Matteo E. Mangoni

Genesis and Regulation of the Heart Automaticity

The heart automaticity is a fundamental physiological function in higher organisms. The spontaneous activity is initiated by specialized populations of cardiac cells generating periodical electrical oscillations. The exact cascade of steps initiating the pacemaker cycle in automatic cells has not yet been entirely elucidated. Nevertheless, ion channels and intracellular Ca(2+) signaling are necessary for the proper setting of the pacemaker mechanism. Here, we review the current knowledge on the cellular mechanisms underlying the generation and regulation of cardiac automaticity. We discuss evidence on the functional role of different families of ion channels in cardiac pacemaking and review recent results obtained on genetically engineered mouse strains displaying dysfunction in heart automaticity. Beside ion channels, intracellular Ca(2+) release has been indicated as an important mechanism for promoting automaticity at rest as well as for acceleration of the heart rate under sympathetic nerve input. The potential links between the activity of ion channels and Ca(2+) release will be discussed with the aim to propose an integrated framework of the mechanism of automaticity.

Functional role of L-type Cav1.3 Ca2+ channels in cardiac pacemaker activity

The spontaneous activity of pacemaker cells in the sino-atrial node (SAN) controls the heart rhythm and rate under physiological conditions. Pacemaker activity in SAN cells is due to the presence of the diastolic depolarization, a slow depolarization phase that drives the membrane voltage from the end of an action potential to the threshold of a new action potential. SAN cells express a wide array of ionic channels, but we have limited knowledge about their functional role in pacemaker activity and we still do not know which channels play a prominent role in the generation of the diastolic depolarization. It is thus important to provide genetic evidence linking the activity of genes coding for ionic channels to specific alterations of pacemaker activity of SAN cells. Here, we show that target inactivation of the gene coding for α1D (Cav1.3) Ca2+ channels in the mouse not only significantly slows pacemaker activity but also promotes spontaneous arrhythmia in SAN pacemaker cells. These alterations of pacemaker activity are linked to abolition of the major component of the L-type current (ICa,L) activating at negative voltages. Pharmacological analysis of ICa,L demonstrates that Cav1.3 gene inactivation specifically abolishes ICa,L in the voltage range corresponding to the diastolic depolarization. Taken together, our data demonstrate that Cav1.3 channels play a major role in the generation of cardiac pacemaker activity by contributing to diastolic depolarization in SAN pacemaker cells.

Specific pattern of ionic channel gene expression associated with pacemaker activity in the mouse heart.

Even though sequencing of the mammalian genome has led to the discovery of a large number of ionic channel genes, identification of the molecular determinants of cellular electrical properties in different regions of the heart has been rarely obtained. We developed a high‐throughput approach capable of simultaneously assessing the expression pattern of ionic channel repertoires from different regions of the mouse heart. By using large‐scale real‐time RT‐PCR, we have profiled 71 channels and related genes in the sinoatrial node (SAN), atrioventricular node (AVN), the atria (A) and ventricles (V). Hearts from 30 adult male C57BL/6 mice were microdissected and RNA was isolated from six pools of five mice each. TaqMan data were analysed using the threshold cycle (Ct) relative quantification method. Cross‐contamination of each region was checked with expression of the atrial and ventricular myosin light chains. Two‐way hierarchical clustering analysis of the 71 genes successfully classified the six pools from the four distinct regions. In comparison with the A, the SAN and AVN were characterized by higher expression of Navβ1, Navβ3, Cav1.3, Cav3.1 and Cavα2δ2, and lower expression of Kv4.2, Cx40, Cx43 and Kir3.1. In addition, the SAN was characterized by higher expression of HCN1 and HCN4, and lower expression of RYR2, Kir6.2, Cavβ2 and Cavγ4. The AVN was characterized by higher expression of Nav1.1, Nav1.7, Kv1.6, Kvβ1, MinK and Cavγ7. Other gene expression profiles discriminate between the ventricular and the atrial myocardium. The present study provides the first genome‐scale regional ionic channel expression profile in the mouse heart.

Bradycardia and Slowing of the Atrioventricular Conduction in Mice Lacking CaV3.1/α1G T-Type Calcium Channels

The generation of the mammalian heartbeat is a complex and vital function requiring multiple and coordinated ionic channel activities. The functional role of low-voltage activated (LVA) T-type calcium channels in the pacemaker activity of the sinoatrial node (SAN) is, to date, unresolved. Here we show that disruption of the gene coding for Cav3.1/&agr;1G T-type calcium channels (cacna1g) abolishes T-type calcium current (ICa,T) in isolated cells from the SAN and the atrioventricular node without affecting the L-type Ca2+ current (ICa,L). By using telemetric electrocardiograms on unrestrained mice and intracardiac recordings, we find that cacna1g inactivation causes bradycardia and delays atrioventricular conduction without affecting the excitability of the right atrium. Consistently, no ICa,T was detected in right atrium myocytes in both wild-type and Cav3.1−/− mice. Furthermore, inactivation of cacna1g significantly slowed the intrinsic in vivo heart rate, prolonged the SAN recovery time, and slowed pacemaker activity of individual SAN cells through a reduction of the slope of the diastolic depolarization. Our results demonstrate that Cav3.1/T-type Ca2+ channels contribute to SAN pacemaker activity and atrioventricular conduction.

Loss of Ca v 1.3 ( CACNA1D ) function in a human channelopathy with bradycardia and congenital deafness

Deafness is genetically very heterogeneous and forms part of several syndromes. So far, delayed rectifier potassium channels have been linked to human deafness associated with prolongation of the QT interval on electrocardiograms and ventricular arrhythmia in Jervell and Lange-Nielsen syndrome. Cav1.3 voltage-gated L-type calcium channels (LTCCs) translate sound-induced depolarization into neurotransmitter release in auditory hair cells and control diastolic depolarization in the mouse sinoatrial node (SAN). Human deafness has not previously been linked to defects in LTCCs. We used positional cloning to identify a mutation in CACNA1D, which encodes the pore-forming α1 subunit of Cav1.3 LTCCs, in two consanguineous families with deafness. All deaf subjects showed pronounced SAN dysfunction at rest. The insertion of a glycine residue in a highly conserved, alternatively spliced region near the channel pore resulted in nonconducting calcium channels that had abnormal voltage-dependent gating. We describe a human channelopathy (termed SANDD syndrome, sinoatrial node dysfunction and deafness) with a cardiac and auditory phenotype that closely resembles that of Cacna1d−/− mice.

Properties of the hyperpolarization-activated current (If) in isolated mouse sino-atrial cells

OBJECTIVE We have investigated the properties of the hyperpolarization-activated (I(f)) current in pacemaker cells from the mouse sino-atrial node (SAN). METHODS The I(f) current was studied in cells isolated enzymatically from the SAN region of adult C57BL6/J mice. The whole-cell variation of the patch-clamp technique was employed to investigate the basic properties of I(f). RESULTS In mouse SAN cells, the I(f) current density at -120 mV was 18+/-2 pA/pF (n=23). I(f) was not detected in cells showing atrial-like morphology that were also found in SAN preparations (n=7). I(f) was blocked by 5 mM Cs(+), was inhibited by application of 5 microM acetylcholine, and was increased by 10 microM noradrenaline. The I(f) current reversal potential was -31+/-2 mV under physiological concentration of Na(+) and K(+) ions. Lowering the extracellular Na(+) concentration reduced I(f) amplitude, while increased when the extracellular K(+) concentration was augmented. I(f) voltage for half activation was -87+/-1 mV (n=6). CONCLUSIONS We conclude that the native I(f) current in mouse SAN cells shows functional properties that are similar to I(f) described in rabbit SAN tissue. This study opens the possibility of investigating the involvement of I(f) in the regulation of heart rate in genetically modified mice.

CHANGE IN MEMBRANE PERMEABILITY INDUCED BY PROTEGRIN 1 : IMPLICATION OF DISULPHIDE BRIDGES FOR PORE FORMATION

Protegrin 1 (PG‐1) is a naturally occurring cationic antimicrobial peptide that is 18 residues long, has an aminated carboxy terminus and contains two disulphide bridges. Here, we investigated the antimicrobial activity of PG‐1 and three linear analogues. Then, the membrane permeabilisation induced by these peptides was studied upon Xenopus laevis oocytes by electrophysiological methods. From the results obtained, we concluded that protegrin is able to form anion channels. Moreover, it seems clear that the presence of disulphide bridges is a prerequisite for the pore formation at the membrane level and not for the antimicrobial activity.

Functional Properties of a Newly Identified C-terminal Splice Variant of Cav1.3 L-type Ca2+ Channels

An intramolecular interaction between a distal (DCRD) and a proximal regulatory domain (PCRD) within the C terminus of long Cav1.3 L-type Ca2+ channels (Cav1.3L) is a major determinant of their voltage- and Ca2+-dependent gating kinetics. Removal of these regulatory domains by alternative splicing generates Cav1.342A channels that activate at a more negative voltage range and exhibit more pronounced Ca2+-dependent inactivation. Here we describe the discovery of a novel short splice variant (Cav1.343S) that is expressed at high levels in the brain but not in the heart. It lacks the DCRD but, in contrast to Cav1.342A, still contains PCRD. When expressed together with α2δ1 and β3 subunits in tsA-201 cells, Cav1.343S also activated at more negative voltages like Cav1.342A but Ca2+-dependent inactivation was less pronounced. Single channel recordings revealed much higher channel open probabilities for both short splice variants as compared with Cav1.3L. The presence of the proximal C terminus in Cav1.343S channels preserved their modulation by distal C terminus-containing Cav1.3- and Cav1.2-derived C-terminal peptides. Removal of the C-terminal modulation by alternative splicing also induced a faster decay of Ca2+ influx during electrical activities mimicking trains of neuronal action potentials. Our findings extend the spectrum of functionally diverse Cav1.3 L-type channels produced by tissue-specific alternative splicing. This diversity may help to fine tune Ca2+ channel signaling and, in the case of short variants lacking a functional C-terminal modulation, prevent excessive Ca2+ accumulation during burst firing in neurons. This may be especially important in neurons that are affected by Ca2+-induced neurodegenerative processes.

Control of heart rate by cAMP sensitivity of HCN channels

“Pacemaker” f-channels mediating the hyperpolarization-activated nonselective cation current If are directly regulated by cAMP. Accordingly, the activity of f-channels increases when cellular cAMP levels are elevated (e.g., during sympathetic stimulation) and decreases when they are reduced (e.g., during vagal stimulation). Although these biophysical properties seem to make f-channels ideal molecular targets for heart rate regulation by the autonomic nervous system, the exact contribution of the major If-mediating cardiac isoforms HCN2 and HCN4 to sinoatrial node (SAN) function remains highly controversial. To directly investigate the role of cAMP-dependent regulation of hyperpolarization activated cyclic nucleotide activated (HCN) channels in SAN activity, we generated mice with heart-specific and inducible expression of a human HCN4 mutation (573X) that abolishes the cAMP-dependent regulation of HCN channels. We found that hHCN4–573X expression causes elimination of the cAMP sensitivity of If and decreases the maximum firing rates of SAN pacemaker cells. In conscious mice, hHCN4–573X expression leads to a marked reduction in heart rate at rest and during exercise. Despite the complete loss of cAMP sensitivity of If, the relative extent of SAN cell frequency and heart rate regulation are preserved. Our data demonstrate that cAMP-mediated regulation of If determines basal and maximal heart rates but does not play an indispensable role in heart rate adaptation during physical activity. Our data also reveal the pathophysiologic mechanism of hHCN4–573X–linked SAN dysfunction in humans.

Piezo1 plays a role in erythrocyte volume homeostasis

Mechanosensitivity is an inherent property of virtually all cell types, allowing them to sense and respond to physical environmental stimuli. Stretch-activated ion channels represent a class of mechanosensitive proteins which allow cells to respond rapidly to changes in membrane tension; however their identity has remained elusive. The piezo genes have recently been identified as a family of stretch-activated mechanosensitive ion channels. We set out to determine the role of piezo1 during zebrafish development. Here we report that morpholino-mediated knockdown of piezo1 impairs erythrocyte survival without affecting hematopoiesis or differentiation. Our results demonstrate that piezo1 is involved in erythrocyte volume homeostasis, disruption of which results in swelling/lysis of red blood cells and consequent anemia.