Effect of Shenfu Injection on Differentiation of Bone Marrow Mesenchymal Stem Cells into Pacemaker-Like Cells and Improvement of Pacing Function of Sinoatrial Node.

Sick sinus syndrome (SSS), a complex type of cardiac arrhythmia, is a major health threat to humans. Shenfu injection (SFI), a formula of traditional Chinese medicine (TCM), is effective in improving bradyarrhythmia. However, the underlying mechanism of SFI's therapeutic effect is subject to few systematic investigations. The purpose of the present research is to examine whether SFI can boost the differentiation effectiveness of bone marrow mesenchymal stem cells (BMSCs) into pacemaker-like cells and whether the transplantation of these cells can improve the pacing function of the sinoatrial node (SAN) in a rabbit model of SSS. BMSCs from New Zealand rabbits were extracted, followed by incubation in vitro. The flow cytometry was utilized to identify the expression of CD29, CD44, CD90, and CD105 surface markers. The isolated BMSCs were treated with SFI, and the whole-cell patch-clamp method was performed to detect hyperpolarization-the activated cyclic nucleotide-gated potassium channel 4 (HCN4) channel current activation curve. The SSS rabbit model was established using the formaldehyde wet dressing method, and BMSCs treated with SFI were transplanted into the SAN of the SSS rabbit model. We detected changes in the body-surface electrocardiogram and recorded dynamic heart rate measurements. Furthermore, transplanted SFI-treated BMSCs were subjected to HE staining, TUNEL staining, qPCR, western blotting, immunofluorescence, immunohistochemistry, and enzyme-linked immunosorbent assay to study their characteristics. Our results indicate that the transplantation of SFI-treated BMSCs into the SAN of SSS rabbits improved the pacing function of the SAN. In vitro data showed that SFI induced the proliferation of BMSCs, promoted their differentiation capacity into pacemaker-like cells, and increased the HCN4 expression in BMSCs. In vivo, the transplantation of SFI treated-BMSCs preserved the function of SAN in SSS rabbits, improved the expression of the HCN4 gene and gap junction proteins (Cx43 and Cx45), and significantly upregulated the expression of cAMP in the SAN, compared to the SSS model group. In summary, the present research demonstrated that SFI might enhance the differentiation capacity of BMSCs into pacemaker-like cells, hence offering a novel approach for the development of biological pacemakers. Additionally, we confirmed the effectiveness and safety of pacemaker-like cells differentiated from BMSCs in improving the pacing function of the SAN.

Dexmedetomidine Exerts a Negative Chronotropic Action on Sinoatrial Node Cells Through the Activation of Imidazoline Receptors.

ABSTRACT: Dexmedetomidine (DEX), an α2-adrenoreceptor (α2-AR) and imidazoline receptor agonist, is most often used for the sedation of patients in the intensive care unit. Its administration is associated with an increased incidence of bradycardia; however, the precise mechanism of DEX-induced bradycardia has yet to be fully elucidated. This study was undertaken to examine whether DEX modifies pacemaker activity and the underlying ionic channel function through α2-AR and imidazoline receptors. The whole-cell patch-clamp techniques were used to record action potentials and related ionic currents of sinoatrial node cells in guinea pigs. DEX (≥10 nM) reduced sinoatrial node automaticity and the diastolic depolarization rate. DEX reduced the amplitude of hyperpolarization-activated cation current (If or Ih) the pacemaker current, even within the physiological pacemaker potential range. DEX slowed the If current activation kinetics and caused a significant shift in the voltage dependence of channel activation to negative potentials. In addition, efaroxan, an α2-AR and imidazoline I1 receptor antagonist, attenuated the inhibitory effects of DEX on sinoatrial node automaticity and If current activity, whereas yohimbine, an α2-AR-selective antagonist, did not. DEX did not affect the current activities of other channels, including rapidly and slowly activating delayed rectifier K+ currents (IKr and IKs), L-type Ca2+ current (ICa,L), Na+/Ca2+ exchange current (INCX), and muscarinic K+ current (IK,ACh). Our results indicate that DEX, at clinically relevant concentrations, induced a negative chronotropic effect on the sinoatrial node function through the downregulation of If current through an imidazoline I1 receptor other than the α2-AR in the clinical setting.

Tongyang Huoxue Decoction (TYHX) Ameliorating Hypoxia/Reoxygenation-Induced Disequilibrium of Calcium Homeostasis and Redox Imbalance via Regulating Mitochondrial Quality Control in Sinoatrial Node Cells.

Sick sinus syndrome (SSS) is a disease with bradycardia or arrhythmia. The pathological mechanism of SSS is mainly due to the abnormal conduction function of the sinoatrial node (SAN) caused by interstitial lesions or fibrosis of the SAN or surrounding tissues, SAN pacing dysfunction, and SAN impulse conduction accompanied by SAN fibrosis. Tongyang Huoxue Decoction (TYHX) is widely used in SSS treatment and amelioration of SAN fibrosis. It has a variety of active ingredients to regulate the redox balance and mitochondrial quality control. This study mainly discusses the mechanism of TYHX in ameliorating calcium homeostasis disorder and redox imbalance of sinoatrial node cells (SANCs) and clarifies the protective mechanism of TYHX on the activity of SANCs. The activity of SANCs was determined by CCK-8 and the TUNEL method. The levels of apoptosis, ROS, and calcium release were analyzed by flow cytometry and immunofluorescence. The mRNA and protein levels of calcium channel regulatory molecules and mitochondrial quality control-related molecules were detected by real-time quantitative PCR and Western Blot. The level of calcium release was detected by laser confocal. It was found that after H/R treatment, the viability of SANCs decreased significantly, the levels of apoptosis and ROS increased, and the cells showed calcium overload, redox imbalance, and mitochondrial dysfunction. After treatment with TYHX, the cell survival level was improved, calcium overload and oxidative stress were inhibited, and mitochondrial energy metabolism and mitochondrial function were restored. However, after the SANCs were treated with siRNA (si-ß-tubulin), the regulation of TYHX on calcium homeostasis and redox balance was counteracted. These results suggest that ß-tubulin interacts with the regulation of mitochondrial function and calcium release. TYHX may regulate mitochondrial quality control, maintain calcium homeostasis and redox balance, and protect SANCs through ß-tubulin. The regulation mechanism of TYHX on mitochondrial quality control may also become a new target for SSS treatment.

Bidirectional flow of the funny current (If) during the pacemaking cycle in murine sinoatrial node myocytes.

Sinoatrial node myocytes (SAMs) act as cardiac pacemaker cells by firing spontaneous action potentials (APs) that initiate each heartbeat. The funny current (If) is critical for the generation of these spontaneous APs; however, its precise role during the pacemaking cycle remains unresolved. Here, we used the AP-clamp technique to quantify If during the cardiac cycle in mouse SAMs. We found that If is persistently active throughout the sinoatrial AP, with surprisingly little voltage-dependent gating. As a consequence, it carries both inward and outward current around its reversal potential of -30 mV. Despite operating at only 2 to 5% of its maximal conductance, If carries a substantial fraction of both depolarizing and repolarizing net charge movement during the firing cycle. We also show that ß-adrenergic receptor stimulation increases the percentage of net depolarizing charge moved by If, consistent with a contribution of If to the fight-or-flight increase in heart rate. These properties were confirmed by heterologously expressed HCN4 channels and by mathematical models of If Modeling further suggested that the slow rates of activation and deactivation of the HCN4 isoform underlie the persistent activity of If during the sinoatrial AP. These results establish a new conceptual framework for the role of If in pacemaking, in which it operates at a very small fraction of maximal activation but nevertheless drives membrane potential oscillations in SAMs by providing substantial driving force in both inward and outward directions.

Impaired regulation of heart rate and sinoatrial node function by the parasympathetic nervous system in type 2 diabetic mice.

Heart rate (HR) and sinoatrial node (SAN) function are modulated by the autonomic nervous system. HR regulation by the parasympathetic nervous system (PNS) is impaired in diabetes mellitus (DM), which is denoted cardiovascular autonomic neuropathy. Whether blunted PNS effects on HR in type 2 DM are related to impaired responsiveness of the SAN to PNS agonists is unknown. This was investigated in type 2 diabetic db/db mice in vivo and in isolated SAN myocytes. The PNS agonist carbachol (CCh) had a smaller inhibitory effect on HR, while HR recovery time after CCh removal was accelerated in db/db mice. In isolated SAN myocytes CCh reduced spontaneous action potential firing frequency but this effect was reduced in db/db mice due to blunted effects on diastolic depolarization slope and maximum diastolic potential. Impaired effects of CCh occurred due to enhanced desensitization of the acetylcholine-activated K+ current (IKACh) and faster IKACh deactivation. IKACh alterations were reversed by inhibition of regulator of G-protein signaling 4 (RGS4) and by the phospholipid PIP3. SAN expression of RGS4 was increased in db/db mice. Impaired PNS regulation of HR in db/db mice occurs due to reduced responsiveness of SAN myocytes to PNS agonists in association with enhanced RGS4 activity.

A detailed characterization of the hyperpolarization-activated "funny" current (If) in human-induced pluripotent stem cell (iPSC)-derived cardiomyocytes with pacemaker activity.

Properties of the funny current (If) have been studied in several animal and cellular models, but so far little is known concerning its properties in human pacemaker cells. This work provides a detailed characterization of If in human-induced pluripotent stem cell (iPSC)-derived pacemaker cardiomyocytes (pCMs), at different time points. Patch-clamp analysis showed that If density did not change during differentiation; however, after day 30, it activates at more negative potential and with slower time constants. These changes are accompanied by a slowing in beating rate. If displayed the voltage-dependent block by caesium and reversed (Erev) at - 22 mV, compatibly with the 3:1 K+/Na+ permeability ratio. Lowering [Na+]o (30 mM) shifted the Erev to - 39 mV without affecting conductance. Increasing [K+]o (30 mM) shifted the Erev to - 15 mV with a fourfold increase in conductance. pCMs express mainly HCN4 and HCN1 together with the accessory subunits CAV3, KCR1, MiRP1, and SAP97 that contribute to the context-dependence of If. Autonomic agonists modulated the diastolic depolarization, and thus rate, of pCMs. The adrenergic agonist isoproterenol induced rate acceleration and a positive shift of If voltage-dependence (EC50 73.4 nM). The muscarinic agonists had opposite effects (Carbachol EC50, 11,6 nM). Carbachol effect was however small but it could be increased by pre-stimulation with isoproterenol, indicating low cAMP levels in pCMs. In conclusion, we demonstrated that pCMs display an If with the physiological properties expected by pacemaker cells and may thus represent a suitable model for studying human If-related sinus arrhythmias.

[Electrophysiological Effects of Ionophore-induced Increases in Intracellular Na+ in Cardiomyocytes].

Na ionophores increase intracellular Na+ ([Na+]i). Membrane potentials and currents were measured using microelectrode and whole-cell patch-clamp techniques. Monensin (10-6-3×10-5 M) reduced the slope of the pacemaker potentials and shortened the action potential duration (APD) in sino-atrial nodal and Purkinje cells. Monensin (10-5 M) shortened the APD and reduced the amplitude of the plateau phase in ventricular myocytes. Monensin decreased the hyperpolarization-activated inward current (If), and it increased the transient outward potassium current (Ito) in Purkinje cells. In addition, monensin decreased the sodium current (INa), shifting the inactivation curve to the hyperpolarized direction. Moreover, monensin decreased the L-type calcium current (ICa) in ventricular myocytes. The Na+-Ca2+ exchange current (INa-Ca) was augmented particularly in the reverse mode, and the Na+-K+ pump current (INa-K) was also activated by monensin in cardiomyocytes. The ATP-activated potassium current (IK,ATP) could be induced by monensin. Notably, the inward rectifying K+ current (IK1), and the slow delayed outward K+ current (IKs) were not affected evidently by monensin. Collectively, alteration of [Na+]i can influence the activities of various ion channels and transporters. Thus, the significance of altered [Na+]i should be taken into consideration in the action of drugs affecting [Na+]i such as digitalis, Na+ channel blockers, and Na+ channel activating agents.

Decreased cardiac pacemaking and attenuated ß-adrenergic response in TRIC-A knockout mice.

Changes in intracellular calcium levels in the sinus node modulate cardiac pacemaking (the calcium clock). Trimeric intracellular cation (TRIC) channels are counterion channels on the surface of the sarcoplasmic reticulum and compensate for calcium release from ryanodine receptors, which play a major role in calcium-induced calcium release (CICR) and the calcium clock. TRIC channels are expected to affect the calcium clock in the sinus node. However, their physiological importance in cardiac rhythm formation remains unclear. We evaluated the importance of TRIC channels on cardiac pacemaking using TRIC-A-null (TRIC-A-/-) as well as TRIC-B+/-mice. Although systolic blood pressure (SBP) was not significantly different between wild-type (WT), TRIC-B+/-, and TRIC-A-/-mice, heart rate (HR) was significantly lower in TRIC-A-/-mice than other lines. Interestingly, HR and SBP showed a positive correlation in WT and TRIC-B+/-mice, while no such correlation was observed in TRIC-A-/-mice, suggesting modification of the blood pressure regulatory system in these mice. Isoproterenol (0.3 mg/kg) increased the HR in WT mice (98.8 ±â€…15.1 bpm), whereas a decreased response in HR was observed in TRIC-A-/-mice (23.8 ±â€…5.8 bpm), suggesting decreased sympathetic responses in TRIC-A-/-mice. Electrocardiography revealed unstable R-R intervals in TRIC-A-/-mice. Furthermore, TRIC-A-/-mice sometimes showed sinus pauses, suggesting a significant role of TRIC-A channels in cardiac pacemaking. In isolated atrium contraction or action potential recording, TRIC-A-/-mice showed decreased response to a ß-adrenergic sympathetic nerve agonist (isoproterenol, 100 nM), indicating decreased sympathetic responses. In summary, TRIC-A-/-mice showed decreased cardiac pacemaking in the sinus node and attenuated responses to ß-adrenergic stimulation, indicating the involvement of TRIC-A channels in cardiac rhythm formation and decreased sympathetic responses.

Maurocalcin and its analog MCaE12A facilitate Ca2+ mobilization in cardiomyocytes.

Ryanodine receptors are responsible for the massive release of calcium from the sarcoplasmic reticulum that triggers heart muscle contraction. Maurocalcin (MCa) is a 33 amino acid peptide toxin known to target skeletal ryanodine receptor. We investigated the effect of MCa and its analog MCaE12A on isolated cardiac ryanodine receptor (RyR2), and showed that they increase RyR2 sensitivity to cytoplasmic calcium concentrations promoting channel opening and decreases its sensitivity to inhibiting calcium concentrations. By measuring intracellular Ca2+ transients, calcium sparks and contraction on cardiomyocytes isolated from adult rats or differentiated from human-induced pluripotent stem cells, we demonstrated that MCaE12A passively penetrates cardiomyocytes and promotes the abnormal opening of RyR2. We also investigated the effect of MCaE12A on the pacemaker activity of sinus node cells from different mice lines and showed that, MCaE12A improves pacemaker activity of sinus node cells obtained from mice lacking L-type Cav1.3 channel, or following selective pharmacologic inhibition of calcium influx via Cav1.3. Our results identify MCaE12A as a high-affinity modulator of RyR2 and make it an important tool for RyR2 structure-to-function studies as well as for manipulating Ca2+ homeostasis and dynamic of cardiac cells.

Contribution of estrogen to the pregnancy-induced increase in cardiac automaticity.

BACKGROUND: The heart rate progressively increases throughout pregnancy, reaching a maximum in the third trimester. This elevated heart rate is also present in pregnant mice and is associated with accelerated automaticity, higher density of the pacemaker current If and changes in Ca2+ homeostasis in sinoatrial node (SAN) cells. Strong evidence has also been provided showing that 17ß-estradiol (E2) and estrogen receptor α (ERα) regulate heart rate. Accordingly, we sought to determine whether E2 levels found in late pregnancy cause the increased cardiac automaticity associated with pregnancy. METHODS AND RESULTS: Voltage- and current-clamp experiments were carried out on SAN cells isolated from female mice lacking estrogen receptor alpha (ERKOα) or beta (ERKOß) receiving chronic E2 treatment mimicking late pregnancy concentrations. E2 treatment significantly increased the action potential rate (284 ± 24 bpm, +E2 354 ± 23 bpm, p = 0.040) and the density of If (+52%) in SAN cells from ERKOß mice. However, If density remains unchanged in SAN cells from E2-treated ERKOα mice. Additionally, E2 also increased If density (+67%) in nodal-like human-induced pluripotent stem cell-derived cardiomyocytes (N-hiPSC-CM), recapitulating in a human SAN cell model the effect produced in mice. However, the L-type calcium current (ICaL) and Ca2+ transients, examined using N-hiPSC-CM and SAN cells respectively, were not affected by E2, indicating that other mechanisms contribute to changes observed in these parameters during pregnancy. CONCLUSION: The accelerated SAN automaticity observed in E2-treated ERKOß mice is explained by an increased If density mediated by ERα, demonstrating that E2 plays a major role in regulating SAN function during pregnancy.

Inhibition of G protein-gated K+ channels by tertiapin-Q rescues sinus node dysfunction and atrioventricular conduction in mouse models of primary bradycardia.

Sinus node (SAN) dysfunction (SND) manifests as low heart rate (HR) and is often accompanied by atrial tachycardia or atrioventricular (AV) block. The only currently available therapy for chronic SND is the implantation of an electronic pacemaker. Because of the growing burden of SND in the population, new pharmacological therapies of chronic SND and heart block are desirable. We developed a collection of genetically modified mouse strains recapitulating human primary SND associated with different degrees of AV block. These mice were generated with genetic ablation of L-type Cav1.3 (Cav1.3-/-), T-type Cav3.1 (Cav3.1-/-), or both (Cav1.3-/-/Cav3.1-/-). We also studied mice haplo-insufficient for the Na+ channel Nav1.5 (Nav1.5+/) and mice in which the cAMP-dependent regulation of hyperpolarization-activated f-(HCN4) channels has been abolished (HCN4-CNBD). We analysed, by telemetric ECG recording, whether pharmacological inhibition of the G-protein-activated K+ current (IKACh) by the peptide tertiapin-Q could improve HR and AV conduction in these mouse strains. Tertiapin-Q significantly improved the HR of Cav1.3-/- (19%), Cav1.3-/-/Cav3.1-/- (23%) and HCN4-CNBD (14%) mice. Tertiapin-Q also improved cardiac conduction of Nav1.5+/- mice by 24%. Our data suggest that the development of pharmacological IKACh inhibitors for the management of SND and conduction disease is a viable approach.

Clinical efficacy of potassium canreonate-canrenone in sinus rhythm restoration among patients with atrial fibrillation - a protocol of a pilot, randomized, double -blind, placebo-controlled study (CANREN-AF trial).

BACKGROUND: Atrial fibrillation (AF) is the most frequent cardiac arrhythmia which increases the risk of thromboembolic complications and impairs quality of life. An important part of a therapeutic approach for AF is sinus rhythm restoration. Antiarrhythmic agents used in pharmacological cardioversion have limited efficacy and potential risk of proarrhythmia. Simultaneously, underlying conditions of AF should be treated (e.g. electrolyte imbalance, increased blood pressure, neurohormonal disturbances, atrial volume overload). There is still the need for an effective and safe approach to increase AF cardioversion efficacy. This randomized, double-blind, placebo-controlled, superiority clinical study is performed in patients with AF in order to evaluate the clinical efficacy of intravenous canrenone in sinus rhythm restoration. METHODS: Eighty eligible patients with an episode of AF lasting less than 48 h are randomized in a 1:1 ratio to receive canrenone or placebo. Patients randomized to a treatment intervention are receiving canrenone intravenously at a dose of 200 mg within 2-3 min. Subjects assigned to a control group obtain the same volume of 0.9% saline within the same time. The primary endpoint includes return of sinus rhythm documented in the electrocardiogram within 2 h after drug or placebo administration. Other endpoints and safety outcomes analyses, due to expected lack of statistical power, are exploratory. DISCUSSION: Current evidence supports renin-angiotensin-aldosterone system (RAAS) inhibition as an upstream therapy in AF management. Excess aldosterone secretion results in proarrhythmic effects. Among the RAAS inhibitors, only canrenone is administered intravenously. Canrenone additionally increases the plasma level of potassium, lowers blood pressure and reduces preload. It has been already used in primary and secondary hyperaldosteronism in the course of chronic liver dysfunction and in heart failure. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03536806. Registered on 25 May 2018.

The effect of forskolin on membrane clock and calcium clock in the hypoxic/reoxygenation of sinoatrial node cells and its mechanism.

BACKGROUND: In this study, we investigated the effect of forskolin (FSK, a selective adenylate cyclase agonist) on the automatic diastolic depolarization of sinus node cells (SNC) with hypoxia/reoxygenation (H/R) injury. METHODS: The SNC of the newborn rat was randomly assigned into the control group, the H/R (H/R injury) group, or the H/R + FSK (H/R injury + FSK treatment) group. Patch-clamp was performed to record the action potential and electrophysiological changes. The cellular distribution of intracellular calcium concentration was analyzed by fluorescence staining. RESULTS: Compared with the control cells, spontaneous pulsation frequency (SPF) and diastolic depolarization rate (DDR) of H/R cells were reduced from 244.3 ± 10.6 times/min and 108.7 ± 7.8 mV/s to 130.5 ± 7.6 times/min and 53.4 ± 6.5 mV/s, respectively. FSK significantly increased SPF and DDR of H/R cells to 208.3 ± 8.3 times/min and 93.2 ± 8.9 mV/s (n = 15, both p < 0.01), respectively. H/R reduced the current densities of If, ICa,T and inward INCX, which were significantly increased by 10 µM FSK treatment (n = 15, p < 0.01). Furthermore, reduced expression of HCN4 and NCX1.1 channel protein were significantly increased by FSK. Inhibitor studies showed that both SQ22536 (a selective adenylate cyclase inhibitor) and H89 (a selective protein kinases A [PKA] inhibitor) blocked the effects of FSK on SPF and DDR. CONCLUSIONS: H/R causes pacemaker dysfunction in newborn rat sinoatrial node cells leading to divergence of the DD and the slow of spontaneous APs, which change can be dramatically reversed by FSK through increasing INCX and If current in H/R injury.

Beyond Heart Failure and Ischemic Heart Disease: A Scoping Review of Novel Uses of Ivabradine in Adults.

Ivabradine lowers heart rate by inhibiting the hyperpolarization-activated current in pacemaker cells, and its use for the treatment of heart failure (HF) and ischemic heart disease (IHD) is well described. Ivabradine may be an attractive treatment option for other conditions for which a reduction in heart rate is desirable but less is known about its role in these settings. The primary objective was to perform a scoping review summarizing the literature evaluating novel uses for ivabradine other than HF and IHD in adults. PubMed and EMBASE were searched for articles for all dates through September 2019. Search strategies combined terms generic, commercial/trade, and international names for ivabradine. Manual search of references was also performed to identify additional articles. Studies were included if they were published in English, evaluated the efficacy of ivabradine for indications other than HF or IHD in patients aged 18 years or older, and the primary outcome included clinically relevant end points. Articles were screened first by title and abstract followed by full-text screening of the remaining articles. After removal of duplicates, 1807 records were screened for inclusion and 84 studies were included in this scoping review. Novel uses of ivabradine were reported for various tachyarrhythmias, valvular heart disease, premedication for coronary computed tomography angiography, perioperative risk reduction, sepsis with and without multi-organ dysfunction syndrome, cor pulmonale, reactive airway disease, and erectile dysfunction. This scoping review identified several potential novel uses for ivabradine in adults. This review may help to identify existing gaps where further research is needed to elucidate the role of ivabradine for indications beyond HF and IHD.

Mechanisms of Chronotropic Incompetence in Heart Failure With Preserved Ejection Fraction.

BACKGROUND: Chronotropic incompetence is common in heart failure with preserved ejection fraction (HFpEF) and is associated with impaired aerobic capacity. We investigated the integrity of cardiac ß-receptor responsiveness, an important mechanism involved in exertional increases in HR, in HFpEF and control subjects. METHODS: Thirteen carefully screened patients with HFpEF and 13 senior controls underwent exercise testing and graded isoproterenol infusion to quantify cardiac ß-receptor-mediated HR responses. To limit autonomic neural influences on heart rate (HR) during isoproterenol, dexmedetomidine and glycopyrrolate were given. Isoproterenol doses were increased incrementally until HR increased by 30 beats per minute. Plasma levels of isoproterenol at each increment were measured by liquid chromatography with electrochemical detection and plotted against HR. RESULTS: Peak VO2 and HR (117±15 versus 156±15 beats per minute; P<0.001) were lower in HFpEF than senior controls. Cardiac ß-receptor sensitivity was lower in HFpEF compared to controls (0.156±0.133 versus 0.254±0.166 beats per minute/[isoproterenol ng/mL]; P<0.001). Seven of 13 HFpEF subjects had ß-receptor sensitivity similar to senior controls but still had lower peak HRs (122±14 versus 156±15 beats per minute; P<0.001). CONCLUSIONS: Contrary to our hypothesis, patients with HFpEF displayed impaired cardiac ß-receptor sensitivity compared with senior controls. In the 7 out of 13 patients with HFpEF with age-appropriate ß-receptor sensitivity, peak HR remained low, suggesting impaired sinus node ß-receptor function may not fully account for low exercise HR response. Rather in some patients with HFpEF, chronotropic incompetence might reflect premature cessation of exercise before maximal sinus node activation. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02524145.

The bitter taste receptor agonist-induced negative chronotropic effects on the Langendorff-perfused isolated rat hearts.

Bitter taste receptors (Tas2rs), the members of the G-protein-coupled receptors, mediate the bitter taste and express in extra-oral tissues. Previous studies have shown that Tas2r mRNAs are expressed in the whole heart and cultured cardiomyocytes of neonatal rats. This study aimed to determine the expression of Tas2rs and their function in the adult rat hearts by using RT-qPCR techniques, Langendorff-perfused isolated hearts, and isolated sinoatrial (SA) nodes. The data presented here revealed the mRNA expression of Tas2rs and their coupled G-protein subunits in the SA node and left ventricle of adult rat hearts. Tas2r agonists, quinine and chloroquine, decreased the heart rate and increased the RR interval and QRS duration in Langendorff-perfused isolated rat hearts; they reduced the spontaneous beating rate of isolated SA nodes with pEC50 values of 4.907 ± 0.045 and 4.968 ± 0.030, respectively. The blockade of Tas2r108 with abscisic acid, the inhibition of phosphodiesterases (PDEs) with 3-isobutyl-1-methylxanthine (IBMX), or the selective inhibition of PDE3 and PDE4 with a cocktail of cilostamide and rolipram, attenuated the negative chronotropic effects of quinine and chloroquine on the SA node. Furthermore, quinine and chloroquine suppressed the tachycardia effect of isoprenaline on the SA node and shifted the concentration-response curve of isoprenaline rightward. In summary, we provided a few lines of evidence that Tas2r agonists, quinine and chloroquine, decreased the heart rate by prolonging ventricular depolarization, and by attenuating the SA node pace in a PDE-dependent manner; they can counteract with ß-adrenergic receptor activation and eliminate isoprenaline-induced tachycardia.

Aberrant sinus node firing during ß-adrenergic stimulation leads to cardiac arrhythmias in diabetic mice.

AIM: Cardiovascular complications, including cardiac arrhythmias, result in high morbidity and mortality in patients with type-2 diabetes mellitus (T2DM). Clinical and experimental data suggest electrophysiological impairment of the natural pacemaker of the diabetic heart. The present study examined sinoatrial node (SAN) arrhythmias in a mouse model of T2DM and physiologically probed their underlying cause. METHODS: Electrocardiograms were obtained from conscious diabetic db/db and lean control db/+ mice. In vivo SAN function was probed through pharmacological autonomic modulation with isoprenaline, atropine and carbachol. Blood pressure stability and heart rate variability (HRV) were evaluated. Intrinsic SAN function was evaluated through ex vivo imaging of spontaneous Ca2+ transients in isolated SAN preparations. RESULTS: While lean control mice showed constant RR intervals during isoprenaline challenge, the diabetic mice experienced SAN arrhythmias with large RR fluctuations in a dose-dependent manner. These arrhythmias were completely abolished by atropine pre-treatment, while carbachol pretreatment significantly increased SAN arrhythmia frequency in the diabetic mice. Blood pressure and HRV were comparable in db/db and db/+ mice, suggesting that neither augmented baroreceptor feedback nor autonomic neuropathy is a likely arrhythmia mechanism. Cycle length response to isoprenaline was comparable in isolated SAN preparations from db/db and db/+ mice; however, Ca2+ spark frequency was significantly increased in db/db mice compared to db/+ at baseline and after isoprenaline. CONCLUSION: Our results demonstrate a dysfunction of cardiac pacemaking in an animal model of T2DM upon challenge with a ß-adrenergic agonist. Ex vivo, higher Ca2+ spark frequency is present in diabetic mice, which may be directly linked to in vivo arrhythmias.

In silico study of the effects of anti-arrhythmic drug treatment on sinoatrial node function for patients with atrial fibrillation.

Sinus node dysfunction (SND) is often associated with atrial fibrillation (AF). Amiodarone is the most frequently used agent for maintaining sinus rhythm in patients with AF, but it impairs the sinoatrial node (SAN) function in one-third of AF patients. This study aims to gain mechanistic insights into the effects of the antiarrhythmic agents in the setting of AF-induced SND. We have adapted a human SAN model to characterize the SND conditions by incorporating experimental data on AF-induced electrical remodelling, and then integrated actions of drugs into the modified model to assess their efficacy. Reductions in pacing rate upon the implementation of AF-induced electrical remodelling associated with SND agreed with the clinical observations. And the simulated results showed the reduced funny current (If) in these remodelled targets mainly contributed to the heart rate reduction. Computational drug treatment simulations predicted a further reduction in heart rate during amiodarone administration, indicating that the reduction was the result of actions of amiodarone on INa, IKur, ICaL, ICaT, If and beta-adrenergic receptors. However, the heart rate was increased in the presence of disopyramide. We concluded that disopyramide may be a desirable choice in reversing the AF-induced SND phenotype.

Chronic heart failure increases negative chronotropic effects of adenosine in canine sinoatrial cells via A1R stimulation and GIRK-mediated IKado.

AIMS: Bradycardia contributes to tachy-brady arrhythmias or sinus arrest during heart failure (HF). Sinoatrial node (SAN) adenosine A1 receptors (ADO A1Rs) are upregulated in HF, and adenosine is known to exert negative chronotropic effects on the SAN. Here, we investigated the role of A1R signaling at physiologically relevant ADO concentrations on HF SAN pacemaker cells. MAIN METHODS: Dogs with tachypacing-induced chronic HF and normal controls (CTL) were studied. SAN tissue was collected for A1R and GIRK mRNA quantification. SAN cells were isolated for perforated patch clamp recordings and firing rate (bpm), slope of slow diastolic depolarization (SDD), and maximum diastolic potential (MDP) were measured. Action potentials (APs) and currents were recorded before and after addition of 1 and 10 µM ADO. To assess contributions of A1R and G protein-coupled Inward Rectifier Potassium Current (GIRK) to ADO effects, APs were measured after the addition of DPCPX (selective A1R antagonist) or TPQ (selective GIRK blocker). KEY FINDINGS: A1R and GIRK mRNA expression were significantly increased in HF. In addition, ADO induced greater rate slowing and membrane hyperpolarization in HF vs CTL (p < 0.05). DPCPX prevented ADO-induced rate slowing in CTL and HF cells. The ADO-induced inward rectifying current, IKado, was observed significantly more frequently in HF than in CTL. TPQ prevented ADO-induced rate slowing in HF. SIGNIFICANCE: An increase in A1R and GIRK expression enhances IKAdo, causing hyperpolarization, and subsequent negative chronotropic effects in canine chronic HF at relevant [ADO]. GIRK blockade may be a useful strategy to mitigate bradycardia in HF.