR and Western blot were used to elucidate underlying mechanism that senescent adipose derives LEVs exacerbates myocardial metabolism.ResultsSA-β-gal staining and RT-qPCR identified the presence of adipose tissue senescence in STZ mice. Through surgical as well as pharmacological means we show that senescent adipose tissue participates in the pathogenesis of DCM in STZ mice by exacerbates myocardial metabolism through secretion of LEVs. Specifically, expression of miRNA-326-3p was up-regulated in LEVs isolated from senescent adipose tissue, circulation, and cardiomyocytes of STZ mice. Up-regulation of miRNA-326-3p coincided with myocardial transcriptomic changes in metabolism. Functionally, we demonstrate that miRNA-326-3p inhibited the expression of Rictor and resulted in impaired mitochondrial and contractile function in cardiomyocytes.ConclusionWe demonstrate for the first time that senescent adipose derived LEVs exacerbates myocardial metabolism through up-regulated miRNA-326-3p which inhibits Rictor in cardiomyocytes. Furthermore, reducing senescence burden in adipose tissue is capable of relieving myocardial metabolism disorder in diabetes mellitus.

Hypoglycemia-Exacerbated Mitochondrial Connexin 43 Accumulation Aggravates Cardiac Dysfunction in Diabetic Cardiomyopathy.

BackgroundDiabetic cardiomyopathy (DCM) is a complex multifaceted disease responsible for elevated heart failure (HF) morbidity and mortality in patients with diabetes mellitus (DM). Patients with DCM exhibit subclinical diastolic dysfunction, progression toward systolic impairment, and abnormal electrophysiology. Hypoglycemia events that occur spontaneously or due to excess insulin administration threaten the lives of patients with DM—with the increased risk of sudden death. However, the molecular underpinnings of this fatal disease remain to be elucidated.Methods and ResultsHere, we used the established streptozotocin-induced DCM murine model to investigate how hypoglycemia aggravates DCM progression. We confirmed connexin 43 (Cx43) dissociation from cell–cell interaction and accumulation at mitochondrial inner membrane both in the cardiomyocytes of patients with DM and DCM murine. Here, we observed that cardiac diastolic function, induced by chronic hyperglycemia, was further aggravated upon hypoglycemia challenge. Similar contractile defects were recapitulated using neonatal mouse ventricular myocytes (NMVMs) under glucose fluctuation challenges. Using immunoprecipitation mass spectrometry, we identified and validated that hypoglycemia challenge activates the mitogen-activated protein kinase kinase (MAPK kinase) (MEK)/extracellular regulated protein kinase (ERK) and inhibits phosphoinositide 3-kinase (PI3K)/Akt pathways, which results in Cx43 phosphorylation by Src protein and translocation to mitochondria in cardiomyocytes. To determine causality, we overexpressed a mitochondrial targeting Cx43 (mtCx43) using adeno-associated virus serotype 2 (AAV2)/9. At normal blood glucose levels, mtCx43 overexpression recapitulated cardiac diastolic dysfunction as well as aberrant electrophysiology in vivo. Our findings give support for therapeutic targeting of MEK/ERK/Src and PI3K/Akt/Src pathways to prevent mtCx43-driven DCM.ConclusionDCM presents compensatory adaptation of mild mtCx43 accumulation, yet acute hypoglycemia challenges result in further accumulation of mtCx43 through the MEK/ERK/Src and PI3K/Akt/Src pathways. We provide evidence that Cx43 mislocalization is present in hearts of patients with DM hearts, STZ-induced DCM murine model, and glucose fluctuation challenged NMVMs. Mechanistically, we demonstrated that mtCx43 is responsible for inducing aberrant contraction and disrupts electrophysiology in cardiomyocytes and our results support targeting of mtCx43 in treating DCM.

TRANSCRIPTOMIC HETEROGENEITY OF THE POSTNATAL ATRIOVENTRICULAR CONDUCTION SYSTEM AT A SINGLE-CELL RESOLUTION

BACKGROUND The atrioventricular conduction system (AVCS) is a network of specialized cardiomyocytes responsible for coordinated spreading of electrical impulses throughout the ventricles to allow synchronized contractions. Specifically, it is composed of the AV node (AVN), which delays the electrical signal, and the ventricular conduction system (VCS) (i.e. His bundle, bundle branches and Purkinje fibers), which rapidly propagates the signal throughout the ventricles. Different AVCS regions possess different electrophysiological properties, such as conduction velocity. Overlapping and nonoverlapping expression patterns of some key developmental transcription factors (TFs), including Irx3, Tbx3, Etv1 and Nkx2.5 within the AVCS, suggest heterogeneous gene expression profiles. Although these structures of the AVCS were discovered >100 years ago, our understanding about its molecular constituents remains largely limited. METHODS AND RESULTS AVCS cells, which make up ∼0.05% of myocytes (∼500 cells/1 million cardiomyocytes), were isolated from early postnatal hearts (P1 to P4) of fluorescent reporter mice (Cntn2-Cre;Rosa26TdTomato), purified using fluorescence-activated cell sorting, and were subjected to single-cell RNA-sequencing. Unbiased cluster analysis on ∼7000 AVCS cells demonstrated distinct transcriptomic profiles in the AVN, proximal-VCS (i.e. His bundle and bundle branches) and distal-VCS (i.e. Purkinje fibers). Conforming to previous studies, well-known conduction markers, such as Etv1 and Kcne1, were present throughout the entire AVCS, whereas a VCS-specific TF, Irx3, was expressed throughout only the VCS. Additionally, Tbx3, Pcp4, Nppa and Lyz2 were enriched in the proximal, not distal, VCS. Sub-clustering analysis of AVN and proximal-VCS further identified unique expression patterns of molecular markers, such as Shox2 (exclusively in compact AVN) and Rspo3 (likely in lower nodal region connecting AVN and proximal-VCS). Moreover, we uncovered a potential regulatory relationship between Irx3 and Tbx3 in mediating their downstream targets, gap junction Gja5 (Cx40) and Gja1 (Cx43). In Irx3- and Tbx3-positive proximal-VCS, Gja5 and Gja1 expressions were lower compared to Irx3-positive/Tbx3-negative distal-VCS. In only Irx3-negative/Tbx3-positive AVN, both Gja5 and Gja1 expressions were undetected. Importantly, this regulatory relationship was recapitulated in cultured neonatal mouse atrial and ventricular myocytes overexpressing Irx3 and/or Tbx3. We also identified a cluster of cells which co-expressed conductive genes (i.e. Irx3, Kcne1, Gja5, Etv1) as well as G2/M cell cycle genes (i.e. Birc5, Ube2c, Cdk1, Ccnb1/2), suggesting the presence of committed conduction cells undergoing proliferation after birth. CONCLUSION Our study provides a comprehensive view of the cellular and molecular heterogeneity within the postnatal AVCS at a high-resolution and identifies potential novel markers with unique and overlapping transcriptomic patterns. The atrioventricular conduction system (AVCS) is a network of specialized cardiomyocytes responsible for coordinated spreading of electrical impulses throughout the ventricles to allow synchronized contractions. Specifically, it is composed of the AV node (AVN), which delays the electrical signal, and the ventricular conduction system (VCS) (i.e. His bundle, bundle branches and Purkinje fibers), which rapidly propagates the signal throughout the ventricles. Different AVCS regions possess different electrophysiological properties, such as conduction velocity. Overlapping and nonoverlapping expression patterns of some key developmental transcription factors (TFs), including Irx3, Tbx3, Etv1 and Nkx2.5 within the AVCS, suggest heterogeneous gene expression profiles. Although these structures of the AVCS were discovered >100 years ago, our understanding about its molecular constituents remains largely limited. AVCS cells, which make up ∼0.05% of myocytes (∼500 cells/1 million cardiomyocytes), were isolated from early postnatal hearts (P1 to P4) of fluorescent reporter mice (Cntn2-Cre;Rosa26TdTomato), purified using fluorescence-activated cell sorting, and were subjected to single-cell RNA-sequencing. Unbiased cluster analysis on ∼7000 AVCS cells demonstrated distinct transcriptomic profiles in the AVN, proximal-VCS (i.e. His bundle and bundle branches) and distal-VCS (i.e. Purkinje fibers). Conforming to previous studies, well-known conduction markers, such as Etv1 and Kcne1, were present throughout the entire AVCS, whereas a VCS-specific TF, Irx3, was expressed throughout only the VCS. Additionally, Tbx3, Pcp4, Nppa and Lyz2 were enriched in the proximal, not distal, VCS. Sub-clustering analysis of AVN and proximal-VCS further identified unique expression patterns of molecular markers, such as Shox2 (exclusively in compact AVN) and Rspo3 (likely in lower nodal region connecting AVN and proximal-VCS). Moreover, we uncovered a potential regulatory relationship between Irx3 and Tbx3 in mediating their downstream targets, gap junction Gja5 (Cx40) and Gja1 (Cx43). In Irx3- and Tbx3-positive proximal-VCS, Gja5 and Gja1 expressions were lower compared to Irx3-positive/Tbx3-negative distal-VCS. In only Irx3-negative/Tbx3-positive AVN, both Gja5 and Gja1 expressions were undetected. Importantly, this regulatory relationship was recapitulated in cultured neonatal mouse atrial and ventricular myocytes overexpressing Irx3 and/or Tbx3. We also identified a cluster of cells which co-expressed conductive genes (i.e. Irx3, Kcne1, Gja5, Etv1) as well as G2/M cell cycle genes (i.e. Birc5, Ube2c, Cdk1, Ccnb1/2), suggesting the presence of committed conduction cells undergoing proliferation after birth. Our study provides a comprehensive view of the cellular and molecular heterogeneity within the postnatal AVCS at a high-resolution and identifies potential novel markers with unique and overlapping transcriptomic patterns.

MitoWave: Spatiotemporal analysis of mitochondrial membrane potential fluctuations during I/R

Mitochondria exhibit unstable inner membrane potentials (ΔΨm) when subjected to stress, such as during ischemia/reperfusion (I/R). Understanding the mechanism of ΔΨm instability involves characterizing and quantifying this phenomenon in an unbiased and reproducible manner. Here, we describe a simple analytical workflow called “MitoWave” that combines wavelet transform methods and image segmentation to unravel dynamic ΔΨm changes in the cardiac mitochondrial network during I/R. In vitro ischemia was affected by placing a glass coverslip on a monolayer of neonatal mouse ventricular myocytes for 1 h and removing the coverslip to allow for reperfusion, revealing complex oscillatory ΔΨm. MitoWave analysis was then used to identify individual mitochondrial clusters within the cells and track their intrinsic oscillation frequencies over the course of reperfusion. Responses segregated into five typical behaviors were quantified by MitoWave that were corroborated by visual inspection of the time series. Statistical analysis of the distribution of oscillating mitochondrial clusters during reperfusion showed significant differences between the five different outcomes. Features such as the time point of ΔΨm depolarization during I/R, area of mitochondrial clusters, and time-resolved frequency components during reperfusion were determined per cell and per mitochondrial cluster. Mitochondria from neonatal mouse ventricular myocytes subjected to I/R oscillate in the frequency range of 8.6–45 mHz, with a mean of 8.73 ± 4.35 mHz. Oscillating clusters had smaller areas ranging from 49.8 ± 1.2 μm2, whereas nonoscillating clusters had larger areas 66 ± 1.5 μm2. A negative correlation between frequency and mitochondrial cluster area was observed. We also observed that late ΔΨm loss during ischemia correlated with early ΔΨm stabilization after oscillation on reperfusion. Thus, MitoWave analysis provides a semiautomated method to quantify complex time-resolved mitochondrial behavior in an easy-to-follow workflow, enabling unbiased, reproducible quantitation of complex nonstationary cellular phenomena.

Knockout of interleukin-17A diminishes ventricular arrhythmia susceptibility in diabetic mice via inhibiting NF-κB-mediated electrical remodeling.

Interleukin-17A (IL-17), a potent proinflammatory cytokine, has been shown to participate in cardiac electrical disorders. Diabetes mellitus is an independent risk factor for ventricular arrhythmia. In this study, we investigated the role of IL-17 in ventricular arrhythmia of diabetic mice. Diabetes was induced in both wild-type and IL-17 knockout mice by intraperitoneal injection of streptozotocin (STZ). High-frequency electrical stimuli were delivered into the right ventricle to induce ventricular arrhythmias. We showed that the occurrence rate of ventricular tachycardia was significantly increased in diabetic mice, which was attenuated by IL-17 knockout. We conducted optical mapping on perfused mouse hearts and found that cardiac conduction velocity (CV) was significantly decreased, and action potential duration (APD) wasprolonged in diabetic mice, which were mitigated by IL-17 knockout. We performed whole-cell patch clamp recordings from isolated ventricular myocytes, and found that the densities of Ito, INa and ICa,L were reduced, the APDs at 50% and 90% repolarization were increased, and early afterdepolarization (EAD) was markedly increased in diabetic mice. These alterations were alleviated by the knockout of IL-17. Moreover, knockout of IL-17 alleviated the downregulation of Nav1.5 (the pore forming subunit of INa), Cav1.2 (the main component subunit of ICa,L) and KChIP2 (potassium voltage-gated channel interacting protein 2, the regulatory subunit of Ito) in the hearts of diabetic mice. The expression of NF-κB was significantly upregulated in the hearts of diabetic mice, which was suppressed by IL-17 knockout. In neonatal mouse ventricular myocytes, knockdown of NF-κB significantly increased the expression of Nav1.5, Cav1.2 and KChIP2. These results imply that IL-17 may represent a potential target for the development of agents against diabetes-related ventricular arrhythmias.

Silica Nanoparticles Disturb Ion Channels and Transmembrane Potentials of Cardiomyocytes and Induce Lethal Arrhythmias in Mice.

BackgroundThe toxicity of silica nanoparticles (SiNPs) on cardiac electrophysiology has seldom been evaluated.MethodsPatch-clamp was used to investigate the acute effects of SiNP-100 (100 nm) and SiNP-20 (20 nm) on the transmembrane potentials (TMPs) and ion channels in cultured neonatal mouse ventricular myocytes. Calcium mobilization in vitro, cardiomyocyte ROS generation, and LDH leakage after exposure to SiNPs in vitro and in vivo were measured using a microplate reader. Surface electrocardiograms were recorded in adult mice to evaluate the arrhythmogenic effects of SiNPs in vivo. SiNP endocytosis was observed using transmission electron microscopy.ResultsWithin 30 min, both SiNPs (10−8–10−6 g/mL) did not affect the resting potential and IK1 channels. SiNP-100 increased the action potential amplitude (APA) and the INa current density, but SiNP-20 decreased APA and INa density. SiNP-100 prolonged the action potential duration (APD) and decreased the Ito current density, while SiNP-20 prolonged or shortened the APD, depending on exposure concentrations and increased Ito density. Both SiNPs (10−6 g/mL) induced calcium mobilization but did not increase ROS and LDH levels and were not endocytosed within 10 min in cardiomyocytes in vitro. In vivo, SiNP-100 (4–10 mg/kg) and SiNP-20 (4–30 mg/kg) did not elevate myocardial ROS but increased LDH levels depending on dose and exposure time. The same higher dose of SiNPs (intravenously injected) induced tachyarrhythmias and lethal bradyarrhythmias within 90 min in adult mice.ConclusionSiNPs (i) exert rapid toxic effects on the TMPs of cardiomyocytes in vitro largely owing to their direct interfering effects on the INa and Ito channels and Ca2+ homeostasis but not IK1 channels and ROS levels, and (ii) induce tachyarrhythmias and lethal bradyarrhythmias in vivo. SiNP-100 is more toxic than SiNP-20 on cardiac electrophysiology, and the toxicity mechanism is likely more complicated in vivo.

Mitophagy inhibitor liensinine suppresses doxorubicin-induced cardiotoxicity through inhibition of Drp1-mediated maladaptive mitochondrial fission

Doxorubicin (DOX) is one of the most effective antineoplastic drugs. However, its clinical application has been greatly limited due to the development of cardiotoxicity with DOX utilization. A number of theories have been postulated for DOX-induced cardiotoxicity with a pivotal contribution from unchecked (excess) mitophagy and mitochondrial fission. Liensinine (LIEN), a newly identified mitophagy inhibitor, strengthens the antineoplastic efficacy of DOX although its action on hearts remains elusive. This study was designed to examine the effect of LIEN on DOX-induced cardiotoxicity and the underlying mechanisms involved with a focus on mitochondrial dynamics. Our data revealed that LIEN alleviated DOX-induced cardiac dysfunction and apoptosis through inhibition of dynamin-related protein 1 (Drp1)-mediated excess (unchecked) mitochondrial fission. LIEN treatment decreased Drp1 phosphorylation at Ser616 site, inhibited mitochondrial fragmentation, mitophagy (assessed by TOM20 and TIM23), oxidative stress, cytochrome C leakage, cardiomyocyte apoptosis, as well as improved mitochondrial function and cardiomyocyte contractile function in DOX-induced cardiac injury. In DOX-challenged neonatal mouse ventricular myocytes (NMVMs), LIEN-suppressed Drp1 phosphorylation, mitochondrial fragmentation, and apoptosis were blunted by Rab7 overexpression, the effect of which was reversed by the ERK inhibitor U0126. Moreover, activation of ERK or Drp1 abolished the protective effects of LIEN on cardiomyocyte mechanical anomalies. These data shed some lights towards understanding the role of LIEN as a new protective agent against DOX-associated cardiotoxicity without compromising its anti-tumor effects.

MiR‐181c Activates Mitochondrial Calcium Uptake by Regulating MICU1 in the Heart

BackgroundTranslocation of miR‐181c into cardiac mitochondria downregulates the mitochondrial gene, mt‐COX1. miR‐181c/d−/− hearts experience less oxidative stress during ischemia/reperfusion (I/R) and are protected against I/R injury. Additionally, miR‐181c overexpression can increase mitochondrial matrix Ca2+ ([Ca2+]m), but the mechanism by which miR‐181c regulates [Ca2+]m is unknown.Methods and ResultsBy RNA sequencing and analysis, here we show that hearts from miR‐181c/d−/− mice overexpress nuclear‐encoded Ca2+ regulatory and metabolic pathway genes, suggesting that alterations in miR‐181c and mt‐COX1 perturb mitochondria‐to‐nucleus retrograde signaling and [Ca2+]m regulation. Quantitative polymerase chain reaction validation of transcription factors that are known to initiate retrograde signaling revealed significantly higher Sp1 (specificity protein) expression in the miR‐181c/d−/− hearts. Furthermore, an association of Sp1 with the promoter region of MICU1 was confirmed by chromatin immunoprecipitation‐quantitative polymerase chain reaction and higher expression of MICU1 was found in the miR‐181c/d−/− hearts. Conversely, downregulation of Sp1 by small interfering RNA decreased MICU1 expression in neonatal mouse ventricular myocytes. Changes in PDH activity provided evidence for a change in [Ca2+]m via the miR‐181c/MICU1 axis. Moreover, this mechanism was implicated in the pathology of I/R injury. When MICU1 was knocked down in the miR‐181c/d−/− heart by lentiviral expression of a short‐hairpin RNA against MICU1, cardioprotective effects against I/R injury were abrogated. Furthermore, using an in vitro I/R model in miR‐181c/d−/− neonatal mouse ventricular myocytes, we confirmed the contribution of both Sp1 and MICU1 in ischemic injury.ConclusionsmiR‐181c regulates mt‐COX1, which in turn regulates MICU1 expression through the Sp1‐mediated mitochondria‐to‐nucleus retrograde pathway. Loss of miR‐181c can protect the heart from I/R injury by modulating [Ca2+]m through the upregulation of MICU1.

Thyroid Hormone Diminishes Ca2+ Overload Induced by Hypoxia/Reoxygenation in Cardiomyocytes by Inhibiting Late Sodium Current and Reverse-Na+/Ca2+ Exchange Current

Background and Purpose: Intracellular calcium concentration ([Ca²⁺]i) overload occurs in myocardial ischemia and ­reperfusion. The augmentation of the late sodium current (I_NaL) causes intracellular Na⁺ accumulation and subsequent [Ca²⁺]i overload via the reverse mode of the Na⁺/Ca²⁺ exchange current (reverse-I_NCX), which can lead to arrhythmia and cardiac dysfunction. Thus, inhibition of I_NaL is a potential therapeutic approach for ischemic heart disease. The aim of this study was to investigate the effects of thyroid hormone on augmented I_NaL, reverse-I_NCX, altered action potential duration (APD), and [Ca²⁺]i concentration in hypoxia/reoxygenation (H/R)-induced ventricular myocytes in vitro. Methods: The transient Na⁺ current (I_NaT), I_NaL, reverse-I_NCX, and APs were recorded using a whole-cell patch-clamp technique in neonatal mouse ventricular myocytes. [Ca²⁺]i concentration alteration were, respectively, observed by confocal microscopy and flow cytometry. Results: Triiodothyronine (T3) pretreatment decreased the I_NaL in a concentration-dependent manner. H/R injury aggravated the I_NaL, I_NaT, and reverse-I_NCX in cardiomyocytes and increased the continuous accumulation of [Ca²⁺]i (p < 0.05). The application of T3 prior to H/R injury significantly decreased the increased I_NaL, I_NaT, and reverse-I_NCX and blunted the [Ca²⁺]i increase. Furthermore, T3 pretreatment shortened the APD induced by H/R injury. Conclusion: T3 inhibited H/R-increased I_NaL and reverse I_NCX augmentation, shortened the APD, and diminished [Ca²⁺]i overload, indicating a potential therapeutic use of T3 as an I_NaL inhibitor to maintain Ca²⁺ homeostasis and protect cardiomyocytes against H/R injury.

Calcium-calmodulin gating of a pH-insensitive isoform of connexin43 gap junctions.

Intracellular protons and calcium ions are two major chemical factors that regulate connexin43 (Cx43) gap junction communication and the synergism or antagonism between pH and Ca2+ has been questioned for decades. To assess the ability of Ca2+ ions to modulate Cx43 junctional conductance (gj) in the absence of pH-sensitivity, patch clamp experiments were performed on Neuroblastoma-2a (N2a) cells or neonatal mouse ventricular myocytes (NMVMs) expressing either full-length Cx43 or the Cx43-M257 (Cx43K258stop) mutant protein, a carboxyl-terminus (CT) truncated version of Cx43 lacking pH-sensitivity. The addition of 1 μM ionomycin to normal calcium saline reduced Cx43 or Cx43-M257 gj to zero within 15 min of perfusion. This response was prevented by Ca2+-free saline or addition of 100 nM calmodulin (CaM) inhibitory peptide to the internal pipette solution. Internal addition of a connexin50 cytoplasmic loop calmodulin-binding domain (CaMBD) mimetic peptide (200 nM) prevented the Ca2+/ionomycin-induced decrease in Cx43 gj, while 100 μM Gap19 peptide had minimal effect. The investigation of the transjunctional voltage (Vj) gating properties of NMVM Cx43-M257 gap junctions confirmed the loss of the fast inactivation of Cx43-M257 gj, but also noted the abolishment of the previously reported facilitated recovery of gj from inactivating potentials. We conclude that the distal CT domain of Cx43 contributes to the Vj-dependent fast inactivation and facilitated recovery of Cx43 gap junctions, but the Ca2+/CaM-dependent gating mechanism remains intact in its absence. Sequence-specific connexin CaMBD mimetic peptides act by binding Ca2+/CaM non-specifically and the Cx43 mimetic Gap19 peptide has negligible effect on this chemical gating mechanism.

Mitochondrial Membrane Potential Oscillations Persist during Reperfusion After Ischemia in MCU Knockout Cardiomyocytes

Ca2+ entry via Mitochondrial Calcium Uniporter (MCU) participates in energetic adaption to workload under physiological conditions but is thought to contribute to cell death during ischemia-reperfusion (I/R) injury. Previous work has shown that mitochondrial membrane potential (ΔΨm) instability contributes to early-reperfusion arrhythmias and contractile dysfunction; however, the role of mitochondrial Ca2+ uptake in triggering ΔΨm oscillation versus sustained permeability transition pore (PTP) opening is unclear. We hypothesized that MCU-mediated Ca2+ uptake is required to trigger irreversible ΔΨm loss mediated by PTP but is not required for ΔΨm oscillation during early reperfusion, involving reactive oxygen species (ROS)-induced ROS release (RIRR). A spinning-disk confocal microscope was used to image ΔΨm(with TMRM) in monolayers of neonatal mouse ventricular myocytes (NMVM) during I/R (1hr/1hr). Acute MCU knockout (KO) was achieved by transducing floxed MCU NMVMs with an adenovirus expressing CRE-recombinase. ΔΨm response to I/R was analyzed at cellular level by segmentation analysis (ImageJ) and a new wavelet transform method was developed to characterize the dominant frequency of ΔΨm oscillation (Matlab). We found that surprisingly, the rate of ΔΨm loss during ischemia was greater in MCU-KO compared to control, yet the recovery of ΔΨm was more sustained, lacking a late decline after 30 min reperfusion. ΔΨm oscillations during reperfusion persisted in MCU-KO myocytes and wavelet analysis revealed a dominant frequency ranging from 0.022Hz-0.0088Hz in both WT and KO. These results suggest that MCU-mediated Ca2+ uptake is not required for RIRR induced ΔΨm oscillation but is involved in late reperfusion-induced sustained ΔΨm collapse; and that MCU-mediated Ca2+ uptake during reperfusion contributes to PTP-mediated ΔΨm collapse during reperfusion. However, ΔΨm oscillations during early reperfusion are independent of MCU-mediated Ca2+ entry, and likely to underlie mechanical and electrophysiological dysfunction observed during this period.

Exogenous IL-19 attenuates acute ischaemic injury and improves survival in male mice with myocardial infarction.

Myocardial infarction (MI) is one of the leading causes of death in China and often results in the development of heart failure. In this work, we tested the therapeutic role of Interleukin-19 (IL-19) in mice with MI and investigated the underlying molecular mechanism. Mice were subjected to MI by ligation of left anterior descending coronary artery (LAD) and treated with IL-19 (10ng g-1 ; i.p.). Protein expression of IL-19 and its receptor in myocardium were upregulated 24 hrs post-MI in male mice. IL-19 treatment decreased infarct and apoptosis in myocardium, accompanied by enhanced haem oxygenase-1 (HO-1) activities and reduced malondialdehyde (MDA) formation. Pretreatment with IL-19 upregulated HO-1 expression in cultured neonatal mouse ventricular myocytes and attenuated oxygen-glucose deprivation (OGD)-induced injuries in vitro. Furthermore, IL-19 preserved cardiac function and improved survival of mice with MI. IL-19 reduced inflammatory infiltrates and suppressed formation of TNF-α, IL-1β, and IL-6. More importantly, IL-19 inhibited polarization toward proinflammatory M1 macrophages and stimulated M2 macrophage polarization in myocardium of mice with MI. IL-19 enhanced protein levels of vascular endothelial growth factor (VEGF) and promoted angiogenesis in myocardium of mice with MI. In addition, IL-19 treatment increased DNA-binding of the transcription factor STAT3 in myocardium of mice with MI. Treatment with exogenous IL-19 attenuated acute ischemic injury and improved survival of mice with MI. The mechanisms underlying these effects involved induction of HO-1, M2 macrophage polarization, angiogenesis, and STAT3 activation.