Cardiac Pacemaker Activity and Aging.

A progressive decline in maximum heart rate (mHR) is a fundamental aspect of aging in humans and other mammals. This decrease in mHR is independent of gender, fitness, and lifestyle, affecting in equal measure women and men, athletes and couch potatoes, spinach eaters and fast food enthusiasts. Importantly, the decline in mHR is the major determinant of the age-dependent decline in aerobic capacity that ultimately limits functional independence for many older individuals. The gradual reduction in mHR with age reflects a slowing of the intrinsic pacemaker activity of the sinoatrial node of the heart, which results from electrical remodeling of individual pacemaker cells along with structural remodeling and a blunted ß-adrenergic response. In this review, we summarize current evidence about the tissue, cellular, and molecular mechanisms that underlie the reduction in pacemaker activity with age and highlight key areas for future work.

Culture and adenoviral infection of sinoatrial node myocytes from adult mice.

Pacemaker myocytes in the sinoatrial node of the heart initiate each heartbeat by firing spontaneous action potentials. However, the molecular processes that underlie pacemaking are incompletely understood, in part because of our limited ability to manipulate protein expression within the native cellular context of sinoatrial node myocytes (SAMs). Here we describe a new method for the culture of fully differentiated SAMs from adult mice, and we demonstrate that robust expression of introduced proteins can be achieved within 24-48 h in vitro via adenoviral gene transfer. Comparison of morphological and electrophysiological characteristics of 48 h-cultured versus acutely isolated SAMs revealed only minor changes in vitro. Specifically, we found that cells tended to flatten in culture but retained an overall normal morphology, with no significant changes in cellular dimensions or membrane capacitance. Cultured cells beat spontaneously and, in patch-clamp recordings, the spontaneous action potential firing rate did not differ between cultured and acutely isolated cells, despite modest changes in a subset of action potential waveform parameters. The biophysical properties of two membrane currents that are critical for pacemaker activity in SAMs, the "funny current" (If) and voltage-gated Ca(2+) currents (ICa), were also indistinguishable between cultured and acutely isolated cells. This new method for culture and adenoviral infection of fully-differentiated SAMs from the adult mouse heart expands the range of experimental techniques that can be applied to study the molecular physiology of cardiac pacemaking because it will enable studies in which protein expression levels can be modified or genetically encoded reporter molecules expressed within SAMs.

Cyclic AMP reverses the effects of aging on pacemaker activity and If in sinoatrial node myocytes.

Aerobic capacity decreases with age, in part because of an age-dependent decline in maximum heart rate (mHR) and a reduction in the intrinsic pacemaker activity of the sinoatrial node of the heart. Isolated sinoatrial node myocytes (SAMs) from aged mice have slower spontaneous action potential (AP) firing rates and a hyperpolarizing shift in the voltage dependence of activation of the "funny current," If Cyclic AMP (cAMP) is a critical modulator of both AP firing rate and If in SAMs. Here, we test the ability of endogenous and exogenous cAMP to overcome age-dependent changes in acutely isolated murine SAMs. We found that maximal stimulation of endogenous cAMP with 3-isobutyl-1-methylxanthine (IBMX) and forskolin significantly increased AP firing rate and depolarized the voltage dependence of activation of If in SAMs from both young and aged mice. However, these changes were insufficient to overcome the deficits in aged SAMs, and significant age-dependent differences in AP firing rate and If persisted in the presence of IBMX and forskolin. In contrast, the effects of aging on SAMs were completely abolished by a high concentration of exogenous cAMP, which restored AP firing rate and If activation to youthful levels in cells from aged animals. Interestingly, the age-dependent differences in AP firing rates and If were similar in whole-cell and perforated-patch recordings, and the hyperpolarizing shift in If persisted in excised inside-out patches, suggesting a limited role for cAMP in causing these changes. Collectively, the data indicate that aging does not impose an absolute limit on pacemaker activity and that it does not act by simply reducing the concentration of freely diffusible cAMP in SAMs.

Phosphodiesterases 3 and 4 Differentially Regulate the Funny Current, If, in Mouse Sinoatrial Node Myocytes.

Cardiac pacemaking, at rest and during the sympathetic fight-or-flight response, depends on cAMP (3',5'-cyclic adenosine monophosphate) signaling in sinoatrial node myocytes (SAMs). The cardiac "funny current" (If) is among the cAMP-sensitive effectors that drive pacemaking in SAMs. If is produced by hyperpolarization-activated, cyclic nucleotide-sensitive (HCN) channels. Voltage-dependent gating of HCN channels is potentiated by cAMP, which acts either by binding directly to the channels or by activating the cAMP-dependent protein kinase (PKA), which phosphorylates them. PKA activity is required for signaling between ß adrenergic receptors (ßARs) and HCN channels in SAMs but the mechanism that constrains cAMP signaling to a PKA-dependent pathway is unknown. Phosphodiesterases (PDEs) hydrolyze cAMP and form cAMP signaling domains in other types of cardiomyocytes. Here we examine the role of PDEs in regulation of If in SAMs. If was recorded in whole-cell voltage-clamp experiments from acutely-isolated mouse SAMs in the absence or presence of PDE and PKA inhibitors, and before and after ßAR stimulation. General PDE inhibition caused a PKA-independent depolarizing shift in the midpoint activation voltage (V1/2) of If at rest and removed the requirement for PKA in ßAR-to-HCN signaling. PDE4 inhibition produced a similar PKA-independent depolarizing shift in the V1/2 of If at rest, but did not remove the requirement for PKA in ßAR-to-HCN signaling. PDE3 inhibition produced PKA-dependent changes in If both at rest and in response to ßAR stimulation. Our results suggest that PDE3 and PDE4 isoforms create distinct cAMP signaling domains that differentially constrain access of cAMP to HCN channels and establish the requirement for PKA in signaling between ßARs and HCN channels in SAMs.

Mammalian γ2 AMPK regulates intrinsic heart rate.

AMPK is a conserved serine/threonine kinase whose activity maintains cellular energy homeostasis. Eukaryotic AMPK exists as αßγ complexes, whose regulatory γ subunit confers energy sensor function by binding adenine nucleotides. Humans bearing activating mutations in the γ2 subunit exhibit a phenotype including unexplained slowing of heart rate (bradycardia). Here, we show that γ2 AMPK activation downregulates fundamental sinoatrial cell pacemaker mechanisms to lower heart rate, including sarcolemmal hyperpolarization-activated current (I f) and ryanodine receptor-derived diastolic local subsarcolemmal Ca2+ release. In contrast, loss of γ2 AMPK induces a reciprocal phenotype of increased heart rate, and prevents the adaptive intrinsic bradycardia of endurance training. Our results reveal that in mammals, for which heart rate is a key determinant of cardiac energy demand, AMPK functions in an organ-specific manner to maintain cardiac energy homeostasis and determines cardiac physiological adaptation to exercise by modulating intrinsic sinoatrial cell behavior.

Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice.

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). These pacemaker APs reflect the coordinated activity of numerous membrane currents and intracellular calcium cycling. However the precise mechanisms that drive spontaneous pacemaker activity in SAMs remain elusive. Acutely isolated SAMs are an essential preparation for experiments to dissect the molecular basis of cardiac pacemaking. However, the indistinct anatomy, complex microdissection, and finicky enzymatic digestion conditions have prevented widespread use of acutely isolated SAMs. In addition, methods were not available until recently to permit longer-term culture of SAMs for protein expression studies. Here we provide a step-by-step protocol and video demonstration for the isolation of SAMs from adult mice. A method is also demonstrated for maintaining adult mouse SAMs in vitro and for expression of exogenous proteins via adenoviral infection. Acutely isolated and cultured SAMs prepared via these methods are suitable for a variety of electrophysiological and imaging studies.

High-efficiency reprogramming of fibroblasts into cardiomyocytes requires suppression of pro-fibrotic signalling.

Direct reprogramming of fibroblasts into cardiomyocytes by forced expression of cardiomyogenic factors, GMT (GATA4, Mef2C, Tbx5) or GHMT (GATA4, Hand2, Mef2C, Tbx5), has recently been demonstrated, suggesting a novel therapeutic strategy for cardiac repair. However, current approaches are inefficient. Here we demonstrate that pro-fibrotic signalling potently antagonizes cardiac reprogramming. Remarkably, inhibition of pro-fibrotic signalling using small molecules that target the transforming growth factor-ß or Rho-associated kinase pathways converts embryonic fibroblasts into functional cardiomyocyte-like cells, with the efficiency up to 60%. Conversely, overactivation of these pro-fibrotic signalling networks attenuates cardiac reprogramming. Furthermore, inhibition of pro-fibrotic signalling dramatically enhances the kinetics of cardiac reprogramming, with spontaneously contracting cardiomyocytes emerging in less than 2 weeks, as opposed to 4 weeks with GHMT alone. These findings provide new insights into the molecular mechanisms underlying cardiac conversion of fibroblasts and would enhance efforts to generate cardiomyocytes for clinical applications.

Levels of Ca(V)1.2 L-Type Ca(2+) Channels Peak in the First Two Weeks in Rat Hippocampus Whereas Ca(V)1.3 Channels Steadily Increase through Development.

Influx of calcium through voltage-dependent channels regulates processes throughout the nervous system. Specifically, influx through L-type channels plays a variety of roles in early neuronal development and is commonly modulated by G-protein-coupled receptors such as GABA(B) receptors. Of the four isoforms of L-type channels, only Ca(V)1.2 and Ca(V)1.3 are predominately expressed in the nervous system. Both isoforms are inhibited by the same pharmacological agents, so it has been difficult to determine the role of specific isoforms in physiological processes. In the present study, Western blot analysis and confocal microscopy were utilized to study developmental expression levels and patterns of Ca(V)1.2 and Ca(V)1.3 in the CA1 region of rat hippocampus. Steady-state expression of Ca(V)1.2 predominated during the early neonatal period decreasing by day 12. Steady-state expression of Ca(V)1.3 was low at birth and gradually rose to adult levels by postnatal day 15. In immunohistochemical studies, antibodies against Ca(V)1.2 and Ca(V)1.3 demonstrated the highest intensity of labeling in the proximal dendrites at all ages studied (P1-72). Immunohistochemical studies on one-week-old hippocampi demonstrated significantly more colocalization of GABA(B) receptors with Ca(V)1.2 than with Ca(V)1.3, suggesting that modulation of L-type calcium current in early development is mediated through Ca(V)1.2 channels.