Claudia M. Moreno

BACKGROUND: Every heartbeat is initiated by a spontaneous electrical signal generated inside the cardiac pacemaker. This signal depends on the coordinated activity of ion channels, where voltage-gated L-type calcium channels play a central role. All mammals experience a progressive decline in pacemaker rate with age, which in humans can become pathological and drive the need for artificial pacemaker implantation. Yet the mechanisms underlying this age-associated slowdown remain incompletely understood. METHODS: We combined patch-clamp electrophysiology, single-channel recording, calcium imaging, immunocytochemistry, and super-resolution microscopy to investigate L-type calcium channel density, organization, and function in sinoatrial node pacemaker cells from young and old mice. RESULTS: Aging reduced L-type calcium channel density at the plasma membrane, disrupted channel clustering, and decreased open probability, collectively diminishing calcium current density by 50%. Pharmacological enhancement of channel open probability with Bay K 8644 was sufficient to accelerate diastolic depolarization and restore pacemaker rate in old cells to young levels. These channel alterations were paralleled by a decline in caveolin-3 expression. Disrupting caveolae in young cells recapitulated the loss of channel clustering, pointing to caveolae as a structural basis for the age-associated channel alterations. CONCLUSIONS: Aging impairs L-type calcium channel organization and function in cardiac pacemaker cells. This impairment is mediated by an age-associated reduction of caveolae, key membrane microdomains for channel organization. The resulting decline in L-type calcium channel density and activity is an important driver of the age-associated slowdown of the cardiac pacemaker.

Summary: BK channels form nanoscale clusters, but the membrane and cytoskeletal mechanisms that organize these structures remain poorly understood. Using super-resolution imaging and FRAP, we dissect how cholesterol, PtdIns(4,5)P ₂ , and actin govern BK channel organization expressed heterologously in tsA-201 cells and expressed endogenously in INS-1 cells. Cholesterol depletion produced opposite outcomes across these systems, enlarging clusters in heterologous cells but reducing both size and density in INS-1 cells. PtdIns(4,5)P ₂ displayed a biphasic regulatory profile: both enrichment and depletion enlarged clusters, revealing that BK assemblies are tuned to a narrow PtdIns(4,5)P ₂ set point. Actin disruption consistently reduced cluster size and density. Together, these findings show that cholesterol, PtdIns(4,5)P ₂ , and actin are not only functional regulators of BK channels but also key determinants of their nanoscale organization. Across perturbations, changes in channel density strongly predicted changes in cluster size, identifying density as a central organizing principle. Significance statement: This study shows that the nanoscale organization of BK channels is dynamically controlled by membrane lipids and the cytoskeleton, demonstrating that ion channel organization is not fixed but actively shaped by the local membrane environment. Across perturbations, cluster size scales with channel density, underscoring density as a major driver of BK nanoscale organization.

Calcium binding to BK channels lowers BK activation threshold, substantiating functional coupling with calcium-permeable channels. This coupling requires close proximity between different channel types, and the formation of BK-Ca _V 1.3 hetero-clusters at nanometer distances exemplifies this unique organization. To investigate the structural basis of this interaction, we tested the hypothesis that BK and Ca _V 1.3 channels assemble before their insertion into the plasma membrane. Our approach incorporated four strategies: (1) detecting interactions between BK and Ca _V 1.3 proteins inside the cell, (2) identifying membrane compartments where intracellular hetero-clusters reside, (3) measuring the proximity of their mRNAs, and (4) assessing protein interactions at the plasma membrane during early translation. These analyses revealed that a subset of BK and Ca _V 1.3 transcripts are spatially close in micro-translational complexes, and their newly synthesized proteins associate within the endoplasmic reticulum (ER) and Golgi. Comparisons with other proteins, transcripts, and randomized localization models support the conclusion that BK and Ca _V 1.3 hetero-clusters form before their insertion at the plasma membrane.

Overactivity of the sympathetic nervous system is a hallmark of aging. The cellular mechanisms behind this overactivity remain poorly understood, with most attention paid to likely central nervous system components. In this work, we hypothesized that aging also affects the function of motor neurons in the peripheral sympathetic ganglia. To test this hypothesis, we compared the electrophysiological responses and ion-channel activity of neurons isolated from the superior cervical ganglia of young (12 weeks), middle-aged (64 weeks), and old (115 weeks) mice. These approaches showed that aging does impact the intrinsic properties of sympathetic motor neurons, increasing spontaneous and evoked firing responses. A reduction of M current emerged as a major contributor to age-related hyperexcitability. Thus, it is essential to consider the effect of aging on motor components of the sympathetic reflex as a crucial part of the mechanism involved in sympathetic overactivity.

Overactivity of the sympathetic nervous system is a hallmark of aging. The cellular mechanisms behind this overactivity remain poorly understood, with most attention paid to likely central nervous system components. In this work, we hypothesized that aging also affects the function of motor neurons in the peripheral sympathetic ganglia. To test this hypothesis, we compared the electrophysiological responses and ion-channel activity of neurons isolated from the superior cervical ganglia of young (12 weeks), middle-aged (64 weeks), and old (115 weeks) mice. These approaches showed that aging does impact the intrinsic properties of sympathetic motor neurons, increasing spontaneous and evoked firing responses. A reduction of M current emerged as a major contributor to age-related hyperexcitability. Thus, it is essential to consider the effect of aging on motor components of the sympathetic reflex as a crucial part of the mechanism involved in sympathetic overactivity.

Sinoatrial node (SAN) cells are the heart’s primary pacemaker. Their activity is tightly regulated by β-adrenergic receptor (β-AR) signaling. Adenylyl cyclase (AC) is a key enzyme in the β-AR pathway that catalyzes the production of cAMP. There are current gaps in our knowledge regarding the dominant AC isoforms and the specific roles of Ca ²⁺ -activated ACs in the SAN. The current study tests the hypothesis that distinct AC isoforms are preferentially expressed in the SAN and compartmentalize within microdomains to orchestrate heart rate regulation during β-AR signaling. In contrast to atrial and ventricular myocytes, SAN cells express a diverse repertoire of ACs, with AC _I as the predominant Ca ²⁺ -activated isoform. Although AC _I - KO ( AC _I ^–/– ) mice exhibit normal cardiac systolic or diastolic function, they experience SAN dysfunction. Similarly, SAN-specific CRISPR/Cas9-mediated gene silencing of AC _I results in sinus node dysfunction. Mechanistically, hyperpolarization-activated cyclic nucleotide-gated 4 (HCN4) channels form functional microdomains almost exclusively with AC _I , while ryanodine receptor and L-type Ca ²⁺ channels likely compartmentalize with AC _I and other AC isoforms. In contrast, there were no significant differences in T-type Ca ²⁺ and Na ⁺ currents at baseline or after β-AR stimulation between WT and ACI ^–/– SAN cells. Due to its central characteristic feature as a Ca ²⁺ -activated isoform, AC _I plays a unique role in sustaining the rise of local cAMP and heart rates during β-AR stimulation. The findings provide insights into the critical roles of the Ca ²⁺ -activated isoform of AC in sustaining SAN automaticity that is distinct from contractile cardiomyocytes.

Every heartbeat is initiated by a spontaneous electrical signal generated inside the cardiac pacemaker. The generation of this electrical signal depends on the coordinated opening and closing of different ion channels, where voltage-gated L-type calcium channels play a central role. Despite the reliability of the pacemaker, all mammals experience a linear slowdown of the pacemaker rate with age. In humans, this slowing can become pathological and constitutes the main cause for the requirement of the implantation of artificial pacemakers. However, the mechanisms behind the age-associated slowdown of the pacemaker are not well understood. Here, we show that age alters L-type calcium channels in pacemaker cells from mice. The age-associated alterations include: i) a reduction in the density of the channels at the plasma membrane, ii) a reduction in the clustering of the channels, and iii) a decrease in channel open probability. Altogether, these age-associated alterations result in a global reduction of the L-type calcium current density and in a slowdown of the pacemaker diastolic depolarization. Remarkably, increasing the open probability of L-type calcium channels pharmacologically was enough to restore pacemaker rate in old cells to the same levels observed in the young. Overall, our findings provide evidence that proper organization and function of L-type calcium channels is impaired by aging and that this dysfunction contributes to the slowdown of pacemaker cells in old animals.

Heart rate is accelerated to match physiological demands through the action of noradrenaline on the cardiac pacemaker. Noradrenaline is released from sympathetic terminals and activates β1-and β2-adrenergic receptors (ΑRs) located at the plasma membrane of pacemaker cells. L-type calcium channels are one of the main downstream targets potentiated by the activation of β-ARs. For this signaling to occur, L-type calcium channels need to be located in close proximity to β-ARs inside caveolae. Although it is known that aging causes a slowdown of the pacemaker rate and a reduction in the response of pacemaker cells to noradrenaline, there is a lack of in-depth mechanistic insights into these age-associated changes. Here, we show that aging affects the formation and function of adrenergic signaling microdomains inside caveolae. By evaluating the β1 and β2 components of the adrenergic regulation of the L-type calcium current, we show that aging does not alter the regulation mediated by β1-ARs but drastically impairs that mediated by β2-ARs. We studied the integrity of the signaling microdomains formed between L-type calcium channels and β-ARs by combining high-resolution microscopy and proximity ligation assays. We show that consistent with the electrophysiological data, aging decreases the physical association between β2-ARs and L-type calcium channels. Interestingly, this reduction is associated with a decrease in the association of L-type calcium channels with the scaffolding protein AKAP150. Old pacemaker cells also have a reduction in caveolae density and in the association of L-type calcium channels with caveolin-3. Together the age-dependent alterations in caveolar formation and the nano-organization of β2-ARs and L-type calcium channels result in a reduced sensitivity of the channels to β2 adrenergic modulation. Our results highlight the importance of these signaling microdomains in maintaining the chronotropic modulation of the heart and also pinpoint the direct impact that aging has on their function.

The cardiac pacemaker ignites and coordinates the contraction of the whole heart, uninterruptedly, throughout our entire life. Pacemaker rate is constantly tuned by the autonomous nervous system to maintain body homeostasis. Sympathetic and parasympathetic terminals act over the pacemaker cells as the accelerator and the brake pedals, increasing or reducing the firing rate of pacemaker cells to match physiological demands. Despite the remarkable reliability of this tissue, the pacemaker is not exempt from the detrimental effects of aging. Mammals experience a natural and continuous decrease in the pacemaker rate throughout the entire lifespan. Why the pacemaker rhythm slows with age is poorly understood. Neural control of the pacemaker is remodeled from birth to adulthood, with strong evidence of age-related dysfunction that leads to a downshift of the pacemaker. Such evidence includes remodeling of pacemaker tissue architecture, alterations in the innervation, changes in the sympathetic acceleration and the parasympathetic deceleration, and alterations in the responsiveness of pacemaker cells to adrenergic and cholinergic modulation. In this review, we revisit the main evidence on the neural control of the pacemaker at the tissue and cellular level and the effects of aging on shaping this neural control.