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The sinoatrial node (SAN) maintains a rhythmic heartbeat; therefore, a better understanding of factors that drive SAN development and function is crucial to generation of potential therapies, such as biological pacemakers, for sinus arrhythmias. Here, we determined that the LIM homeodomain transcription factor ISL1 plays a key role in survival, proliferation, and function of pacemaker cells throughout development. Analysis of several Isl1 mutant mouse lines, including animals harboring an SAN-specific Isl1 deletion, revealed that ISL1 within SAN is a requirement for early embryonic viability. RNA-sequencing (RNA-seq) analyses of FACS-purified cells from ISL1-deficient SANs revealed that a number of genes critical for SAN function, including those encoding transcription factors and ion channels, were downstream of ISL1. Chromatin immunoprecipitation assays performed with anti-ISL1 antibodies and chromatin extracts from FACS-purified SAN cells demonstrated that ISL1 directly binds genomic regions within several genes required for normal pacemaker function, including subunits of the L-type calcium channel, Ank2, and Tbx3. Other genes implicated in abnormal heart rhythm in humans were also direct ISL1 targets. Together, our results demonstrate that ISL1 regulates approximately one-third of SAN-specific genes, indicate that a combination of ISL1 and other SAN transcription factors could be utilized to generate pacemaker cells, and suggest ISL1 mutations may underlie sick sinus syndrome.

BackgroundNeural crest defects lead to congenital heart disease involving outflow tract malformation. Integrin-linked-kinase (ILK) plays important roles in multiple cellular processes and embryogenesis. ILK is expressed in the neural crest, but its role in neural crest and outflow tract morphogenesis remains unknown.ResultsWe ablated ILK specifically in the neural crest using the Wnt1-Cre transgene. ILK ablation resulted in abnormal migration and overpopulation of neural crest cells in the pharyngeal arches and outflow tract and a significant reduction in the expression of neural cell adhesion molecule (NCAM) and extracellular matrix components. ILK mutant embryos exhibited an enlarged common arterial trunk and ventricular septal defect. Reduced smooth muscle differentiation, but increased ossification and neurogenesis/innervation were observed in ILK mutant outflow tract that may partly be due to reduced transforming growth factor β2 (TGFβ2) but increased bone morphogenetic protein (BMP) signaling. Consistent with these observations, microarray analysis of fluorescence-activated cell sorting (FACS)-sorted neural crest cells revealed reduced expression of genes associated with muscle differentiation, but increased expression of genes of neurogenesis and osteogenesis.ConclusionsOur results demonstrate that ILK plays essential roles in neural crest and outflow tract development by mediating complex crosstalk between cell matrix and multiple signaling pathways. Changes in these pathways may collectively result in the unique neural crest and outflow tract phenotypes observed in ILK mutants.

Left ventricular (LV) diastolic function during atrial fibrillation (AF) remains poorly understood due to the complex interaction of factors and beat-to-beat variability. The purpose of the present study was to elucidate the physiological determinants of beat-to-beat changes in LV diastolic function during AF. The RR intervals preceding a given cardiac beat were measured from the right ventricular electrogram in 12 healthy open-chest mongrel dogs during AF. Doppler echocardiography and LV pressure and volume beat-to-beat analyses were performed. The LV filling time (FT) and early diastolic mitral inflow velocity-time integral ([E.sub.vti]) were measured using the pulsed Doppler method. The LV end-diastolic volume (EDV), peak systolic LV pressure (LVP), minimum value of the first derivative of LV pressure curve (dP/[dt.sub.min]), and the time constant of LV pressure decay ([tau]) were evaluated with the use of a conductance catheter for 100 consecutive cardiac cycles. Beat-to-beat analysis revealed a cascade of important causal relations. LV-FT showed a significant positive linear relationship with [E.sub.vti] (r = 0.87). Importantly, there was a significant positive linear relationship between the RR interval and LV-EDV in the same cardiac beat (r = 0.53). Consequently, there was a positive linear relationship between LV-EDV and subsequent peak systolic LVP (r = 0.82). Furthermore, there were significant positive linear and negative curvilinear relationships between peak systolic LVP and dP/[dt.sub.min] (r = 0.95) and [tau] (r = -0.85), respectively, in the same cardiac beat. In addition, there was a significant negative curvilinear relationship between dP/[dt.sub.min] and [tau] (r = -0.86). We have concluded that the determinants of LV diastolic function in individual beats during AF depend strongly on the peak systolic LVP. This suggests that the major benefit of slower ventricular rate appears related to lengthening of LV filling interval, promoting subsequent higher peak systolic LVP and greater LV relaxation. filling interval; left ventricular pressure

Although the beneficial effects of ventricular rate (VR) slowing during atrial fibrillation (AF) are axiomatic, the precise relationship between VR and hemodynamics has not been determined. We hypothesized that selective atrioventricular node (AVN) vagal stimulation (AVN-VS) by varying the nerve stimulation intensity could achieve precise graded slowing and permit evaluation of an optimal VR during AF. The aims of the present study were the following: 1) to develop a method for computerized vagally controlled VR slowing during AF, 2) to determine the hemodynamic changes at each level of VR slowing, and 3) to establish the optimal anterograde VR during AF. AVN-VS was delivered to the epicardial fat pad that projects parasympathetic nerve fibers to the AVN in 14 dogs. Four target average VR levels, corresponding to 75%, 100%, 125%, and 150% of the sinus cycle length (SCL), were achieved by computer feedback algorithm. VR slowing resulted in improved hemodynamics and polynomial fit analysis found an optimum for the cardiac output at VR slowing of 87% SCL. We conclude that this novel method can be used to maintain slow anterograde conduction with best hemodynamics during AF. heart rhythm; arrhythmia; hemodynamics; autonomic control; atrioventricular node

Echinacoside (ECH) is a compound derived from the natural herbs Cistanche and Echinacea, which has considerable protective effects on heart failure (HF). HF is characterized by myocardial damage and abnormal ferroptosis. Glutathione peroxidase 4 (GPX4) is an important regulator of ferroptosis, which plays a role in ferroptosis-related diseases. Despite this, the therapeutic mechanisms of ECH against HF remain unknown. Therefore, the aim of the present study was to investigate the cardioprotective effect and underlying mechanisms of ECH in the treatment of doxorubicin (DOX)-induced chronic HF (CHF). Cell proliferation was assessed using a CCK-8 assay. Furthermore, cardiac cell injury and oxidative stress were determined by measuring the lactate dehydrogenase (LDH), malondialdehyde (MDA), and glutathione (GSH) levels. The levels of Fe2+ and lipid reactive oxygen species (ROS), and expression of the biomarkers of ferroptosis, including GPX4 and prostaglandin-endoperoxide synthase 2 (PTGS2), were measured to examine cardiomyocyte ferroptosis. Additionally, RNA interference was used to silence Gpx4. In vitro and in vivo, ECH considerably reduced the MDA and LDH levels and increased the GSH level, thereby attenuating DOX-induced cardiac injury and oxidative stress. Meanwhile, ECH treatment decreased the lipid ROS levels and PTGS2 expression while increasing GPX4 expression, thereby alleviating DOX-induced cardiomyocyte ferroptosis. Moreover, knockdown of Gpx4 inhibited the protective effects of ECH on DOX-induced accumulation of lipid ROS in cardiomyocytes. These findings indicate that ECH can reduce DOX-induced cardiac injury by inhibiting ferroptosis via GPX4, highlighting its value as a potentially valuable therapeutic target in the management of CHF. [ABSTRACT FROM AUTHOR]

Although the atrioventricular node (AVN) plays a vital role in blocking many of the atrial impulses from reaching the ventricles during atrial fibrillation (AF), a rapid irregular ventricular rate nevertheless persists. The goals of the present study were to explore the feasibility of novel epicardial selective vagal nerve stimulation for slowing of the ventricular rate during AF and to characterize the hemodynamic benefits in vivo. Electrophysiological-echocardiographic experiments were performed on 11 anesthetized open-chest dogs. Hemodynamic measurements were performed during three distinct periods: 1) sinus rate, 2) AF, and 3) AF with vagal nerve stimulation. AF was associated with significant deterioration of all measured parameters (P [is less than] 0.025). The vagal nerve stimulation produced slowing of the ventricular rate, significant reversal of the pressure and contractile indexes (P [is less than] 0.025), and a sharp reduction in one-half of the abortive ventricular contractions. The present study provides comprehensive evidence that slowing of the ventricular rate during AF by selective ganglionic stimulation of the vagal nerves that innervate the AVN successfully improved the hemodynamic responses. ventricular rate; epicardial fat pads; localized nerve stimulation

The clinical assessment of left ventricular (LV) systolic function during atrial fibrillation (AF) is unreliable and difficult because of beat-to-beat variability. We evaluated an index for the estimation of LV systolic function in AF that is based on the relationship between the preceding (R-[R.sub.1]) and prepreceding (R-[R.sub.2]) R-R intervals. LV Doppler stroke volume (SV), ejection fraction (EF), peak aortic flow rate (AoF) and the maximum value of the first derivative of the LV pressure curve (dP/[dt.sub.max]) were evaluated in 13 healthy open-chest dogs during triggered AF. All parameters showed a significantly strong positive linear relationship with the ratio of R-[R.sub.1]/R-[R.sub.2] (r = 0.65, 0.74, 0.75, and 0.70 for SV, EF, AoF, and dP/[dt.sub.max], respectively). The calculated value of LV systolic parameters at R-[R.sub.1]/ R-[R.sub.2] -- 1 in the linear regression line showed a good relationship and an agreement with the measured average value of the parameter over all cardiac cycles (SV, 12.1 vs. 12.8 ml; EF, 49.6 vs. 51.2%; AoF, 1.37 vs. 1.48 ]/min; and dP/[dt.sub.max], 2,323 vs. 2,454 mmHg/s). Using the LV systolic parameters estimated at R-[R.sub.1]/ R-[R.sub.2] = 1 in the linear regression line allows the LV contractile function to be accurately and reproducibly evaluated during AF and obviates the less-reliable process of averaging multiple cardiac cycles. preload; afterload; contractility; R-R interval; echocardiography; left ventricular