Aging Alters the Formation and Functionality of Signaling Microdomains Between L-type Calcium Channels and ß2-Adrenergic Receptors in Cardiac Pacemaker Cells.

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 ß1-and ß2-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 ß-ARs. For this signaling to occur, L-type calcium channels need to be located in close proximity to ß-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 ß1 and ß2 components of the adrenergic regulation of the L-type calcium current, we show that aging does not alter the regulation mediated by ß1-ARs but drastically impairs that mediated by ß2-ARs. We studied the integrity of the signaling microdomains formed between L-type calcium channels and ß-ARs by combining high-resolution microscopy and proximity ligation assays. We show that consistent with the electrophysiological data, aging decreases the physical association between ß2-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 ß2-ARs and L-type calcium channels result in a reduced sensitivity of the channels to ß2 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.

Slowing down as we age: aging of the cardiac pacemaker's neural control.

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.

Concomitant genetic ablation of L-type Cav1.3 (α1D) and T-type Cav3.1 (α1G) Ca2+ channels disrupts heart automaticity.

Cardiac automaticity is set by pacemaker activity of the sinus node (SAN). In addition to the ubiquitously expressed cardiac voltage-gated L-type Cav1.2 Ca2+ channel isoform, pacemaker cells within the SAN and the atrioventricular node co-express voltage-gated L-type Cav1.3 and T-type Cav3.1 Ca2+ channels (SAN-VGCCs). The role of SAN-VGCCs in automaticity is incompletely understood. We used knockout mice carrying individual genetic ablation of Cav1.3 (Cav1.3-/-) or Cav3.1 (Cav3.1-/-) channels and double mutant Cav1.3-/-/Cav3.1-/- mice expressing only Cav1.2 channels. We show that concomitant loss of SAN-VGCCs prevents physiological SAN automaticity, blocks impulse conduction and compromises ventricular rhythmicity. Coexpression of SAN-VGCCs is necessary for impulse formation in the central SAN. In mice lacking SAN-VGCCs, residual pacemaker activity is predominantly generated in peripheral nodal and extranodal sites by f-channels and TTX-sensitive Na+ channels. In beating SAN cells, ablation of SAN-VGCCs disrupted late diastolic local intracellular Ca2+ release, which demonstrates an important role for these channels in supporting the sarcoplasmic reticulum based "Ca2+ clock" mechanism during normal pacemaking. These data implicate an underappreciated role for co-expression of SAN-VGCCs in heart automaticity and define an integral role for these channels in mechanisms that control the heartbeat.

G protein-gated IKACh channels as therapeutic targets for treatment of sick sinus syndrome and heart block.

Dysfunction of pacemaker activity in the sinoatrial node (SAN) underlies "sick sinus" syndrome (SSS), a common clinical condition characterized by abnormally low heart rate (bradycardia). If untreated, SSS carries potentially life-threatening symptoms, such as syncope and end-stage organ hypoperfusion. The only currently available therapy for SSS consists of electronic pacemaker implantation. Mice lacking L-type Cav1.3 Ca(2+) channels (Cav1.3(-/-)) recapitulate several symptoms of SSS in humans, including bradycardia and atrioventricular (AV) dysfunction (heart block). Here, we tested whether genetic ablation or pharmacological inhibition of the muscarinic-gated K(+) channel (IKACh) could rescue SSS and heart block in Cav1.3(-/-) mice. We found that genetic inactivation of IKACh abolished SSS symptoms in Cav1.3(-/-) mice without reducing the relative degree of heart rate regulation. Rescuing of SAN and AV dysfunction could be obtained also by pharmacological inhibition of IKACh either in Cav1.3(-/-) mice or following selective inhibition of Cav1.3-mediated L-type Ca(2+) (ICa,L) current in vivo. Ablation of IKACh prevented dysfunction of SAN pacemaker activity by allowing net inward current to flow during the diastolic depolarization phase under cholinergic activation. Our data suggest that patients affected by SSS and heart block may benefit from IKACh suppression achieved by gene therapy or selective pharmacological inhibition.