Pacemaker Channels and the Chronotropic Response in Health and Disease.

Loss or dysregulation of the normally precise control of heart rate via the autonomic nervous system plays a critical role during the development and progression of cardiovascular disease-including ischemic heart disease, heart failure, and arrhythmias. While the clinical significance of regulating changes in heart rate, known as the chronotropic effect, is undeniable, the mechanisms controlling these changes remain not fully understood. Heart rate acceleration and deceleration are mediated by increasing or decreasing the spontaneous firing rate of pacemaker cells in the sinoatrial node. During the transition from rest to activity, sympathetic neurons stimulate these cells by activating ß-adrenergic receptors and increasing intracellular cyclic adenosine monophosphate. The same signal transduction pathway is targeted by positive chronotropic drugs such as norepinephrine and dobutamine, which are used in the treatment of cardiogenic shock and severe heart failure. The cyclic adenosine monophosphate-sensitive hyperpolarization-activated current (If) in pacemaker cells is passed by hyperpolarization-activated cyclic nucleotide-gated cation channels and is critical for generating the autonomous heartbeat. In addition, this current has been suggested to play a central role in the chronotropic effect. Recent studies demonstrate that cyclic adenosine monophosphate-dependent regulation of HCN4 (hyperpolarization-activated cyclic nucleotide-gated cation channel isoform 4) acts to stabilize the heart rate, particularly during rapid rate transitions induced by the autonomic nervous system. The mechanism is based on creating a balance between firing and recently discovered nonfiring pacemaker cells in the sinoatrial node. In this way, hyperpolarization-activated cyclic nucleotide-gated cation channels may protect the heart from sinoatrial node dysfunction, secondary arrhythmia of the atria, and potentially fatal tachyarrhythmia of the ventricles. Here, we review the latest findings on sinoatrial node automaticity and discuss the physiological and pathophysiological role of HCN pacemaker channels in the chronotropic response and beyond.

mRNA trans-splicing dual AAV vectors for (epi)genome editing and gene therapy.

Large genes including several CRISPR-Cas modules like gene activators (CRISPRa) require dual adeno-associated viral (AAV) vectors for an efficient in vivo delivery and expression. Current dual AAV vector approaches have important limitations, e.g., low reconstitution efficiency, production of alien proteins, or low flexibility in split site selection. Here, we present a dual AAV vector technology based on reconstitution via mRNA trans-splicing (REVeRT). REVeRT is flexible in split site selection and can efficiently reconstitute different split genes in numerous in vitro models, in human organoids, and in vivo. Furthermore, REVeRT can functionally reconstitute a CRISPRa module targeting genes in various mouse tissues and organs in single or multiplexed approaches upon different routes of administration. Finally, REVeRT enabled the reconstitution of full-length ABCA4 after intravitreal injection in a mouse model of Stargardt disease. Due to its flexibility and efficiency REVeRT harbors great potential for basic research and clinical applications.

Hyperpolarization-activated cyclic nucleotide-gated cation channel 3 promotes HCC development in a female-biased manner.

Sex differences in hepatocellular carcinoma (HCC) development are regulated by sex and non-sex chromosomes, sex hormones, and environmental factors. We previously reported that Ncoa5+/- mice develop HCC in a male-biased manner. Here we show that NCOA5 expression is reduced in male patient HCCs while the expression of an NCOA5-interacting tumor suppressor, TIP30, is lower in female HCCs. Tip30 heterozygous deletion does not change HCC incidence in Ncoa5+/- male mice but dramatically increases HCC incidence in Ncoa5+/- female mice, accompanied by hepatic hyperpolarization-activated cyclic nucleotide-gated cation channel 3 (HCN3) overexpression. HCN3 overexpression cooperates with MYC to promote mouse HCC development, whereas Hcn3 knockout preferentially hinders HCC development in female mice. Furthermore, HCN3 amplification and overexpression occur in human HCCs and correlate with a poorer prognosis of patients in a female-biased manner. Our results suggest that TIP30 and NCOA5 protect against female liver oncogenesis and that HCN3 is a female-biased HCC driver.

Paradigm shift: new concepts for HCN4 function in cardiac pacemaking.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are the molecular correlate of the If current and are critically involved in controlling neuronal excitability and the autonomous rhythm of the heart. The HCN4 isoform is the main HCN channel subtype expressed in the sinoatrial node (SAN), a tissue composed of specialized pacemaker cells responsible for generating the intrinsic heartbeat. More than 40 years ago, the If current was first discovered in rabbit SAN tissue. Along with this discovery, a theory was proposed that cyclic adenosine monophosphate-dependent modulation of If mediates heart rate regulation by the autonomic nervous system-a process called chronotropic effect. However, up to the present day, this classical theory could not be reliably validated. Recently, new concepts emerged confirming that HCN4 channels indeed play an important role in heart rate regulation. However, the cellular mechanism by which HCN4 controls heart rate turned out to be completely different than originally postulated. Here, we review the latest findings regarding the physiological role of HCN4 in the SAN. We describe a newly discovered mechanism underlying heart rate regulation by HCN4 at the tissue and single cell levels, and we discuss these observations in the context of results from previously studied HCN4 mouse models.

MicroRNA-365 regulates human cardiac action potential duration.

Abnormalities of ventricular action potential cause malignant cardiac arrhythmias and sudden cardiac death. Here, we aim to identify microRNAs that regulate the human cardiac action potential and ask whether their manipulation allows for therapeutic modulation of action potential abnormalities. Quantitative analysis of the microRNA targetomes in human cardiac myocytes identifies miR-365 as a primary microRNA to regulate repolarizing ion channels. Action potential recordings in patient-specific induced pluripotent stem cell-derived cardiac myocytes show that elevation of miR-365 significantly prolongs action potential duration in myocytes derived from a Short-QT syndrome patient, whereas specific inhibition of miR-365 normalizes pathologically prolonged action potential in Long-QT syndrome myocytes. Transcriptome analyses in these cells at bulk and single-cell level corroborate the key cardiac repolarizing channels as direct targets of miR-365, together with functionally synergistic regulation of additional action potential-regulating genes by this microRNA. Whole-cell patch-clamp experiments confirm miR-365-dependent regulation of repolarizing ionic current Iks. Finally, refractory period measurements in human myocardial slices substantiate the regulatory effect of miR-365 on action potential in adult human myocardial tissue. Our results delineate miR-365 to regulate human cardiac action potential duration by targeting key factors of cardiac repolarization.

Implantation of Combined Telemetric ECG and Blood Pressure Transmitters to Determine Spontaneous Baroreflex Sensitivity in Conscious Mice.

Blood pressure (BP) and heart rate (HR) are both controlled by the autonomic nervous system (ANS) and are closely intertwined due to reflex mechanisms. The baroreflex is a key homeostatic mechanism to counteract acute, short-term changes in arterial BP and to maintain BP in a relatively narrow physiological range. BP is sensed by baroreceptors located in the aortic arch and carotid sinus. When BP changes, signals are transmitted to the central nervous system and are then communicated to the parasympathetic and sympathetic branches of the autonomic nervous system to adjust HR. A rise in BP causes a reflex decrease in HR, a drop in BP causes a reflex increase in HR. Baroreflex sensitivity (BRS) is the quantitative relationship between changes in arterial BP and corresponding changes in HR. Cardiovascular diseases are often associated with impaired baroreflex function. In various studies reduced BRS has been reported in e.g., heart failure, myocardial infarction, or coronary artery disease. Determination of BRS requires information from both BP and HR, which can be recorded simultaneously using telemetric devices. The surgical procedure is described beginning with the insertion of the pressure sensor into the left carotid artery and positioning of its tip in the aortic arch to monitor arterial pressure followed by the subcutaneous placement of the transmitter and ECG electrodes. We also describe postoperative intensive care and analgesic management. After a two-week period of post-surgery recovery long-term ECG and BP recordings are performed in conscious and unrestrained mice. Finally, we include examples of high-quality recordings and the analysis of spontaneous baroreceptor sensitivity using the sequence method.

cAMP-dependent regulation of HCN4 controls the tonic entrainment process in sinoatrial node pacemaker cells.

It is highly debated how cyclic adenosine monophosphate-dependent regulation (CDR) of the major pacemaker channel HCN4 in the sinoatrial node (SAN) is involved in heart rate regulation by the autonomic nervous system. We addressed this question using a knockin mouse line expressing cyclic adenosine monophosphate-insensitive HCN4 channels. This mouse line displayed a complex cardiac phenotype characterized by sinus dysrhythmia, severe sinus bradycardia, sinus pauses and chronotropic incompetence. Furthermore, the absence of CDR leads to inappropriately enhanced heart rate responses of the SAN to vagal nerve activity in vivo. The mechanism underlying these symptoms can be explained by the presence of nonfiring pacemaker cells. We provide evidence that a tonic and mutual interaction process (tonic entrainment) between firing and nonfiring cells slows down the overall rhythm of the SAN. Most importantly, we show that the proportion of firing cells can be increased by CDR of HCN4 to efficiently oppose enhanced responses to vagal activity. In conclusion, we provide evidence for a novel role of CDR of HCN4 for the central pacemaker process in the sinoatrial node.

A gene therapy for inherited blindness using dCas9-VPR-mediated transcriptional activation.

Catalytically inactive dCas9 fused to transcriptional activators (dCas9-VPR) enables activation of silent genes. Many disease genes have counterparts, which serve similar functions but are expressed in distinct cell types. One attractive option to compensate for the missing function of a defective gene could be to transcriptionally activate its functionally equivalent counterpart via dCas9-VPR. Key challenges of this approach include the delivery of dCas9-VPR, activation efficiency, long-term expression of the target gene, and adverse effects in vivo. Using dual adeno-associated viral vectors expressing split dCas9-VPR, we show efficient transcriptional activation and long-term expression of cone photoreceptor-specific M-opsin (Opn1mw) in a rhodopsin-deficient mouse model for retinitis pigmentosa. One year after treatment, this approach yields improved retinal function and attenuated retinal degeneration with no apparent adverse effects. Our study demonstrates that dCas9-VPR-mediated transcriptional activation of functionally equivalent genes has great potential for the treatment of genetic disorders.

Abolishing cAMP sensitivity in HCN2 pacemaker channels induces generalized seizures.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are dually gated channels that are operated by voltage and by neurotransmitters via the cAMP system. cAMP-dependent HCN regulation has been proposed to play a key role in regulating circuit behavior in the thalamus. By analyzing a knockin mouse model (HCN2EA), in which binding of cAMP to HCN2 was abolished by 2 amino acid exchanges (R591E, T592A), we found that cAMP gating of HCN2 is essential for regulating the transition between the burst and tonic modes of firing in thalamic dorsal-lateral geniculate (dLGN) and ventrobasal (VB) nuclei. HCN2EA mice display impaired visual learning, generalized seizures of thalamic origin, and altered NREM sleep properties. VB-specific deletion of HCN2, but not of HCN4, also induced these generalized seizures of the absence type, corroborating a key role of HCN2 in this particular nucleus for controlling consciousness. Together, our data define distinct pathological phenotypes resulting from the loss of cAMP-mediated gating of a neuronal HCN channel.

Protein kinase A regulates inflammatory pain sensitization by modulating HCN2 channel activity in nociceptive sensory neurons.

Several studies implicated cyclic adenosine monophosphate (cAMP) as an important second messenger for regulating nociceptor sensitization, but downstream targets of this signaling pathway which contribute to neuronal plasticity are not well understood. We used a Cre/loxP-based strategy to disable the function of either HCN2 or PKA selectively in a subset of peripheral nociceptive neurons and analyzed the nociceptive responses in both transgenic lines. A near-complete lack of sensitization was observed in both mutant strains when peripheral inflammation was induced by an intradermal injection of 8br-cAMP. The lack of HCN2 as well as the inhibition of PKA eliminated the cAMP-mediated increase of calcium transients in dorsal root ganglion neurons. Facilitation of Ih via cAMP, a hallmark of the Ih current, was abolished in neurons without PKA activity. Collectively, these results show a significant contribution of both genes to inflammatory pain and suggest that PKA-dependent activation of HCN2 underlies cAMP-triggered neuronal sensitization.

Comprehensive multilevel in vivo and in vitro analysis of heart rate fluctuations in mice by ECG telemetry and electrophysiology.

The normal heartbeat slightly fluctuates around a mean value; this phenomenon is called physiological heart rate variability (HRV). It is well known that altered HRV is a risk factor for sudden cardiac death. The availability of genetic mouse models makes it possible to experimentally dissect the mechanism of pathological changes in HRV and its relation to sudden cardiac death. Here we provide a protocol that allows for a comprehensive multilevel analysis of heart rate (HR) fluctuations. The protocol comprises a set of techniques that include in vivo telemetry and in vitro electrophysiology of intact sinoatrial network preparations or isolated single sinoatrial node (SAN) cells. In vitro preparations can be completed within a few hours, with data acquisition within 1 d. In vivo telemetric ECG requires 1 h for surgery and several weeks for data acquisition and analysis. This protocol is of interest to researchers investigating cardiovascular physiology and the pathophysiology of sudden cardiac death.